The Relationship Between D Hordein and Malting Quality in Barley

Journal of Cereal Science 24 (1996) 47–53

The Relationship Between D Hordein and Malting Quality in Barley K. A. Howard∗, K. R. Gayler∗, H. A. Eagles† and G. M. Halloran‡ ∗The University of Melbourne, Russell Grimwade School of Biochemistry and Molecular Biology, Parkville, Victoria, Australia, 3052, †Victorian Institute for Dryland Agriculture, Horsham, Victoria, Australia, 3400 and ‡The University of Melbourne, Department of Agriculture, Parkville, Victoria, Australia, 3052 Received 23 March 1995

ABSTRACT The contribution of grain protein to the malting quality of barley (Hordeum vulgare L.) was investigated by comparing the hordein composition and the malting quality in barley produced under a range of field conditions. Two malting cultivars, Schooner and Arapiles, and one feed cultivar, Galleon, were grown under five nitrogen regimes in each of two years. Hordein composition of the grain was determined at maturity using a combination of sodium dodecyl sulphate-polacrylamide gel electrophoresis and laser densitometry. Malt extract was determined on all samples after micromalting. Variation in growth conditions resulted in a wide range of grain protein contents and malt extract values, as well as variation in the proportions of the individual B, C and D hordeins in the grain. D hordein in particular varied over a 10-fold range. High levels of all protein fractions were associated with low malt extract. Total protein, as expected, displayed a strong, negative correlation with malt extract. The relationship was cultivar specific and separate regression lines were generated for each cultivar. Of the individual hordein fractions, D hordein displayed the strongest negative correlation with malt extract and its relationship to malt extract was independent of cultivar. A consistent relationship between D hordein and malt extract was observed across seasons, treatments and cultivars that was indicative of a causal relationship between D hordein and malting quality. D hordein therefore offers an alternative measurement to total protein for the prediction of malting quality over a wide range of environmental conditions and cultivars.  1996 Academic Press Limited

Keywords: barley, environment, hordein, malting.

INTRODUCTION

rently used to predict potential malting quality at the farm gate. It is sensitive to seasonal fluctuations in weather conditions and shows a negative correlation with malting quality as measured by malt extract1,2. While this correlation provides a useful guide to potential malting quality, it does not always explain the variation in malting quality experienced between seasons. In addition, while total protein shows high correlation with malt extract within particular cultivars, the relationship is less significant when comparisons are made between cultivars3–5. Grain protein comprises a complex mixture of polypeptides that are classified by their extractability characteristics. The alcohol-extract-

One of the difficulties facing the malting barley industry in Australia is maintaining quality from season to season. It is commonly accepted throughout the industry that seasonal fluctuations in climatic conditions are responsible for changes in malting quality. However, the specific effects that climatic factors have on grain development and subsequent malting quality are still not fully understood. Total protein is one grain characteristic cur-

Corresponding author: Dr K. R. Gayler 0733–5210/96/040047+07 $18.00/0

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 1996 Academic Press Limited

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able fraction of barley grain contains the main storage proteins, the hordeins. The hordein fraction comprises 30–50% of the total grain protein, the exact proportion depending on cultivar and nutrition6–8. Hordein comprises two main subfractions; B hordein (sulphur-rich), C hordein (sulphur-poor); and two minor sub-fractions, c hordein (sulphur-rich) and D hordein (high molecular weight), distinguishable by their electrophoretic mobilities and amino acid compositions9,10. The B and C hordeins account for 70–80% and 10–20% of the hordein fraction, respectively, while c and D hordein form less than 5% of the total hordein fraction7,9,11. The proportions of the different hordein fractions present in mature grain are affected by growth conditions, particularly N nutrition8. Hordein is synthesised during mid- to late grain filling12 and is deposited in protein bodies that distort and rupture during grain filling to form a protein matrix within the endosperm cells13. The hordein in the protein matrix is degraded during germination to provide substrates for synthesis of proteins in the growing embryo14. The mobilisation of hordein is dependent on the synthesis and activity of peptidases and proteases and the passage of these enzymes through the endosperm cells to the hordein15. Its mobilisation is considered necessary for hydrolytic enzymes to access starch grains for starch mobilisation16. The objective of this study was to examine how changes in hordein composition affect malting quality. Barley was grown under field conditions with varying amounts of applied nitrogen to produce grain samples with differences in hordein composition and protein content. Changes in hordein composition were then compared with malt extract to assess their relationship with malting quality. EXPERIMENTAL Barley samples Three Australian barley cultivars were selected from the stubble rotation of a trial grown at the Victorian Institute for Dryland Agriculture (VIDA) in Horsham, Victoria. The cultivars, Schooner and Arapiles were chosen to represent high malting quality and Galleon to represent low malting quality. The three cultivars were grown under five nitrogen regimes (0, 20, 40, 80 and 160 kg/ha applied N). The nitrogen was applied as am-

monium nitrate. Each treatment was replicated three times and the trial was conducted over two seasons (1990 and 1991).

Extraction of proteins Grain was ground in a Falling Number mill to pass through a 0·5 mm sieve. Protein fractions were extracted from samples (1–3 g) by procedures based on the methods of Giese and Hejgaard17 and Shewry et al.7,18. Salt-extractable proteins were extracted twice for 1 h each with 0·15  potassium phosphate, pH 8·0; 5 m DTT (10–25 ml) at room temperature. All extractions were homogenised for 10–30 s and continuously agitated. Unextracted material was collected by centrifugation at 1000 g for 8 min, washed once with water and the supernatants containing the extracted proteins combined. The hordeins were similarly extracted three times for 1 h each with 55% (v/v) propan-2-ol, 1% (v/v) acetic acid and 2% (v/v) 2-mercaptoethanol (15–30 ml) at 60 °C. The residual proteins, the glutelins, were extracted three times for 1 h each with 0·05  sodium borate, pH 10; 2% (v/v) 2mercaptoethanol; 1% (w/v) sodium dodecyl sulphate (15–30 ml) at room temperature. Extracted proteins were exhaustively dialysed against water and freeze dried to determine dry weight. The hordein fraction was reduced with 1% (v/v) 2mercaptoethanol and alkylated with 1·5% (v/v) 4vinyl-pyridine19 to disrupt permanently the disulphide bonds within and between the B and D hordeins.

Protein separation Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS– PAGE) in 10 and 12% acrylamide slab gels at pH 8·8 using the discontinuous buffer system of Laemmli20. Gels were fixed in 15% (w/v) trichloroacetic acid for 1 h, stained in methanol: acetic acid:water (25:9:65) (v/v/v) containing 0·5% (w/v) Coomassie Brilliant Blue R250 for 45 min at 45 °C and destained in several changes of 7·5% (v/v) acetic acid at 60 °C. Rahman et al.12 have previously shown that the B, C and D hordeins all bind Coomassie blue to a similar extent, enabling their concentrations to be estimated accurately using scanning of stained proteins.

D Hordein and malting quality

Quantitation of hordeins The total amount of hordein was determined from the dry weight of pooled freeze dried extracts. The proportions of individual hordeins were determined by densitometry of total extracts separated by SDS–PAGE (Molecular Dynamics Densitometer). The amount of B, C and D hordein in each sample was expressed as mg per gram of flour. The c hordeins were included in the estimation of B hordein as both these types of hordein migrated to the same region on the gels. Malt analysis Samples (50 g) were micromalted in an automated micromalting facility (Phoenix systems) using the following schedule; steep: 2:2:6:3:9 h 15 °C (wet: dry:wet:dry:wet), germination: 101 h at 16 °C, kiln: 30–40 °C 8 h, 40–60 °C 3 h, 60–70 °C 2 h, 70–80 °C 6 h. Malt extract was measured using a modified I.O.B. method21. Grain protein was determined by Kjeldahl N22 and multiplied by 6·25. Protein content was expressed on a dry weight basis. RESULTS Extraction and quantitation of hordeins Sequential extraction procedures were chosen to achieve complete and separate extraction of each of the classes of proteins: the albumins and globulins, the prolamins (hordeins) and the glutelins, as shown in Figure 1. Only the hordein fraction was used for further analysis. Incomplete extraction of hordein, especially B and D hordeins, has previously posed a problem when analysing hordein18,23,24. To extract the hordeins completely, three sequential extractions with 55% (v/v) propan-2-ol, 1% (v/v) acetic acid and 2% (v/v) 2mercaptoethanol at 60 °C for one hour each were used. This was necessary, especially in the samples that contained high total protein. Analysis of the residual proteins extracted after hordein (glutelins) did not show evidence of residual hordein, indicating that the hordein fraction and in particular D hordein had been extracted completely (Fig. 1). Relationships of grain characters with malting quality Seasonal differences in growth conditions, combined with the range of fertilisation treatments,

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resulted in barley samples with a wide range of malt extract values and protein contents (Table I). There was also a wide range in the amounts and the proportions of the B, C, and D hordein components. The pool size of D hordein in particular in grain samples varied over a 10-fold range (Table I). Malt extract was compared with each of the individual hordeins to detect relationships between particular proteins and malting quality. Correlations between malt extract and the various grain protein fractions for all samples are shown in Table II. Each of the components of grain protein displayed significant negative correlations with malt extract across seasons, nitrogen treatments and cultivars (Table II). The correlation of C hordein with malt extract, although significant, was low enough to suggest that C hordein has little direct relationship with malting quality as measured by malt extract. The analysis of total hordein and B hordein suggested that each of these measurements simply reflected total protein levels, and this is expected as these fractions form the majority of total protein in the grain (Table I). Although total protein and D hordein predicted malt extract with similar precision overall, there were differences within the trial that suggested that D hordein might be the more significant measurement. This was highlighted particularly in the 1991 season with the analysis of the relationship between malt extract and protein for each cultivar (Table III). For grain grown during the 1991 season, the correlation of protein with malt extract was much higher within each cultivar (−0·92 to −0·96) than the equivalent correlation (−0·55) obtained when the data from all three cultivars was combined (Table III). This suggested that the relationship between total protein and malt extract was essentially cultivar-dependent. Separate regression lines were fitted for each cultivar relating total protein and malt extract in the 1991 grain samples (Fig. 2). Analysis of grain grown in the 1990 season showed a similar trend towards cultivar-specific correlations between total protein and malt extract but with much greater scatter of the data (data not shown). In contrast to the cultivar-specific trends observed between total protein and malt extract, the relationship between D hordein and malt extract was independent of cultivar and season. The correlation coefficients for the individual cultivars were almost identical to that for the combined

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Figure 1 SDS–PAGE patterns of sequentially extracted barley proteins from cvs Arapiles, Schooner and Galleon, Lane (a) Arapiles salt-extractable proteins, (b) Schooner salt-extractable proteins, (c) Galleon salt-extractable proteins, (d) Arapiles hordein, (e) Schooner hordein, (f ) Galleon hordein, (g) Arapiles glutelin, (h) Schooner glutelin and (i) Galleon glutelin. The positions of B, C and D hordeins in lanes d to f are indicated.

Table I Variation in protein, malt extract and hordein components observed in the two seasons studied Range Difference Range of Difference of values (fold) values (fold) (%) (mg/g flour) Malt extract Protein Total Hordein B Hordein C Hordein D Hordein

53.3–81·0a 8·1–18·2b 26·9–51·9c 76·2–86·7d 10·4–18·1d 2·5–103d

1·5 2·3 — — — —

24·6–81·3 20·3–64·6 3·4–12·0 0·7–6·9

3·3 3·2 3·5 9·9

a b c ˜ of malt dry weight, % of grain dry weight, % of total protein, d % of total hordein.

Table II

Correlation coefficients (r) for malt extract with protein fractions Correlation coefficienta (r)

Total grain protein Total hordein B hordein C hordein D hordein a

−0·67 −0·70 −0·70 −0·42 −0·77

Correlations significant P<0·001.

population (Fig. 3 and Table III). Samples from the different treatments and cultivars were shown

Table III Correlation coefficients (r) for total protein (Fig. 2) and D hordein (Fig. 3) with malt extract for each cultivar compared to the overall correlation in the season 1991 Cultivar Arapiles Schooner Galleon 1991 (all cultivars) a

Total proteina

D hordeina

−0·92 −0·93 −0·96 −0·55

−0·76 −0·78 −0·69 −0·74

Correlations significant P<0·001.

to cluster within the different seasons but, ultimately, all fitted the same regression line (Fig. 3). The range of variation together with the observation of cultivar independence suggests a causal relationship between D hordein and malt extract. This property of D hordein was considered advantageous when considering its use as a predictor across seasons and cultivars. DISCUSSION In Australia, protein content is a key grain characteristic used for accepting or rejecting barley for malting. Ideally, an acceptable grain sample should be plump, clean, bright and free of disease and contain a level of protein that does not exceed the industry standard for that season. While allowance is made for the effect of season on the

D Hordein and malting quality

85

Malt extract (%)

80

75

70

65

60

0

7

8

9

10 11 12 13 Protein (%)

14

15

16

Figure 2 The relationship of malt extract and total protein for samples from the cultivars Schooner (Ε), Galleon (Χ) and Arapiles (Μ) grown under five nitrogen regimes in the season of 1991. Lines are calculated from least squares regression analysis.

85 80

Malt extract (%)

75 70 65 60 55 50

0

1

2

4 6 3 5 D hordein (mg/g flour)

7

8

Figure 3 The relationship between D hordein and malt extract for all three cultivars grown under five nitrogen regimes in both seasons; Schooner 1991 (Ε), Galleon 1991 (Χ), Arapiles 1991 (Μ), Schooner 1990, Galleon 1990 (Β) and Arapiles 1990 (Α). The line is calculated from least squares regression analysis.

protein content of the grain, all cultivars are judged on the basis of the same protein range. This ignores the effect that different cultivars have on the relationship between protein and malt extract ob-

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served in this study and in previous studies3–5. The results of the present study suggest that measurement of D hordein would be a more appropriate single measurement than protein for the prediction of malting quality when samples span a range of cultivars, climatic and agronomic conditions. The physiological basis for the relationship between D hordein and malting quality is complex and still not fully understood. Hordeins predominate in the protein matrix that surrounds the starch granules within the cells of the endosperm16. Degradation of the hordein in this matrix during malting is necessary to allow starch degrading enzymes access to the starch, facilitating complete starch hydrolysis16,25. If hordein is only partially degraded during malting and mashing, it may form a physical barrier to the passage of the starch degrading enzymes and limit starch hydrolysis16. Undegraded hordein may also form disulphide bonded complexes during mashing which can cause wort filtration problems, resulting in decreased malt extract24. Experimental evidence describing the roles that particular hordeins play in these complexes has been difficult to obtain. While hordein polypeptide patterns have been used successfully for cultivar identification7,26, no particular patterns of B and C hordeins have been linked successfully with malting quality27,28. The capacity of some hordeins to form aggregates through intra- and inter-molecular disulphide bonds has lead to the isolation of these hordeins as separate fractions, such as Hm hordein25, hordein-II10 and gel protein29–33. The amount of these aggregated hordeins extracted from grain has been shown to be dependent on the protein content of the grain and the cultivar10,25,30. Miflin et al.10 found that the amount of aggregated hordein extracted from any sample was generally higher in poor malting cultivars compared with good malting cultivars of the same total protein content. Furthermore, a negative correlation has been shown between the amount of gel protein (aggregated hordein) and both malt extract30,32 and wort filtration rate32. Gel protein has been shown to comprise both B and D hordeins31, disulphide-bonded together in long chains with D hordein as the backbone32. Comparisons between the amounts of B and D hordeins in the grain with gel protein indicated that, while B hordein is present in both aggregated and nonaggregated hordein fractions, D hordein is found nearly exclusively in the gel protein (aggregated hordein) fraction30,34. This suggests that the limiting

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factor in gel protein formation is the amount of D hordein in the grain30. Measurement of the amount of D hordein in the grain would therefore offer an estimate of the amount of gel protein that could theoretically form during malting and should correlate with malting quality. The results presented in this paper support the proposition that the amount of D hordein in the grain offers a more accurate guide to malting quality than the use of total protein alone. Unlike total protein, the relationship between D hordein and malt extract was not influenced by cultivar and therefore could be applied to samples from a range of environmental conditions and cultivars.

11.

12.

13.

14.

15.

Acknowledgements We would like to thank Joe Panozzo and Alan Bedggood at VIDA, Horsham, for their significant contribution to this project in providing the source material for analysis and the micro malting data. We would also like to thank Lachlan Ingram for his technical assistance at the University of Melbourne. This project was funded by the Grains Research and Development Corporation.

16. 17. 18.

19.

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