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→Biogenic Amines
The ''[[Saccharomyces]]'' dominated stage of fermentation is followed by prolonged and gradual acid and flavor development accompanied by the final points of attenuation, which lasts anywhere from 2 to 10 months <ref name="Roos_2018" />. This stage is dominated by lactic acid bacteria (LAB), primarily ''[[Pediococcus]]'' and sometimes ''[[Lactobacillus]]''. Several organic acids are produced during this stage with the majority of them being lactic acid and acetic acid, resulting in the pH of the beer dropping to below 3.5 <ref name="Van Oevelen et al., 1977" /><ref name="Bongaerts_2021" /><ref name="Roosa_2024"/>. Other sources describe the acidification and maturation phases as one extended maturation phase with acidification from ''Pediococcus'' and ''Brettanomyces'' growth occurring simultaneously <ref name="Spitaels et al., 2015" /><ref name="Bokulic et al., 2012" /><ref name="Spitaels et al., 2014" />. When the wort is pre-acidified, the acidification and maturation phases overlap <ref name="Roos_2018" />. Other yeasts such as ''Candida'', ''Cryptococcus'', and ''Torulopsis'' species have also been isolated from mature lambic, although their impact other than possibly being involved in the formation of a pellicle is unknown <ref>[https://onlinelibrary.wiley.com/doi/abs/10.1002/j.2050-0416.1977.tb03825.x MICROBIOLOGICAL ASPECTS OF SPONTANEOUS WORT FERMENTATION IN THE PRODUCTION OF LAMBIC AND GUEUZE. D. Van Oevelen M. Spaepen P. Timmermans H. Verachtert. 1977. DOI: https://doi.org/10.1002/j.2050-0416.1977.tb03825.x.]</ref>. As many of the flavor and aroma characteristics that we associate with spontaneously fermented beer are produced during this slow maturation/acidification phase, allowing sufficient aging time is important when producing spontaneously fermented beers <ref name="Van Oevelen et al., 1976" /><ref name="Spaepen et al., 1978" />. Specifically, the ratio of lactic acid to acetic acid greatly impacts the flavor profile of the beer. Lactic acid can range from 1.5 to 10 g/l, where as acetic acid is hopefully limited to 1.5 g/l due to he more harsh acidic flavor of acetic acid <ref name="Bongaerts_2021" />. Homebrewer Caleb Buck reported data on several batches of homebrewed spontaneously fermented beer and observed a slower drop in gravity for some batches than others over about a 7 month period (see [http://www.archaicpursuit.com/2018/08/2017-coolship-experiment-hopping-rate.html?m=1 this graph for details]). De Roos et al. (2018) reported a gradual increase in glucose, maltose, and maltotriose from week 7 to month 6 due to the degradation of maltooligosaccharides (higher chain sugars) <ref name="Roos_2018_2" />.
The acidification phase is also accompanied by the growth of acetic acid bacteria (AAB), which can be undesirable if this growth is excessive since it leads to greater [[Acetic Acid|acetic acid]] production (in high quantities, acetic acid smells and tastes like vinegar and is very harsh on the palate and throat) as well as acetoin. These microbes include species from the genera of ''Acetobacter'' and ''Gluconobacter''. The species diversity of these genre is lower than during the primary stage due to acid and ethanol selecting for species that are more tolerant to these harsher conditions. For example, De Roos et al. (2018) reported high numbers of ''Acetobacter pasteurianus'', which contains extra genes that code for acid and ethanol tolerance more so than other species of ''Acetobacter'', in lambic from month 3 to month 6, with it disappearing around month 9-13 as ''Pediococcus damnosus'' took its place. ''Acetobacter lambici'' is another species found in lambic during this stage and is well adapted to the lambic environment due to its ability to break down maltooligosaccharides (dextrins) via maltooligosyl trehalose synthase. These microbes are dependent on oxygen in order to metabolize ethanol into acetic acid (with acetaldehyde produced as an intermediary step) and acetoin from lactic acid and are found on the surface of the wort where oxygen is available. The beer/air interface (or surface of the beer that interfaces with the air above it) is also where higher concentrations of acetic acid, ethyl acetate, and acetoin are found due to the AAB being present there rather than deeper within the beer (this is similar to [[Flanders Red Ale]]) <ref name="Roos_2018_2" /><ref name="Roos_2018" /><ref name="Bongaerts_2021" />. With the flavor threshold of acetic acid in beer being 90 ppm <ref>[https://www.aroxa.com/beer/beer-flavour-standard/acetic-acid Aroxa website. "Acetic Acid". Retrieved 11/19/2018.]</ref>, and the levels of acetic acid in Belgian gueuze/lambic being reported in the range of 727-2240 ppm, acetic acid levels in this range is an important flavor compound in spontaneously fermented beers <ref>[http://beachwoodbbq.com/pdf/BBAIBLTBLENDERY.pdf Ryan Fields. "Brewing Beer in America Inspired By the Belgian Lambic Tradition". 2018.]</ref><ref name="Spitaels et al., 2015" />. During a second phase of growth of acetic acid bacteria starting at week 7 in lambic casks, significantly more acetoin (moldy/must flavor when above 50 ppm <ref>[https://www.morebeer.com/articles/Fatty_Flavors_Diacetyl "Fatty Flavors and Diacetyl - Should Your Beer Be Fat-Free?". MoreBeer website. Scott Bickham. Retrieved 04/04/2023.]</ref>) was found in the top portion of the lambic casks above flavor threshold. Acetic acid bacteria has been shown to reduce lactic acid into acetoin, and in another study by De Roose et. al (2023) the researchers found that ''Acetobacter lambici'' present in lambic samples that were tested had the genetic capability to utilize lactic acid as a food source <ref name="Roosa_2024"/>. Acetoin was gradually reduced (presumably metabolized by ''Brettanomyces'') below flavor thresholds at month 9 until it reached near 0 ppm around month 18 <ref name="De_roos_AAB_2018" />.
====Fourth Stage: Maturation====
Loret et al (2005) examined the levels of biogenic amines in Belgian beers, including lagers, traditional ales, bottle conditioned ales, and spontaneously fermented beer (presumably lambic). They found that spontaneously fermented beers, which were 42 samples out of the total 297 samples and from 10 different breweries, generally contained the most biogenic amines, namely tyramine (associated with hypertension), histamine (associated with hypotension), and cadaverine. However, not all of these breweries produced higher levels of amines, suggesting that processing in some breweries is limiting the biogenic amine production (this process was not identified in the Loret et al study, but the process is likely to be the lowering of the wort pH to 4.5 to limit the enterobacteria phase). For example, 6 of the spontaneous fermentation breweries had levels of histamine as an average between 20-45 mg/l, and levels of tyamine between 30-60 mg/l, which is higher than the upper limit that is generally considered to be safe for consumers (this upper limit was stated as 10 mg/l by Loret et al.). The other 4 breweries had levels of histamine and tyramine between 0-20 mg/L, as an average. Interestingly, they also found that a small number of ales and bottle conditioned ales, 21 out of 220 samples, also had levels of tyamine above the upper limit of 10 mg/l. None of the lagers sampled had biogenic amines above 10 mg/l <ref name="loret_2005" />. Another study in Portugal measured biogenic amines in 5 Portuguese craft beers (4 ales and 1 lager; no mixed fermentation or sour beers) and reported that the total biogenic amine content was less than 10 mg/l for all of the beers <ref>[https://www.mdpi.com/1424-8220/23/1/343 Gil, R.L.; Amorim, C.M.P.G.; Amorim, H.G.; Montenegro, M.d.C.B.S.M.; Araújo, A.N. Influence of Brewing Process on the Profile of Biogenic Amines in Craft Beers. Sensors 2023, 23, 343. https://doi.org/10.3390/s23010343.]</ref>.
De Roos et al. (2018) measured biogenic amines over the fermentation lifespan of Belgian lambic beers that were pre-acidified to a pH of 4.5 before being cooled in a coolship and spontaneously fermented. They found that the initial wort had low concentrations of some biogenic amines, such as agmatine (9 mg/l), putrescine (8 mg/l), and cadaverine (3 mg/l). In one cask, the agmatine remained stable while in the second cask the agmatine declined to zero during the maturation phase, and then slightly increased to less than 5 mg/l. Cadaverine was produced during the first three weeks of fermentation and remained steady throughout the fermentation process at about 30 mg/l. Histamine was produced during the acidification phase by ''Pediococcus damnosus'' between 3 and 9 months and ended up at around 15 mg/l. Tyramine had final concentrations of around 30-40 mg/l and was formed either during the acidification phase (6 months) or the late maturation phase (18-24 months), potentially by ''P. damnosus'' or some other LAB that was at too low of a population to detect, or maybe as a result of autolysis of dead yeast cells. 2-Phenylethylamine and tryptamine were never found in the lambic beers <ref name="Roos_2018_2" />. A second study by the same group of researchers in 2024 confirmed that ''P. damnosus'' was responsible for producing histamine in lambic samples that were tested at one brewery; the total biogenic amines remained below the regulatory levels deemed safe for consumption in Europe <ref name="Roosa_2024" />.
The biogenic amines content of spontaneously fermented beer that is not acidified before fermentation is not likely to be high enough to cause serious physical harm. Large amounts of beer must be consumed before advisable limits are reached. However, the additive effect of different amines consumed with a large amount of beer might cause health related reactions in some people. See [http://suigenerisbrewing.com/index.php/2019/01/22/biogenic-amines/ "Fact or Fiction – Biogenic Amines in Beer" by Dr. Bryan Heit; an analysis of biogenic amines in spontaneously fermented beer and associated health concerns] for more information.
Landschoot et al. (2015) attempted to recover yeasts and bacteria sampled from the brewery walls, ceilings, and coolship in an industrial brewery in West-Flanders that also produces lambic. They were unable to recover any yeasts or bacteria from these surfaces. The team did recover several species of microbes from the air of the coolship room such as ''Klebsiella oxytoca'', ''Bacillus'', and ''Staphylococcus'', but these microbes were not found in the wort after cooling in the coolship nor in the foeders during the sample times of 1, 2 and 3 weeks. ''S. cerevisiae'', ''S. pastorianus'', ''D. bruxellensis'', ''P. damnosus'', and a diverse range of acetic acid bacteria were the dominate species of microbes found during the fermentation of lambic in this brewery. After boiling, the brewery cooled its wort to 40°C using an industrial heat exchanger before pumping it into the coolship to reside overnight for 24 hours. The foeders in this brewery are cleaned with a pressure washer and are no attempt was made to pasteurize or sanitize the foeders with heat or chemicals. The inside surfaces of the foeders were sampled and these samples were the only samples that contained microbe species that were found during the fermentation of the lambic. The authors concluded that the microbes fermenting lambic at this brewery are introduced into the wort via the wooden inner surfaces of the foeders <ref name="Landschoot_2015"/>.
De Roosa et al. (2024) reported finding a very diverse set of species in wort sampled from coolships after cooling (100-200 mL samples). Using DNA sequencing techniques and in order of abundance, they found ''H. uvarum'', ''S. cerevisiae'', ''Penicillium roqueforti'', ''Acinetobacter guillouiae'', ''Lactococcus raffinolactis'', ''Geotrichum candidum'', ''Chryseobacterium bovis'', ''Flavobacterium hibernum'', ''Yarrowia lipolytica'', and ''Trichococcus flocculiformis''. However, the only species from the coolship samples that were found during fermentation in two wooden casks Italian wine barrels were ''H. uvarum'' and ''S. cerevisiae''. The authors concluded that the wooden casks themselves, despite heavy cleaning methods, contributed the majority of impactful microbe species during fermentation and aging, including ''BrettanmoycesBrettanomyces'' <ref name="Roosa_2024"/>.The authors of the study write: <blockquote>''The present study further contributed to the role of wooden barrels in the spontaneous inoculation of the fermenting wort and maturing beer during lambic beer production. Although shotgun metagenomics revealed that the cooled wort had the highest diversity and evenness, its harboring species did not contribute to the wort fermentation and beer maturation in the casks.'' <ref name="Roosa_2024"/></blockquote>
Various brewer anecdotes from experiments appear to contradict the published scientific literature. James Howat of Black Project Spontaneous Ales reported conducting an experiment that showed a similar fermentation profile between a barrel fermented spontaneous beer and samples taken from the coolship and aged in glass flasks <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/1977772602250944/?comment_id=1977929225568615&reply_comment_id=1978520655509472&comment_tracking=%7B%22tn%22%3A%22R%22%7D James Howat. Milk The Funk thread on the source of ''Brettanomyces'' in lambic. 02/05/2018.]</ref>. The spontaneous beers at Black Project were produced for a few years by placing a kettle on the roof outside of the brewery to collect microbes from the air and then racked directly to barrels, so there was no influence of microbes living within the brewery (Black Project now uses a coolship inside the brewery). This led James to conduct this experiment to see how much the barrels were influencing the microbiome of the beers even after they were steamed to the [[Barrel#Sanitizing|point of possible pasteurization]]. James reported that the sensory characteristics between the barrel aged beers and the flask-aged beers were very similar, other than the obvious differences that the oak would have provided, which led him to believe that air inoculation provides a significant contribution to the microbial load of spontaneously fermented beers. Additionally, Brasserie-Brouwerij Cantillon (ref needed) and Oud Beersel <ref>[https://soundcloud.com/craftbeerbrew/podcast-episode-21-new-belgiums-wood-cellar-director-blender-lauren-limbach Lauren Limbach. Craft Beer and Brewing Magazine Podcast. Episode 21. 02/16/2018.]</ref> (~42 minutes in) are known to steam clean their barrels which might be enough to [[Barrel#Sanitizing|sanitize them]], although former Cantillon brewer and saison expert Yvan De Beats maintains that Cantillon barrels are not heated enough to be pasteurized <ref>[https://www.crowdcast.io/e/saison-ale-myths-yvan-baets/1 De Beats, Ybsn. Doug pipers Crowdcast. 08/26/2021.]</ref>. Pierre Tilquin reported that different worts brewed and cooled by different lambic brewers present different fermentation and flavor profiles when barrel fermented in his blendery, particularly when he began steam cleaning emptied barrels <ref>[https://beerandbrewing.com/podcast-episode-234-pierre-tilquin-of-gueuzerie-tilquin-makes-lambic-and/ Pierre Tilquin. Interview on Craft Beer & Brewing Podcast. Episode 234. 04/15/2022.]</ref>(~15 and ~27 mins in). Mitch Ermatinger of Speciation Artisan Ales reported moving wort cooled overnight in a coolship to a stainless fermenter, and the wort began showing signs of visual fermentation four days later <ref>[https://www.facebook.com/groups/MilkTheFunk/permalink/3012223975472463/?comment_id=3012239425470918&reply_comment_id=3012428892118638 Mitch Ermatinger. Milk The Funk Facebook thread on sources for microbes in spontaneous fermentation. 10/28/2019.]</ref>. Such anecdotes deserve further investigation using the full scientific process.