The influence of starch pasting properties and grain protein content on water uptake in barley

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1 Research article Received: 7 May 2013 nstit Revised: 27 October 2013 Accepted: 30 November 2013 of Brewing & Distilling Published online in Wiley Online Library: 8 January 2014 (wileyonhinelibrary.com) DOl /jib.1 09 The influence of starch pasting properties and grain protein content on water uptake in barley D. Cozzolino,* S. Roumeliotis and i. K. Eglinton Steeping is the first operation of malting and its purpose is to increase the water content of the grain up to 43-46%; however, such a simple step encompasses several metabolic processes that affect germination and the final malt quality. The aim of this study was to evaluate the effect of initial grain protein (GP) content and starch pasting properties, measured using the Rapid Visco Analyser (RVA) on water uptake in different barley varieties. The partial least squares (P152) regression algorithm was used to compare the two data matrices, the independent block of variables X (RVA data and GP) and the dependent block Y (water uptake at different steeping times). The first two P152 loadings explained 65 and 23% of the total variation in water uptake in the barley samples. Water uptake at 1 and 2 h was poorly explained by the P152 models, while after 5 h the models explained more than 40% of the variability. The results from this study showed that, although a relationship between GP and water uptake exists, it is not universal for all the varieties, indicating that the relationship between GP and water uptake is varietal dependent. Keywords: water uptake; barley; protein; starch pasting; RVA; PLS2 ntroduction The uptake of water by seeds is an essential and initial step towards germination. For malting barley, this is represented by the first stage of the malting process, often referred to as steeping (1-5). Steeping is the first operation of malting and its purpose is to increase the water content of the grain up to 43-46%; however, such a simple step encompasses several metabolic processes that affect germination and the final malt quality (1-4,6,7). The initial hours of barley steeping are critical to the development of components such as enzyme activity and hormonal development and release, which will determine the quality of the malt (1-4,6,8). Kernel morphology, chemical composition (e.g. protein, starch, lipids, cell wall composition), genetics and environmental factors (e.g. temperature) influence the physiology of the kernel during these initial hours (3-5,7-12). Variations in water imbibitions rates may also lead to inefficient hydration and therefore to over- or under-modification of the endosperm for industrial use (e.g. mafting) (3,4,7-11). However, a general comprehensive understanding of the biophysical, physiological and biochemical determinants of seed germination and their interactions is still lacking (13). More information regarding the pathway(s) and dynamics of water penetration in barley and the corresponding initiation of metabolism is required in order to produce barley varieties in a sustainable manner, as well as improve their mafting quality (1-4,13,14). Overall, few and/or contradictory studies have been found on the factors that influence water uptake in barley, in particular the effect of grain protein (GP) content (1-4,9,14). t has been reported that protein content and fl-hordein content of the endosperm of barley can impede water movement into the endosperm (7,9,15,16). n a 2008 study, Gamlatha and collaborators (14) found no relationship between total protein content of the endosperm and water uptake of several barley kernels during steeping. Similar results had been reported in 2002 by Leach and co-workers (17). Other factors such as endosperm physical characteristics and chemical composition were found to be responsible for water J. nst. Brew. 2074; 120: uptake (3,4,1 1,15,16,18,19).lt has been reported thatthe physical characteristics of the barley endosperm can have a significant impact on the processing behaviour of barley, where its compactness (structure of the endosperm is referred to as steeliness and mealiness) has been regarded as a useful indicator of malting quality in barley (20). Loosely packed endosperm gives a soft (mealy) structure and facilitates better moisture and enzyme movements in the endosperm. n contrast, steeliness (hard endosperm) appears to be a major factor restricting the uptake and distribution of water across the endosperm during steeping. Overall, the higher GP content may not be an important factor in controlling water uptake of the kernel, since high nitrogen content does not necessarily correlate with the steely areas (18). Other characteristics of the endosperm, such as cell walls, also have an important impact on malt quality (11). t has been reported that hardness of the endosperm, or water uptake during steeping, may be influenced by cell wall organization and the composition of the endosperm (1 1,14,16,18). The aim of this study was to evaluate the effect of initial grain protein content and starch pasting properties, measured using the Rapid Visco Analyser (RVA), on water uptake in different barley varieties. Materials and methods Samples Whole barley samples (Hordeum vuigare L) having different grain protein (GP) content and pasting properties were sourced from * Correspondence to: D. Cozzolino, School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1 Glen Osmond SA 5064, Australia. d.cozzolino@adelaide.edu.au School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1 Glen Osmond SA, 5064, Australia

2 nfluence of starch pasting properties and protein content on water uptake commercial varieties: Commander (n =10), Schooner (n = 6), Gairdner (n=6), Admiral (n= 10), Navigator (n= 10) and Vlamingh (n= 9). Samples were harvested in 2010, at two localities in South Australia (Roseworthy and Charlick) and in 2012, from two localities in Western Australia (Scaddan and Corrigin). More details about the commercial varieties can be found on the Barley Australia website (httpf/ ). Water uptake measurements Barley samples (1.5g±0.01 g) were placed (in triplicate) in a sample holder (40x32x5mm), and soaked at 22 C (± 1 C) in a water bath. Samples were taken every hour from 1 to 8 h, and after 24, 28, 30, 48 and 50h in order to simulate the water uptake as suggested by other authors (5). Before weighing, excessive water on the sample holder was removed by shaking, and from the grain surface using a dry filter paper. Water uptake was then calculated using the equation reported elsewhere and expressed as a percentage of grain weight (5,12). Samples were analysed in duplicate for both moisture (M) and grain protein (GP) using near infrared (NR) spectroscopy (Unity Scientific SpectraStar" 2500, Unity Scientific Asia Pacific, Australia) as a reference method. Rate of water uptake was calculated following the procedure reported elsewhere (5,12). lnstute of Brewing & Distilling Statistical analysis Mean differences in water uptake between samples were analysed using ANOVA (imp, version 4; imp is a business unit of SAS, Copyright , SAS nstitute nc., USA). The partial least squares (PLS2) regression algorithm was used to compare the two data matrices, the independent block of variables X (RVA data and GP) and the dependent block V (water uptake at different steeping times). The PLS method is a covariance-based method of projection of large sets of data into their latent structures (22). The use of PLS allows for an efficient multivariate calibration by identifying the latent structure of the signal (response) and concentration (predictor) matrices and linking them in a calibration relationship. Score vectors extracted by PLS operate as a new predictor representation that regresses the response variables on these new predictors (22). As opposed to the PLS1 algorithm that does not reflect the relation between the response and predictor matrices, the PLS2 algorithm was used in this work as an exploratory tool. Both principal component analysis (PCA) and PLS2 were performed using The Unscrambler (version X, CAMO, Norway). The data was mean centred and weighted (autoscaled) by the standard deviation (1/STD) prior to the PCA and PLS2 regression. Models were developed using full cross-validation (leave one out). The PLS2 were developed using the root mean square of the standard error in cross validation (RMSECV) and the coefficient of determination (R2 22). ) ( Measurement of starch pasting properties - Rapid Visco Analyser Results Samples were milled using an UDY Cyclone Mill (Fort Collins, CO. USA) through a 0.8 mm screen, before RVA analysis. Ground barley flour samples (4.0 g ± 0.01 g of flour corrected using the moisture content of the sample) were slurried with distilled water (25.0 g ± 0.1 g, as function of the amount of adjusted sample) in an aluminium canister. The pasting properties of the slurries were determined with a RVA instrument in duplicate (TecMaster, Perten nstruments, NSW, Australia). The RVA test profile used was reported in a previous study (21). The RVA properties calculated using the software TCW3 (Thermocline, Perten nstruments, NSW, Australia) were pasting viscosity (PV), trough, breakdown, final viscosity, setback, time to peak and pasting temperature (21). Table 1 shows the mean, range, standard deviation and coefficient of variation of water uptake values at 8 and 24 h steeping times on the barley samples analysed. Note that only water uptake by variety at these two steeping times was considered for statistical analysis. Statistical significant differences (p < 0.05) in water uptake at 8 and 24 h were observed between the different commercial varieties analysed. Figure 1 illustrates the effect of the initial GP content on the water uptake between 1 and 8 h, in the different commercial varieties and analysed. t was observed for Navigator (Fig. 1 C) and Vlamingh (Fig. 1D) that, the lower the protein content in the grain was, the higher the water uptake by the sample. These results are in agreement with those reported by Molina-Cano and Table 1. Mean, standard deviation and range in percentage water uptake after 8 and 24h of steeping for different commercial barley varieties Commander (n=12) 8h Mean (%) SD Range (%) CV 24 h Mean (%) SD Range (%) CV 535a a Gairdner(n=6) 442" " Schooner(n=6) 42.9" " Navigator(n=10) 47.03c a Vlamingh (n=9) 573 a a N, number of samples; SD, standard deviation; CV, coefficient of variation. Numbers in the same row connected letter showed no statistical differences (p < 0.05). J. nst. Brew. 2074; 120: Admiral (n=10) 47.8c " with the same wileyonlinelibrary.com/journal/jib

3 nstitute of Brewing & Distilling D. Cozzolino et al. A B iyief Time (,) E F 11 ri r is Time (i) Figure 1. Water uptake of commercial barley varieties with varying initial grain protein content, during the first 8h of steeping. A, Admiral; B, Commander; C, Navigator; D, Vlamingh; E, Gairdner; F, Schooner. collaborators (7). However, varieties such as Admiral (Fig. 1A), Commander (Fig. 113), Gairdner (Fig. 1E) and Schooner (Fig. if) did not show the same trend in relation to protein and water uptake. These varieties showed either an inverse or no relationship between GP and water uptake. Figure 2 shows the water uptake in Navigator (Fig. 2A) and Admiral (Fig. 213) after 24h and up to 50h of steeping. Similar trends to those found during the first hours of steeping were observed after longer steeping times. These results showed that some varieties tend to follow the reported relationship between GP and water uptake; however, other grain characteristics such as hardness, starch or cell wall characteristics might play a role in explaining the water uptake by the endosperm. However, these characteristics were not measured in this study. Figure 3 illustrates the water uptake of two commercial varieties, Gairdner and Schooner, having similar GP levels (10.7% GP dry basis (db). The initial water uptake was very similar in both genotypes. However, as the steeping time progressed, differences could be observed after 2 h of steeping, where Gairdner tended to take up more water compared with Schooner. Considering that the varieties had a similar GP content, such differences might be explained by differences in other grain chemical or biophysical characteristics. Figure 4 shows the rates of water uptake at a single time point in the Navigator (Fig. 4A) and Admiral (Fig. 413) samples, which had a different GP content. The sample from Navigator had the highest GP content and showed lower rates of water uptake compared with those sourced from Admiral. Conversely, samples of Navigator had a lower GP content and showed a high rate of water uptake. n addition, a reduction in the rate of water uptake was observed in both varieties between 3 and 5 h. n particular, an increase in the rate of water uptake at 3 h was observed in the samples having a high GP content. n order to further study the effect of genotype and to evaluate other endosperm characteristics, rather than GP content on the water uptake, starch pasting properties measured using the RVA method and derived parameters were analysed. n general, the RVA results indicated that varieties with low water uptake values displayed a high RVA profile compared with those that had a high water uptake, independent of the protein level. These trends were observed for the RVA profiles of Schooner (PV = 2390 cp; GP =10.5% db; water uptake 8 h = 40%), Gairdner (PV= 2035 cp; GP = 93% db; water uptake 8 h = 43%), Commander (PV= 1964 cp; GP = 9.1% db; water uptake 8 h = 39%) and Navigator (range of PV= 1500 cp; GP = 9.7% db; water uptake 8 h = 44%; see Fig. 5A). wileyonhinelibrary.com/journal/jib J. nst. Brew. 2074; 120: 38-44

4 nfluence of starch pasting properties and protein content on water uptake A lnstute of Brewing & Distilling barley grain. Figure 6 summarizes in a PCA biplot (eigenvectors and scores) the influence of the different quality parameters [i.e. diastatic power, free amino nitrogen, GP, attenuation limit (AAL) and hot water extract (HWE)] in the commercial barley varieties and analysed from the same locality and harvest. These results indicated that samples from Navigator tended to produce malts with a high HWE. while Admiral tended to yield malts with both high fermentability (high AAL values) and high HWE. 90 so Discussion CL ] Time (b) Figure 2. Water uptake in different barley genotypes having similar protein content after 24 h and up to 50 h of steeping. A, Navigator B, Admiral. However, samples from Admiral did not show the same trend. The RVA profiles of samples sourced from Admiral and Navigator, with a different GP content and similar water uptake values, are shown in Fig. 5(B). Table 2 shows the f?2 and RMSECV for the PLS2 models developed for the prediction of water uptake using the GP and RVA data. The first two PLS2 loadings explained 65 and 23% of the total variation in water uptake in the barley samples. Water uptake between 1 and 3 h is poorly explained by the PLS2 models (R2 < 030), while for water uptake after 5 h, 40% of the variability is explained by the combination of starch properties and the GP content of the grain. These results confirm that not only protein, but also other parameters were affecting water uptake by the 9.1 Tr1e lfi Figure 3. Comparison of water uptake in two barley genotypes (Schooner and Gairdner) having similar grain protein content (10.7% db) during the first 8 h of steeping. J. nst. Brew. 2074; 120: From the data presented in Table 1, it was observed that samples sourced from the Vlamingh variety showed the highest values in water uptake, while samples from the Schooner variety showed the lowest values. These differences might be associated with different endosperm structures, grain porosity and starch content as well as other biophysical characteristics (hardness, friability, lipids, cell walls) of the grain that influence water uptake and diffusion into the grain, as suggested by other authors (5,7,12,23-25). t has been reported by other authors that grains with lower protein content tend to absorb water and to malt more rapidly than their high-gp counterparts (7). This trend was observed when the rate of water uptake was compared between samples sourced from Navigator and Admiral varieties (see Fig. 4A and B). Considering that water uptake is a process comprising two overlapping phases, water uptake can be modulated by imbibitions of water by the outer grain layers and the internal redistribution of water in the endosperm (7,9). Figure 4 also shows different phases in the rate of water uptake, independent of the GP content. The results from this study suggest that it is not only the GP content of the sample that is responsible for the differences in the total and rate of water uptake between varieties (genotypes). The combination of GP and other endosperm characteristics, such as starch pasting properties, may additionally explain the differences in water uptake into the grain. Therefore, in order to explain the basis for the differences in water uptake, other endosperm characteristics should be considered or measured. t has been also reported by other authors that starching pasting properties and in particular RVA profiles (e.g. peak viscosity) are associated with amylose, amylopectin and starch content, biophysical characteristics of the endosperm (e.g. granules) as well as a tendency to produce malts with better modification (26). Bryce and collaborators (26) also reported that high PV can be associated with starch being incompletely hydrolysed. t is evident from the data derived from the RVA profiles, indicated by the PV, that physical or biophysical characteristics of the endosperm related with starch (e.g. amylose and amylopectin) might have an additional effect on the water uptake by the grain regardless of the initial GP content of the grain (Fig. 5). Samples from the Navigator variety, having different levels of GP (range 9-13% db), tend to yield similar and high RVA profiles compared with those generated by samples from Admiral. n previous studies, samples from Admiral tended to have a higher amylose content compared with Navigator (12). Water uptake is generally thought to be more rapid in smaller grains or in those with lower nitrogen content. This is supported by the fact that cavities and/or channels in the surface of the starch granule will result in a reduction in size of the amylose and amylopectin polymers, resulting in lower viscosities (18,19). Consequently, a mealy (soft) endosperm is more easily degraded wileyonlinelibrary.com/journal/jib

5 m nstitute of Brewing & Distilling D. Cozzolino etal. A '[ C -c 1 1i[ 13%GF Wi Wi Wi W W.. we, V.5 i0 ' } j i i 5 -. DM%3P i ADM L1%GP S 2 1 Wi W2 W3 W W5 Vvb W Time (h) Figure 4. Rate of water uptake at a single time point with two levels of grain protein (GP) comparing two barley varieties. A, Navigator; B, Admiral Time (sec) by hydrolytic enzymes during malting (27). The endosperm of steely (hard) kernels is tougher, denser in structure and shows a slower rate of water uptake during steeping and a poorer distribution of enzymes during malting (18). t has also been reported that mealy kernels might absorb water more rapidly, requiring less time to attain a predetermined moisture content, and thus tend to malt faster than steely kernels (10,28-30). Therefore, the structure of the endosperm (e.g. steeliness) appears to be a major factor restricting the uptake and distribution of water across the endosperm during steeping, whereas a higher total protein content may not be the sole factor in controlling water uptake of the kernel, since high nitrogen content does not necessarily correlate with the steely areas that act as water barriers in the endosperm (10,18,29). From this data, it is evident that genotype plays a role in determining the amount of water uptake by the grain, as well as in the distribution efficiency and rates of water uptake by the endosperm. Similar results have been observed by other authors (8,10,29). To further understand the effect of genotype Table 2. Partial least squares (PLS2) regression coefficients for samples sourced from different steeping times Steeping time Wi W2 W3 W4 W5 W6 W Time heci Figure S. Rapid Visco Analyser (RVA) profiles of barley samples having different water uptake values. RMSECV f? W, water uptake; RMSECV, root mean square of cross validation;!?2, coefficient of determination. wileyonhinelibrary.com/journal/jib J. nst. Brew. 2074; 120: 38-44

6 nfluence of starch pasting properties and protein content on water uptake lnstute of Brewing & Distilling GP MAV 0.9 NV 05 VT AW VT ADM 01 M*d r4. 0 o -Q 3AR VT ADPA VT com GAR il -05 U.T co V tlr.o O -01 'fl 42.1 a D 04 OS 06 a? First principa' component {42%} Figure 6. Scores and eigenvectors biplot of the principal component analysis of chemical information of commercial barley varieties analysed. SP, specific gravity; GP, grain protein; HWE, hot water extract; K, Kolbach index; FAN, free amino nitrogen; AAL, attenuation limit; DP, diastatic power Beta, beta amylase; Alpha, alpha amylase; VT ADM, Admiral; COM, Commander GAR, Gairdner SCHO, Schooner; NAy, Navigator. on water uptake by the grain, separate analyses were carried out (see Figs 3-5). Results presented in Fig. 4 showed that the rate of water uptake at a single steeping point was higher in the samples containing a low GP, compared with those having high GP content. This was observed for the Navigator and Admiral samples analysed. t has also been reported that the large amount of insoluble proteins present in the grain matrix might act as a barrier to hydration and as a consequence these varieties tend to absorb less water (7). t is well known that the relationship between endosperm modification and fermentability is complex, where in some cases fermentable extract might be reduced under modification, owing to restricted access of enzymes to the starch substrate. However, this could act on the extract component of fermentable extract (measured as HWE), rather than the fermentability component (measured as AAL). n order to produce malt of good quality, the cell walls of the endosperm, as well as a part of the small starch granules and surrounding protein matrix, should be broken down during the malting process. This can also be indicated by the degradation of endosperm reserves, which involves a parallel action of the enzymes hydrolysing GP, starch and cell wall structures. We can only speculate that it is not only the total GP content, but also the quality of the GP (e.g. hordeins profile), that might play a role in regulating water uptake by the grain in different varieties. Conclusions The results from this study have shown that, although a relationship between GP and water uptake exists, it is not universal for all of the varieties, indicating that the relationship between GP and water uptake is varietal dependent. We can only speculate that it is not only the total GP content, but also the quality of the protein (e.g. hordein profile), that plays a role in regulating water uptake by the grain, and that this is directly related to the varietal characteristics. The RVA profiles analysed also showed that the properties of the starch (e.g. pasting or J. nst, Brew. 2074; 120: gelatinization) influenced water uptake by the grain. These results suggest that the combination of GP and other endosperm characteristics, such as starch pasting properties, may additionally explain water uptake in the grain. Acknowledgements This project was supported by Australia's grain growers through their investment body the Grain Research and Development Corporation, with matching funds from the Australian government. The authors also thank the reviewers for their helpful comments. References Agu, R. C., and Palmer, G. H. (1998) Some relationships between the protein nitrogen of barley and the production of amylolytic enzymes during malting, J. nst. Brew., 104, Agu, R. C., and Palmer, G. H. (2001) The effect of nitrogen level on the performance of malting barley varieties during germination, J. nst. Brew., 107, Agu, R. C. (2003) Some relationships between malted barleys of different nitrogen levels and the wort properties, J. nst. Brew., 109, Agu, R. C. (2005) Quality assessment and performance of malted barley for food processing, Tech. Q. Master Brew. Assoc. Am., 42, Mayolle, J. E., Lullien-Pellerin, V., Corbineau, F., Boivin, P., and Guillard, V. (2012) Water diffusion and enzyme activities during malting of barley grains: A relationship assessment, J. Food Eng., 109, Swanston, J. S., and Taylor, K. (1990) The effects of different steeping regimes on water uptake, germination rate, milling energy and hot water extract, J. nst. Brew., 96, 3-6. Molina-Cano, J. L, Sopena, A., Polo, J. P., Bergareche, C., Moralejo, M. A., Swanston, J. S., and Glidewell, S. M. (2002) Relationships between barley hordeins and malting quality in a mutant of cv. Triumph.. Genetic and environmental effects on water uptake, J. Cereal Sd, 36, McEntyre, E., Ruan, R., and Fulcher, R. G. (1998) Comparison of water absorption patterns in two barley cultivars using magnetic resonance imaging, Cereal Chem., 75, Brookes, P. A., Lovett, D. A., and MacWilliam,. C. (1976) Steeping of barley - review of metabolic consequences of water uptake, and their practical implications,). nst. Brew., 82, wileyonlinelibrary.com/journal/jib

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