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Bacteria and 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. 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. One study 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:
* [https://www.facebook.com/groups/MilkTheFunk/permalink/1710242802337260/ Another MTF thread on sanitation.]
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>. 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 on surfaces <ref>Private correspondence with Dr. Bryan Heit by Dan Pixley. 04/12/2018.</ref>.
'''Five Star Star San''' Five Star Chemicals product Star San is a popular acid anionic sanitizer sold to homebrewers because of its relative safety and ease of use. Claims that acid anionic sanitizers are not effective at killing yeast have been made on various internet forums <ref>[https://www.homebrewersassociation.org/forum/index.php?topic=24447.msg312961#msg312961 User 'S. cerevisiae'. American Homebrewers Association forums. 10/05/2015. Retrieved 04/11/2018.]</ref><ref>[https://www.homebrewersassociation.org/forum/index.php?topic=4576.msg52894#msg52894 User 'richardt'. American Homebrewers Association forums. 11/15/2010. Retrieved 04/11/2018.]</ref>. These claims are based on the food science textbooks, "Principles of Food Sanitation," by Norman G. Marriott and Robert B. Gravani (2006) and "Basic Food Microbiology" by George Banward (1989), which contain conflicting information about the effectiveness of acid anionic sanitizers, and neither source contains experimental data nor references to experimental data. Furthermore, the provided explanation, which is that acid anionic sanitizers supposedly don't work effectively against yeast and molds is because acid anionic sanitizers are negatively charged and yeast are also negatively charged yet bacteria is killed because it is positively charged, is biologically incorrect. According to [https://www.youtube.com/watch?v=0JC9n50RdVo Dr. Bryan Heit of Sui Generis blog], both yeast and bacteria have negatively charged cell walls, and this fact has been well established in microbiology since the 1940's (Dr. Heit has published several [https://scholar.google.com/citations?user=8yxqYNgAAAAJ&hl=en&oi=ao peer-reviewed scientific studies on cell wall polarity]). While we are not aware of any publicly available published studies on the efficacy of StarSan, several studies with other acid anionic sanitizers have confirmed that they are effective against non-spore forming yeast. [https://onlinelibrary.wiley.com/doi/full/10.1111/j.1750-3841.2007.00496.x 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'' was 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 <ref>[https://onlinelibrary.wiley.com/doi/full/10.1111/j.1750-3841.2007.00496.x Efficacy of Two Acidic Sanitizers for MicrobialReduction on Metal Cans and Low-Density Polyethylene Film Surfaces. J. L EE , M.J. G UPTA , J. L OPES , AND M.A. P ASCALL. 2007.]</ref>. Five star [http://www.fivestarchemicals.com/wp-content/uploads/Star-San-HB4.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 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>.
See [https://www.facebook.com/groups/MilkTheFunk/permalink/1436419659719577/ this MTF thread] for a more extensive explanation of why skepticism should be applied to the claim that acid anionic sanitizers are not effective at killing yeast.
Tips for using Star San:
# Completely remove all soils as soon as possible from equipment after use using an effective cleaning agent.
# Apply Star San before using the already cleaned equipment.
# Dilute the Star San in distilled or reverse osmosis water so that the pH is not buffered by chemicals in tap water.
# Leave in contact with surfaces for 5 minutes or more.
# Optional: warm the Star San and water solution to 40°C/104°F.
==Quality Control==