Changes

While most beer styles are fermented using one culture of ''Saccharomyces cerevisiae'' or ''S. pastorianus'', Flanders Red Ales are fermented with a [[Mixed Fermentation|mixed culture fermentation]]. At one brewery (presumed to be Rodenbach) studied by Martens et al., two beers were produced using mixed fermentation and blended together. The first "light beer" was 11°P and was less acidic, while the second "heavy beer" was 13°P and served as an Old Ale if unblended. Both beers were inoculated with an acid washed yeast slurry that was harvested from a previous fermentation of the "light beer", and contained about 5% lactic acid bacteria. The fermentation of these beers followed three stages: 1. a seven day ethanol fermentation that is dominated by ''Saccharomyces'', 2. a four to five week lactic acid fermentation that was dominated by ''Lactobacilli'', and 3. a twenty to twenty-four month fermentation that was dominated by ''Brettanomyces'', ''Lactobacilli'', ''Pediococcus parvulus'', and acetic acid bacteria. The development of the ''Brettanomyces'' and ''Pediococcus'' stage was similar to the development of these microbes in [[Lambic]] fermentation. The "light beer" was never allowed to go through the third phase of fermentation, and was instead chilled to 0°C and then used to blend with previous batches of the "heavy beer" <ref name="Martens">[http://onlinelibrary.wiley.com/doi/10.1002/j.2050-0416.1997.tb00939.x/abstract MICROBIOLOGICAL ASPECTS OF A MIXED YEAST—BACTERIAL FERMENTATION IN THE PRODUCTION OF A SPECIAL BELGIAN ACIDIC ALE. H. Martens, D. Iserentant andH. Verachtert. 1997.]</ref>. Interestingly, and perhaps frustratingly, Flanders red and brown ales have been the subject of published studies far less than Belgian lambic beers, so the following information is based off of the Martens et al. study (see reference).

The same study by Martens et al. looked at two casks during the third fermentation, where the "heavy beer" beer was transferred from the secondary fermentation vessel to age for two years. At the beginning of the third phase of fermentation, ''Saccharomyces'' cell counts began to drop while the appearance of ''Brettanomyces'' began. After 10 weeks in the casks, ''Lactobacilli'' greatly decreased, giving rise to strains of ''Pediococcus parvulus''. After 12 weeks for Cask A and 18 weeks for Cask B, the beer no longer contained ''Saccharomyces'', and ''Brettanomyces'' dominated. Specifically, ''B. lambicus'' (now classified as a strain of ''B. bruxellensis'') and ''B. bruxellensis'' were the dominate species, but much smaller counts of ''B. intermedius'' (now classified as ''B. anomala'') and ''B. custersianus'' were also found. The ''Brettanomyces'' species continued to be the primary microorganisms for 36 weeks in Cask A and 50 weeks in Cask B. After this time period, ''P. parvulus'' began to dominate. Acetic acid bacteria also began to make an appearance in the casks, being detected at 27 weeks in Cask A and 40 weeks in Cask B. The exact numbers of the acetic acid bacteria were not reported by this study since they mostly grow on the surface of the beer inside the cask, and possibly on the walls of the cask where diffused oxygen could reach them more easily, and samples were not taken from these sections of the casks. The difference in the time periods for the microbial populations was determined to be affected by the casks themselves, which differed in age, size, and possibly different microbe colonization inside them before they were filled <ref name="Martens"></ref>. During the third fermentation phase, lactic acid greatly increases from ~600 ppm to ~4500 ppm after 35 weeks, and then slowly increases to ~5200 ppm at 60 weeks, at which time L-lactic is only slightly less (~48%) than D-lactic acid <ref name="Verachtert"></ref>. Acetic acid levels reached 1600 ppm by the end of the third phase. The final pH of the "heavy beer" is around 3.2-3.5 <ref name="snauwaert"></ref>.