Interactions of barley endosperm proteins with starch and their effects on functional properties

Abstract

Many factors can alter starch degradation rate, with interactions with proteins being one of the most important factors. Although barley protein content is currently a major parameter for predicting the quality of a barley genotype in brewing, the mechanisms underlying the effects of barley protein on grain utilization are poorly understood.This project aims to explore these mechanisms of these effects on starch utilization in both food and brewing industries to provide a better way for brewers and food producers to choose a suitable barley genotype for beer and food production. As part of this aim, mechanistic explanations of the observations will also be obtained.Protein content is currently an important parameter used to choose a suitable barley genotype, it is of value to find out if there are any relations among barley protein contents with other grain parameters, like, the grain size and starch properties. 30 different barley samples were used. Size- exclusion chromatography (SEC) and fluorophore- assisted carbohydrate electrophoresis (FACE) were used together to investigate starch molecular structure, while both amylose and amylopectin chain-length distributions (CLDs) being fitted with biosynthesis-based models to find statistically meaningful correlations.In order to understand the mechanism concerning how barley protein affects starch degradation, in vitro starch digestion measurements were carried out, using raw barley flour as substrate. Pepsin and porcine pancreatic α-amylase were used, and both logarithm of slopes and NLLS- methods used to fit the data, the latter being devised here for this purpose. Confocal microscopy was used to visualize the absorption of a- amylase on barley protein.Another research goal is to investigate and understand how barley protein affects starch degradation during mashing (65 °C) as well as fermentable sugar productions (glucose, maltose, maltotriose). Ten barley samples with varied protein contents were used to do malting followed by mashing. For malting which is conducted based on industrial standards, both scanning electron microscopy (SEM), SEC with MALLS (multiple-angle laser light scattering) detection and differential scanning calorimetry (DSC) were used to visualize and to determine starch morphological and structural changes. Debranched starch amylose and amylopectin CLDs were fitted using biosynthesis-basedmodels, reducing data to a small number of biologically-meaningful parameters for subsequent correlation analysis. For mashing, high-performance liquid chromatography (HPLC) was used to determine effects of barley protein on starch degradation during mashing and therefore on altering the content of fermentable sugars in wort. Our results showed that: (1) increased protein content significantly negatively correlated with the amounts of amylose with longer chains (degree of polymerization, DP 1600– 40000) but significantly positively correlated with the proportion of longer chains of amylopectin (DP 34–100); (2), barley proteins, especially water-insoluble components, were found to slow down starch degradation rate by α-amylase through binding with α-amylase at an in vitro digestion environment (37 °C); (3), during malting, starch content decreased significantly, while no significant change with both amylose and amylopectin CLDs have been observed. However, malting resulted in a significant decrease in the weight- average molecular weight of all starch polymers while the average size (Rg) did not change significantly, thus indicating that malting removes whole chains throughout the whole starch molecules; (4), after mashing, both total and insoluble barley protein significantly negatively correlated with fermentable sugar content. DSC results showed that protein may retard starch hydrolysis by inhibiting granule swelling during mashing; (5), amylose content, in both unmalted and malted barley significantly negatively correlated with fermentable sugar content; (6), by parameterizing starch structural data with biosynthesis- based model, it was found that barleys with a higher amount of short- chain amylose molecules produce more fermentable sugars after mashing, as more small amylose molecules leaching out and bother becoming available for enzymatic degradation and also loosening the structure of the remaining starch; (7) results showed that barley protein can inhibit starch hydrolysis, particularly on starch polymers with hydrodynamic radius Rh> 100 nm, through affecting the leaching of starch amylose molecules with short chain length during mashing. The research findings of this whole project have clarified systematically the mechanism beneath the effects of barley protein on starch utilization in both brewing and food industries: the binding interactions between barley protein (water- insoluble) with amylase at 37 °C and the inhibition of barley protein on starch gelatinization during mashing (65 °C). Barley protein content is currently a widely-useful parameter for the prediction of barley brewing quality, with a higher protein content malted barley genotype tends to produce a less content of fermentable sugars after mashing. The importance of barley starch structural effects has also been examined, particularly the CLDs of amylose and amylopectin. During mashing, except for protein inhibition on starch gelatinization, effects of amylose molecular size and CLD, is also useful for determining the release of fermentable sugars. This further provides more targeting information for barley breeders to grow barley with industrially preferred barley genotype.