Session I - Tech#4 Muhlenchemie

Esterase in Baking
Dr. Lutz Popper, Mühlenchemie GmbH & Co. KG, Ahrensburg, Germany
Esterases split ester bonds into acids and alcohols, binding water in the process. This
reaction is known as hydrolysis, and the esterases are therefore classified as hydrolases. In
spite of this common feature the subclass of the esterases (EC 3.1) is distinguished by
numerous enzymes, some of which catalyse the reaction of very different biomolecules in a
highly specific manner. Tab. 1 shows examples of esterases that are also used in the
production of foods.
Tab. 1: Some esterases and their applications in food technology
No. IUB No. Enzyme Reaction Catalysed Applications
1 EC 3.1.1.3 Lipase
(triacylglycerol
lipase)
Splits fats and lipids into
fatty acids and glycerol
or other alcohols
Maturing of cheese;
emulsifier production;
interesterification of
2 EC 3.1.1.4 Phospholipase A 2
3 EC 3.1.1.32 Phospholipase A 1
4 EC 3.1.1.5 Lyso-phospholipase
Hydrolyses
phospholipids (lecithin)
5 EC 3.1.1.26 Galactolipase Splits fatty acids off
galactolipids
6 EC 3.1.1.6 Acetyl esterase Splits off acetyl groups,
e.g. from pectin or xylan
7 EC 3.1.1.11 Pectin esterase Splits methyl groups off
pectin
8 EC 3.1.11 –
EC 3.1.31
Exo- and
endonucleases
Splits nucleic acid
between phosphate and
nucleobase
9 EC 3.1.1.73 Feruloyl esterase Splits off ferulic acid,
e.g. from wheat xylans
10 EC 3.1.1.?? Coumaroyl esterase Splits off cumaric acid,
e.g. from wheat xylans
11 EC 3.1.3.8, Phytase
Removes phosphoric
EC 3.1.3.26
acid from phytate
fats; baking
Improvement of
emulsifying power
(e.g. egg yolk);
degumming
Improvement of
emulsifying power;
baking
Baking; fruit juice
Clarification of fruit
juice; gel formation;
stabilizing of fruit
Flavour; yeast extract
Flavouring; baking
Flavouring; baking
Digestibility &
bioavailability
1 Lipolytic esterases
Esterases especially important in the food sector are lipolytic enzymes, i.e. enzymes that
split fats and similar substances (Nos. 1 – 5 in Tab. 1).
The function of the lipolytic enzymes lies chiefly in converting flour lipids into more strongly
polar and more hydrophilic molecules, i.e. triglycerides into diglycerides, lecithins into lysolecithins
and galactolipids into lyso-galactolipids, by splitting off the long, uncharged fattyacid
chains (Figs. 1-3).

Fig. 1: Effect of lipases on triglycerides Fig. 2: Effect of phospholipases on
phosphatidylcholine (lecithin
fraction)
Bread volume [ml/100 g flour]
200
180
160
140
polar lipids
total lipids
non-polar lipids
volume prior to baking
0 200 200 300 400 500 600 700 800 900
1000
Re-added wheat lipid [mg/100 g flour]
Fig. 3:
Effect of galactolipase on
monogalactosyl diglyceride
Fig. 4:
Effect of polar and non-polar
wheat lipids on the volume yield
of defatted wheat flour (modified
from MacRitchie and Gras, 1973)
The resulting structures with a greater affinity with water interact strongly with the proteins of
the gluten. In the dough phase the lipids are already largely associated with the protein, so
after their modification they are already in the right position for an optimum effect on the
protein.
Over the past few years, lipolytic enzymes have steadily gained significance in flour
standardization – especially in the field of baking improvers and baking premixes. In the
premixes these enzymes can partially or wholly replace emulsifiers, making it possible to
reduce dosages, simplify label declarations and cut costs.
A suitable product series for this purpose is Alphamalt EFX. In developing it, Mühlenchemie
took the heterogeneous nature of the wheat lipids into account (Tab. 2).

Tab. 2: Lipids in wheat flour (0.405 % ash)
Total lipids (14 % m.b.) 1,280
Non-polar lipids 457
Polar lipids 823
Phosphatides (lecithin) 250
Phosphatidyl acid 30
Phosphatidyl glycerol 51
Phosphatidyl cholin 27
Phosphatidyl ethanolamine
traces
Phosphatidyl serine 15
Lyso-phosphatidyl cholin 117
Lyso-phosphatidyl ethanolamine 10
Galactolipids 249
Other polar lipids 320
The result is an esterase with which the fatty-acid residues can be split off from a wide range
of substrates. However, the enzyme has a preference for longer chains (over C8), which
means that even in the presence of butter fat there is very little risk of an off-taste caused by
the release of volatile fatty acids.
If an enzyme is to act on the lipids it first has to gain access to them by diffusion. For this
reason the Alphamalt EFX series is most suitable for baking methods with long fermentation
times, e.g. baguettes (as shown for Alphamalt EFX Super in Fig. 5 and 6), although the
effects are already visible with short proof times such as those for split rolls.
Basic treatment:
FAA, 1 SKB/g
ADA, 40 ppm
Asc., 160 ppm
SSL, 0.3 %
0 ppm 10 ppm 25 ppm 50 ppm
743 748 787 856
1.5 h,
normal proof
Volume yield,
mL/100 g flour
803 852 882 935
2 h,
over-proof 1
863 937 965 1015
2.5 h,
over-proof 2
Fig. 5:
Effect of Dosage and Proof Time on Baguette Rolls with
Alphamalt EFX Super

1.5 h,
normal proof,
no EFX Super
1.5 h,
normal proof
2 h,
over-proof 1
2.5 h,
over-proof 2
Basic treatment:
FAA, 1 SKB/g
ADA, 40 ppm
Asc., 160 ppm
SSL, 0.3 %
743 856 935 1015
Volume yield,
mL/100 g flour
Fig. 6:
Effect of Proof Time on Baguette Rolls with Alphamalt
EFX Super (50 ppm)
For applications with short proof times, especially, it is advisable to test combinations with
emulsifiers such as DATEM or SSL. It is often possible to enhance the baking properties or
reduce the dose of emulsifier. With Alphamalt EFX, dough stability and fermentation
tolerance are constantly adjusted to the requirements of the baking process: the longer the
proof time, the more chance Alphamalt has to develop its activity.
So it is that the user has a flexible, adaptable flour improvement system at his disposal that
makes it possible to control dough stability and volume yield.
The dosage is only 5 – 50 ppm (0.5 – 5 g to 100 kg of flour). Large doses should only be
used in conjunction with short proof times. An overdose does not increase the effect. On the
contrary: it may even result in a slight loss of volume yield.
Wheat flour contains about 1.5% lipids (fat-like substances) which fall into two categories:
non-polar lipids that are not miscible with water, and polar lipids. The polar lipids include the
lecithins and compounds of sugars and fats, for instance galactolipids. The effects of polar
and non-polar lipids on the volume yield of flour were already described by MacRitchie and
Gras in 1973. According to these authors it is only the polar lipids that bring about an
increase in volume (Fig. 4).
At first glance it may seem odd that the use of (totally non-polar) fats in baking usually
increases the volume of the baked goods, at least at low dosages (up to about 10%). But we
have to remember that in contrast to the trials that led to Fig. 4, the fat is added to an intact
flour in which polar and non-polar lipids are present in their natural ratio and in their usual
positions. In this case the addition of polar lipids evidently results in increased volume. One
might compare the effect to that of a lubricant that makes the dough more pliant, allowing the
“layers” to slide over each other.
2 Xylanolytic esterases
The function of other esterases consists in splitting off aromatic acid residues from the xylans
(pentosans) of the grain (Fig. 7). In the xylans, ferulic acid and cumaric acid are bound to the
pendant arabinose groups. Through them the xylan chains can be linked with each other and
also with proteins (Fig. 8 and 9). In a reaction called “oxidative gel formation” they thus
increase the water-binding capacity of the dough and result in higher viscosity and reduced
extensibility.

Fig. 7:
Enzymatic hydrolysis sites in
wheat xylan
Fig. 8:
Cross-linking of wheat gluten and
xylan (modified from Hoseney
and Faubion, 1981)
Fig. 9:
Interaction of feruloyl side-chains in
arabinoxylan (modified from Williamson et al.,
1998)
Splitting of the bonds with xylan by the esterases leads to a breakdown of gel-forming
structures and thus to softening of the dough. Single released molecules of these acids are
important precursors of flavour, for example in the maturing of sour dough and also in the
biochemical (“natural”) synthesis of vanilla. A further application is total liquefaction, for
example in the production of ethanol from grain.
A ferulic acid esterase (FAE) from Streptomyces werraensis is described by José-Luis Copa
Patiño et. al. When developing this enzyme for marketing we found – not surprisingly – that
the micro-organism produces a large number of ligno- and hemicellulolytic enzymes (Tab. 3)
of which very few had so far been investigated for their effect on dough and baking
properties.

Ferulic acid esterase will shortly be available commercially as Alphamalt FSR. Its main
function will lie in optimizing the rheological properties of dough (Fig. 10) and in reducing the
cost of enzymes through combination with other xylanolytic enzymes.
160
Extensibility L (mm)
150
140
130
120
110
100
90
80
0.00 0.05 0.10 0.15 0.20 0.25
FAE dosage (%)
Fig. 10: Effect of feruloyl esterase (crude extract) on
the extensibility in the alveogram
Tab. 3: Enzymatic activity in the culture supernatant of Streptomyces werraensis
UAH 44 in various different nutrient media
Substrate
Enzyme
Medium
1 2 3 4 5 6 7 8 9 10
AZCL-amylose Amylase + - - + - + ± ++++ - -
AZCL-arabinan (debranched) Endo-1,5-a-L-arabinase + ++ + +++++ +++ +++ +++ +++ ++++ ++++
AZCL-arabinoxylan (wheat) Endo-xylanase ++++ ++++ ++++ +++ +++ +++ +++ ++ +++ +++
AZCL-HE cellulose Endo-cellulase - + - ++ - - - - +++ +
AZCL-curdlan Endo-1,3-b-glucanase - +++ ± +++ + + + ++ ++++ +++
AZCL-dextran Endo-1,6-a-glucanase - - - - - - - - - -
AZCL-galactan (potato) Endo-1,4-b-galactanase - ± - ++++ ++ ++ ++ + ++++ +++
AZCL-galactomannan b-mannanase - - - + - - - - + ±
AZCL-b-glucan Malt b-glucanase ++ +++ ++++ +++ ++++ +++ ++++ +++ +++ ++
AZCL-pullulan Limit dextrinase - - - - - - - - - -
AZCL-xylan(oat) Endo-xylanase ++ ++++ ++ ++++++ ++++++ + +++++ ++ ++++++ +++
Skimmed milk Protease - + - +++ +++ - ++ - +++ +++
3 Proteases with esterase activity
Esterase activity is sometimes found in unexpected places – for example in some proteases,
where it is a genuine side activity of the protease molecule. The reason is that the reaction
catalysed by proteases is very similar to ester cleavage (Fig. 9).
Protease: -R 1 CH-CO-NH-CR 2 - + H 2 O ↔ -R 1 CH-COO - + + H 3 N-CR 2 -
Esterase: R 1 -CO-O-R 2 + H 2 O ↔ R 1 -COOH + R 2 -OH
Fig. 11: Representations of reactions catalysed by protease
and esterase
The active centre of serine proteases (subtilisin, trypsin, chymotrypsin) is similar to that of the
lipases. Both contain the same catalytic triad, the amino acid asparagine, histidine and

serine. Papain also has an unspecific effect on lipids, whereas metallo-proteases show no
esterase activity.
The “side effect” plays a certain positive role in the maturing of cheese, where it aids the
development of flavour, but our experience shows that it has no significance at all in baking.
In other applications it can be a great nuisance, for example in the production of precious
vegetable oils, where proteases are used to increase the yield.
4 Phytase
Phytases have found wide applications in feed rations. They degrade phytate, an antinutritive
factor found in cereals and legumes. Phytic acid binds iron, calcium and other
bivalent metal ions, necessary as growth factors and stabilizing agents for enzymes. Phytic
acid also inhibits microorganisms including yeasts, probably by its ion binding properties.
Phytate
Myo-inositol
6-phytase
cereals
H
H 2 O 3 PO
H
H 2 O 3 PO
H
OPO 3 H 2
OPO 3 H 2
H 2 O 3 PO
H
H
OPO 3 H 2
H 6 H 2 O
6 H 3 PO 4
HO
H
HO H
H
OH
OH
HO
H
H OH
H
3-phytase
microorganisms
Fig. 12: Action of phytases on phytate
Phytases split the ester bond between phosphoric acid and inositol, hence depriving phytic
from is ion binding capability (Fig. 12).
Although cereals and legumes also contain phytases, those are not able to break down all
the phytate under normal conditions of processing and digestions. In particular wholegrain
cereal preparations contain a high amount of phytate, which is sterically separated from the
grain’s own phytase (Fig. 13). Furthermore, the level of phytase underlies large natural
fluctuations. Therefore, microbial phytase can be used to improve the nutritive value of food
(and feed) from cereals and legumes.

Phytate
Phytase
2%
13%
85%
8%
15%
34%
40%
Aleuron
Germ
Endosperm
Scutellum
Other
3%
Fig. 13: Distribution of phytate and phytase in wheat
(modified from Zimmermann et al., 2000)
In baking, phytases are able to contribute to dough stability and volume yield, probably due
to a better availability of ions for dough stabilization and microbial (yeast) activity .
5 Other esterases
As far as we know, the enzymes listed as items 6 to 8 in Tab. 1 are not used directly in
baking applications. Although acetyl esterase has a certain effect on wheat xylan, this is
without significance as yet. Nucleases have only been put to technical use indirectly in
baking to produce yeast hydrolysates that have the effect of intensifying the flavour or
replacing salt.
6 References
Copa-Patiño, J.L., Caballero, A., Popper, L., Arenas, M., Soliveri, J., 2007. Empleo de un
complejo enzimático producido por una cepa de actinomicetos en procesos de
panificación. Poster presentation, XXI Congreso Nacional de Microbiología, Sevilla,
Spain, September 17-20.
Hoseney,R.C., Faubion, J.M., 1981. A mechanism for the oxidative gelation of wheat flour
water-soluble pentosans. Cereal Chem. 58(5), 421-424.
MacRitchie, F., and Gras, P. W. 1973. The role of flour lipids in baking. Cereal Chem. 50(3),
292-302.
Williamson, G., Kroon, P.A. and Faulds, C.B., 1998. Hairy plant polysaccharides: a close
shave with microbial esterases. Microbiol. 144, 2011-2023.
Zimmermann, B., Krämer, K., Biesalski, H.-K., Drochner, W., 2000. Zur Bedeutung
pflanzlicher und mikrobieller Phytasen in der Humanernährung. Teil 1: Charakteristik
und Wirkung von Phytinsäure und Phytasen. Ernährungs-Umschau 47(11), 423-427.