Compositional Changes and Baking Performance of Rye Dough As

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Compositional Changes and Baking Performance of Rye Dough as Affected by Microbial Transglutaminase and Xylanase Isabel Grossmann, Clemens Doering, Mario Jekle, Thomas Becker, and Peter Koehler J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01545 • Publication Date (Web): 28 Jun 2016 Downloaded from http://pubs.acs.org on June 29, 2016

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Journal of Agricultural and Food Chemistry

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Compositional Changes and Baking Performance of Rye Dough as

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Affected by Microbial Transglutaminase and Xylanase

3 Isabel Grossmann1, Clemens Döring2, Mario Jekle2, Thomas Becker2, Peter Koehler1*

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1

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Deutsche Forschungsanstalt für Lebensmittelchemie, Leibniz Institut, Lise-Meitner-Straße 34, 85354 Freising, Germany

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Technische Universität München, Institute of Brewing and Beverage Technology, Research Group Cereal Process Engineering, Weihenstephaner Steig 20, 85354 Freising,

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Germany

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Corresponding author: phone +49 8161 712928; fax +49 8161 712970; e-mail: [email protected] 1 Environment ACS Paragon Plus


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ABSTRACT

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Doughs supplemented with endoxylanase (XYL) and varying amounts of microbial

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transglutaminase (TG) were analyzed by sequential protein extraction, quantitation of protein

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fractions and protein types, and determination of water-extractable arabinoxylans. With

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increasing TG activity, the concentration of prolamins and glutelins decreased and increased,

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respectively, and the prolamin-to-glutelin ratio strongly declined. The overall amount of

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extractable protein decreased with increasing TG level showing that cross-linking by TG

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provided high-molecular-weight protein aggregates. The decrease of the high-molecular-

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weight arabinoxylan fraction and the concurrent increase of the medium-molecular-weight

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fraction confirmed the degradation of arabinoxylans by XYL. However, XYL addition did not

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lead to significant improved cross-linking of rye proteins by TG. Volume and crumb hardness

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measurements of bread showed increased protein connectivity induced by XYL and TG.

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Significant positive effects on the final bread quality were especially obtained by XYL

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addition.

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KEYWORDS:

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Rye proteins

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Rye arabinoxylans

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Transglutaminase

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Endoxylanase

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2 Environment ACS Paragon Plus

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INTRODUCTION

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Several studies have been conducted to analyze the effects of microbial transglutaminase (TG;

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protein-glutamine:amine γ-glutamyl-transferase, EC 2.3.2.13) on the composition of cereal

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proteins and the resulting changes of the baking performance of flours and flour mixtures. TG

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catalyzes an acyl-transfer between Lys and Gln side chains of proteins thereby forming

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covalent cross-links due to ε-(γ-Gln)-Lys bonds (“isopeptide bonds”). Whereas the

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bioavailability of the essential amino acid Lys is not impaired by the isopeptide bond, the

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properties of dough and baked goods e. g. loaf volume are affected. In wheat breadmaking,

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the effects of TG are highly dependent on the applied activity. Whereas in poor flour qualities

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an increase of the loaf volume at low TG concentrations might occur, high TG activities

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usually lead to a drop of the loaf volume.1-3 The effect of TG has not only been studied in

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wheat but also in barley and soybean4, in wheat-millet mixtures5, and in oats6. TG had a

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positive effect on the quality of wheat breads supplemented with barley7 and millet5 but not

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on breads from wheat/soy flour mixtures7.

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In contrast to wheat, rye has not yet been in the focus of interest for the application of TG. By

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now only one study has been conducted on the effects of TG on the rheology and baking

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performance of rye dough. Beck et al. (2011) showed an increase in size of microscopic

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protein particles upon addition of TG resulting in altered dough rheological properties.

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Positive effects on loaf volume and crumb structure were shown after addition of up to 500 U

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TG/kg of flour, and higher levels had detrimental effects.8 The distribution of rye proteins as

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affected by TG has not been studied yet. The consistency of rye dough is characterized by

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viscosity-enhancing polysaccharides, in particular arabinoxylans (AX). Recent work on rye

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model doughs has highlighted the importance of AX and, in particular, the significance of

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high-molecular-weight complexes of AX, protein, and starch for the technological properties

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of the dough and the texture of the bread.9,10 Proteins are thought to contribute only to a minor 3 Environment ACS Paragon Plus


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extent to the dough properties, however, a recent study showed that extensive hydrolysis of

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rye proteins in rye flour by gluten-specific peptidases did not yield dough and bread with the

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same properties as bread made from native rye flour.11

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The gas holding capacity of rye dough may be attributed to the AX that cause high viscosity

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of the aqueous phase.12 Besides the positive effects, AX are also supposed to hinder protein

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(gluten) aggregation by forming slimy layers around the flour and protein particles. Another

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recent study of rye model dough systems (i. e. doughs made only from flour, water and NaCl)

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showed that protein network formation was significantly influenced by rye arabinoxylans13.

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One possible approach for improving protein aggregation of rye dough would be to remove

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AX layers around protein particles by adding endoxylanases (XYL). Depending on their

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specificity, XYL cleave the glycosidic bond of the linear (14)-β-D-xylopyranose backbone

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of AX thereby reducing the molecular weight of water-extractable (WE) and/or water-

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unextractable (WU) AX, thus reducing the viscosity. Previous work showed that the addition

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of XYL increased the dough volume after proofing and left the bread volume unaffected. In

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the XYL-containing bread the cell walls were more fragmented than in the control.14

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Caballero et al. (2007) combined the addition of TG and XYL to wheat dough and showed

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interactive effects of the enzymes on the bread quality.3 The same effect was shown by for

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wheat/millet bread (50/50, w/w).5 These studies showed an increase of the loaf volume by

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25% upon combined addition of TG and XYL. We assume that a partial degradation of AX by

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XYL in rye dough might improve the accessibility of the proteins for TG due to reduced

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viscosity of the aqueous dough phase around the protein particles. While network formation is

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an intrinsic property of wheat gluten, cross-linking of rye proteins could be improved by


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combined addition of XYL and TG and, thus, dough properties and crumb structure could be

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improved. However, this has not been studied before.

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Therefore, the aim of the present study was to investigate the effects of various amounts of

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TG and of combined addition of XYL and TG on the protein distribution of rye dough, on the

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molecular weight distribution of water-extractable AX, and on the texture of the resulting

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bread. This should contribute to improving rye bread quality and open the possibility for

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producing novel types of rye-based baked goods.

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MATERIALS AND METHODS

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Chemicals

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All chemicals were purchased from SIGMA Aldrich (Steinheim, Germany) or VWR

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(Darmstadt, Germany) at analytical or higher grade. Water was deionized by an Arium 611VF

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water purification system (Sartorius, Göttingen, Germany).

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Flour

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Rye flour (Type 1150; mean ash content 1.15% in dry mass) was obtained from a commercial

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mill (Rosenmühle GmbH, Ergolding, Germany). Rye flour with these specifications is

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typically used for rye bread production in Germany. The water content of the flour was 12.7%

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and the crude protein content was 9.1% in dry mass (Dumas method; N 5.7; TruSpec N

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nitrogen analyzer; Leco, Moenchengladbach, Germany).

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Enzymes

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A concentrate of bacterial transglutaminase derived from Streptoverticillium sp. (Veron TG)

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was from AB Enzymes, Darmstadt, Germany. The enzyme activity determined with the

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hydroxamate method15 was 354.3 ± 6.1 U/g powder (1 U = 1 µmol of hydroxamate/min, pH 5 Environment ACS Paragon Plus


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4.2, 30 °C). SDS-PAGE revealed that TG was the only protein in the powder with a relative

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molecular weight (Mr) of about 40,000. A concentrate of bacterial xylanase derived from

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Bacillus subtilis (Veron RL) was from AB Enzymes, Darmstadt, Germany. Endoxylanase

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activity determined with the method of Bailey et al. (1992) was 12.2 ± 0.2 U/mg (pH 4.2,

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30°C).16 Xylosidase and arabinofuranosidase activities were determined by the method of

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Poutanen and Puls (1998) using 4-nitrophenyl-β-D-xylopyranoside and 4-nitrophenyl-α-L-

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arabinofuranoside as substrates, respectively17. Feruloyl esterase activity was determined

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according to the method of Mastihuba et al. (2002) with 4-nitrophenyl ferulate as substrate18.

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4-Nitrophenyl ferulate was synthesized according to Hegde et al. (2009)19 with the

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modification that the synthesis was carried out stirring overnight and that the completion of

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the reaction was assessed only after16 h and not during the process. α-Amylase activity was

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determined with the Ceralpha Kit (Megazyme, Bray Ireland). No xylosidase,

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arabinofuranosidase, feruloyl esterase, and α-amylase activities were detected in the XYL and

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the TG sample. For improving WEAX extraction thermostable α-amylase (Sigma A 4551)

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and amyloglucosidase (Sigma 10115) were used.

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Dough preparation, breadmaking, and crumb analysis

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Doughs containing varying amounts of TG and a combination of TG and XYL were made

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from rye flour (1000 g; 860 g dry mass), NaCl (15 g; Südsalz, Heilbronn, Germany), dry

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instant yeast (10 g; Fermipan Red, Casteggio Lieviti, Casteggio, Italy) and water (760 mL).

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The pH value of the dough was adjusted to 4.2 with lactic acid (8.59 mL; 90% (w/v);

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AppliChem GmbH, Darmstadt, Germany) to simulate sourdough processing. TG (0, 50, 100,

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250, 500, 750, 1000 mg/kg flour; 0 - 354.3 U/kg flour) without or with XYL (25 mg/kg flour;

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610.0 U/kg flour) were suspended in the 760 mL water added to the dough. These dosages of

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TG were chosen based on the results of preliminary tests, in which TG-induced changes of the

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protein were not detectable via the Osborne fractionation/HPLC method (see following 6 Environment ACS Paragon Plus

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section) with TG concentrations below 50 mg/kg of flour. The dosage of XYL was chosen

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according to the recommendation of the supplier. All ingredients were mixed for 4 min at 100

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rpm and 1 min at 200 rpm in a Diosna SP 12 A-4 laboratory spiral kneader (Dierks & Soehne

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GmbH, Osnabrück, Germany). The dough temperature at the end of the mixing process was

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adjusted to 28 ± 2 °C by the water temperature. Immediately after mixing, 25 g of the dough

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was frozen with liquid nitrogen, lyophilized, ground in a laboratory mill (IKA A 10, Staufen,

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Germany), and stored at -18 °C until analysis. The rest of the dough was scaled into 300 g

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pieces, hand-molded, placed into baking-tins (height/width/depth: 11.0 cm/7.0 cm/8.0 cm),

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and proofed (30 °C, 80% rel. humidity, 45 min). Breads were baked in a rack oven (Matador

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Store, Werner & Pfleiderer, Germany; 0.5 L steam injection; baking time 40 min; top heat

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230 °C falling to 200 °C in 40 min; bottom heat 200 °C). After baking, the breads were

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cooled to room temperature (RT; ≈ 20 °C) for 2 h before monitoring weight, volume, and

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crumb firmness (hardness). Volume was determined on a laser device (BVM L370

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Volumeter, TexVol Instruments, Viken, Sweden). Hardness was analyzed on a texture

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analyzer (TVT 300 XP, TexVol Instruments AB, Viken, Sweden) according to AACC

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International method 74 10A. Four slices of single bread from three different sets of baking

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were analyzed and averaged.

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Protein composition of flour and dough

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The content of albumins/globulins (ALGL), prolamins (PROL), and glutelins (GLUT) was

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determined by an extraction/reversed-phase high-performance liquid chromatography (RP-

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HPLC) method as described by Thanhaeuser et al. (2014)20 except that the PROL fraction was

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additionally analyzed in its reduced form (1% (w/v) dithioerythritol, 60 °C, 30 min). Briefly,

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flour or dough powder (100 mg) was extracted stepwise with 2 × 1 mL 0.4 mol/L NaCl /

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0.067 mol/L HKNaPO4, pH 7.6 at RT (ALGL), with 3 × 0.5 mL 60% (v/v) ethanol at RT

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(PROL), and with 2 × 1.0 mL 50% (v/v) 1-propanol / 0.05 mol/L Tris-0.05 mol/L HKNaPO4, 7 Environment ACS Paragon Plus


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pH 7.5 / 1% (w/v) dithioerythritol at 60 °C under argon (GLUT). After centrifugation (4235 ×

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g, 20 min, RT) the corresponding supernatants were combined, diluted to 2.0 mL with the

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respective solvent, and filtered through a 0.45 µm cellulose acetate membrane (FP30,

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WhatmanTM, GE Healthcare, Life Science, Darmstadt. Germany). Half of the amount of the

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PROL supernatant was reduced with dithioerythritol (1% (w/v), 60 °C, 30 min) prior to

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analysis. For RP-HPLC, a Jasco-X-LC with autosampler X-LC 3059 AS, ternary gradient unit

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LG-2080-02, 3-line degasser DG-2080-53, pump PU-2085 Plus, column oven X-LC 3067

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CO, UV detector X-LC 3075 UV and Chrompass software (version 1.8.6.1) were used (Jasco,

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Gross-Umstadt, Germany). The column was an Acclaim Nucleosil 300 C18 with 3 µm particle

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size, 30 nm pore size and the dimensions were 2.1 × 150 mm (Dionex, Idstein, Germany).

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The column was operated at 60°C and 20 µL were injected for ALGL, reduced PROL, and

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GLUT, whereas 10 µL were injected for PROL. Water/trifluoroacetic acid (TFA) (999/1, v/v)

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was solvent A and acetonitrile/TFA (999/1, v/v) was solvent B. The gradient for ALGL

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started at 20% B, was raised to 60% B within 7 min and was kept at 60% B for 10 min. The

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gradient for PROL, reduced PROL, and GLUT, started at 24% B, was raised to 56% B within

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20 min and was kept at 56% B for 10 min. A flow rate of 0.2 mL/min was applied and the

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effluent was monitored at 210 nm. For external calibration, reference gliadin (2.5 mg/mL)

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from the Working Group on Prolamin Analysis and Toxicity (PWG-gliadin)21 was dissolved

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in 60% (v/v) ethanol, 5, 10, 15, and 20 µL were injected and analyzed. The residue of the

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flour/dough extraction was lyophilized and the crude protein content determined as described

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above. All determinations were made in triplicate.

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Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)

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SDS-PAGE was performed on a 10% Bis-Tris Gel (Novex by Life Technologies, Carlsbad,

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CA, USA) as described by Lagrain et al. (2012).22 Dough samples made from rye flour, salt,

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yeast, and water with addition of TG (0 - 1000 mg/kg of flour) and with addition of a 8 Environment ACS Paragon Plus

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combination of XYL (25 mg/kg of flour) and TG (0 - 1000 mg/kg of flour were adjusted to

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pH 4.2 with lactic acid, frozen immediately after mixing (5 min) and lyophilized. Lyophilized

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dough powder (30 mg) was dissolved in sample buffer (1 mL), and 15 µL of sample solution

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was applied onto the SDS gel. All determinations were made in triplicate.

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Isolation of WEAX from rye dough

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WEAX were extracted from dough powder following a method described by Ragaee et al.

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(2001) with some modifications23. Freeze dried dough (1.5 g) was extracted with water (15

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mL) for 90 min at RT under continuous stirring. The slurry was centrifuged (3500 × g, 30

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min, RT) and phosphate buffer (0.3 mL; 0.05 mol/L, pH 6.9) was added to the supernatant. To

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remove remaining starch, the supernatant was incubated with thermostable α-amylase (90 U;

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0.4 mg; Sigma A 4551) at 95 °C for 60 min. The mixture was cooled to RT, centrifuged (3500

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× g, 30 min, RT), acetate buffer (0.3 mL; 1 mol/L, pH 5.0) and amyloglucosidase (6 U; 0.1

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mg; Sigma 10115) were added to the supernatant, and the solution was incubated at 60 °C

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overnight. The mixture was again centrifuged (3500 × g, 30 min, RT) and the supernatant was

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brought to pH 3 with hydrochloric acid (1 mol/L). To remove proteins, the supernatant was

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stirred with Montmorillonite (0.6 g; Sigma 69904) for 30 min at RT. Before centrifugation

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(3500 × g, 30 min, RT) the slurry was adjusted to pH 7 with sodium hydroxide (2 mol/L). The

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supernatant was filtered through a 0.45 µm cellulose acetate membrane (see above) and used

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for gel-permeation HPLC with refractive index detection (GP-HPLC/RI). All determinations

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were made in triplicate.

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GP-HPLC/RI of WEAX solutions

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The Jasco-X-LC system described above with a Jasco RI-2031 Intelligent Refractive Index

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Detector and Agilent Galaxie software (version 1.10.0.5590) was used for GP-HPLC. The

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column was a Yarra 3µ SEC-2000 with dimensions of 300 x 7.8 mm, 3 µm particle size, a 9 Environment ACS Paragon Plus


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separation range of 1000 – 300,000 and a security guard precolumn GFC-2000 with

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dimensions of 30 x 4 mm (Phenomenex Aschaffenburg, Germany). The column was operated

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at RT using 0,1 mol/L NaCl as solvent and the flow rate was 1 mL/min. 100 µL sample were

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injected and the RI of the effluent was monitored. Chromatograms were divided into a high-

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molecular-weight and a medium-molecular-weight fraction and the peak areas under the curve

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were determined. The lower Mr limit of the medium-molecular-weight fraction was defined

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by running maltoheptaose (Mr = 1152). The areas of the control sample without enzyme

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addition were set to 100%, and the areas of the samples were expressed relative to the control.

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All determinations were made in triplicate.

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Statistical analysis

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Data was statistically analyzed with the Software Statgraphics (Statpoint Technologies, Inc.

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Warrenton, Virginia 20186, USA) and are expressed as mean values ± standard deviation of

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triplicates. Differences were considered significant for P < 0.05.

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RESULTS AND DISCUSSION

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Protein composition of rye dough as affected by TG and XYL

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SDS-PAGE

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Firstly, the protein pattern of the doughs was analyzed by SDS-PAGE after reduction. This

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cleaved disulfide bonds but TG-induced isopeptide bonds remained intact. The protein band

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pattern of all doughs looked similar regardless of the TG concentration (not shown).

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Quantitation of protein fractions

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In this study, the classification of rye storage proteins as PROL and GLUT is based on the

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definition according to solubility (“Osborne-fractions”) and not on the biochemical origin.24

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Storage proteins soluble in aqueous ethanol are designated as PROL, whereas GLUT are only 10 Environment ACS Paragon Plus

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soluble in aqueous ethanol after reduction of disulfide bonds. The contents of the protein

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fractions are presented in Table 1. In doughs supplemented with TG, the PROL content

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decreased from 3.3 to 2.0 g/100 g flour with increasing dosage of TG. The decrease was

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significant (P < 0.05) for a TG level of 100 mg/kg flour or beyond. In TG + XYL doughs the

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effect was somewhat stronger with a significant decrease from 50 mg TG/kg flour. This can

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be interpreted by TG-induced cross-linking of PROL and formation of protein aggregates that

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were no longer soluble in 60% ethanol thus causing a decrease of the PROL content with

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increasing TG concentration. The more pronounced decrease of PROL in the TG + XYL

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doughs in comparison to the TG doughs may have resulted from a better accessibility of the

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proteins for TG-induced transamidation due to partial degradation of AX by XYL. This can

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be assumed from microscopic studies showing that rye flour particles in water are surrounded

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by viscous layers that remained unstained.25 Studies on wheat dough2, wheat flour / water

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suspensions26, and TG-treated wheat kernels27 also showed a decrease in the PROL (gliadin)

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content upon addition of TG, whereas Gerrard et al. (2001)28 found an increase of the gliadin

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content in TG-supplemented wheat flour dough. Steffolani et al. (2010) reported an increase

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of the gliadin content in XYL-containing wheat doughs compared to control dough without

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enzyme addition, which they attributed to enhanced protein extraction due to a reduced size of

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the AX.2 This result could not be confirmed in the present study because both rye doughs (0

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and 25 mg XYL/kg flour) had the same PROL content. However, these authors used 70 %

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isopropanol for PROL extraction, which might have dissolved a small portion of low-

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molecular-weight GLUT into the PROL fraction.

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The content of GLUT in TG-containing doughs increased from 1.1 to 1.5 g/100 g flour upon

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addition of up to 1000 mg TG/kg flour (Table 1). The increase was only significant for

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doughs with 750 and 1000 mg TG/kg flour. In TG + XYL doughs the GLUT content

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increased from 0.8 g/100 g in flour without TG to 1.2 g/100 g in flour with 1000 mg TG/kg 11 Environment ACS Paragon Plus


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flour. The increase was significant for the TG levels beyond 250 mg/kg flour. The GLUT

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content of doughs with TG + XYL was significantly lower than of TG doughs for TG levels

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of 50 to 1000 mg/kg flour, but the relative increase (∆ = 0.4 mg/100 g flour) was the same.

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The action of XYL on the AX might have caused a better accessibility of the proteins for TG

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resulting in large protein aggregates that were not extractable under reducing conditions. In

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wheat flour / water suspensions, the GLUT content only increased at low TG concentration

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and decreased when high TG levels were added.26 The increase of the GLUT compensated for

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cross-linked PROL that were no longer soluble in 60% ethanol and ended up in the GLUT

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fraction. In contrast to these results, several groups reported a decrease of the GLUT content

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of wheat dough and flour suspensions with TG.2,27,28 This effect can be explained by the

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formation of cross-linked aggregates of a size that were not soluble in any of the employed

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extraction solutions, thus leading to a decrease in the GLUT content, which was not

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compensated by cross-linked PROL. Upon addition of XYL, a decrease of the GLUT content

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in wheat dough was attributed to interactions between soluble AX and proteins through ferulic

280

acid cross-links.2 However, no significant difference between the GLUT content of doughs

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with and without XYL was found in the present study.

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The decrease of the PROL and the increase of the GLUT content were reflected in the

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PROL/GLUT ratio (Table 1), which decreased with increasing TG concentration from 3.0 to

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1.4 in TG doughs and from 3.9 to 1.5 in TG + XYL doughs. In wheat, the gliadin/glutenin

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ratio is negatively correlated with dough extensibility.29 Thus, a low gliadin/glutenin ratio has

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been considered favorable for the baking properties of wheat and einkorn flours.30 If this

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concept would be applied to rye, TG addition would decrease the PROL/GLUT ratio and,

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therefore, improve the baking performance.

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The unextractable proteins accounted for about one third of the total protein content of the

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doughs (Table 1). This showed that the solvents used for the Osborne extraction were not

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capable of dissolving as much of the rye proteins as it has been described for wheat doughs.26

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Upon addition of TG to rye dough, the content of unextractable protein increased from 3.1

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g/100 g flour in flour without TG to 3.6 g/100 g in flour with 1000 mg TG/kg, but the

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increase was only significant for 1000 mg TG/kg flour. Comparable results were obtained for

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TG + XYL doughs. As described above, the increase of the content of unextractable protein

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was most likely due to the formation of cross-linked proteins with very high molecular weight

299 300

Addition of TG or TG + XYL only caused a small decrease of the content of the ALGL

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fraction (Table 1). These small changes indicated that this protein fraction did not play an

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important role in protein cross-linking.31 In contrast to these findings, Steffolani et al. (2010)

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found a decrease of the ALGL content in wheat dough with increasing TG levels, which can

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be explained by a slightly different extraction protocol.2

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Extractable protein from the modified Osborne fractionation and residual protein were

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summed up and compared to the crude protein content of the dough powder (Table 1). The

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sum of the proteins was between 90 and 101% of the crude protein content. This showed that

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quantitative monitoring of the protein distribution of rye dough was well possible by a

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combination of Osborne fractionation and Dumas analysis of the residue.

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Quantitation of protein types

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The detailed evaluation of the RP-chromatograms enabled quantitation of single protein types

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in the reduced PROL and the GLUT fractions. The PROL fraction was reduced before RP-

315

HPLC to be able to distinguish γ-75k- and γ-40k-secalins as outlined by Gellrich et al.

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(2003).32 Protein types in the reduced PROL fraction were HMW-secalins + ω-secalins, γ13 Environment ACS Paragon Plus


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75k-secalins, and γ-40k-secalins; in the GLUT fraction HMW-secalins, γ-75k-secalins, and γ-

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40k-secalins were found. Typical chromatograms of the fractions obtained from doughs

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treated with 0 and 500 mg TG/kg flour shown in Figure 1 were already different upon visual

320

comparison. The corresponding quantitative data was normalized to the control dough without

321

enzyme (each protein type set as 100%) and is shown in Table 2. Only a slight decrease of 5

322

to 15% was detected for HMW+ω-secalins and γ-75k secalins in the reduced PROL fraction.

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Instead, the relative amount of γ-40k secalins increased with increasing amount of TG up to

324

29.1% (1000 mg of TG) and 14.7% (1000 mg of TG + XYL), respectively.

325 326

In contrast to the PROL fraction, a strong decrease of the concentration of the HMW-secalins

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in the GLUT fraction was found. With 70.7% (1000 mg of TG) and 71.6% (1000 mg of TG +

328

XYL) of the original value (0 mg of TG, 100%), respectively, the decrease was much more

329

pronounced than for the PROL fraction. γ-40k secalins decreased up to 12% with increasing

330

amount of TG. Only the γ-75k secalins increased by up to 22.7% (1000 mg of TG) and 25.9%

331

(1000 mg of TG + XYL), respectively, with increasing amount of TG.

332 333

In wheat flour and dough, a decrease of the content of HMW-glutenin subunits, which are

334

homologous to HMW-secalins of rye, and of ω-gliadins, homologous to ω-secalins of rye,

335

was reported upon addition of TG, whereas LMW-glutenin subunits, corresponding to γ-75k

336

secalins in rye, increased. In wheat flour and dough, the content of α/β- and γ-gliadins

337

decreased but in rye dough the concentration of the homologous γ-40k-secalins

338

increased.27,31,33 In wheat, the different reactivity of the protein subunits in the presence of TG

339

is not due to the Gln content (Gln is abundant in all gluten proteins with 34 - 56 mol%) but

340

due to the varying Lys content (0.2 - 1.1 mol%).26 In rye, the Lys content of protein subtypes


Page 14 of 31

Page 15 of 31


341

is 0.4 - 2.0 mol%.33 HMW-secalins, with 2.0 mol% having the highest Lys content of the

342

protein subunits, showed the strongest decrease with increasing TG concentration.

343 344

WEAX content of rye dough as affected by TG and XYL

345

WEAX were isolated from lyophilized rye dough according to a standard protocol for WEAX

346

isolation including elimination of proteins from the WEAX extract by Montmorillonite. The

347

yield was 4.3%.34 Although Buksa et al (2016) showed that rye proteins might be associated

348

with AX and postulated that ferulic acid could play a significant role in this interaction,10 we

349

are in doubt about that. Previous work has shown the presence of covalent AX-protein cross-

350

links, however, the content was so low that a functional effect is questionable.35 The isolated

351

WEAX were separated and analyzed by GP-HPLC/RI. According to the elution pattern, two

352

relevant peaks were assessed, the first one containing high-molecular-weight WEAX and the

353

second one medium-molecular-weight WEAX (Figure 2). Maltoheptaose was run as a marker

354

for low-molecular-weight oligosaccharides that were not considered in this study also non-

355

carbohydrate materials such as buffer salts were eluted at higher retention times. The relevant

356

WEAX peaks were integrated and the areas normalized to the control dough without TG

357

(= 100%). The relative peak areas are summarized in Table 3. In doughs with varying

358

amounts of TG but no addition of XYL the areas for the high-molecular-weight and the

359

medium-molecular weight peak were not significantly different. This was expected as no

360

XYL had been added. The XYL + TG doughs partially showed an increase of both the area of

361

the high-molecular-weight and medium-molecular-weight WEAX but no dependence on the

362

dosage of TG was established. Concerning the addition of XYL, preferably high-molecular-

363

weight WEAX were degraded by XYL. The decrease of the area was less pronounced with

364

increasing amounts of TG added. With decreasing area of the peak of the high-molecular-

365

weight WEAX a concurrent increase of the area of the medium-molecular-weight WEAX was

366

obtained when XYL was added. This shift in the distribution of molecular weight fractions of 15 Environment ACS Paragon Plus


367

WEAX confirmed the effect of the XYL. The experimental setup did not allow the conclusion

368

whether the increase of the medium-molecular-weight WEAX fraction was caused by

369

degradation of high-molecular-weight WEAX or by degradation of WUAX, as the XYL could

370

have acted on both fractions

371 372

Quality characteristics of rye bread

373

The influence of altered protein cross-linking in rye dough induced by TG or TG + XYL in

374

baked breads were investigated for volume and crumb firmness. The specific volumes of the

375

control rye bread (no added enzymes), rye bread containing XYL as well as rye bread treated

376

with different levels of TG and TG + XYL are illustrated in Table 4. The addition of XYL (25

377

mg/kg flour) increased the specific volume significantly by 6.6% (from 1.49 ± 0.02 to 1.59 ±

378

0.03 mL/g) compared to the control bread. The increase in the specific volume is in

379

accordance with the findings of Autio et al.,14 in which the effect of different XYL

380

concentrations on baking characteristics of rye dough were analyzed. The positive effect of

381

XYL on the bread volume could be related to an increase of the content of WEAX that

382

stabilized the liquid films around gas cells and, consequently, the foam structure by increasing

383

the viscosity of the dough aqueous phase as described by Courtin and Delcour36, Autio et

384

al.14, and Kühn and Grosch37 for wheat and rye bread. A further explanation could be the

385

release of bound water due to the hydrolysis of AX presence of XYL. With more free water

386

the dough becomes softer and up to a specific optimum the bread volume increases. This

387

effect is based on the plasticizing effect of the water molecules and therefore reflecting

388

increased dough extensibility as described by Jekle and Becker.38 The effect of additional free

389

water on bread volume and crumb hardness caused by AX hydrolysis can be countered for by

390

water adjustment.

391

For a comprehensive comparison of the primary effects of enzymatic treatment on the dough

392

and bread functionality an adjustment of the water content was consciously avoided. In case 16 Environment ACS Paragon Plus

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393

of TG addition, no significant influence on the specific volume was found for concentrations

394

up to 500 mg/kg flour. These results are in accordance with the results of Beck et al. (2011),

395

in which the effect of different TG concentrations on final rye bread volume (without XYL

396

addition) was reported.8 With increasing TG concentration (750 - 1000g/kg flour), a

397

significant decrease in specific volume was shown. The decrease in specific volume can be

398

explained by an excessive cross-linking in proteins, which prevent gas bubbles from

399

expanding.8 It is worth to mention that a decrease in specific volume could be also related to a

400

decrease in the free amount of water as deliberated later. Nevertheless, the combination of

401

both enzymes (XYL + TG) led to a significant increase of the specific volume up to a TG

402

concentration of 1000 mg/kg flour compared to the control bread (Table 4). The combination

403

of XYL and TG showed a synergistic effect on the bread volume in particular at low TG

404

concentrations. It can be postulated that the addition of XYL led to a modification of the AX

405

structure, whereby improved protein connectivity induced by TG can be assumed on the basis

406

of protein modifications described earlier. Improved protein connectivity improved the

407

stabilization of the dough structure and prevented coalescence of gas cells, thus leading to an

408

increased dough and bread volume. The detrimental effect on the specific volume observed at

409

higher TG levels could be related to excessively cross-linked proteins, which prevented gas

410

bubbles from expanding.8 It could also be related to a lack of water due to the newly formed

411

cross-links between the proteins. It has been described that highly cross-linked proteins need

412

more water for proper dough development than weakly cross-linked proteins.39

413 414

Additionally, the crumb firmness of rye bread was determined (Table 4) by texture profile

415

analysis. For the breads treated with TG, no significant differences in crumb firmness up to

416

500 mg/kg flour were shown. TG concentrations beyond 500 mg/kg flour increased the crumb

417

firmness significantly. The combination of XYL and TG led to weaker crumb firmness for all

418

breads compared to the breads without XYL addition. These results have to be considered in 17 Environment ACS Paragon Plus


419

relation to the bread volume, because increased bread volume exhibits lower crumb hardness

420

due to the increased porosity. Furthermore, the bread volume depends on the amount of free

421

water. Therefore, the crumb hardness is indirectly affected by the amount of free water. In

422

summary, the addition of both enzymes (optimum: XYL + TG 100 mg/kg flour) caused a

423

significant decrease of the crumb firmness. However, increasing TG concentrations increased

424

the crumb hardness, which was most likely related to the formation of additional isopeptide

425

cross-links between rye proteins.

426 427

Significance of TG-/XYL-addition in rye breadmaking

428

The addition of TG to rye dough has positive technological effects. Modified Osborne

429

fractionation provides an explanation regarding proteins because it clearly shows a shift of

430

soluble protein fractions (ALGL, PROL) towards insoluble ones (GLUT, residual protein) due

431

to cross-linking. As a consequence, the PROL/GLUT ratio decreases, which may be

432

associated with a contribution to higher dough strength.

433 434

The addition of XYL does not affect the protein distribution but the effect of XYL on the

435

molecular weight distribution of WEAX appears to cause a beneficial effect on the baking

436

performance.

437 438

If XYL and TG are used in combination, the bread volume strongly increases suggesting a

439

synergistic effect of XYL and TG. A possible reason could be increased strength of the

440

protein network induced by TG combined with the facilitated formation of AX-protein

441

complexes, because of increased mobility of WEAX due to (partial) depolymerization

442

induced by XYL. AX-protein complexes have recently been postulated to positively affect the

443

baking performance of rye dough.10

444 18 Environment ACS Paragon Plus

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445

Thus, future work will be focused on using diverse XYL samples with different substrate

446

selectivities to achieve optimal AX degradation for subsequent protein cross-linking induced

447

by TG and complex formation. Finally, standardization of the water addition and mixing time

448

for enzyme-treated dough will be considered, since the modification of AX by XYL leads to a

449

shift of the water absorption and the viscoelastic behavior of rye dough.

450 451

NOTES

452

The authors declare no competing financial interest.

453 454

ACKNOWLEDGMENT

455

This research project was supported by the German Ministry of Economics and Technology

456

(via AIF) and the FEI (Forschungskreis der Ernährungsindustrie e. V., Bonn). Project

457

17315 N.

458 459



460

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29. Wieser, H.; Kieffer, R. Correlations of the amount of gluten protein types to the

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31. Medina-Rodríguez, C.L.; Torres, P.; Martínez-Bustos, F.; Ramirez-Wong, B.; Paredes-

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López, O.; Castaño-Tostado, E. Effect of microbial transglutaminase on dough proteins

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of the storage protein (secalin) types in rye flour. Cereal Chem. 2003, 80, 102-109.

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33. Bushuk, W. ,Ed. Rye: Production, Chemistry and Technology, second ed., AACC, St. Paul, 2001. 34. Großmann, I.; Koehler, P. Fractionation-reconstitution studies to determine the functional properties of rye flour constituents. J. Cereal Sci. 2016, 70, 1-8.

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35. Piber, M.; Koehler, P. Identification of dehydro‐ferulic acid‐tyrosine in rye and wheat:

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Evidence for a covalent cross‐link between arabinoxylans and proteins. J. Agric. Food

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36. Courtin, C.M.; Delcour, J.A. Arabinoxylans and endoxylanases in wheat flour breadmaking. J. Cereal Sci. 2002, 35, 225-243. 37. Kühn, M.C.; Grosch, W. Influence of the enzymatic modification of the nonstarchy

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polysaccharide fractions on the baking properties of reconstituted rye flour. J. Food Sci.

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1988, 53, 889-895.

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38. Jekle, M.; Becker, T. Dough microstructure: Novel analysis by quantification using confocal laser scanning microscopy. Food Res. Int. 2011, 44, 984-991. 39. Gerrard, J.A.; Fayle, S.E.; Wilson, A.J.; Newberry, M.P.; Ross, M.; Kavale, S. Dough

562

properties and crumb strength of white pan bread as affected by microbial

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transglutaminase. J. Food Sci. 1998, 63, 472-475.

564 565



566

FIGURE CAPTIONS

567 568

Figure 1. RP-HPLC of reduced prolamin (a, b) and glutelin (c, d) fractions from rye dough

569

supplemented with transglutaminase (TG). Concentration of TG (mg/kg of flour): 0 (a, c) and

570

500 (b, d). Protein types are marked: ω+HMW, ω-secalins containing minor amounts of

571

HMW-secalins; γ-75k, γ-75k-secalins; γ-40k, γ-40k-secalins; HMW, HMW-secalins.

572 573

Figure 2. GP-HPLC of water-extractable arabinoxylans from rye dough supplemented with

574

xylanase (XYL) and transglutaminase (TG). (a) - - - control without enzymes; ─── XYL, 25

575

mg/kg flour; TG, 0 mg/kg flour. (b) - - - - XYL, 0 mg/kg flour; TG, 1000 mg/kg flour;

576

─── XYL, 25 mg/kg of flour; TG, 1000 mg/kg of flour. Fractions of high- (HMW-AX) and

577

medium-molecular-weight water-extractable arabinoxylans (MMW-AX) are indicated.

578


Page 24 of 31

Page 25 of 31


TABLES Table 1. Crude protein content, content of the protein fractions albumins/globulins (ALGL), prolamins (PROL), glutelins (GLUT), residual proteins, the sum of extractable and residual proteins (Σ), and prolamin/glutelin ratio (PROL/GLUT) of rye dough as affected by the addition of transglutaminase (TG) and xylanase (XYL) Concentration (g/100 g flour) of

Enzyme (mg/kg flour)

PROL/GLUT

XYL

ALGLa

PROLa

GLUTa

Residuea

Σ

Crude protein

0

0

2.0 ± 0.0 A

3.3 ± 0.0 A

1.1 ± 0.2 A

3.1 ± 0.0 A

9.4

9.4 ± 0.0 A

3.1

50

0

1.9 ± 0.0 A

3.2 ± 0.1 A

1.0 ± 0.0 A

3.0 ± 0.1 A

9.2

9.2 ± 0.2 A

3.2

100

0

1.9 ± 0.0 B

3.2 ± 0.0 B

1.0 ± 0.1 A

3.1 ± 0.3 A

9.2

9.5 ± 0.1 A

3.1

250

0

1.9 ± 0.0 B

3.0 ± 0.1 B

1.1 ± 0.0 A

3.3 ± 0.1 A

9.3

9.2 ± 0.1 B

2.7

0

1.8 ± 0.0

B

2.5 ± 0.1

B

1.3 ± 0.0

A

3.3 ± 0.1

A

9.0

9.2 ± 0.2

A

1.9

1.8 ± 0.0

B

2.3 ± 0.0

B

1.4 ± 0.0

A

3.2 ± 0.1

A

9.2 ± 0.3

A

1.6

1.8 ± 0.0

B

2.0 ± 0.1

B

1.5 ± 0.1

B

3.6 ± 0.0

B

8.9

9.2 ± 0.2

A

1.4

TG

500 750

0

8.7

1000

0

0

25

2.0 ± 0.0 A

3.3 ± 0.1 A

0.8 ± 0.0 A

3.0 ± 0.2 A

9.1

9.1 ± 0.0 A

3.9

50

25

2.0 ± 0.0 A

3.0 ± 0.1 B

0.8 ± 0.0 A

3.0 ± 0.2 A

8.8

9.1 ± 0.0 A

3.8

100

25

2.0 ± 0.0 A

2.9 ± 0.0 B

0.9 ± 0.0 A

3.0 ± 0.1 A

8.8

9.2 ± 0.1 A

3.4

250

25

1.9 ± 0.0 A

2.8 ± 0.0 B

1.0 ± 0.0 B

3.1 ± 0.1 A

8.8

9.2 ± 0.1 A

2.8

25

2.0 ± 0.0

A

2.4 ± 0.0

B

1.0 ± 0.0

B

3.3 ± 0.1

A

8.7

9.3 ± 0.1

A

2.4

1.9 ± 0.0

B

2.1 ± 0.1

B

1.1 ± 0.1

B

3.4 ± 0.1

B

9.2 ± 0.1

A

2.0

1.9 ± 0.0

B

1.8 ± 0.0

B

1.2 ± 0.0

B

3.6 ± 0.1

B

9.3 ± 0.2

A

1.5

500 750 1000

25 25

a

8.6 8.4

mean value of three determinations ± standard deviation. Values associated with different capital letters are significantly different to the control doughs within the same column (one-way ANOVA, Tukey test, P < 0.05)

25 ACS Paragon Plus Environment


Page 26 of 31

Table 2. Relative content of prolamin (PROL) and glutelin (GLUT) types of rye dough as affected by the addition of transglutaminase (TG) and xylanase (XYL). The PROL fraction was reduced prior to RP-HPLC separation Enzyme (mg/kg flour) TG

XYL

PROL type (% of control)a

GLUT type (% of control)a

ω+HMWb

γ-75kb

HMWb

=100.0 ± 0.6

=100.0 ± 0.7

=100.0 ± 1.3

=100.0 ± 6.4

=100.0 ± 4.5

=100.0 ± 7.1

A

A

A

A

A

A

γ- 40kb

γ-75kb

γ-40kb

0

0

50

0

98.8 ± 0.4 A

99.9 ± 0.2 A

100.9 ± 0.3 A

108.1 ± 1.9 A

98.1 ± 0.6 A

99.9 ± 3.1 A

100

0

97.8 ± 1.3 B

99.2 ± 0.5 B

101.4 ± 1.5 B

105.4 ± 3.6 A

98.1± 1.2 A

102.5 ± 4.2 A

250

0

94.2 ± 0.8 B

98.5 ± 0.2 B

105.6 ± 1.1 B

102.3 ± 0.9 A

104.2 ± 1.2 A

94.9 ± 0.3 A

500

0

88.2 ± 0.6 B

95.1 ± 0.6 B

114.9 ± 1.1 B

88.1 ± 2.5 A

114.4 ± 1.2 A

88.2 ± 1.3 A

750

0

86.3 ± 0.7 B

91.4 ± 0.4 B

119.9 ± 1.2 B

79.8 ± 1.4 A

118.4 ± 0.8 A

87.6± 1.3 A

1000

0

85.3 ± 0.0 B

85.2 ± 0.5 B

129.1 ± 1.1 B

70.7 ± 0.6 A

122.7 ± 0.1 B

87.8 ± 0.2 A

0

25

=102.9 ± 0.8 A

=100.0 ± 2.5 A

=100.0 ± 3.6 B

=109.6 ± 0.9 A

=100.0 ± 0.6 A

=100.0 ± 0.5 A

50

25

98.8 ± 2.3 A

102.2 ± 3.4 A

98.4 ± 10.4 B

104.3 ± 1.7 A

97.3 ± 1.0 A

105.1 ± 2.9 A

100

25

97.6 ± 0.6 A

104.6 ± 0.1 A

93.8 ± 0.7 B

103.6 ± 1.1 A

101.1 ± 0.3 A

101.0 ± 3.0 A

250

25

93.7 ± 0.0 A

103.9 ± 0.3 A

97.2 ± 0.3 B

97.7 ± 0.7 B

107.5 ± 0.7 B

96.2 ± 0.4 A

500

25

92.6 ±3.5 A

100.8 ± 1.8 A

100.1 ± 2.3 B

89.5 ± 1.4 A

114.6 ± 0.9 B

90.6 ± 2.9 A

750

25

88.0 ± 0.6 A

100.0 ± 0.2 A

106.6 ± 1.3 B

81.3 ± 1.8 A

120.4 ± 0.7 B

88.6 ± 4.5 A

1000

25

85.6 ± 0.4 B

94.3 ± 1.0 A

114.7 ± 1.0 B

71.6 ± 0.9 B

125.9 ± 0.5 B

87.9 ± 1.9 B

a

mean value of three determinations ± standard deviation. Values associated with different capital letters are significantly different to the control doughs within the same column (one-way ANOVA, Tukey test, P < 0.05) b ω+ΗΜW, ω-secalins containing minor amounts of HMW-secalins; γ-75k, γ-75k-secalins; γ-40k, γ-40k-secalins; HMW, HMW-secalins


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Table 3. Relative area under the peak of the high- (HMW-AX) and medium-molecular weight fraction of water-extractable arabinoxylans (MMW-AX) of rye dough as affected by the addition of transglutaminase (TG) and xylanase (XYL) Enzyme (mg/kg flour)

Relative area (%) of

Area remaining after XYL addition (%)

HMW-AXa

MMW-AXa

HMW-AXa

0

100.0 ± 11.7 A

100.0 ± 27.8 A

50

0

108.7 ± 4.9

A

A

none added none added

100

0

111.6 ± 8.7 A

75.0 ± 6.9 A

none added

none added

250

0

100.4 ± 7.4 A

88.4 ± 7.9 A

none added

none added

500

0

101.8 ± 4.9 A

75.7 ± 1.1 A

none added

none added

750

0

97.8 ± 9.2 A

63.9 ± 4.0 A

none added

none added

1000

0

94.2 ± 11.1 A

65.0 ± 20.7 A

none added

none added

0

25

100.0 ± 25.4 A

100.0 ± 4.6 A

36.8 ± 9.4 A

103.1 ± 4.7 A

50

25

105.9 ± 12.5 A

154.0 ± 8.0 A

35.9 ± 4.2 A

191.9 ± 10.0 B

100

25

120.6 ± 13.4 A

104.7 ± 6.7 A

39.8 ± 4.4 A

144.0 ± 9.2 B

250

25

154.1 ± 5.4 A

99.4 ± 3.6 A

54.6 ± 1.9 B

116.0 ± 4.2 B

500

25

107.0 ± 7.4 A

104.3 ± 12.7 A

38.8 ± 2.7 A

142.0 ± 17.3 B

750

25

216.3 ± 62.6 A

139.1 ± 10.5 A

81.4 ± 23.6 B

224.3 ± 16.9 B

1000

25

180.9 ± 7.4 A

102.7 ± 11.3 A

73.9 ± 5.2 B

158.6 ± 15.9 B

TG

XYL 0

82.7 ± 8.2

a

MMW-AXa none added none added

mean value of three determinations ± standard deviation. Values associated with different capital letters are significantly different to the control doughs within the same column (one-way ANOVA, Tukey test, P < 0.05)



Table 4. Specific volume and crumb firmness of rye bread as affected by transglutaminase (TG; different concentrations), xylanase (XYL; 25 mg/kg flour), and TG (different concentrations) in combination with XYL (25 mg/kg flour). Enzyme (mg/kg flour) TG

XYL

Bread propertya Specific volume (mL/g)

Crumb firmness (N)

A

18.47 ± 1.37 A

0

0

1.49 ± 0.02

50

0

1.47 ± 0.02 A

17.35 ± 1.68 A

100

0

1.47 ± 0.02 A

18.31 ± 1.08 A

250

0

1.48 ± 0.01 A

17.68 ± 0.78 A

500

0

1.47 ± 0.01 A

18.69 ± 0.72 A

750

0

1.45 ± 0.02 A

20.86 ± 1.57 B

1000

0

1.44 ± 0.02 B

22.51 ± 1.23 B

0

25

1.59 ± 0.03 B

14.06 ± 1.06 B

50

25

1.74 ± 0.03 B

9.92 ± 0.70 B

100

25

1.77 ± 0.01 B

9.62 ± 0.88 B

250

25

1.73 ± 0.06 B

12.06 ± 1.92 B

500

25

1.75 ± 0.02 B

10.33 ± 2.30 B

750

25

1.61 ± 0.02 B

12.89 ± 1.57 B

1000

25

1.60 ± 0.02 B

14.41 ± 0.75 B

a

mean value of four slices of bread from three different sets of baking ± standard deviation. Values associated with different capital letters are significantly different to the control bread within the same column (one-way ANOVA, Tukey test, P < 0.05)


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UV-Absorbance at 210 nm

800 (a)

γ-75k

600 400

300

γ-40k

ω+HMW

γ-75k

200

200

100

0

0

800 (b)

(c)

HMW

γ-40k

γ-75k

300 (d)

600 γ-75k

400 200

ω+HMW

200 γ-40k

100

HMW

γ-40k

0

0 10

15 20 Time (min)

25

10

Figure 1


15 20 Time (min)

25

HMWAX

MMWAX

RI-Signal (mV)

RI-Signal (mV)


Time (min)

Figure 2 30 Environment ACS Paragon Plus

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GRAPHIC FOR TABLE OF CONTENTS


Compositional Changes and Baking Performance of Rye Dough As

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