Amylolytic Enzymes and Bread Firming
Hans Goesaert*, Louise Slade and Jan A. Delcour*
*Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and
Nutrition Research Centre (LFoRCe), [Link], Leuven, Belgium, and Food Polymer Science Consultancy, Morris Plains, New Jersey, USA
ESEGP-5 Norwich, April 1 2008
Laboratory of Food Chemistry and Biochemistry
Outline
Introduction Starch and gluten in breadmaking Networks in dough and bread Amylases in breadmaking Experimental work Conclusions
Laboratory of Food Chemistry and Biochemistry
Outline
Introduction Starch and gluten in breadmaking Networks in dough and bread Amylases in breadmaking Experimental work Conclusions
Laboratory of Food Chemistry and Biochemistry
Introduction
Quality and freshness are very important for bakery products Bread is an important constituent of an equilibrated diet Bread staling is an important phenomenon
Loss of aroma Loss of crust crispness Crumb firming
Laboratory of Food Chemistry and Biochemistry
Outline
Introduction Starch and gluten in breadmaking Networks in dough and bread Amylases in breadmaking Experimental work Conclusions
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking From wheat (100 %) to flour (ca 70 %) Wheat flour constituents - starch (ca. 70 %) - protein (ca. 12 %) - nonstarch polysaccharides (ca. 2 %) - lipids (ca. 1%) - water (ca. 14%)
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking Starch Reserve polysaccharide Granular structure - sizes: - shape: 2 to 10 , 20 to 35 round and lenticular
Milling causes some starch damage
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking Starch granular structure
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking Starch composition
Poly--D-glucose: - amylose (ca 25 %) - amylopectin (ca 75 %)
Amylose :
- -1,4 - hardly -1,6 - ca 2,000 glucose units - essentially linear - linkages: - amorphous
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking Starch composition
Amylopectin: - linkages: - ca 5% -1,6 - ca 95% -1,4
- ca 500,000 glucose units - heavily branched - amorphous/crystalline A-type crystals
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking Amylopectin structure
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking
Starch related phenomena occurring at dough stage Native starch: water uptake - 30% water - 5% volume increase Damaged starch: - huge water uptake - amylose and amylopectin entanglement
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking
Starch related phenomena occurring during baking Irreversible water uptake - swelling - (limited) amylose leaching - granule remnants - swollen - enriched in amylopectin - loss of crystallinity Increase in viscosity
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking
Gluten and protein Osborne fractions: Albumins Globulins Prolamins (gliadins): 70% ethanol extractable Glutelins (glutenins): dilute acid extractable Unextractable glutenin Prolamin + glutenin - majority of protein - together form gluten
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking
Gliadin: Monomeric protein: - MM 30 - 80 kDa - intramolecular S-S -, -, - Types Form gluten together with glutenin
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking
Glutenin: Polymeric protein consisting of subunits - HMW - GS (ca 15%): MM 65 - 90 kDa - LMW - GS (ca 85%): MM 30 - 60 kDa Intermolecular and intramolecular S-S Form gluten together with gliadin
Laboratory of Food Chemistry and Biochemistry
Starch and gluten in breadmaking
Gluten: Gluten - forms transient network during dough mixing - responsible for dough - visco-elastic properties - gas holding properties Gluten functionality impacted by its size Gluten polymerises during baking and forms permanent network
Laboratory of Food Chemistry and Biochemistry
Outline
Introduction Starch and gluten in breadmaking Networks in dough and bread Amylases in breadmaking Experimental work Conclusions
Laboratory of Food Chemistry and Biochemistry
Networks in dough and bread
Networks necessary for coherent dough/crumb structures Networks can be transient , based on - entanglement - dynamic exchange covalent bonds permanent, based on - covalent linkages - (partial) crystallinity
Laboratory of Food Chemistry and Biochemistry
Networks in dough and bread
Networks in dough are of transient type and consist of Amylose and amylopectin from damaged starch Glutenin - dynamic exchange of disulfide bonds - entanglement Gliadin does not entangle (globular)
Laboratory of Food Chemistry and Biochemistry
Networks in dough and bread
Networks in baked fresh warm bread are of transient and permanent types: Gelatinized starch is entangled Glutenin network is permanent as a result of thermosetting and has (some) gliadin incorporated
Laboratory of Food Chemistry and Biochemistry
Networks in dough and bread
Networks in baked fresh cooled bread are of transient and permanent types: Gelatinized starch is entangled Glutenin network is permanent as a result of thermosetting and has (some) gliadin incorporated Amylose network is permanent as a result of chain folded crystallisation leading to partial crystallinity
Laboratory of Food Chemistry and Biochemistry
Networks in dough and bread
Networks in baked stored bread are Networks in baked fresh bread + Permanent amylopectin network which itself is the result of amylopectin intermolecular crystallisation
Laboratory of Food Chemistry and Biochemistry
Networks in dough and bread
Networks in baked stored cool bread are increasingly of the permanent type: permanent amylopectin B-type crystals incorporate water from the amorphous phase, making gluten e.g. less plasticized The net result is bread firming and loss of resilience
Laboratory of Food Chemistry and Biochemistry
Outline
Introduction Starch and gluten in breadmaking Networks in dough and bread Amylases in breadmaking Experimental work Conclusions
Laboratory of Food Chemistry and Biochemistry
Amylases in breadmaking
-Amylase -Amylase Maltogenic amylase
Laboratory of Food Chemistry and Biochemistry
Amylases in breadmaking
-Amylase
active on native but much more on gelatinized starch virtually absent in sound flour generates maltose for fermentation reduces viscosity during starch gelatinization and thus increases volume antifirming effects, many times poor crumb resilience malt, fungal or bacterial origin
-Amylase
active on gelatinized starch abundantly present in sound flour inactivated before starch gelatinization
Maltogenic amylase
active on gelatinized starch antifirming effects, good crumb resilience
Laboratory of Food Chemistry and Biochemistry
Amylases in breadmaking
The ideal antifirming amylases have a right window of activity, i.e. they
are not inactivated before starch gelatinization are active during/after starch gelatinization are denatured during further baking and/or are no longer active when the bread is stored lead to soft crumb structures lead to resilient crumb structures by ensuring water retention by gluten
Laboratory of Food Chemistry and Biochemistry
Amylases in breadmaking -amylases
hydrolyze -(14)-bonds of starch internally
Laboratory of Food Chemistry and Biochemistry
Amylases in breadmaking -amylases -amylase
hydrolyze -(14)-bonds of starch internally from the non-reducing ends
maltogenic amylase
Laboratory of Food Chemistry and Biochemistry
Amylases in breadmaking
BREAD: water, flour, yeast, sugar and salt Amylases
Starch
Dextrins
Amylose
Amylopectin
Earlier hypothesis 1: The formed dextrins - interfere with gluten-starch-interactions - influence retrogradation of the residual starch fraction
Laboratory of Food Chemistry and Biochemistry
Amylases in breadmaking
BREAD: water, flour, yeast, sugar and salt Amylases
Starch
Dextrins
Amylose
Amylopectin
Earlier Hypothesis 2: The resulting amylose and amylopectin have a changed retrogradation behaviour
Laboratory of Food Chemistry and Biochemistry
Outline
Introduction Starch and gluten in breadmaking Networks in dough and bread Amylases in breadmaking Experimental work Conclusions
Laboratory of Food Chemistry and Biochemistry
Experimental work
Experiments were designed to explain the
Positive impact of amylase on crumb firming and the negative impact on resilience Positive impacts of BStA (maltogenic amylase) on both crumb firming and resilience
Laboratory of Food Chemistry and Biochemistry
Experimental work
Amylases Characterised amylases
Straight-dough procedure
Thermal stability Mode of action
Breadmaking
Bread characteristics
Amylose (SEC) Starch characteristics Amylopectin (HPAEC) Dextrins (HPAEC)
Laboratory of Food Chemistry and Biochemistry
Experimental work
Amylase characteristics
Amylase Mode of action Thermal stability intermediate
BSuA
Bacillus subtilis
endo
PPA
Porcine pancreatic
endo
low
BStA
Bacillus stearothermophilus
maltogenic
intermediate
Laboratory of Food Chemistry and Biochemistry
Experimental work
Characterised amylases
Straight-dough procedure
Breadmaking
Firmness/resilience Digital image analysis
Amylose (SEC)
Starch characteristics
Amylopectin (HPAEC) Dextrins (HPAEC)
Laboratory of Food Chemistry and Biochemistry
Experimental work
Bread characteristics: Firmness/resilience
Initial firmness
2000 Firmness (g) 1500 1000 500 0
ro l EU EU EU EU on t 09 7 11 9 17 0 4
Firmness after 6 days of storage
EU
EU
5, 06
68 .
,1
0,
0,
0,
10
BS uA
BS uA
BS uA
Low elastic properties
BS tA
BS tA
Good elastic properties
Laboratory of Food Chemistry and Biochemistry
BS tA
PP A
20
,2
EU
Experimental work
Bread characteristics: digital image analysis
Control BSuA 0.119 EU/ g flour BStA 5.06 EU/ g flour
Mean Cell Area (mm) Control BSuA 0.119 EU/ g flour BStA 5.06 EU/ g flour 0.31 0.41 0.33
Cells/ cm 74.7 56.2 72.9
Cells to total area ratio 27.7 25.9 27.5
Laboratory of Food Chemistry and Biochemistry
Experimental work
Characterised amylases
Straight-dough procedure
Breadmaking
Bread characteristics
Amylose (SEC)
Starch characteristics Amylopectin (HPAEC) Dextrins (HPAEC)
Laboratory of Food Chemistry and Biochemistry
Experimental work
Samples withdrawn
The centre of bread loaf (oven temp. 210C)
100 90 T emp eratu re (C) 80 70 60 50 40 30 20 0 10 20 30 40 50 60 Time (min) 70 80 90 100 110
Samples 00 10 20 30 40 (30C) (35C) (80C) (97C) (98C)
Influence during down Cooling bread making ??
Heating step
d0 after 4h of cooling d6 after 6 days of storage
Laboratory of Food Chemistry and Biochemistry
Experimental work
Amylose MW distribution during bread making and storage
Sepharose Cl-2B (Amylose distribution during bread making)
0.25
0.2
Absorption (A)
0.15
AM AM AM AM AM AM AM AM
Legat Native BSuA 0.119EU BSuA 0.119EU BSuA 0.119EU BSuA 0.119EU BSuA 0.119EU BSuA 0.119EU BSuA 0.119EU
00 10 20' 30' 40' d0 d6
40 30 20 10
98C 97C 80C 35C
0.1
0.05 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125
Elution volume (ml)
Laboratory of Food Chemistry and Biochemistry
Experimental work
Amylose MW distribution during bread making and storage
0.25
Se pharose Cl-2B (Amylose distribution during bre ad making)
AM AM AM AM AM AM AM AM Legat native BStA 5.06EU BStA 5.06EU BStA 5.06EU BStA 5.06EU BStA 5.06EU BStA 5.06EU BStA 5.06EU 00' 10' 20' 30' 40' d0 d6
40 30 20 10
98C 97C 80C 35C
0.2 Absorption (A)
0.15
0.1
0.05 40 45 50 55 60 65 70 75 80 85 90 Elution volum e (m l) 95 100 105 110 115 120 125
Laboratory of Food Chemistry and Biochemistry
Experimental work
Amylases Characterised amylases
Straight-dough procedure
Breadmaking
Bread characteristics
Amylose (SEC) Starch characteristics
Amylopectin (HPAEC)
Dextrins (HPAEC)
Laboratory of Food Chemistry and Biochemistry
Experimental work
Molecular properties of amylopectin: side chain distribution
18 16
Control BSuA 0.097 EU d0 PPA 68.4 EU d0
Relative distribution (%)
14 12 10 8 6 4 2 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Degree of polymerisation (DP)
Laboratory of Food Chemistry and Biochemistry
Experimental work
Molecular properties of amylopectin: side chain distribution
18 16
Control BSuA 0.097 EU d0 PPA 68.4 EU d0 BStA 5.06EU d0 BStA 10.1 EU d0
Relative distribution (%)
14 12 10 8 6 4 2 0 0 5 10 15 20 25 30 35 40
45
50
55
60
Degree of polymerisation (DP)
Laboratory of Food Chemistry and Biochemistry
Experimental work
Molecular properties of AP (general concept)
Endo-action Single attack (low enzyme activity) mechanism
BSuA TAKA
BStA
Maltogenic action Exo/endo mechanism (high enzyme activity)
+ DP2,3,4
Laboratory of Food Chemistry and Biochemistry
Experimental work
Characterised amylases
Straight-dough procedure
Breadmaking
Bread characteristics
Amylose (HPSEC) Starch characteristics Amylopectin (HPAEC)
Dextrins (HPAEC)
Laboratory of Food Chemistry and Biochemistry
Experimental work
Molecular properties of dextrins & residual amylase activity
Amylase concentration (EU/ g flour) Control BSuA 0.097 EU BSuA 0.119 EU PPA 68.4 EU BStA 5.06 EU BStA 10.1 EU Average DP Day 0 2.1 5.1 6.2 5.5 2.8 2.6 Day 6 2.0 5.4 nd 5.3 2.8 2.7 Soluble carbohydrate (%) Day 0 9.7 17.9 21.2 14.2 21.6 22.3 Day 6 10.0 22.9 27.1 14.3 21.9 22.2
Laboratory of Food Chemistry and Biochemistry
Outline
Introduction Starch and gluten in breadmaking Networks in dough and bread Amylases in breadmaking Experimental work Conclusions
Laboratory of Food Chemistry and Biochemistry
Conclusions BSuA
not active on crystallisable outer amylopectin branches loss of starch entanglement network because of endo action and hence antifirming effect continues action after baking crystallization of outer branches and moisture loss from gluten amorphous phase to crystals continues poor resilience of resultant bread
Laboratory of Food Chemistry and Biochemistry
Conclusions BStA (maltogenic amylase)
active on crystallisable outer amylopectin branches loss of amylopectin crystallisation network because of exo action and hence antifirming effect does not continue action after baking prevents moisture loss from gluten excellent resilience of resultant bread
Laboratory of Food Chemistry and Biochemistry
Conclusions BStA action during bread making:
BStA is mainly active after the structure is set (~70-80C)
no influence on loaf volume and crumb structure
A significant portion of starch fraction is degraded in situ formation of high levels of dextrins (DP 2-3)
significant reduction of amylose MW
mainly hydrolysed at high temperature and during cooling
extensive degradation of external amylopectin chains
mainly exo-type degradation
Laboratory of Food Chemistry and Biochemistry
Conclusions
BStA increases initial firmness
increased mobility of the monodisperse amylose population of lower MW may lead to enhanced network formation
BStA is an efficient antistaling enzyme
specific amylopectin degradation inhibits the formation of double helices and hence amylopectin recrystallization formation of high levels of maltose reduces the mobility of amylopectin chains hinders formation of double helices (antiplasticizing effect) reduces crumb RH and water migration to the crust
Laboratory of Food Chemistry and Biochemistry
