VLB - Versuchs- und Lehranstalt für Brauerei in Berlin

Brewing Report: Mash and Boiling Acidification

Gustavo Trentini Hamester
International Brewmaster Course 2022

List of Contents

Introduction ........................................................................................................................ 1

pH in Brewing .................................................................................................................... 2

Water and pH .................................................................................................................... 4

Mash Acidification and its Effect on Wort and Beer ............................................................ 5

Boiling Acidification and its Effect on Wort and Beer .......................................................... 9

Subject and Assignment .................................................................................................. 13

Material and Method ........................................................................................................ 13

Results ............................................................................................................................ 15

Discussion ....................................................................................................................... 26

Nitrogen Fractions ........................................................................................................ 27

Fermentable Sugars ..................................................................................................... 28

Brewhouse Yield .......................................................................................................... 28

Hop Utilization .............................................................................................................. 30

Volatile Sulphureous Compounds and Maillard Products ............................................. 30

Summary ......................................................................................................................... 32

Bibliography ..................................................................................................................... 33

Appendix.......................................................................................................................... 37
List of Figures

Figure 1 – Main enzymes group activity during mashing with respect to pH and temperature
according to Narziss8. Source: braukaiser.com/wiki/index.php/How_pH_affects_brewing.. 3
Figure 2 - Effect of different acidifying agents on main water chemistry parameters. Reiter,
200810. ............................................................................................................................... 5
Figure 3 - pH behavior at run-off wort when treat with Calcium. Taylor, 199016. ................. 7
Figure 4 - Wort composition related to increasing mash liquor Calcium content. Taylor,
199016. ............................................................................................................................... 9
Figure 5. Mashing procedure used in all trials. ................................................................. 14
Figure 6. Mash pH and amount of acid added per group. ................................................ 15
Figure 7. Wort pH behavior during boiling and amount of acid added. ............................. 16
Figure 8. Lautering diagram from group 1 showing turbidity, extract and steps. ............... 16
Figure 9. Lautering diagram from group 2 showing turbidity, extract and steps. ............... 17
Figure 10. Lautering diagram from group 3 showing turbidity, extract and steps. ............. 17
Figure 11. Lautering diagram from group 4 showing turbidity, extract and steps. ............. 18
Figure 12. Initial apparent extract for final attenuation analysis. ....................................... 18
Figure 13. Apparent extract at the end of forced fermentation.......................................... 19
Figure 14. Final attenuation analysis. ............................................................................... 19
Figure 15. Photometric iodine reaction analysis. .............................................................. 20
Figure 16. Wort density. ................................................................................................... 20
Figure 17. Free amino nitrogen analysis. ......................................................................... 21
Figure 18. Total nitrogen analysis. ................................................................................... 21
Figure 19. Total protein analysis. ..................................................................................... 22
Figure 20. Coagulable protein analysis. ........................................................................... 22
Figure 21. Wort viscosity. ................................................................................................. 23
Figure 22. Total dimethylsulphide content split into free DMS and PDMS content. .......... 23
Figure 23. Wort Color....................................................................................................... 24
Figure 24. Wort bitterness units. ...................................................................................... 24
Figure 25. Hop yield from boiling to final wort calculated. ................................................. 25
Figure 26. Brewhouse yield using Munich Formula and Overall Formula. ........................ 25
Figure 27. Evaporation rate using volume and extract. .................................................... 26
 1

Introduction

The beer market is increasingly competitive. Every brand wants to be the most
recognized in its niche. To reach it, quality standards in production must be at highest level
in brewery. With that in mind, brewmaster have been developing research and innovating to
increase control and predictability of the processes. One remarkable achievement was the
pH, or concentration of hydrogen ions, effect in brewing.
The beer industry is intensively based on biochemistry since grains growth where
the plant develops its metabolism using nutrients, light and water; malting where the grains
are shortly stimulated to germinate; brewing where desired nutrients are extract and
separated from the grains in a way to function as food for the yeast; fermentation where the
yeast metabolism produces remarkable flavors; and finally ageing where its stability is tested
to the limits. Just from these statements and from short understanding of chemistry is
possible to imagine the implications of pH.
This work discusses how increasing the concentration of hydrogen ions in the
brewhouse affects several parameters in the mash, wort, beer and final product. On top of
that, it is discussed how pH affects the efficiencies of the unit operations during the process.
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pH in Brewing

The pH stands for the concentration of hydrogen ions in a sample. It is a

mathematical solution to express the extremely low concentration of ions in a day-to-day
parameter which is defined as the negative log10 of the concentration of hydrogen ions.
These straightforward definitions were claimed in 1909 by Soren Sorensen, a Danish
researcher of the Carlsberg Laboratory, that was studying the role of acidity and basicity on
the scope of proteins in the beer production process1.
From the fact that pH was defined in a beer research laboratory it is clear to
understand that it has significant importance, but it is not simple to describe in which extent.
All the processes in the beer industry have in common the dependency on organic and life
chemistry which requires the understanding of pH in several aspects2,3.
The first aspect is the water. It is the main solvent known and composes more than
90% of the beer weight. The brew water quality depends on the source of water, the
temperature and the water treatment. The source defines the concentration of carbonates,
Ca2+, Mg2+ and Na1+ present and along with the temperature it defines the state of this water
which is crucial when a brewmaster is setting up a recipe. These compounds interact as a
buffer system, give different dissociation responses and results in variations of the pH when
mashing in4,5.
The second are the buffering compounds of the grains. Barley malt is the main
source of starch, but it also contains polypeptides, peptides, FAN and phosphates that are
actively exchanging hydrogen ions when dissolved in water6. But, nevertheless, distinct
types of grains have different nutrient composition; therefore, they result in a different
response in pH. When aminoacids are in solution they can assume a protonated (low pH)
or deprotonated (high pH) structure. In the process of protonation or deprotonation the
aminoacids, depending also on its side chain, have a capacity of balancing the hydrogen
ions with slight changes in its concentration in the media. When this capacity is over, the pH
changes with higher rate. The phosphates have a similar behavior. Therefore, a buffer
system with a determined buffer capacity is created.
The third aspect are the enzymes. They are active proteins that are used in the
metabolism to reduce the activation energy of a reaction when the substrate binds to the
active center. However, the response to hydrogen ions in aminoacids changes the
intermolecular interaction between them and the media and directly affects the stereo
structure of the active center. Hence, enzymes show an optimal activity when the
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intermolecular interactions, driven by the concentration of hydrogen ions and temperature,

are in the best conformation for the desired reaction to occur7.

Figure 1 – Main enzymes group activity during mashing with respect to pH and temperature
according to Narziss8. Source: braukaiser.com/wiki/index.php/How_pH_affects_brewing.
The fourth is the brewing process design and control. A brewmaster might be able
to change from the plantation soil pH to the bottling beer pH, but every change has its
consequences. The most studied changes in pH in a maltery and brewery are the
acidification of the: malt during steeping, water in the water treatment plant, mash during
mash in, wort during lautering, and wort at the start and end of boiling. Every point and
moment of addition will affect the final beer properties3,9,10.
The last aspect is the pH adjusting agent. Several acidifying agents have been used
for example: phosphoric acids, lactic acid, sulphuric acid, hydrochloric acid, calcium
sulphate and calcium chloride. And, also alkalizing agents as dibasic ammonium sulphate,
calcium carbonate, sodium carbonate or alkali hydroxide. Mainly in a brewery the goal is to
increase the hydrogen ion concentration. Each of these components interact differently with
the existing components of the water and the grist due to electronegativity, volume of dosage
and concentration of the solution. This interaction will affect directly key brewing indicators
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as final attenuation, total extract, soluble proteins, coagulable proteins, FAN, color, lipids,
fatty acids, polyphenols, DMS, bitter units, aldehydes and hot break formation9,10.

Water and pH

Pure water dissociation into hydrogen and hydroxide ions is used to define the
concept of neutrality which occurs at pH of 7. It means that at 25°C the pure water has a
concentration of 1x10-7 hydrogen and hydroxide ions. When the pH is higher than 7 the
solution is considered alkaline, and when it is lower than 7 the solution is considered acidic.
This dissociation behavior of water is to be traced back to the fact that in each equilibrium
state of a reaction there is a constant of equilibrium that determines the ratio between
products (dissociated ions) and reactants (water)5. However, water is not found in a pure
state, the anions and minerals composition shift the solution to a different equilibrium.
Carbon dioxide dissociation in water is the most important buffer system present in the pH
range of the brewing water, mash and beer, from 3,8 to 6,0. In this range other acids as
nitric, sulphuric and chlorine are completely dissociated and the bases like ammonia and
other hydroxide salts are completely undissociated. The only exception is the phosphoric
acid which its buffer system occurs in this pH range, but the concentration in water sources
is negligible compared to carbonates4.
Carbon dioxide dissociates into two different buffer systems resulting in
hydrogencarbonate and carbonate. This process depends on the mineral content and
temperature that will lead to a concentration of CO2 dissociated which is called total
alkalinity. The more carbonates present, the higher the total alkalinity. Another key concept
is the concentration of calcium and magnesium ions which is called the total hardness. In
cases where the total alkalinity is higher than the total hardness the carbonates are bounded
to sodium, and it is called apparent hardness which is related to soft water4,5,9.
Kolbach, 195311, stated the carbonates have a pH increase effect in wort and the
calcium and magnesium concentration have a pH decrease effect due to interactions with
the phosphates from the malt. In the same research he defined a key indicator to express
the alkalinity left after the totality of the present hardness reacted, and, which the called the
residual alkalinity (RA). It is the total alkalinity (from titration) minus the calcium and
magnesium effect, where Ca2+ concentration has double the effect as Mg2+. Therefore, water
with negative or zero RA have no carbonate buffer effect and the pH can be adjusted more
easily. On the other hand, deLange, 200512, developed a theoretical model to describe the
 5

effect of Ca2+ and phosphate concentration on the pH which, according to his assumptions,
resulted in a lower Ca2+ demand to neutralize the same amount of alkalinity when the
phosphate concentration increased. In the end rising a question about Kolbach’s equation.
Residual alkalinity can be either accepted as it is in the water source or designed by
the brewmaster using a water treatment plant. It is possible to treat the water with addition
of flocculants (alumina) to decrease carbonated hardness, addition of calcium hydroxide to
decrease total alkalinity and total hardness, treat with weak ion exchanger to decrease
carbonated hardness, treat with strong ion exchanger to decrease total hardness and total
alkalinity or reverse osmosis for complete desalinization4.
Reiter, 200810, tested the effectiveness of hydrochloric acid, phosphoric acid,
sulphuric acid and lactic acid as acidifying agents of the water prior to mash-in to
decarbonate the water without decreasing hardness. The results in Figure 2 showed that all
agents, except phosphoric acid, reacted according to the calculated dissociation
equivalency. Phosphoric delivered a reduction of 5,6 dH when 10 dH was expected. This
relates to the dissociation pH range of the phosphoric acid protons, phosphoric acid has
three and the other acids have one, but these hydrogen ions are not completely dissociated
since the phosphoric acid has three buffer systems10.

Figure 2 - Effect of different acidifying agents on main water chemistry parameters. Reiter,
200810.

Mash Acidification and its Effect on Wort and Beer

The mashing is a solid-liquid extraction and a bioreactor at the same time.

Temperature, water to grist ratio, stirring rate, time and pH are the driving factors. Among
these, the pH is by far the most complex to describe and with equal importance to the others.
 6

The mashing procedure must work in perfect synergy with the lautering process that works
as a solid-liquid separation to deliver the desired wort. Therefore, studies have been
developed to understand the effect of pH on enzymes activity, the effect of pH on the
solubility of compounds from raw materials and the behavior of pH on different mixtures.
An extensively used method for acidification of the mash is the addition of calcium
ions as sulphate or chloride to increase water hardness and decrease its RA9,13. Calcium in
solution reacts with phosphates to release hydrogen ions and calcium phosphate that
precipitates, moreover the lower pH increases the phosphatases activity which will result in
an increase of breakdown of the phytic acid and increase of the buffer capacity13.
Additionally, calcium can react with oxalic acid, increasing beer stone formation before
lautering, bind with bigger particles as high molecular proteins and granules from the grist,
increasing the lauterability of the mash, and act as a stabilizer for α-amylase9,14. Later on in
the process, it improves yeast metabolism and flocculation15.
Taylor, 199016, studied the influence of calcium concentration on the run-off wort pH.
Figure 3 shows a step change in the wort pH in the first 2 liters when Ca2+ concentration
was increased from 50 ppm to 100 ppm, and not significant changes when more than 200
ppm was added. On top of that, the sparging with untreated water resulted in an increase of
in pH from 4,9 to 6,1 when comparing the mash with 300 and 0 ppm of Ca2+ addition,
respectively. In the end, the overall run-off wort pH varied from 5,10 to 5,45 depending on
the Ca2+ addition, this means that the hydrogen ion concentration dropped from 7,94x10-6 to
3,55x10-6 which is more than 50% decrease16. This comparison is often forgotten due to the
logarithmic scale of pH.
Through his study is also clear that changes in the mash pH have a combined effect
in the lautering process. Sparging the lauter bed without treating the water increases the pH
but can be overcome with increased hardness of the sparging water. This is shown by
Taylor, on Figure 3. At the same time, the combination of the mash treated with 50 ppm with
untreated sparging results in even higher pH in the last runnings than both untreated
processes due to faster wash out of the buffering compounds in the bed. Additionally, the
pH increase, difference between mash and last runnings, was higher in both mashes treated
with calcium but untreated sparging water. This should trace back to the different wort
composition after mashing with a lower pH, which increases the importance of lowering the
pH, or compensate the Ca2+ dosage in the mash, of the sparging water to avoid extracting
polyphenols from husks which are more soluble at higher pH due to its weak acid nature17.
 7

Figure 3 - pH behavior at run-off wort when treat with Calcium. Taylor, 199016.

Bamforth, 20019, compared the results of studies with different acidifying agents.
From the comparison is possible to see different “optimal” pH values for fermentable extract,
total extract, lauterability, soluble nitrogen and FAN. For example, Briggs, 198118, stated that
mashes with pH lower than 4,7 are “impossible to filter”, but Bamforth disagreed stating that
4,4-4,6 was the optimal pH to increase the bed permeability. This reassures the importance
of the synergy added by Ca2+ ions compared to other agents. On the other hand, Li, 201519,
pointed out that wort only treated with Ca2+ has limit in the pH reduction and then phosphoric,
lactic or acetic acid can be applied in combination to balance the reduction of the buffering
capacity and total acidity. But one must be aware that each acid promoted a different
buffering capacity and the combination of calcium with acids can quickly drop the pH.
Acidification with mineral, organic or biological acids have similar effect in the pH
reduction of the mash. Biologically obtained acids from lactic acid bacteria (LAB) of barley
husks have the advantage to comply with the German Purity Law and give more rounded
taste beer3. Mineral acids as sulphuric and hydrochloric are stronger and completely
dissociated at mash pH. Phosphoric is weaker and is not completely dissociated in the mash
pH which increases the buffering capacity with the decrease in pH. Organic acids as acetic,
lactic and others, were studied by Li, 201519, who found that acetic acid increases the
buffering capacity the most and lactic acid (LA) increases 30% less than acetic acid.
Biological LA is produced from a certain strain of bacteria that can be directly applied
to the main wort or applied to an unhoped wort that is later added to the main wort. Reiter,
showed that lowering the pH with LAB increased the zinc content of the wort and lowered
 8

the viscosity10. Lowe, 200420, compared different strains of LAB and chemical LA. The
differences were a very slight improvement in FAN, fermentable extract and viscosity which
varied from one strain to the other. Therefore, tests and optimization are recommended
depending on the desired application.
Holzmann, 197721, studied the solubility of metal ions in wort from different malt
varieties, malt modification, mash temperature, time at different temperatures, mash
concentration and mash pH. His work showed more pronounced solubility of Calcium,
Magnesium, Manganese and Zinc when lowering the pH. Iron and Potassium also increased
solubility with lower pH but more discrete. Copper peaked between 5,5 and 5,7. And Sodium
decreased in solubility.
Barth and Zaman, 201522, studied the effect of the grist composition on the mash
pH. The research compared ale, Munich and pilsner malt when added to different water
hardness and alkalinity. From their experiment was possible to state that different malts
show different increase or decrease in mash pH that follow the Kolbach’s RA equation with
a R2 of 0,63. These malts mainly differ on the protein content, extent of the modification and
coloring substances. On top of that, they proposed a new model to predict the pH in the
mash according to the malt and, what they named, adjusted alkalinity22. This is an
improvement to the RA equation which is related to changes in pH in the cast-out wort.
Another correlation was tested by Troester, 200923, where he found that malt colors and
malt acidity have two clusters, base and caramel malts acidity increase with color increase,
and the roasted malts not showing correlation in acidity with color increase but still showing
higher acidity than base malts. Wheat showed lower acidity as expected due to higher
protein content.
According to Taylor’s report in Figure 4, the more calcium added and the lower the
pH, the extract content, total soluble nitrogen and FAN increased. Reiter, 200810, reported
the same behavior but acidifying with lactic acid. The increase in FAN corresponds to a
better activity of the carboxypeptidase and endopeptidase at lower pH. The lower pH also
improves the solubility of nitrogen compounds which explains the increase in extract24.
Increasing the FAN content is better for the fermentability of the wort and can increases
color formation but increasing too much the TSN can cause higher turbidity of the wort and
chill-haze from the proline interaction with polyphenols in the final product. Unbalanced
increase of FAN with respect to soluble nitrogen will also decrease the foam stability25,26.
And Peyer, 20176, shows in his work that the more FAN is present in the wort, the higher in
the buffering capacity.
 9

Figure 4 - Wort composition related to increasing mash liquor Calcium content. Taylor,
199016.
On the amylolytic side, a lower pH has the tendency to increase the fermentable
sugars content. According to Stenholm, 199927, this is not because an increase of α- or β-
amylases, but due to an increase of the limit dextrinases (pullulanases7) activity. A decrease
in pH from 5,7 to 5,4 increased the fermentability from 83% to 86%. In boiling it can result in
a higher color formation. In fermentation the yeast tends to produce more volatile esters and
will increase the alcohol content of the final product.
Working with a mash at lower pH also contributes to decrease the lipoxygenase
activity. LOX is a group of thermosensitive enzymes that, if supplied with O2, breakdown
fatty acids into staling flavors, for example (E)-2-nonenal, which is derived from linoleic acid
found in barley malts28. Kobayashi, 199329, showed that lowering the pH from 5,5 to 5,0
reduced the formation of hydroperoxy fatty acids that later are converted into (E)-2-
nonenal30. However, acidification of the mash must be balanced with the buffer capacity
since low pH in the final product can triple the rate of flavor staling reactions as found by
Kaneda, 199731.

Boiling Acidification and its Effect on Wort and Beer

Boiling is an evaporation process and a chemical reactor. It mainly promotes the

evaporation of unwanted volatiles as dimethyl sulphide (and conversion from precursor),
solubilization and isomerization of hops α-acids, coagulation of protein, formation of hot
break (protein-polyphenols-lipids), Maillard reactions, sterilization and concentration of the
wort. The a few drivers of this process are temperature of the wort, pressure inside the kettle,
agitation, time and pH. Although not as important as temperature, pH plays a role in
solubilization and in equilibrium of chemical reactions that will produce a different wort
depending on the moment of acidification during boiling. Normally the wort experiences a
decrease in pH from the start to the end of boiling around 0,1-0,3 due to Maillard products
and decrease in protein content.
 10

Protein coagulation is important for enhancing the colloidal stability. This process
comes from the fact that proteins have a structural conformation that depends on the
hydrogen bonds, di-sulphide linkages, ionic bond, hydrophilic and hydrophobic interactions.
Exposing proteins to temperature increase leads these links to become unstable, and the
protein changes its shape. When this happens the hydrophobic interactions that are
normally within a protein, are now opened to bind with other components in the wort. Another
important characteristic is the overall charge of a protein. In cases where the concentration
of hydrogen ions in the wort reaches a state which the protein has no overall charge, its
solubility largely decreases because it is the least polar conformation of the protein. This is
called isoelectric point. Both effects increase the formation of clumps, specially from proteins
containing the aminoacids proline32.
Leather, 199733, reported that at pH of 5,0 the best coagulation was reached and
below 4,7 the wort was strongly more turbid. Important to notice that slight changes in pH
can cause severely changes in coagulation, especially when working with different wort
compositions. Therefore, increased quality in the earlier processes is critical to standardize
the coagulation for several brews since the cleaner of high molecular proteins (>10 kDa)
and polyphenols the wort is, the less interaction will occur in the final product avoiding beer
haze formation.
The formation of hot break mainly occurs from the interaction of protein and
polyphenols. These polyphenols can come from barley husk, ferulic acid and hops. Phenols
have a weak acidic behavior that interacts with weak base sites of protein promoting the
formation of clumps. It is recommended to boil the wort for a few minutes without adding
hops to increase the interaction of proteins with barley polyphenols because hop
polyphenols react better with protein than barley32. Additionally, polyphenols are
antioxidants in beer by reacting with free radicals. Promoting low molecular polyphenols
increase flavor stability with smaller risk of haze formation in the final product28.
To increase the hot break separation from wort the brewers have developed
solutions as the use of k-carrageenan and tannic acid where regulations permit its use. K-
Carrageenan is a complex polysaccharide that enhance the flock formation, and its dosage
has to be optimized with respect to wort pH, soluble protein concentration and metal cations
as presented by Dale, 199634. On the other hand, tannic acid is a gallotannin that binds to
proteins to format bigger flocks and sediment. Nonetheless, the already mentioned methods
of wort acidification as Ca2+ and acids can also be applied to decrease the pH and enhance
hot break formation.
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On the lipid side of break formation, Schisler, 198235, compared the effect between
high trub removal e low trub removal. The trub contained 13,4 mg of lipids/g dry matter while
the wort only 0,26 mg/g dm, 100x difference. He states that wort with higher trub content
can develop faster attenuation profiles, higher yeast cell counts and better flocculation.
However, too high content of trub can also decrease foam stability, promote pre-mature
flocculation of yeast, and lower the yeast cell area, thus not reaching the final attenuation.
Converting SMM into DMS is highly depending on temperature, but the DMSO,
formatted during malt kilning, is also present in wort. While DMS is volatile, DMSO is heat
stable and goes to the fermenter. During fermentation the yeast can convert DMSO into
DMS that stays soluble due to low agitation and pressurized processes downstream. The
rate of conversion of DMSO into DMS can be 11x (1100%) lower in a wort with lower pH as
presented by Anness, 198236. Another effect is the possible contamination of the wort with
bacteria that can increase the conversion of DMSO to DMS. Dickenson, 197837, and
Bamforth, 201438, compared the SMM half-life at pH 5,2 and 5,5 and found that with the
lower pH the SMM half-life increased only 17%. Therefore, the higher SMM conversion, the
higher DMS evaporation and the lower pH will promote a beer with lower DMS content.
Maillard reactions are responsible for the color increase during boiling and are known
to be highly sensitive to pH39,40. Vanderhaegen, 2004, and De Schutter, 2008, reported a
continuous increase in the formation of volatile compounds when lowering the pH. The more
volatiles were removed the lesser reactants were available for melanoidin formation, thus
color formation was decreased. De Schutter, 200839, also reported a catalytic effect when
acidifying the wort with phosphoric acid and stating that in his trials he used sulphuric acid.
At the same study, was reported that varying the wort pH in the range from 5,0 to 6,0 no
relevant change in the thiobarbituric acid index developed.
Jaskula, 201041, showed that the utilization of bitter substances from hops decreased
with a decrease of pH. At pH 7,0 the utilization was 81,3% and at pH 4,8 it was 32,8%. This
is particularly due to the isomerization process from α-acids to iso-α-acids that requires
anionic format of α-acids but the pKa is between 5,0 to 5,5. One possibility to increase the
utilization was tested by pre-dissolving the hops in ethanol prior to addition which resulted
in 4% increase in the utilization at pH 5,2, but no positive result at other pH tested. Adding
to that, Verzele, 196542, reported that dosing Ca2+ in this range helped to increase the hop
utilization, but Kappler, 201043, reported a decrease. This difference might come from the
fact that different buffer capacities and wort composition were used. Another finding by
Kappler was the increased degradation of iso-α-acids at lower pH that resulted in increased
 12

harsh bitterness and lingering aftertaste. Besides isomerization, the solubility of humulones
and lupulones were studied by Spetsig, 195544, where he showed that the solubility
increases with higher temperature but it decreases with lower pH and the decrease in
solubility of humulones occurs at a lower pH than lupulones which explains Kunze, 20043,
statement that lower pH results in better bitterness.
With all these impacts from different pH, the moment of acidification during the boiling
process is to be decided upon each type of beer, amount of pH decrease, type of agent and
equipment technology available. When acidifying in the start compared to doing in the end
it is expected to: increase protein coagulation, higher hot break formation, better colloidal
stability, lower lipid content, higher SMM content, higher DMS in the end of cooling, lower
color, better flavor stability, lower harsh bitterness, lower lingering, but lower hop utilization.
Beer with high bitterness in the recipe may be better to optimize the moment of acidification.
Another possible solution is the use of pre-isomerized hop pellets or pre-iso hop extract to
avoid having this process during boiling, the main limitation is the regulation for recipes
brewed under the German Purity Law since pre-isomerization requires MgO addition.
The effect of low wort pH in fermentation will also depend on the wort buffering
capacity. This property comes from the FAN content of the wort. The higher the FAN content
the higher is the buffering capacity. But, as discussed by Taylor, 199016, increased FAN
content will also increase yeast multiplication, thus increasing the release of organic acids
and lowering the pH. From his work is possible to theorize that depending on the type of
aminoacids in the FAN content it can result in more uptake or less uptake of a fraction of the
total FAN because yeast has different preference of aminoacids. This traces back to the
importance of the quality of raw materials. pH will also effect the yeast flocculation but is
difficult to settle on a tendency since other factors work simultaneously15. The lower the pH
will also increase the conversion of α-acetolactate into diacetyl during fermentation and is
stated as the main contributor along with the temperature45. Moreover, the microbiological
stability is increase against several types of bacteria when pH is below 4,346.
In the final product the changes of pH done in the brewhouse are balanced during
fermentation due to the buffering capacity thus not directly correlating a low pH in the wort
to a lower pH in the beer9,10,16. The flavor stability decreases with lower pH in the final product
mainly due to (E)-2-nonenal release28,47. To summarize the beer final taste according to pH
Taylor16 used the following words: “At low pH values (less than 4.0), beers tend to taste more
sharp and acidic, with increased drying after-palate and a tendency for perceived bitterness
to be enhanced. Above pH 4.0, the palate effects relate to increased mouth-coating, with
 13

enhanced scores for biscuity, toasted characters. In the experimental flavor series, at very
low pH (3.7 and below), the sharp, bitter, drying effects increased in intensity rapidly, with
markedly enhanced metallic after-palate. Above 4.4, the cloying, mouth-coating effects
became increasingly more accentuated, with soapy, caustic characters becoming apparent.”

Subject and Assignment

As discussed in the literature review, the pH change can significantly alter the
extraction efficiency, hops utilization, flavor, aroma and stability of the beer. However,
depending on the type of acidifying agent, acidifying agent dosage, pH change, moment of
addition and point of addition, the results of the final wort and, consequently, the final beer
can show different behavior. Thus, to have a better understanding of the possible changes,
four trials were conducted in this work.
The goal of this work is to assess the influence of acidification on the main
parameters of the cooled wort for a typical Pilsener beer. The trials were divided in one per
each group following: no acidification (NA), only mash acidification (MA), mash acidification
and before boiling acidification (MA+BBA), mash acidification and after boiling acidification
(MA+ABA) procedure. After brewing and analyzing the wort, the data was compiled for
comparison of the results between the groups and with the literature.

Material and Method

The trials were conducted in the VLB’s pilot plant using a base recipe and only
changing the acidification moment. The recipe consisted in using 80 kg of pilsener malt from
IREKS at 1:3 ratio for brewing water during mashing in. The malt was milled using a 4-roller
mill right before mashing-in. To the mashing-in water, 150 mg of CaCl2 per liter of brewing
water were added to reduce residual alkalinity. After mashing-in, a sample of the mash was
taken to measure the pH of the mash. For the groups acidifying the mash the sample was
titrated with a solution of H3PO4 8% until pH of 5,50 to determine the volume of phosphoric
acid to be added as soon as possible. The mashing procedure followed the diagram on
Figure 5: mashing-in at 62°C for approximately 15 minutes, increasing to 64°C to rest for 35
minutes, increasing to 72°C to rest for 25 minutes and, after confirmation of the iodine test,
mash-out at 78°C.
 14

Figure 5. Mashing procedure used in all trials.

The mash was filtered using a lauter tun. Brewing water without calcium chloride
addition was used for sparging. The mash rested for 10 minutes and later circulated until the
inline turbidity measurement fell below 50 EBC. During lautering, every 10 minutes the
extract was measured using a saccharometer. For all groups the lautering was performed
until the kettle extract reached approximately 11,0°P due to the 4,8% expected evaporation
rate.
After lautering the wort was boiled for 60 minutes at 100°C in a kettle with internal
boiler. Group 3 titrated the kettle full wort and adjusted the pH with phosphoric acid to 5,20.
Group 4 titrated the wort after boiling and adjusted the pH with phosphoric acid to 5,20.
Groups 1 and 2 did not acidify the wort. The hop product used was Hallertau Hallertauer
Magnum Extract from HVG, and it was calculated for a concentration of 100 mg of α-acids
per liter of cast-out wort and added 10 minutes after the start of boiling. If the extract at the
end of boiling was above the target of 11,5%, then water had to be added to adjust back to
11,5%. In sequence, the wort was sent to the whirlpool to rest and sediment for 20 minutes.
Once the rest time ended, the wort was cooled down to 10°C with a plate heat
exchanger and aerated. The cold wort samples were collected at the middle of the cooling
process. Exclusively the DMS samples were collected in an ice bath. All the samples were
sent to the VLB laboratory for the following analysis: PDMS, DMS, final attenuation, density,
FAN, total nitrogen, coagulable nitrogen, total protein, photometric iodine reaction, bitterness
units, pH, viscosity and color. The complete data used for discussion is available in the
Appendix section.
 15

Results

In this section is presented the wort analysis and relevant process data for each trial.
Group 1 did not acidify the mash nor the wort (G1 NA). Group 2 did only mash acidification
(G2 MA). Group 3 did mash acidification plus before boiling acidification (G3 MA+BBA).
Group 4 did mash acidification plus after boiling acidification (G3 MA+ABA).

Figure 6. Mash pH and amount of acid added per group.

The Figure 6 shows the pH at the start of mashing, how much of acid was added
and the target mash pH after the addition of phosphoric acid according to the titration of the
mash. Even though the recipes were the same, a variation from 5,62 to 5,65 was found for
the mash pH at the start and more acid had to be added in the G4 trial than in G2 and G3.
 16

Figure 7. Wort pH behavior during boiling and amount of acid added.

The Figure 7 shows the pH behavior up to 5 distinct stages of the process depending
on the available data. The pH kettle full refers to the pH before the start of boiling and any
acid treatment. The pH start of boiling is the moment right after acidification for G3, and the
same as kettle full for the other trials. pH end of boiling refers to the pH at the end of boiling
but before adding phosphoric acid in G4 and dilution in the other groups. The pH cast out is
after adding phosphoric acid in G4 and after dilution, this analysis is the only one performed
in the VLB Lab from the wort sample. Finally, the pH fermenter is the pH measured from the
fermenter right after finishing filling the fermenter. The pH of G1 trial dropped 0,19 during
boiling, similar behavior was seen on G4 dropping from 5,58 to 5,43. The pH of G3 dropped
0,38 when 472 mL of phosphoric acid was added, and during boiling it further decreased to
5,13 and it confirmed 5,12 on the fermenter. However, G3 sample in the lab showed pH of
5,17. By analyzing the other trials, there’s a tendency to have a higher pH in the cast-out
wort than in the fermenter or end of boiling which could be related to the pHmeter used and
homogeneity of the sampling point. G4’s pH decreased from 5,43 to 5,24 after adding 300
mL of phosphoric acid.

Figure 8. Lautering diagram from group 1 showing turbidity, extract and steps.

On the Figure 8, the lautering diagram of group 1, no acidification, shows a 50 EBC

turbidity in the beginning that drops fast. The extract has a slightly decrease in the beginning
of the first sparge, but only starts to decrease faster after 60 minutes. A variation in the
turbidity can also be seen between 60 to 75 minutes.
 17

Figure 9. Lautering diagram from group 2 showing turbidity, extract and steps.

On the Figure 9, the lautering diagram of group 2, acidified mash, shows a 30 EBC
turbidity in the beginning that drops fast just as G1. The extract follows the same trend as
G1.

Figure 10. Lautering diagram from group 3 showing turbidity, extract and steps.

On the Figure 10, the lautering diagram of group 3, acidified mash, shows a 12 EBC
turbidity in the beginning that drops fast just as G1 and G2, but increases after 75 minutes.
The extract follows the same trend as G1 and G2.
 18

Figure 11. Lautering diagram from group 4 showing turbidity, extract and steps.

On the Figure 11, the lautering diagram of group 4, acidified mash, shows a 50 EBC
turbidity in the beginning that drops slower than the other trials, fluctuates between 30 and
45 minutes and increases towards the end. The extract follows a higher concentration than
other trials until the second part of the second sparging and then drops quickly.

Figure 12. Initial apparent extract for final attenuation analysis.

On the Figure 12, the initial apparent extract was within the range of the targeted
11,50% and within the range reported by Bamforth for pilsener from 11,3 to 12,0%48.
Between trials there were no significant variations.
 19

Figure 13. Apparent extract at the end of forced fermentation.

On the Figure 13, the final apparent extract was the same for G1, G2 and G4, only
G3 showed a 0,4% higher final apparent extract.

Figure 14. Final attenuation analysis.

On the Figure 14, the final apparent degree of attenuation was 83,3% for G1, G2
and G4, only G3 showed 79,9%. This is expected since the result comes from the difference
between initial and final apparent extract, however no trend according to acidification
procedure was found. The value is in the range reported by Kunze from 75,2% to 85,6%
which is a wide range that most of the beer styles lies within3. Another source, Bamforth,
201648, reports that the final degree of attenuation for a Pilsener should be from 84,0% to
85,0%, therefore classifying our trial as not in accordance to the style.
 20

Figure 15. Photometric iodine reaction analysis.

On the Figure 15, the photometric iodine reaction test showed a higher value for G2,
0,676. Followed by G1, G4 and G3. No tendency is shown according to the different
acidification procedures. Kunze reports that normally found ranges starts from 0,19 to 1,86,
therefore all the value are within range. However, according to Bamforth the iodine value
should be below 0,30 for Pilsener.

Figure 16. Wort density.

On the Figure 16, just as in Figure 12, all the trials reached close to the 1,04436
g/cm³ target that corresponds to 11,5°P.
 21

Figure 17. Free amino nitrogen analysis.

The Figure 17 shows the free amino nitrogen content for different trials. The values
have a maximum difference of 9 mg/L which is below the 28 mg/L reported variation from
the analysis according to MEBAK49, therefore there are no significant differences between
trials. In the same reference, it is also reported that values are between 200 to 250 mg/L
which means that all trials worked in the lower limit values for wort.

Figure 18. Total nitrogen analysis.

The Figure 18 shows the total nitrogen values for each trial. The results show a
slightly higher content for G1 than the others. G3 has 24 mg/L more nitrogen than G4 and
10 mg/L more than G2. And, G2 has 14 mg/L more than G4. In the MEBAK the range is
reported between 362 to 1195 mg/L which all trials are inserted49. Bamforth reports that for
 22

Pilsener 800 to 1000 mg/L is acceptable, only wheat beer would reach the range from 1000
to 1200 mg/L48.

Figure 19. Total protein analysis.

On the Figure 19, the protein analysis, which is derived from the total nitrogen
(6,25*N), shows no significant differences in the final wort between all trials. Following the
same range from the total nitrogen, the total protein should be between 0,23 and 0,75
g/100mL.

Figure 20. Coagulable protein analysis.

The Figure 20 shows the results for the coagulable fraction of the nitrogen content. The G1
and G3 content are higher than the other groups. But no tendency related to the acidification
is clear. MEBAK reports that the precision is 8 mg/L, therefore the only significant difference
 23

is G1 when compared to G2 and G449. Also, it reports that the range is between 15 and 25
mg/L which means that all trials are above the acceptable coagulable nitrogen content.

Figure 21. Wort viscosity.

The Figure 21 shows the results for viscosity of each trial. According to Kunze the
range for cast-out wort is from 1,65 mPa*s to 1,83 mPa*s, but the precision according to
MEBAK is 0,02 mPa*s49. Therefore, all trials have no significant difference in viscosity.

Figure 22. Total dimethylsulphide content split into free DMS and PDMS content.

On the Figure 22, the wort results of total DMS are split in free and precursor DMS.
G3 showed a significant higher PDMS compared to the other trials, but similar free DMS. It
was followed by G4 and G2 which were also acidified during mashing. G1 presented the
lowest value of 35 μg/L PDMS and 83 μg/L total DMS. G2 showed the lowest free DMS
 24

content. MEBAK only reports values for total DMS, which should be below 100 μg/L.
Therefore, only G1 and G2 are according to the standard DMS values that is related to the
flavor threshold.

Figure 23. Wort Color.

The Figure 23 shows the results for wort color. G1, no acidification, showed a 1,2
EBC higher color than G3, followed by 1,5 and 1,6 EBC from G4 and G2, respectively.
Bamforth reports that the color should be between 7 to 10 EBC48. Based on that, all the trials
are within range for the style.

Figure 24. Wort bitterness units.

On the Figure 24, the bittering units for every trial are shown. G1 presented a higher
bittering unit than the other trials. The lowest value was 49 IBU for the G3 trial, which means
that the highest difference between each trial was 7 IBU. According to Bamforth48, a typical
 25

Pilsener wort IBU is between 25 and 45. From that is possible to conclude that the bitterness
is higher than recommended for the style.

Figure 25. Hop yield from boiling to final wort calculated.

The Figure 25 shows the hop yield of the wort for each trial. It is calculated as the
ratio of grams of iso-α-acids in the wort per grams of α-acids added from hops, Equation 1.
G1 showed the highest yield, followed by G2, G3 and G4 in this sequence. The results
tendency were similar to the reported in the literature by Jaskula41, the lower the pH the
lower the yield. However, a higher difference was expected between G3 and G4.
Wort IBU[mg⁄L]*Vol. Cast Out[L]*0,96/1000
Hop Yield=
Mass Hops[g]*Hop α-acids[g of α⁄g of hops]
Equation 1. Hop yield calculation.

Figure 26. Brewhouse yield using Munich Formula and Overall Formula.
 26

On the Figure 26 the brewhouse yield calculated by Munich and Overall formulas
are presented. The overall considers the malt extract content, while the Munich formula only
considers the amount of malt weighted. Independent of which method is used, both showed
that G1 had a better yield from the added malt than the other trials. G2 and G3 presented
the same yield of 93,6%.
Ext. Cast Out[kg/hL] ∗ Vol. Cast Out[L]*0,96/1000
Munich Brewhouse Yield =
Grist Load[kg]
Equation 2. Munich brewhouse yield.

Munich Brewhouse Yield

Overall Brewhouse Yield =
Extract Malt[%]
Equation 3. Overall brewhouse yield.

Figure 27. Evaporation rate using volume and extract.

The Figure 27 presents the evaporation rate for each trial calculated by the ratio of
volume and extract at the start and end of boiling. G1 had the lowest evaporation rate, even
though it had the same boiling time. G2 had the highest evaporation rate, 6,25%. The
average difference between the ER calculated by extract and volume was 0,14%. Therefore,
any of them can be used as reference.

Discussion

The following discussion starts addressing the process parameters used, the
variability of the process and expected final wort results comparing with the literature. In
sequence it addresses the nitrogen fractions, fermentable sugars, brewhouse yield, hop
 27

utilization, volatile sulphureous compounds and Maillard reactions from the final wort
analysis.
The mashing procedure used in this work focused on the β-amylase and α-amylase
rests since the malt was well modified. The assumption of well modified malt comes from
malt analysis sheet: friability 92,8%, viscosity 1,46 mPa*s, Kolbach Index 43,9% and total
protein 10,3%. This was confirmed for all trials by the viscosity results on the lower range of
the recommended values, Figure 21, and the total nitrogen on the upper limit of the
recommended values for a pilsener, Figure 18.
The assessment of the different acidification procedure had two main expected
results. First, the difference in acidifying the mashing or not acidifying should have a direct
effect on the brewhouse yield and fermentable sugars since lowering the pH would cause a
decrease in the α- and increase in the β-amylase activity and an increase in the solubility of
nitrogen compounds. Second, the difference in acidifying the wort before and after boiling
would cause a difference in the hop utilization and the total DMS content since lowering the
pH would cause a decrease in the solubility of bitter substances from the hops and increase
in conversion of PDMS to DMS.

Nitrogen Fractions
As said before, the mashing procedure was not focused on the endo- or carboxy-
peptidases rest due to the well modified malt. At the mash-in temperature the enzymes for
protein breakdown were already denatured. Thus, changes in the nitrogen content and
fractions came mostly from the solubility and interaction with other components of the wort
as polyphenols and carbohydrates. This is the reason why FAN, total nitrogen and
coagulable nitrogen had no straight correlation with the acidification procedure. Other
process parameters had most probably a higher effect as for example the total hot time,
evaporation rate and whirlpool rest time. All the FAN results were in the range of variability
of the analysis thus, is only possible to state that the acidification had no effect since it was
expected to have a higher FAN for the acidified mashes due to the higher solubility at lower
pH. For the total nitrogen, the tendency is the opposite as expected in the literature, since
G1-NA showed a higher content but all the others that acidified the mash showed 16 to 40
mg/L less total nitrogen. This can be related to the size of the protein fractions since G1-NA
and G3-MA+BBA showed both higher total nitrogen and higher coagulable nitrogen, even
though there is no correlation with the acidification procedure, the correlation of coagulable
nitrogen with the evaporation rate is R² 0,903 meaning that the higher evaporation rate the
 28

lower the coagulable nitrogen content. This supports that the boiling process highly
influences the colloidal stability and the statement that other variables of the process
affected more than the acidification procedure.

Fermentable Sugars
The solubilization of starch depends on the mashing in procedure, for example an
increased formation of lumps will decrease the yield. The amylolytic activity depends on the
synergy of α- and β-amylase, therefore even though they have optimum activity at different
pH values the brewer must adjust the pH in a value in between. The β-amylase works better
at lower molecular weight carbohydrates that can be delivered by a good α-amylase activity.
The synergy of these two enzymes also depends on the barley variety which can have
different content of types of α- and β-amylase with different pH and temperature optimum
according to Henson50.
The mash-in pH varied from 5,62 to 5,66, thus lowering the pH to 5,50 was expected
to have an effect of increasing the β-amylase activity, optimum around 5,4, and decrease
the α-amylase activity, optimum around 5,8. From this, the final attenuation for the acidified
mashes was expected to be higher than G1-NA, but what was found is that only G3-
MA+BBA had a final attenuation of 79,9%, Figure 14, and the other groups 83,3%. On top
of that, the similarity of values found for final apparent extract, Figure 13, calls attention to
the precision of the analysis. Therefore, no correlation was found with the acidification
procedure. Another measurement for the amylolytic activity was the photometric iodine
reaction (PIR) that showed no correlation with the acidification procedure because although
G3-MA+BBA and G4-MA+ABA had lower PIR than G1-NA, the G2-MA had a higher PIR
than G1-NA. Again, other processes might have affected this value as for example the lower
pH of the wort during the whirlpool rest might have increased the interaction of large
carbohydrates with proteins and polyphenols of the trub. To have a more in-depth
comparison of the amylolytic activity it is recommended to perform a sugar profile analysis
to understand what the carbohydrates fractions after mashing are.

Brewhouse Yield
The best pH for brewhouse yield showed an opposite trend than Briggs18, but within
the ranges stated by Bamforth9. Comparing with the Munich formula, the G1-NA showed 2,1
percentage points higher yield than the second largest yield, G2-MA and G3-MA+BBA, and
3,6 than the lowest yield, G4-MA+ABA. Aspects as solubilization of starch, solubilization of
 29

nitrogen fractions, amylolytic activity, lautering process and losses during the transferring
process could have affected this result. The extract is composed by all the solutes present
in the sample, and not only sugar, that is why the solubilization of nitrogen fractions, which
is higher with the lower pH9, can increase the brewhouse yield.
The lautering process is critical for the brewhouse yield. It is affected mainly by the
factors present in the Darcy’s Law: pressure difference, depth of the filtration bed, viscosity,
permeability and filtration area. Bed depth and filtration area are related to the grist load and
equipment used which were the same for all trials. However, viscosity, permeability and
pressure difference are more closely related to the milling and mashing results and are
interconnected. The final wort viscosity, Figure 21, showed no significant difference between
trials. The permeability could be affected by a poor lauter rest, but the groups rested for 9 to
10 minutes; late start of sparging, but all groups started sparging before the wort level
dropped below the bed level; and poor turbidity circulation, but no change correlated with
the mash acidification was found. When comparing the lauter diagram between groups a
few characteristics calls attention. First is the initial turbidity varying from 12 to 47 EBC,
which could not be explained by the milling, mashing, acidification, mash transfer and lauter
rest information available. Second, the lower the turbidity at the start of lautering the less
turbidity fluctuations were perceived. G1-NA and G4-MA+ABA showed fluctuations when in
the beginning and in the end of the first sparge. G2-MA and G3-MA+BBA showed a
fluctuation in turbidity just in the end when all trials showed variations. Third, the extract
curve of G4 showed a faster drop than the other groups. Fourth, the total filtration time of
G4 was 10 minutes shorter than the other groups which could explain the lower kettle full
volume and the lower extract yield. And fifth, the average flow rate for G3 and G4 were 2,23
and 2,26 hL/h, while G1 and G2 it was 2,16 and 2,19 hL/h which could explain the slightly
longer filtration times for G1 and G2.
In the end, the points of measurement when calculating the yield must be precise
because dilution and interfaces of wort and water can mask results for yield. For example,
G1 showed 37 liters more as cast out wort than G4, but the homogeneity of this volume to
be multiplied by the extract can be a topic for further discussion. But most certainly,
shortening the lauter process for G4 influenced the results, because the kettle full volume
was 20 to 24 liters less than all groups. When manually integrating the extract
measurements every 10 minutes higher extract values were found specially for groups 2
and 3 where the extract in °P raised to 12,27 and 12,38 respectively, Table 1 in Appendix.
 30

To sum up, the expected general trend of higher yield for acidified mashes were not
found and the non-acidified mash delivered higher extract yield. Thus, it is recommended to
perform the trials one more time and reduce the variability of lautering parameters as total
filtrated volume.

Hop Utilization
The hop utilization shown on Figure 25 followed the literature results from Jaskula41
and Spetzig44 that the lower the pH, the lower the solubility of bitter substances. In this
experiment G1-NA had a pH of 5,65 at the start of boiling and the hop yield was 55%. G2-
MA did not present any result for the boiling pH, but assuming that the drop in pH exclusively
due to the boiling process was in the range of 0,15 to 0,19 as in G1 and G4, the G2-MA pH
at the start of boiling was between 5,55 to 5,59, then 0,10 to 0,06 less than G1 and showed
a hop yield of 51%. G3-MA+BBA had a pH of 5,62 when kettle full and phosphoric acid was
added before the start of boiling, then after acidification the pH was 5,25 and the hop yield
was 48%. The last trial was G4-MA+ABA that had a pH of 5,58 at the start of boiling, after
the boiling the pH dropped to 5,43 only due to the boiling process. After boiling, phosphoric
acid was added to wort and dropped the pH to 5,24 and the hop yield was 47%.
From that is possible to state that acidifying before or after boiling has no direct effect
on the IBU of the wort. On top of that, lowering the pH after boiling can have a continuous
effect on the solubilization of iso-α-acids and bitter substances which comes from the fact
the G2 and G4 had the similar conditions for solubilization and isomerization of the bitter
substances, but most probably after lowering the pH of the G4 wort the bitter substances
came out of solution because they had more affinity to the substances in the trub.
Furthermore, according to Spetzig44 and MEBAK49 is important to understand that the
measurement of IBU using iso-octane can also extract bitter substances other than iso-α-
acids, then a higher hop yield found on G1 and G2 does not mean that this increase came
from the same fraction extracted by G3 and G4 since harsher bitter substances have lower
solubility with lower pH. Therefore, to assess the bitterness quality a HPLC analysis of the
bitter substances and sensory analysis is recommended.

Volatile Sulphureous Compounds and Maillard Products

The volatile sulphureous discussed here are the PDMS and DMS which are
dependent on the protein content of the malt, total hot time (from mash-out to middle of
cooling), evaporation rate, whirlpool rest time and pH.
 31

In this work the total hot time varied from 295 to 351 minutes. This has an effect of
increased conversion of PDMS into DMS, and, depending on the moment this time was
extended, it can reflect into a lower total DMS. When breaking down the total hot time, it is
possible to see that the time from the start of boiling to the middle of cooling had no
significant difference varying from 122 to 128 minutes, but the time from mash-out to start
of boiling varied from 173 to 225 minutes. This means that more PDMS was converted into
DMS for G1, then G3, G2 and G4, in this sequence. When more DMS is present before
boiling it can be faster evaporated since the conversion time was already spent before
boiling.
The evaporation rate can also influence the removal of total DMS. The higher the
evaporation rate (ER) the better conversion of PDMS into DMS and removal of free DMS.
In this work the ER calculated by volume varied from 2,46% for G1 to 6,10% for G2. G3 and
G4 were in the middle with 4,38% and 5,15%, respectively. This is another contribution to
the variation of total DMS which builds complexity for the assessment of the acidification
impact on the DMS. G1 was expected to have the highest total DMS and G2 the lowest total
DMS.
The whirlpool rest time was also not constant for all the trials and can convert more
PDMS into DMS but without an evaporation step, thus the DMS can stay in the final product
with higher concentration. In this work G4 rested for 12 minutes while the other groups varied
from 19 to 20 minutes. From this it could be expected that the value for free DMS would be
higher for G4 than the other groups, but this was not true when comparing to G3.
The last variable was the pH that was almost the same for G1, G2 and G4 at the
beginning of boiling and lower for G3 after acidification before boiling. According to
Bamforth38, the lower the pH the better is the conversion of PDMS into DMS, therefore it
was expected to have a better conversion in the G3 trial. However, G3 presented the highest
total DMS content and PDMS to total DMS ratio, but since several factors were varying at
the same time it is complex enough to state that other trials are necessary to confirm any
theoretical behavior.
Following similar influences as the DMS, the color was highly affected by the total
hot time (THT) since G1 with the highest THT resulted in the highest color while G2 and G4
in the lower range of the THT had the lowest color. This behavior is expected since the more
time spent at higher temperatures the more interactions of carbohydrates and low molecular
nitrogen fractions causing Maillard reactions are present. The effect of pH change on the
color formation was no perceived due to other more influencing processes.
 32

Summary

In this work was possible to assess the behavior of the brewing process when
different acidification strategies were applied to a typical Pilsener recipe and using the same
equipment. On top of that, not only it was also possible to see how sensible the process is
to several factors occurring simultaneously, but also how the analysis performance and
sampling is important to the result.
The literature extensively reports that acidifying the mash provides a better synergy
of the amylolytic activity, but this was not perceived in this work. The non-acidified wort
delivered higher yield between all the groups. Due to the sensibility of the enzymatic process
any minute at different conditions can deliver different results, therefore if one group took
one minute more than the other group to acidify the mash it would have impacted the results
for example.
On the hop utilization side, it was clear how the acidification affects the solubility of
bitter substances. The reported behavior in the literature was found, then the lower pH the
lower the hop utilization. However, more can be explored from the actual fractions of α-acids,
β-acids and oils extracted at different pH. It could be assessed using state-of-the-art
technologies for the hop analysis as High-Pressure Liquid Column or Gas Chromatography.
In the end the DMS results showed how the brewing process is continuous and any
dead or longer times between main processes will influence the wort analysis. The literature
trend which states the lower the pH the better the conversion of PDMS into DMS was
masked by variation of other influential factors.
Therefore, to have a clearer view of the acidification on the brewing process it is
recommended to repeat the trials accounting specially for variations on side processes and
increasing the pH difference between the control trial and the acidified trials.
All things considered, this work was a generous moment to understand the effects
taking charge on the brewing process, sample analysis and conclude that there is much
more to be explored and controlled than we already know.
 33

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Appendix

Table 1. Losses calculation.

G1 G2 G3 G4
Brewhouse Yield Unit
NA MA MA+BBA MA+ABA
Volume Kettle Full L 529 525 525 505
Extract Kettle Full °P, g/100g 11,50 11,25 11,30 11,50
Average Lautering Flowrate hL/h 2,16 2,19 2,23 2,26
Extract KF Integrated °P, g/100g 11,97 12,27 12,38 12,20
Grist Load kg 80 80 80 80
Extract End of Boiling °P, g/100g 11,80 12,00 11,80 12,10
Extract Cast Out kg/hL 12,34 12,56 12,34 12,67
Volume Cast Out L 516 493 502 479
Extract Malt % 79,4% 79,4% 79,4% 79,4%
Overall BY % 96,2% 93,6% 93,6% 91,7%
Total Extract Losses % 3,8% 6,4% 6,4% 8,3%
Munich BY % 76,4% 74,3% 74,3% 72,8%
Diff OBY vs MBY % 19,8% 19,3% 19,3% 18,9%
Table 2. Processes elapsed time.
G1 G2 G3 G4
Process Times Unit
NA MA MA+BBA MA+ABA
Total Lautering Time min 147 144 141 134
Pre-boiling Hot Time min 225 196 216 173
Post-boiling Hot Time min 126 128 128 122
Total Hot Time min 351 324 344 295
Whirlpool Rest Time min 20 19 20 12
Table 3. Wort quality results.
G1 G2 G3 G4
Wort Analysis Unit
NA MA MA+BBA MA+ABA
Dimethylsulphide Precursor (PDSM) µg/L 35 42 68 54
Free Dimethylsulfide (DMS) µg/L 48 45 49 50
Final Apparent Extract °P 2,0% 2,0% 2,4% 2,0%
Final Attenuation, apparent % 83,3% 83,3% 79,9% 83,3%
Free Amino Nitrogen mg/L 199 202 204 195
Total Nitrogen mg/L 1095 1069 1079 1055
Total Protein g/100 mL 0,68 0,67 0,67 0,66
Photometric Iodine Reaction ΔE 0,539 0,676 0,413 0,420
Bitterness Units BU, mg i-α-a/L 56 53 49 50
pH Cooled Wort - 5,41 5,4 5,17 5,24
Wort Color EBC 10 8,4 8,8 8,5
Initial Apparent Extract °P 11,53% 11,49% 11,52% 11,49%
Density g/cm³ 1,04447 1,04431 1,04445 1,04431
Viscosity mPa*s, cP 1,639 1,641 1,621 1,628
Coagulable Nitrogen mg/L 39 27 35 28