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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), including polysaccharides and proteins, 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 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 found that alcohol-based disinfectants (ethanol and isopropyl alcohol) 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" />.
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), 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. 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">[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>.
====Cleaning and Sanitizing====