Brewery Laboratory Manual
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Brewery Laboratory Manual
Our highly praised lab handbook has been fully overhauled and updated with powerful content, rich graphics, and must-have information for successful brewing. This handbook helps you do just that, while respecting the fact that you want to spend time in the brewery, not in the lab. I have read and accept the Wiley Online Library Terms and Conditions of Use Shareable Link Use the link below to share a full-text version of this article with your friends and colleagues. Learn more. Copy URL During fermentation, yeast cells convert cereal?derived sugars into ethanol and CO 2. Yeast also produces a wide array of aroma compounds that influence beer taste and aroma. The complex interaction between all these aroma compounds results in each beer having its own distinctive palette. This article contains all protocols needed to brew beer in a standard lab environment and focuses on the use of yeast in beer brewing. More specifically, it provides protocols for yeast propagation, brewing calculations and, of course, beer brewing.Beer is traditionally made from four key ingredients: malted cereals (barley or other), water, hops, and yeast. Each of these ingredients contributes to the final taste and aroma of beer. The main goal of malting is to activate enzymes within the grain. These enzymes will hydrolyze starch and other compounds within the kernels during mashing (Goldammer, 2008; Kunze, 2004 ). During malting, barely kernels are soaked in water and periodically aerated, the so?called steeping and germination phase. Next, in the drying and kilning phase, kernels are dried and heated. This stops germination, arrests enzymatic activity within the kernels, reduces spoilage risks, and determines the impact of malt on the final aroma and color of the beer.The main goal is to convert insoluble malt or grain material into a soluble and fermentable extract. This mash is kept at specific temperatures and pH to ensure proper enzymatic conversion of starch and proteins.
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At this temperature, proteases are activated and degrade proteins to short peptides and amino acids, that will form the major nitrogen source for yeast during fermentation. Beta?amylases will cleave off maltose from starch molecules.The remaining extract (wort) is transferred to the boiling vessel. These contribute to bitterness and aroma of the final beer. More specifically, hops contain alpha acids and during boiling, these acids will isomerize into iso?alpha acids, the major bittering substances in beer. Aroma hops are typically only added towards the end of the boil, or in the dry?hopping of green beer to reduce the stripping of aroma?active compounds.The boiled wort is then transferred to a whirlpool to remove the aggregated protein and insoluble hop components (hot trub). Finally, the wort is cooled, aerated, and transferred into the fermentor, where yeast is added. At the same time, hundreds of secondary metabolites that influence the aroma and taste of beer are produced. Variation in these metabolites across different yeast strains is what allows yeast to so uniquely influence beer flavor. Examples of typical yeast?derived beer flavors are the esters isoamylacetate (banana aroma), ethyl acetate (solvent?like aroma), and ethyl hexanoate (pineapple aroma).During this maturation, the remaining yeast are still metabolically active and can produce additional CO 2 and ethanol as well as reduce off?flavors such as diacetyl (buttery, rancid aroma).Two main types of yeast are used in brewing: Saccharomyces cerevisiae is a top?fermenting yeast used to make ales while Saccharomyces pastorianus is a bottom?fermenting yeast used in lager brewing. The latter is actually a hybrid yeast and combines phenotypes of both its parental species: the high fermentation capacity of S. cerevisiae and the cold tolerance of S. eubayanus (Libkind et al., 2011 ).
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These kinds of fermentations usually involve a mix of different yeast species (and bacteria as well) that appear sequentially over time, giving the beer an added complexity.More specifically, this article describes the different steps in producing your own beer, starting from some basic brewing calculations to yeast propagation and chemical analysis of the end product. We deliberately only included one beer recipe, so you can experiment with how different yeast strains can influence beer aroma. Since the brewing world is full of its own jargon, we have included a glossary of brewing terminology and abbreviations (Table 1 ). The methods provided in this protocol are designed to have a minimal equipment requirement and is similar to a small?scale home brewing approach. Far more advanced brewing setups are available but are often costly and vary greatly in the specifics of usage and are, thus, beyond the scope of this protocol unit. Each of the brewing?specific materials can be purchased from various suppliers, we have listed some of them in Table 2.Important for the production of unsaturated fatty acids and sterols needed for yeast membrane production. Show a low solubility in water but isomerize during the boiling process into water soluble iso???acids. The maximum attenuation is determined by wort composition whereas the actual attenuation is largely determined by the yeast strain. Usually constitutes the bulk of the grain bill. During dry hopping predominantly aroma oils dissolve into the wort, while almost no bitterness is added. The abbreviation EBC is also used as the unit for the color of malts and wort.Depends on the boiling time and wort gravity. Also includes the remaining hop material. Contains yeast cells, protein, and hop resins. A highly modified malt is typically suitable for direct mash?in at amylase rest temperatures without a previous protein rest. Period after the end of fermentation during which the beer is rounding off its flavor profile, e.g.
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, by reduction of diacetyl by yeast. Caused by phenolic compounds, most prominently 4?vinylguaiacol (4?VG).Usually no enzymatic potential, cell walls, proteins, and starch are well degraded, free sugars are caramelized during kilning and subsequent roasting. For this, we refer readers to, for example Goldammer, 2008; Snyder, 1997, and to the Internet Resources listed at the end of the article.Making a wort recipe from scratch allows complete control of this process. This can be important if the experiments in question require specific modifications to wort composition. Here we provide a basic wort recipe starting from whole grains that can easily be scaled or modified as needed. It is not designed to mimic a specific beer style but rather produce a lightly hopped wort that can be used as a growth medium for fermentation assays. Individual styles are beyond the scope of this method but there are many resources and software available for developing recipes within individual beer style guidelines, see Internet Resources.For yeast propagation and simple fermentation tests, wort prepared according to Alternate Protocol 1 can also be used.It should also still include unshredded husks to create a stable grain bed during the lautering and prevent a stuck mash (see also Troubleshooting). During this time, gently stir the mash every 10 min. The wort's viscosity is also decreased at higher temperatures, which helps in the subsequent lautering.A low flow rate is needed to avoid collapse or creation of channels through the grain bed. As the lautering progresses keep track of the wort gravity (see Basic Protocol 2 or Alternate Protocol 2 ).The bitterness of a beer can be affected by, among others, the alcohol level, residual sugars or the use of roasted malts. Alpha acid greatly depends on hop variety, harvest year, and hop storage conditions. It can be estimated using the following formulaIt is unique to each brewing setup and recipe and has to be determined empirically.
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However, a 60 brewhouse yield can be used as a reasonable estimate to calculate the first brew. For more details on brewhouse yield estimation, see Kunze ( 2004 ).This protocol describes how to check if the starch has been sufficiently hydrolyzed. Dried malt extract (DME) wort offers a very reproducible recipe that can be easily prepared without the specialized equipment required for an all grain?derived wort (see Basic Protocol 1 ). DME, or spray malt, is available in a range of colors and comes in hopped and unhopped varieties. Here we use light unhopped DME, but the recipe can be readily adapted using other DME variants. This recipe can also be used as a concentrated stock if lower gravity wort is required (e.g., for yeast propagation).Because of this, twice the volume is prepared to ensure there is sufficient clarified wort harvested in the end. Traditionally, wort density is measured using a hydrometer. The value can be determined as the point to which the stem sinks into the fluid. A hydrometer will sink deeper into a sample with lower density and vice versa. Extract values measured after the fermentation is finished are called residual extract (RE) or FG, respectively. Pull it up again and then lower it gently until the expected value is almost reached. Please check the user manual. The working principle of a density meter is based on an oscillating U?tube made of glass. The U?tube gets excited to vibrate at its characteristic frequency. Depending on the density of the tested sample this characteristic frequency changes, which allows calculation of the actual density (DMA 35 Portable Density Meter, Instruction Manual, Anton Paar GmbH, Austria, 2009). This calculation is automatically done by density meter devices for beverage use (e.g., Anton Paar DMA 35). To this end, aspire the liquid into the U?tube using the built?in sample tube and pump lever or inject the sample with a plastic syringe. Then dispose of the liquid by depressing the pump lever.
Nowadays, different brewing yeasts are commercially available. These yeast strains are most often supplied as liquid yeast slurries or as active dried yeast powder. For use of these yeast starter cultures, we refer to the manufacturers protocol. Please check Critical Parameters and Troubleshooting if the cell counts are much lower than expected.Volumes larger than 5 ml should be grown in Erlenmeyer flasks in a shaking incubator. The pH of the starter wort should be around 5. All yeast cells take up the dye, but only viable cells can reduce it to a colorless product.Cell counts and viability measurements can also be automated, for example by using a TC20 Automated Cell Counter (Bio?Rad). Use a lint?free paper tissue to remove excess water. Usually a 1:100 or a 1:1000 dilution of your original culture works well.Avoid air bubbles under the coverslip. It basically comprises a chamber, created by a grid of perpendicular lines that are carved into glass (upper panel). The area bound by each line, as well as the volume of each chamber that is created in this way, is known. By counting cells in a specific area of the grid (e.g.,, in the five indicated areas in lower panel), it is possible to calculate the original overall cell concentration in the suspension that was pipetted in the chamber. In general, pale violet cells and budding yeast cells are considered as viable cells. Each counted square holds the precise volume of 4 nl. If you counted 5 squares, you have the cell count for a total volume of 20 nl. To determine cell count in 1 ml, multiply by 50,000 (5 ? 10 4 ). Please note that volume of counting chamber is given for a Neubauer?improved hemocytometer. If you are using a different type, please check the manufacturer's instructions on how to determine total cell count.This process will take around 6 days for an ale and 10 days for a lager.
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Lower the bottling wand to the bottom of the bottle and gently depress to slowly begin filling while being careful of avoid aeration of the beer. Often it is useful to take samples throughout the fermentation process to measure metabolite production or track fermentation kinetics. Here we describe a setup where samples can be taken while avoiding contamination of the beer (see also Fig. 3 ). Full?scale industrial fermentations take place under very different physical conditions compared to lab?scale fermentations. There is a number of ways to mimic the conditions of larger scale fermentations. One method is to simply use stirred fermentations. This protocol is described below.The septum allows for sampling without risk of contamination.In this case, the stopper and airlock should be sanitized with 70 ethanol prior to use. Do not use more than 80 of the total volume to avoid over foaming. In some cases, with yeast that ferment heavily or produce large krausen even smaller volumes should be used. The given protocol will lead to total pitch rates of 1.35 billion cells for ale yeasts and 2.7 billion cells for lager yeasts. EBC can be converted to standard reference method (SRM), which is used in the Americas.In case the measured absorbance is too high, use a dilution of the sample. In contrast to the wort, which can be considered as a mixture of water and dissolved extract, the final beer also includes alcohol. Therefore, only the apparent attenuation can be determined based on density measurements. Variation in these metabolites across different yeast strains is what allows yeast to so uniquely influence beer flavor. The protocol below describes how to detect several of the most important yeast?produced flavor compounds, namely ester compounds. Decarboxylation of ferulic acid by yeast during fermentation leads to formation of 4?vinyl guaiacol (4?VG). 4?VG has a very distinctive smoky, clove?like aroma.
In most beers, except for particular styles such as Belgian witbier and German hefeweizen, 4?VG is considered a yeast?derived off?flavor; often also referred to as a phenolic off?flavor (POF). This is to avoid false results due to gas bubbles adhering to measuring devices (e.g., hydrometer) or emerging during an actual measurement (e.g., density meter). For all protocols handling fermenting wort or beer, this degassing protocol should be followed.Release the pressure carefully by lifting the stopper from time to time. Discard the first 20 ml of flow through and collect the rest into the second Erlenmeyer flask. Because of this spontaneous process, fermentation results often varied a lot, with big differences in flavor and aroma between different fermentation rounds. To create more consistency and predictability in the quality of the end product, early brewers started using a small fraction of a previously fermented batch to inoculate a new batch; effectively (but unknowingly) recycling the microbes from the previous fermentation round to the next. This practice is called backslopping and promoted adaptation of microbes to the fermentation environment, a process called domestication.Driven by this demand, brewers and researchers are currently investigating the potential of alternative yeast species (both Saccharomyces and non.Follow the tips below to avoid or solve the problem.Avoid a high flour percentage. It helps to fully open the tap of the lauter tun two to three times in the beginning for a short time (?2 s) to remove bigger particles from underneath the false bottom.This will most likely just worsen the problem by contracting the filter bed. Instead, lower the flow rate and carefully cut the filter bed with a long knife in a rhombus like pattern. Don't cut all the way down to the false bottom.In this case extend the incubation time. Beware that this can also lead to lautering problems (see above).
Allow the mash to equilibrate and take the first measurement after 10 min. A typical mash pH should be between 5.2 and 5.5, but depends on the malt and water used.Adjust by boiling more vigorously, increasing the duration or by reducing the sparge water volume. Check the following points if the fermentation stops with an unexpectedly low ADA.Strains unable to ferment this trisaccharide typically yield ADA around 60. Insufficient aeration limits cell membrane production can lead to unhealthy yeast. Moderately modified malts may require a protease rest while mashing to release enough FAN to the wort. Cool hot wort as fast as possible. Do not cover pot during the boil. Minimize oxygen exposure of the finished beer. Please check the Critical Parameters and Troubleshooting section if unanticipated results occur. All of these characteristics are influenced by multiple parameters of the brewing process, e.g., malt composition, fermentation temperature, hop variety, and yeast. Using controlled conditions, a standard wort, and consistent fermentation conditions, these measurements can be used to characterize a given yeast strain. These measurements can be further used, for example, to check if the resulting beer falls within specific style guidelines.If the compounds are present in beer below the detection limit of the used setup, they are often represented as “not detected” (N.D.). Table 9 contains data of these aroma compounds for two “standard” types of beer, as well as the concentration ranges in beer reported for each of these compounds, found in literature (Chemists).Production of POF is undesirable for most beer yeast strains and beer styles, with a few exceptions. In some Belgian specialty beers POF is tolerated and for Wit as well as German Hefeweizen, it is even a characteristic of the style.KV is supported by a FWO postdoctoral fellowship.
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Original research in the lab of KV is supported by KU Leuven Program Financing, European Research Council (ERC) Consolidator Grant CoG682009, VIB, European Molecular Biology Organization (EMBO) Young Investigator program, FWO, and VLAIO (Vlaams Agentschap Innoveren en Ondernemen). Stuttgart, Germany: Verlag Eugen Ulmer.Retrieved from Berlin, Germany: Versuchs.New York, NY: William Morrow Cookbooks. Please upgrade your browser to improve your experience. This is done by increasing process visibility, reducing risk and minimizing destroyed product. When used effectively, it should help to protect a brewery’s reputation. In today’s Colorado craft beer market, your reputation is as valuable as ever. Labs in our industry range from roller tables in brewhouse corners to large designated rooms filled with rows of laminar flow hoods, instruments, and microscopes. There’s no right answer, as every brewery has different priorities, but I firmly believe that something is better than nothing. Start with what you can afford, and expand as you are able. Note that this blog post is inherently scientific. Feel free to reach out with questions. There are volumes of information on each of the items listed below. This is simply meant to help you organize your thoughts and quickly put some quality control measures in place. Once all or most of Tier I is up and running, move on to Tier II, and so on. Or mix and match!! Also, be sure to use control samples to check your sampling and inoculation technique, and reduce false readings. See references (2) and (3) for more information. They’re so easy, there’s no reason every brewer can’t do them. In this article, we’ll put some of this lab equipment to use with some simple lab tests that I have adapted from a number of sources. All of them are easy to run, require a minimum of equipment, and provide useful information on your beer and brewing procedures.
Finally, I include several easy tests for assaying whether your beer is subject to increased diacetyl levels, what your final gravity will be, whether your sweet wort is sufficiently clear, and whether your yeast has mutated to unacceptable levels. This test is so ridiculously simple that you can (and should) run it every time you brew. Check it every 12 hours or so. If the wort remains clear and shows no signs of growth for more than 48 hours, your sanitization is probably good enough; whatever contaminants may exist in your wort are probably at such low levels that yeast pitched at a decent rate would probably overwhelm and outcompete the contaminants. If the sample stays clear for 72 hours or more, pat yourself on the back for excellent attention to cleaning and sanitization. On the other hand, if the wort turns cloudy or shows other signs of growth within 24 hours, you’ve got major problems with your sanitization methods. To test for contaminants in your slurry or pitching methods, you need to kill off the brewers yeast but leave the contaminants alive. Fortunately, cycloheximide (also known as Actidione) will do just that. Cycloheximide is heat-sensitive, so store your solution in a refrigerator (but keep in mind that cycloheximide is nasty stuff — please keep it away from children!). Using the syringe with the filter attached, add the cycloheximide solution to the sample at the rate of 1 mL per 100 mL of pitched wort. They syringe filter keeps the cycloheximide solution from being a potential source of contaminants. Mark this sample as poison — you don’t want to sip this by mistake. Incubate it the same way as in the wort stability test. Cycloheximide kills brewers yeast, so if anything grows in your sample it’s not something you typically want (unless you’re brewing lambic, of course). If the sample stays clear and shows no signs of growth for 72 hours or more, all is well.
If your wort stability test comes out OK but the pitched wort stability test fails, then it’s likely that your yeast is infected or you otherwise introduced an infection at pitching. If the wort stability test fails but the pitched wort test comes out OK, then the contaminant is something sensitive to cycloheximide — such as brewers yeast. This force test can alert you to such problems many days or weeks before they are noticeable in the beer. If you plan to sample beer in kegs or serving tanks, sanitize the sample port or serving hose first, then take two samples. If testing bottled beer, wipe the crown and neck down with alcohol, uncap, flame the bottle mouth, and quickly pour samples into the two sterile containers. In either case, add cycloheximide to one sample and mark it, and incubate both samples in a warm, dark place. If the plain sample shows growth but the cycloheximide sample does not, your contamination is an organism that’s sensitive to cycloheximide — such as brewers yeast. In that case, either the beer wasn’t quite finished fermenting before going to the bright tank, or you have some sort of mutant or wild yeast that’s eating dextrins in the beer. If both samples show growth, you’ve probably got a bacterial infection. It is a special growth medium developed and patented by S.Y. Lee of Coors Brewing Company in the 1970s. It contains cycloheximide and therefore will not support the growth of brewers yeast, but will support and encourage the growth of most common brewery bacteria better than Universal Beer Agar (UBA). I’ve discovered that it can also be used to test for certain black molds endemic to the Texas Gulf Coast (a common contaminant in my brewery). For example, a colony of Lactobacillus will look different from one of Pediococcus, and both will look different from acetic acid bacteria colonies, based on color and texture (see box, “Identifying Common Brewery Bacteria,” above).
Further, while anaerobic (oxygenless) incubation is needed to grow colonies of Zymomonas, Megasphaera, and other true anaerobes, most of the bacteria of interest to brewers — including facultative anaerobes such as Lactobacillus and Pediococcus — will grow on LMDA under aerobic conditions. If you want to be really thorough, you can inoculate two plates of LMDA and incubate one under normal aerobic conditions and the other in a sealed container with an activated GasPak Plus envelope to create an anaerobic environment. To be completely thorough, let the wort stand for 2 hours to assess the true amount of sediment. Of course, this is rarely practical for 5-gallon batches, but it will give you a good data point about your procedures for future batches. It’s possible your beer had a Pediococcus infection, but it might have fallen victim to a much simpler problem instead. The yeast can, over time, reabsorb the diacetyl and reduce it to an almost flavorless compound, butanediol. The oxidation (oxidative decarboxylation) of alpha-acetolactate to diacetyl takes place more rapidly at higher temperatures, which is one reason why a diacetyl rest works — it accelerates this reaction at a time when lots of active yeast are available to reabsorb and reduce the diacetyl. If a significant amount of alpha-acetolactate is present after primary fermentation, however, it can remain in the beer and oxidize into diacetyl. This happens rapidly if the beer is ever subjected to warm temperatures (such as might be encountered in shipping), and more slowly as the beer ages. Armed with this information, you will know whether to conduct (or continue) a diacetyl rest to facilitate its elimination before racking the beer off the yeast. (Thanks again to George DePiro for this test.) If you take a sample to assess the beer’s gravity at this point, you can just use the beer from the hydrometer jar; this test doesn’t require aseptic sampling. Pour the sample into two flasks or glasses. Taste each one.
If the heated sample tastes significantly more “buttery” than the other, then your beer contains a substantial amount of alpha-acetolactate and a diacetyl rest may be prudent. If the two samples are roughly equivalent and the diacetyl level is not objectionable, then no diacetyl rest is needed. At elevated populations, these mutated yeast cells can cause increased levels of diacetyl and fusel alcohols, among other problems (6). One of the most common off-flavors is caused by mutants’ production of 4-vinyl guaiacol, a compound that imparts a phenolic, Weizenbier-like flavor. While not especially difficult, these methods are sufficiently inconvenient to discourage many brewers from using them. Fortunately, an easier way exists (courtesy of the Brewing-Science Institute): a special prepared media known as Respiratory Deficient Mutant Agar (RDMA), which allows “one-step” screening for petite mutants. The only minor drawback is that RDMA is heat- and light-sensitive (as is TTC), but this is no real problem if you keep your RDMA plates wrapped in foil and refrigerated until needed. Scoop up a very tiny bit of yeast slurry and streak it out on the RMDA plate. (The procedure is exactly the same as streaking an agar plate with a yeast sample from a slant. Incubate until yeast colonies are visible (usually about 72 hours). If you have less than 1 mutants, things are fine; if the mutant count exceeds 10, you have probably just identified the source of the problems you are having. All rights reserved. No part of this document or the related files may be reproduced or transmitted in any form, by any means (electronic, photocopying, recording, or otherwise) without the prior written permission of the publisher. The program includesStudent learn proper brewing techniques at the laboratory andWhile you will learnIn addition to the hands-onCheck out this story as the brewery was being built on campus.
Go find that information and learn moreCoupled with any major on campus, the minor can be a good start to a career in this rapidly growing industry.Included as well is tuition for CHEM 395 - Brewing ScienceGreeley, CO 80639. Editor Karl Ockert has assembled a talented group of expert contributors from within the pub, craft, and large brewing communities, who write from their own experience and knowledge to bring you the know-how you need in order to answer real-life questions quickly and easily. Practical knowledge is the objective for this handbook series which stresses useful applications over theory.
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