The Biology and Chemistry of Brewing: An Interdisciplinary Course

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The Biology and Chemistry of Brewing: An Interdisciplinary Course Paul D. Hooker,*,† William A. Deutschman,† and Brian J. Avery‡ †

Chemistry Department, Westminster College, Salt Lake City, Utah 84105, United States Biology Department, Westminster College, Salt Lake City, Utah 84105, United States

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S Supporting Information *

ABSTRACT: For the past nine years, we have been offering an interdisciplinary course for science majors: The Biology and Chemistry of Brewing. This course is primarily laboratory- and inquiry-based; from a total of 24 h of student/instructor contact time, approximately 6 h are devoted to lecture, and the other 18 h are divided between laboratory exercises, research, and field trips. Here, we describe course development as well as a summary of the laboratory assignments, with complete details of laboratory exercises available in the supporting information. This course is appropriate for students who have one year of general chemistry and general biology. KEYWORDS: Second-Year Undergraduate, Interdisciplinary/Multidisciplinary, Inquiry-Based/Discovery Learning, Agricultural Chemistry, Laboratory Instruction, Analytical Chemistry, Applications of Chemistry, Enzymes

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CONTEXT AND STUDENT BODY In its present form, this course is offered in a short but intensive four-week “semester”. It provides two elective credit hours for chemistry and biology students and has consistently filled all sections that have been offered, each section having a cap of 20 students. The prerequisite classes for students are one year of general chemistry and one year of general biology. The instructor goals of the course are to: 1. Expose science majors to instrumental methods and analytical techniques used in modern chemistry and biology. 2. Engage students in science by applying the concepts learned in general chemistry and general biology to a complex system. 3. Share information on a subject in which students are interested, but for the most part, not very knowledgeable. 4. Promote writing and research skills, with an emphasis on how to keep a lab notebook as a record of laboratory activity, by incorporating inquiry based laboratory exercises.

he necessity of covering required course content in introductory chemistry and biology courses rather limits diversions into areas of applied chemistry and biology that are often of more appeal to students. The Biology and Chemistry of Brewing, a rigorous course developed as a collaborative effort between a chemistry program and a biology program, was developed to address this issue. The main theme of the course is one that resonates with many college-age students. In times when the interest in learning science out of curiosity appears to be declining,1,2 having engaged and attentive students the first day of class allows the instructor to present subject material in greater depth. The students might believe that they are simply in the class to learn how to brew beer. However, the instructor is able to harness student interest and lead them to deeper critical and analytical thinking than is often possible in typical introductory chemistry and biology classes, as well as expose them to instrumental methodologies traditionally found in more advanced courses. Previous interdisciplinary courses discussed in this Journal have been developed for nonscience majors,3−5 education science students,6 and upper-division chemistry majors.7 A similarity that these courses share, along with this one, is that a theme is selected that crosses traditional scientific disciplines. A course that focuses on the chemistry of winemaking has been described in this Journal,8 as well as a module with beer as the central topic designed for high school students.9 The number of papers describing various chemical aspects of mashing, brewing, and beer that have appeared in this Journal alone are indicative of the interest of this topic to scientists.10−15 © 2014 American Chemical Society and Division of Chemical Education, Inc.

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COURSE DESCRIPTION The course includes a variety of components. The informational lecture component of the course is detailed in Box 1. The laboratory component consists of monitoring two fermentations throughout the duration of the course, as well as the completion of four other laboratory exercises. To replicate the brewing of beer on a small scale, two 1-gal fermentations are set up by the students. One is started at the Published: February 14, 2014 336

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handling quantities of material proportional to the scale of the fermentations they are undertaking. The chemistry laboratory exercises are chosen to expose the students, many of whom having just completed their first year of college, to a variety of instrumental techniques. Comprehensive details as to how to implement each of the laboratory exercises are included in the Supporting Information. These include instructor notes with guidance on selecting suitable equipment for the fermentations, expected results (where relevant), and student handouts with answers to supplementary questions.

Box 1. Course Material Presented in Lectures Lecture Topics An Introduction to Brewing Beer The Role of Water in the Brewing Process The Role of Hops in the Brewing Process The Role of Yeast in the Brewing Process The Role of Malt in the Brewing Process Brewing from All Grain Industrial Scale Brewing Beer Styles

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CHEMISTRY-FOCUSED LABS The five chemistry-focused laboratory exercises are summarized below.

beginning of the class and one begun half way through the class. The progression of each of these is monitored daily throughout the course. The fermentation started on the first day of class uses dried malt extract (DME), water, yeast, and hops. All students use the same type and quantity of DME and yeast, but a different variety of hops. Samples are collected daily and stored at 4 °C until the required measurements are made. For the first brew, the specific gravity is measured using a hydrometer and the “Brix”, the mass ratio of dissolved sugar to water in a liquid, is measured using a refractometer. The rate of fermentation is estimated from the number of CO2 bubbles being produced per minute. The second fermentation is from an “all-grain” extraction and requires extra mashing steps in which the enzymes present in the malted barley break down the starch into maltose and other sugars.16 The students design their own recipe, depending on the yeast and hop variety they have selected, using research tools and information readily available in home-brewing resources.17 To monitor the fermentation progress, the specific gravity of samples collected from this all-grain brew is measured, as well as a daily yeast count performed using a hemocytometer and microscope. The four laboratory exercises that students complete are chosen from the list in Box 2. To ensure the students are

Analysis of α- and β-Acids in Hop Pellets by HPLC

The main bittering components of finished beer are α-acids; these are extracted from hops (and isomerized) during a protracted boil in the preparation of the wort (beer before fermentation).14 Accurate analysis of the α-acid content of hops is essential before being added to the boiling wort as there are many hop varieties with a wide range of α-acid content. In this exercise, the hop acids are extracted into ether, diluted in methanol, separated in a 25 min isocratic HPLC run, and compared to a commercially available standard mixture of hop acids. Table 1 lists results (N = 1) collected from six pairs of Table 1. Student Results for Analysis of α-Acids by HPLC Hop Variety

α-Acid Content, %

Student Results, %

Cascade Perle Northern Brewer Cascade Hallertau Chinook

6.9 7.6 7.0 6.2 4.6 12.1

6.5 6.2 7.5 6.0 3.9 10.5

Box 2. Laboratory Exercises students who undertook this experiment in May 2010, and are typical of those obtained by students who successfully complete the exercise.

Chemistry 1. Analysis of α- and β-Acids in Hop Pellets by HPLC 2. Determining the Acidity of Beer Using an Automatic Titrator 3. Determination of the Calcium Content of Beer and Water Using Atomic Absorption Spectroscopy 4. Determination of Beer Bitterness by UV Spectrophotometry 5. Determination of the Alcohol Content of Beer Using Gas Chromatography Biology 6. Yeast Flocculation Assay 7. Yeast Genotyping Using Delta Sequence PCR 8. Determining the Amylase Activity in Malted Barley

Determination of the Acidity of Beer Using an Automatic Titrator

Beer is naturally acidic owing mainly to byproducts of yeast metabolism, the major component being lactic acid. Titration to a pH of 8.20, as prescribed by the ASBC, gives a measure of the lactic acid content of the beer. Too much lactic acid present indicates possible bacterial spoiling or poor yeast performance. The use of an automatic titrator allows the rapid determination of the acidity of several samples and does not need a colorchanging indicator to be present. Reported student results are in the range 0.10−0.40 wt % of lactic acid, depending on the type of beer analyzed. Typical values expected for beer are between 0.10 and 0.20%.19 Determination of the Calcium Content of Beer and Water Using Atomic Absorption Spectroscopy

exposed to a sufficient variety, they are required to select laboratories from both chemistry and biology. The majority of these laboratories have been developed from procedures detailed in Methods of Analysis published by the American Society of Brewing Chemists, ASBC.18 This publication provides a resource for large-scale breweries and so most analyses used in this course are scaled down so that students are

The mineral composition of the water used to make beer imparts unique characteristics to the final brew. Calcium is the most important metal ion to be considered, as it must be present for proper yeast metabolism and flocculation. Student results will vary as to the specific water supply; we have observed increases of calcium content with respect to the water (brewing liquor) in the first few days of fermentation, 337

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electrophoresis to generate a collection of PCR products that allows differentiation of yeast varieties. Students are asked to decide which types of yeast can or cannot be discriminated based on their results. Typically, students are able to differentiate between lager and ale strains quite easily and can often find ways to identify and group closely related strains within those two categories based on the presence or absence of certain sizes of PCR products.

presumably because it is present in the added DME. There is also a decrease in calcium content toward the end of the fermentation, that is, after the majority of the yeast has flocculated. Determining the Bitterness of Beer Using UV Spectroscopy

The isomerized α-acid content of beer, the measure of beer bitterness, can be estimated by extraction of the acids into 2,4,4-trimethylpentane and measuring the absorbance at 275 nm.20 Simply multiplying the absorbance by 50 gives the approximate concentration of isomerized α-acids in ppm, expressed in international bittering units (IBU). Ten separate student results for a single beer (English bitter style) ranged between 15.0 and 19.6 IBU. Bitterness measurements on the fermenting brews showed a marked decrease as the fermentation proceeded.

Determining the Amylase Activity in Malted Barley

Malted barley contains enzymes that are essential for converting the starch in the grain to fermentable sugars during the mashing process. Students extract enzymes from the malt and incubate them with a starch substrate and iodine indicator. The absorbance of the solution, monitored spectrophotometrically at 600 nm, decreases as the starch is metabolized and the blue iodine/starch complex decreases in concentration. After selecting an appropriate blank, the students design their own series of simple experiments by proposing a simple hypothesis, which they then test. Example hypotheses often center on the role of calcium ions, pH, or temperature in the activity of the amylase enzymes. Students also often examine the amylase activity of the more darkly roasted grains as compared to the pale or base malts to examine how resistant amylase is to inactivation by the heating and drying processes that are part of malt production. Upon successful completion of the course, students have gained experience in instrumental analytical techniques not usually encountered in introductory chemistry classes, while learning to document their laboratory work in a laboratory notebook. Students have also applied their knowledge of chemistry and biology to a complex system, and learned about the brewing of beer on a small scale and on an industrial scale.

Determination of the Alcohol Content of Beer Using Gas Chromatography

The ethanol in beer can be separated by direct injection of diluted beer onto a packed or capillary column and quantified using a flame ionization detector or a thermal conductivity detector.21 To compensate for the variability of injecting 1 μL samples into the instrument, an internal standard is used, 1-propanol, which is added to all standards and samples. Standards ranging from 0.025% to 0.250% (v/v) ethanol and with 0.250% (v/v) of the internal standard are prepared in water. Appropriately diluted beer or wort samples are filtered through a 0.45 μm frit prior to injection, if necessary, to ensure no solid particles enter the column. No detrimental performance of the gas chromatograph or column has been observed after many hundreds of injections. Student results typically show an increase in ethanol content of their wort as the fermentation proceeds, and a good correlation between commercial samples and their reported alcohol contents. Ethanol is even detected in “nonalcoholic” beers, albeit below the 0.50% (v/v) allowed for a beer to be considered “nonalcoholic”.

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ASSOCIATED CONTENT

S Supporting Information *

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Instructor notes and student handouts for each of the laboratory assignments. This material is available via the Internet at http://pubs.acs.org.

BIOLOGY-FOCUSED LABS The three biology-focused laboratory exercises are summarized below.

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Yeast Flocculation Assay

AUTHOR INFORMATION

Corresponding Author

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Using an adaptation of the Helm assay, students monitor the flocculation (precipitation) of different strains of yeast by comparison with a control and measuring the absorbance spectrophotometrically at 600 nm. The control is a nonflocculating sample of yeast, carefully washed to remove all media and Ca2+ ions, a metal ion essential for flocculation. The flocculating yeast samples are suspended in a Ca2+(aq) solution, and after a given time that allows for the larger yeast aggregates to settle, the solution is sampled just below the meniscus. Absorbance measurements allow for the relative concentration of individual yeast cells that remain suspended to be estimated. Typical student experiments compare the flocculation properties of ale and lager yeasts, for example, or yeast strains used in the production of wheat beers.

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to acknowledge the generosity of the Willard Eccles Foundation, and Greg Schirf, owner of Wasatch Beers, for providing financial support for the brewing program at Westminster.

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Yeast Genotyping Using Delta Sequence PCR

There are several hundred yeast types that have been developed by the brewing industry to impart special characteristics into the final product. These all look very similar under a microscope and genetic fingerprinting is often required for proper strain identification. Using an adaptation of the method developed by Coakley et al.,23 students use PCR and 338

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