Beer Brewing

1244 Journal of Chemical Education

_Vol. 87 No. 11 November 2010

_pubs.acs.org/jchemeduc

_r2010 American Chemical Society and Division of Chemical Education, Inc.

10.1021/ed100442b Published on Web 09/27/2010

In the Laboratory

Brewing Beer in the Laboratory: Grain Amylasesand Yeast's Sweet ToothBlake Gillespie*Chemistry Program, California State University Channel Islands, Camarillo, California 93012*[email protected]

William A. DeutschmanChemistry Department, Westminster College, Salt Lake City, Utah 84105

Glycolysis and fermentation are ubiquitous components ofthe undergraduate biochemistry curriculum, and many impor-tant methods of teaching these energy production pathways havebeen presented in this and other journals (1-9). In the instruc-tional laboratory, however, these models of metabolism and itsregulation often prove difficult to address. The popularity ofbeer-related laboratories, first noted in this Journal over 30 yearsago (2), provides a bridge to that metabolism content. We havefound that coupling even this basic brewing activity to standardmetabolism class content adds a powerful educational experienceand helps cement the course's basic learning objectives.

Saccharomyces cerevisiae is capable of electron transport-dependent ATP synthesis under aerobic conditions but turns toalcoholic fermentation in the absence of O2. Under theseconditions, glycolysis becomes the yeast's primary energy sourcebecause oxidative phosphorylation fails (for an overview of thispathway see, e.g., Voet and Voet in ref 10). Overall, 2 equiv ofATP are produced for each glucose molecule that enters theglycolytic pathway; the anaerobic fermentation of pyruvate toethanol provides glycolysis with the NADþ normally regener-ated during mitochondrial electron transport. Thus, brewingbeer provides an instructional key to energy metabolism: glyco-lysis requires recycling of NADþ, whether aerobically by electrontransport or anaerobically by fermentation of glycolytic productsto ethanol (e.g., brewing beer).

This exercise also shows how grain amylases control thepathways that the carbohydrates enter. The amylases are barleyenzymes that degrade starch into the less complex sugars that feedplant as well as yeast fuel oxidation. R-Amylase, β-amylase, R-glucosidase, and limit dextrinase act on starch granules in theendosperm, hydrolyzing glycosidic bonds at different positionsand resulting in a diverse profile of carbohydrates available to theyeast for fermentation (Figure 1) (11).

Brewing also gives students hands-on experience with avariety of laboratory techniques. Thin-layer chromatography(TLC) provides a measure of fermentable sugar productionand consumption by qualitative inspection of developed platesfor the presence or absence of sugars, or more precisely byquantifying spot densities using digital imaging methods. Usingthe mono- and trisaccharides glucose and raffinose, respectively,as standards for amylase products, students observe whichamylases are most active at a given temperature. As well, theysee the nutrient preferences of S. cerevisiae as each sugar isdepleted in turn. The liberation of CO2 gas is another measureof fermentation because the volume of gas released over timepeaks and decreases as sugars are consumed. Finally, students use

changes in specific gravity or refractive index to quantify howmuch carbon was actually lost and what remains behind asethanol.

We have expanded upon existing simple carbohydrateextraction laboratory activities (e.g., ref 9) to include the char-acterization of barley-extract sugar composition as well as yeastfermentation of these extracts. Students extract complex poly-saccharides from barley and examine the enzyme-mediatedconversion of these into fermentable sugars. Qualitative andquantitative methods are used to determine which types of sugarsare preferred substrates for yeast metabolism. The exercise alsosuggests further explorations of the factors controlling starchmobilization, the interplay between sugar metabolism and yeastgrowth, and the metabolic fates of carbohydrates in aerobic andanaerobic metabolic pathways.

General Procedure

Brewing may be broken down into three phases (see ref 12for a comprehensive discussion of brewing methods andterminology). In the “mash-in” period, starch is solubilized andgrain amylases degrade it into simple sugars; this solution isreferred to as the “wort”. Second, the grains are “lautered” and“sparged” (filtered and extracted with boiling water). Theextracted wort is boiled to denature the grain amylases, lockingin the wort's profile of fermentable sugars. Finally, after rapidcooling to 25 �C, the wort is inoculated with yeast (the yeast is“pitched”) and fermentation begins. This experiment gives thestudent experience with each of these steps, using each toillustrate specific biochemical and metabolic lessons, as outlinedbelow. The experiment is designed to span two 3-h lab periodsseparated by 1 week.

Students begin by crushing barley, warming both the drygrain and∼400mL of distilled water in a water bath, and mixingthe two to initiate the mash-in. During the mash-in, studentsperiodically stir the mixture, taking temperature readings andremoving samples for TLC analysis at 10-30 min intervals. Theenzyme activity in these samples is immediately halted by briefboiling. The samples are cleared of solids in a microcentrifugeand frozen for later analysis. After 60-90 min of mashing, thewort is boiled to inactivate the amylases, filtered, and then cooledin an ice bath. Optionally, the wort can be centrifuged to removeparticulates before sterilizing. Finally, the yeast is pitched and theflask 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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In the Laboratory

meeting periods to measure CO2 output, refractive index, and toremove samples for subsequent TLC analysis. In such a smallvolume, fermentation peaks after a day or two and then trails off.

The following week, students spot each sample onto TLCplates. Sugars are separated with an isopropyl alcohol/watermobile phase. TLC plates are developed using the colorimetricorcinolmethod inwhich carbohydrates, dehydrated with sulfuricacid, produce furfurals that then condense with orcinol to formbrownish products, depending on the specific sugars present(8 and references therein). TLC runwith carbohydrate standardsallows students to monitor sugar profile development duringmash in as well as the sequence of consumption during fermen-tation. While their TLC is in progress, students plot theirrefractive index and CO2 evolution as measures of sugar con-centration and ethanol production, respectively. As well, pre- andpostfermentation refractometer or hydrometer readings allowcalculation of the volume percentage of ethanol produced (12, 13).

Hazards

Hot liquids present the risk of scalding and severe burns. Therunning and development of the TLC plates are this experiment'smost significant safety concern. Methanol and isopropyl alcohol arevolatile organic solvents, and inhalation of methanol vapors cancause severe health effects. Orcinol is a white powder that causesirritation and redness upon exposure or ingestion. Sulfuric acid is acorrosive liquid that can cause severe burns anddeathupon ingestion.Inhalation of sulfuric acid vapors will cause severe, acute irritation ofmucous membranes. Fume hoods and appropriate clothing, gloves,and eye protection should be used at all steps of this exercise.

Results

TLC illustrates both sugar profile development duringmash-in, as well as the progression of sugars utilized by the yeastduring fermentation (Figure 2). Students clearly observe thatthe disaccharide maltose is the predominant product, indicat-ing that β-amylase is the most active amylase at a mash-intemperature of 64 �C; glucose, produced by R-glucosidase, ispresent at a far lower concentration. This ratio can be altered byusing higher or lower mash temperatures and provides anexcellent basis for experimentation and discussion betweenstudent groups. In terms of fermentation, students note thatyeast uses glucose as its primary fuel source, switching to themore abundant maltose only when the glucose concentrationdrops. Only when these two sources are exhausted are trisac-charides consumed.

As shown in Figure 3A, this cascade of sugar utilization iseasily quantified by measuring spot intensity in TLC plateimages. This analysis was performed in Microsoft WindowsXP using the Scion Image program (14). Likewise, studentsmeasure refractive index changes and CO2 evolution as afunction of time postinoculation (Figure 3B). These two ob-servables track closely and comparison with the TLC resultsshow that the yeast are growing and reducing pyruvate to ethanoland CO2 so long as there is fermentable sugar available. Con-sumption of sugar during fermentation and at the end point isalso measured by refractive index or hydrometer. These measure-ments allow students to determine the percentage of ethanol inthe product, as well as the efficiency of the fermentation. Thedensity of the wort depends on the concentration of sugar; the

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

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_r2010 American Chemical Society and Division of Chemical Education, Inc.

In the Laboratory

change in density is a measure of the quantity of carbon lostto CO2.

Conclusions

The correlation between yeast fuel preferences and thechanges in sugar profile observed by TLC analysis is striking.By coupling chromatographic measurements to observations ofevolved CO2 and changes in refractive index and specific gravity,the exercise provides students with a wide array of qualitative and

quantitative metrics. Beyond improving laboratory skills andpractice, students rate this exercise as highly useful because itsupports classroom content. Pedagogically, laboratory experi-ences that link to classroom content enhance students' achieve-ment of learning objectives (15, 16). Students also find thecooking-class atmosphere to be comfortable, while maintainingthe quantitative rigor of the biochemistry laboratory. Instructorsmay go further, considering how unfermentable carbohydratescan make a beer sweeter or more viscous, how Maillard brown-ing reactions between amino acids and reducing sugars can

Figure 2. TLC of mash-in and fermentation. (A) Samples removed from the mash-in at regular intervals show that simple sugars are extracted completelyby 90 min and that disaccharides (most likely maltose) are by far the most abundant sugar in the wort. (B) Samples removed during fermentation showa clear progression of carbohydrate consumption, beginning with monosaccharides, proceeding through disaccharides to trisaccharides. Samples andstandards were resolved using a 91:9 v/v isopropyl alcohol/water solution, and plates were developed using the orcinol method in which acid-treatedsugars 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 thequalitative 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 withthe TLC-observed depletion of fermentable sugars.

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_Journal of Chemical Education 1247

In the Laboratory

contribute to the wide range of beer colors, or how diastaticpower, or enzyme activity, determines its alcohol content.Regardless of how the lab is configured, brewing places metabo-lism in a familiar context, and the intersection of course contentand real-world experiences dramatically magnifies the lesson.

Literature Cited

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10. Voet, D. J.; Voet, J. G. Biochemistry, 3rd ed.; Wiley: New York,2004; Chapter 16.

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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 fromNIH Image for theMacintosh by

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Supporting Information Available

Detailed lists of hardware and reagents; instructor protocols andteaching points; a student handout and procedure. This material isavailable via the Internet at http://pubs.acs.org.

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Beer Brewing