Quality Assurance

(In progress)

Quality Assurance refers to the process if developing standard operating procedures for proactively avoiding quality problems . In the brewing industry, this includes avoiding off-flavors from contamination, dissolved oxygen in beer, fermentation and ingredient issues, etc.

Avoiding Cross Contamination

General

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 form biofilms and survive in beer and make a potential impact on the beer's flavor by producing acidity, phenols, and turbidity with just a few surviving cells. Adaption to the brewing environment also makes them more able to survive the harsh environment of beer. These species include Brettanomyces species, numerous Lactobacillus species, Pediococcus damnosus, Pectinatus cerevisiphilus, P. frisingensis, Megasphaera cerevisiae, Selenomonas lactifex, and Saccharomyces cerevisiae var. diastaticus. In sour beers with a pH below 4.3, only the lactic acid bacteria, Brettanomyces, and some wild Saccharomyces have the potential for unwanted growth, while beers with low alcohol, a small amount of hops, lower CO² volumes (cask ales and beers dispensed with nitrogen, for example), and higher pH (4.4-4.6) are the most susceptible to contamination. Other species of microbes do not grow in beer but can become contaminants earlier on in the brewing process (for example during kettle souring). These species include enterobacteria such as Clostridium species, Obesumbacterium proteus and Rahnella aquatilis, and wild Saccharomyces that might not be able to grow in finished beer. Other species are considered "indicator" species because they do not directly cause spoilage of beer, but indicate that there is a hygiene problem. These include Acetobacter, Gluconobacter, and Klebsiella species, as well as aerobic yeasts .

Biofilm forming spoilage organisms include a much wider range and higher frequency in beer tap systems than in brewhouses. This is due to the availability of oxygen and higher temperatures at certain points in the tap system, as well as poorer hygiene in tap systems as well as the difficulty to effectively clean plastic hoses. Of particular concern here is the ability of E. coli serotype O157:H7 to survive in tap systems, which has had a couple of documented occurrences in contaminated apple cider. Another study showed that aerobic yeasts were able to grow in dispensing lines, as well as L. brevis, and in many cases the draft lines were re-contaminated after just one week of cleaning, indicating that a contamination in draft lines is difficult to remove .

Sources for contamination in breweries can occur as "primary" contaminations (yeast pitching, and brewhouse related contaminations), or as "secondary" contaminations (packaging and cellaring), as well as in tap systems. They are usually not sudden occurrences, but a result of continued growth of microorganisms in a problem area. Historically, re-pitching yeast was often a source of contamination, however, more recently this has become less of a source for contaminations due to better education and techniques. Typical sources for contamination also include unclean equipment such as thermometers, manometers, valves, dead ends, gas pipes, leaks in any part of the system (especially at heat exchangers), wort aeration equipment, and even worn floor surfaces. More than half of documented contaminations come from the packaging system. These are typically the sealer (35%), the filler (25%), the bottle inspector (10%), dripping water from the bottle washer (10%), and the environment close to the filler and sealer (10%). In regards to the environment as a source of contamination, this has been found to be from airborne contaminants near the filler and crowner. The higher the humidity and the more airflow, the more chances of airborne contamination. In tap systems at taverns, 'one-way' valves that are attached to kegs have been found to be a source of contamination, as well as the dispensing line .

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), including polysaccharides and proteins, similar to a 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 . 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 .

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 "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 "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 . 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 . 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 .

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) .

See also:

• Time lapse of biofilm formation.

Spores

Some species of fungi and bacteria can form spores. Fungi form 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 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 . Spore-forming species of bacteria, however, are not considered beer spoilers . 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, mold formation during fruit fermentation or barrel aging, and the long storage of unfermented wort.

Methods For Avoiding Contamination

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), 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 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 .

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) . 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 . 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 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 cerevisiae var. diastaticus in canning lines .

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 . Below is a typical CIP process according to 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:

Action	Temperature	Duration
Pre-rinse	cold or hot	5-10 min
Alkali cleaning with 1.5-4% sodium hydroxide (caustic)	cold or hot (60-85°C)	10-60 min
Rinse	cold	10-30 min
Acid wash (phosphoric, nitric, or sulphuric acid)	cold	10-30 min
Rinse	cold	10-30 min
Disinfection (chemical such as peracetic acid or hot water at 85-90°C)	cold (or hot if using 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 .

See also:

• Joe Idoni's heat sanitation based SOP.
• MTF thread on brewing mixed fermentation beers with clean beers in a commercial brewery.
• Another MTF thread on sanitation.

Efficacy of Cleaning Agents

Commercial cleaners and disinfectants

Sodium hydroxide (caustic), 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 . 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 . 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 , 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 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) . 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 kills within seconds on surfaces .

Homebrew cleaners and disinfectants

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 . 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 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 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. 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). Five star 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. 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) .

See 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.

Quality Control

(To do)

Quality Control is the process of identifying quality problems in the product, and is a reactive process aimed at correcting a detected problem .

Detection methods :

• ATP bioluminescence system monitors for contaminants.
• Swabbing and cultivating on agar and PCR.
• Direct epifluorescence filter technique (DEFT)/microcolony method.
• Direct impedimetry.
• Flow cytometry.

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. Malt agar has been shown to be more effective at showing growth than other types of agar (Dekkera Medium, Universal Beer Agar, Potato Dextrose Agar, and Beer Agar). 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 , 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 (see also 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 for the recipes for these agar types.

Quality Improvement

(To do)

See Also

Additional Articles on MTF Wiki

• Mixed Fermentation