Changes

* 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>.

* [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 Risk Risks of Non-Conventional Yeast Yeasts in Breweries.," bachelors thesis for Ronja Eerikäinen.pdf(yeast study only)]; see also an interview with Eerikäinen on the [https://brulosophy.com/podcasts/the-bru-lab/ The Brü Lab podcast, Episode 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.]

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" />. 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.

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''', 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>.

"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'']]

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>.