MALTING BIOCHEMISTRY - American Brewers Guild: … · · 2015-01-27Malting Biochemistry In the lecture on malting technology we described the process as it affects a batch of

Malting Biochemistry In the lecture on malting technology we described the process as it affects a batch of grains. We can now view what happens in a single grain during the entire malting process. Steeping The first stage of malting involves soaking the grain in water. If the water is too warm then bacterial growth could inhibit the malting process so cool water is generally used (50-55 F0). Maltsters also wish to avoid prolonged anaerobic conditions so air is added to the water by bubbling it through the bottom of the tank, and occasionally all the water is drained and the grain allowed to sit in air for a period before more water is added. This air rest is known as “couching”. Water and Oxygen enter the grain at the proximal end of the grain where there is a gap in the waterproof pericarp/testa layer. This area is known as the micropyle. Once the average moisture content of the kernel is at about 30% (the embryo may be at 70%) the embryo “wakes up”, and begins to secrete enzymes and plant hormones. Barley in the steep eventually rises to between 43% and 48% moisture content and swells to nearly 1 ½ times its original size. Water flows from the embryo slowly across the skutellar membrane and more quickly around the living cells of the aleurone layer from the proximal toward the distal end. The water hydrates the interior of the endosperm from the out side toward the inside of the grain. Water uptake

In 1890 Horace Brown discovered that if he separated the embryo from the endosperm and soaked the embryo in water it could degrade starch, but the endosperm would not. At the same time Haberkandt discovered that if you separated the embryo from the endosperm after soaking for 24 hours and remove the embryo the endosperm will continue to produce enzymes. Therefore there must be an intermediate factor in enzyme production. It wasn’t until the late 50’s that this factor was identified as the plant hormone gibberellic acid (GA). The embryo produces some enzymes (Brown had pulled off a small piece of aleurone layer) but mainly GA, which stimulates the aleurone layer to produce enzymes. The GA is carried along with the water flowing through the aluerone layer, and the enzymes produced in the aleurone layer are carried along by the water as it soaks into the endosperm.

Malting Biochemistry Page 1 of 12

Malting Biochemistry Gibberillic Acid flow

Out of the steep the barley has just produced the first signs of a root structure known as a “chit” and the malt is known as “chit” malt

photograph courtesy of Briess Malt


Malting Biochemistry Germination Once the barley has reached its target moisture level it is transferred to the germination area for the remainder of the modification process. Germination in the malthouse is a delicate balancing act between the efficient degradation of the starchy endosperm and the demands of the brewer. The brewer needs a malt with the right amount of enzymes present, the β glucan and protein broken down and solubilised to the correct degree, and the small starch granules degraded while the large granules remain largely intact. The β glucan solubilase releases β glucan from its binding protein to render it available for β glucanase to begin breaking it down into smaller more soluble pieces. The larger β glucan molecules can cause haze problems in beer and wort viscosity problems, while the smaller molecules provide positive benefits to both foam and mouthfeel. The breakdown of the cell wall material opens up the protein matrix to degradation. Too much enzyme action will break down the soluble proteins into pieces that are too small causing a lack of foam positive material in the malt. Too little will provide a malt that will produce hazes and poor yeast nutrition. Starch degradation is also a matter of balance. The small starch granules with their high gelatinisation temperature must be completely degraded or beer with starch hazes will result, but over modification will lead to loss of extract potential that the brewer needs. Approximately 5% of the total dry weight of the barley is lost to respiration in the germination chamber. The maltster uses a variety of tools to judge the progress of the modification including the senses. Rubbing a sample between the finger and thumb to visually and tactily judge the modification is a key technique.

photograph courtesy of Briess Malt


Malting Biochemistry Enzymes The embryo produces a few enzymes which rapidly attack starch molecules to produce glucose and energy to fuel the earliest phases of growth 1) Phosphorylase. In the presence of phosphate breaks α 1-4 link to produce glucose-1-phosphate, which can rapidly produce energy 2) α glucosidase. In the presence of water this enzyme breaks α 1-4 and α 1-6 bonds to produce glucose. The aluerone layer produces the main enzymes responsible for the solubilisation and breakdown of the starchy endosperm

1. α amylase breaks the α 1-4 bond of amylose and amylopectin at random points to produce glucose, maltose, branched dextrins etc

2. Cytases a) β glucanases which break large β glucan molecules into smaller ones b) pentosanases which break large pentosan molecules into smaller ones.

3. Proteases which break down and solubalise proteins in the cell wall and matrix.

There are two types of enzyme differentiated based on their point of attack. exo proteases BREAK POINT

AA—AA—AA—AA—AA—AA—AA—AA—AA—AA—AA—AA—AA—AA— endo proteases BREAK POINT

AA—AA—AA—AA—AA—AA—AA—AA—AA—AA—AA—AA—AA—AA—

4. Limit dextrinase which cleaves the α 1-6 bond in amylopectin (debranching enzyme)

One other important enzyme is already present in barley but is bound up in the starch granules. β amylase is actually activated by the action of a protease enzyme form the aleurone layer after modification has begun. Ultimately the barley kernel is doing to grow into a barley plant utilizing all of the components of the endosperm as an energy source and as a source of material to create the new plant.


Malting Biochemistry Barley Starch The starch granules are very different in composition as well as size. In malting the small granules are nearly all coverted to simple sugar and you can see that this can account for the 10% loss of starch during malting. Early research postulated that the large granules are surrounded by a membrane made up of various proteins and lipids which takes longer to degrade, indeed may require the heat and water of mashing to break down. It may be that the small granules are more densely packed. Small granules left in a poorly modified malt will survive the mashing process due to the high gelatinization temp and cause haze and viscosity problems later on.

Barley starch granules large granules 25 µ small granules 5 µ by # % 13 87 by wt % 89 11 protein % 2 1.5 gel temp C 61 92 amylose % 24 40.5 amylopectin% 74 58

The starch in Barley is made up of 2 different types of molecule called amylose and amylopectin. Amylose is a coiled, linear, unbranced chain polysaccharide made up of 1000-4000 glucose molecules linked together with α 1-4 bonds.It has one reducing and one non-reducing end. Amylopectin is a branched polysaccharide made up of around 2500 glucose units linked together in straight chains with α 1-4 bonds and branched, on average, every 26 units with α 1-6 bonds. It has one reducing but many non-reducing ends. Amyl

ose α 1-4 linked glucose chain


Malting Biochemistry

mylopectin α 1-4 linked glucose chain with α 1-6 branches A



ction of the three main enzymes on amylose and amylopectin A

Limit dextrinase cleaves the α 1-6 bond at the branch points on the amylopectin molecule, but will not cleave the 1-6 bond if there are less than 2 groups on the side chain. Amyloglucosidase is an artificial debranching enzyme and it can be added to wort or beer to produce fermentable sugars.

Comparison of malt α amylase and β amylase

α amylase β amylase

1 attack on starch ranmolecules

domly an endo-enzyme

releases maltose an exo-enzyme

lucosidic link attacked

ups

5 70 C 60 C

6 presence during malting not present in barley present but inactive in barley

2 g α 1-4 α 1-4

3 # of reducing groproduced one one

4 optimum pH 5.5 5.2

optimum temperature


Malting Biochemistry Protein Protein in barley has historically been differentiated into four groups based on old fashioned wet chemistry definitions according to their solubility. Albumins are soluble in water and h ul te salt water and represent around 5%. Hordeins are soluble in dilute alcohol solutions and

or 40% of the total protein. Glutelins are present at about 35% and are soluble in torage

en

rrounding the starch granules • break down the protein connections in the cell wall releasing β glucan (β glucan

ules produce free amino acids in malt.

The a e they act on a long chain proteins cleaving randomly in the middle of the molecule to produce peptides and polypeptides. There are two types of exo enzymes, carb y ch attack the carboxyl and amino ends

spectively, of the protein or the peptide molecule producing amino acids.

tidase

H2 ----------------------------------- ----------------------------------------COOH

amino acids + polypeptides + peptides + amino acids

represent about 10% of t e total protein. Glob ins are soluble in dilu

account fweak alkali or acid solutions. A better way to differentiate would probably be into sproteins represented by the hordeins and the globulins, and non storage proteins which would be the albumins and the glutelins, which represent the enzymes and the structural proteins respectively. In a well modified malt around 40% of the total protein is brokdown into soluble components Protease enzymes Protease enzymes fulfill a variety of functions. They;

• break down the matrix su

solubilase) • activate β amylase inside the starch gran•

re re at least five different endo acting enzymes or endo-peptidases so called becaus

ox peptidase and aminopeptidase, whire Protein with exo and endo acting enzyme. aminopeptidase endopeptidase carboxypep N ----------------- peptides



ell Wall he cell wall contains 75 % β glucan , 20% pentosan and 5% protein.

β 1-4 linked glucose molecules terspersed with short chains of β1-3 linked glucose molecules. β glucanases are bstantially degraded in kilning and so β glucans must be significantly reduced during

hey can cause problems in the mash. There is also an enzyme that solubalises attacks the links

heir

β 1-4

CTβ glucan is a complex carbohydrate made up ofinsumalting or tthem, β glucan solubilase which survives kilning to a limited extent and between proteins and β glucans. Generally β glucans are reduced to 10-20% of toriginal level during malting. β 1-4, and β 1-3 links

β 1-3

(diagrams from Scientific Principles of Malting and Brewing by CW Bamforth)


Malting Biochemistry As in the case with proteins, β glucan is acted on by endo, and exo enzymes, to produce smaller soluble dextrins, and a small amount of glucose. Pentosans These are made up of 5 carbon sugars called xylose linked in chains with β 1-4 links and side chains of arabinose linked at the 2 or 3 position. They are insoluble and therefore of no interest to brewers but petosanases do break a portion of them down to xylose, arabinose, and pentosan polymers. Lipids Fatty acids and glycerides or lipids released during modification are another factor in

alt quality of interest to the brewer. Lipids are largely insoluble and so their effect is limited to h as

eir negative effect on foam formation. They are also important in yeast nutrition during rmentation. A lot of attention has recently been paid to a particular system involving

dehydes. This is only likely to

in lightly kilned malts since the enzymes are destroyed, and the compounds

nt in the husk, and in the cell wall of endosperm ey can become oxidized, and in that form combine to form complexes with

m their physical effect on wort and beer components in the brewhouse suc

thfethe enzyme lipoxygenase and its ability to oxidize unsaturated fatty acids such as linoleicand linolenic acid to hydroperoxides, and hence staling albe a problemreduced in the heat of the kiln. Long term storage of malt also results in the reduction of levels of this enzyme. Polyphenols These complex compounds are presecells. Thprotein. Pattern of modification While it is possible to describe each of the actions separately it should be stressed that during conversion all the reactions are occurring at the same time and so there are many interactions between systems. The physical structure of the kernel, the flow of water around the aluerone layer and through the endosperm, result in a very specific pattern to the modification of the endosperm.

(diagram from Brewing by Lewis and Young)


Malting Biochemistry The pattern of modification follows a route from the proximal to the distal end and from the outside to ward the center of the kernel. There is also a slight bias from the dorsal to the ventral side. The modification process is halted by transferring the malt to the kiln, before parts of the kernel become overmodified and extract is lost. This can result in an unmodified portion or “steely tip” toward the distal end of the kernel. Rapid modification in the germination chamber, perhaps due to higher temperatures, can result in a larger “steely tip”. It is possible to even out the pattern by adding extraneous gibberillic acid to the grain after it has been treated to allow uptake of water at other points around the exterior of the kernel. This involves breaching the husk at various other positions and applying fungal gibberillic acid to allow germination to begin from a number of starting points. This technique is sometimes used in the U.K. but never in the U.S.A. Green or even “chit” malt can be used for brewing when purpose dictates.

Germany “Chitted” malt can be used to circumvent the Rheinheitsgebot which prevents the use of barle

ce

point where it can be easily removed by simple agitation and screening. The

e plex compounds

lavor uced

lavor compounds, ranging from mild caramel to acrid bitter nd this forms the basis for specialty malt production discussed later. Simple

ation reactions can also occur during kilning. Dimethyl sulphide can be a

om

ore

Iny.

Kilning At the appropriate degree of modification the “green” malt is transferred to the kiln. Hereit is subjected to a flow of warm and eventually hot air, at various rates, in order to reduthe moisture content, and then arrest the activity and finally protect and stabilize the enzymes developed during malting. The moisture drops rapidly at first and the low moisture content serves to protect the enzymes from denaturation. Kilning dries the rootlet to the rootlet has no brewing value. Under favorable conditions of heat, reducing sugars will combine with amino acids to form compounds known as reductones. Reductones formed as intermediates have a role in protecting beer from the effects of oxidation by acting as oxygen scavengers. These compounds can polymerize to form melanoidins, which arresponsible for darker color, and are flavorful. They include comincluding pyrazines, furfurals, maltol and isomaltol. The latter two are the fcompound responsible for the “malty” flavor of malt. They can become further redby heat to form a range of facaramelizpositive flavor component in some beer styles or a fault in others. It manifests itself as a corn like flavor at low levels but can approach onion or garlic at high levels. It is produced by the breakdown of its precursor S-methyl methionine, which is formed frproteins during malting. The decomposition is thermal and occurs during high temperature kilning. Lightly kilned malts, heated less to avoid color formation are mprone to contain the precursor. During kettle boil the precursor is converted to DMS, which is highly volatile and is boiled off. If all of the precursor is not converted and removed as DMS, then the DMS can be formed in the whirlpool or hop back. Lipoxygenase enzyme is largely denatured by kilning and this helps protect against potential beer staling precursors being formed in the brewery mash.



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Malting Biochemistry Page 12

(diagrams courtesy of David Kuske with Briess Malting)

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MALTING BIOCHEMISTRY - American Brewers Guild: … · · 2015-01-27Malting Biochemistry In the lecture on malting technology we described the process as it affects a batch of