Thursday 11 August 2016

Fact or Fiction? Can Pathogens Survive in Beer - Mould Edition.

The topic of pathogens in beer is a persistent one; two years ago I wrote an extensive post on the topic (the answer is, yes, pathogens can survive in beer, but is a thankfully rare issue). More recently a similar theme consistently comes up in the various brewing forums I participate in. The new theme regards moulds (molds, for my US readers). There is no question that mould can grow on beer - indeed, most of us have seen them at one time or another. Rather, the question is if they are dangerous and whether they can be prevented.


What Exactly Is Mould?

Contrary to what many people believe, moulds are not bacteria - evolutionary speaking they are far closer to us than to bacteria. Rather, moulds are the close cousins of yeast, both of which are fungi. Yeast and moulds are very similar in their genetics, cell structure, and even some aspects of their lifestyles. There are two major features which separate yeast from moulds. The first is that moulds are almost exclusively obligate aerobes - meaning they only grow in the presence of oxygen. Some yeast are also obligate aerobes, but the yeast we use in brewing are capable of some degree of anaerobic metabolism - AKA fermentation - and thankfully so, or there'd be no alcohol in our beer. 

The second difference is how mould versus yeast cells assemble. Yeast cells are individually living cells, meaning that each cell is its own fortress and takes care of itself and no one else. Even when yeast form into filaments, they are merely "glued" together. Moulds are the opposite - moulds always form filaments, with each cell in the filament connected to its neighbouring cells such that they can share nutrients, energy, and waste.


Are Moulds Dangerous?

The answer here is "often, but not always". Moulds were (and in undeveloped areas of the world, remain) a serious issue in food safety. Even in the brewing world, moulds were an issue upto the 1930's, and its only because of our food safety measures that they've remained a historical issue. Historically, the primary fungal issue brewers faced was ergot, a fungal infection of barley (and other cereal grains) which can cause an oft-fatal disease called ergotism. This toxicity is caused by the production of an LSD-like molecule by the fungus, which when ingested could cause issues ranging from mild digestive discomfort, through to convulsions, gangrene, and far too often, death. Today this is largely a non-issue as improved grain production and harvest methods have eliminated ergot from the food chain, outside of a few small scale producers and the developing world. Ergotism was a frequent complaint (and/or preferred feature) of many beers in early European history. It was a common problem in the Anglo-Saxon era, and may even have been a "feature" of shamanic beers produced by the vikings.

Another serious historical issue, although it was not appreciated at the time, was other, more insidious mould infections. Many moulds (as well as some yeasts) produce toxins - biological products with poisonous effects. Mould-derived toxins (mycotoxins) are very different from those made by bacteria; most bacterial toxins are proteins and are readily destroyed by factors such  as the boiling, acidity and alcohol present in beer production. Mycotoxins are very different - most are small stable organic chemicals which are impervious to conditions encountered in beer production. Some of these toxins even have cumulative effects, meaning that multiple exposures to levels with no immediate toxic effects could ultimately be deadly. This often manifested itself as cancer - indeed, until the widespread use of refrigeration, stomach cancer was the most common cancer in the western world - a cancer caused almost solely by fungal toxins in improperly stored foods. A combination of refrigeration and antifungal pesticides has purged this scourge from our food supply, albeit, not soon enough to save my grandfather who fought (and ultimately lost) a 15-year battle with stomach cancer that was almost certainty caused by fungal toxins.

In terms of the toxins themselves, how long they take to form and how toxic they are is extremely variable. Gliotoxin, produced by Aspergillis (as well as other fungi and even some yeast) is produced almost immediately upon initiation of cell division. Other toxins may even be present in the spores, while yet others won't be produced until significant amounts of fungi are present. Aflatoxins,one of the most common types of fungal toxins, and commonplace in many grain (and home) loving fungi, is the predominant toxin responsible for stomach cancer. Other long-term effects of mycotoxin exposure can include immunosuppression, liver and kidney damage and reproductive issues.

So fungal toxins are dangerous - but how common are they? The answer there is complex; of all fungi, those which produce mycotoxins that harm humans are relatively rare. However, the toxins are common in the fungi which tend to thrive in our foods and in our homes. If you see a mould in your home, chances are better than 50-50 that it makes a toxin which can harm you. As a general "rule", pigmented fungi are more likely to produce toxins than are unpigmented (white) fungi, but that is not a universal rule. Indeed, the mould used to make blue cheese is intensely pigmented and yet is harmless to us (unless you are allergic to penicillin), whereas nonpigmented fungi are responsible for 2/3rds of fungal eye infections.


I Have Mould In My Beer - What Should I Do?

What should be done if there is mould on your beer is not an easy question to answer. Mould is not uncommon when brewing fruit beers; fruits often carry mould spores, and since fruit tends to float, it carries to mould to the top of the beer where it is exposed to oxygen and can grow. A small amount of growth under this situation is probably harmless, and can be easily managed (see section below). Mould on a non-fruit beer is more problematic, as it indicates that the beer was contaminated at some point after brew-day, and that the beer has been exposed to significant levels of oxygen. Some mould growth is not uncommon in the first few weeks of a wild ferment; mould lasting past that point, or appearing a any time point in a conventionally brewed beer, is something I personally would consider to be a sign that the beer should be dumped.

Preventing And Managing Mould

Preventing mould in conventional brewing is easy - normal brewing practices should kill any spores present in the grain or hops, and limiting oxygen exposure once primary fermentation is complete will prevent the germination and growth of any spores which enter the beer after brew-day.

If adding fruit, "punching down" any floating fruit (keeping the fruit below the level of the liquid), only adding fruit to secondary in an oxygen-impermeable fermenter (e.g. a carboy with an airlock ), and purging the airspace with CO2, will prevent mould from growing. As an added precaution, fruit can be washed in a mixture of water and hydrogen peroxide prior to adding to beer. To do this, add ~1/4 cup of 3% peroxide (from your local pharmacy) to a sink full of cold water. Soak intact fruit for ~5 minutes, then rinse, freeze/puree (or whatever you do prior to adding fruit to your beer), and add to the beer as normal.

For wild ferments (coolship ales, etc), preventing mould is equally simple. Ferment in a carboy or other oxygen impermeable container, using an airlock once primary fermentation is complete to exclude oxygen. Purging the headspace with CO2 after any transfers or sampling will further limit any mould growth, and has the added advantage of preventing acetic acid formation. In simple terms, if you can prevent Acetobacter from turning your beer into vinegar, and Brettanomyces from turning your beer into nail polish remover, than you can keep mould from growing.

If mould begins to form your options are more limited Fruit beers with mild contamination can be punched down and the headspace purged; if the mould doesn't return you should be OK. For other beers any mould growth should be considered a serious problem - simply removing "floaties" will not help as a small mould island will be connected to an extensive network of near-invisible fibrils which will remain behind and continue to grow. Personally, I would dump any conventionally brewed beer with mould on the surface (after confirming it is mould and not yeast or trub islands). Again, prevention through limiting oxygen ingress is a better choice than trying to remove it later.

Barrels can be particularly hard to deal with, as their higher oxygen permeability aids in mould growth. Suflating empty barrels, keeping them properly filled, and using either a tightly fitting bung or quality airlock, will prevent mould growth in most cases. Commercial brewers and vinters will usually toss barrels that develop mould; a practice we homebrewers should emulate.

In Conclusion

Long story made short, you cannot tell whether a mould is toxic or not, short of subjecting it to laboratory identification. Given that mycotoxins can have cumulative effects, the ease of preventing mould growth, and the relatively low costs of homebrew-sized batches of beer, best practices are simply to dump any mouldy beers...with the possible exception of fruit and wild beers, as discussed above (and even then, an ounce of prevention is worth a pound of cure).

12 comments:

  1. Thanks for the thorough write up, Bryan! I linked to it on the MTF "mold" wiki page, and changed references that pointed to your MTF thread to this blog article instead. Cheers!

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  2. Loving the more regular content, especially the videos, at first I thought you were going to be banking small 1.5-2ml ones though like in your yeast bank!

    I was looking at if it's possible to use gelatin vs agar online and I saw this relatively new channel I thought you might enjoy: https://www.youtube.com/channel/UC8_kozGERzHnjVIizhnh43g/videos

    Similar content, I'm jealous of his spectrophotometer haha. I got some isopropanol for a spirit lamp so cheers for the suggestion!

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    1. I didn't do the small frozen tubes for the video as it requires equipment most brewers wouldn't have access too (centrifuges, cryogenic freezer, etc). The goal of my video series is home-based lab methods, so that kind of stuff doesn't fit the channel.

      Cool channel.

      In terms of gelatin, yes you can use it, but it may not always work. Agar is made of an undigestable carbohydrate, so its essentially an inert substrate for yeast/bugs to grow on. Gelatin is a protein, and so many organisms will digest it - including some strains of lacto. Gelatin also tend to be more expensive.

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  3. Was just searching for yeast on the microbiology subreddit and noticed your video came up!

    https://www.reddit.com/r/microbiology/comments/3lcnnb/cooling_inoculation_loop_in_alcohol_vs_on_agar/

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  4. Hi Bryan, sorry to bother you with so many questions. I was wondering if you could help me out.

    How hard is it to separate a culture containing the same yeast?

    For example 3 belgian yeast strains of Saccharomyces C., Brett (different types) and Saccharomyces C + P. Some blends from yeast companies contain 2-5+ strains and if you were to isolate these it would become a lot more attractive economically (plus fun!). I understand you can use different medias for bacteria and yeast but it is puzzling to me how people can get different strains from lambic bottles, is it to do with microscopy and colony morphology?

    Also, I ordered some new petri dishes from ebay, any idea what the clearance between the side of the lid and dish should be?

    I gained a lot more respect for your lab videos after seeing how fluid you are with yours, these are fiddly!

    Thanks again for your time, I'm really looking forward to ranching some wild yeast, if I find any which are distinct or unique I'll be sure to send them.

    Cheers!

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    1. It is possible to separate strains, but it is not trivial. The process is:

      1) Plate out the source material in a fashion where you get single colonies on the plate spread out enough to make picking single colonies possible (e.g. by streak plates)

      2) Grab individual colonies off of this plate, and use them to make pure cultures (either by streaking out on a clean plate, or by innoculating tubes of wort).

      3) Identify what each colony is. This is the hard part - you can use DNA sequencing if you have the resources, otherwise you'll need to use a combination of fermentation characteristics and cell morphology (under a microscope)

      I have a series of video's which can help with this:
      http://suigenerisbrewing.blogspot.ca/search/label/sui%20generis%20videos

      Specifically, I'd recommend the videos on streak plating, "capturing wild yeast" parts II and III (which are equally applicable to non-wild cultures), and my microscopy video.

      I'm not sure what you mean by the clerance on the petri dishes - they are usually ~5 mm deep; there is an airgap between the lid and the edge of the plate, but it is quite small.

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    2. Thanks, I really appreciate it. I guess I'll have to do a lot of mini fermentations with ~50ml of wort.

      In step 2 is streaking from one petri dish to another redundant or is it to minimise contamination?

      Yeah that's what I meant, I just wanted to double check I was supplied with the right dishes.

      Thanks very much for your time, I'm a big fan of your YouTube series.
      Hope I'm not hassling you too much I know you're a very busy person!

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    3. Re-streaking is a good way to ensure you don't loose the organisms. If you look at part II of the capturing wild yeast video you'll see a plate I setup with a grid of ~16 organisms. That plate was made from single colonies off of streak plates, and acted as a source for those yeasts. I then used the "grid" plate to make mini-beers to test each yeast. By keeping the yeast on the 'grid' I could be sure of maintaining pure cultures, free of infection (or accidental drinking) during the mini-beer phase.

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    4. Fantastic, I'll do that. Thanks again for everything!

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  5. Bryan,

    Thanks for the detailed write-up! Much appreciated.

    I recently found a dark mold spot growing on the inside of my plastic hydrometer case. If I haven't had mold growing in any fermenting beers, does that suggest mold spores didn't make it in to the beer? I dump post-boil hydrometer samples, so the only time the hydrometer would have touched the beer/wort is for my pre-boil gravity samples.

    Thanks in advance!

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    1. Your beer should be fine - anything pre-boil would be killed by the boil, and the resulting dead spores won't contain enough toxin (assuming they have any) to be an issue in the beer.

      A short soak (30 seconds) using a 10% vol/vol bleach solution will rid you of that mould.

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