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→Cleaning and Sanitizing
==Overview==
Any microorganism that is introduced into a beer unintentionally and can survive in the beer is considered a "beer spoiler". Beer can be affected by contaminants via raw ingredients, poor sanitation, incorrect pasteurization, brewery environment air pollution, and inadequate ethanol production <ref>[https://www.mdpi.com/2304-8158/11/17/2693 Ciont, C.; Epuran, A.; Kerezsi, A.D.; Coldea, T.E.; Mudura, E.; Pasqualone, A.; Zhao, H.; Suharoschi, R.; Vriesekoop, F.; Pop, O.L. Beer Safety: New Challenges and Future Trends within Craft and Large-Scale Production. Foods 2022, 11, 2693. https://doi.org/10.3390/foods11172693.]</ref>. One survey of 38 craft beers in the Spanish market found that 68% of them had some presence of unwanted microbes, with beers under 5% ABV being more susceptible than higher ABV beers, indicating that craft breweries in particular may have a high degree of contamination issues <ref>[https://www.jmbfs.org/issue/june-july-2020-vol-9-no-6/jmbfs_2132_garcia-lopez/?issue_id=7366&article_id=25 CONTAMINANT MICROBIOTA IN CRAFT BEERS. Marta García López, Elena Rocheb, Encarnación Rodríguez. 2020.]</ref>. While most microorganisms cannot survive in beer due to the hops, low pH, alcohol content, relatively high carbon dioxide, and shortage of nutrients, certain species are considered to be beer spoilage organisms due to their ability to adapt to brewing conditions (namely hops, ethanol, and low pH) and sometimes form biofilms that help them resist cleaning. Some are able to survive in beer and make a potential impact on the beer's flavor by producing acidity, phenols, turbidity/ropiness via exopolysaccharides (EPS), and/or super-attenuation (which can cause gushing or in extreme cases exploding bottles/cans) with just a few surviving cells. These effects can sometimes manifest days or even weeks after packaging, and longer storage or non-refrigerated storage can increase the potential for beer spoilers to negatively impact the beer. Bacteria species that have adapted to the brewing environment tend to be hop tolerant, but strains of the same species found outside of breweries are not tolerant of brewing conditions. It is thought that these species evolved to carry the genes to adapt to brewing conditions during the 5th to 9th centuries when hops were first being used in brewing, and that this evolution gave them a specialized adaption to the brewing environment where few competitors can survive <ref name="Suzuki_2012">[https://onlinelibrary.wiley.com/doi/abs/10.1002/j.2050-0416.2011.tb00454.x 125th Anniversary Review: Microbiological Instability of Beer Caused by Spoilage Bacteria. Ken Suzuki. 2012. DOI: https://doi.org/10.1002/j.2050-0416.2011.tb00454.x]</ref>. Hop tolerant lactic acid bacteria have been found on the surfaces of many places in the brewing environment, including the fermentation area, bottling area, and cold storage. Hop tolerant lactic acid bacteria have been isolated from the air in at least one brewery in the fermentation and bottling areas <ref>[https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2017-4294-01?src=recsys Distribution of Lactobacillus and Pediococcus in a Brewery Environment. Jorge Hugo Garcia-Garcia, Luis J. Galán-Wong, Benito Pereyra-Alférez, Luis C. Damas-Buenrostro, Esmeralda Pérez, and Juan Carlos Cabada. 2017. DOI: https://doi.org/10.1094/ASBCJ-2017-4294-01.]</ref>.
Species of yeast and bacteria that are considered beer spoilers include [[Brettanomyces|''Brettanomyces'']] species, numerous [[Lactobacillus|''Lactobacillus'']] species, ''Pediococcus damnosus'', ''Pectinatus'' species (anaerobe responsible for 20-30% of bacterial contaminations that produces acetic acid, [https://en.wikipedia.org/wiki/Propionic_acid propionic acid], acetoin, and 'rotten egg' like odors in contaminated beer), ''Megasphaera cerevisiae'' (7% of bacterial contaminations; inhibited below pH 4.1 and 2.8% ABV but can produce considerable amounts of [[Butyric Acid|butyric acid]] along with smaller amounts of acetic acid, caproic acid, [[Isovaleric Acid|isovaleric acid]], acetoin, and hydrogen sulphide), ''Selenomonas lactifex'', ''Zymophilus'' spp., [[Saccharomyces#Diastatic_strains_of_Saccharomyces_cerevisiae|diastatic strains of ''Saccharomyces cerevisiae'']], and some species from the ''Candida'' and ''Pichia'' genera. Most wild yeasts that can grow in beer in lab conditions are not considered largely impactful because of their limitatons to growing in the presence of ethanol or lack of oxygen, but they can become impactful on barrel aged beers where oxygen is present (''Candida'' species, ''Pichia'' species, ''Torulaspora delbrueckii'', ''Issatchenkia orientalis'', ''Kluyveromyces marxianus'', ''Debaryomyces hansenii'', ''Zygosaccharomyces bailii'', ''Zygosaccharomyces bisporus'', ''Schizosaccharomyces pombe'', and ''Kloeckera apiculata'') <ref name="Bokulich_2013">[http://mmbr.asm.org/content/77/2/157.full The Microbiology of Malting and Brewing. Nicholas A. Bokulich and Charles W. Bamforth. 2013. DOI: 10.1128/MMBR.00060-12]</ref>. Hop tolerant lactic acid bacteria make up the majority of contamination issues in breweries, with ''L. brevis'' making up more than half of the reported contaminations, and all lactic acid bacteria making up 60-90% of reported contaminations. A new species of ''Lactobacillus'' was recently identified called ''L. acetotolerans'' and was [https://www.facebook.com/groups/MilkTheFunk/permalink/1363048380390039/ responsible for contaminating Goose Island's Bourbon County Stout], which is 60 IBU and 11% ABV. In sour beers with a pH below 4.3, only some lactic acid bacteria, ''Brettanomyces'', and some wild ''Saccharomyces cerevisiae'' strains (which sometimes produce phenols, haze, over-attenuation, and/or over-carbonation) have the potential for unwanted growth, while beers with low alcohol, a small amount of hops, lower CO<sup>2</sup> volumes (cask ales and beers dispensed with nitrogen, for example), and higher pH (4.4-4.6) are the most susceptible to contamination <ref name="Vaughan_2005">[https://onlinelibrary.wiley.com/doi/full/10.1002/j.2050-0416.2005.tb00221.x Enhancing the Microbiological Stability of Malt and Beer — A Review. Anne Vaughan, Tadhg O'Sullivan, Douwe Van Sinderen. 2005. DOI: https://doi.org/10.1002/j.2050-0416.2005.tb00221.x.]</ref>. ''Pectinatus'' and ''Megasphaera'' are Gram-negative anaerobic species that produce a number of off-flavors in ales but not lagers (probably due to their preference for warmer temperatures). They are somewhat tolerant of hops (they can grow in beers with IBU's as high as 33-38 with one strain isolated from pickles reported to grow in beer up to 5% ABV and 80 IBU <ref>[https://www.sciencedirect.com/science/article/pii/S0740002020300514 Comparative genetic and physiological characterisation of Pectinatus species reveals shared tolerance to beer-associated stressors but halotolerance specific to pickle-associated strains. Timo Kramer, Philip Kelleher, Julia van der Meer, Tadhg O’Sullivan, Jan-Maarten A.Geertman, Sylvia H. Duncan, Harry J. Flint, Petra Louis. 2020. DOI: https://doi.org/10.1016/j.fm.2020.103462.]</ref>) and often survive within the biofilms of other species in the brewing environment where the biofilm creates an anaerobic environment for them. They are sometimes found contaminating low ABV beers (under 5.2%) during packaging. They are not tolerant of pH below 4 and are killed at relatively low temperatures (58–60°C for one min) <ref name="Suzuki_2012" />. ''Zymomonas mobilis'' is a microaerophilic Gram-negative acetic acid bacteria that can withstand hops and can grow in bottled beer or casks where priming sugar is added and small amounts of air is present and produces high levels of acetaldehyde and hydrogen sulphide <ref name="Vaughan_2005" />. While the Gram-positive ''Staphylococcus xylosus'' bacteria, which grows on the skin of humans and animals, is not normally considered a beer contaminant, one strain was isolated from craft beer and was identified as the cause of increased turbidity, lactic acid, and succinic acid. It could grow at a pH between 3-7 (although only produced turbidity at a pH of 4-7), a temperature between 4–37°C, and as much as 8% ABV. This demonstrates that it is possible for species to adapt to living in beer other than the more typical beer spoilers <ref>[https://onlinelibrary.wiley.com/doi/pdf/10.1002/fsn3.1256 Beer‐spoilage characteristics of Staphylococcus xylosus newly isolated from craft beer and its potential to influence beer quality. Zhimin Yu, Qiuying Luo, Li Xiao, Yumei Sun, Rong Li, Zhen Sun, Xianzhen Li. 2019. DOI: 10.1002/fsn3.1256.]</ref>.
Sources for contamination in breweries can occur as "primary" contaminations (yeast pitching, and brewhouse related contaminations), or as "secondary" contaminations (packaging and cellaring), as well as in tap systems. They are usually not sudden occurrences, but a result of the continued growth of microorganisms in a problem area. Historically, re-pitching yeast was often a source of contamination; however, more recently this has become less of a source for contaminations due to better education and techniques. Typical sources for contamination also include unclean equipment such as thermometers, manometers, valves, dead ends, gas pipes, leaks in any part of the system (especially at heat exchangers), wort aeration equipment, and even worn floor surfaces. More than half of the documented contaminations come from the packaging system. These are typically the sealer (35%), the filler (25%), the bottle inspector (10%), dripping water from the bottle washer (10%), and the environment close to the filler and sealer (10%). In regards to the environment as a source of contamination, this has been found to be from airborne contaminants near the filler and crowner in open bottles on their way from the bottle washer to the filler and from the filler to the capper. The higher the humidity and the more airflow, the more chances of airborne contamination <ref name="storgards_2000" /><ref name="Vaughan_2005" />. For example, ''Pectinatus'', while mostly found in lubrication oils, water systems, floors, water condensed on ceilings, etc., it can also survive on [https://en.wikipedia.org/wiki/Aerosol aerosols] in the air and is thought to possibly transferred to beer that is being packaged via the air <ref name="Suzuki_2012" />. In tap systems at taverns, 'one-way' valves that are attached to kegs have been found to be a source of contamination, as well as the dispensing line <ref name="storgards_2000" /><ref name="Vaughan_2005" />.
Thompson et al. (2024) divided the brewing environment into three "zones": areas that have direct contact with beer, such as the brewhouse, areas that are adjacent to beer production areas that have no physical barrier from the beer production area, and areas that have a physical barrier between the beer production areas, such as offices, tap rooms, etc. They took samples from all areas in three small microbreweries. The study found that racking arm valves were one of the worst offenders of housing contaminating microbes, although the results were skewed by one of the breweries having a higher load of contaminants in general between the three microbreweries. Proper cleaning, sanitation, and maintenance of racking arms is recommended to reduce biofilm formation. Other areas that harbored bacteria included the bottom valve, carb stone valve, spray ball valve, and the yeast pitch. All areas of the brewery that were swabbed show at least some bacterial growth, demonstrating that all areas of the brewery are potential sources of contamination. This study noticed that one brewery in particular had a higher occurrence of spoilage microbes; the study speculated that this might be due to cleaning and sanitation protocols during packaging, and heavy loads of contaminants were found in the packaging system <ref>[https://academicjournals.org/journal/JBD/article-full-text-pdf/705951B71856 Alex R. Thompson, Julie K. Northcutt and Paul Dawson. Bacterial contamination and surface hygiene in the microbrewery environment. Journal of Brewing and Distilling. Vol. 13(1), pp. 1-10, January-June 2024. DOI: 10.5897/JBD2024.0060.]</ref>.
See also:
Yeast washing is the practice of exposing a yeast slurry to extreme acidic conditions in order to destroy bacteria contaminants in the slurry, and it has been used for over a century in the brewing industry to help reduce the potential for lactic acid bacteria spoilage. Although techniques might vary throughout the brewing industry, the most typical technique is to add phosphoric acid to a slurry of yeast until a pH of 2 is reached, and then the slurry is stored for 2 hours at 5°C (41°F). While phosphoric acid is a good choice for acid washing because of being inexpensive compared to other acids, its tendency to not kill yeast, and its lack of affecting beer flavor, it also does not kill some contaminants such as ''Shimwellia pseudoproteus''. It has also been proposed that chlorine dioxide, a disinfectant that is often used in the vegetable, meat, and water treatment industries, can be successfully used to wash a yeast slurry, with the first study on this reporting that a concentration of 78 mg/L (concentration value is for the entire slurry) and stored for 30 minutes at 8°C (46.4°F) was effective <ref>[https://link.springer.com/article/10.1007/s00253-020-10534-x Modeling the inactivation of Lactobacillus brevis DSM 6235 and retaining the viability of brewing pitching yeast submitted to acid and chlorine washing. Munford, A.R.G., Chaves, R.D., Granato, D. et al. Appl Microbiol Biotechnol (2020). https://doi.org/10.1007/s00253-020-10534-x.]</ref>.
Lysozyme, an enzyme that is often extracted from hen egg whites, is known to inhibit Gram-negative bacteria such as ''Lactobacillus'' but not Gram-positive bacteria such as ''Acetobacter'', and has been shown to be an enzyme that can help inhibit spoilage bacteria in wine and cider fermentations <ref>[https://en.wikipedia.org/wiki/Lysozyme "Lysozyme". Wikipedia. Retrieved 0319/2020.]</ref>. Lysozyme is normally added to wine with a stuck fermentation or to limit malolactic fermentation, and several yeast companies offer a lysozyme-based product <ref>[https://scottlab.com/content/files/Documents/Handbooks/ScottlabsHandbook2018.pdf 2018 Fermentation Handbook. Scott Laboratories. Retrieved 03/19/2020.]</ref><ref>[https://www.academia.edu/16244381/Lysozyme_in_Wine_An_Overview_of_Current_and_Future_Applications?email_work_card=title Lysozyme in Wine: An Overview of Current and Future Applications. Marco Esti, Ilaria Benucci. Comprehensive Reviews in Food Science and Food Safety. 2014.]</ref>. It has also been suggested to be useful for limiting lactic acid bacteria in yeast slurries, but one experiment reported that the sensitivity of different species of lactic acid bacteria varies, with ''Pediococcus inopinatus'', ''Lactobacillus brevis'', ''Lactobacillus brevisimilis'' showing similar levels of sensitivity, but ''L. linderi'' showing less sensitivity. Bacteria were inhibited more at 22°C than at 4°C. At 300 mg/L, although lactic acid bacteria was inhibited, it was not killed completely <ref>[https://www.researchgate.net/publication/293048096_Antibacterial_properties_of_hen_egg_white_lysozyme_against_beer_spoilage_bacteria_and_effect_of_lysozyme_on_yeast_fermentation/citation/download Van Landschoot, Anita & Villa, A. (2005). Antibacterial properties of hen egg white lysozyme against beer spoilage bacteria and effect of lysozyme on yeast fermentation.]</ref>. Nisin has also been proposed as a potential preservative that can be added to wort during boiling or cooling as well as to the fermenter in order to limit the growth of lactic acid bacteria by up to 90% <ref>[https://onlinelibrary.wiley.com/doi/full/10.1002/jib.233 Müller-Auffermann, K, Grijalva, F, Jacob, F, and Hutzler, M (2015), Nisin and its usage in breweries: a review and discussion. J. Inst. Brew., 121, 309–319. doi: 10.1002/jib.233.]</ref>.
The homebrew practice of mixing distilled or sanitized water into a yeast slurry, letting the slurry settle into three layers, and then removing the bottom and top layer and re-pitching or saving the middle layer, is different than "yeast washing". This process is known as "yeast rinsing", and is primarily employed by homebrewers who wish to separate trub material from their yeast slurries before reusing the yeast slurry. This might have the benefit of removing unwanted flavors from the slurry (although there is a lack of evidence that we know of for this claim <ref>[http://brulosophy.com/2015/03/02/sloppy-slurry-vs-clean-starter-exbeeriment-results/ "Sloppy Slurry vs. Clean Starter". Brulosophy website. 2015. Retrieved 03/19/2020.]</ref>) or hop material that could inhibit yeast growth, but it does not inhibit lactic acid bacteria or any other contaminants (in fact, this process increases the chances of contaminating the yeast slurry). See [https://www.homebrewersassociation.org/how-to-brew/yeast-washing-yeast-rinsing-whats-difference/ this AHA article] for more details on yeast rinsing.
See also:
* [https://www.masterbrewerspodcast.com/253 MBAA Podcast Episode 253 CIP Fundamentals.]
* [https://www.masterbrewerspodcast.com/318 MBAA Podcast Episode 318 on Autoclaves.]
* [[Barrel#Sanitizing|Barrel Sanitizing]].
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1891215887573283/ Joe Idoni's heat sanitation based SOP.]
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There are two types of pasteurization methods used in brewing: tunnel pasteurization and flash pasteurization. In tunnel pasteurization, which is more widely used in breweries, cans or bottles of packaged beer is moved slowly through a tunnel of fixed temperatures. In flash pasteurization (or plate pasteurization), large quantities of beer are pasteurized at the same time via a heat exchanger and is usually performed before the beer is packaged <ref name="Vaughan_2005" />. Since thermal death rates for beer spoilage organisms has been identified to be 140°F (60°C) for 15 minutes <ref name="Haas_1960" /><ref>[https://onlinelibrary.wiley.com/doi/pdf/10.1002/j.2050-0416.1946.tb01593.x THERMAL DEATH POINTS OF MICRO-ORGANISMS IN BEER. Aage Lund. 1947.]</ref>, this is the baseline temperature and time for pasteurization, although higher temperatures and shorter times are used for some pasteurization methods (see the below links). The complete thermal death of ''Brettanomyces'' in wines has been reported to be 50°C for 5 minutes. <ref>[https://pubmed.ncbi.nlm.nih.gov/15996781/ Thermal inactivation of the wine spoilage yeasts Dekkera/Brettanomyces. José António Couto, Filipe Neves, Francisco Campos, Tim Hogg. 2005. DOI: 10.1016/j.ijfoodmicro.2005.03.014.]</ref><ref name="Nunes de Lima 2020" />. ''Pediococcus'' is generally not tolerant of temperatures over 45°C (see [[Pediococcus#Growth_and_Environment|''Pediococcus'']]). Some strains of ''Lactobacillus'' have been shown to potentially survive pasteurization temperatures for at least some amount of time; see [[Lactobacillus#Tolerance_of_Extreme_Temperature|''Lactobacillus'' Heat Tolerance]] for more information.
Microfiltration is an alternative technology to heat pasteurization that can be used to pasteurize beer. Microfiltration uses a set of membranes, usually in the 0.45–0.65 μm range, for filtering bacteria and yeast. Bacteria have a cell size of about 5-10 μm and yeast species have a cell size of about 5–16 μm, while flavor compounds such as phenols are filtered out when using a smaller diameter filter such as 0.2 μm. One study by Bernardi et al. (2019) found that filtration with polyethermide membranes removed around 1-2 IBU, ~30% of yeast-produced phenolic compounds (most polyphenols from hops were not filtered out), and larger tannins (which were only a small portion of the total polyphenol content). The antioxidant activity was largely not impacted. After filtration, the beers were 26%-33% lighter in color, depending on the style of the beer, and were 100% clearer. The filtration that was used, which was 1.2 μm, also produced fully pasteurized beers <ref>[https://www.sciencedirect.com/science/article/pii/B9780128152584000135 Microfiltration for Filtration and Pasteurization of Beers. Guilherme dos Santos Bernardi, Jacir Dal Magro, Marcio A. Mazutti, J. Vladimir Oliveira, Marco Di Luccio, Giovani Leone Zabot, Marcus V. Tres. 2019. DOI: https://doi.org/10.1016/B978-0-12-815258-4.00013-5.]</ref>.
* [https://www.craftbrewingbusiness.com/packaging-distribution/preserve-product-quality-flash-pasteurization/ "Is flash pasteurization right for your craft beer?" by Chris Crowell in Craft Brewing Business website (details case studies for temperatures and times).]
* [https://www.masterbrewerspodcast.com/240 "Understanding the Risk of Can Pressure Failures" interview with Jim Kuhr on MBAA Podcast episode #240.]
* [https://www.masterbrewerspodcast.com/314 MBAA Podcast "Pasteurization At Goose Island".]
* [https://www.homebrewtalk.com/forum/threads/easy-stove-top-pasteurizing-with-pics.193295/?fbclid=IwAR3Glsqo-mWT70l4mY9AhmYa9SFKpfxo8gJAi8wJixlOlyccHVU5VCzn3cQ Example homebrew method for heat pasteurization by Pappers_ on HomebrewTalk '''(do not attempt this with highly carbonated beverages; bottles will break)'''.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/2350583941636473/ MTF thread on using sulfites and sorbate to stabilize fermentation in beer.]
* [https://www.masterbrewerspodcast.com/045 MBAA Podcast interview with Eric Jorgenson about his approach to microbiology and his quick reference guide of significant bacteria found in the brewery environment.]
* [https://www.masterbrewerspodcast.com/271 MBAA Podcast interview with Lauryn Rivera and Tess Downer about Comprehensive Quality at Odell's.]
* [https://www.masterbrewerspodcast.com/episodes MBAA Podcast interview with Jack Van Paepeghem about troubleshooting and identifying ''Pectinatus'' (viscous and hydrogen sulfide in lagers) contamination in a CFT 10 head 2 seamer.]
* "Quality Management: Essential Planning for Breweries" by Mary Pellettieri (Brewers Publications), 2015.
* "Illustrated Guide to Microbes and Sediments in Wine, Beer & Juice" by Charles G. Edwards (WineBuggs LLC), 2005 - A microscope companion book that includes over 30 different species of yeast, bacteria and mold commonly associated with beverages, as well as frequently encountered sediments.