Microbiology and Biochemistry: Difference between revisions

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Enteric bacteria are responsible for the production of [[acetic acid]], and the pH of the wort falls from around 5 to 4.5 in the first week of fermentation. The 40 to 120 mg/L acetic acid found in the wort after the first week is very close to the amount found in the final product.<ref name=Oevelen77 />
Enteric bacteria are responsible for the production of [[acetic acid]], and the pH of the wort falls from around 5 to 4.5 in the first week of fermentation. The 40 to 120 mg/L acetic acid found in the wort after the first week is very close to the amount found in the final product.<ref name=Oevelen77 />
<ref name=sour> J. Edwards and A. DiCaprio. [http://www.process-nmr.com/Presentation/Edwards%20-%20SMASH%202014%20-%20MNova%20Users%20Meeting%20-%209-7-14.pdf | When Beer Goes Sour: An NMR Investigation], Mestrelab
<ref name=sour> J. Edwards and A. DiCaprio. [http://www.process-nmr.com/Presentation/Edwards%20-%20SMASH%202014%20-%20MNova%20Users%20Meeting%20-%209-7-14.pdf | When Beer Goes Sour: An NMR Investigation], Mestrelab
MNova Users Meeting, SMASH – Atlanta, GA, September 7, 2014</ref> Significant changes to the concentration of acetic acid should not occur until the ethanol has a chance to oxidize in aging in the bottle over many years or even decades.<ref name=Vanderhaegen1> B. Vanderhaegen, H. Neven, H. Verachtert, G. Derdelinckx [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCAQFjAA&url=http%3A%2F%2Fwww.researchgate.net%2Fprofile%2FGuy_Derdelinckx%2Fpublication%2F222839054_The_chemistry_of_beer_aging__a_critical_review%2Flinks%2F0c960523339c4b25a6000000.pdf&ei=Tq3IVKmfFcGyogSs_YLQCA&usg=AFQjCNFaBrvqDGjqEV2I9uQ73dYh_ParXg&sig2=Z8dY4iDHozbT1eb9JeAdrw&bvm=bv.84607526,d.cGU | The chemistry of beer aging – a critical review], 2006</ref><ref name = Werner> Werner Van Obberghen, '''2. Het algemene productieproces van bier'''</ref> The pellicle that forms on the top of the wort may be the product of acetobacteria during the enteric phase,[9] though most other sources inidcate that the pellicle is the result of Brettanomyces (with Pichia and Candida).<ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref>
MNova Users Meeting, SMASH – Atlanta, GA, September 7, 2014</ref> Significant changes to the concentration of acetic acid should not occur until the ethanol has a chance to oxidize in aging in the bottle over many years or even decades.<ref name=Vanderhaegen1> B. Vanderhaegen, H. Neven, H. Verachtert, G. Derdelinckx [http://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&ved=0CCAQFjAA&url=http%3A%2F%2Fwww.researchgate.net%2Fprofile%2FGuy_Derdelinckx%2Fpublication%2F222839054_The_chemistry_of_beer_aging__a_critical_review%2Flinks%2F0c960523339c4b25a6000000.pdf&ei=Tq3IVKmfFcGyogSs_YLQCA&usg=AFQjCNFaBrvqDGjqEV2I9uQ73dYh_ParXg&sig2=Z8dY4iDHozbT1eb9JeAdrw&bvm=bv.84607526,d.cGU | The chemistry of beer aging – a critical review], 2006</ref><ref name = Werner> Werner Van Obberghen, '''2. Het algemene productieproces van bier'''</ref> The pellicle that forms on the top of the wort may be the product of acetobacteria during the enteric phase,[9] though most other sources indicate that the pellicle is the result of Brettanomyces (with Pichia and Candida).<ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref>


Low pH (below ~4.5) and an ethanol concentration higher than ~2% by volume is a hostile environment to the enterobacteria, and Saccharomyces species are able to dominate the flora in the wort once these conditions occur around 30 to 60 days into fermentation.
Low pH (below ~4.5) and an ethanol concentration higher than ~2% by volume is a hostile environment to the enterobacteria, and Saccharomyces species are able to dominate the flora in the wort once these conditions occur around 30 to 60 days into fermentation.
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Brettanomyces has been implicated in producing most of the aroma compounds in Lambic.<ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref> Sensory-significant quantities of ethyl acetate and ethyl lactate form at this time from ethanol entering into an ester bond with [[Acetic acid|acetic]] and [[lactic acid]], respectively. In addition, ethylphenols formed from hydroxycinammic acid -- found in the grain used to make the wort -- contribute an odor often described as "horse sweat", "barnyard", or "leather" <ref name=Crauwels1> S. Crawels et. al. [http://link.springer.com/article/10.1007%2Fs00253-015-6769-9 | Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains], 2015</ref> <ref name=Lentz1> M. Lentz and C. Harris. [https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0ahUKEwirpvaJlerMAhVBz2MKHXxYB_kQFggnMAE&url=http%3A%2F%2Fwww.mdpi.com%2F2304-8158%2F4%2F4%2F581%2Fpdf&usg=AFQjCNHZB4IHgQasVxVL3JjdmMcWjdFIUw&sig2=pJ8f-mmJKAYHIfO5xj7GhQ | Analysis of Growth Inhibition and Metabolism of Hydroxycinnamic Acids by Brewing and Spoilage Strains of Brettanomyces Yeast], 2015</ref>. The esterization process is greatly helped by the enzyme esterase provided by Brettanomyces. However, the enzymatic esterization is highly reversible and esters found in high concentrations in the lambic prior to the presence of the esterase will often achieve a lower equilibrium at the end of fermentation. This is the case with iso-amyl acetate, which is produced by Saccharomyces and is a characteristic odor compound in many other beers.
Brettanomyces has been implicated in producing most of the aroma compounds in Lambic.<ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref> Sensory-significant quantities of ethyl acetate and ethyl lactate form at this time from ethanol entering into an ester bond with [[Acetic acid|acetic]] and [[lactic acid]], respectively. In addition, ethylphenols formed from hydroxycinammic acid -- found in the grain used to make the wort -- contribute an odor often described as "horse sweat", "barnyard", or "leather" <ref name=Crauwels1> S. Crawels et. al. [http://link.springer.com/article/10.1007%2Fs00253-015-6769-9 | Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains], 2015</ref> <ref name=Lentz1> M. Lentz and C. Harris. [https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0ahUKEwirpvaJlerMAhVBz2MKHXxYB_kQFggnMAE&url=http%3A%2F%2Fwww.mdpi.com%2F2304-8158%2F4%2F4%2F581%2Fpdf&usg=AFQjCNHZB4IHgQasVxVL3JjdmMcWjdFIUw&sig2=pJ8f-mmJKAYHIfO5xj7GhQ | Analysis of Growth Inhibition and Metabolism of Hydroxycinnamic Acids by Brewing and Spoilage Strains of Brettanomyces Yeast], 2015</ref>. The esterization process is greatly helped by the enzyme esterase provided by Brettanomyces. However, the enzymatic esterization is highly reversible and esters found in high concentrations in the lambic prior to the presence of the esterase will often achieve a lower equilibrium at the end of fermentation. This is the case with iso-amyl acetate, which is produced by Saccharomyces and is a characteristic odor compound in many other beers.


Tetrahydropyridines (THPs) produced by Brettanomyces (as well as some Lactobacilli) have a wide variety of odors and give lambic much of its "mousey" aroma, as well as cider- and horse-like aromas, though the concentrations and thus smells of THPs are variable.<ref name=Heresztyn1> T. Heresztyn [http://ajevonline.org/content/37/2/127.short | Formation of Substituted Tetrahydropyridines by Species of Brettanomyces and Lactobacillus Isolated from Mousy Wines], 1986</ref> Other important odor and flavor compounds produced by Brettanomyces include 4-ethylphenol, 4-ethylguaiacol, and isovaleric acid. 4-ethylphenol produces barnyard and horsey flavors which can taste like Band-aids in higher concentrations. 4-ethylguaiacol lends spicier flavors of bacon and cloves and can be smoky, while isovaleric acid gives lambic its sweaty and cheesy flavors and odors.
[[Tetrahydropyridines]] (THPs) produced by Brettanomyces (as well as some Lactobacilli) have a wide variety of odors and give lambic much of its "mousey" aroma, as well as cider- and horse-like aromas, though the concentrations and thus smells of THPs are variable.<ref name=Heresztyn1> T. Heresztyn [http://ajevonline.org/content/37/2/127.short | Formation of Substituted Tetrahydropyridines by Species of Brettanomyces and Lactobacillus Isolated from Mousy Wines], 1986</ref> Other important odor and flavor compounds produced by Brettanomyces include 4-ethylphenol, 4-ethylguaiacol, and isovaleric acid. 4-ethylphenol produces barnyard and horsey flavors which can taste like Band-aids in higher concentrations. 4-ethylguaiacol lends spicier flavors of bacon and cloves and can be smoky, while isovaleric acid gives lambic its sweaty and cheesy flavors and odors.


Around 16 months after the start of fermentation, during this stage, the pH of the beer reaches a minimum of about 3.0, which then rises slightly in the following months to ~3.2 to 3.4,<ref name = EtF> [http://embracethefunk.com/ph-readings-of-commercial-beers/ | Embrace the Funk's list of beer pH]</ref><ref name=Oevelen77 /><ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref> perhaps due to the enzymatic esterification of organic acids by Brettanomyces.
Around 16 months after the start of fermentation, during this stage, the pH of the beer reaches a minimum of about 3.0, which then rises slightly in the following months to ~3.2 to 3.4,<ref name = EtF> [http://embracethefunk.com/ph-readings-of-commercial-beers/ | Embrace the Funk's list of beer pH]</ref><ref name=Oevelen77 /><ref name="Guinard">Jean-Xavier Guinard, [[Books#Classic Beer Styles: Lambic|Classic Beer Styles: Lambic]], 1990</ref> perhaps due to the enzymatic esterification of organic acids by Brettanomyces.
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The microbes found in lambic may come from a variety of sources, as nearly every surface and even the air found in the brewery are teeming with life. While the air above the [[koelschip]] is often cited as the source of the microorganisms in lambic, other sources are now known to play a significant role.
The microbes found in lambic may come from a variety of sources, as nearly every surface and even the air found in the brewery are teeming with life. While the air above the [[koelschip]] is often cited as the source of the microorganisms in lambic, other sources are now known to play a significant role.


While there are many potential places that the wort can aquire its characteristic flora, some primary reservoirs to consider are:
While there are many potential places that the wort can acquire its characteristic flora, some primary reservoirs to consider are:


# The air over the wort and in the cellar where the wort is aged.
# The air over the wort and in the cellar where the wort is aged.
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Little research exists correlating the season of brewing to changes in the microbiology and chemistry of lambic; however, a delay in the appearance of the late-fermentation bacterial flora in lambic was observed when fermentation started earlier in the brewing season, leading to cooler fermentation temperatures.<ref name=Spitaels /> The flora were indistinguishible after 18 months.
Little research exists correlating the season of brewing to changes in the microbiology and chemistry of lambic; however, a delay in the appearance of the late-fermentation bacterial flora in lambic was observed when fermentation started earlier in the brewing season, leading to cooler fermentation temperatures.<ref name=Spitaels /> The flora were indistinguishible after 18 months.


Similarly, a study on spontaneously fermented ales in the United States revealed marked differences between ales brewed in the spring versus those in the winter.<ref name=AWAs /> The flora broadly follow the same pattern of succession regardless of the season of innoculation, although genetic analysis showed distinct differences between the flora responsible for fermentation occurring in the spring versus the winter. The differences between the organisms found in the wort innoculated at different seasons were largest in the early stages of fermentation and by 36 weeks, there was no longer a significant difference in the flora of in either season's wort.
Similarly, a study on spontaneously fermented ales in the United States revealed marked differences between ales brewed in the spring versus those in the winter.<ref name=AWAs /> The flora broadly follow the same pattern of succession regardless of the season of inoculation, although genetic analysis showed distinct differences between the flora responsible for fermentation occurring in the spring versus the winter. The differences between the organisms found in the wort inoculated at different seasons were largest in the early stages of fermentation and by 36 weeks, there was no longer a significant difference in the flora of in either season's wort.


=Other spontaneous fermentations=
=Other spontaneous fermentations=