Hello Everyone.
Welcome to the brewing science and technology
series.
I'm Amul Ghimire.
Today we shall discuss about the mashing theory
and the biochemistry behind the mashing.
Mashing is a stage in the wort production
in which the grist is mixed with brew water
and the resulting mash is held for a predetermined
period at different controlled temperatures
and controlled pH.
Mashing is very crucial because it impacts
yeast performance, filtration, beer flavor,
alcohol content and foam stability.
Why do we need to mash?
So, Mashing has two purpose.
One is to dissolve as much of insoluble compounds
to the soluble compounds and get more yield
from the raw material that we use.
This is performed by continue degradation
process that were started during malting which
includes breakdown of starch, protein and
beta-glucans.
Second objective of mashing is to get better
quality of extract.
Some of the desirable components such as sugar
and protein are extracted more whereas the
undesirable components such as tannins in
the husk are extracted less.
What are the goals during mashing?
We should have Optimal mixing of malt grist,
adjunct and water for homogenous mixture.
The agitator speed of the mash tun needs to
be optimized so that the solution is homogenized.
However, care must be taken to avoid the ingress
of oxygen due to the vortex effect formed
at high agitator speed.
We should have maximum enzymatic reactions
for the conversion of complex molecules to
the simpler dissolved form which shall ultimately
results in higher yield.
The mash is held at right temperature and
pH for sufficient time so that the enzymatic
reactions are completed.
Mashing affect various downstream process.
Mashing impacts the spectrum of the sugars
present in wort which has final effect on
the yeast performance, alcohol content and
flavor profile.
Higher maltose will result in higher attenuation
of the wort resulting in higher alcohol production
during fermentation.
Similarly, the spectrum of peptides effect
the foam stability and colloidal stability.
Mashing at higher agitation conditions may
solubilize the β-glucan from the grist and
may consequently increase the viscosity and
the run-off time during the wort filtration
in lauter-tun or mash-filter.
Therefore mashing is optimized in order to
have positive influence on the downstream
processes.
The final wort composition depends on the
time-temperature profile.
The goal when choosing a suitable time-temperature
profile is to produce a wort with the desired
properties and flavor.
Mashing involves heating at various temperature
for varying time and is very energy-intensive.
Different approaches such as use of hot water
and other renewable energy are explored for
heating in order to be energy-sufficient.
So, in bigger picture, Mashing is mixing of
water and malt to produce spent grain and
wort.
During mashing, malt grist, solid adjuncts,
and water are mixed at a suitable temperature
and time for the malt enzymes to convert the
various cereal components into fermentable
sugar and other nutrients.
Spent grains constitute the solid residue
that remains after wort has been separated
from the mash.
Spent grains contain a high proportion of
polysaccharide cell wall material from the
malt and adjuncts with some lipids and proteins.
In addition, a proportion of entrained wort
always remains in the spent grains.
The liquid, separated from the spent grains,
containing the sugars and yeast nutrients
is referred to as wort or extract.
So, what is extract?
Extract is a measure of wort concentration.
Extract includes the substance dissolved in
water from brewing material such as malt and
adjunct by the mashing process with suitable
time-temperature profile through the action
of enzymes.
Extract contain the substate for the yeast
such as fermentable sugars, free amino nitrogen,
minerals, vitamins and other yeast nutrients.
And other than the substrate for the yeast,
it also contain other wort constituents to
produce desired beer which includes unfermentable
sugars and dextrins, soluble protein, inorganic
substances and flavor and color compounds.
Each style of beer demands its own wort composition
and thus the mashing process needs to be modified
to achieve so.
So where do all that grain go?
Here we see where does all that grain that
we use for mashing go.
Major portion (almost 80%) of the grain is
soluble as extract that constitute the fermentable
and unfermentable extract.
Almost 40-45% of the grain is fermentable
extract which is utilized by the yeast and
almost 25% of the grain is unfermentable extract
which gives body and mouthfeel to the finished
beer.
Nearly 20% of the grain is separated from
the wort as spent grain which include the
husk and insoluble protein.
Some extract is always entrapped in the spent
grain and some are lost during the transfer
and pipeline push-out to different vessels.
In total, the extract loss accounts for 5-10%
of the total grain solids.
We see here different units for the measurement
of the extract.
Balling, Brix and Plato are three different
units that are used interchangeably but all
of them refer the same thing.
They all express the weight % of sucrose solution.
Here sucrose mean table sugar or the common
sugar that we use.
Balling was published in 1843 as a unit of
concentration for wort and sugar solutions
based on density measured at 17.5°C whereas
Degree Brix is also used as a measure of dissolved
solids in weight basis but it was originally
measured at 15.5°C, however these days it
is measured at 20°C. It was based on a recalculation
of the tables of Balling.
The scale is used principally for expressing
the concentrations of sugar solutions as in
the case of the syrups commonly used in brewing.
It is widely used in fruit juice concentration
and winery but limitedly used in brewing.
Plato scale is a revision of that of Balling
and made by Plato in 1918 since the degree
Balling was erroneous.
As with the Balling table the concentration,
usually of wort, is compared to the value
of sucrose solution measured at 20°C. So
the difference here is Balling is measured
at 17.5 degree centigrade whereas Plato is
measured at 20 degree centigrade or 68 degree
Fahrenheit.
The concentration in degree Plato is numerically
roughly equivalent to a quarter of the present
gravity as depicted by the equation here.
So if you see the specific gravity can be
calculated by the formula 1 plus Plato into
4 by 1000.
For example, for 12 degree Plato the specific
gravity would be calculated as 1.048.
So the equation can be fairly used as estimate
between specific gravity and Plato.
Understanding of extract is very crucial in
brewing profession.
Extract like we saw before is measure of wort
concentration.
The concentration of all soluble constituents
in wort before the fermentation starts is
called original extract.
For example, here.
However, once the fermentation starts, the
presence of yeast and the resultant formation
of ethanol have effect on extract measurements
that require correction.
Since ethanol is less dense than water, it
exerts a depressing effect, and for this reason
uncorrected values, lower than the actual
extract, are referred as apparent extracts.
Concentration of extract measured predictably
after the removal of ethanol by distillation
and correction for volume and temperature
are referred to as real extracts which is
this value here.
So if you see, apparent extract is somewhere
in the range of 2.5 to 3 , the real extract
is in the range of around 6.
So real extract is always higher than the
apparent extract.
Apparent extract is lower because of the depressing
effect of ethanol formed during the fermentation
process.
This is the relation between the original
extract, real extract and alcohol percentage
on weight by weight basis.
This equation here also been defined by Balling
in 1843 and can be used as fair estimate to
calculate the relation between original extract,
real extract and alcohol percentage.
In this equation, "p" here is the original
extract, "ER" is the real extract and "A"
is the alcohol content on weight by weight
basis.
We shall now see the biochemistry of mashing
at the molecular level.
Carbohydrate accounts for 90-92% of the total
solids in wort.
Maltose (2-units of glucose polymer) is the
most abundant sugar in wort which accounts
for almost 40% of the total solids.
Maltotriose and maltotetrose which are respectively
three-glucose and four-glucose carbohydrate
form about 20% of the total solids.
Simple fermentable carbohydrates such as glucose,
fructose and sucrose composite about 15% of
the total solids while the longer chains dextrins,
glucans and pentosans (which is a C-5 sugar)
composite around 20% of the total solid.
The same is also seen here in this graph.
If you see, maltose, it composites the most
of the extract wherease the glucose, sucrose,
fructose and maltotriose form smaller proportions.
The non fermentable extract which includes
dextrin and other insoluble proteins composites
about 20% of the total solids.
Carbohydrates are the biological molecule
consisting of Carbon, Hydrogen and Oxygen
atoms.
Depending on the number of carbon atoms, carbohydrates
are classified as monosaccharides (1-sugar
molecule), disaccharides(2 sugar molecule),
oligosaccharides (3-9 sugar molecule) and
polysaccharides (>9 sugar molecule).
Monosachharides are simplest units with the
general formula of (CH2O)n where n is between
3 and 6.
If n=3, its triose, if n=4, its tetrose, if
n=5, its pentose and if n=6, its hexose.
For n=6 (hexose sugar such as glucose and
fructose), molecular formula would be C6H12O6.
Easy way to differentiate between fructose
and glucose is that glucose makes a six carbon
ring, six membered ring whereas fructose makes
a five membered ring.
Lets see here 1,2,3,4,5 membered ring for
fructose and for glucose, its 1,2,3,4,5,6
membered ring.
Glucose are called alpha and beta based on
the arrangement of hydroxyl group.
So if you see here these hydroxyl group here,
these and these.
If both hydroxyl group are on same side, its
called alpha for example in this case alpha
glucose whereas if these hydroxyl groups is
present on the other side and since this hydroxyl
group and these hydroxyl group are on the
different side, it would be called beta glucose.
Disaccharides are two monosaccharides linked
together by glycosidic bonds.
Example of disaccharides are sucrose and maltose.
Sucrose consist of one glucose and one fructose
unit.
for example here six cerbon ring glucose and
five carbon ring fructose, they are bonded
together by glycosidic bond and this is called
sucrose.
It is found in small quantity and can be added
as adjunct to the kettle.
Maltose is two glucose unit for example here
glucose and glucose.
Most important sugar in the wort that makes
up around 60% of wort sugar.
Maltose is found in germinating seed example
barley as they break down their starch stores
to use for food.
Trisaccharides and Dextrin.
Similarly maltotriose are oligosaccharides
with 3 glucose units and they are partially
utilized by the brewing yeast.
These are three glucose units and therefore
called trisaccharides.
Dextrins represent approximately 90% of the
nonfermentable residue in beer.
Some 40–50% of wort dextrins contain 4–9
glucose units; while the remaining higher
dextrins contain 10 or more glucose units.
Dextrins are glucose polymers and can be linear
with α -(1-4) linkage or branched with α
-(1-6) linkage. for example here one, this
is one, this is numbered as one carbon, 1,2,3,4,5
and 6.
This is always numbered in this way in glucose.
1,2,3,4,5, and 6 so if you see the bond here
the glycosidic bond, it is formed between
one and four and therefore called 1,4 glycosidic
bond.
Similarly if this bond is formed between one
and six carbon atom this is one and this is
six carbon carbon atom, this bond would be
called 1,6 glycosidic bond.
So, 1,4 or 1,6 are bonds which depicts the
numbers of the carbon atoms involved in bonding.
Dextrins are not fermented by the brewing
yeast and they persist in beer where they
contribute mouthfeel and body.
Concentration of dextrin in wort range from
2.5 – 4.5 g per 100ml of wort.
Starch is major carbon storage material of
green plants.
Chemically it is a polymer of glucose in which
maltose is the repeating unit.
The sources of starch are cereals that we
use for brewing purpose for example barley,
rice, wheat, corn and other cereals.
Starch consist of two types of molecules,
one is amylose and another is amylopectin.
Amylose is the internal substance of the starch
granule.
It constitutes 20 to 30% of the structure
by weight.
It's in the lower proportion than amylopectin.
Its unbranched helical chain.
Its alwyas with alpha 1-4 bond so whatever
is unbranched is always with alpha 1-4 bond.
If it is branched, it will contain alpha 1-6
bond like in amylopectin.
So amylopectin is the outer layer of the starch
granule.
It constitute 70-80% of the structure by weight,
so major portion of starch is amylopectin.
It contains up to 6,000 glucose units with
both alpha 1-4 unbranched helical chains alpha
1-6 branches.
The branches are always in between 15 to 30
glucose units.
So if you see here, amylose is small 200-400
glucose units whereas amylopectine are larger
with both helical unbranched and branched
chain.
Amylose are simpler molecule in small proportion
of around 20-30% and is only helical with
unbranched chain.
In the figure if you see here, like before,
this is always if you see this oxygen atom
here 1,2,3,4,5 and 6.
Similarly here 1 2 3 4 5 & 6, so the amylose
is always between 1 & 4 carbon atom so this
glycosidic bond is called alpha 1-4 bonds
and amylose only has alpha 1-4 bond whereas
in case of amylopectin, if you see here, this
is the 1-4 bond whereas this here is 1-6 bond.
for example 1 2 3 4 5 6, the glycosidic bond
here is between one carbon atom and six carbon
atom so this is alpha 1-6 bond and these bond
the alpha 1-6 bond are generally between every
15-30 glucose units and are responsible for
branching of amylopectin.
Hemicelluoses are non-starch polysaccharide
polymers that form part of the structure of
plant cell wall.
They have a relatively amorphous cross-linked
structure and form the matrix of cell wall
being interposed within the cellulose component.
They are not entirely composed of glucose
residues; therefore is called heteropolymer.
Other sugars are also present, for example
pentosans or pentoses – therefore they are
called heteropolymers.
During malting the β -glucan component is
preferentially degraded and in consequence
the proportion of residual pentosans (such
as arabinose and xylose) increases.
The pentose sugar increases.
Residual pentosans that remain undegraded
after the mashing may contribute to problems
associated with very viscous wort.
Pentosans can bind water and form gum.
And therefore the complex of pentosans when
present at sufficient concentration may impede
wort run-off during wort filtration.
This is the structure of hemicellulose so
if you see here, these are all with glucose
units but they have 1-3 bond like you see
here 1,2,3 and 1-3 bond or they have 1-4 bond
like this here.
Amino acids - peptides and protein.
So amino acid is basic building block of protein.
There are 20 different amino acids in proteins
and they are composed of amine group, carboxylic
group COOH and the side chain specific to
each amino acid.
These amino acids are essential for yeast
growth.
The peptide are shorter chain of amino acid
monomers which are linked together by peptide
bond.
The shortest peptides are dipeptides with
2 amino acids.
Peptide are distinguished from the protein
based on the size.
Peptide has nearly 50 or fewer amino acids
whereas protein are longer chain of amino
acids.
Proteins form the structural and the catalytic
function.
Catalytic function are due to the enzymes
so enzymes are also proteins.
If you see here these small group, these small
circle here, they are individual amino acids.
These amino acids for example these amino
acids - they have amino group NH2 group, carboxylic
group COOH group and a side chain which are
different for each different kind of amino
acids.
These amino acids are linked together by the
peptide bond so these bonds here are peptide
bonds.
The primary protein structure is the chain
of amino acids that make up the protein - so
this complete structure - group of amino acids
forms a protein.
Enzymes : So enzymes are protein that are
catalytic function.
They catalyze chemical reaction - facilitate
and speed the reaction.
They are not changed or consumed by the reaction.
The molecule at the beginning of the process
called substrate are converted into different
molecules called products.
So enzymes are specific - each enzyme catalyzes
just one reaction.
The enzyme name reflect the reaction they
facilitate.
For example if they breakdown amylose its
called amylase - this is a complex structure
of amylase; if they breakdown protein, they
are called protease; if they breakdown glucan,
they are called glucanase.
So each enzyme reflect the name of the substrate
that they act on.
The structure of enzymes and their chemistry
are very complex - like this one here.
The enzymes are sensitive to biochemical environment
for example temperature, pH, minerals and
other compounds.
Lock and key principle implies that the substrate
is like a key - the substrate here is like
the key that fits into a lock exclusively
on the active site on the enzyme surface.
Enzyme function by lowering the activation
energy of the reaction that they catalyze.
In order for any chemical reaction to occur
sufficient energy must be available to drive
it.
This is termed the activation energy.
The reactant in enzyme-catalyzed reactions,
termed substrate, first bind to the enzyme.
Substrates bind to particular sites on the
enzyme molecule, termed active site.
They are made up of a unique combination of
functional chemical group with a particular
arrangement such that one or a very limited
number of reactant substrate molecule are
able to bind.
The enzyme–substrate complex reduces the
activation energy and the particular reaction
can proceed.
The product are released, returning the enzyme
to its initial state and free to catalyze
another reaction.
The ability of the enzyme to bind one or a
limited number of substrate, based upon the
configuration of the active site, explains
enzyme specificity.
More recently new hypothesis is developed
called induced fit hypothesis which is based
on substrate specificity rather than enzyme
specificity.
Enzyme activity may depend on co-factor.
Cofactor may be essential for activity but
are not permanently bound.
They are also called coenzymes.
They are neither the part of the enzyme nor
the substrate.
They function as intermediaries by binding
particular groups that are subsequently involved
in the enzyme-catalyzed reaction.
E.g., calcium for alpha amylase.
Various factors affect the enzyme activity
such as pH, temperature and concentration
of enzymes and substrate.
Each enzyme has specific range of pH and temperature
where the enzyme functions at optimal activity
and stability.
Extremely high or low pH values generally
result in complete loss of activity for most
enzymes.
Similarly, this is the optimal temperature
- higher temperature than the optimal temperature
will result in denaturation of enzyme where
the enzyme is irreversibly changed in it’s
structure and is no more able to function
at all.
Boiling destroys all enzyme - the enzymes
are completely denatured at boiling temperature.
Similarly the substrate concentration also
impact the enzyme activity.
If we have diluted substrate or diluted enzyme
concentration, the activity are lowered.
The substrate concentration and enzyme concentration
have to be optimal for maximum enzyme activity.
Therefore, the grist water ratio is very important
to maintain for optimum substrate concentration
or optimum enzyme concentration in order to
achieve maximum degradation of the substrate.
Enzymes in Brewing.
So brewer can use two types of enzyme - one
is endogenous - native to barley malt.
Most brewers use only endogenous enzymes.
Some enzymes are not found in raw barley and
are developed during the malting process example
alpha-amylase.
Wherease the other enzyme are exogenous from
other sources.
These enzymes are added externally for special
situations like in Low carbohydrate light
beer - beta amylase and limit dextrinase are
used for complete degradation of starch.
For High adjunct percentage - the inherent
alpha amylase and beta amylase would not be
sufficient to breakdown the starch in high
adjunct mash, so externally alpha and beta
amylases are used.
To reduce beta-glucan content - if the malt
is not friable, is not well modified then
in that case the beta glucan can impede the
wort run-off and therefore beta-glucanase
are used externally in the mash.
In order to produce the Gluten free beer certain
endo-proteases are used externally.
So all the enzymes that we use externally
are exogenous.
Special care are made for German-purity law
or Rheinhetsgebot where the exogenous use
of enzymes are not permitted.
Enzymes in brewing are majorly categorized
into cytolytic enzymes, proteolytic enzyme
and amylolytic enzyme.
Cytolysis describes the degradation of the
supporting and structural cell wall substances
in the coating of the starch - bearing cells
of the endosperm.
They make the proteolytic and the amylolytic
enzyme to act inside the cell wall so for
these enzyme to act, cytolytic enzyme have
to first breakdown the cell wall.
Certain cytolytic enzymes are beta glucan
solubilase, beta glucanase.
These enzymes are usually active at lower
temperature and therefore if the malt is not
friable enough during mashing, brewers can
maintain certain rest time at low temperature
to activate the inherent glucanases which
shall breakdown the cell wall and reduce the
viscosity of the mash for proteolytic and
amylolytic enzyme to work.
Proteolysis describes the degradation of the
grain protein.
They assist cytolytic enzyme by making starch
more accesible for amylolytic enzyme.
Amylolytic enzymes are responsible for breakdown
of starch and sugar.
We shall see these in more details.
Cytolytic enzymes attack the structural proteins
and cell wall polysaccharides, especially
β - glucan.
A wide degradation of structural substance
during malting enables an easier enzymatic
attack of the starch in the course of mashing
and, consequently, a better yield in the brewhouse.
, under-modified cell walls or undermodified
malt causes, on the one hand, yield losses
and, on the other hand a transfer of a large
amount of high – molecular β - glucans
in dissolved form.
Mainly insufficiently degraded, high molecular
β - glucan originating from the cell wall
of the endosperm contribute to high viscosity.
In the picture here you see the large starch
granule and these small starch granule, so
the cell wall has be broken down for these
starch granules to be accessible for amylolytic
enzymes.
Proteolytic enzymes.
Barley protein is stored mostly in endosperm.
Its combined with hemicellulose in the cell
walls that encapsulate starch.
Therefore like cell wall the protein breakdown
is also essential to access these starch.
Its composition in wort affect the fermentation
and beer quality.
Nitrogen in Brewing.
Free amino nitrogen (FAN) is the sum of the
individual wort amino acids, ammonium ions
and small peptides which is the critical source
of nitrogen for the brewing yeast.
Low FAN results in low yeast propagation and
the development of undesired fermentation
byproducts such as diacetyl.
However, excessive proteolysis results in
too strong degradation of high molecular protein.
The lack of sufficient quantities of high
molecular proteins as well as the backlog
of mid molecular compound and certain amino
acids ( such as lysine, arginine and histidine)
has a negative influence on foam stability.
These wort and beer tend to have high colors,
and a surplus of certain amino acids could
cause an off - flavor and poor flavor stability.
In addition, beers with high amino acids content
are very susceptible to beer - spoiling microorganism.
Therefore the proteolytic enzyme are very
important in order to breakdown certain protein
and also preserve certain protein.
The proteolytic enzymes basically work at
around 45 to 50 degree centigrade so if we
want to have protein breakdown, we have give
rest at this temperature of around 45 to 50
degree centigrade.
Amylolytic enzymes are starch degrading enzymes
and are also called diastase.
However for the amylolytic enzyme to act,
certain preliminary condition needs to be
fulfilled for example modification, gelatinization
and liquefaction.
Modification involves the breakdown of cell
wall by cytolytic and proteolytic enzyme for
the starch to be accessible by the amylolytic
enzyme.
During gelatinization, starch takes on water,
swells, stretches and lose their organized
structure.
The mash become more viscous.
Malt starch gelatinizes above 60 degree centigrade
whereas adjuncts such as rice, maize and sorghum
require higher gelatinization temperature
of about 75 - 80 degree centigrade and is
performed using separate cereal cooker and
then returned to the main malt mash.
After the gelatinization, liquefaction steps
taken in where enzymes start to break down
the starch and reduce the viscosity.
Two enzyme degrade the starch producing sugars
- one is alpha amylase and other is beta amylase.
Alpha amylase are not found in barley and
are formed during the malting process.
They are endo-enzyme and acts in the interior
of the starch granule.
They produces mostly dextrins.
Alpha amylase attacks anywhere whereas beta
amylase - they are exo-enzyme.
They attack the unbranched end, produces only
maltose.
Beta take bites.
So alpha amylase is at 65-70 degree centigrade.
Generally the rest is given at 72 degree centigrade
for alpha amylase - also called sachharification
rest whereas beta amylase is active at around
60 to 65 degree centigrade where it acts on
starch to produce the maltose.
beta amylase increases the attenuation of
the beer.
Rest at around 65 degree centigrade for longer
period will produce highly attenuated wort
with lower limit extract, more alcohol and
light body beer.
So beta amylase, if you see here beta amylase,
they cannot hydrolyze α -(1,4) linkages that
are close to α -(1,6) branch points.
So if you remember the branching is alpha
1-6 bond and the other linear chain has alpha
1-4 bond.
Both alpha and beta - they act on alpha 1-4
bond whereas alpha attack anywhere so they
go inside and attack whereas beta bites - so
they produce maltose - they always starts
from the terminal end and produces maltose.
Some part of the starch, like this one here,
near the branching point always remains and
those remaining starch are called limit dextrin.
Limit dextrinase is another group of starch
degrading enzyme also known as debranching
enzyme - so they act on these branched portion.
Where worts with high fermentability are required
it is common to add a supplement of a preparation
of a microbial limit dextrinase.
Endogenous limit dextrinase during mashing
is highly dependent upon the conditions imposed
during kilning and barley variety.
However, some activity of limit dextrinase
persists into mashing at lower temperature
of around 55-60 deg C. So if we want to have
highly attenuated beer, the rest is given
at around 55-60 degree centigrade for some
inherent limit dextrinase to act.
To confirm the completion of starch hydrolysis,
Iodine test is most practiced and easiest
method.
Starch has a powerful color reaction with
iodine solution: amylose yields an intense
deep blue to black color because the iodine
incorporates itself easily into the helical
structure of amylose whereas amylopectin gives
considerably less intense blue to violet color
because it lacks the extensive helical regions.
Exhaustive action of beta-amylase on amylopectin
leaves limit dextrins which give a brick-red
or brown color with iodine.
Total mash saccrification causes no change
in the color of iodine.
So if you see in the initial stage, if we
put drop of iodine on the mash, it gives very
dark blue to black color whereas in the inetrmediate
stage, the color fades and at the end of the
saccharification, iodine maintain its color
and there is no change or very slight change
in the color of iodine.
This confirms that saccharification has been
completed and this test is one of the most
used test to confirm the completion of the
starch degrading enzymes.
So by this time, you should be able to answer
all of these questions.
I hope we were able to cover all these topics
in this presentation.
If you guys have any questions, you may email
at brewing.distilling.professionals@gmail.com.
Hope you guys enjoyed.
Hope it was a informative session.
Thank you all.
Cheers.
