Fact or Fiction Part VI – Nitrosamines in Beer.

Most of my “Fact or Fiction” articles cover relatively common claims regarding brewing risks. Today’s article is a little different, and covers a more historical (e.g. largely dealt with) problem – nitrosamines.

Unfortunately for this article (but good for brewers), this is a “solved” problem. Sadly, many of the sources I am using are not available on the net. If you can access it, Brewing Yeast and Fermentation by Boulton and Quain has a good overview on this issue. Most of the information in this article comes from that source, with additional information thrown in where available.

Nitrosamines – What Are They?

Nitrosamines are a class of chemicals which form when nitrites such as nitrous acid react with amino acids (proteins). They are known carcinogens (cancer-causing chemicals), which is why they are worth worrying about. The most common source of nitrosamine exposure is the cooking preserved meats (e.g. bacon). Nitrosamines can be found in other sources – e.g. tobacco smoke, but have otherwise been largely eliminated from the food chain. Nitrosamines are also formed when reactions occur between nitrites and biogenic amines – a class of chemicals formed during spontaneous fermentation.

Historically, nitrosamines were common in beer and other fermented products. These chemicals came from two sources: volatile forms were produced during the killining of malt, and non-volatile forms were produced during fermentation. The former were eliminated by changes to killing practices introduced in the early 1970’s. The source and control of the later was more difficult to identify.

In the late 1970’s the source of the non-volatile nitrosamines (commonly abbreviated as ATNC – apparent total N-nitroso compounds) was finally identified. ANTC’s were a fermentation product created by a common contaminant of yeast strains. This bacterium, identified as Obesumbacterium proteus, was found in many yeast strains from breweries and yeast suppliers. It went largely undetected before this point, as it did not create off-flavours unless present in high amounts (>1% of cells).

Later, an additional bacterial source of ATNC’s was identified – Bacillus coagulans, which could produce ATNC’s in hot wort (as high as 70C!). B. coagulans is a rare contaminant of breweries, and is most often present as a biofilm on poorly cleaned equipment. Because it is easily controlled, and unlikely to be encountered in a brewery using proper cleaning and sanitation techniques, the rest of this article will focus on Obesumbacterium proteus.

Obesumbacterium proteus

O. proteus is a member of the Enterobacterales, a large group of Gram-negative bacteria that include many food-poisoning associated organisms (E. coli and Salmonella). They are facultative anaerobes, meaning that they can use oxygen to grow, but can also grow without it oxygen. Their ability to make nitrosamines is due to the presence of a nitrate reductase, which converts nitrates into reactive nitrites – which then react with amino acids to form nitrosamines. Outside of this, their impact on beer is minimal, especially at the contamination levels historically found in breweries.

The amount of O. proteus that needs to be present to generate levels of nitrosamines considered potentially carcinogenic (>20 ug/L) is on-par with the levels of contaminating bacteria required to generate off-flavours – typically >0.05% of all cells present. O. proteus doesn’t show any other overt signs of its presence at these levels, but at higher levels may impart a cabbage-like aroma. Luckily, most of the nitrite produced by these bacteria is off-gassed rather than converted into ATNC’s. Otherwise, much lower contamination rates could be hazardous.

As mentioned earlier, O. proteus was identified in the 1970’s as the primary source of ATNC’s in fermented beer. Soon thereafter it was eliminated from beer through a combination of better yeast production processes and acid washing. While this is great for brewers and consumers, it has hurt our scientific understanding of this organism.

Because of this, very little is known of O. proteus. We don’t know what its environmental source is. It’s likely from soil, but I was unable to find confirmation of this. It does not come from humans or other mammals – indeed, it is unable to grow at temperatures above 35C. It is not a human pathogen. Outside of this, very little is known of this rare and elusive bacteria.

When O. proteus isn’t O. proteus

While research on O. proteus has been pretty minimal since the 1970’s, one piece of interesting research has been done. O. proteus was first identified in the late 1960’s. But even then, it was apparent to scientists that bacteria in this “species” tended to fall into two groups. It’s been proposed to split the O. proteus “species” into two to account for this, but until 2010 there wasn’t enough evidence to support this split.

In 2010 a genetic analysis was performed of 5 Enterobacterales genes. Based on this analysis, the evolutionary positioning of this bacterium was determined. As expected, O. proteus falls into two distinct groups:

The first of these groups was the original O. proteus species, described back in the 1960’s. It is closely related to Hafnia alvei, a bacteria sometimes used in cheese making. Group 2 isn’t O. proteus, but rather is a different species (Escherichia blattae) that had been mistaken for O. proteus.

It is unclear of the Group 2 (Escherichia blattae) “strains” of O. proteus also produce ATNC’s.

Risk in Modern Brewing

Hopefully it is obvious that commercial or home brewer using yeast purchased from a yeast supplier are at low risk. Essentially, commercially prepared yeast plus good sanitation and the occasional replacement with fresh yeast stocks is sufficient to eliminate this risk from conventional brewing

But what about spontaneous (wild) fermentation? The good news is that none of the studies which have investigated the microbial communities present during spontaneous fermentation reported the presence of O. proteus [example 1, 2, 3], nor could I find reports of nitrosamines or ATNC’s as a detected metabolite in spontaneously fermented beers. O. proteus is known to be very sensitive to acidic conditions, meaning that its growth in a mixed fermentation would be quite limited, and would cease soon after a spontaneous fermentation begins. Of course, this comes back to my frequent recommendation that you pre-acidify wort before attempting any spontaneous fermentation. Likewise, if purifying yeast/bacteria from wild sources, using a bootcamp-style approach is not a bad idea, as this should eliminate any O. proteus you happen to collect.