Breeding of Saccharomyces cerevisiae

Chuan YANG　Jinglong LI

Abstract With three Saccharomyces cerevisiae strains collected by Institute of Modern Brewing Apparatus and Technology of Qilu University of Technology as experimental materials， an excellent S. cerevisiae strain， R2b， was selected by the plate separation culture method through the selection of separation medium， observation of appearance of yeast colonies， cell morphology and cell size， and the determination of cell death rate， diacetyl content， reducing power and fermentation power. The yeast strain can meet the demand for fermentation of fresh beer.

Key words Yeast; Separation and purification; Beer; Breeding

The brewing of beer is a complex process. From raw material selection to wort production， and finally to tank fermentation， each step is extremely important. The fermentation process of wort is the process of yeast metabolism. Yeast consumes nutrients in the wort and not only produces ethanol and CO2， but also produces flavor substances such as acids， phenols， aldehydes and sulfides， which not only form the taste of beer， but also have an important influence on the appearance of beer and the durability of the foam， and the dedicate degree[1]. Therefore， the quality of beer is largely determined by the quality of the yeast. It is not an exaggeration to call yeast the soul of beer. Yeast is generally referred to as singlecell fungi that can consume sugar. Such fungi can break down sugar into alcohol and carbon dioxide， and they are widely distributed in nature. They can not only perform aerobic respiration， but also survive in an environment without oxygen. Yeast is a natural starter. At the earliest time of human beings， yeast was used to produce beer. With the development of technology， people know more about yeast. The importance of yeast for beer brewing is irreplaceable. The taste of beer brewed by different yeast varieties varies widely[2]， and even the same yeast strain can produce different flavors of beer because of different brewing processes. Therefore， the selection of a highquality Saccharomyces cerevisiae strain is of decisive significance for the production of beer and the improvement of product quality.

For the general brewing enterprises， not only is the introduction of good yeast strains for fermentation production every time costly， but whether the yeast varieties are suitable for the production capacity in the current environment should be considered. Therefore， the isolation and cultivation of a yeast strain suitable for the taste of corresponding beer not only solves the problem of yeast survival adaptability， but also greatly saves the brewing cost. In this study， three kinds of preserved S. cerevisiae strains were selected from the Institute of Modern Brewing Apparatus and Technology  of Qilu University of Technology， and after amplification culture， primary screening was performed according to cell morphology in the solid medium first， according to the colony size and color. After the primary screening， the fermentation characteristics of yeast cells such as death rate， flocculability， fermentation degree， fermentation power， and postfermentation diacetyl content were tested for secondary screening of the yeast strains. The death rate of yeast cells indicates the number of yeast cells that die within a certain period of time， which is an important index to measure the survival time of yeast. Yeast flocculability plays a prominent role particularly in production， and different flocculability causes differences in precipitation speed. It is well known that yeast has certain taste， and for fresh wines that are not directly sold without filtration， if the yeast has poor flocculability， it can directly affect the taste of fresh beer. Furthermore， due to the suspension of yeast， the appearance of fresh beer is relatively cloudy， which affects sales[3]. The fermentation power of yeast indicates the rate of yeast fermentation in a certain period of time， and yeast with high fermentation power can be cooled in advance， thereby shortening the brewing time. In this study， based on the principle of low death rate， strong flocculability and high fermentation power， an excellent S. cerevisiae strain， R2b， was selected.

Materials and Equipment

Materials and reagents

Agar powder （pure）， Hangzhou Baisi Biotechnology Co.， Ltd.;yeast extract （BR）， Hangzhou Baisi Biotechnology Co.， Ltd.; peptone （BR）， Hangzhou Baisi Biotechnology Co.， Ltd.; sodium citrate （analytically pure）， Sinopharm Chemical Reagent Co.， Ltd.; methylene blue （analytically pure）， Sinopharm Chemical Reagent Co.， Ltd.; dipotassium hydrogen phosphate （analytically pure）， Sinopharm Chemical Reagent Co.， Ltd.; yeast extract powder （BR）， Hangzhou Baisi Biotechnology Co.， Ltd.; sodium acetate （analytically pure）， Sinopharm Chemical Reagent Co.， Ltd.; glucose （analytically pure）， Sinopharm Chemical Reagent Co.， Ltd.; magnesium sulfate （analytically pure）， Sinopharm Chemical Reagent Co.， Ltd.; manganese sulfate （analytically pure），  Sinopharm Chemical Reagent Co.， Ltd.; ginger beer wort （selfmade）， Institute of Modern Brewing Apparatus and Technology.

Experimental instruments

BXM50VE autoclave， Medical Equipment Factory of Shanghai Boxun Industry & Commerce Co.， Ltd.; SWCJ2D clean bench， Jiangsu Tongjing Purification Equipment Co.， Ltd.; standard blood counting chamber， Shanghai Qijing Biochemical Reagent Instrument Co.， Ltd.; SHP150 constant temperature incubator， Shanghai Jinghong Experimental Equipment Co.， Ltd.; SYG1220 water bath， Crystal Technology & Industries， Inc.; KH23A centrifuge， Hunan Kaida Scientific Instrument Co.， Ltd.; AUW320 Analytical Balance， Shimadzu Corporation， Japan; HWS20 constant temperature water bath， Shanghai Lixian Instrument Co.， Ltd.; TU1810 ultraviolet spectrophotometer， Beijing Haiguang Instrument Co.， Ltd.; WD800 microwave oven， Galanz microwave oven electric appliance Co.， Ltd.; ZWY211B shaker， Shanghai Zhicheng Analytical Instrument Manufacturing Co.， Ltd; H550S multifunction microscope， Nikon Corporation， Japan; 5 ml pipette， Eppendorf China Limited.

Experimental strains

The experimental strains used in this study were the strains isolatedand preserved for many years by the Institute of Modern Brewing Apparatus and Technology  of Qilu University of Technology： S. cerevisiae 1， S. cerevisiae 2 and S. cerevisiae 3.

Angel S. cerevisiae （SC） was used as a control.

Media

Walt extract medium： 10°P ginger beer wort was added with 2% of agar. The mixture was heated and melted， followed by sterilization in an autoclave at 0.1 MPa for 20 min. The sterilized mixture was then poured into sterilized plates for late use.

Yeast extract peptone dextrose medium （YPD）： A certain amountof yeast extract （10 g） and a certain amount of peptone （20 g）were dissolved in 900 ml of distilled water， and added with 20 g of agar powder. The mixture and 100 ml of glucose solution were sterilized in an autoclave at 0.1 MPa for 20 min. The sterilized glucose was then added to the mixture， the pH of which was then adjusted to 6.0. The obtained mixture was finally poured into sterilized plates for later use.

Broth medium： Into 300 ml of distilled water， ingredients were added as follows： peptone： 1%， beef extract powder： 0.5%， yeast extract powder： 0.4%， glucose： 2%， dipotassium hydrogen phosphate： 0.2%， sodium acetate： 0.5%， magnesium sulfate： 0.02%， and manganese sulfate： 0.005%. Finally， the pH was adjusted to 6.2-6.4.

Experimental Methods

Primary screening

S. cerevisiae 1， S. cerevisiae 2 and S. cerevisiae 3 were inoculated to YPD medium[4]. The UV lamp of a clean bench was turned on half an hour in advance for sterilization. After the clean bench was ventilated， an alcohol lamp was ignited， and the following operations were carried out near the alcohol lamp. The palms， nail gaps and wrists where bacteria can attach to easily were wiped with alcohol to sterilize them. Inoculating rings were washed， and in use， they were burnt to red on the flame of an alcohol lamp and employed after cooling immediately to pick S. cerevisiae 1， S. cerevisiae 2 and S. cerevisiae 3 separately from the strain preservation slant to the broth culture medium to activate them. Then， the activated S. cerevisiae 1， S. cerevisiae 2 and S. cerevisiae 3 were transferred to YPD medium with pipettes. The yeast strains were then smeared onto plates with coating sticks which had been sterilized on the flame of an alcohol lamp， and the plates were rapidly covered and labeled as R1， R2 and R3， respectively. The inoculated yeast strains were cultured at 25 ℃ in a constant temperature incubator for 72-96 h， followed by observation every day. The primary screening was carried out according to the bacterial morphology， colony size and color of the yeast in the solid medium. Three colonies which were fullbodied， moderately large and uniform in size， full and lustrous were selected from each of the three media， R1， R2 and R3， and inoculated to the wort slant medium. They were labeled as R1a， R1b， R1c， R2a， R2b， R2c， R3a， R3b and R3c， respectively， and cultured at 25 ℃ in a constant temperature incubator for 48-72 h.

Secondary screening

Secondary screening theory[5]： The corresponding colonies were transferred to the wort medium at 25 ℃ for activation culture. According to the yeast culture condition， the morphology and size were determined， and the yeast characteristic tests were carried out. Excellent strains with low death rate， strong fermentation ability and good flocculability were selected from the tests for calculating the death rate， fermentation degree and flocculability.

Secondary screening experiment： The yeast strains to be tested were inoculated into a liquid wort medium test tube， respectively， and cultured at 25 ℃ for 24 h. The cultures were diluted with distilled water to 40-70 yeast cells per check for later use.

Yeast morphology and size determination

The size of the cells was examined by microscopy and was measured with a micrometer.

Total number of yeast cells and death rate determination

Preparation of 0.01% methylene blue dyeing solution： A certain amount of sodium citrate （2 g） was added into a small beaker， and 20 ml of distilled water was measured with a measuring cylinder and added into the beaker， followed by heating to dissolve the sodium citrate in the beaker. Then， 0.01 g of methylene blue was added with stirring， obtaining the solution which was finally diluted with distilled water to 100 ml.

Into a test tube， 1 ml of the yeast to be tested was added， and 1 ml of the 0.01% methylene blue dye solution was then added， followed by mixing and standing for 5 min.

A clean blood cell counting plate was covered with a cover glass， and a drop of the mixture was dropped on the edge of the cover glass to allow the yeast cells to flow naturally into the countingplate， without generating bubbles in the counting chamber. After standing for 2 min， the checks were first found with a low power lens， which was then converted to a high power lens for counting.

The yeast cells and dead cells （blue） in five middle checks were counted. Each sample was counted for 3 times， and the results were averaged.

Death rate of Saccharomyces （%）=Number of dead cells in five middle checks/Total number of cells in five middle checks×100%[6]

Determination of yeast cell flocculability

Determination of yeast cell flocculability by Burns method [7]： The selected yeast was inoculated into 200 ml of sterilized medium， cultured in a constant temperature incubator at 25 ℃ for 48 h， and centrifuged at 600 r/min for 2 min. Then， 1 g of the yeast slurry was transferred to a centrifuge tube with a volume of 15 ml， which was then added with 10 ml of acetic acid buffer with a pH of 4.5. The mixture was kept at 20 ℃ for 20 min. The volume of the precipitate at the bottom of the centrifuge tube was recorded， and the value obtained was the Burns value.

Determination of yeast fermentation degree

Determination principle： Fermentation degree is determined according to the sugar consumed by yeast fermentation， that is， the reduction of wort sugar in the medium before and after fermentation.

Determination method： A certain amount of wort （300 ml） with a sugar content of 10°P （denoted as P） was added into a 500 mlconical flask. After sterilization， 5 ml of yeast sample solution was added into the flask， and culture was then performed in a constant temperature incubator at 25 ℃ for 48 h， with shaking once every 6 h. Finally， the remaining sugar degree was measured and recorded as M[8].

Fermentation degree =[（P-M）/P]×100%

Determination of yeast fermentation power

Fermentation principle： During the fermentation process， CO2 is produced， so the total mass of the fermentation liquid is lowered， and the mass of escaped CO2 is measured by the weight loss method and expressed as fermentation power.

A certain amount of wort （300 ml） was added into a 500 ml conical flask. After sterilization， 5 ml of the yeast sample solution was added into the flask， and the total mass was measured as m1. The conical flask was then put in a constant temperature incubator for 24 h of culture at 25 ℃， with shaking once every 6 h. The total mass was measured and recorded as m2[9].

Fermentation power=m2-m1

Diacetyl formation and determination of reduction capacity

Determination method： According to the national standard GB/T 4928， the content of diacetyl in the fermentation liquid was measured by the ophenylenediamine colorimetric method. The diacetyl in the sample was distilled out by a distillation device， and added with ophenylenediamine. The hydrochloride of diacetyl has a maximum absorption peak at the wavelength of 335 nm of ultraviolet light， and its content can be determined accordingly. Therefore， the absorbance of the sample was measured at the wavelength of 335 nm with an ultraviolet spectrophotometer and recorded as A335[10].

Diacetyl （mg/L）=A335×1.2

Results and Methods

Yeast cell and colony morphology

The observed morphology and sizes of the primarily screened yeast are shown in Table 1. According to the morphological sizes of the nine yeast strains， such four strains as R1a， R2a， R3b and R3c were weeded under following standards： long axis≥7.5，  short axis≥6.25， and the ratio of the two in the range of （1.3-1.1）∶1.The colonies of R1a were grayish white， which disaccords with the color of normal yeast which is milky yellow and milky white， so it was weeded out. The colonies of R2a were nontransparent with irregular edges. The colonies of R3b had untidy edges with a long axis not meeting the screening criteria. The colonies of R3c were nontransparent， and the cells long axis/short axis was relatively small， so it was also eliminated. Five yeast strains were selected， namely R1b， R1c， R2b， R2c and R3a. These five yeast strains met the selection criteria， regardless of colony morphology or cell size， so they were selected. They were then measured for death rate and total number of cells.

Chuan YANG et al. Breeding of Saccharomyces cerevisiae

Total number of yeast cells and death rate

The results of the total number of yeast cells and death rate are shown in Table 2. By measuring the total number of cells and death rate， it was found that the death rate was highest in R1c yeast and the lowest in R2b， and the total number of yeast cells was the highest in R1c and the lowest in R2c. Therefore， R1c and R2c were excluded， and the fermentation performance of the remaining yeast strains R1b， R2b and R3a were compared. Angel S. cerevisiae was used as a control group.

Fermentation performance of yeast

The fermentation performance of the selected yeast strains was measured， including flocculability， fermentation degree， fermentation power and diacetyl content. The determination results are shown in Table 3. It can be seen from the table that the three test strains and the control strain showed the fermentation degree order of R2b>R3a>R1b>SC; the fermentation power ranked as R3a>R2b>R1b>SC; the flocculability was in order of R2b>R1b>R3a>SC; and the diacetyl contents exhibited the order of R2b<R3a<SC<R1b.

In summary， the results of all screening tests showed that the fermentation power of fermentation strain R2b was strong， which also had a high fermentation speed and strong flocculability. Therefore， fermentation strain R2b was selected in this study and subjected to amplification culture after two times of activation.

Conclusions

Through above selection of the yeast colonies， isolation and purification of yeast species， strain proliferation and measurement of fermentation degree， it can be seen that R2b was prominent in the indexes such as colony morphology， cell number， death rate， proliferation and fermentation power. Therefore， this strain was identifiedas an excellent usable yeast strain.

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Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU