Euphytica 124: 55–58, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Genotypic variation in polyphenol content of barley grain

Masaya Fujita1,3, Kazuyoshi Takeda2, Noriko Kohyama1, Yoshinori Doi1 &Hitoshi Matsunaka1

1Shikoku National Agricultural Experiment Station, Zentsuji, Kagawa 765-8508 Japan; 2Research Institute forBioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan; 3Present address: National Institute ofCrop Science, Tsukuba, Ibaraki 305-8518, Japan (E-mail: mfujita@affrc.go.jp)

Received 28 December 2000; accepted 4 May 2001

Key words: browning reaction, Hordeum vulgare, polyphenol, variation

Summary

The polyphenol content in pearl barley, which is highly correlated to a browning reaction after heat treatment,was investigated using 1,347 cultivated barley varieties (H. vulgare) and two wild accessions (H. vulgare subsp.spontaneum) collected from different areas of the world. The polyphenol content in the cultivated barley shows awide variation ranging from 0.19 to 0.75 mg/g with a nearly normal frequency distribution. The polyphenol contentin the hulless varieties from Japan and Korea was low. On the other hand, the polyphenol content in wild barleywas about two times higher than the average value recorded in cultivated barley. Based on HPLC analysis, fivelowest-polyphenol content local varieties do not represent proanthocyanidin-free mutants.

Abbreviations: PA – proanthocyanidin; PDB3 – prodelphinidin B3; PCB3 – procyanidin B3

Introduction

Barley has been used for human food for a long timeand has positive effects on human health (Bhatty,1992). In Japan, hulless barley is usually used as apearl barley or as an ingredient of miso, a traditionalfermented food. However, barley is susceptible to abrowning reaction after heating that occurs when pearlbarley is cooked with rice. This is one of the con-straints on the use of barley for human food. Thetendency of barley to turn brown when heated is cor-related to its polyphenol content (Sato, 1995; Fujitaet al., 2000). Therefore, it may be possible to addressthis problem by genetically reducing the polyphenolcontent in barley grain. In this study, we investigatedthe variation of polyphenol content in barley grainusing 1,347 cultivated barley varieties and two wildaccessions collected from different areas of the world,and found some gene sources for low-polyphenolbreeding.

Materials and methods

Plant materials

SV collection that was 247 barley varieties selectedfrom ca. 8,000 accessions of an international col-lection at Okayama University, 1,065 local hullessvarieties recently collected from Nepal (NI), Tibet(CT) and the Sichuan Province of China, and twowild accessions (H. vulgare subsp. spontaneum) col-lected from West Asia by Kyoto Univ., were harvestedfrom experimental plots at Kurashiki, Okayama in1995. Thirty-two improved Japanese barley cultivars,two proanthocyanidin (PA)-free mutants (ant13-152and ant17-148, Jende-Strid, 1993) and their originalcultivar ‘Triumph’ were harvested from experimentalplots at Zentsuji, Kagawa in 1996 (Japanese cultivars)and 1999 (PA-free mutants and their original cultivar)(Table 1).

10-gram of grains were pearled to a 6.0g in the caseof the hulless barley, or to a 5.5 g in the case of thehulled barley using a small pearling machine (Kett,

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Table 1. Variation of polyphenol content in pearl barley grain

Origin or var. No. Polyphenol content

tested (mg/g)

Avg. Max. Min.

SV collection

USA & Others 3 0.55 0.58 0.51

North Africa 11 0.46 0.60 0.40

China 17 0.49 0.61 0.28

(China hulless) ( 6) (0.42) (0.50) (0.28)

Ethiopia 37 0.44 0.57 0.31

(Ethiopia hulless) ( 2) (0.48) (0.49) (0.46)

Southwest Asia 35 0.50 0.70 0.28

(SW Asia hulless) ( 4) (0.57) (0.70) (0.40)

Japan 29 0.43 0.61 0.28

(Japan hulless) ( 13) (0.38) (0.44) (0.29)

Korea 30 0.41 0.55 0.23

(Korea hulless) ( 7) (0.37) (0.45) (0.23)

Nepal 19 0.47 0.68 0.32

(Nepal hulless) ( 9) (0.42) (0.53) (0.32)

Turkey 17 0.44 0.63 0.29

Europe 49 0.44 0.73 0.19

(Europe hulless) ( 2) (0.49) (0.56) (0.41)

Sub total 247 0.45 0.73 0.19

(hulless sub total) ( 43) (0.42) (0.70) (0.23)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Tibet, China (CT) (316) (0.48) (0.75) (0.34)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sichuan Provence, China (323) (0.45) (0.63) (0.30)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nepal (NI) (426) (0.47) (0.71) (0.25)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Improved Japanese cultivars 32 0.44 0.64 0.34

(Impr. Japanese hulless cv.) ( 13) (0.39) (0.42) (0.34)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ant13-152 (induced in Triumph) 0.20

ant17-148 (induced in Triumph) 0.32

‘Triumph’ 0.58

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

H. vulgare subsp. spontaneum 2 0.98 1.09 0.87

(): hulless varieties.

Tokyo, Japan). The furrow width of a pearl grain inthe SV collection was measured and classified into 5categories by observation: (1) very narrow, (2) narrow,(3) middle, (4) wide and (5) very wide.

Assay for polyphenol content

The pearl barley was milled using a Cyclotec 1093sample mill (Tecator, Sweden). The barley flour wasextracted with methanol, and the total polyphenol

Figure 1. Frequency distribution of polyphenol content in cultivatedbarley (n = 1,347).

content was measured with the Prussian blue assay(Yanagisawa and Amano 1996). Polyphenols wereexpressed as the (+)-catechin equivalent per 1-grambarley flour.

HPLC Analysis

The barley flour was extracted with 75% acetone, andthe separations were carried out on a TSK gel ODS-80Ts QA column, 4.6 × 250 mm (Tosoh Co., Tokyo,Japan) at 30 ◦C, with solvent A (2.5% acetic acidin water) and solvent B (2.5% acetic acid in meth-anol), the solvent gradient being increased from 0%to 30% B in 30 min. The elution was monitored witha L-7450 diode array detector (Hitachi Ltd., Tokyo,Japan) at 280 nm. Known amounts of kojic acid usedas an internal standard and (+)-catechin were used forcalibration. Peaks of (+)-catechin, prodelphinidin B3(PDB3) and procyanidin B3 (PCB3) were identifiedby comparison with the authentic sample purchasedfrom Sigma Chemical Co. (St. Louis, MO, USA).

Results and discussion

The polyphenol content in cultivated barley has awide variation ranging from 0.19 to 0.75 mg/g anda frequency distribution with a nearly normal curve(Figure 1). Among the SV collection, about 17% of theentries (43/247) were hulless. The polyphenol contentof the Japanese and Korean varieties was relativelylow, and this was especially remarkable for the hullessvarieties from these areas (Table 1). The polyphenolcontent of improved Japanese hulless cultivars wasalso low, but the local hulless varieties recently col-lected from Tibet, Nepal and China represent a widevariation in polyphenol content.

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Table 2. The polyphenol constitution of low-polyphenol varieties

Collection Origin Polyphenol (+)-catechin PCB3 PDB3

No. or var. content (mg/g)

SV158 Europe 0.19 + + +

SV251 China 0.23 + + +

NI260 Nepal 0.25 + + +

NI462 Nepal 0.26 + + +

NI411 Nepal 0.27 + + +

ant13-152 Carlsberg 0.20 ± – –

ant17-148 Carlsberg 0.32 ± – –

‘Triumph’ Carlsberg 0.58 + + +

–: not detected, ±: traces, +: as in the wild type.

Figure 2. Relationship between polyphenol content and furrowwidth in the SV collection.

On the other hand, the average polyphenol contentof two wild barley was about two times higher than theaverage value recorded in cultivated barley (Table 1).

In Japan and Korea, hulless barley is primarilycooked with rice for a long time. There is a possibilitythat Japanese and Korean people have unconsciouslychosen to eat low-polyphenol barley because of itspleiotropic effect on browning.

The furrow width of a pearl grain in the SV col-lection had a significant correlation to the polyphenolcontent (Figure 2, r = 0.31∗∗). According to Kohyama& Fujita (2000), polyphenols were mainly found in theperipheral part of grains. The furrow was a part of thepericarp. Therefore the width of the furrow is relatedto the polyphenol content in pearl grain.

The polyphenol content of PA-free mutants wasclearly lower than that of their original cultivar ‘Tri-umph’. We found that the polyphenol content of some

local varieties were as low as those of the PA-freemutants, ant13-152 and ant17-158 (Table 1).

We analyzed the polyphenol constitution of twoPA-free mutants, their mother cultivar and five lowest-polyphenol content (under 0.27 mg/g) local varieties.Based on HPLC analysis, the PA-free mutants con-tained no or very little PDB3, PCB3 or (+)-catechin.On the other hand, the five lowest-polyphenol con-tent local varieties contained measurable amounts ofthese polyphenols, showing that they were not PA-freemutants (Table 2).

According to Skadhauge et al. (1997), PA-freemutants (ant13-152 and ant17-148) were more sens-itive to Fusarium attack than the wild types. Riiset al. (1995) also reported that non-pigmented PA-free breeding lines were more infected by Fusariumthan the normal lines. In Japan, Fusarium head blightcaused by Fusarium sp. is a serious problem. There-fore PA-free mutants like ant 13-152 and ant 17-148 were difficult to use as gene sources for low-polyphenol barley breeding in Japan. On the contrary,low-polyphenol local varieties, which are not PA-free,are important gene sources.

Acknowledgements

We thank Dr B. Jende-Strid, Carlsberg ResearchLaboratory, Denmark, for kindly providing the seedsof the proanthocyanidin-free mutants, and Dr AthicoIlye for reading the manuscript.

References

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