Beer Poster
1
The effects of varying pH and temperature on the activity of amylases involved in beer production Justin G. Tedeschi, Joseph R. Falcone, Kirk A. Bartholomew, and Geffrey F. Stopper Department of Biology, Sacred Heart University Introduction Methods Results, Discussion, & Ongoing Work References Mashes and subsequent fermentations were performed in two separate batches on separate dates, with the procedures for a single batch given here. Five water baths were filled with tap water until the depth of the water was 55 mm and then the water baths were set to the target temperatures, each of which was set to 8 °C above the target mash temperature based on prior experiments measuring the dispersion of heat through samples of they type used here. About 1.6 kg Briess brand two row malted barley was cracked in a hand-driven malt mill. 100 grams of the crushed malted barley was added to each of fifteen 500 mL Erlenmeyer flasks, and 250 mL of cold tap water was added to each flask to prepare mashes. Each flask was then swirled to mix the contents. The pH of each mash sample was taken with litmus paper. A 1.5 mL sample was removed from each flask and put into a 1.5 mL micro centrifuge tube, boiled in a water bath for 5 minutes, then stored at -20 °C for later analysis. Three flasks were placed in each water bath for 60 minutes. After 60 minutes, each flask was removed and placed in an ice bath to cool. At this time, another 1.5 mL sample was taken from each flask and put into a 1.5 mL micro centrifuge tube, boiled in a water bath for 5 minutes, then stored at -20 °C for later analysis. After the mashes reached room temperature, fifteen empty 250 mL Erlenmeyer flasks were massed. One drop of fermcap-S anti-foaming agent and approximately 150-250 mg of dry Danstar Nottingham Ale Yeast was added to each flask. Cheesecloth was placed over a funnel, which was placed in a 250 mL graduated cylinder. The contents of the 500 mL flasks were poured into the cheesecloth over the funnel. The cheesecloth was then squeezed so the liquid filtered through the cheesecloth and into the graduated cylinder. The liquid in the graduated cylinder was then poured into the appropriately labeled 250 mL Erlenmeyer flask. This was repeated for each sample. Each full 250 mL flask was then massed. Airlocks were placed on each 250 ml flask and flasks were left to ferment for seven days at room temperature. Following fermentation, the mass of each flask was measured without the airlock. A 1.5 mL sample was taken from each flask and put into a 1.5 mL micro centrifuge tube, boiled in a water bath for 5 minutes, then stored at -20 °C for later analysis. Final density values for each sample were measured using an Aton-Paar alcolyzer. Amylose and amylopectin are two starches that make up the vast majority of carbohydrates found in unmalted and malted barley. Amylose is a long string of glucoses that exhibit 1-4 glycosidic linkages (Figure 1). Amylopectin also has 1-4 linkages, but is branched and so also has 1-6 linkages (Figure 1). Barley naturally has two enzymes—alpha amylase and beta amylase—that break the starches down. Brewing takes advantage of these enzymes to break down the starches into yeast-fermentable sugars by soaking and incubating the grains at temperatures that support fast enzymatic action; this process is called mashing. Alpha amylase breaks 1-4 glycosidic linkages at random locations anywhere along a polysaccharide, but it can’t work very close to a 1- 6 linkage. It also has a much higher affinity for long polysaccharides with 1-4 linkages and very little affinity for short ones, so it tends to leave short polysaccharides behind. (MacGregor et al., 1992; MacGregor et al., 1994). Because yeast can only break down starches of 3 monosaccharides long or less (Table 1), alpha amylase can often leave some sugars that are unfermentable. Beta amylase breaks 1-4 glycosidic linkages methodically from the non-reducing end of a polysaccharide chain only, and always breaks the chain two bonds from the end, leaving a two-glucose maltose molecule. In this way it basically eats its way down a polysaccharide chain producing maltose subunits until it reaches the other end of the polysaccharide (in the case of the straight amylose) or until it is stopped by a 1-6 branch point (in the case of branching amylopectin). Here we set out to test the effects of specific temperature and pH conditions on the total sugars extracted during mashing and the fermentability (size composition of resultant carbohydrates) of wort (pre-fermented beer). We used 67 °C as a midpoint mash temperature that is frequently used by brewers to balance the action of both alpha amylase and beta amylase (Figure 2). As temperature increased from this value, we found that starting specific gravity (representative of the total amount of soluble carbohydrate extracted from the malted barley) remained high (Figure 3). This is consistent with the theory-based prediction that high alpha amylase activity should break down both amylose and amylopectin into smaller carbohydrates that are water-soluble (long carbohydrates are not water-soluble, but short carbohydrates are water-soluble). Final specific gravity, on the other hand, steadily increased with increasing temperature (Figure 3). This is consistent with the prediction that reduced beta amylase activity should result in carbohydrates that are small, but less frequently small enough to be fermented by S. cerevisiae. These increasing mash temperatures result in a beer that is progressively sweeter and fuller-bodied, but relatively lower in alcohol due to unfermented carbohydrates in the finished beer (Figure 4). As temperature decreased from the typical mash temperature, starting specific gravity decreased (Figure 3). This is consistent with the prediction that decreased alpha amylase activity will result in more carbohydrates remaining very long and insoluble, therefore being discarded with grain and not dissolved in the liquid wort. The final specific gravity, on the other hand, decreases slightly as temperature is decreased slightly. This is consistent with the prediction that increased beta amylase activity should result in increased proportions of fermentable maltose. These slightly decreasing mash temperatures result in a beer that is progressively drier, and higher in alcohol relative to the starting specific gravity (Figure 4), due to the increased proportion of carbohydrates that are fermentable due to beta amylase making fermentable maltose. As temperatures decrease even further, the total starting specific gravity decreases further, as predicted (Figure 3). The final specific gravity, however, increases. This was unexpected, and the reason for this is currently unclear, though it is plausible that, even though beta amylase activity is high, the decreasing alpha amylase activity is leaving carbohydrates that are soluble, but with an increased tendency to leave longer strings of glucoses on the reducing side of 1-6 glycosidic linkages. We are in the process of carrying out similar experiments with variable pH. The data analyzed here represent only measures of the total mass of soluble carbohydrates before and after fermentation, and what proportion of the mass of those carbohydrates falls into fermentable vs. non-fermentable categories. To further understand the composition of sugars before and after fermentation, we have reserved samples on which we will perform a colorimetric reducing sugar assay to determine the number of reducing ends of carbohydrates in the solution. There is a single reducing site on each molecule of amylose, amylopectin, and all of the molecules that result from the action of alpha and beta amylase on these starches, regardless of the size of the carbohydrate. Thus, this assay will give us a quantification of the number of carbohydrate molecules at each sampling point, which can help us better understand the molecular structure composition of the carbohydrates produced under each mash condition. Janson LW. 1996. Brew Chem 101: The Basics of Homebrewing Chemistry. North Adams: Storey Publishing. MacGregor AW, Morgan JE, MacGregor EA. 1992. The action of germinated barley alpha-amylases on linear maltodextrins. Carbohydr Res 227:301-313. MacGregor EA, MacGregor AW, Macri LJ, Morgan JE. 1994. Models for the action of barley alpha-amylase isozymes on linear substrates. Carbohydr Res 257:249-268. http://www.dailykos.com/story/2009/08/04/761297/-WHEE-Carbohydrates-glycemic-index-and-glycemic-load Carbohydrate Molecule Number of monosaccharides Metabolized by Saccharomyces cereviseae? Structure Typical percent of carbohydrates in unmalted grain Typical percent of carbohydrates in wort Amylopectin 4 through thousands No …G-G-G… | G-G-G… 70-90 25 – 35 Amylose 4 through thousands No …G-G-G… 10-30 < 1 Raffinose 3 Variable by strain G-G-F < 1 < 1 Maltotriose 3 Yes G-G-G < 1 12 – 18 Maltose 2 Yes G-G < 1 46 – 50 Sucrose 2 Yes G-F < 1 4 – 8 Glucose 1 Yes G < 1 8 – 10 Fructose 1 Yes F < 1 1 – 2 Table 1. Carbohydrate constituents of grain and wort. Table includes the most important carbohydrates contributing to unmalted grain and wort, but is not an exclusive list of carbohydrates present. G indicates glucose, F indicates fructose, a monosaccharide with the same chemical formula as glucose (Janson, 1996). Temperature or pH Enzyme Activity Alpha Amylase Beta Amylase Acknowledgments We thank Two Roads Brewing Company, Stratford, CT, for use of their alcolyzer to measure densities of fermented samples Figure 1. Hypothesized relative enzyme activity of alpha and beta amylase based on temperature and pH Figure 1. Structure of amylose (left) and amylopectin (right) indicating the location of the reducing end on each molecule. Images from dailykos.com Figure 3. Average starting specific gravity (after mashing, before fermentation; blue) and average final specific gravity (after fermentation; red) for each temperature group. 67 °C is represented twice because it was used as a base condition in each of the two trials to confirm that the results were reproducible between experiments. All data points represent the average of three samples except 70 °C, where one outlier was removed. Error bars represent standard deviations. Figure 4. Average alcohol (% alcohol by volume) for each temperature group. 67 °C is represented twice because it was used as a base condition in each of the two trials to confirm that the results were reproducible between experiments. All data points represent the average of three samples except 70 °C, where one outlier was removed. Error bars represent standard deviations.
-
Upload
justin-tedeschi -
Category
Documents
-
view
54 -
download
0
Transcript of Beer Poster
The effects of varying pH and temperature on the activity of amylases involved in beer production
Justin G. Tedeschi, Joseph R. Falcone, Kirk A. Bartholomew, and Geffrey F. Stopper
Department of Biology, Sacred Heart University Introduction
Methods Results, Discussion, & Ongoing Work
References
Mashes and subsequent fermentations were performed in two separate batches on separate dates, with the procedures for a single batch given here. Five water baths were filled with tap water until the depth of the water was 55 mm and then the water baths were set to the target temperatures, each of which was set to 8 °C above the target mash temperature based on prior experiments measuring the dispersion of heat through samples of they type used here. About 1.6 kg Briess brand two row malted barley was cracked in a hand-driven malt mill. 100 grams of the crushed malted barley was added to each of fifteen 500 mL Erlenmeyer flasks, and 250 mL of cold tap water was added to each flask to prepare mashes. Each flask was then swirled to mix the contents. The pH of each mash sample was taken with litmus paper. A 1.5 mL sample was removed from each flask and put into a 1.5 mL micro centrifuge tube, boiled in a water bath for 5 minutes, then stored at -20 °C for later analysis. Three flasks were placed in each water bath for 60 minutes. After 60 minutes, each flask was removed and placed in an ice bath to cool. At this time, another 1.5 mL sample was taken from each flask and put into a 1.5 mL micro centrifuge tube, boiled in a water bath for 5 minutes, then stored at -20 °C for later analysis. After the mashes reached room temperature, fifteen empty 250 mL Erlenmeyer flasks were massed. One drop of fermcap-S anti-foaming agent and approximately 150-250 mg of dry Danstar Nottingham Ale Yeast was added to each flask. Cheesecloth was placed over a funnel, which was placed in a 250 mL graduated cylinder. The contents of the 500 mL flasks were poured into the cheesecloth over the funnel. The cheesecloth was then squeezed so the liquid filtered through the cheesecloth and into the graduated cylinder. The liquid in the graduated cylinder was then poured into the appropriately labeled 250 mL Erlenmeyer flask. This was repeated for each sample. Each full 250 mL flask was then massed. Airlocks were placed on each 250 ml flask and flasks were left to ferment for seven days at room temperature. Following fermentation, the mass of each flask was measured without the airlock. A 1.5 mL sample was taken from each flask and put into a 1.5 mL micro centrifuge tube, boiled in a water bath for 5 minutes, then stored at -20 °C for later analysis. Final density values for each sample were measured using an Aton-Paar alcolyzer.
Amylose and amylopectin are two starches that make up the vast majority of carbohydrates found in unmalted and malted barley. Amylose is a long string of glucoses that exhibit 1-4 glycosidic linkages (Figure 1). Amylopectin also has 1-4 linkages, but is branched and so also has 1-6 linkages (Figure 1). Barley naturally has two enzymes—alpha amylase and beta amylase—that break the starches down. Brewing takes advantage of these enzymes to break down the starches into yeast-fermentable sugars by soaking and incubating the grains at temperatures that support fast enzymatic action; this process is called mashing. Alpha amylase breaks 1-4 glycosidic linkages at random locations anywhere along a polysaccharide, but it can’t work very close to a 1-6 linkage. It also has a much higher affinity for long polysaccharides with 1-4 linkages and very little affinity for short ones, so it tends to leave short polysaccharides behind. (MacGregor et al., 1992; MacGregor et al., 1994). Because yeast can only break down starches of 3 monosaccharides long or less (Table 1), alpha amylase can often leave some sugars that are unfermentable. Beta amylase breaks 1-4 glycosidic linkages methodically from the non-reducing end of a polysaccharide chain only, and always breaks the chain two bonds from the end, leaving a two-glucose maltose molecule. In this way it basically eats its way down a polysaccharide chain producing maltose subunits until it reaches the other end of the polysaccharide (in the case of the straight amylose) or until it is stopped by a 1-6 branch point (in the case of branching amylopectin). Here we set out to test the effects of specific temperature and pH conditions on the total sugars extracted during mashing and the fermentability (size composition of resultant carbohydrates) of wort (pre-fermented beer).
We used 67 °C as a midpoint mash temperature that is frequently used by brewers to balance the action of both alpha amylase and beta amylase (Figure 2). As temperature increased from this value, we found that starting specific gravity (representative of the total amount of soluble carbohydrate extracted from the malted barley) remained high (Figure 3). This is consistent with the theory-based prediction that high alpha amylase activity should break down both amylose and amylopectin into smaller carbohydrates that are water-soluble (long carbohydrates are not water-soluble, but short carbohydrates are water-soluble). Final specific gravity, on the other hand, steadily increased with increasing temperature (Figure 3). This is consistent with the prediction that reduced beta amylase activity should result in carbohydrates that are small, but less frequently small enough to be fermented by S. cerevisiae. These increasing mash temperatures result in a beer that is progressively sweeter and fuller-bodied, but relatively lower in alcohol due to unfermented carbohydrates in the finished beer (Figure 4). As temperature decreased from the typical mash temperature, starting specific gravity decreased (Figure 3). This is consistent with the prediction that decreased alpha amylase activity will result in more carbohydrates remaining very long and insoluble, therefore being discarded with grain and not dissolved in the liquid wort. The final specific gravity, on the other hand, decreases slightly as temperature is decreased slightly. This is consistent with the prediction that increased beta amylase activity should result in increased proportions of fermentable maltose. These slightly decreasing mash temperatures result in a beer that is progressively drier, and higher in alcohol relative to the starting specific gravity (Figure 4), due to the increased proportion of carbohydrates that are fermentable due to beta amylase making fermentable maltose. As temperatures decrease even further, the total starting specific gravity decreases further, as predicted (Figure 3). The final specific gravity, however, increases. This was unexpected, and the reason for this is currently unclear, though it is plausible that, even though beta amylase activity is high, the decreasing alpha amylase activity is leaving carbohydrates that are soluble, but with an increased tendency to leave longer strings of glucoses on the reducing side of 1-6 glycosidic linkages. We are in the process of carrying out similar experiments with variable pH. The data analyzed here represent only measures of the total mass of soluble carbohydrates before and after fermentation, and what proportion of the mass of those carbohydrates falls into fermentable vs. non-fermentable categories. To further understand the composition of sugars before and after fermentation, we have reserved samples on which we will perform a colorimetric reducing sugar assay to determine the number of reducing ends of carbohydrates in the solution. There is a single reducing site on each molecule of amylose, amylopectin, and all of the molecules that result from the action of alpha and beta amylase on these starches, regardless of the size of the carbohydrate. Thus, this assay will give us a quantification of the number of carbohydrate molecules at each sampling point, which can help us better understand the molecular structure composition of the carbohydrates produced under each mash condition.
Janson LW. 1996. Brew Chem 101: The Basics of Homebrewing Chemistry. North Adams: Storey Publishing. MacGregor AW, Morgan JE, MacGregor EA. 1992. The action of germinated barley alpha-amylases on linear
maltodextrins. Carbohydr Res 227:301-313. MacGregor EA, MacGregor AW, Macri LJ, Morgan JE. 1994. Models for the action of barley alpha-amylase isozymes on
linear substrates. Carbohydr Res 257:249-268. http://www.dailykos.com/story/2009/08/04/761297/-WHEE-Carbohydrates-glycemic-index-and-glycemic-load
Carbohydrate Molecule
Number of monosaccharides
Metabolized by Saccharomyces
cereviseae?
Structure Typical percent of carbohydrates in unmalted
grain
Typical percent of carbohydrates in
wort Amylopectin 4 through thousands No …G-G-G…
| G-G-G…
70-90 25 – 35
Amylose 4 through thousands No …G-G-G… 10-30 < 1 Raffinose 3 Variable by strain G-G-F < 1 < 1 Maltotriose 3 Yes G-G-G < 1 12 – 18 Maltose 2 Yes G-G < 1 46 – 50 Sucrose 2 Yes G-F < 1 4 – 8 Glucose 1 Yes G < 1 8 – 10 Fructose 1 Yes F < 1 1 – 2
Table 1. Carbohydrate constituents of grain and wort. Table includes the most important carbohydrates contributing to unmalted grain and wort, but is not an exclusive list of carbohydrates present. G indicates glucose, F indicates fructose, a monosaccharide with the same chemical formula as glucose (Janson, 1996).
Temperature or pH
Enzy
me
Activ
ity
Alpha Amylase Beta Amylase
Acknowledgments We thank Two Roads Brewing Company, Stratford, CT, for use of their alcolyzer to measure densities of fermented samples
Figure 1. Hypothesized relative enzyme activity of alpha and beta amylase based on temperature and pH
Figure 1. Structure of amylose (left) and amylopectin (right) indicating the location of the reducing end on each molecule. Images from dailykos.com
Figure 3. Average starting specific gravity (after mashing, before fermentation; blue) and average final specific gravity (after fermentation; red) for each temperature group. 67 °C is represented twice because it was used as a base condition in each of the two trials to confirm that the results were reproducible between experiments. All data points represent the average of three samples except 70 °C, where one outlier was removed. Error bars represent standard deviations.
Figure 4. Average alcohol (% alcohol by volume) for each temperature group. 67 °C is represented twice because it was used as a base condition in each of the two trials to confirm that the results were reproducible between experiments. All data points represent the average of three samples except 70 °C, where one outlier was removed. Error bars represent standard deviations.
