AU2021106385A4 - Wine yeast with low yield of hydrogen sulfide and ethyl carbamate and its screening method and application - Google Patents

Abstract

The invention discloses wine yeast with low yield of hydrogen sulfide (H2S) and ethyl carbamate (EC) and screening method and application. The name of the strain is Saccharomyces cerevisiae CEC LFN524, which has been preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC NO.4299 and the preservation date of November 5th, 2010. The strain was isolated and screened from the natural fermentation process of small seedless white grapes in Loulan Winery of Xinjiang, China. WLN (Wallerstein Laboratory Nutrient) culture medium was used to separate and purify the strains. The strain with low H2S production was screened by lead acetate test paper method and re-screened by H2S detection tube method, and the content of H2S and EC in the fermentation liquor was detected. Finally, the strain ware screened and obtained, and can be used in the wine making industry.

Description

Wine yeast with low yield of hydrogen sulfide and ethyl carbamate and its screening

method and application

TECHNICAL FIELD

The invention relates to the field of fermentation engineering, in particular to a wine yeast

with low yield of hydrogen sulfide and ethyl carbamate and its screening method and

application.

BACKGROUND

Hydrogen sulfide (H2S) is a volatile sulfide, which has an important effect on the flavor of

wine. In the fermentation process of wine, trace amounts of H2S are produced by sulfur

metabolism of S. cerevisiae. H2S is a volatile compound and has unpleasant odor, which is

usually described as rotten egg, skunk, garlic or onion flavor, and its odor threshold is 10

[g/L. If hydrogen sulfide is further combined with alcohol substances, mercaptan can

be formed. The influence of mercaptans on wine quality is two-sided. On the one hand,

some mercaptans are the components of wine aroma characteristics, for example, 4

mercapto -4- methyl -2- pentanone (4-MMP) can bring blackcurrant, boxwood and broom

aroma to Cabernet Sauvignon dry red wines and typical blackcurrant bud and cat urine

smell to Sauvignon Blanc dry white wines. On the other hand, lower mercaptan is toxic

and harmful to health, which destroy the flavor of wine and give people an unpleasant

feeling. Although their production level is only a few tens to several hundred micrograms

per liter, their sensory stimulation is obvious, which has a destructive effect on the flavor

of wine. Moreover, once its taste is formed, it is difficult to be removed simply by

ventilation and other general methods. In wine making, this kind of influence is common.

Ethyl Carbamate (EC, also known as Urethane) was proved to be a carcinogen as early as

1943. Studies show that EC is a multi-site carcinogen to rodents, which can cause lung

cancer, lymphoma, liver cancer and skin cancer. Ethanol can promote the carcinogenicity

of EC. In 1971, Lofroth and Gejvall discovered EC in wine, and in 1976, Ough detected

EC in yogurt and wine. The formation of EC in wine is related to the concentration of

arginine and urea in grape juice, nutritional additives of grape juice (such as diammonium

hydrogen phosphate) and the brewing technology of wine.

SUMMARY

The purpose of the invention is to provide wine yeast with low yield of H2S and EC and its

screening method and application, so as to solve the problems existing in the prior art.

To achieve the above purpose, the present invention provides the following scheme:

The wine yeast with low yield of H2S and EC is S cerevisiae CEC LFN524, which is

deposited at CGMCC for the preservation of microbial strains, with the preservation

number CGMCC NO.4299, and the preservation date of November 5th2010.

The invention also provides a screening method for wine yeast with low H2S and EC

production, which comprises the following steps: picking Xinjiang small seedless white

grape, taking fermentation juice in the natural fermentation process, adding 9 mL sterile

water into 1 mL fermentation juice sample for gradient dilution, coating WLN plate,

culturing at 28°C for 5 days. Then the single colonies of S. cerevisiae with smooth cream

color, conical protrusion in the center, opaque and creamy single colony with dark yellow

or light green color ring around were selected according to colony characteristics of S.

cerevisiaeon WLN, and then the single colony of S. cerevisiaeis selected from the purified

WLN culture medium and transferred to YEPD liquid medium, and after being cultured at

28°C for 2 days, the strain of S. cerevisiae is preserved by glycerol freezing method.

Marking YEPD culture solution on YEPD solid medium with inoculation ring, culturing at

28°C for 3 days, extracting yeast DNA, amplifying the 26S D1/D2 region of rDNA gene

by polymerase chain reaction, sequencing the PCR amplification products, and comparing

the sequencing results with the Nucleotide collection database in the national

biotechnology information center, identifying as S. cerevisiae strain. The preserved S.

cerevisiae strain was activated and then inoculated into the fermentation medium. The

liberation of H2S during fermentation was detected by lead acetate test paper, H2S detection

tube and methylene blue spectrophotometry, and the content of EC after the fermentation

was detected by GC-MS. The S. cerevisiae CEC LFN524 with low yield ofH2S and EC

was screened from 47 indigenous Saccharomyces strais.

The invention also provides an application of the wine yeast strain of S. cerevisiae CEC

LFN524 with low H2S and low EC yield in producing wine with high quality and health

security by low H2S and low EC yield.

The invention also provides an application of S. cerevisiae CEC LFN524 with low

production of H2S and EC in dry white wine and dry red wine making industry.

The invention discloses the following technical effects:

The wine yeast strain with low H2S and EC yield screened by the invention is separated

and screened from the natural fermentation process of small seedless white grapes in

Loulan Winery, Xinjiang, China. The wine yeast strain has excellent wine making

characteristics, has the functions of low H2S and EC production, and can be effectively

applied to the wine making industry, optimizes the wine process, and has important

significance for improving the quality of wine.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the colony characteristics of S. cerevisiae on WLN medium;

Fig. 2 is an Interdelta fingerprint of the S. cerevisiae CEC LFN524, the present invention;

Fig. 3 shows the fermentation curve of Xinjiang chardonnay wine made by S. cerevisiae

CEC LFN524 in 2010.

Fig.4 shows the sensory tasting score of industry scale wines fermenting by CEC LFN524

and commercial strains in 2010.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention will now be described in detail,

which should not be regarded as a limitation of the present invention, but rather as a more

detailed description of certain aspects, characteristics and embodiments of the present

invention.

Example 1 Screening of Wine Saccharomyces cerevisiae with Low H2S and EC yield.

1 Method

1.1 Isolation, purification, identification and preservation of yeast

100 g fresh grapes with good maturity are aseptically weighed, put into an aseptic 500 mL

triangular flask, crushed with an aseptic hammer, and cultured at 27C. 1 mL of

fermentation broth is taken every 24-72 hours, and then added into 9 mL of sterile water

for gradient dilution. 50 pL of dilution is put into WLN solid medium, coated uniformly

with an aseptic spatula, and cultured at 28°C for 5 days. According to the characteristics of

single colony of S. cerevisiae on WLN medium, the surface is smooth and creamy, with a

cone-shaped protrusion in the center, dark yellow or light green color ring around, opaque

and creamy. Fig. 1 is the map of S. cerevisiae on WLN medium in the experiment. The single colony of S. cerevisiae was selected and further purified on WLN culture medium.

The purified strain was transferred to YEPD liquid culture medium for 2 days at 28°C, and

the strain was preserved by glycerol freezing method (40% glycerol : yeast culture =1:1,

°C).

1.1.1 Extraction of yeast DNA

The YEPD culture solution was streaked and cultured on YEPD solid medium for 3 days

at 28°C, and then proper colony was selected to extract total DNA.

1.1.2 Amplification of D1/D2 region and 5.8S-ITS region of 26s rDNA

The 26S rDNA D1/D2 region was amplified using primers NL1 (5

gcatatcaaagcggaggaaag-3) and NL4 (5-ggtccggtttcaagacgg-3).

PCR cycle is: pre-denaturation at 95°C for 5 minutes, denaturation at 94°C for 1 minute,

annealing at 52°C for 1 minute, and extension at 72°C for 1 minute, with 36 cycles Maintain

temperature at 72°C for 8 minutes.

PCR reaction system (50 L system): 10*PCR buffer 5 L ; 25 mM MgCl2 3 pL ; 10

mM dNTP 1 L ; 10 M primers, 1 L each; 1.5 U Taq enzyme, 10 ng/L template DNA

1 L; At last, add double distilled water to constant volume of 50 pL .

The primers itsl (5-tccgtagtgaacctgcgg-3)/its4 (5-tcccgcttattgatatgc-3) were used to

amplify the 5.8S-ITS region gene of the strain. PCR cycle is 95°C for 95°C 5 minutes, 95°C

for 1 minute, 52°C for 2 minutes, 72°C for 2 minutes, with 35 cycles, and finally extended

for 10 minutes at 72°C. Total volume of PCR reaction system is 50 L, and PCR reaction

system is 10*PCR buffer (Taq buffer with KCl) 5 L; 25 mM MgCl2 6pL; 10 mM dNTPs

1 L; 2.5 L each of 10 M primers; Taq enzyme 2.0 U, template DNA 1l1; Add double

distilled water to 50 L).

Electrophoretic detection of PCR reaction products: take 5 L of PCR amplification stock

solution and put it on1% agarose gel (1*TAE buffer) for electrophoresis. After staining

with ethidium bromide (EB), it is preliminarily determined whether the desired fragment

is expanded under the irradiation of ultraviolet lamp. After successful amplification,

sequencing was carried out. Sequencing results were compared with the Nucleotide

collection(nr/nt) database of National Biotechnology Information Center (NCBI), and

whether the yeast strain was identified

1.1.3 Interdelta sequence amplification.

(1) Using genomic DNA as template, using optimized Interdelta primers for PCR

amplification; The forward and reverse primer sequences are as follows (Legras

& Karst,2003):

delta12(5'-TCAACAATGGAATCCCAAC-3');

delta21(5'-CATCTTAACACCGTATATGA-3');

(2) The PCR reaction system is 25 L, and 1* PCR buffer (10 mmol/L Tris-C1 pH 8.4, 50

mM KCl) is added to each tube; 2.5 mM MgC2; 0.5 pmol/L delta primer; 200 M dNTPs;

Template DNA30-100 ng, Taq enzyme 2.0 U, ddH20 was replenished to the reaction

system.

(3) PCR cycle is pre-denatured at 95°C for 4 minutes, denatured at 95°C for 30 s,

renaturated at 46°C for 30 s, extended at 72°C for 90 s, and the cycle times are 35 times.

Finally, 72°C for 10 minutes;

(4) The amplified product is electrophoresed on 2% agarose gel for 2.5 hours at 100V and

mA; Observe and take pictures on the ultraviolet imager.

Interdelta amplification map is the molecular fingerprint of the strain.

2 Results and analysis

2.1 Results of molecular identification of yeast strains

The 26S D1-D2 region sequence (577bp) is shown in SEQ ID No.1;

The similarity with related model strain NRRL Y-12632 was 99.30%, and it was identified

as S. cerevisiae.

Partial sequence of 5.8S-ITS (818bp) is shown in SEQ ID No.2;

Similarity with related model strain MUCL51208: 99.13%.

It was identified as S. cerevisiaeby molecular identification. As shown in Fig 1, the colony

characteristics of the S. cerevisiae was appeared on WLN medium.

2.1.3. Interdelta fingerprint:

Interdelta sequence amplification

With genomic DNA as template, the optimized Interdelta primers (delta12, delta2l) were

used for PCR amplification. The Interdelta fingerprint of strain CEC LFN524 is shown in

fig. 2.

2.2 Screening of low-yield hydrogen sulfide and ethyl carbamate

2.2.1 Screening and identification of strain CEC LFN524 with low H2S production.

The tested strains were fermented with triple M simulated juice, and the amount of H2S

produced was detected by H2S detection tube method for screening and identification. The

results showed that the H2S production of CEC LFN524 was 1.08 [g/L, which was far

lower than that of high-yield industrial yeast UCD522 (302.40 g/L) and low-yield

industrial yeast UCD819 (2.16 [tg/L). Therefore, according to the screening and

identification results, the strain CEC LFN524 is a S. cerevisiae strain with low hydrogen

sulfide production.

2.2.2 Strain CEC LFN524 produced low yield of EC

In order to promote the formation of EC, the temperature of simulated wine fermentation

was 28°C. The EC content of two strains with low H2S production were detected. The EC

content of CEC LFN524 was 9.66 [g/L, and that of UCD819 with low H2S production was

11.92 [g/L, both of which were lower than the EC content limit of 15 g/L. It belonged to

low EC strains. Up to now, CEC LFN524 has the characteristics of low production of

hydrogen sulfide and EC.

Example 2 Small-scale fermentation test of Chardonnay grape juice of wine S. cerevisiae

CEC LFN524 strain with low H2S and EC yield was optimized.

The fresh berries of white grape variety Chardonnay were juiced, 6% sulfurous acid was

added according to the total S02 content of 50 mg/L. The total sugar content of grape juice

was 210 g/L, the total acid was 7.5 g/L, the fermentation volume was 500 mL, and each

bottle was inoculated with the activated yeast solution of grape juice according to the

inoculation amount of 5*105 cfu/mL (count the strains cultured in logarithmic phase by

using blood cell counting plate to control the inoculation amount), and the control was

carried out. Because of the large amount of fermentation, it is impossible to install the H2S

detection tube device on the fermentation bottle. Therefore, the detection of H2S in wine

samples refers to GB/T16489-1996, by methylene blue spectrophotometry. Solid/liquid

extraction combined with GC-MS analysis was applied to determine EC. The fermentation

is finished when the reducing sugar content is less than 2 g/L, and the alcohol content, total

sugar, total acid (in tartaric acid) and volatile acid (in acetic acid) are detected according to

GB/T 15038-2006 after the alcohol fermentation.

The results showed that the H2S production of industrial strain UCD819 with low H2S

production was 65.51 [g/L, and that of strain UCD522 with high H2S production was

177.05 [g/L. However, the H2S content produced by the selected Saccharomyces

cerevisiae strain CEC LFN524 was 33.47 [g/L, which is far lower than that produced by

the industrial strain with low H2S yield, and has low yield characteristics. The content of

EC in Chardonnay dry white wine after fermentation was detected. It can be seen from the

physical and chemical parameters that the screened S. cerevisiae CEC LFN524 can be

finish fermentation completely, reaching the basic parameters of dry wine in national wine

standard GB15037-2006. The alcohol content of wine samples is >11%v/v, the total sugar

is <4.0 g/L, and the volatile acid is < 1.2 g/L; Meet the requirements of industrial

fermentation, so the strain CEC LFN524 can be used as the final screening target S.

cerevisiae strain.

Example 3 Winery fermentation test of wine S. cerevisiaestrain with low H2S and EC yield

was optimized.

In 2010, 2011 and 2012, the selected optimal strain CEC LFN524 with low H2S and EC

yield was fermented in industry scale in the wineries of Yuma, Ningxia, Rongzi, Shanxi

and Mogao, Gansu, respectively. To study its wine making characteristics, the fresh wine

grapes were broken after stem removed, and sulfurous acid was added until the free S02

content reached 45 mg/L. After 30 minutes, 20 mg/L pectinase was added. After mixing,

the total acid and sugar content of grape juice were determined. Fresh sulfur-adjusted grape

juice was used for strain activation. The test strains were inoculated into proper grape juice

with 3% inoculation amount, cultured in a constant temperature incubator at 28°C for 24

hours, and then the activated test strains were added into the fermentation tank with 106 cfu/mL inoculation amount, with two parallel strains for each strain to be tested. The fermenter was pumping over three times in the morning, in the middle and in the evening every day. After pumping over, samples were taken to monitor the temperature and specific gravity of the fermentation must. When the specific gravity reached 0.990-0.996, the reducing sugar was measured. When the reducing sugar content is less than 2 g/L, the fermentation is finished. After alcohol fermentation, alcohol content, total sugar, total acid, volatile acid and dry extract are measured according to GB/T 15038-2006. The fermentation curve of chardonnay grape CEC LFN524 produced in Ningxia in 2010 is shown in Figure

3.

The fermentation test for three consecutive years showed that CEC LFN524 has stable wine

making characteristics, with alcohol content >11%v/v and total sugar < 4. The hydrogen

sulfide content of CEC LFN524 wine was significantly lower than that of commercial

control, which belonged to low-yield hydrogen sulfide strain, and EC was not detected, and

its performance was recognized by wine making industry. As showed in Figure 4, the

wines fermented by CEC LFN524 were significantly better than that of the wines

fermented by commercial strains, evaluated both for the sensory tasting scores and for the

sensory characters. Years of multi-point wine brewing experiments confirmed that this

preferred strain had great potential for application and promotion in wine industry.

The above embodiments only describe the preferred mode of the invention, but do not limit

the scope of the invention. On the premise of not departing from the design spirit of the

invention, various modifications and improvements made by ordinary technicians in the

field to the technical scheme of the invention shall fall within the protection scope

determined by the claims of the invention.

Claims (4)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A wine yeast (Saccharomyces cerevisiae CEC LFN524) with low yield of

hydrogen sulfide (H2S) and ethyl carbamate (EC) is characterized, and the preservation

address is the CGMCC, the preservation number is CGMCC NO.4299, and the

preservation date is November 5th2010.

2. An application of the Saccharomyces cerevisiae CEC LFN524 according to

claim 1 is used for producing wine with low hydrogen sulfide (H2S) and ethyl carbamate

(EC) yield.

3. An application of the Saccharomyces cerevisiae CEC LFN524 according to

claim 1 in dry white wine making industry.

4. An application of the Saccharomyces cerevisiae CEC LFN524 according to

claim 1 in dry red wine making industry.