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→Cleaning and Sanitizing
'''(In progress)Quality Assurance'''refers to the process if developing standard operating procedures for proactively avoiding quality problems <ref name="Diffen">[https://www.diffen.com/difference/Quality_Assurance_vs_Quality_Control "Quality Assurance vs. Quality Control". Diffen website. Retrieved 03/28/2018.]</ref>. In the brewing industry, this includes avoiding off-flavors from contamination, dissolved oxygen in beer, fermentation and ingredient issues, etc. Although quality assurance in the brewing industry covers a wide range of approaches such as verifying yeast health and pitching rates, recording fermentation data, performance tracking, etc. (see the [[Quality_Assurance#External_Resources|External Resources]] below and [[Laboratory Techniques]]) <ref>Zachary Taggart. "Quality Assurance for Small Breweries: The Key to Growth". CBC 2018.</ref>, this article will focus primarily on avoiding microbial contamination through cleaning and disinfecting, particularly in the area of sharing a space for both pure culture fermentation and [[Mixed Fermentation|mixed fermentation]] beer.
==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'''Quality Assurance''' refers 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 process if developing standard operating procedures for proactively avoiding quality problems brewing environment where few competitors can survive <ref name="DiffenSuzuki_2012">[https://wwwonlinelibrary.diffenwiley.com/differencedoi/Quality_Assurance_vs_Quality_Control "Quality Assurance vsabs/10.1002/j.2050-0416.2011.tb00454.x 125th Anniversary Review: Microbiological Instability of Beer Caused by Spoilage Bacteria. Quality Control" Ken Suzuki. Diffen website2012. Retrieved 03DOI: https:/28/2018doi.org/10.1002/j.2050-0416.2011.tb00454.x]</ref>. In Hop tolerant lactic acid bacteria have been found on the surfaces of many places in the brewing industryenvironment, this includes avoiding off-flavors including the fermentation area, bottling area, and cold storage. Hop tolerant lactic acid bacteria have been isolated from contamination, dissolved oxygen the air in at least one brewery in beer, the fermentation and ingredient issuesbottling 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, etcand Juan Carlos Cabada. 2017. DOI: https://doi.org/10. 1094/ASBCJ-2017-4294-01.]</ref>.
See also:* The efficacy of different chemicals to kill microbes within a biofilm isn't widely studied in the brewing or wine industries, partly because testing procedures are laborious and difficult to standardize[[Barrel#Sanitizing|Barrel]] wiki page.* [http://mmbr.asm. One study found that alcohol-based disinfectants (ethanol and isopropyl alcohol) were effective at killing microbes within a biofilm, and peracetic acid disinfectants were not as effectiveorg/content/77/2/157/F3. A higher concentration of peracetic acid (from 0expansion.25% to 1% html Diagram overview of products containing 4-15%) was required to be more effective than lower concentrations. However, these disinfectants did not kill bacterial and fungal species reported at all stages of the cells without a cleaning regiment first. Yeast biofilms, in general, are more susceptible to cleaning chemicals than bacteria biofilms. Biofilms that are formed under static conditions (still or dried up liquid) are more resistant to disinfectants than biofilms that form under flow conditions (movement of liquid) <ref name="Wirtanen_2001" />beer production.]
===Spores===Some species of fungi and bacteria can form spores. Fungi form spores Microbe Populations in order to reproduce sexually. Their sporulated forms are not a mode of protection from disinfectants and are therefore killed by normal sanitation methods. Bacteria form spores as a mode of survival. For example, some dangerous types ''Clostridium botulinum'' spores require 250°F (121°C) for 3 minutes to be killed, which is the requirement for canned goods <ref>[http://www.jfoodprotection.org/doi/abs/10.4315/0362-028X-45.5.466?codeMixed Fermentation Breweries=fopr-site Differences and Similarities Among Proteolytic and Nonproteolytic Strains of Clostridium botulinum Types A, B, E and F: A Review. RICHARD K. LYNT*, DONALD A. KAUTTER and HAIM M. SOLOMON. 1982.]</ref><ref>[http://beerandwinejournal.com/botulism/ Chris Colby. "Storing Wort Runs the Risk of Botulism". Beer and Wine Journal Blog. 04/17/2014. Retrieved 04/04/2018.]</ref>. Spore-forming species of bacteria, however, are not considered beer spoilers <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2047716495256554/?comment_id=2047776558583881&reply_comment_id=2048688798492657&comment_tracking=%7B%22tn%22%3A%22R%22%7D Bryan Heit. Milk The Funk Facebook thread on yeast and bacteria spores and brewery hygiene. 04/04/2018.]</ref>. Thus, the challenge of killing yeast or bacteria spores is irrelevant in most beer and wine production. There are some extraneous brewing methods where bacteria spores should be considered, for example [[Wild_Yeast_Isolation#Safety|wild yeast isolation safety]], [[mold]] formation during fruit fermentation or barrel aging, and the [http://beerandwinejournal.com/botulism/ long storage of unfermented wort].
Seasonality played a minor role in the populations of microbes throughout the brewery, with ale yeast and ''Candida santamariae'' spreading from the fermentation and packaging area to the rest of the brewery from fall to summer (it was proposed that the warming from fall to summer played a role in the spread of ale yeast and ''Candida santamariae'' throughout the brewery). ''Micrococcus'' and ''Kocuria'' were found in more localized areas such as the floors and other surfaces in the barrel room, cellar, and packaging room. ''Acetobacter'' and ''Lactobacillus'' were found specifically in areas where a lot of wort or beer was being processed (conveyor belts and floors below the packaging equipment, hotside and cellar area sinks, and sample ports on kegs and fermenters). ''Lactobacillus'' was more common on surfaces where sour beer production was (fermenters and barrel surfaces), with floor surfaces having a more diverse mixture of LAB species <ref name="Bokulich_2015" />. Microbes with hop tolerant genes were found more abundantly in the fermenter and packaging areas (filler heads, below the bottling line belt, packaging sink, and a keg faucet) compared to microbes found on pellet hops (which were determined to not be a source of contamination), kegs, or barrel bungs, and was associated with where beer was being processed, particularly mixed fermentation and spontaneously fermented sour beer <ref name="Bokulich_2015" />. In the case of mixed cultures or contaminations that contain ''Pediococcus damnosus''., ''Lactobacillus brevis'', or ''Lactobacillus lindneri'', and possibly other species of these two genera, it is possible for the cells of these bacteria to adhere to the cells of brewers yeast. This can cause the yeast to prematurely drop out of suspension during fermentation, resulting in under attenuated beer. This function of the bacteria is thought to contribute to the slower development of the bacteria after the yeast has dropped out of solution. Other species of ''Lactobacillus'' such as ''L. casei/paracasei'', ''L. coryniformis'' and ''L. plantarum'' as well as species of ''Leuconostoc'' are very intolerant of hops and are therefore only considered a threat against beer that is very lowly hopped <ref name="Suzuki_2012" />. ==Biofilms==Many microorganisms can form ''biofilms'' which is defined as a community of cells of one or more species that are attached to each other and/or a surface and are embedded in a matrix of extracellular polymeric substances (EPS). EPS consists of polysaccharides and proteins that are produced by the microorganisms and expelled out of the cells, similar to a [[Pellicle|pellicle]]. Biofilms allow microbes to survive less vigorous cleaning and sanitizing regiments and chemicals and has become a concern in the food industry as well as in the brewing and winemaking industries <ref>[https://onlinelibrary.wiley.com/doi/abs/10.1111/1541-4337.12087 The Paradox of Mixed‐Species Biofilms in the Context of Food Safety. Iqbal Kabir Jahid and Sang‐Do Ha. 2014.]</ref>. Biofilms most often form in the packaging system somewhere, but can also be found on side rails, wearstrips, conveyor tracks, drip pans, and in-between chain links <ref name="storgards_2000" />. Bacteria and wild yeast form a biofilm in two stages, which are determined by a number of variables. In the first stage, the microbes remain in their [http://www.dictionary.com/browse/planktonic|"planktonic"] form (floating around in the liquid), but they begin to adhere on surfaces and to each other as those surfaces. Other species of microbes can also be adhered to during this phase. The second stage is where the microbes start producing exopolysaccharides (EPS) which helps them bind together in a matrix, along with any available proteins and exopolymers produced by the bacteria. A large portion of biofilms is actually water (80-80%) as this allows the microbes to remove waste and consume nutrients. This matrix helps the microbes resist antibiotics, UV radiation, and cleaning chemicals. Gene exchange also occurs more frequently. At the end of this second stage, the microbes become attached to surfaces in such a way that is permanent without the use of cleaning chemicals. This is known as the microbe's [http://www.dictionary.com/browse/sessile|"sessile"] form (immobile). Bacteria in this form continue to multiply, and upon maturation of the biofilm, eventually, planktonic cells begin to be produced and released from the biofilm to find new homes. They also display different phenotypes, which might contribute to their ability to resist cleaning chemicals. Rough surfaces, scratched surfaces, jagged edges, and pores are more prone to biofilm formation due to the higher surface area. Hydrophobic surfaces, such as Teflon and other plastics, are more prone to biofilm formation than hydrophilic surfaces (glass and stainless steel). Nitrile butyl rubber (NBR) was found to inhibit biofilm formation when new, but as the material breaks down biofilms are able to grow <ref>Biofilms in the Food and Beverage Industries. P M Fratamico, B A Annous, N W Guenther. Elsevier, Sep 22, 2009. Pp 4-14.</ref>. Biofilm formation is strain specific rather than species specific; some strains can form thicker biofilms than others within the same species and faster, and some strains of lactic acid species are not good biofilm producers <ref name="Wirtanen_2001" />. Wild strains of ''S. cerevisiae'' that carry the "Flo11p" gene tend to form biofilms which suggests that this ability is important for survival in the wild, but domesticated strains have mostly lost this ability probably due to evolving under nutrient-rich environments (human-controlled fermentation), and their planktonic form may give them an advantage in nutrient-rich liquids, especially during spontaneous fermentation where their ability to be mobile might help them compete against other species of microbes <ref>[https://www.nature.com/articles/s41467-018-05106-7 The origin and adaptive evolution of domesticated populations of yeast from Far East Asia. Shou-Fu Duan, Pei-Jie Han, Qi-Ming Wang, Wan-Qiu Liu, Jun-Yan Shi, Kuan Li, Xiao-Ling Zhang & Feng-Yan Bai. 2018.]</ref><ref>[https://www.ncbi.nlm.nih.gov/pubmed/11157168 Bakers' yeast, a model for fungal biofilm formation. Reynolds and Fink. 2011. DOI: 10.1126/science.291.5505.878.]</ref>. Full biofilms can form within 2-4 days for some strains, while 10 days is required for significant biofilm formation in other strains. For example, one strain of ''Lactobacillus brevis'' isolated from draft beer did not form any biofilm, while another strain of ''L. brevis'' tested was a strong biofilm producer. Similar results were observed for ''Brettanomyces'' strains. In general, mixed cultures form stronger biofilms than single cultures. The presence of soil (biological residue) encourages biofilm formation <ref name="Wirtanen_2001" />. The presence of sweeteners or sugar also encourages the formation of biofilms. In one study (Storgårds 2006), biofilm forming species were found to begin attaching themselves to brand new sterile stainless steel surfaces within 2-12 hours after the new equipment was used for production <ref name="Storgårds_2006">[https://www.researchgate.net/publication/279707988_Microbial_attachment_and_biofilm_formation_in_brewery_bottling_plants Microbial attachment and biofilm formation in brewery bottling plants. Erna Storgårds, Kaisa Tapani, Peter Hartwall, Riitta Saleva & Maija-Liisa Suihko. 2006. DOI: https://doi.org/10.1094/ASBCJ-64-0008.]</ref>. The efficacy of different chemicals to kill microbes within a biofilm isn't widely studied in the brewing or wine industries, partly because testing procedures are laborious and difficult to standardize. Studies have found that alcohol-based disinfectants (ethanol and isopropyl alcohol) and hydrogen peroxide-based disinfectants were effective at killing microbes within a biofilm, and peracetic acid disinfectants were not as effective. A higher concentration of peracetic acid (from 0.25% to 1% of products containing 4-15%) was required to be more effective than lower concentrations. However, these disinfectants did not kill all of the cells without a cleaning regiment first. Yeast biofilms, in general, are more susceptible to cleaning chemicals than bacteria biofilms. Biofilms that are formed under static conditions (still or dried up liquid) are more resistant to disinfectants than biofilms that form under flow conditions (movement of liquid) <ref name="Wirtanen_2001" /><ref name="Wirtanen_2003">[https://link.springer.com/article/10.1023/B:RESB.0000040471.15700.03 Disinfection in Food Processing – Efficacy Testing of Disinfectants. G. Wirtanen, S. Salo. 2003.]</ref>. See also:* [[Brettanomyces#Biofilm|''Brettanomyces'' biofilm]]* [https://twitter.com/socialmicrobes/status/983764240254341125?s=04 Time lapse of biofilm formation.] ==Spores==Some species of fungi and bacteria can form spores. Yeast generally forms spores in order to reproduce sexually. Their sporulated forms are not a mode of protection from disinfectants and are therefore killed by normal sanitation methods. Bacteria and molds form spores as a mode of survival. For example, some dangerous types ''Clostridium botulinum'' spores require 250°F (121°C) for 3 minutes to be killed, which is the requirement for canned goods <ref>[https://www.cdc.gov/mold/faqs.htm#mold "What are molds?". CDC website. Retrieved 01/25/2019.]</ref><ref>[http://www.jfoodprotection.org/doi/abs/10.4315/0362-028X-45.5.466?code=fopr-site Differences and Similarities Among Proteolytic and Nonproteolytic Strains of Clostridium botulinum Types A, B, E and F: A Review. RICHARD K. LYNT*, DONALD A. KAUTTER and HAIM M. SOLOMON. 1982.]</ref><ref>[http://beerandwinejournal.com/botulism/ Chris Colby. "Storing Wort Runs the Risk of Botulism". Beer and Wine Journal Blog. 04/17/2014. Retrieved 04/04/2018.]</ref>. Spore-forming species of bacteria, however, are not considered beer spoilers <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2047716495256554/?comment_id=2047776558583881&reply_comment_id=2048688798492657&comment_tracking=%7B%22tn%22%3A%22R%22%7D Bryan Heit. Milk The Funk Facebook thread on yeast and bacteria spores and brewery hygiene. 04/04/2018.]</ref>. Thus, the challenge of killing yeast or bacteria spores is irrelevant in most beer and wine production. There are some extraneous brewing methods where bacteria spores should be considered, for example [[Wild_Yeast_Isolation#Safety|wild yeast isolation safety]], [[mold]] formation during fruit fermentation or barrel aging, and the [http://beerandwinejournal.com/botulism/ long storage of unfermented wort]. ''Lactobacillus'' species [https://www.researchgate.net/post/How_to_prepare_spore_forming_media_for_lactobacillus do not form spores]. ==Hygienic Methods For Avoiding Contamination=====General Approaches===* Clean and disinfect all equipment that comes into contact with the beer or wort, including beer dispensing systems, and keep them in prime condition.* Use the maximum concentrations, exposure times, and hottest temperatures (considering temperature limitations of plastics and glass) suggested by the manufacturers of any given cleaning/disinfectant product.* Clean first using an effective cleaner, and then apply a disinfectant/sanitizer as a second step.* Use 180°F (82°C) hot water for 60 minutes to disinfect stainless steel and other heat tolerant materials (check with your manufacturer to make sure that the vessel is rated to withstand fast hot/cold cycles; vacuum or pressure relief valves should be used in order to avoid imploding due to fast temperature shifts for some equipment). * 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#Diastatic_strains_of_Saccharomyces_cerevisiae|diastatic strains of ''Saccharomyces cerevisiae'']]''.* Replace rubber and plastic parts such as gaskets as often as recommended by the manufacturer or when wear is apparent.* When operating a commercial brewery, invest in a quality control lab and procedures to identify inefficient hygiene practices. * Use a separate packaging system for sour beer unless the packaging system can be sanitized with hot water or caustic (foam disinfectants that are often used in packaging lines have been reported to be not as effective against removing biofilms).* The more surface area that equipment has, the more prone it is to biofilm formation. Horizontal surfaces are more prone than vertical surfaces to biofilm formation.* Heat 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>. ===Reducing Microorganisms===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#Diastatic_strains_of_Saccharomyces_cerevisiae|diastatic strains of ''Saccharomyces cerevisiae'']] 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>. Some other methods have been proposed by scientists as being novel ways to reduce unwanted microorganisms. These include exploiting naturally produced toxins. For example, some lactic acid bacteria [[Lactobacillus#Bacteriocins|produce bacteriocins]] which can kill other bacteria. Some strains of wine yeast can [[Saccharomyces#Killer_Wine_Yeast|produce zymocins]] that kill other species of yeast. Such methods are viewed as being fairly extreme. Advances in genetic engineering techniques make these approaches technically possible, however, there currently exists a commercial stigma against genetic modification. Additionally, there are many types of toxins which target only specific species, so anticipating which species should be targeted could be challenging <ref name="Vaughan_2005" />. ====Yeast Washing====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. ===Cleaning and Sanitizing====The goal of cleaning is to remove as much biomaterial as possible, while the goal of sanitizing is to reduce the population of viable microbes as much as possible and prevent them from growing on surfaces during the non-production time. It's been shown that chemical cleaners are better at removing biofilms than sanitizers and disinfectants, and sanitizers that kill cells in suspension may not be effective at killing cells within biofilms. Complete removal of unwanted microbes within biofilms can be achieved by first using a cleaning agent to remove the biomass followed by a sanitizing /disinfecting agent. CIP procedures may not be enough to remove biofilms without high turbulent flow with spray nozzles and the use of heat (low cleaning temperatures are not effective at removing biofilms). Chlorinated alkaline detergents were found to be the most effective at removing biofilms <ref name="Wirtanen_2001" />. Below is a typical CIP process according to [http://www.vtt.fi/inf/pdf/publications/2000/P410.pdf Erna Storgårds (2000)]; CIP processes at room temperatures are not adequate enough to remove biofilms, so use hot temperatures when applicable. Use the highest chemical concentrations recommended by the vendor. Also, the higher the velocity of the cleaning fluid through the system, the more efficient it is at removing biofilms:
{| class="wikitable sortable"
| Rinse || cold || 10-30 min
|-
| Disinfection (chemical such as peracetic acid or hot water at 85-90°C) || cold (or hot if using just water) || 10-30 min with chemical, or 45-60 min with hot water
|-
| Rinse (might contain a low concentration disinfectant) || cold || 5-10 min
Open surfaces such as bottle inspectors, fillers, and conveyor belts in the packaging line should be first rinsed with water, then cleaned with a foaming agent, rinsed again with water, and then sprayed with a disinfectant solution and a final rinse. Components that cannot be visually inspected should be dismantled and inspected. Rubber gaskets and sealings have been found to house biofilms, especially after deteriorating, and so they should be inspected and replaced as needed. NBR rubber has been found to inhibit biofilms when new, and EPDM rubber has been found to be anti-bacterial towards some bacteria <ref name="Wirtanen_2001" />.
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.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1952066318154906/ MTF thread on brewing mixed fermentation beers with clean beers in a commercial brewery.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1710242802337260/ Another MTF thread on sanitation.]
* [https://www.facebook.com/groups/MilkTheFunk/permalink/2461516447209888/ MTF thread on using one keg cleaner to clean regular beers and sour beers in commercial breweries.]
* [https://byo.com/article/a-clean-fight-the-science-of-hygienic-brewing/ "A Clean Fight: The Science of Hygienic Brewing" by Colin Kaminski in Brew Your Own Magazine.]
*[https://www.facebook.com/groups/MilkTheFunk/permalink/4279204685441046/ Brian Hall demonstrates steam sanitizing a corny keg.]
* [https://www.theseus.fi/bitstream/handle/10024/348696/Assessing%20Contamination%20Risk%20of%20Non-Conventional%20Yeast%20in%20Breweries.Ronja%20Eerik%C3%A4inen.pdf?sequence=2 "Assessing Contamination Risks of Non-Conventional Yeasts in Breweries," bachelors thesis for Ronja Eerikäinen (yeast study only)]; see also an interview with Eerikäinen on the [https://brulosophy.com/podcasts/the-bru-lab/ The Brü Lab podcast episode 16].
* [https://www.birkocorp.com/wp-content/uploads/2017/08/Birko_ReducingDissolvedOxygen_WhitePaper.pdf "Reducing Dissolved Oxygen: Acid and Detergent Cleaning of Brite Tanks," white paper by Dana Johnson Technical Director, Craft Brewing, Birko. As appeared in The New Brewer, July/August 2011.]
===Oak Barrels===
See the [[Barrel#Sanitizing|Barrel]] wiki page.
===Pasteurization===
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 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>.
Diastatic strains of ''Saccharomyces cerevisiae'' can have a wide range of temperature tolerance. Strains that can form ascospores or vegetative cells can be more heat tolerant. One thesis paper reported it taking 9 minutes at 60°C to kill 90% of a strain of diastatic ''S. cerevisiae'' in ascospore form <ref>[https://www.sciencedirect.com/science/article/pii/S2214799322000170#bib0135 Spoilage yeasts in beer and beer products. Inge M Suiker, Han AB Wösten. Current Opinion in Food Science. Microbiology, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. 02/19/2022.]</ref>.
See also:
* [[Barrel#Sanitizing|Barrel Sanitizing]].
* [http://wiki.zero-emissions.at/index.php?title=Pasteurization_in_beer_production "Pasteurization in Beer Production"] and [http://wiki.zero-emissions.at/index.php?title=Process_info:_Pasteurization_in_beer_production "Pasteurization in Breweries"]; AEE - Institut für Nachhaltige Technologien wiki.
* [http://milkfacts.info/Milk%20Processing/Heat%20Treatments%20and%20Pasteurization.htm Heat Treatments and Pasteurization standards for milk processing.]
* [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.]
* [[Cider#Keeving|"Keeving," a traditional method of achieving carbonation in sweet ciders (not pasteurization, but potentially a related application).]]
===Draught Line Cleaning===
See the Brewers Association [https://www.brewersassociation.org/educational-publications/draught-beer-quality-manual/ Draught Beer Quality Manual].
==Quality Control==
(To do)
===Quality Control=== '''Quality Control''' is the process of identifying quality problems in the product, and is a reactive process aimed at correcting a detected problem <ref name="Diffen" />.
Detection methods <ref name="Wirtanen_2001" /><ref name="Bokulich_2018" />:* [http://cdn.intechopen.com/pdfs/27440/InTech-Use_of_atp_bioluminescence_for_rapid_detection_and_enumeration_of_contaminants_the_milliflex_rapid_microbiology_detection_and_enumeration_system.pdf ATP bioluminescence system monitors for contaminants.] This method uses light to detect organic matter, but cannot identify the microorganisms that are present. It also indicates whether the biological matter is low enough to employ disinfectant.
* Swabbing and cultivating on agar and PCR.
* Direct epifluorescence filter technique (DEFT)/microcolony method.
* Direct impedimetry.
* Flow cytometry.
* DGGE/TGGE (separation of heterologous sequences by chemical/heat denaturation).
* Clone Libraries (Sanger sequencing of transformed ''E. coli'' clones containing diagnostic gene amplicons).
* qPCR
* [http://illumina.com/ Illumina] Sequencing
* 454 Life Sciences Pyrosequencing
* [https://europepmc.org/abstract/med/29703269 PCR DNA dipstick for rapid detection at different stages of the brewing process].
* Disposable electrochemical biosensors for ''Brettanomyces bruxellensis'' in wine <ref>[https://www.sciencedirect.com/science/article/pii/S0925400518317271 Disposable electrochemical biosensors for Brettanomyces bruxellensis and total yeast content in wine based on core-shell magnetic nanoparticles. María L. Villalong, Boryana Borisova, Christian B. Arenas, Anabel Villalonga, María Arévalo-Villena, Alfredo Sánchez, José M. Pingarrón, Ana Briones-Pérez, Reynaldo Villalong. 2018.]</ref>
* [https://www.scribd.com/document/395419129/Micro-Identification?secret_password=yFUCiiLek4p72XWmAzMK&fbclid=IwAR1y4-yrXgpoqtyPsSTt8scfM41OBMX3hhRi0InSlLUNIIeRA8yS-3ezs_k PDF from Richard Preiss with a summary mostly derived from Jorgenson, E. (2017). An Overview of Bacteria Found in Brewing Ecosystems. Master Brew. Assoc.]
* [https://onlinelibrary.wiley.com/doi/pdf/10.1111/1751-7915.13422 RNA-FISH (Fluorescense ''In Situ'' Hybridization) technique for detecting ''Brettanomyces'' contamination should use a red-emitting fluorophore.]
* [https://www.sciencedirect.com/science/article/pii/S0030402619318546 Surface enhanced Raman spectroscopy phylogenetictree for genosensing of ''Brettanomyces bruxellensis'' yeast on nanostructured ultrafine glass supports.]
Beer adapted strains of ''Brettanomyces'' and lactic acid bacteria have been found to be more difficult to culture on agar than when they are not adapted to the environment<ref name="Suzuki_2012" />. Malt agar has been shown to be more effective at showing growth than some other types of agar (Dekkera Medium, Universal Beer Agar, Potato Dextrose Agar, and Beer Agar). [[Laboratory_Techniques#Saccharomyces|YPD]] with 10 ppm Cycloheximide was not tested in the study, but Nick Impellitteri, the owner of [[The Yeast Bay]], reports that this media works well for him <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/2047731471921723/?comment_id=2047735648587972&reply_comment_id=2047763545251849&comment_tracking=%7B%22tn%22%3A%22R0%22%7D Nick Impellitteri. Milk The Funk Facebook post on the ability of different media to grow beer adapted ''Brettanomyces''. 04/03/2018.]</ref>, and DBDM media has also been reported to work well for growing ''Brettanomyces''. In addition, it is recommended that 100 mL samples are taken since beer adapted contaminants are harder to grow on media, and at least 7 days of incubation time should be allowed for ''Brettanomyces'' to show signs of growth <ref>[https://www.tandfonline.com/doi/abs/10.1094/ASBCJ-2008-0917-01 Effects of Beer Adaptation on Culturability of Beer-Spoilage Dekkera/Brettanomyces Yeasts. Koji Suzuki, Shizuka Asano, Kazumaru Iijima, Tomoo Ogata, Yasushi Kitagawa & Tsunehiro Ikeda. 2018.]</ref> (see also [https://www.facebook.com/groups/MilkTheFunk/permalink/2047731471921723/ this MTF thread)].
Advanced beer-spoiler detection medium (ABD) has been shown as a more effective growth medium compared to other media when attempting to grow beer-adapted bacteria such as hop tolerant strains of ''Lactobacillus'' that don't grow well on other media. See [[Laboratory_Techniques#Lactobacillus.2FPediococcus|Lab techniques]] for more information<ref name="Suzuki_2012" />.
See also :* [[Laboratory Techniques]]for the recipes for these agar types.*[https://www.brewersassociation.org/directories/suppliers Brewers Association list of lab services (filter by "Services", then "Contamination" or "Laboratory services").]* [https://drive.google.com/drive/folders/1IsB_deiFyJfwKbHiKFf5-YCWwEufHQk1 Kevin McGabe's HomebrewCon 2018 Seminar "Bringing a Brewery Quality Control Lab Into Your Home".]* [https://www.mbaa.com/publications/tq/tqPastIssues/2017/Documents/TQ-54-2-0408-01.pdf MBAA guide to common spoilage bacteria and their morphology.]* [https://www.mbaa.com/publications/tq/tqPastIssues/2020/Pages/TQ-57-1-0222-01.aspx MBAA TQ article, "Building a Quality Control Lab: An Introduction to Starting and Growing a Quality Program" by Melissa Antone.]* [https://file.scirp.org/pdf/AiM_2016032215023444.pdf Review of Gram negative bacteria in brewing.]* [http://suigenerisbrewing.com/index.php/2019/12/04/contamination-detection-3/ Dr. Bryan Heit of Sui Generis blog explains his PCR method for detecting 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===="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'']] =====VBNC In Yeast=====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 SO<sub>2</sub>) 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 SO<sub>2</sub>, 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 SO<sub>2</sub> 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 SO<sub>2</sub> is the only form of SO<sub>2</sub> 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 SO<sub>2</sub> (for example when wineries use much higher concentrations of SO<sub>2</sub> 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 SO<sub>2</sub> 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>. ===Sensory Analysis===Sensory analysis is the process of implementing a standardized method for collecting data on finished beer through sensory evaluation. Sensory analysis can be a low tech approach to first steps in identifying potential quality issues <ref>[https://beerandbrewing.com/dictionary/nB20s1RI6j/ "Sensory Evaluation" Craft Beer & Brewing website. Retrieved 02/14/2020.]</ref>. See also:* [https://beerandbrewing.com/dictionary/nB20s1RI6j/ "Sensory Evaluation" on Craft beer & Brewing website.]* [https://www.morebeer.com/articles/Beer_Sensory_Analysis "An Introduction to Sensory Analysis" by Scott Bickham on the Morebeer website.]* [https://www.youtube.com/channel/UCql1o9GpzeDqWHgd52Zxm9Q BrewEssence product videos.] ===Supply Chain Management===* [https://www.brewersassociation.org/presentations/supply-chain-management-for-craft-brewers/ Supply Chain Management on the BA Website.]
==Quality Improvement==
(To do)
'''Quality improvement''' is a formal and systematic practice of analyzing ongoing processes and identifying problems or places for improvement.
* [https://info.moravia.com/blog/quality-assurance-quality-control-and-quality-improvement-explained "Confused? Don’t Be! Quality Assurance, Control and Improvement Explained" by Lee Densmer.]
==See Also==
===Additional Articles on MTF Wiki===
* [[Laboratory Techniques]]
* [[Wild_Yeast_Isolation#Safety|Wild Microbe Safety]]
* [[Mold]]
* [[Grain#Microbial_Populations_on_Barley|Microbial Populations on Grain and Malted Barley]]
* [[Mixed Fermentation]]
===External Resources=== * [https://www.youtube.com/watch?v=TyBiDvphGHs Cleaning and Sanitation with Rick Theiner - BeerSmith Podcast #187.]* [http://masterbrewerspodcast.com/115-todays-challenges-in-beer-quality MBAA Podcast interview with Mary Pellettieri, author of "Quality Management: Essential Planning for Breweries".]* [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.* [http://my.asbcnet.org/ItemDetail?iProductCode=96346 "QUALITY SYSTEMS: Practical Guides for Beer Quality", by Dr. Charlie Bamforth, 2019.]* [http://my.mbaa.com/ItemDetail?iProductCode=72697 "Brewery Cleaning: Equipment, Procedures, and Troubleshooting" by Richard J. Rench.]* [https://jp.hach.com/asset-get.download.jsa?id=50544340479 Industry standards of dissolved oxygen levels in beer throughout the brewing process, by Hach.]* [https://eurekabrewing.wordpress.com/yeast-cultivation/ Eureka Brewing Blog on lab techniques, yeast handling, etc.]* "Yeast: A Practical Guide to Beer Fermentation" by Chris White and Jamil Zainesheff, 2010.* "Brewing Yeast and Fermentation" by Boulton and Quain, 2006.* [https://www.asbcnet.org/Pages/default.aspx American Society of Brewing Chemists.]* [https://brewingscience.com/brewers-lab-handbook/ "Brewers' Laboratory Handbook: Brewing Without the Blindfold™", by Brewing Science Institute.]* [https://bkyeast.wordpress.com/2012/02/19/selective-media-part-i/ "Selective Media Part 1" by BKYeast blog.]* [https://bkyeast.wordpress.com/2013/05/06/pouring-plates-and-making-slants/ "Pouring Plates and Making Slants" by BKYeast blog.]* [https://www.escarpmentlabs.com/single-post/2019/06/17/Quality-management-at-Escarpment-Labs Quality Management at Escarpment Labs.]* [http://www.terifahrendorf.com/Lab-Manual.pdf Teri Fahrendorf’s "Small Brewery Lab Procedures Manual"]* [https://coloradobeer.org/tech-safety-post/so-you-want-to-add-a-brewing-lab/ Colorado Brewers Guild "So You Want to Add a Brewing Lab?"]* [https://www.youtube.com/watch?v=bW-UkOHtx9k Brew Strong podcast interview with Dr. Jon Hughes on Building A Quality Control Lab On A Budget (Aug 6, 2021).]* [https://journals.asm.org/doi/pdf/10.1128/jmbe.00336-21 Bootleg Biology: a Semester-Long CURE Using Wild Yeast to Brew Beer.]
==References==