Enzymes in Baking Prof. K. Lösche BILB Bremerhaven

Introduction Enzyme Basics Enzymes are proteins that function as biological catalysts Enzymes are specific, both in the substrate they modify and in the reactions they catalyze Enzymes work quickly and under mild conditions Enzymes derived from microbial sources are produced by fermentation Current EU legislation considers Enzymes as processing aids The protein produced by the fermentation is added to the formulation, NOT the microorganism itself

Enzymes for Baking Indigenous Enzymes At harvest: plant Amylase Exogenous Enzymes By microorganisms in situ: lipase, phosphatase, amylases etc. Produced by Yeast Maltogenase, protease, alcohol dehydrogenase, etc. Endogenous Enzymes Commercial enzymes added during process: amylases, pentosanases, lipase, proteases etc.

Fungal α-Amylase Action Effect in bread degrades alpha-1.4 links in amylose and amylopectin produces di- and trisaccharides and dextrins endo-activity Effect in bread larger oven spring, i.e larger volume improves crumb structure and texture brown crust colour (Maillard-Reaktion) Synergistic effects with other enzymes like xylanase, lipase, glucose oxidase and maltogenic α-amylase.

Amyloglucosidase Action Effect in bread Degrades maltose to glucose Exo-activity Effect in bread Browning due to Maillard-reaction Synergistic effects with other enzymes like xylanase, lipase, glucose oxidase and maltogenic α-amylase.

Xylanases / Hemicellulases Effect in bread improved dough stability and mixing tolerance, tolerance to overfermentation increased oven spring larger volume, better crumb structure crumb softness due to larger volume and improved crumb structure crust crispiness Action degrade and modify nonstarch- polysaccharides fraction Change interaction between wheat arabinoxylans and gluten proteins Synergistic effects with other enzymes like α -amylase, lipase, glucose oxidase and maltogenic α -amylase

Glucose Oxidase Action Effect in bread Glucose is oxidized to Gluconolacton and H2O2 H2O2 interlinks the free sulfhydrylgroups (-SH) in the gluten to disulfidbonds (-S-S-) Interlinkage of Arabinoxylan through oxidation of Ferulic acid Effect in bread Reduction of dough stickiness, Gluten strengthening Increases bread volume, especially in weak flours Increases water absorption Synergistic effects with other enzymes like α-amylase, xylanase, lipase and maltogenic α-amylase

Mechanism of Glucose Oxidase H2O2 oxidizes the Gluten network H2O2 oxidizes the sulfhydrylgroup (-SH) of the amino acid Cysteine from wheat gluten, forming Disulfide bonds within the gluten network. This leads to dough strengthening!

Synergies of Glucose Oxidase with Xylanase and Amylase Hemicellulase Amylase Amylase Hemicellulase 100 U Glucose Oxidase per kg of flour Control Procedure: Straight dough pan bread Flour: European Flour all doughs contained 40ppm ascorbic acid and the optimal dosage of Amylase and Xylanase

Replacement of oxidants with Glucose Oxidase in French baquette 25% ascorbic acid 112.5 Units of Glucose Oxidase per kg flour 100% ascorbic acid Procedure: baguette bread Fermentation time: 2hours All bread contain 50ppm Amylase/ Xylanase 100% ascorbic acid = 60ppm ascorbic acid

Replacement of oxidants with Glucose Oxidase in Pan Bread 40ppm ADA 100 Units Glucose Oxidase per kg flour ADA = Azodicarbonamide Procedure: Sponge Dough Flour: US HRW flour Both loaves contain 50ppm ascorbic acid and the optimal level of Fungal Amylase and Xylanase

Partial or full replacement of extra wheat gluten with Glucose Oxidase The loaves shown were fermented for 2.5 hours and contain the optimal dosage of fungal alpha-Amylase and Xylanase along with 30ppm of ascorbic acid. Flour: French Flour 150 U Glucose Oxidase per kg of flour 1% Additional Wheat Gluten

Maltogenic α-Amylase Action Effect in bread degrades alpha-1.4 links in amylose and amylopectin produces mainly maltose, but also mono- and oligosaccharides (DP2-7) active above starch gelatinisation deactivated at the end of baking Effect in bread reduces starch retrogradation, i.e. reduces staling rate unique effect on maintaining crumb elasticity and softness no effect on dough characteristics nor on volume or crumb structure Synergistic effects with other enzymes like α-amylase, xylanase, lipase and glucose oxidase

Dosage Response of Maltogenic Amylase on Crumb Texture of White Pan bread Reference 350 MANU maltogene Amylase 750 MANU maltogene Amylase Process: Sponge & Dough maltogenic alpha-Amylase added on top

Effect of Different Amylases on Crumb Softness and Elasticity in pan bread Monoglycerides = 0.5% Maltogenic alpha-Amylase, 450 U/kg flour Thermostable Bacterial alpha-Amylase, 1.5 U/kg flour Sponge & dough procedure using American flour, differences of loaf volume: max. +/- 3%

Synergistic effect of enzymes on crumb softness and elasticity Fungal alpha-amylase 12.5 FAU/kg flour Xylanase 112.5 FXU/kg flour Maltogene alpha-Amylase (450 und 750 MANU) Straight dough process Controlled volume, 3% soy fat

Influence of maltogenic alpha-Amylase on shelf life characteristics of mixed wheat/rye bread Control 1350 Units maltogenic Amylase 200 Units acidic maltogenic Amylase 350 Units acidic maltogenic Amylase US Rye and wheat flour (50/50) Straight dough process Bread crumb pH = 4.9

Maltogenic Amylase in Unproofed Frozen Dough 7 Weeks Frozen Storage at -18°C DMG = Destilled Monoglycerides MANU = Maltogenic Amylase

Effect of maltogenic alpha-Amylase on Shelf Life of Panettone crumb softness crumb elasticity Panettone cake was produced according to traditional recipe (European flour; sponge dough method) also with high levels of eggs and margerine, no dried fruits included basic recipe contains 15 U fungal alpha- Amylase, 90 U Xylanase per kg flour and 40ppm ascorbic acid

Effect of Different Amylases on Crumb Softness and Elasticity in gluten-free bread Control 600 U maltogenic alpha-Amylase 900 U maltogenic alpha- Amylase 600 U maltogenic alpha- Amylase + 12.5 U fungal alpha-Amylase

The Present Staling Mechanism Model

Action and deactivation temperatures of different Amylases during the baking process A Intact starch granules are inaccessible for enzymes B Stach granules start to swell C Amylose starts to leach into intergranular space D Bulk of starch is gelatinised; optimal temperature for the degradation of amylose and amylopectin

Action Pattern on Amylopectin by different

Lipases with different specificity towards native flour lipids Effect in bread Assures better dough consistency and stability, thereby increasing fermentation tolerance, reduction of dough stickiness Increased volume of the baked product with fine, regular crumb structure. Mainly the Lipase with broad substrate specificity is an alternative to dough strengthening emulsifiers 1,3-specific Lipase hydrolyzes non-polar lipids f.e. 1,3 ester bonds of triglycerides Lipase with broad substrate specificity Modifies triglycerides but also polar lipids like f.e. Lecithin by which they become more polar and improve their surface active function.

Synergy: Combination of Amylase/Xylanase with 1,3-specific Lipase Enzyme: 1,3-specific Lipase combined with Fungal Amylase and Xylanase Improved bread volume and bloom Uniform and regular crumb structure Whiter crumb structure

Combination of Amylase or Xylanase with Lipase in Hard Rolls and Pan Bread

Use of dual specificity Lipase in European white pan bread, straight dough process control Reference 25ppm Lipase 0,3% Datem European flour, reference contains optimal dosage of amylase and xylanase

Use of dual specificity Lipase in Turkishstyle Bread, straight dough process control 0,3% Datem 6ppm Lipase European flour Xylanase / Amylase included

Use of dual specificity Lipase in Pan bread, sponge dough process 0.375% SSL 30ppm Lipase 30ppm Lipase 0.375% SSL Winter patent flour; rotary dough divider Xylanase/Amylase included

Use of dual specificity Lipase in high speed mixing, under vacuum 0.5% SSL 0.4% Datem 65ppm Lipase 0.5% SSL 0.125% SSL + 65 ppm Lipase European flour Xylanase/Amylase included American flour Xylanase/Amylase included

Use of dual specificity Lipase in Wholemeal pan bread, straight dough process 0.12% 0.24% 30 ppm DATEM DATEM Lipase 60% wholemeal flour, 40% white (T550) flour, Xylanase/Amylase included

Use of dual specificity Lipase in Maraquetta control 0,24% Datem 0,2 % SSL 30ppm Lipase European flour contains Xylanase/Amylase/Ascorbic Acid

Use of dual specificity Lipase in French Baguette, straight dough process Control 15ppm Glucose 15ppm Glucose Oxidase Oxidase Procedure: Baquette bread procedure Fermentation time: 2.5h, Flour: French Flour 20ppm ascorbic acid Standardized Amylase & Xylanase

Actions towards native flour lipids

Lipase activity towards triglycerides and lecithin

HPLC profile of lipids from dough made with and without dual specificity Lipase Treatment indicates that DGDG and lecithin peaks decrease, DGMG, lysolecithin and FFA peaks increase Lipids extracted from dough using water saturated butanol at 25°C

Stages of gas cells during baking Gan, et al, 1995