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Barley Grain Colour – Objective Measurement and Biochemical Studies

Glen Fox1, Maria Sulman1, Kevin Young2 and Andy Inkerman1

1Barley Quality Laboratory, QDPI, Toowoomba, Queensland
Agriculture Western Australia, Esperance, Western Australia


Barley grain colour is a major grain quality trait that is effected by environmental conditions which in turn has limited the total malting barley intake in Western Australia, NSW and Queensland. Since 1998, studies have been undertaken to establish an objective method to measure grain colour (brightness – “L”) at delivery through the application of NIR. In addition, we investigated the possibility of any biochemical interactions in the development of grain brightness or yellowing (“b”).

The outcome for the first objective has been very successful with NIR calibrations for grain brightness being adopted for Qld and NSW (a calibration existed in WA). For the second objective, barley genotypes from four growing seasons in Northern Australia were evaluated for (i) grain colour (“L” and “b”) (ii) the effect of pre-harvest rain on grain appearance and quality, and (iii) for changes in total grain pigment. Preliminary evaluation of the data suggested there was relationship between grain brightness and pre-harvest rain. However, there was no relationship between “L” and pre-harvest sprouting (Falling Number method). There were significant (p < 0.05) environmental and genotype effects for “b”, grain pigment and Falling Number; while for “L” environmental conditions had a significant effect.


Australian malting barley producers lose around $10 million each year due to malting barley being down graded as a result of discolouration. The primary effect is rain just prior to harvest which results in a dark, stained appearance on the surface of the grain. In some cases, in particular in the United States of America, this staining is also attributed to fungal staining, caused by Fusarium graminearum. The disease caused by the fungus has been called Fusarium Head Blight. The F. graminearum produces a toxin, deoxynivalenol. Studies have indicated severe crop losses and down grading of malting barley crops as grain has major staining problems (de la Pena et al. 1999). In Australia, no linkages have been made to malting barley staining and any Fusarium, but rather to saprophytic fungi such as Alternaria, spp. In addition to the acceptable appearance of the grain has been decreased, what other traits have been affected due to the environmental cause of the staining, ie. pre-harvest rain?

In this study, we have examined a number of genotypes taken from a range of environments. These environments were sites in Queensland and northern New South Wales where breeding trials were grown as part of the Northern Barley Improvement Program. In particular, pre-harvest sprouting (Falling Number) and grain colour components, Lightness (“L) and Hue (“b”) were measured. We compared these against environment and genotype to observe if either had any significant effects on grain quality in terms of pre-harvest sprouting or grain colour.

Materials and Methods

Samples were collected from Stage IV trials from the Northern Barley Improvement Program from 1996 to 1999. For pre-harvest sprouting measurements, Falling Number tests were conducted. In this method approximately 100 g of barley was milled. A portion of flour, depending upon samples moisture was mixed with water and placed in a boiling bath. A plunger was used to stir the slurry for 60 seconds. The plunger was released from a pre-determined height. The time the plunger takes to fall through the mixture was recorded as Falling Number (AACC, 1985).

Grain colour was measured using a Minolta CR310 colour meter. For the colour measurement, a subsample of grain was poured into the Granular Materials Attachment. The Lightness (“L”) value was recorded. The Minolta was calibrated using the white tile provided.

Grain pigments using the outer fractions of barley grain that was pearled. The grist was mixed with isobutanol and extracted overnight. The solution was centrifuged at 4000 rpm for 10 min. The absorbance was measured at 440nm.

Additional tests were included for the 1999 trials where grain size and thousand grain weights were measured (Fox et al. 2001).

All statistical analysis was carried out in Genstat (5th edition).

Results and Discussion

Rainfall prior to harvest has been associated with weather damage and now with grain discolouration, in the form of mould appearing on the surface of the grain. The data analysed in the study covered a range of environment over four years.

The initial analysis included eight genotypes that were continued in the Stage IV trial over four years. The relationships between the parameters measured appears in Table I. There appear to be very little of a relationship between many of the parameters measured in this set of data.

Table I. Correlation coefficients for –pre-harvest weathering and grain colour components






Falling Number












Barley Pigment





However, when either sites or genotypes was considered separately, the relationship between Falling Number (FN), as a measure of weather damage, and grain discolouration (“L”) was significant (p <0.001) (Table II). In addition, the level of pre-harvest weathering also had an significant effect (p <0.001) on the yellow appearance and barley pigment levels (Table II). Additional method where the grain pigments were extracted from just husk and pericarp layers revealed significant effects between, Falling Number “L”, and “b” values. The method used to extract grain pigment was initially used to extract pigments from wheat flour for noodle production (AACC, 1985). Preliminary studies with HPLC-MS on the extract derived from this procedure on barley, suggest that xanthophyll and similar compounds were increasing as the grain increased in yellow appearance.

Table II. Levels of significance and Adjusted R2 values for Falling Number and “b”


Response Variate



Falling Number


*** (79.1)

*** (21.0)



*** (74.9)

*** (13.0)



*** (42.7)

*** (13.4)



*** (76.7)

ns (2.5)



*** (53.8)

*** (25.1)



*** (59.6)

*** (59.9)

ns not significant, *** p < 0.001

Comparison of grain quality and discolouration

Grain size and thousand grain weight measurements were compared with pre-harvest weathering levels (Falling Number) and grain discolouration, specifically grain lightness (“L), to determine if any relationships between this traits existed. The summary of results is presented in Table III. An important point to report prior to discussing these results is that for each of these sites, no disease was present during the growing season. When Falling Number was correlated with thousand grain weight and grain discolouration, there were low correlations of 0.384 and 0.299, respectively. The stronger relationship between Falling Number and grain discolouration would be due to pre-harvest rain.

Table III Summary of 1999 Stage IV site data



















Hermitage Irrigated


















lsd (p < 0.05)






Sites had a significant effect (p < 0.05) on all variables measured, whereas the effect of genotypes was not as strong. The results suggested that sites, ie. growing environment, had a significant impact on grain size and thousand grain weight. Emerald and Pirrinuan had the lowest average grain size and thousand grain weights. While Falling Number and grain discolouration was also effected by site, this would a result of a short-term effect or single climatic event rather than a growing season effect. Supporting this point is that the effect is not the same for each trait. While Emerald had the highest Falling Number value, ie. no pre-harvest sprouting, Emerald did not achieve the highest “L” value. Also at the sites where pre-harvest rain occurred, the site most effected, Pirrinuan, failed to achieve the lowest “L” value. A number of variables impact of each of these traits, but one critical point in terms of pre-harvest sprouting would be the maturity of the grain at the time of the rain fall event. Others related to this include the volume of rain as well as the dormancy of the genotype.


Grain colour remains an important attribute for malting barley growers as well as grain handlers and marketers. Pre-harvest rain is the single most important parameters that impacts on grain appearance. While the most apparent impact is the darkening of the grain caused by increased fungal contamination on the surface of the while a more subtle effect is the yellowing of the grain. The results from this study suggest that the yellowing appearance was a result of an increase in grain pigments in the out layers of the grain resulting from environment effects prior to harvest. Genotype had a significant effect of both “L” and “b”, suggesting that selection through breeding will improved the grain appearance of future varieties. Both grain discolouration and yellowing was a direct result of climatic events just prior to harvest. In regards to the relationship between grain discolouration and grain quality, it would appear that the most obvious was the level of pre-harvest sprouting, when no disease is present. However, further work may be required to link the relationships between grain quality, pre-harvest sprouting, grain discolouration and plant disease levels.


Karyn Only-Watson is thanked for carrying out the Falling Number tests. Kerry Bell is thanked for assistance in the statistical analysis. The GRDC is gratefully acknowledged for their financial support. The team of the Northern Barley Improvement program is thanked for their support in supplying samples.


1. Amercian Association of Cereal Chemists (1985) Methods of analysis.

2. Fox, G., Onley-Watson, K., Ferguson, R., Skerman, A., Poulsen, D., Johnston P., and Inkerman, A. (2001) Proceedings of the 10th Australian Barley Technical Symposium, Canberra.

3. de la Pena R., Smith K., Capettini F., Muehlbauer, G., Gallo-Meagher ,M., Dill_Macky,D., Somers D. Rasmusson, D (1999) Theor. Appl. Genet, 99, 561.

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