Changes

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#Saccharomyces_cerevisiae_var._diastaticusDiastatic_strains_of_Saccharomyces_cerevisiae|diastatic strains of ''Saccharomyces cerevisiae'' var. ''diastaticus'']], 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 CO² 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>.

* For any plastics that cannot be treated with heat, especially tubing, keep separate plastics for use with potential contaminants such as ''[[Lactobacillus]]'', ''[[Pediococcus]]'', ''[[Brettanomyces]]'', and ''[[Saccharomyces#Saccharomyces_cerevisiae_var._diastaticusDiastatic_strains_of_Saccharomyces_cerevisiae|diastatic strains of ''Saccharomyces cerevisiae'' var. ''diastaticus]]''.

* Boil beer instead of heat pasteurizing at below boiling temperaturesHeat pasteurize, and store beer and yeast at low temperatures. Beer filtration and pasteurization are effective ways to reduce the chance of contamination.* Non-alcoholic beer and beer under 1.3% ABV grew bacteria 2-5 times more than 4.5% ABV lager beer in one study, and the authors concluded that long-draw draft lines should not be used to serve these types of beers <ref>[https://onlinelibrary.wiley.com/doi/abs/10.1002/jib.670 Quain, D. E. (2021) The enhanced susceptibility of alcohol-free and low alcohol beers to microbiological spoilage: implications for draught dispense, J. Inst. Brew., XXX, doi: https://doi.org/10.1002/jib.670.]</ref>.

Several generalized procedures are used for limiting the number of unwanted microorganisms. These include acid washing yeast that is re-pitched (kills bacteria but not wild yeast that is tolerant of low pH), keeping beer cool (slows the growth of microbes in general), filtration (removes yeast), pasteurization (kills vegetative cells in the finished beer, but not spores - most beer spoilers are killed at 15 [http://wiki.zero-emissions.at/index.php?title=Pasteurization_in_beer_production pasteurization units (PU)] and all are killed at 30 PU using a recommended pasteurization temperature of 66°C ), and aseptic or hygienic packaging. Note that some strains of ''Lactobacillus'' have been shown to survive pasteurization temperatures; see [[Lactobacillus#Tolerance_of_Extreme_Temperature|''Lactobacillus'' Heat Tolerance]]. Packaging systems should be frequently flooded with hot water between 80-95°C or saturated steam (every 2 hours in the summer and every 4 hours in the winter). UV light or disinfecting chemicals are also used. The filler and crowner should be disinfected frequently as well. Packaging in an aseptic room with HEPA filtration and higher air pressure within the room compared to outside, along with special clothing, is another method that larger breweries use to remain aseptic <ref name="storgards_2000" />.

Most brewing equipment should be designed for good hygiene. Pits and crevices should be avoided, and all surfaces should be smooth when possible. All equipment and pipelines should be self-draining. Valves are a typical source of contamination because they are not easily CIP'ed, especially plug valves and ball valves (although butterfly, gate, and globe valves are also difficult to CIP) <ref name="storgards_2000" />. Horizontal surfaces and wet surfaces are more prone to biofilm formation. In one study that compared biofilm formation in bottling lines versus canning lines, it was found that canning lines develop less microbial biofilms and contaminations than bottling lines due to not having rinsing stations, labeling stations, and simpler constructions than the bottling lines that were studied <ref name="Storgårds_2006" />. However, some canning lines cannot use caustic for cleaning, or it is not common practice, but use foaming agents instead which are less effective at removing biofilms (see [[Quality_Assurance#Efficacy_of_Cleaning_Agents|efficacy of cleaning agents below]]). The lack of use of caustic cleaners in canning lines has been identified as a source of contamination issues with [[Saccharomyces#Saccharomyces_cerevisiae_var._diastaticusDiastatic_strains_of_Saccharomyces_cerevisiae|diastatic strains of ''Saccharomyces cerevisiae'' var. ''diastaticus'']] in canning lines <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1561762887185253/?comment_id=1791471917547681&reply_comment_id=2017381731623364&comment_tracking=%7B%22tn%22%3A%22R9%22%7D Caroline Smith from Lallemand. Milk The Funk Facebook group post on diastaticus contamination. Feb 2018.]</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.]* [[Barrel#Sanitizing|Barrel Sanitizing]].

Pasteurization is measured in terms of "pasteurization units" (PU). One PU is equal to exposure of 60°C (140°F) for 1 minute. The total PU is determined by plotting time against temperature in degrees Fahrenheit. A total of 15 PU's has been given as the target for pasteurizing beer. The following equation can be used to calculate PU's using different temperatures and times <ref name="Haas_1960">[https://www.sciencedirect.com/science/article/abs/pii/S0065216408701242 Gerhard J. Haas. "Microbial Control Methods in the Brewery". Editor(s): Wayne W. Umbreit. Advances in Applied Microbiology. Academic Press, Volume 2, 1960. Pages 113-162. ISSN 0065-2164. ISBN 9780120026029.]</ref>:  [[File:PU calculation.jpg|none|360px|]] 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 under 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.

Sodium hydroxide (caustic), [https://en.wikipedia.org/wiki/Ethylenediaminetetraacetic_acid EDTA (ethylene diaminetetra-acetic acid)], chlorinated disinfectants, and hydrogen peroxide-based disinfectants such as Pur-Ox from Birko or Lerasept-O from Loeffler are effective at breaking up biofilms when used in their highest recommended concentrations <ref name="Wirtanen_2001" /><ref>Brandon Jones. Private correspondence with Dan Pixley. 04/02/2018.</ref><ref>[https://www.reddit.com/r/TheBrewery/comments/6hqnvf/mtkettle_cleaning/dj0zd0s/ Levader on Reddit.com. "The Brewery". Retrieved 04/02/2018.]</ref>. Foaming agents that are often used in packaging lines for cleaning, however, might not be as effective. One study found that one foaming agent (VK10 Shureclean, which is sodium alkylbenzenesulphonate) required two times the maximum concentration that is recommended by the manufacturer to completely remove biofilms. In comparison, all of the sodium hydroxide (caustic) based cleaners that were tested were effective at completely removing biofilms in concentrations that were below the vendors' recommended maximum concentrations <ref>[https://link.springer.com/article/10.1007/s13213-010-0085-5#Bib1 Susceptibility of wine spoilage yeasts and bacteria in the planktonic state and in biofilms to disinfectants. Mariana Tristezza, António Lourenço, André Barata, Luísa Brito, Manuel Malfeito-Ferreira, Virgílio Loureiro. 2010.]</ref>. Another study tested six different types of cleaners were tested to see how well they removed the biofilms: keytones + surfactant detergent, quaternary ammonia + surfactant detergent, sodium hydroxide (caustic soda), sodium carbonate (soda ash), sodium hydroxide + surfactant (alkaline detergent), and chlorine (sanitizer, not a detergent). They found that only caustic soda was consistently efficient at removing the biofilm (the caustic, ammonia + surfactant cleaner, and the quaternary ammonia + surfactant detergent all efficiently removed cells that were adhered to a surface over 6 hours and were determined to be good for stopping biofilm formation from occurring in the first place) <ref>[https://www.researchgate.net/publication/235411588_Adhesion_and_biofilm_production_by_wine_isolates_of_Brettanomyces_bruxellensis Adhesion and biofilm production by wine isolates of Brettanomyces bruxellensis. C. M. Lucy Joseph, Gagandeep Renuka Kumar, Gagandeep Renuka Kumar, Edward Su, Linda F Bisson. 2006. American Journal of Enology and Viticulture 58(3):373-378.]</ref>. Peracetic acid (PAA) has also been shown to be effective against biofilms in the highest recommended concentrations but isn't as effective as the previously mentioned cleaners and should be used after a caustic cleaning cycle <ref>[https://www.researchgate.net/publication/273439407_Disinfectant_testing_against_brewery-related_biofilms Disinfectant testing against brewery-related biofilms. Storgårds, Erna & Närhi, Mikko & Wirtanen, Gun. 2001.]</ref><ref>[https://www.researchgate.net/publication/244994186_COMMERCIAL_SANITIZERS_EFFICACY_-_A_WINERY_TRIAL COMMERCIAL SANITIZERS EFFICACY – A WINERY TRIAL. Duarte, Filomena & López, Alberto & Alemão, Filomena & Santos, Rodrigo & Canas, Sara. 2011.]</ref>, but its effectiveness decreases below 20°C. Chlorine and iodine-based disinfectants destroy microbe at colder temperatures, however, they are less effective in the presence of wort or other residues. Chlorine-based disinfectants can cause pitting in stainless steel if left in contact for too long, and [https://ssbrewtech.zendesk.com/hc/en-us/articles/205602399-DO-NOT-USE-BLEACH-OR-CHLORINATED-CHEMICALS- some stainless steel manufacturers] recommend not using chlorine-based disinfectants at all (refer to your equipment and chemical manufacturers). Hot water is one of the most effective disinfectants, however, dry heat is not as effective at killing bacteria (one strain of ''L. brevis'' was able to withstand 80°C dry heat for 60 minutes) <ref name="Wirtanen_2001" />. Dry heat at higher temperatures will sterilize at 170°C for 1 hour or 190°C for 12 minutes and can be used to sterilize many metal and glass instruments. Flaming surfaces kills within seconds <ref>Private correspondence with Dr. Bryan Heit by Dan Pixley. 04/12/2018.</ref>.

Star San has only been officially tested by Five Star Chemicals against the pathogenic bacteria species ''E. coli'' and ''S. Aureus'', which is the minimum baseline required by the EPA to be labeled a "sanitizer" <ref>[https://www.homebrewersassociation.org/forum/index.php?topic=31537.msg409346#msg409346 Conn, Denny. American Homebrew Association forums. 04/08/2018. retrieved 07/09/2018.]</ref>. While we are not aware of any publicly available published studies on the efficacy of StarSan to kill yeast, several studies with other acid anionic sanitizers have confirmed that they are effective against yeast. Lee et al. (2007) found that an acid sanitizer very similar to Star San that uses citric acid instead of phosphate but the same surfactant (sodium dodecylbenzene sulfonate) took 5 minutes to kill ''Saccharomyces cerevisiae'', ''E. coli'', and ''Listeria innocua'' at room temperature (some species were killed faster than others with the ''E. coli'' actually being more resistant than the yeast), and one minute if the sanitizer was heated to 40°C on both metal and LDPE plastic (they compared the acid anionic sanitizer to 35% hydrogen peroxide, which killed all organisms with 15 seconds, indicating that this acid anionic sanitizer is effective at killing yeast, but it takes longer than a stronger chemical such as hydrogen peroxide). This study did not make mention of biofilms, however, the cultures were allowed to grow and dry overnight which could have allowed for biofilm formation <ref>[https://onlinelibrary.wiley.com/doi/full/10.1111/j.1750-3841.2007.00496.x Efficacy of Two Acidic Sanitizers for Microbial Reduction on Metal Cans and Low-Density Polyethylene Film Surfaces. J. LEE, M.J. GUPTA, J. LOPES, AND M.A. PASCALL. 2007.]</ref>. Five star Star Chemicals [httphttps://www.fivestarchemicalsbsgcraft.com/wp-contentResources/Craftbrewing/PDFs/uploadsProduct_Spec_and_Data_Sheets/Star-San-HB4Product_Spec_Sheets/StarSan_Tech.pdf also recommends 5 minutes of contact time with Star San]. Winniczuk et al. (1997) found that three phosphoric acid anionic sanitizers ("CS-100" and "CS-101-lf" by Chemical Systems of Florida, and "Clear-Clean" by Pelican Brand) were less effective at killing yeast than bacteria in the timeframe tested (1 minute contact time), but they were still effective at killing yeast at high concentrations (peracetic acid also required a higher concentration to kill yeast than bacteria). However, one of the acid anionic sanitizers tested was more effective than the other two, indicating that the chemical makeup of the particular acid anionic sanitizer has an impact on how effective it is as a sanitizer relative to other acid anionic sanitizers. Additionally, they found that peracetic acid, iodophor, and chlorine dioxide required less concentration than the acid anionic sanitizers to be effective (again, tested at 1 minute exposure time) <ref>[http://lp7lc5er8n.scholar.serialssolutions.com/?sid=google&auinit=PP&aulast=Winniczuk&atitle=Minimum+inhibitory+concentrations+of+antimicrobials+against+micro-organisms+related+to+citrus+juice&id=doi:10.1006/fmic.1997.0103&title=Food+microbiology&volume=14&issue=4&date=1997&spage=373&issn=0740-0020 Minimum inhibitory concentrations of antimicrobials against micro-organisms related to citrus juice. P.P Winniczuk, M.E Parish. 1997.]</ref>.

[http://masterbrewerspodcast.com/096-efficacy-of-sanitizers-in-the-brewery Elliot Parcells & Josh Pohlmann from Bells Brewery] tested the efficiency of various sanitizers to kill ''Lactobacillus'', ''Brettanomyces'', and diastatic strains of ''Saccharomyces cerevisiae'' var. ''diastaticus'', and claimed that iodophor was ineffective at concentrations of 25 ppm, which is the maximum concentration of iodophor for it to be considered a no-rinse sanitizer. The methodology of this experiment has [https://suigenerisbrewing.com/index.php/2022/01/20/how-not-to-test-a-sanitizer/ received some criticism ] that might invalidate its results. They diluted yeast/bacteria cultures on a 1:10 ratio into iodophor, thus diluting the iodophor below its recommended dilution and exposing it to concentrations of microbes far beyond what it was designed to kill. Testing the ability to sterilize a bulk solution does not translate to the ability of the sanitizer to sanitize trace surface microbes after proper cleaning <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2172245389470330/?comment_id=2172685769426292&comment_tracking=%7B%22tn%22%3A%22R%22%7D Dr. Bryan Heit. Milk The Funk Facebook thread on an MBAA podcast about the efficacy of iodohor as a sanitizer. 07/10/2018.]</ref>.

* [https://www.brewingsciencembaa.com/identification_keypublications/tq/tqPastIssues/2017/Documents/TQ-54-2-0408-01.htm Bacteria colony formation examples for several pdf MBAA guide to common spoilage species by BSIbacteria and their morphology.]* [https://www.mbaa.com/publications/tq/tqPastIssues/20172020/DocumentsPages/TQ-5457-21-04080222-01.pdf?fbclid=IwAR12IMUpwpF5ZXXVVEtxhFjd3QVckmJMmtOoSTR3hok21vzmYsI1WetC8uo aspx MBAA guide TQ article, "​Building a Quality Control Lab: An Introduction to common spoilage bacteria Starting and their morphologyGrowing a Quality Program" by Melissa Antone.]

* [https://www.carllibri.com/colouratlas "Colour Atlas and handbook of Beverage Biology" by Dr. Werner.]* [http://suigenerisbrewing.com/index.php/2019/12/04/contamination-detection-3/ Dr. Bryan Heit of Sui Generis blog explains his PCR method for detecting diastaticus diastatic yeast.]* [https://www.facebook.com/groups/MilkTheFunk/posts/5310978085597029 Zach Taggart offers QC tips on testing ''Lactobacillus'' growth using modified MRS agar plating and recording morphology.]* [https://www.masterbrewerspodcast.com/196 MBAA Podcast with Goose Island on how they traced an elusive wild yeast infection in their brewery.]* Books:** [https://my.mbaa.com/ItemDetail?iProductCode=52206 "Illustrated Guide to Microbes and Sediments in Wine, Beer and Juice" by George G. Edwards.]** [https://www.carllibri.com/colouratlas "Colour Atlas and handbook of Beverage Biology" by Dr. Werner.]

"Viable but nonculturable" ('''VBNC''') is a newly identified state for bacteria that are not able to grow or form colonies on typical growth media (i.e., lack of cell division), but they remain viable (alive) and retain a limited level of metabolic activity (reduced nutrient transport, respiration, and synthesis of compounds) while sometimes being able to regain their population when returned to a more ideal environment. The cells often exhibit dwarfing, and they can remain in this state without dying for 4-12+ months depending on the species. An appropriate viability test for a given species can be performed to show that the cells are not dead, even though they don't grow on typical growth media (for example, intracellular hydrolysis of CTC or reduction of INT as an indication of metabolic activity, by establishing the presence of an intact cytoplasmic membrane via BacLight® or propidium iodide, or by multi-parameter flow cytometry). Cells enter this state as a way to survive some sort of stress in their environment (for example, osmotic stress, too much oxygen exposure, exposure to white light, etc.). Treatments such as pasteurization in milk and chlorination of wastewater have also been shown to induce VBNC. A number of species have been found to be able to enter the VBNC state, including ''E. coli'', ''Lactobacillus plantarum'', ''L. lactis'', ''L. linderi'', ''L. casei'', ''L. plantarum'', ''L. paracollinocides'', ''L. acetotolerans'', and several species of ''Salmonella''. Early studies on VBNC microbes were not able to fully show that the resuscitation was truly from VBNC cells rather than a very small number of culturable cells, but later studies were able to show that some bacteria can be resuscitated from a VBNC state, although most bacteria that enter a VBNC state have not been shown to be able to be resuscitated <ref>[https://pdfs.semanticscholar.org/e661/934dca6bb0dbb31a8781f3193232b7b5a8a4.pdf The Viable but Nonculturable State in Bacteria. James D. Oliver. The Journal of Microbiology. 2005.]</ref><ref name="Liu_2018">[https://www.frontiersin.org/articles/10.3389/fmicb.2018.02076/full#B28 Induction and Recovery of the Viable but Nonculturable State of Hop-Resistance Lactobacillus brevis. Junyan Liu, Yang Deng, Thanapop Soteyome, Yanyan Li, Jianyu Su, Lin Li, Bing Li, Mark E. Shirtliff, Zhenbo Xu, and Brian M. Peters. Front. Microbiol. 2018. DOI: https://doi.org/10.3389/fmicb.2018.02076.]</ref>. The concept of VBNC cells is somewhat controversial in microbiology; some experts argue that there is no difference between so-called "VBNC" cells and persister cells <ref>[https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.15463 Song, S. and Wood, T.K. (2021), ‘Viable but non-culturable cells’ are dead. Environ Microbiol, 23: 2335-2338. https://doi.org/10.1111/1462-2920.15463.]</ref>. See also this [https://www.facebook.com/groups/MilkTheFunk/posts/6036200423074788/ MTF thread by Dr. Bryan Heit].

A couple of published studies have reported inducing the VBNC state in bacteria strains that were isolated from contaminated beer. Liu et al. (2017) were able to induce a VBNC state (meaning that they were not able to grow on growth media for up to 14 days) in a strain of ''Lactobacillus lindneri'', a species that is responsible for 15-25% of spoiled beer reports, and determined VBNC via a Live/Dead BacLight® bacterial viability kit. They induced this state by storing the cells in beer at 0°C without shaking for 190 days. They also found that storing the VBNC cells at -80°C in glycerol stocks was the best way to maintain the cells. They were able to resuscitate the cells by growing them on MRS media that had 500-1000-μL of the enzyme catalase spread onto them (trying higher temperatures did not work to resuscitate, nor using higher concentrations of MRS), thus showing that brewers can use catalase to help grow VBNC state ''L. lindneri'' cells on MRS media (and perhaps other species of ''Lactobacillus'' as well). It took 3-4 days to begin showing signs of growth on the catalase supplemented MRS media. It was also demonstrated that VBNC cells could grow in beer after 30 days of incubation, and showed final cell counts similar to normal ''L. lindneri'' and resuscitated cells <ref>[https://www.sciencedirect.com/science/article/pii/S0882401017303030?via%3Dihub First study on the formation and resuscitation of viable but nonculturable state and beer spoilage capability of Lactobacillus lindneri. Junyan Liu, Lin Lia, Bing Li, Brian M. Peters, Yang Deng, Zhenbo Xua, Mark E. Shirtliff. Microbial Pathogenesis, Vol 107. 2017. DOI: https://doi.org/10.1016/j.micpath.2017.03.043.]</ref>. Lui et al. (2018) reproduced these results with a hop tolerant strain of ''L. brevis'' <ref name="Liu_2018" />.The genes that are associated with VBNC were also found in a beer contaminating strain of ''Lactobacillus acetolerans'' <ref>[https://www.tandfonline.com/doi/abs/10.1080/03610470.2021.1997280 Chunguang Luan, Weihua Cao, Na Luo, Jingxia Tu, Jianqin Hao, Yihong Bao, Feike Hao, Deliang Wang & Xin Jiang (2021) Genomic Insights into the Adaptability of the Spoilage Bacterium Lactobacillus acetotolerans CN247 to the Beer Microenvironment, Journal of the American Society of Brewing Chemists, DOI: 10.1080/03610470.2021.1997280.]</ref>. See also:* [[Pediococcus#VBNC|VBNC in ''Pediococcus'']]

While the VBNC state has mostly only been studied in detail for bacteria, it has also been suggested that this state is also possible with eukaryotes (yeast). It has been reported that although there are many methods for detecting ''Brettanomyces'' in winemaking, there are cases when ''Brettanomyces'' is not found using culturing techniques, but years later have still infected wine. Agnolucci et al. (2010) found that sulfur dioxide induces the VBNC state in 7 ''Brettanomyces'' strains isolated from wine at concentrations of 0.2 mg/L (molecular SO₂) for 5 out of the 7 strains to 0.4 mg/L for the other 2 strains after 24 hours of incubation in a synthetic wine medium that was supplemented with various amounts of sulfur dioxide. At 0.4 mg/L they found that all but one strain had 0% culturable cells, and 4-26% VBNC cells depending on the strain. At 0.8 mg/L, no strains had culturable cells, but they all had at least 4.6-17% VBNC cells (percent of the original number of cells before being exposed to the sulfur dioxide). Even at 1 mg/L of sulfur dioxide levels, there were 2.9-15% VBNC cells, depending on the strain (the ability for the ''Brettanomyces'' to remain viable at 1mg/L of sulfur dioxide might have also been due to the pH only being 3.5, and ethanol only being 13%). They also found that 2.1 mg/L was required to reduce the VBNC state of cells to zero after 55 days of incubation and limit the amount of ethyl phenols produced by ''Brettanomyces''. They reported that trypan blue was the best method for detecting VBNC cells <ref>[https://www.sciencedirect.com/science/article/pii/S0168160510003958?via%3Dihub Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/Dekkera bruxellensis. Agnolucci M, Rea F, Sbrana C, Cristani C, Fracassetti D, Tirelli A, Nuti M. 2010. DOI: https://doi.org/10.1016/j.ijfoodmicro.2010.07.022.]</ref>.

The Agnolucci et al. (2010) study did not provide a method or data for resuscitating the VBNC ''Brettanomyces'' cells so that they can again divide and grow colonies, and this has been criticized because resuscitation of VBNC cells is considered an important aspect of strengthening the conclusion that the cells are indeed VBNC. Serpaggi et al. (2012) found similar results with using 0.8 mg/L of molecular SO₂, which resulted in all ''Brettanomyces'' cells from one wine strain to not be culturable on YPD after being incubated in synthetic wine medium that was supplemented with SO₂ for 2 days, while the viability of the cells remained high for as long as 11 days (they did not check viability after 11 days, and the viability count remained constant from day 2 to day 11; viability was determined by staining with fluorescein diacetate which stains when certain metabolic esterase activity is present). They were able to resuscitate the cells by adding NaOH to the media to bring the pH up from 3.5 to 4.0 in order to effectively eliminate the molecular sulfur dioxide (molecular SO₂ is the only form of SO₂ that is significantly effective at inhibiting microbes, and it is only stable at very low pH's). They noted that VBNC cells were about 20% smaller in size than culturable cells. The cells in the VBNC state did not produce 4EG phenol but did produce a very small amount of 4EP phenol <ref>[https://www.ncbi.nlm.nih.gov/pubmed/22365358 Characterization of the "viable but nonculturable" (VBNC) state in the wine spoilage yeast Brettanomyces. Serpaggi V, Remize F, Recorbet G, Gaudot-Dumas E, Sequeira-Le Grand A, Alexandre H. 2012. DOI: 10.1016/j.fm.2011.12.020.]</ref>. It has also been demonstrated that the presence of minerals and vitamins, as well as p-coumaric acid, can assist in resuscitating so-called VBNC cells of ''Brettanomyces'' <ref>[https://www.mdpi.com/2306-5710/9/3/69 Chandra M, Branco P, Prista C, Malfeito-Ferreira M. Role of p-Coumaric Acid and Micronutrients in Sulfur Dioxide Tolerance in Brettanomyces bruxellensis. Beverages. 2023; 9(3):69. https://doi.org/10.3390/beverages9030069.]</ref>.

It is worth noting that the VBNC state in ''Brettanomyces'' has not been tested against a higher concentration of SO₂ (for example when wineries use much higher concentrations of SO₂ solutions as a sanitizer), other chemical sanitizers, and pasteurization-level temperatures. For example, it has been proposed that steaming barrels in order for them to reach 140°F (60°C) for 20 minutes is enough to sanitize them (see [[Barrel#Sanitizing|Barrel Sanitizing]] for more information). However, Nunes de Lima et al. (2020) inoculated wines with various strains of ''Brettanomyces'' after raising the pH of the wine to 3.8, which rendered the amount of free SO₂ in the wines completely ineffective, and many of the strains entered a VBNC state. This indicates that other unknown factors can induce a VBNC state in ''Brettanomyces''. It has been documented that other factors, such as nutrient starvation, extreme temperatures, osmotic pressure and oxygen, has caused a VBNC state in other microorganisms (VBNC has been mostly studied in bacteria) <ref name="Nunes de Lima 2020">[https://www.sciencedirect.com/science/article/abs/pii/S0740002020302069 Survival and metabolism of hydroxycinnamic acids by Dekkera bruxellensis in monovarietal wines. Adriana Nunes de Lima, Rui Magalhães, Francisco Manuel Campos, José António Couto. 2020. DOI: https://doi.org/10.1016/j.fm.2020.103617.]</ref>.

* [https://www.brewingsciencemasterbrewerspodcast.com/PDF045 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://BSI_brewers_lab_handbookwww.pdf Brewers’ Laboratory Handbookmasterbrewerspodcast.com/271 MBAA Podcast interview with Lauryn Rivera and Tess Downer about Comprehensive Quality at Odell's.]* [https: Brewing Without The Blondfold™//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.]

* [httphttps://www.brewingscience.com/PDFbrewers-lab-handbook/BSI_brewers_lab_handbook.pdf "Brewers' Laboratory Handbook: Brewing Without the Blindfold™", by Brewing Science Institute.]