How Does the Preparation of Rye Porridge Affect Molecular Weight Distribution of Extractable Dietary Fibers?

Extractable dietary fiber (DF) plays an important role in nutrition. This study on porridge making with whole grain rye investigated the effect of rest time of flour slurries at room temperature before cooking and amount of flour and salt in the recipe on the content of DF components and molecular weight distribution of extractable fructan, mixed linkage (1→3)(1→4)-β-d-glucan (β-glucan) and arabinoxylan (AX) in the porridge. The content of total DF was increased (from about 20% to 23% of dry matter) during porridge making due to formation of insoluble resistant starch. A small but significant increase in the extractability of β-glucan (P = 0.016) and AX (P = 0.002) due to rest time was also noted. The molecular weight of extractable fructan and AX remained stable during porridge making. However, incubation of the rye flour slurries at increased temperature resulted in a significant decrease in extractable AX molecular weight. The molecular weight of extractable β-glucan decreased greatly during a rest time before cooking, most likely by the action of endogenous enzymes. The amount of salt and flour used in the recipe had small but significant effects on the molecular weight of β-glucan. These results show that whole grain rye porridge made without a rest time before cooking contains extractable DF components maintaining high molecular weights. High molecular weight is most likely of nutritional importance

Comparative in situ analyses of cell wall matrix polysaccharide dynamics in developing rice and wheat grain

Cell wall polysaccharides of wheat and rice endosperm are an important source of dietary fibre. Monoclonal antibodies specific to cell wall polysaccharides were used to determine polysaccharide dynamics during the development of both wheat and rice grain. Wheat and rice grain present near synchronous developmental processes and significantly different endosperm cell wall compositions, allowing the localisation of these polysaccharides to be related to developmental changes. Arabinoxylan (AX) and mixed-linkage glucan (MLG) have analogous cellular locations in both species, with deposition of AX and MLG coinciding with the start of grain filling. A glucuronoxylan (GUX) epitope was detected in rice, but not wheat endosperm cell walls. Callose has been reported to be associated with the formation of cell wall outgrowths during endosperm cellularisation and xyloglucan is here shown to be a component of these anticlinal extensions, occurring transiently in both species. Pectic homogalacturonan (HG) was abundant in cell walls of maternal tissues of wheat and rice grain, but only detected in endosperm cell walls of rice in an unesterified HG form. A rhamnogalacturonan-I (RG-I) backbone epitope was observed to be temporally regulated in both species, detected in endosperm cell walls from 12 DAA in rice and 20 DAA in wheat grain. Detection of the LM5 galactan epitope showed a clear distinction between wheat and rice, being detected at the earliest stages of development in rice endosperm cell walls, but not detected in wheat endosperm cell walls, only in maternal tissues. In contrast, the LM6 arabinan epitope was detected in both species around 8 DAA and was transient in wheat grain, but persisted in rice until maturity

The contribution of wheat to human diet and health

Wheat is the most important staple crop in temperate zones and is in increasing demand in countries undergoing urbanization and industrialization. In addition to being a major source of starch and energy, wheat also provides substantial amounts of a number of components which are essential or beneficial for health, notably protein, vitamins (notably B vitamins), dietary fiber, and phytochemicals. Of these, wheat is a particularly important source of dietary fiber, with bread alone providing 20% of the daily intake in the UK, and well-established relationships between the consumption of cereal dietary fiber and reduced risk of cardio-vascular disease, type 2 diabetes, and forms of cancer (notably colo-rectal cancer). Wheat shows high variability in the contents and compositions of beneficial components, with some (including dietary fiber) showing high heritability. Hence, plant breeders should be able to select for enhanced health benefits in addition to increased crop yield

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