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. This article will focus on microbial contamination issues and methods, particularly in the area of sharing a space for both pure culture fermentation and mixed fermentation of beer.

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, turbidity, and/or super-attenuation 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, Zymophilus spp., Saccharomyces cerevisiae var. diastaticus, and some species from the Candida and Pichia genera. Hop tolerant lactic acid bacteria make up the majority of contamination issues in breweries, with L. brevis making up more than half of the reported contaminations. 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, all of which usually don't have an impact when present unless oxygen is also present .

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 one week after 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 .

Microbe Populations in Mixed Fermentation Breweries

Bokulich et al. (2015) studied the microbial populations throughout a brewery (presumably Allagash) that produces clean beer, mixed fermentation sour beer, and spontaneously fermented coolship ale. They found that most of the microbes living in breweries were introduced from the ingredients such as malted barley and hops, and many populations were confined to specific rooms or areas within the brewery. Some species did spread to other rooms, presumably through human and insect vectors. Beer resistant lactic acid bacteria spread throughout the brewery (although more abundantly found near packaging equipment and fermenters that were filled with sour beer), but the clean beer was largely uncontaminated. Physical partitions and walls appeared to help inhibit the spread of microbes from room to room.

Ale yeast was found throughout the brewery, especially in the fermentation cellar. Malted grains were determined to be the highest source of potentially contaminating microbes (mostly Pediococcus, although the PCR method used in the study was not adequate for detecting microbes on grain) in the hotside areas of the brewery compared to other microbe sources such as human skin, outdoor air, soil, saliva, feces, water from the plumbing, etc. Hops were determined to be the highest source of microbes in the cellar fermentation areas (Pediococcus spp, Lactobacillus lindneri, and L. brevis were detected on pellet hops, although the presence of hop tolerant genes was not detected from microbes found on hops), and yeast was the highest population in fermenter and packaging areas. While human skin was a minor contributor to microbe populations found in the brewery, oak barrel surfaces were populated with microbes from unknown sources. Therefore, it was determined in this study that raw materials are the major source for potential contaminants in a brewery, although minor sources such as sinks are still potential problems and hop tolerant species were linked to the purposeful inoculation of souring microbes in the sour beer production itself.

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.

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 .

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. 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) .

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

General Approaches

• 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.
• For any plastics that cannot be treated with heat, especially tubing, keep separate plastics for use with Lactobacillus, Pediococcus, Brettanomyces, and Saccharomyces cerevisiae var. diastaticus.
• 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.

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.

Oak Barrels

See the Barrel wiki page.

Efficacy of Cleaning Agents

Commercial cleaners and disinfectants

Advantages and disadvantages of disinfectants in food processing. Source: "Disinfection in Food Processing – Efficacy Testing of Disinfectants". G. Wirtanen and S. Salo. 2003.

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 surfaces kills within seconds .

Homebrew cleaners and disinfectants

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 . 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). This study did not make mention of biofilms, however, the cultures were allowed to grow and dry overnight . 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 (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) .

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.

Tips for using Star San:

1. Completely remove all soils as soon as possible from equipment after use using an effective cleaning agent.
2. Apply Star San before using the already cleaned equipment.
3. Dilute the Star San in distilled or reverse osmosis water so that the pH is not buffered by chemicals in tap water.
4. Leave in contact with surfaces for 5 minutes or more.
5. Optional: warm the Star San and water solution to 40°C/104°F.

Five Star PBW

PBW is a replacement for caustic and is much safer to use and dispose of. It is non-corrosive and safe to use on stainless steel and plastics. We are not aware of any studies that examine the efficacy of P.B.W. to remove biofilms, nor any other percarbonate-based cleaners efficacy in a brewing environment.

Tips for using PBW on equipment that may have had biofilm formation :

1. For circulated cleaning, use 3 ounces per gallon of 180°F for 30 minutes (if using this product on plastics, heat the water that is mixed with the PBW to be as hot as the manufacturer of the plastics recommends, and extend the circulation time to an hour or two).
2. For non-circulated cleaning, use 8 ounces per gallon of 180°F water (if using this product on plastics, heat the water that is mixed with the PBW to be as hot as the manufacturer of the plastics recommends), and soak overnight.
3. Rinse thoroughly with hot water (cold water may not remove some of the residual chemicals).

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. This method uses light to detect organic matter, but cannot identify the microorganisms that are present.
• 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
• Illuminia Sequencing
• 454 Life Sciences Pyrosequencing

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 some 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)

Quality improvement is a formal and systematic practice of analyzing ongoing processes and identifying problems or places for improvement.

• "Confused? Don’t Be! Quality Assurance, Control and Improvement Explained" by Lee Densmer.

See Also

Additional Articles on MTF Wiki

• Mixed Fermentation