Brewing Beer in the Laboratory: Grain Amylases and Yeast's Sweet

In the Laboratory

Brewing Beer in the Laboratory: Grain Amylases and Yeast's Sweet Tooth Blake Gillespie* Chemistry Program, California State University Channel Islands, Camarillo, California 93012 *[email protected] William A. Deutschman Chemistry Department, Westminster College, Salt Lake City, Utah 84105

Glycolysis and fermentation are ubiquitous components of the undergraduate biochemistry curriculum, and many important methods of teaching these energy production pathways have been presented in this and other journals (1-9). In the instructional laboratory, however, these models of metabolism and its regulation often prove difficult to address. The popularity of beer-related laboratories, first noted in this Journal over 30 years ago (2), provides a bridge to that metabolism content. We have found that coupling even this basic brewing activity to standard metabolism class content adds a powerful educational experience and helps cement the course's basic learning objectives. Saccharomyces cerevisiae is capable of electron transportdependent ATP synthesis under aerobic conditions but turns to alcoholic fermentation in the absence of O2. Under these conditions, glycolysis becomes the yeast's primary energy source because oxidative phosphorylation fails (for an overview of this pathway see, e.g., Voet and Voet in ref 10). Overall, 2 equiv of ATP are produced for each glucose molecule that enters the glycolytic pathway; the anaerobic fermentation of pyruvate to ethanol provides glycolysis with the NADþ normally regenerated during mitochondrial electron transport. Thus, brewing beer provides an instructional key to energy metabolism: glycolysis requires recycling of NADþ, whether aerobically by electron transport or anaerobically by fermentation of glycolytic products to ethanol (e.g., brewing beer). This exercise also shows how grain amylases control the pathways that the carbohydrates enter. The amylases are barley enzymes that degrade starch into the less complex sugars that feed plant as well as yeast fuel oxidation. R-Amylase, β-amylase, Rglucosidase, and limit dextrinase act on starch granules in the endosperm, hydrolyzing glycosidic bonds at different positions and resulting in a diverse profile of carbohydrates available to the yeast for fermentation (Figure 1) (11). Brewing also gives students hands-on experience with a variety of laboratory techniques. Thin-layer chromatography (TLC) provides a measure of fermentable sugar production and consumption by qualitative inspection of developed plates for the presence or absence of sugars, or more precisely by quantifying spot densities using digital imaging methods. Using the mono- and trisaccharides glucose and raffinose, respectively, as standards for amylase products, students observe which amylases are most active at a given temperature. As well, they see the nutrient preferences of S. cerevisiae as each sugar is depleted in turn. The liberation of CO2 gas is another measure of fermentation because the volume of gas released over time peaks and decreases as sugars are consumed. Finally, students use 1244

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changes in specific gravity or refractive index to quantify how much carbon was actually lost and what remains behind as ethanol. We have expanded upon existing simple carbohydrate extraction laboratory activities (e.g., ref 9) to include the characterization of barley-extract sugar composition as well as yeast fermentation of these extracts. Students extract complex polysaccharides from barley and examine the enzyme-mediated conversion of these into fermentable sugars. Qualitative and quantitative methods are used to determine which types of sugars are preferred substrates for yeast metabolism. The exercise also suggests further explorations of the factors controlling starch mobilization, the interplay between sugar metabolism and yeast growth, and the metabolic fates of carbohydrates in aerobic and anaerobic metabolic pathways. General Procedure Brewing may be broken down into three phases (see ref 12 for a comprehensive discussion of brewing methods and terminology). In the “mash-in” period, starch is solubilized and grain amylases degrade it into simple sugars; this solution is referred to as the “wort”. Second, the grains are “lautered” and “sparged” (filtered and extracted with boiling water). The extracted wort is boiled to denature the grain amylases, locking in the wort's profile of fermentable sugars. Finally, after rapid cooling to 25 °C, the wort is inoculated with yeast (the yeast is “pitched”) and fermentation begins. This experiment gives the student experience with each of these steps, using each to illustrate specific biochemical and metabolic lessons, as outlined below. The experiment is designed to span two 3-h lab periods separated by 1 week. Students begin by crushing barley, warming both the dry grain and ∼400 mL of distilled water in a water bath, and mixing the two to initiate the mash-in. During the mash-in, students periodically stir the mixture, taking temperature readings and removing samples for TLC analysis at 10-30 min intervals. The enzyme activity in these samples is immediately halted by brief boiling. The samples are cleared of solids in a microcentrifuge and frozen for later analysis. After 60-90 min of mashing, the wort is boiled to inactivate the amylases, filtered, and then cooled in an ice bath. Optionally, the wort can be centrifuged to remove particulates before sterilizing. Finally, the yeast is pitched and the flask capped with a bubble hook airlock. After a few hours, fermentation commences. For the most complete set of results, students should return to the lab in the week between the regular

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Vol. 87 No. 11 November 2010 pubs.acs.org/jchemeduc r 2010 American Chemical Society and Division of Chemical Education, Inc. 10.1021/ed100442b Published on Web 09/27/2010

In the Laboratory

Figure 1. Cartoon of amylase degradation of amlyopectin. Amylopectin's glucose monomer units are represented as light ovals, while bold ovals represent free glucose molecules. The bonds hydrolyzed by each particular amylase are labeled with asterisks. R-Glucosidase hydrolyzes R-1,4 glycosidic bonds of starch polymers at their nonreducing ends, liberating glucose monomers. R-Amylase produces complex limit dextrins by hydrolyzing R-1,4 glycosidic bonds at branch points. Limit dextrinase degrades these products by hydrolyzing R-1,6 glycosidic bonds. Smaller fragments, such as the disaccharide maltose generated by β-amylase, are then degraded to glucose by the action of R-glucosidase.

meeting periods to measure CO2 output, refractive index, and to remove samples for subsequent TLC analysis. In such a small volume, fermentation peaks after a day or two and then trails off. The following week, students spot each sample onto TLC plates. Sugars are separated with an isopropyl alcohol/water mobile phase. TLC plates are developed using the colorimetric orcinol method in which carbohydrates, dehydrated with sulfuric acid, produce furfurals that then condense with orcinol to form brownish products, depending on the specific sugars present (8 and references therein). TLC run with carbohydrate standards allows students to monitor sugar profile development during mash in as well as the sequence of consumption during fermentation. While their TLC is in progress, students plot their refractive index and CO2 evolution as measures of sugar concentration and ethanol production, respectively. As well, pre- and postfermentation refractometer or hydrometer readings allow calculation of the volume percentage of ethanol produced (12, 13). Hazards Hot liquids present the risk of scalding and severe burns. The running and development of the TLC plates are this experiment's most significant safety concern. Methanol and isopropyl alcohol are volatile organic solvents, and inhalation of methanol vapors can cause severe health effects. Orcinol is a white powder that causes irritation and redness upon exposure or ingestion. Sulfuric acid is a corrosive liquid that can cause severe burns and death upon ingestion. Inhalation of sulfuric acid vapors will cause severe, acute irritation of mucous membranes. Fume hoods and appropriate clothing, gloves, and eye protection should be used at all steps of this exercise.

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Results TLC illustrates both sugar profile development during mash-in, as well as the progression of sugars utilized by the yeast during fermentation (Figure 2). Students clearly observe that the disaccharide maltose is the predominant product, indicating that β-amylase is the most active amylase at a mash-in temperature of 64 °C; glucose, produced by R-glucosidase, is present at a far lower concentration. This ratio can be altered by using higher or lower mash temperatures and provides an excellent basis for experimentation and discussion between student groups. In terms of fermentation, students note that yeast uses glucose as its primary fuel source, switching to the more abundant maltose only when the glucose concentration drops. Only when these two sources are exhausted are trisaccharides consumed. As shown in Figure 3A, this cascade of sugar utilization is easily quantified by measuring spot intensity in TLC plate images. This analysis was performed in Microsoft Windows XP using the Scion Image program (14). Likewise, students measure refractive index changes and CO2 evolution as a function of time postinoculation (Figure 3B). These two observables track closely and comparison with the TLC results show that the yeast are growing and reducing pyruvate to ethanol and CO2 so long as there is fermentable sugar available. Consumption of sugar during fermentation and at the end point is also measured by refractive index or hydrometer. These measurements allow students to determine the percentage of ethanol in the product, as well as the efficiency of the fermentation. The density of the wort depends on the concentration of sugar; the

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Figure 2. TLC of mash-in and fermentation. (A) Samples removed from the mash-in at regular intervals show that simple sugars are extracted completely by 90 min and that disaccharides (most likely maltose) are by far the most abundant sugar in the wort. (B) Samples removed during fermentation show a clear progression of carbohydrate consumption, beginning with monosaccharides, proceeding through disaccharides to trisaccharides. Samples and standards were resolved using a 91:9 v/v isopropyl alcohol/water solution, and plates were developed using the orcinol method in which acid-treated sugars are converted to reactive furfurals that polymerize with orcinol, forming a brown pigment (8).

Figure 3. Quantification of fermentation. (A) Spot intensities were quantified using Scion Image and normalized to the first time-point. As in the qualitative analysis, the progression from mono- to trisaccharide is clear. (B) Refractive index and CO2 evolution rate were measured at intervals. Refractive index decreases as sugar is consumed, and CO2 peaks then drops off as the yeast consume the available sugars. Both are well correlated with the TLC-observed depletion of fermentable sugars.

change in density is a measure of the quantity of carbon lost to CO2. Conclusions The correlation between yeast fuel preferences and the changes in sugar profile observed by TLC analysis is striking. By coupling chromatographic measurements to observations of evolved CO2 and changes in refractive index and specific gravity, the exercise provides students with a wide array of qualitative and 1246


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quantitative metrics. Beyond improving laboratory skills and practice, students rate this exercise as highly useful because it supports classroom content. Pedagogically, laboratory experiences that link to classroom content enhance students' achievement of learning objectives (15, 16). Students also find the cooking-class atmosphere to be comfortable, while maintaining the quantitative rigor of the biochemistry laboratory. Instructors may go further, considering how unfermentable carbohydrates can make a beer sweeter or more viscous, how Maillard browning reactions between amino acids and reducing sugars can


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In the Laboratory

contribute to the wide range of beer colors, or how diastatic power, or enzyme activity, determines its alcohol content. Regardless of how the lab is configured, brewing places metabolism in a familiar context, and the intersection of course content and real-world experiences dramatically magnifies the lesson. Literature Cited Staudemayer, T. J. Chem. Educ. 1964, 41, 46. Lokken, D. A. J. Chem. Educ. 1975, 52, 379. McClure, D. W. J. Chem. Educ. 1976, 53, 70–73. Vogler, A.; Kunkely, H. J. Chem. Educ. 1982, 59, 25–27. Bering, C. L. J. Chem. Educ. 1988, 65, 519–521. Feinman, R. D. J. Chem. Educ. 2001, 78, 1215–1220. Stewart, G. G. J. Chem. Educ. 2004, 81, 963–968. Paulino, T. P.; Cardoso, M.; Bruschi-Thedei, G. C.; Ciancaglini, P.; Thedei, G. Biochem. Mol. Biol. Educ. 2003, 31, 180–184. 9. Pelter, M. W.; McQuade, J. J. Chem. Educ. 2005, 82, 1811– 1812.

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10. Voet, D. J.; Voet, J. G. Biochemistry, 3rd ed.; Wiley: New York, 2004; Chapter 16. 11. Sun, Z.; Henson, C. A. Arch. Biochem. Biophys. 1991, 284, 298–305. 12. Palmer, J. J. How To Brew: Everything You Need To Know To Brew Beer Right the First Time; Brewers Publications: Boulder, CO, 2006. 13. Ball, D. W. J. Chem. Educ. 2006, 83, 1489. 14. Scion Image program ported from NIH Image for the Macintosh by Scion Corporation and available at http://www.scioncorp.com (accessed Aug 2010). 15. Olson, S.; Loucks-Horsley, S. Inquiry and the National Science Education Standards: A Guide of Teaching and Learning; National Academic Press: Washington, DC, 2000. 16. Hofstein, A. Chem. Educ.: Res. Pract. 2004, 5, 247–264.

Supporting Information Available Detailed lists of hardware and reagents; instructor protocols and teaching points; a student handout and procedure. This material is available via the Internet at http://pubs.acs.org.


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Brewing Beer in the Laboratory: Grain Amylases and Yeast's Sweet

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