Insight into the distribution and variability of endoxylanases in wheat and their functionality during bread making

Variations in wheat quality are of major concern for the wheat processing industry, as they require both small continuous and large year-to-year adaptations of processing conditions and alter end-product quality. Such quality variations are not only caused by genetic differences between wheat varieties, but also often depend strongly on climatological and agronomic circumstances. Because of the great economical importance, considerable effort has already been made to elucidate the effects of genetic, climatological and agronomic conditions on gluten protein contents and quality and on α-amylase activity levels. The impact of varying levels of wheat associated endoxylanases as possible contributors to differences in wheat functionality is not known. This is rather surprising, as their potential importance can be easily demonstrated. On the one hand, microbial endoxylanases are frequently added in diverse wheat processing applications, albeit often on an empirical basis. On the other hand, recent research at the Laboratory of Food Chemistry and Biochemistry (K.U.Leuven) revealed that flour associated endoxylanase levels can vary more than twentyfold and that they are able to impact product quality. Furthermore, some preliminary evidence was provided for the presence of microbial endoxylanases in wheat (flour), and their levels are most likely strongly underestimated due to the large concentrations of endoxylanase inhibitors present in wheat (flour), interfering with activity measurements. The endoxylanase activity levels measured so far in wheat (flour) should therefore be considered as apparent activity levels. To fill this gap and to contribute to a better understanding of the varying functionality of wheat flour in biotechnological applications, the distribution and variability of wheat associated endoxylanases were investigated, as well as their functionality during the bread making process. As all methods commonly used to measure endoxylanase activity levels in wheat were expected to underestimate the actual levels, a method was developed to quantify all wheat associated endoxylanases in an adequate way. The method is based on a physical separation of microbial endoxylanases from endogenous endoxylanases and endoxylanase inhibitors by washing of wheat kernels, followed by classic endoxylanase activity measurements. The total wheat associated endoxylanase activity levels can be calculated as the sum of the endoxylanase activity level in the washing liquid, corresponding to the microbial endoxylanase population, and that in the washed kernels, corresponding to the endogenous endoxylanase population. The developed method does not only allow estimation of the total endoxylanase activity levels, but also assessment of the relative contributions of microbial and endogenous endoxylanases to this total endoxylanase activity level. The results indicated that a large discrepancy exists between the commonly measured apparent endoxylanase activity levels and the total wheat associated endoxylanase activity levels due to the presence of a vast majority of inhibition sensitive microbial endoxylanases in wheat. With the developed method, the variability in both microbial and endogenous endoxylanase activity levels in wheat as a function of genetic, climatological and agronomic factors was analyzed. Genotype was the most important determinant for the variability in microbial endoxylanase activity levels, while the interaction between harvest year and genotype mainly determined the variations in endogenous endoxylanase activity levels. The impact of harvest year on the observed variability is, most likely, due to differences in the climatological conditions preceding the time of harvest. Furthermore, it was clear that agronomic factors, such as fungicide treatment and nitrogen fertilization, only had small effects on the levels of wheat associated endoxylanase activities. The variability in the arabinoxylan (AX) and endoxylanase inhibitor contents was also studied and was found to be mainly caused by genetic differences. Selection of appropriate wheat varieties can hence be an effective tool in reducing the problems associated with such variability. The distribution of endoxylanases over different wheat milling streams was investigated, together with the distribution of AX and endoxylanase inhibitors, and more standard parameters, such as ash, starch and protein contents and α-amylase activity levels. Bran fractions were significantly richer in endoxylanase activity levels and in AX and endoxylanase inhibitor contents than germ and, even more so, than flour fractions. Endoxylanase activity levels varied enormously between the different flour fractions and were strongly positively correlated with ash and negatively with starch content. This indicates that the endoxylanase activity level in wheat flour largely depends on the level of bran contamination and that microbial and/or aleurone specific endoxylanases, located on or in the outer wheat kernel layers, can end up as a contamination in wheat flour. As endoxylanase activity levels vary widely in wheat and wheat flour, the impact of varying levels of endoxylanase activity in wheat flour on the AX population in dough after mixing and resting was studied. AX solubilization during the mixing phase of bread making resulted from physical phenomena. Enzymic solubilization hardly took place, most likely due to the relatively short mixing time. During the dough resting phase, the levels of solubilized AX and reducing xylose formed were correlated well with the endoxylanase activity level of the flour, indicating AX hydrolysis by wheat flour associated endoxylanases. The effects of wheat flour associated endoxylanases on the AX population during dough and bread making were mainly caused by endogenous endoxylanases. The contribution of microbial endoxylanases was generally limited which can be explained by either a limited contamination of wheat flour with microbial enzymes during milling or by an extensive inactivation of these microbial endoxylanases that end up as contamination in wheat flour by endoxylanase inhibitors. The potential effects of all microbial endoxylanases present on wheat kernels were much larger, implying much larger variability problems in whole meal wheat processing applications.VOORWOORD TABLE OF CONTENTS I LIST OF ABBREVIATIONS V LIST OF PUBLICATIONS VII INTERNATIONAL PEER REVIEWED ARTICLES vii INTERNATIONAL PROCEEDINGS viii CONGRESS PAPERS WITH ABSTRACTS viii SUMMARY IX SAMENVATTING XIII CONTEXT AND AIMS OF THE STUDY - 1 - CHAPTER ONE WHEAT: PLANT DEVELOPMENT, QUALITY AND SAFETY - 7 - 1.1 INTRODUCTION - 7 - 1.2 STRUCTURE OF A WHEAT KERNEL - 8 - 1.3 WHEAT PLANT DEVELOPMENT - 10 - 1.3.1 Germination and emergence - 10 - 1.3.2 From seedling growth to ear emergence - 11 - 1.3.3 From anthesis to harvest maturity - 13 - 1.4 WHEAT QUALITY - 14 - 1.4.1 Wheat gluten proteins - 15 - 1.4.2 Wheat starch - 15 - 1.4.3 Less abundant constituents of wheat - 16 - 1.4.4 Enzyme activities and enzyme inhibitors of wheat - 16 - 1.5 MICROBIAL CONTAMINATION OF WHEAT - 17 - 1.5.1 Microbial contamination in the field - 18 - 1.5.2 Microbial contamination during storage - 19 - 1.5.3 Microbial contamination during milling - 20 - 1.6 IMPACT OF CLIMATOLOGICAL AND AGRONOMIC FACTORS ON WHEAT DEVELOPMENT, QUALITY AND SAFETY - 21 - 1.6.1 Temperature - 22 - 1.6.2 Water availability - 23 - 1.6.3 Nitrogen fertilization - 23 - 1.6.4 Fungicide treatment - 24 - 1.7 CONCLUSION - 25 - CHAPTER TWO ARABINOXYLANS, ENDOXYLANASES AND ENDOXYLANASE INHIBITORS - 27 - 2.1 INTRODUCTION - 27 - 2.2 ARABINOXYLAN - 27 - 2.2.1 General structure - 28 - 2.2.2 Physico-chemical properties - 29 - 2.2.3 Enzymic degradation of AX - 31 - 2.3 ENDOXYLANASES - 32 - 2.3.1 Classification - 32 - 2.3.2 Occurrence - 33 - 2.3.3 Biochemical characteristics - 35 - 2.3.4 Industrial use - 38 - 2.4 ENDOXYLANASE INHIBITORS - 38 - 2.4.1 TAXI - 38 - 2.4.2 XIP - 40 - 2.4.3 TLXI - 41 - 2.5 FUNCTIONALITY OF AX, ENDOXYLANASES AND ENDOXYLANASE INHIBITORS DURING BREAD MAKING - 41 - 2.5.1 Effects of AX - 42 - 2.5.2 Effects of endoxylanases - 43 - 2.5.3 Effects of endoxylanase inhibitors - 44 - 2.5.4 Conclusion - 44 - CHAPTER THREE WHEAT KERNEL ASSOCIATED ENDOXYLANASES CONSIST OF A MAJORITY OF MICROBIAL AND A MINORITY OF WHEAT ENDOGENOUS ENDOXYLANASES - 49 - 3.1 INTRODUCTION - 49 - 3.2 MATERIALS AND METHODS - 50 - 3.2.1 Materials - 50 - 3.2.2 Debranning of wheat kernels - 50 - 3.2.3 Sodium hypochlorite surface treatment of wheat kernels - 51 - 3.2.4 Washing of wheat kernels - 51 - 3.2.5 Grinding of wheat kernels - 52 - 3.2.6 Extraction of wheat material - 52 - 3.2.7 Determination of endoxylanase activity levels - 52 - 3.2.8 Determination of endoxylanase inhibitor contents - 52 - 3.2.9 Determination of proteolytic activity levels - 53 - 3.2.10 Microbial analyses - 54 - 3.3 RESULTS AND DISCUSSION - 54 - 3.3.1 Sample heterogeneity and size - 54 - 3.3.2 Distribution of endoxylanases over the wheat kernel - 56 - 3.3.3 Development of a washing procedure - 58 - 3.3.4 Validation of the washing procedure - 60 - 3.3.5 Total endoxylanase activity levels - 62 - 3.3.6 Application of the washing procedure - 64 - 3.3.7 Relevance of the present findings - 64 - 3.4 CONCLUSION - 65 - CHAPTER FOUR IMPACT OF GENOTYPE, HARVEST YEAR AND GENOTYPE-BY-HARVEST YEAR INTERACTION ON THE VARIABILITY OF AX, ENDOXYLANASES AND ENDOXYLANASE INHIBITORS IN WHEAT - 67 - 4.1 INTRODUCTION - 67 - 4.2 MATERIALS AND METHODS - 68 - 4.2.1 Wheat samples - 68 - 4.2.2 Chemicals and reagents - 68 - 4.2.3 Determination of grain yields, specific weights and TKWs - 69 - 4.2.4 Grinding of wheat kernels - 69 - 4.2.5 Determination of protein contents - 69 - 4.2.6 Determination of HFNs - 70 - 4.2.7 Determination of -amylase activity levels - 70 - 4.2.8 Determination of AX contents - 70 - 4.2.9 Determination of endoxylanase activity levels - 71 - 4.2.10 Determination of endoxylanase inhibitor contents - 71 - 4.2.11 Statistical analyses - 71 - 4.3 RESULTS AND DISCUSSION - 72 - 4.3.1 Characteristics of growing conditions and wheat varieties - 72 - 4.3.2 AX contents - 74 - 4.3.3 Endoxylanase activity levels - 77 - 4.3.4 Endoxylanase inhibitors contents - 79 - 4.3.5 Partial correlations between AX contents, endoxylanase activity levels, endoxylanase inhibitor contents and other grain characteristics - 80 - 4.3.6 Relevance of the present findings - 82 - 4.4 CONCLUSION - 83 - CHAPTER FIVE IMPACT OF FUNGICIDE TREATMENT, N-FERTILIZATION AND HARVEST DATE ON THE VARIABILITY OF AX, ENDOXYLANASES AND ENDOXYLANASE INHIBITORS IN WHEAT - 85 - 5.1 INTRODUCTION - 85 - 5.2 MATERIALS AND METHODS - 86 - 5.2.1 Wheat samples - 86 - 5.2.2 Chemicals and reagents - 87 - 5.2.3 Methods - 87 - 5.2.4 Statistical analyses - 88 - 5.3 RESULTS AND DISCUSSION - 88 - 5.3.1 Fungicide treatment - 88 - 5.3.2 N-fertilization - 93 - 5.3.3 Harvest date - 97 - 5.3.4 Relevance of the present findings - 103 - 5.4 CONCLUSION - 103 - CHAPTER SIX INSIGHT INTO THE DISTRIBUTION OF AX, ENDOXYLANASES AND ENDOXYLANASE INHIBITORS IN INDUSTRIAL WHEAT ROLLER MILL STREAMS - 105 - 6.1 INTRODUCTION - 105 - 6.2 MATERIALS AND METHODS - 106 - 6.2.1 Materials - 106 - 6.2.2 Milling - 106 - 6.2.3 Methods - 107 - 6.3 RESULTS AND DISCUSSION - 109 - 6.3.1 Differences in composition of germ, flour and bran fractions - 109 - 6.3.2 PCA and correlations between the different parameters - 116 - 6.3.3 Relevance of the present findings - 121 - 6.4 CONCLUSION - 122 - CHAPTER SEVEN IMPACT OF WHEAT FLOUR ASSOCIATED ENDOXYLANASES ON THE AX POPULATION IN DOUGH AFTER MIXING AND RESTING - 123 - 7.1 INTRODUCTION - 123 - 7.2 MATERIALS AND METHODS - 124 - 7.2.1 Materials - 124 - 7.2.2 Preparation of dough samples - 124 - 7.2.3 Determination of AX contents - 124 - 7.2.4 Determination of reducing end sugar and free sugar contents - 124 - 7.2.5 Determination of apparent endoxylanase activity levels - 125 - 7.2.6 Determination of xylosidase and arabinofuranosidase activity levels - 125 - 7.2.7 Inactivation of enzymes in flour samples - 125 - 7.2.8 Determination of MM by high performance size exclusion chromatography - 126 - 7.3 RESULTS AND DISCUSSION - 126 - 7.3.1 Selection and characterization of flour samples - 126 - 7.3.2 Analysis of soluble AX contents in dough - 127 - 7.3.3 Analysis of reducing end and free sugar contents in dough - 129 - 7.3.4 Analysis of A/X ratio of soluble AX in dough - 132 - 7.3.5 Analysis of MM distribution of soluble AX - 132 - 7.3.6 Relevance of the present findings - 134 - 7.4 CONCLUSION - 135 - CHAPTER EIGHT ASSESSMENT OF THE CONTRIBUTION OF WHEAT ASSOCIATED ENDOGENOUS AND MICROBIAL ENDOXYLANASES TO THE CHANGES IN THE AX POPULATION DURING BREAD MAKING - 137 - 8.1 INTRODUCTION - 137 - 8.2 MATERIALS AND METHODS - 138 - 8.2.1 Materials - 138 - 8.2.2 Sodium hypochlorite surface treatment of wheat kernels - 138 - 8.2.3 Isolation of wheat kernel associated microbial endoxylanases - 138 - 8.2.4 Milling of wheat - 139 - 8.2.5 Standard analyses - 139 - 8.2.6 Bread making - 139 - 8.2.7 Determination of non-cellulosic carbohydrate compositions and contents - 140 - 8.2.8 Determination of reducing end sugar and free sugar contents - 140 - 8.2.9 Determination of enzyme activity levels and inhibitor contents - 141 - 8.2.10 Determination of MM distribution by HPSEC - 141 - 8.2.11 Statistical analysis - 141 - 8.3 RESULTS AND DISCUSSION - 141 - 8.3.1 Milling of surface treated and untreated wheat kernels - 141 - 8.3.2 Isolation of wheat kernel associated microbial endoxylanases - 143 - 8.3.3 Changes in the AX population during bread making - 143 - 8.3.4 Bread making results - 147 - 8.3.5 Relevance of the present findings - 149 - 8.4 CONCLUSION - 149 - GENERAL CONCLUSIONS AND PERSPECTIVES FOR FUTURE WORK - 151 - ANNEX - 157 - REFERENCES - 163 -status: publishe

Grain-associated xylanases: occurrence, variability, and implications for cereal processing

Xylanases (EC 3.2.1.8) hydrolyse the backbone of cereal cell wall arabinoxylans and often have a significant impact on cereal-based processes and end-products. The use of microbial xylanases as processing aids in this respect is well established and has been extensively studied. Much less research has focused on inherently present cereal-associated xylanases and their possible impact. Cereals produce xylanases for remodeling and expansion of cereal cell walls during normal cell growth and for more drastic cell wall degradation during seed germination. Besides these endogenous xylanases, cereals also contain microbial xylanases from micro-organisms populating the outer grain kernels layers. Unfortunately, these microbial xylanases are often inhibited by wheat proteinaceous xylanase inhibitors and they hence escape standard xylanase activity measurements. It is more correct to refer to these activity levels as 'apparent' xylanase activity levels. As a result, the occurrence of cereal-associated xylanases might have been largely underestimated in the past and hence unjustly been disregarded. The levels and the types of cereal-associated xylanases differ strongly between grain species, varieties, and tissues, and are largely affected by grain growing conditions. These variations in the levels of grain-associated xylanase activity affect several cereal-based food and feed applications. This paper provides an overview of the occurrence and variability of cereal-associated xylanases and of their potential impact on bread making, shelf life of refrigerated doughs, brewing, animal feed efficiency, pasta production, and wheat gluten-starch separation.status: publishe

Selectivity for water-unextractable arabinoxylan and inhibition sensitivity govern the strong bread improving potential of an acidophilic GH11 Aureobasidium pullulans xylanase

An acidophilic xylanase of Aureobasidium pullulans (XAPI) was recombinantly produced, characterised and its functionality in bread making compared with that of Bacillus subtilis Xyn A xylanase (XBS), an enzyme frequently used in bread making. Prominent characteristics of XAPI were its high specific activity towards arabinoxylan (AX), its relative preference for hydrolysis of water-unextractable AX (WU-AX), its optimal activity under acidic conditions and its sensitivity towards TAXI (Triticum aestivum xylanase inhibitor) and XIP (xylanase inhibiting protein). Optimally developed dough containing XAPI had a considerably drier feel after mixing and was less sticky after fermentation than dough treated with XBS. Both xylanases improved bread loaf volume by 24% under the conditions of the process. In spite of the higher dosage of XAPI necessary to obtain this result, the similar loaf volume increase coincided with less solubilisation of WU-AX and even lesser degradation of solubilised AX (S-AX) and water-extractable AX (WE-AX) by XAPI than by XBS during the bread making process. This is probably due to different dynamics and extent of xylanase inhibition in the dough matrix for both xylanases. AX solubilisation by the acidophilic XAPI was not boosted by the drop in pH in the dough during fermentation. Results show that significant bread loaf volume increase goes hand in hand with excellent dough properties when a limited solubilisation of WU-AX during mixing is coupled to retention of a high molecular mass of S-AX and WE-AX during the rest of the process. (C) 2010 Elsevier Ltd. All rights reserved.status: publishe

Impact of Wheat Bran Derived Arabinoxylanoligosaccharides and Associated Ferulic Acid on Dough and Bread Properties

The impact of arabinoxylanoligosaccharides (AXOS) with varying bound or free ferulic acid (FA) content on dough and bread properties was studied in view of their prebiotic and antioxidant properties. AXOS with an FA content of 0.1-1.7% caused an increase in dough firmness with increasing AXOS concentration. AXOS with a high FA content (7.2%), on the contrary, resulted in an increase in dough extensibility and a decrease in resistance to extension, similar to that for free FA, when added in levels up to 2%. Higher levels resulted in unmanageable dough. A limited impact on dough gluten network formation was observed. These results suggest that for highly feruloylated AXOS, the FA-mediated dough softening supersedes the firming effect displayed by the carbohydrate moiety of AXOS. The impact of the different AXOS on bread volume, however, was minimal. Furthermore, AXOS in bread were not engaged in covalent cross-linking and significantly increased its antioxidant capacity.status: publishe

Occurrence and functional significance of secondary carbohydrate binding sites in glycoside hydrolases

Non-catalytic carbohydrate binding on independent carbohydrate-binding modules (CBMs) has been reported frequently for glycoside hydrolases (GHs) and reviewed thoroughly. However, various structural studies of GHs have revealed that non-catalytic carbohydrate binding sites can also occur on the surface of the structural unit comprising the active site. Here, the discovery of these sites, referred to as secondary binding sites (SBSs), and their putative roles in different GHs is reviewed for the first time. The majority of the SBSs have been discovered in starch-active enzymes, but there are also many reports of SBSs in various other enzymes. A wide variety of functions has been ascribed to these sites, including (1) targeting of the enzyme towards its substrate, (2) guiding the substrate into the active site groove, (3) substrate disruption, (4) enhancing processivity, (5) allosteric regulation, (6) passing on reaction products, and (7) anchoring to the cell wall of the parent microorganism. A lot of these putative functions are in agreement with the functions ascribed to non-catalytic binding in CBMs. Contrarily to CBMs, SBSs have a fixed position relative to the catalytic site, making them more or less suitable to take up specific functions.status: publishe

The secondary substrate binding site of the Pseudoalteromonas haloplanktis GH8 xylanase is relevant for activity on insoluble but not soluble substrates

Previously, it has been demonstrated that the glycoside hydrolase family 8 xylanase from the psychrophylic bacterium Pseudoalteromonas haloplanktis (XPH) can bind substrate non-catalytically on the surface of its catalytic module. In the present study, the functional relevance of this secondary binding site (SBS) for the enzyme is investigated by site-directed mutagenesis and evaluation of activity and binding properties of mutant variants on a range of structurally different homoxylan and heteroxylan substrates. The SBS had an impact on the activity on insoluble substrates, whereas the activity on soluble substrates remained unaffected. Unexpectedly, the activity on a soluble oligomeric substrate was also affected for some mutants and results on a chromophoric polymeric model substrate were in contrast with the trends observed on the corresponding natural substrate. All in all, results show that the impact of the SBS on the activity of XPH is in part analogous to the functioning of some carbohydrate-binding modules in modular enzymes.status: publishe

Critical assessment of the formation of hydrogen peroxide in dough by fermenting yeast cells

Fermentation of bread dough leads to strengthening of the dough matrix. This effect has previously been ascribed to the action of hydrogen peroxide (H2O2) produced by yeast in dough. In this study, we re-evaluate the production of H2O2 by yeast in dough and aqueous fermentation broth. Results show that the previously reported high levels of H2O2 in fermenting dough were most probably due to the lack of specificity of the potassium dichromate/acetic acid-based method used. Using the chemiluminescent HyPerBlu assay, no yeast H2O2 production could be detected in fermented dough or broth. Even though the formation of low levels of H2O2 cannot be ruled out due to the presence of catalase in flour and the fast reaction of H2O2 with gluten proteins, our results suggest that the changes in dough matrix rheological properties upon fermentation are not due to production of H2O2 by yeast.publisher: Elsevier articletitle: Critical assessment of the formation of hydrogen peroxide in dough by fermenting yeast cells journaltitle: Food Chemistry articlelink: http://dx.doi.org/10.1016/j.foodchem.2014.07.050 content_type: article copyright: Copyright © 2014 Elsevier Ltd. All rights reserved.status: publishe

Quantification and visualization of dietary fibre components in spelt and wheat kernels

This study was undertaken to determine contents and structural characteristics of arabinoxylan (AX), fructan and β-glucan in 28 spelt accessions, and to compare them with those of dietary fibre components in 11 wheat accessions. In addition, microscopic visualization of AX and β-glucan distribution in a selection of these accessions was performed. On average, wheat contained more total AX (TOT-AX; 6.90 versus 5.74%) and water-extractable AX (WE-AX; 0.71 versus 0.59%) than spelt. The overall arabinose to xylose ratio (A/X; 0.72 for wheat and 0.71 for spelt) was similar for both subspecies, but that of water extractable material was higher for spelt than for wheat (1.25 versus 0.97). Fructan content and degree of polymerization (DP) were lower in spelt than in wheat (fructan content; 1.29% versus 1.53% and DP; 3.3 versus 4.5). β-glucan content was similar for both subspecies (0.54% for spelt and 0.51% for wheat). In the spelt accessions, the contents of the different fibre components significantly differed (up to 74% difference) between accessions and countries of origin, but were not affected by accession status and bread making quality. Microscopic visualizations showed that the two subspecies are very similar in terms of dietary fibre distribution over the kernel cell walls.publisher: Elsevier articletitle: Quantification and visualization of dietary fibre components in spelt and wheat kernels journaltitle: Journal of Cereal Science articlelink: http://dx.doi.org/10.1016/j.jcs.2015.01.003 content_type: article copyright: Copyright © 2015 Elsevier Ltd. All rights reserved.status: publishe

Suitability of solvent retention capacity tests to assess the cookie and bread making quality of European wheat flours

A solvent retention capacity (SRC) profile of flour consists of its water RC (WRC), sodium carbonate SRC (SCSRC), sucrose SRC (SuSRC) and lactic acid SRC (LASRC) values. SRC tests have been designed to assess flour quality of North American soft wheats, but the value of these tests for European flour which is generally from harder wheats is rather unclear. We here studied the ability of the SRC values to assess the cookie and bread quality of nineteen European commercial flours and compared their predictive value with that of some conventional flour quality parameters. WRC value was a better parameter to assess the cookie diameter than Farinograph or Mixograph water absorption capacities and Alveograph dough tenacity values. In contrast, Zeleny sedimentation values and some rheological data were better to assess bread volume than LASRC values. When LASRC values were corrected for the contribution of non-glutenin polymers, they could assess bread volume to a comparable extent as the Zeleny sedimentation readings. In conclusion, the SRC tests are good alternative tests to assess the cookie and bread quality of European wheat flours. Furthermore, it is not always necessary to determine the entire SRC profile to sufficiently assess flour quality for a specific end-product.status: publishe