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Abstract The brewing characteristics of three kinds of beer yeasts commonly used in the market were compared and studied. The results showed that the three kinds of yeasts had different characteristics and slightly different fermentation degrees， but the fermentation speeds all were higher and the diacetyl reduction ability was excellent. The finished beers were golden yellow in appearance， rich and delicate in foam， pleasant in aroma， mellow in body and similar in physicochemical indexes， all in line with domestic beer standards.
Key words Strong beer; Ale yeast; Brewing characteristics
Strong ale beer， refers to the high ethanol concentration beer fermented by the top beer yeast[1]， which has the characteristics of rapid fermentation and strong aroma compared with the bottom fermented beer[2]. Strong ale beer has high ethanol concentration， mellow body and rich aroma， which is a top quality brewed beer favored by beer lovers in recent years[3-5]. Generally speaking， beer yeast with low alcohol tolerance will lead to low fermentation of beer[6]. As strong ale beer has high alcohol， the yeast with low alcohol production should be selected[7-9]. The low alcohol tolerance of ordinary beer yeast can lead to low fermentation degree of finished beer. Because of the high alcohol content of strong ale beer， the yeast producing low alcohols should be selected[10].
At the present， there are various kinds of yeasts on the market， but choosing the right yeast is undoubtedly the most important for the quality and productivity of beer. In this study， three kinds of beer yeasts commonly used in the market were selected， and a suitable strong beer yeast was selected by comparing the brewing performance and product index of the beer yeast， in order to provide some guidance for future beer brewing.
Materials and Methods
Materials
Pilsner Malt （4 EBC） and Caramel Malt （25 EBC）： Shandong Sinbach Biotechnology Co.， Ltd; Pelletized （T90） Magnum hops （14% αacids）; Yakima chief （HK） Ltd; Pelletized （T90） Saaz hops （4.5% αacids）： Batterhaas Trading Co.， Ltd; Dried Yeast S04， BE256， S33： Fermentis Yeast Co.， Ltd.
Instrument and equipment
ABF804C11EBC grinder： Buhler， AG; ZM30L vertical automatic fermentation tank： Jinan Zhengmai Machinery Equipment Co.， Ltd; WYTJ Handheld saccharimeter： Chengdu Haochang Photoelectric Instrument Co.， Ltd; SWCJ1BU sterile ultraclean worktable： Suzhou Shangdian Clean Technology Co.， Ltd; UV2350 ultravioletvisible spectrophotometer： especially Nico （Shanghai） Instrument Co.， Ltd. Methods
Malt milling
According to the original gravity of 16°P， the ingredients were crushed. The ratio of the two malts was 95% pilsner malt （4EBC） and 5% caramel malt （25EBC）.
Mashing
The wort was prepared using commercially available 100% malts， the proportion of malt and water was 1.0∶3.5 （kg∶L）. The comminuted malt was added to water at 52 ℃ and keep warm for 30 min， then heating up to 65 ℃ for 40 min， and 72 ℃ for 20 min， finally the temperature was increased to 78 ℃， and the mashing process was finished.
Wort filtration
The saccharified wort was poured into the filter tank， and the wort was filtered after standing for 20 min. Then the wort was washed twice with 78 ℃ water， and the whole process ensures that the wort was clear.
Wort boiling
Magnum hops was added to the first boiling with a rate of 0.3 g/L， and Saaz hops were added after boiling for 55 min with 0.1 g/L[11]. The whole boiling time was 60 min.
Cyclotron precipitation and wort cooling
The wort was transferred to a cyclotron settling tank after boiling， after standing for 20 min， the clarified wort was obtained， and then it was transferred to the fermentor after cooling to 18 ℃. The whole process was oxygenated with wort at a ratio of 8-10 mg/L.
Fermentation
Sterile water was boiled and cooled to 20 ℃， and dried yeast powder was added at a ratio of 0.8 g/L. After 15 min of stationary， yeast liquid was shaken and added to fermentor. Main fermentation was conducted when the wort was filled at 20 ℃. When the sugar content of wort was reduced to 5 °P， the tank was sealed for reducing the content of diacetyl， and the pressure was controlled at 0.14-0.16 MPa[12].
Cooling and storage
When the content of diacetyl was less than 0.08 mg/L， the beer was cooled to 0-1 ℃， and the pressure was controlled to 0.12-0.14 MPa. The beer ripened after storage for one week.
Testing of physical and chemical indexes of beer
During the fermentation process， the determination of sugar， alcohol， diacetyl and other physical and chemical indicators was performed in accordance with GB/T 49272008.
Sensory evaluation of 3 finished beers using trained panelists
Ten trained panelists with previous experience evaluating beer were asked to evaluate the finished beers for both taste and aroma. Judging from the four aspects of appearance， foam， aroma and taste， the full score was 100 points， we took the average score[13]. The Sensory evaluation standard was shown in Table 1. Results and Discussion
The change of sugar content of fermentation broth with fermentation time is shown in Fig. 1. Fig 1 shows that the three dried yeasts had a relatively fast rate of sugar reduction during the first three days， which was the peak period of fermentation; the sugar content could be reduced to 5 °P after 3 or 4 d to seal the tank， and then the rate of sugar reduction significantly slowed down， and tended to be stable. The rate of degrading sugar of S04 and BE256 were faster than S33 during the first 2 d， S33 was faster than S04 on the fourth day， BE256 was superior to the other two yeasts in fermentation speed and fermentation performance.
The change of alcohol content with fermentation time is shown in Fig. 2. Yeast produces alcohol by consuming sugars by anaerobic respiration[14]. As shown in Fig. 2， the rate of degrading sugar was very fast during the first 3 d， more alcohol will be produced， and then the alcohol content will rise slowly and the alcohol content will be stabilized at about 6% Vol. The fermentation degree of the three yeasts is BE256 alcohol > S33 > S04.
The change of diacetyl with fermentation time is shown in Fig. 3. Diacetyl is a flavor substance in beer， which is a sign of beer maturity[15]. It is generally necessary to ensure that the content of diacetyl in beer is less than 0.1 mg/L. As shown in Fig. 3， the diacetyl reduction rate of the three yeasts was faster， and the diacetyl content of the three yeasts could be reduced below 0.1 mg/L on the 8th day. The diacetyl reduction ability of the three yeasts was S04 > BE256 > S33.
The sensory evaluation of three yeast fermented beer is compared with that shown in Fig. 4. The sensory score of BE256 > S04 > S33 is shown in Fig. 4. On the whole， the fermentation characteristics of BE256 yeast is excellent， the sensory score reaches 92， and the comprehensive situation of the finished beer is ideal. The fermentation performance of S04 and S33 yeasts is close.
The physicochemical properties of the beer fermented by the three yeasts are shown in Table 2. The original gravity of the three beers is close， the alcohol content and fermentation degree are slightly different， and the fermentation degree of BE256 yeast is better than the other two. On the whole， the indicators are close to each other and are in line with national standards. The finished beers have golden yellow appearance， rich and delicate foam， pleasant aroma and strong taste， all belong to high quality beer. Conclusions
The fermentation performance of three strong ale yeasts was compared and analyzed. The results showed that the physical and chemical indexes of these three fermented beer met national standards. The finished beers were golden yellow with rich foam， delicate fragrance and strong taste. The three beer yeasts were excellent in reducing sugar speed， fermentation degree and diacetyl reduction ability. On the whole， the fermentation performance of BE256 yeast was better than that of the other two yeasts， which had a certain guiding role in the future production of strong ale beer.
References
[1] WEISENBERGER C. Craft beer boom creates hops shortage[J]. Supermarket News Expert Blog， 2014.
[2] MIGNANI AG， CIACCHERI L， MENCAGLIA AA， et al. Optical measurements and pattern recognition techniques for autheticating topfermented and bottomfermented beers and predicting the alcoholic strength[C]∥ Sensors. IEEE， 2012： 1-4.
[3] LANDSCHOOT AV， VANDERPUTTEN D， STALS I， et al. Spectrum of polysaccharides degradation products of ales and lager beers[C].// European Conference on Food Chemistry. Gesellschaft Deutscher Chemiker， 2005： 615-618.
[4] KRIZEK M. Assesment of biogenic amines in beer[J]. Kvasny Prumysl， 1995.
[5] HUVAERE K， SINNAEVE B， VAN BJ， et al. Photooxidative degradation of beer bittering principles： product analysis with respect to lightstruck flavour formation.[J]. Photochem Photobiol Sci， 2004， 3（9）： 854-858.
[6] MOHREKESH M， SHAHRAKI ADF， GHALAMKARI GR， et al. Effects of three methods of oral seleniumenriched yeast supplementation on blood components and growth in Holstein dairy calves[J]. Animal Production Science， 2018.
[7] QUAN QX， CHEN LQ， WEI RL， et al. Study on the production of seleniumenriched pork by using the seleniumenriched yeast[J]. Food Research & Development， 2016.
[8] WANG Z， ZHANG L， TAN T. High cell density fermentation of Saccharomyces cerevisiae， GS2 for seleniumenriched yeast production[J]. Korean Journal of Chemical Engineering， 2010， 27（6）： 1836-1840.
[9] FREDLUND E， BEERLAGE C， MELIN P， et al. Oxygen and carbon sourceregulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala[J]. Yeast， 2010， 23（16）：1137-1149.
[10] CASAREGOLA S， NGUYEN HV， LAPATHITIS G， et al. Analysis of the constitution of the beer yeast genome by PCR， sequencing and subtelomeric sequence hybridization[J]. Int.j.syst.evol.microbiol， 2001， 51（4）： 1607-1618.
[11] ZHANG XQ. Discussion on addition of hop pellets[J]. Global Alcinfo， 2017（17）： 33-35. （in Chinese）
[12] ZHOU GT. Modern beer technology[M]. Beijing： Chemical Industry Press， 2007： 81-82. （in Chinese）
[13] DONG XL， ZHOU GT， CUI YQ. Sensory evaluation of beer[M]. Beijing： Chemical Industry Press， 2008： 126-131. （in Chinese）
[14] ZHOU B， ZHANG CY， CHEN YF， et al. Effects of high gravity and high temperature on fermentation performance of beer yeast[J]. China Brewing， 2016， 35（2）： 9-12. （in Chinese）
[15] KAN X. Preliminary study on high alcohol beer[D]. Wuxi： Jiangnan University， 2012. （in Chinese）
Key words Strong beer; Ale yeast; Brewing characteristics
Strong ale beer， refers to the high ethanol concentration beer fermented by the top beer yeast[1]， which has the characteristics of rapid fermentation and strong aroma compared with the bottom fermented beer[2]. Strong ale beer has high ethanol concentration， mellow body and rich aroma， which is a top quality brewed beer favored by beer lovers in recent years[3-5]. Generally speaking， beer yeast with low alcohol tolerance will lead to low fermentation of beer[6]. As strong ale beer has high alcohol， the yeast with low alcohol production should be selected[7-9]. The low alcohol tolerance of ordinary beer yeast can lead to low fermentation degree of finished beer. Because of the high alcohol content of strong ale beer， the yeast producing low alcohols should be selected[10].
At the present， there are various kinds of yeasts on the market， but choosing the right yeast is undoubtedly the most important for the quality and productivity of beer. In this study， three kinds of beer yeasts commonly used in the market were selected， and a suitable strong beer yeast was selected by comparing the brewing performance and product index of the beer yeast， in order to provide some guidance for future beer brewing.
Materials and Methods
Materials
Pilsner Malt （4 EBC） and Caramel Malt （25 EBC）： Shandong Sinbach Biotechnology Co.， Ltd; Pelletized （T90） Magnum hops （14% αacids）; Yakima chief （HK） Ltd; Pelletized （T90） Saaz hops （4.5% αacids）： Batterhaas Trading Co.， Ltd; Dried Yeast S04， BE256， S33： Fermentis Yeast Co.， Ltd.
Instrument and equipment
ABF804C11EBC grinder： Buhler， AG; ZM30L vertical automatic fermentation tank： Jinan Zhengmai Machinery Equipment Co.， Ltd; WYTJ Handheld saccharimeter： Chengdu Haochang Photoelectric Instrument Co.， Ltd; SWCJ1BU sterile ultraclean worktable： Suzhou Shangdian Clean Technology Co.， Ltd; UV2350 ultravioletvisible spectrophotometer： especially Nico （Shanghai） Instrument Co.， Ltd. Methods
Malt milling
According to the original gravity of 16°P， the ingredients were crushed. The ratio of the two malts was 95% pilsner malt （4EBC） and 5% caramel malt （25EBC）.
Mashing
The wort was prepared using commercially available 100% malts， the proportion of malt and water was 1.0∶3.5 （kg∶L）. The comminuted malt was added to water at 52 ℃ and keep warm for 30 min， then heating up to 65 ℃ for 40 min， and 72 ℃ for 20 min， finally the temperature was increased to 78 ℃， and the mashing process was finished.
Wort filtration
The saccharified wort was poured into the filter tank， and the wort was filtered after standing for 20 min. Then the wort was washed twice with 78 ℃ water， and the whole process ensures that the wort was clear.
Wort boiling
Magnum hops was added to the first boiling with a rate of 0.3 g/L， and Saaz hops were added after boiling for 55 min with 0.1 g/L[11]. The whole boiling time was 60 min.
Cyclotron precipitation and wort cooling
The wort was transferred to a cyclotron settling tank after boiling， after standing for 20 min， the clarified wort was obtained， and then it was transferred to the fermentor after cooling to 18 ℃. The whole process was oxygenated with wort at a ratio of 8-10 mg/L.
Fermentation
Sterile water was boiled and cooled to 20 ℃， and dried yeast powder was added at a ratio of 0.8 g/L. After 15 min of stationary， yeast liquid was shaken and added to fermentor. Main fermentation was conducted when the wort was filled at 20 ℃. When the sugar content of wort was reduced to 5 °P， the tank was sealed for reducing the content of diacetyl， and the pressure was controlled at 0.14-0.16 MPa[12].
Cooling and storage
When the content of diacetyl was less than 0.08 mg/L， the beer was cooled to 0-1 ℃， and the pressure was controlled to 0.12-0.14 MPa. The beer ripened after storage for one week.
Testing of physical and chemical indexes of beer
During the fermentation process， the determination of sugar， alcohol， diacetyl and other physical and chemical indicators was performed in accordance with GB/T 49272008.
Sensory evaluation of 3 finished beers using trained panelists
Ten trained panelists with previous experience evaluating beer were asked to evaluate the finished beers for both taste and aroma. Judging from the four aspects of appearance， foam， aroma and taste， the full score was 100 points， we took the average score[13]. The Sensory evaluation standard was shown in Table 1. Results and Discussion
The change of sugar content of fermentation broth with fermentation time is shown in Fig. 1. Fig 1 shows that the three dried yeasts had a relatively fast rate of sugar reduction during the first three days， which was the peak period of fermentation; the sugar content could be reduced to 5 °P after 3 or 4 d to seal the tank， and then the rate of sugar reduction significantly slowed down， and tended to be stable. The rate of degrading sugar of S04 and BE256 were faster than S33 during the first 2 d， S33 was faster than S04 on the fourth day， BE256 was superior to the other two yeasts in fermentation speed and fermentation performance.
The change of alcohol content with fermentation time is shown in Fig. 2. Yeast produces alcohol by consuming sugars by anaerobic respiration[14]. As shown in Fig. 2， the rate of degrading sugar was very fast during the first 3 d， more alcohol will be produced， and then the alcohol content will rise slowly and the alcohol content will be stabilized at about 6% Vol. The fermentation degree of the three yeasts is BE256 alcohol > S33 > S04.
The change of diacetyl with fermentation time is shown in Fig. 3. Diacetyl is a flavor substance in beer， which is a sign of beer maturity[15]. It is generally necessary to ensure that the content of diacetyl in beer is less than 0.1 mg/L. As shown in Fig. 3， the diacetyl reduction rate of the three yeasts was faster， and the diacetyl content of the three yeasts could be reduced below 0.1 mg/L on the 8th day. The diacetyl reduction ability of the three yeasts was S04 > BE256 > S33.
The sensory evaluation of three yeast fermented beer is compared with that shown in Fig. 4. The sensory score of BE256 > S04 > S33 is shown in Fig. 4. On the whole， the fermentation characteristics of BE256 yeast is excellent， the sensory score reaches 92， and the comprehensive situation of the finished beer is ideal. The fermentation performance of S04 and S33 yeasts is close.
The physicochemical properties of the beer fermented by the three yeasts are shown in Table 2. The original gravity of the three beers is close， the alcohol content and fermentation degree are slightly different， and the fermentation degree of BE256 yeast is better than the other two. On the whole， the indicators are close to each other and are in line with national standards. The finished beers have golden yellow appearance， rich and delicate foam， pleasant aroma and strong taste， all belong to high quality beer. Conclusions
The fermentation performance of three strong ale yeasts was compared and analyzed. The results showed that the physical and chemical indexes of these three fermented beer met national standards. The finished beers were golden yellow with rich foam， delicate fragrance and strong taste. The three beer yeasts were excellent in reducing sugar speed， fermentation degree and diacetyl reduction ability. On the whole， the fermentation performance of BE256 yeast was better than that of the other two yeasts， which had a certain guiding role in the future production of strong ale beer.
References
[1] WEISENBERGER C. Craft beer boom creates hops shortage[J]. Supermarket News Expert Blog， 2014.
[2] MIGNANI AG， CIACCHERI L， MENCAGLIA AA， et al. Optical measurements and pattern recognition techniques for autheticating topfermented and bottomfermented beers and predicting the alcoholic strength[C]∥ Sensors. IEEE， 2012： 1-4.
[3] LANDSCHOOT AV， VANDERPUTTEN D， STALS I， et al. Spectrum of polysaccharides degradation products of ales and lager beers[C].// European Conference on Food Chemistry. Gesellschaft Deutscher Chemiker， 2005： 615-618.
[4] KRIZEK M. Assesment of biogenic amines in beer[J]. Kvasny Prumysl， 1995.
[5] HUVAERE K， SINNAEVE B， VAN BJ， et al. Photooxidative degradation of beer bittering principles： product analysis with respect to lightstruck flavour formation.[J]. Photochem Photobiol Sci， 2004， 3（9）： 854-858.
[6] MOHREKESH M， SHAHRAKI ADF， GHALAMKARI GR， et al. Effects of three methods of oral seleniumenriched yeast supplementation on blood components and growth in Holstein dairy calves[J]. Animal Production Science， 2018.
[7] QUAN QX， CHEN LQ， WEI RL， et al. Study on the production of seleniumenriched pork by using the seleniumenriched yeast[J]. Food Research & Development， 2016.
[8] WANG Z， ZHANG L， TAN T. High cell density fermentation of Saccharomyces cerevisiae， GS2 for seleniumenriched yeast production[J]. Korean Journal of Chemical Engineering， 2010， 27（6）： 1836-1840.
[9] FREDLUND E， BEERLAGE C， MELIN P， et al. Oxygen and carbon sourceregulated expression of PDC and ADH genes in the respiratory yeast Pichia anomala[J]. Yeast， 2010， 23（16）：1137-1149.
[10] CASAREGOLA S， NGUYEN HV， LAPATHITIS G， et al. Analysis of the constitution of the beer yeast genome by PCR， sequencing and subtelomeric sequence hybridization[J]. Int.j.syst.evol.microbiol， 2001， 51（4）： 1607-1618.
[11] ZHANG XQ. Discussion on addition of hop pellets[J]. Global Alcinfo， 2017（17）： 33-35. （in Chinese）
[12] ZHOU GT. Modern beer technology[M]. Beijing： Chemical Industry Press， 2007： 81-82. （in Chinese）
[13] DONG XL， ZHOU GT， CUI YQ. Sensory evaluation of beer[M]. Beijing： Chemical Industry Press， 2008： 126-131. （in Chinese）
[14] ZHOU B， ZHANG CY， CHEN YF， et al. Effects of high gravity and high temperature on fermentation performance of beer yeast[J]. China Brewing， 2016， 35（2）： 9-12. （in Chinese）
[15] KAN X. Preliminary study on high alcohol beer[D]. Wuxi： Jiangnan University， 2012. （in Chinese）