Effects of the waxy genotype and storage temperature of pearled barley on the volatile compounds after cooking

Abstract

Headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry was utilized to analyze the volatile compounds in cooked barley. A total of 56 compounds were identified, including 18 aldehydes, 13 ketones, 6 alcohols, 6 furans, 5 acids, 2 furfurals, and 6 others. The grains of waxy and nonwaxy barley were pearled and stored at either 5 °C or 40 °C for 5 months prior to cooking. The waxy genotypes exhibited higher levels of key odorants in cooked barley, such as hexanal, 2-octenal, 2,4-nonadienal (E, E), and 2,4-decadienal (E, E), compared to the nonwaxy genotypes. Elevated storage temperatures (40 °C) led to an increase in 42 volatile compounds, particularly 2-butyl-2-octenal in cooked barley. Principal component analysis revealed distinguishable profiles of volatile compounds among the nonwaxy 40 °C storage group, the waxy hull-less 40 °C storage group, the waxy hulled 40 °C storage group, and the 5 °C storage group.

Introduction

Barley (Hordeum vulgare L.) holds the fourth position in global cereal production and is commonly used in various forms, such as pearled barley, rolled barley, and barley flour, which are cooked alongside rice or substituted for wheat flour. Barley grains are particularly rich in dietary fiber, especially β-glucan, which comprises β-(1-3) and (1-4) glycosidic polysaccharides. β-Glucan is soluble in water and exhibits high viscosity in the stomach and intestinal tract, resulting in slower absorption of nutrients and a reduction in excessive increases in blood glucose levels (El Khoury et al., 2012). Consumption of barley β-glucan has been associated with the reduction of visceral fat accumulation (Aoe et al, 2017) and a decreased risk of cardiovascular disease by lowering total and LDL cholesterol levels (Ho et al., 2016). As a result, barley grain is widely recognized as a healthy food with various metabolic syndrome-protective benefits.

Barley exhibits two types of waxy mutants: the indigenous waxy mutant that contains a small percentage of amylose (low-amylose), and the amylose-free mutant that originated from an artificial mutation (Domon et al., 2002). The starch in waxy barley has a lower pasting temperature compared to nonwaxy barley, and the pasting temperature of amylose-free starch is even lower than that of low-amylose starch (Yanagisawa et al., 2006). Waxy barley cultivars also tend to have higher β-glucan content compared to nonwaxy barley cultivars (Cramer et al., 2005; Seki et al., 2018; Yanagisawa, 2019). Consequently, waxy barley has gained attention as a valuable source of high β-glucan content in addition to its desirable soft and sticky texture (Seki et al., 2018).

Unfortunately, barley products often have a distinct and disagreeable odor, such as when pearled and rolled barley is boiled or barley dough is produced. This unpleasant odor may be a deterrent to increasing barley intake as a staple diet. Volatile compounds from cooked rolled barley were investigated using gas chromatography-olfactometry (GC-O) and aroma extract dilution analysis (AEDA) in our prior work (Kaneko et al., 2013). Four aldehydes, hexanal, 2-nonenal, 2,4-nonadienal (E, E), and 2,4-decadienal (E, E), were identified as major odorants with the largest flavor dilution (FD) factor, and additional 19 compounds were proposed to contribute to the barley odor (Kaneko et al., 2013). Takemitsu et al. (2019) recently observed that cooking with superheated steam decreased the odor of barley and volatile compounds, such as acetic acid, 3-methylbutyric acid, and 1-hexanol, when compared to standard cooked barley. Cramer et al. (2005) discovered seven aldehydes as significant odorants in unheated whole barley flours: 3-methylbutanal, 2-methylbutanal, hexanal, 2-hexenal, 2-heptenal, 2-nonenal, and decanal. They also discovered that hulled cultivars had higher levels of volatile compounds (aldehyde, ketone, alcohol, and furan) than hull-less cultivars. The odor-active chemical hexanal was found in greater concentrations in cooked waxy rice, than in cooked nonwaxy rice (Yang et al., 2010; Fukuda et al., 2014). Zhang et al. (2023) recently reported that volatile flavor compounds in whole barley flours differed according to processing methods, such as extrusion puffed, explosion puffed, baked, and fried, before pulverization and storage. However, there are no studies describing the influence of the waxy genotype on the odor of cooked barley.

Solid-phase microextraction (SPME) is a quick, sensitive, solvent-free, and cost-effective technique of sample preparation in flavor analysis (Wardencki et al., 2004). Headspace (HS)-SPME gas chromatography-mass spectrometry (GC-MS) is a common approach for semiquantitative measurement of volatile compounds in meals. Many studies used HS-SPME GC-MS for volatile compound analyses, including analyses of different brewing barley cultivars (Dong et al., 2015), different malts (Wang et al., 2022), rice bran after storage and processing (Gao et al., 2021), rice stored under different conditions (Zhao et al., 2020), and different proso millet varieties (Yang et al., 2022).

Typically, barley products such as pearled and rolled barley are distributed and stored at ambient temperature throughout the year. The purpose of this study is to determine the effects of waxy genotype and storage temperature on the volatile compounds of cooked barley. To clarify the effect of waxy genotype on the volatile compounds, we used combinations of cultivars and lines whose genetic background is identical: ‘Fiber Snow’ and ‘Haneumamochi’, which is an amylose-free type waxy mutant of ‘Fiber Snow’ (Seki et al., 2018), and ‘Shikokuhadaka 84’ and its near-isogenic lines harboring indigenous waxy gene ‘Shikokuhadaka 84 (wxa)’ and amylose-free waxy gene ‘Shikokuhadaka 84 (wxb)’ (Yanagisawa, 2019). Pearled barley from nine cultivars and three lines was stored at 5 °C or 40 °C for five months, and their volatile compounds were evaluated using HS-SPME GC-MS after cooking.

Materials and Methods

Chemicals Authentic standards for analysis, including n-alkane standards, 2-ethylfuran, hexanal, 2-hexenal, 2-heptenal, nonanal, 2-octenal, 1-octen-3-ol, furfural, 2,4-heptadienal (E, E), 2-nonenal, benzaldehyde, 2,4-nonadienal (E, E), and 2,4-decadienal (E, E), were purchased from Sigma-Aldrich (St. Louis, MO). Additionally, 2-methylfuran, 2-methylbutanal, 2-heptanone, 2-octanone, 6-methyl-5-hepten-2-one, 1-hexanol, 2-nonanone, 3-octen-2-one, 1-heptanol, 2-ethyl-1-hexanol, 1-octanol, 5-methylfurfural, hexanoic acid and nonanoic acid were obtained from Tokyo Chemical Industry (Tokyo, Japan). Furthermore, 2-butylfuran, 2-pentylfuran, 2-propyl-2-heptenal, 2-butyl-2-octenal, and 2-methoxy-4-vinylphenol were obtained from Fujifilm Wako Chemicals (Osaka, Japan). Milli-Q grade water was used throughout the experiment.

Plant materials Table 1 shows nine cultivars and three lines of barley grown in the same experimental field in Tsukuba, Japan, and harvested in 2021. Near-isogenic lines containing indigenous waxy gene and amylose-free waxy gene were creations of our institute, by introducing waxy genes to ‘Shikokuhadaka 84’ and crossing back 5 times. Threshed barley grains were dried at 35 °C in a forced convection oven (DKN-912, Yamato Scientific Co., Tokyo, Japan) until grain moisture was less than 12 %, as determined by Riceter F (Kett, Tokyo, Japan). Using a TM-05C test mill (Satake Co., Higashihiroshima, Japan), the resulting barley grains were pearled into 55 % yield for hulled barley and 60 % yield for hull-less barley to remove the outer layers of the grain. Pearled grains of each cultivar and line were separated into two groups: one was kept at 5 °C until use (5 °C group), and the other was held at 40 °C for 5 months before being stored at 5 °C until use (40 °C group) in polyethylene bags. Portions (10 g) of the pearled grains were pulverated for 90 seconds in a TI-100 vibrating sample mill (CMT, Tokyo, Japan).

Table 1. Profile of barley cultivars and lines used.

Variety/line	Abbreviation	Row	Hull	Amylose	β-Glucan (% dw)
Fiber snow	FS	6	Hulled	normal	5.8 ± 0.3	d
Haneumamochi	HM	6	Hulled	amylose-free	8.5 ± 0.4	bc
Kusumochi nijo	K2	2	Hulled	amylose-free	7.6 ± 0.2	c
Kihadamochi	KH	6	Hulled	amylose-free	9.2 ± 0.3	b
White fiber	WF	6	Hulled	low-amylose	8.4 ± 0.2	bc
Setsugenmochi	SM	6	Hulled	low-amylose	8.4 ± 0.2	bc
Daishimochi	DM	6	Hull-less	low-amylose	8.5 ± 0.3	bc
Kirarimochi	KR	2	Hull-less	amylose-free	9.4 ± 0.7	b
Waxy fiber	WxF	2	Hull-less	amylose-free	19.5 ± 0.7	a
Shikokuhadaka 84	S84	2	Hull-less	normal	6.2 ± 0.3	d
Shikokuhadaka 84 (wxa)	wxa	2	Hull-less	low-amylose	9.4 ± 0.8	b
Shikokuhadaka 84 (wxb)	wxb	2	Hull-less	amylose-free	9.5 ± 0.6	b

β-Glucan content of pearled grain was expressed as mean ± standard deviation of triplicates. Different letters represent significant differences (p < 0.05) by Tukey’s analysis.

Chemical composition assay AOAC method 995.16 was used to determine the total β-glucan content of each pearled barley flour using a Megazyme test kit (Bray, Ireland). Each pearled barley flour’s free fatty acids (FFA) were extracted with 10 v/w isopropyl alcohol and their amounts were measured using an FFA quantification fluorometric kit (Bio Vision, Milpitas, CA, USA).

Preparation of cooked barley In a 20-mL glass vial with a screw cap, 4 g of pearled barley were submerged in 8.8 mL of water and incubated at 25 °C for 60 minutes. Following that, the vial was put in an aluminum block heater (ALB-221, Scinics Co., Tokyo, Japan) at 120 °C for 30 minutes to cook the barley, and then cooled in a water bath at 25 °C for 1 minute. The cooked barley samples were replicated four times.

HS-SPME GC-MS analysis Volatile compounds were extracted immediately after cooking using SPME fibers coated with divinylbenzene/carboxen on polydimethylsiloxane (DVB/CAR/PDMS, 50/30 μm; Supelco, Bellfonte, PA, USA) by exposing the fiber to headspace gas in the vial at 60 °C for 60 minutes using a multi-purpose sampler (GERSTEL K.K., Tokyo, Japan). The SPME fiber was inserted into the injection port of the GC/MS (7890B and 5975C, Agilent, Santa Clara, CA, USA), which was maintained at 250 °C for 3 minutes. Desorbed volatile chemicals were separated using an HP-INNOWAX column (60 m × 0.25 mm-i.d., 0.25 μm, Agilent) in an oven with a temperature rise rate of 7 °C/min from 40 °C to 250°C. Helium was used as the carrier gas, with a flow rate of 1 mL/min. The temperature at the interface was fixed at 250 °C. The mass spectrometer was set to electron impact mode, with an electron energy of 70 eV and a scan range of 45–300 m/z (a scan rate of 5.4 scans per second). The temperature of the MS source and quadrupole were tuned to 230°C and 150°C, respectively. Using C₆–C₂₄ n-alkanes, the retention times of each compound were converted to Kovats retention indices (RI). The automated mass-spectral deconvolution and identification system (AMDIS) version 2.72 was used to identify volatile compounds by comparing their mass spectra and RI to those of genuine reference standard compounds, NIST 2011 mass libraries, and the NIST Chemistry WebBook, SRD 69ⁱ⁾. Volatile chemical amounts were reported as the peak area of the quantified ion that was typical of each compound (Table 2).

Table 2. Profile of volatile compounds in cooked barley.

No.	RT	Compound name	RI (calculated / literature)	Quantified ion	Identification	Peak area in 40°C group (× 104)	Peak area in 5°C group (× 104)
Aldehydes
5	6.21	2-methylbutanal	889/903	57	MS, RI, S	1.5 ± 1.0	1.8 ± 0.7
6	6.26	3-methylbutanal	892/906	58	MS, RI	1.8 ± 1.0	2.0 ± 0.8
8	7.11	pentanal	943/982	58	MS, RI	62 ± 26	11 ± 4	*
11	8.79	hexanal	1045/1068	56	MS, RI, S	787 ± 253	317 ± 112	*
15	11.32	2-hexenal	1198/1213	69	MS, RI, S	15 ± 6	15 ± 4
22	13.45	2-heptenal	1328/1333	83	MS, RI, S	118 ± 51	82 ± 37	*
26	14.69	nonanal	1402/1396	57	MS, RI, S	78 ± 22	34 ± 15	*
28	15.46	2-octenal	1449/1442	83	MS, RI, S	259 ± 127	96 ± 40	*
31	16.21	2-propyl-2-heptenal	1494/ND	125	MS, S	17 ± 11	ND	*
35	16.83	2,4-heptadienal (E, E)	1532/1530	81	MS, RI, S	130 ± 34	232 ± 123	*
38	17.40	2-nonenal	1566/1546	83	MS, RI, S	116 ± 36	124 ± 36
39	17.54	benzaldehyde	1575/1545	106	MS, RI, S	259 ± 70	68 ± 22	*
43	18.53	2,4-octadienal (E, E)	1635/1632	81	MS, RI	22 ± 10	7.7 ± 3.0	*
45	19.30	2-decenal	1682/1639	55	MS, RI	35 ± 15	15 ± 8	*
46	19.63	benzeneacetaldehyde	1702/1668	91	MS, RI	67 ± 38	86 ± 27	*
47	19.65	2-butyl-2-octenal	1703/1663	111	MS, RI, S	547 ± 375	1.8 ± 0.8	*
49	20.41	2,4-nonadienal (E, E)	1749/1745	81	MS, RI, S	422 ± 176	144 ± 92	*
50	22.17	2,4-decadienal (E, E)	1855/1827	81	MS, RI, S	2103 ± 844	1818 ± 1163
Ketones
13	10.51	2-heptanone	1149/1158	58	MS, RI, S	63 ± 49	13 ± 12	*
16	11.72	3-octanone	1223/1251	99	MS, RI	7.7 ± 2.8	3.3 ± 2.6	*
18	12.43	2-octanone	1266/1275	58	MS, RI, S	69 ± 19	28 ± 10	*
20	12.89	1-octen-3-one	1294/1315	55	MS, RI	7.6 ± 4.1	3.9 ± 2.8	*
21	13.09	2,3-octanedione	1306/1325	99	MS, RI	36 ± 13	13 ± 4	*
23	13.60	6-methyl-5-hepten-2-one	1336/1343	108	MS, RI, S	20 ± 6	10 ± 3	*
25	14.47	2-nonanone	1389/1385	58	MS, RI, S	20 ± 7	5.0 ± 2.2	*
27	15.07	3-octen-2-one	1426/1429	111	MS, RI, S	220 ± 73	80 ± 43	*
36	16.98	3-nonen-2-one	1541/1522	125	MS, RI	52 ± 26	6.0 ± 2.9	*
37	17.23	3,5-octadien-2-one (E, Z)	1556/1556	95	MS, RI	165 ± 53	252 ± 102	*
41	18.20	3,5-octadien-2-one (E, E)	1615/1610	95	MS, RI	458 ± 109	359 ± 148	*
44	18.61	6-methyl-3,5-heptadien-2-one	1640/1602	109	MS, RI	57 ± 19	16 ± 7	*
48	19.78	acetophenone	1710/1671	105	MS, RI	22 ± 6	9.5 ± 2.5	*
Alcohols
17	12.21	1-pentanol	1252/1260	55	MS, RI, S	72 ± 29	13 ± 5	*
24	14.07	1-hexanol	1365/1356	56	MS, RI, S	41 ± 15	23 ± 9	*
29	15.70	1-octen-3-ol	1463/1456	57	MS, RI, S	312 ± 113	162 ± 61	*
30	15.90	1-heptanol	1476/1458	70	MS, RI, S	8.3 ± 2.3	2.8 ± 0.6	*
34	16.43	2-ethyl-1-hexanol	1508/1490	57	MS, RI, S	77 ± 44	51 ± 30	*
40	17.66	1-octanol	1582/1560	69	MS, RI, S	11 ± 3	4.1 ± 1.3	*
Furans
4	5.72	2-methylfuran	860/881	82	MS, RI, S	4.6 ± 1.5	5.3 ± 2.2
7	6.71	2-ethylfuran	919/936	81	MS, RI, S	81 ± 20	103 ± 30	*
9	7.91	2-propylfuran	992/1011	81	MS, RI	29 ± 7	8.9 ± 4.2	*
12	9.48	2-butylfuran	1087/1112	81	MS, RI, S	55 ± 21	16 ± 4	*
14	11.03	2-pentylfuran	1181/1198	138	MS, RI, S	570 ± 281	320 ± 173	*
19	12.63	2-(2-pentenyl)furan	1278/1282	107	MS, RI	8.1 ± 2.2	13 ± 3	*
Acids
32	16.22	acetic acid	1495/1471	60	MS, RI	27 ± 12	21 ± 11	*
51	22.56	hexanoic acid	1879/1862	60	MS, RI, S	1177 ± 470	218 ± 61	*
53	25.69	octanoic acid	2068/2051	60	MS, RI	120 ± 62	27 ± 12	*
54	27.15	nonanoic acid	2156/2152	60	MS, RI, S	63 ± 18	14 ± 5	*
56	28.53	decanoic acid	2240/2258	73	MS, RI	15 ± 6	7.3 ± 3.8	*
Furfurals
33	16.42	furfural	1507/1483	96	MS, RI, S	59 ± 18	20 ± 10	*
42	18.39	5-methylfurfural	1627/1595	109	MS, RI, S	5.0 ± 1.6	2.3 ± 0.8	*
Others
1	4.51	methanethiol	654/640	47	MS, RI	5.0 ± 2.8	4.2 ± 2.2
2	4.73	carbon disulfide	714/710	76	MS, RI	3.2 ± 1.5	3.0 ± 1.5
3	4.82	dimethyl sulfide	738/746	62	MS, RI	1.9 ± 1.8	12 ± 7	*
10	8.14	toluene	1006/1022	91	MS, RI	15 ± 5	11 ± 3	*
52	24.77	benzothiazole	2012/1996	135	MS, RI	41 ± 10	14 ± 5	*
55	27.88	2-methoxy-4-vinylphenol	2201/2210	150	MS, RI, S	137 ± 48	110 ± 43	*

MS, RI, and S mean compounds identified by mass spectra, retention indeces calculated based on n-alkanes, and genuine standards, respectively. ND means not detected. Peak area of quantified ion was expressed as mean ± standard deviation of 12 barley caltivars and lines with 4 replications. ^* means significant differences between 40 °C and 5 °C groups by Tukey’s analysis (p < 0.05).

Statistical analysis Results are presented as the mean ± standard deviation of four replicates. Differences in volatile compounds and FFA content between waxy and nonwaxy cultivars and lines were determined by Tukey’s test using JMP software version 9.02 (SAS Institute, Carry, MC, USA). Principal component analysis (PCA) of volatile compounds was also conducted using JMP software.

Results and Discussion

Volatile compounds from cooked barley The levels of volatile compounds in cooked barley were examined using HS-SPME GC-MS to compare their intensities in various samples. A total of 56 compounds were tentatively identified after deconvoluted mass chromatogram analysis using AMDIS software, and each peak was estimated by the peak area of the quantified ion (Table 2). Of the volatile compounds identified, there were 18 aldehydes, 13 ketones, 6 alcohols, 6 furans, 5 acids, 2 furfurals, and 6 others. Supplementary Table 1 shows the peak areas of 56 volatile compounds in cooked barley from 24 samples. In our previous study (Kaneko et al., 2013), 23 volatile chemicals were found as odor-active compounds with FD factors greater than one in cooked barley. In this investigation, 16 compounds (Nos. 6, 11, 14, 17, 23, 24, 27, 28, 29, 33, 38, 39, 41, 49, 50, and 51) were identified. Cramer et al. (2005) identified seven aldehydes as significant odorants in whole barley flour, and six of these seven aldehydes were identified in our investigation (Nos. 5, 6, 11, 15, 22, and 38). Even though pyrazines and pyrroles rose when barley grains were roasted (Kohyama et al., 2022), no pyrazines or pyrroles were found in this cooked barley. This observation may be attributable to differences in heating temperatures and water content.

Effect of waxy genotype on volatile compounds from cooked barley Between the waxy and nonwaxy genotypes, the levels of the cooked barley’s odor-active compounds previously reported (Kaneko et al., 2013) were compared (Fig. 1). Among the 16 compounds, 12 compounds (hexanal, 2-pentylfuran, 1-pentanol, 1-hexanol, 3-octen-2-one, 2-octenal, 1-octen-3-ol, furfural, 2-nonenal, 2,4-nonadienal (E, E), 2,4-decadienal (E, E), and hexanoic acid) were more abundant in ‘Haneumamochi’ compared to ‘Fiber Snow’ in the 40 °C group or both the 5 °C and 40 °C groups. Similar relationships or trends were observed in the volatile compounds of ‘Shikokuhadaka 84’ and its near-isogenic lines, ‘Shikokuhadaka 84 (wxa)’ and ‘Shikokuhadaka 84 (wxb)’. Hexanal was more abundant in ‘Shikokuhadaka 84 (wxb)’ compared to ‘Shikokuhadaka 84 (wxa)’, however the other compounds did not show significant differences between the two lines. According to Kaneko et al. (2013), the odor notes of 6-methyl-5-hepten-2-one, 3-octen-2-one, furfural, benzaldehyde, and 3,5-octadien-2-one (E, E) are popcorn, sweet, caramel-like, fragrant, and sweet, respectively; therefore, these compounds wouldn’t significantly add to the disagreeable odor of cooked barley. Hexanal, 2-pentylfuran, 1-pentanol, 1-hexanol, and 2,4-nonadienal (E, E) have a green or grassy odor, in contrast to 2-octenal, 2-nonenal and 2,4-decadienal (E, E), which have a fatty or oily odor. Hexanoic acid has a highly unpleasant smell. Therefore, these substances would be to blame for the bad smell of cooked barley. When waxy barley was cooked, major aldehydes such as hexanal, 2-octenal, and 2,4-decadienal (E, E) were plentiful in comparison to nonwaxy barley at both storage temperatures. Waxy hull-less barley flour contains more aldehydes than nonwaxy hull-less barley flour, according to a prior study by Cramer et al. (2005), which is consistent with our findings. Cooked waxy rice contained 16 times more hexanal than premium-quality nonwaxy rice, which had a weaker overall scent, according to Yang et al. (2010). The overall quantity of volatiles in cooked waxy rice was nearly five times greater than in nonwaxy rice, according to Fukuda et al. (2014). Although the differences between waxy and nonwaxy barley were smaller than in rice, our findings revealed that cooked waxy barley tends to have a stronger odor than cooked nonwaxy barley.

Fig. 1

Odor-active compounds of cooked barley of waxy and nonwaxy genotypes. Data are shown as mean ± standard deviations of peak area at the quantification ion of four replications. Open and solid bars are shown for samples stored at 5 °C and 40 °C, respectively. Different alphabets represent significant differences by Tukey’s analysis (p < 0.05).

Under both storage temperatures, ‘Haneumamochi’ had a considerably higher FFA content than ‘Fiber Snow’ (Fig. 2). Additionally, while the differences were negligible, near-isogenic lines of the waxy genotypes ‘Shikokuhadaka 84 (wxa)’ and ‘Shikokuhadaka 84 (wxb)’ contain greater FFA than ‘Shikokuhadaka 84’ in pearled barley held at 40 °C (Fig. 2). According to Morrison et al. (1984), waxy barley, waxy rice, and waxy maize have far lower lipid concentrations in their starch than nonwaxy grains. Thus, FFA would come from lipids other than starch granules in pearled barley. Among odor-active compounds, many aldehydes (hexanal, 2-octenal, 2-nonenal, 2,4-decadienal (E, E)), alcohols (1-pentanol, 1-octen-3-ol), 2-pentylfuran, and hexanoic acid are derived from hydroperoxides of fatty acids (Kato et al., 2022), which are generated by enzymic or nonenzymic oxidation of unsaturated fatty acids. Therefore, an abundance of their precursors, unsaturated fatty acids, would be the reason for the large amount of odor compounds in cooked waxy barley.

Fig. 2

Contents of FFA of pearled barley flours of waxy and nonwaxy genotypes. Different alphabets represent significant differences by Tukey’s analysis (p < 0.05).

Pearled grains of waxy genotype contain about 1.5 times more β-glucan than those of nonwaxy genotype (Table 1), as previously reported (Seki et al., 2018; Yanagisawa, 2019). There is no significant difference in β-glucan content between low-amylose and amylose-free genotypes. Cooked waxy barley has a more unpleasant odor than non-waxy barley despite the abundance of β-glucan.

Effect of storage temperature before cooking on volatile compounds after cooking When pearled barley was stored at 40 °C for 5 months (40 °C group), the odor of cooked barley grew fatty and stronger compared to that from cold storage (5 °C group). Among 56 volatile compounds, 42 compounds such as pentanal, 1-pentanol, 3-nonen-2-one, and hexanoic acid were significantly more abundant in the 40 °C group compared to the 5 °C group (Table 2). Particularly, 2-butyl-2-octenal was very rare or not detected in cooked barley of the 5 °C group, but it was one of the major peaks in cooked barley of the 40 °C group in all cultivars and lines analyzed. On the other hand, six volatile compounds (dimethyl sulfide, 2-ethylfuran, 2-(2-pentenyl)furan, 2,4-heptadienal (E, E), 3,5-octadien-2-one (E, Z), and benzeneacetaldehyde) were significantly more abundant in the 5 °C group (Table 2). There were no significant differences between the two groups with respect to the contents of eight compounds (methanethiol, carbon disulfide, 2-methylfuran, 2-methylbutanal, 3-methylbutanal, 2-hexenal, 2-nonenal, 2,4-decadienal (E, E)). Zhang et al. (2023) reported that 2-butyl-2-octenal increased in extrusion puffed barley flour during storage at 50 °C. Therefore, 2-butyl-2-octenal could be a marker of odor change resulting from high-temperature storage, and dimethyl sulfide could be a marker of freshness. Though 2-nonenal and 2,4-decadienal (E, E) are reported as the key odorants of cooked barley (Kaneko et al., 2013), storage temperature had little impact on the amounts of these compounds. Thus the key odorants of cooked barley are not the same as the key odorants of deterioration by high-temperature storage.

Fig. 3 shows a PCA biplot of cooked barley based on the contents of 56 volatile compounds. Principal components 1 (PC1) and 2 (PC2) explained 58.2 % and 13.4 % of the total variation of the volatile compounds, respectively. PC1 mainly described changes caused by storage at high temperatures and was prominently correlated with the contents of hexanoic acid (51, r = 0.981), 1-pentanol (17, r = 0.972), 2-butylfuran (12, r = 0.971), 3-nonen-2-one (36, r = 0.970), nonanoic acid (54, r = 0.967), hexanal (11, r = 0.966), 2-propyl-2-heptenal (31, r = 0.966), and 2,4-nonadienal (E, E) (49, r = 0.929). PC2 reflected barley genotypes such as waxy/nonwaxy and hulled/hull-less, and correlated with the contents of 2-methoxy-4-vinylphenol (55, r = 0.784), toluene (10, r = 0.776), 2-nonenal (38, r = −0.745), and 2,4-nonadienal (E, E) (49, r = −0.692). Eigen vectors of aldehydes, ketones, and furans were scattered throughout the plot area; on the other hand, eigen vectors of alcohols, acids, and furfurals were located on the PC1 positive area. Aldehydes that have a fatty or oily odor seemed to be located on the PC2 negative area. Volatile compounds of cooked barley in the 40 °C groups were distinguishable from nonwaxy, waxy hull-less, and waxy hulled, though they were undistinguishable from each other in the 5 °C group. However, both the low-amylose and amylose-free genotypes were included in the same clusters. Changes in volatile compounds in the cooked waxy hull-less barley 40 °C group were smaller than those in the waxy hulled barley 40 °C group. High-temperature storage of pearled barley appears to be one of the reasons for the fatty and stronger odor of cooked barley because oxidized products of unsaturated fatty acids increase in the 40 °C groups.

Fig. 3

PCA of volatile compounds from cooked barley. In score plot, square, circle, rhombus, and triangle represent waxy hull-less, waxy hulled, nonwaxy hull-less and nonwaxy hulled barley, respectively, and open and solid marks represent stored at 5 °C and 40 °C, respectively. The number in the loading plot represents the corresponding volatile compounds in Table 2.

Cooked barley of waxy genotypes includes more unpleasant odor-active compounds compared to nonwaxy genotypes. The volatile compounds abundant in cooked barley of waxy genotypes and those accumulated after high-temperature storage were not the same. However, low-temperature storage of pearled barley appears to suppress increases in unpleasant odors after cooking.

Conclusions

Waxy barley cultivars and lines include more odor-active compounds such as hexanal, 2-octenal, 2,4-nonadienal (E, E), 2,4-decadienal (E, E), and hexanoic acid when pearled barley grains are cooked. High-temperature storage of pearled barley increased the content of 42 volatile compounds in cooked barley, such as 2-butyl-2-octenal, 3-nonen-2-one, pentanal, 1-pentanol, and hexanoic acid. After pearled barley was stored at a high-temperature, volatile compounds in cooked barley were distinguishable between nonwaxy, waxy hull-less, and waxy hulled barley. Storage of pearled barley at a cold temperature should reduce the accumulation of unpleasant odor compounds after cooking.

Conflict of interest There are no conflicts of interest to declare.

Supplemental Table 1 is available on the website.

Supplemental Table 1. Peak area of each volatile compound in cooked barley (× 10⁴)

No.		FS	HM	K2	KH	WF	SM	DM	KR	WxF	S84	wxa	wxb
1	5°C	5.4 ± 1.7	4.6 ± 2.2	6.9 ± 1.8	5.1 ± 1.6	4.9 ± 1.8	5.7 ± 2.0	1.8 ± 1.3	4.0 ± 1.8	5.0 ± 2.8	1.9 ± 0.9	4.3 ± 1.5	2.1 ± 0.8
	40°C	8.8 ± 2.4	8.1 ± 2.6	5.0 ± 2.8	6.5 ± 1.3	5.3 ± 1.4	4.5 ± 0.2	1.7 ± 1.1	3.9 ± 2.3	2.5 ± 0.6	5.0 ± 3.4	3.1 ± 1.1	4.0 ± 2.1
2	5°C	3.8 ± 0.3	5.3 ± 1.6	2.9 ± 0.2	2.8 ± 0.3	3.1 ± 0.2	6.0 ± 0.4	1.7 ± 0.3	2.6 ± 0.2	2.1 ± 0.6	1.0 ± 0.2	2.8 ± 1.4	2.0 ± 0.5
	40°C	5.5 ± 1.6	4.6 ± 0.4	3.4 ± 0.5	2.4 ± 0.1	3.4 ± 0.4	5.0 ± 0.4	1.7 ± 0.8	3.3 ± 0.5	1.8 ± 0.8	1.4 ± 0.2	2.2 ± 0.4	2.8 ± 1.6
3	5°C	11 ± 3	27 ± 8	6.1 ± 0.9	10 ± 1	16 ± 3	8.5 ± 0.5	6.7 ± 2.7	7.9 ± 1.5	17 ± 6	4.0 ± 1.4	11 ± 2	21 ± 6
	40°C	2.5 ± 0.3	6.5 ± 2.4	1.1 ± 0.3	1.8 ± 0.5	2.3 ± 0.8	1.3 ± 0.3	0.6 ± 0.4	0.9 ± 0.3	1.4 ± 0.8	0.7 ± 0.1	2.3 ± 1.1	1.1 ± 0.4
4	5°C	3.9 ± 0.5	4.0 ± 0.6	5.8 ± 0.7	3.7 ± 0.3	4.1 ± 0.3	4.5 ± 0.6	2.5 ± 0.6	6.8 ± 2.0	8.9 ± 1.7	4.3 ± 1.7	8.6 ± 1.6	6.8 ± 2.4
	40°C	4.4 ± 0.4	4.3 ± 0.2	6.6 ± 1.7	5.0 ± 0.4	5.0 ± 0.7	3.9 ± 0.5	1.8 ± 0.2	5.2 ± 0.8	5.6 ± 1.3	4.7 ± 1.3	5.3 ± 1.4	2.1 ± 2.1
5	5°C	2.2 ± 0.4	1.5 ± 0.3	1.8 ± 0.2	0.9 ± 0.0	1.7 ± 0.2	1.9 ± 0.1	0.6 ± 0.2	1.6 ± 0.1	2.6 ± 0.6	1.4 ± 0.3	2.5 ± 0.3	2.4 ± 0.9
	40°C	1.8 ± 0.2	0.7 ± 0.1	1.0 ± 0.2	0.5 ± 0.0	0.9 ± 0.2	0.9 ± 0.3	0.5 ± 0.1	1.3 ± 0.2	2.1 ± 0.4	2.6 ± 1.4	3.2 ± 1.8	1.8 ± 0.6
6	5°C	2.4 ± 0.5	1.7 ± 0.4	2.1 ± 0.2	1.1 ± 0.2	1.9 ± 0.3	2.2 ± 0.0	0.8 ± 0.3	2.2 ± 0.5	3.0 ± 0.7	1.5 ± 0.3	2.9 ± 0.4	2.8 ± 1.2
	40°C	2.3 ± 0.4	1.0 ± 0.2	1.3 ± 0.4	0.7 ± 0.1	1.2 ± 0.0	1.0 ± 0.1	1.0 ± 0.0	1.7 ± 0.0	2.5 ± 0.4	2.9 ± 1.1	3.2 ± 1.7	1.9 ± 0.7
7	5°C	102 ± 13	80 ± 12	105 ± 10	86 ± 9	96 ± 4	97 ± 13	56 ± 11	142 ± 23	131 ± 15	80 ± 17	149 ± 14	109 ± 32
	40°C	89 ± 10	67 ± 6	92 ± 14	84 ± 8	87 ± 11	71 ± 8	47 ± 13	100 ± 10	75 ± 10	90 ± 44	81 ± 13	85 ± 22
8	5°C	6.2 ± 0.6	13 ± 2	15 ± 1	13 ± 1	10 ± 1	10 ± 1	9.8 ± 2.0	12 ± 4	18 ± 4	3.9 ± 0.7	11 ± 3	12 ± 6
	40°C	33 ± 4	79 ± 11	105 ± 16	90 ± 3	90 ± 10	61 ± 8	34 ± 6	67 ± 13	50 ± 7	27 ± 12	47 ± 17	59 ± 12
9	5°C	9.2 ± 1.1	9.6 ± 1.4	15 ± 1	9.5 ± 1.0	0.9 ± 0.1	0.7 ± 0.0	6.1 ± 0.9	13 ± 2	13 ± 1	5.7 ± 1.2	11 ± 1	9.4 ± 2.6
	40°C	29 ± 3	27 ± 3	40 ± 3	33 ± 3	34 ± 4	30 ± 3	19 ± 6	35 ± 4	26 ± 4	22 ± 7	23 ± 3	27 ± 4
10	5°C	15 ± 3	9.9 ± 3.6	12 ± 1	7.8 ± 0.6	11 ± 0	12 ± 1	5.2 ± 0.9	14 ± 1	8.9 ± 1.0	9.8 ± 1.5	14 ± 2	10 ± 3
	40°C	28 ± 3	12 ± 1	18 ± 2	11 ± 1	14 ± 2	13 ± 2	8.7 ± 2.6	15 ± 2	13 ± 2	17 ± 2	16 ± 2	14 ± 4
11	5°C	152 ± 18	427 ± 70	431 ± 28	443 ± 31	304 ± 17	338 ± 28	352 ± 46	282 ± 17	448 ± 43	130 ± 29	112 ± 28	273 ± 46
	40°C	539 ± 37	945 ± 85	1150 ± 132	1094 ± 52	1105 ± 128	856 ± 82	577 ± 103	824 ± 131	697 ± 71	394 ± 68	572 ± 83	693 ± 31
12	5°C	14 ± 2	18 ± 2	17 ± 1	19 ± 2	17 ± 1	16 ± 1	17 ± 2	18 ± 3	20 ± 2	7.8 ± 1.3	14 ± 2	11 ± 3
	40°C	37 ± 3	64 ± 8	82 ± 4	81 ± 6	79 ± 10	64 ± 6	45 ± 13	69 ± 9	42 ± 5	27 ± 8	30 ± 4	42 ± 8
13	5°C	5.7 ± 0.6	7.0 ± 0.8	8.0 ± 0.4	6.1 ± 0.4	8.0 ± 0.5	6.9 ± 0.4	15 ± 12	5.9 ± 0.7	22 ± 18	18 ± 10	30 ± 17	24 ± 12
	40°C	23 ± 2	35 ± 4	44 ± 7	38 ± 2	44 ± 4	30 ± 3	94 ± 20	27 ± 4	119 ± 60	16 ± 4	133 ± 31	137 ± 12
14	5°C	104 ± 102	388 ± 27	126 ± 142	487 ± 45	171 ± 158	342 ± 21	549 ± 58	420 ± 94	499 ± 49	155 ± 29	353 ± 91	294 ± 110
	40°C	243 ± 235	850 ± 92	475 ± 452	499 ± 471	482 ± 405	805 ± 53	592 ± 123	844 ± 110	489 ± 60	378 ± 43	531 ± 59	639 ± 117
15	5°C	15 ± 3	16 ± 4	21 ± 2	18 ± 1	18 ± 2	20 ± 2	10 ± 2	16 ± 1	14 ± 4	9.1 ± 1.9	14 ± 3	14 ± 4
	40°C	13 ± 1	21 ± 2	20 ± 6	24 ± 3	21 ± 1	18 ± 2	9.3 ± 2.3	14 ± 4	9.6 ± 3.3	6.8 ± 1.0	11 ± 4	11 ± 2
16	5°C	2.5 ± 0.6	3.1 ± 0.6	3.6 ± 0.4	2.7 ± 0.1	3.2 ± 0.4	3.7 ± 0.4	0.1 ± 0.0	11 ± 1	2.1 ± 0.3	1.0 ± 0.3	1.9 ± 0.1	1.7 ± 0.6
	40°C	8.2 ± 0.1	9.9 ± 0.8	11 ± 3	10 ± 1	11 ± 2	8.7 ± 0.8	4.8 ± 1.5	8.0 ± 2.0	6.8 ± 1.4	4.1 ± 0.8	5.0 ± 0.5	4.8 ± 0.6
17	5°C	7.6 ± 0.5	13 ± 3	22 ± 1	15 ± 2	15 ± 1	15 ± 3	9.0 ± 4.0	12 ± 4	18 ± 5	5.1 ± 0.9	13 ± 3	14 ± 5
	40°C	48 ± 8	90 ± 17	104 ± 13	109 ± 7	117 ± 14	80 ± 11	38 ± 9	68 ± 8	68 ± 10	33 ± 7	55 ± 18	58 ± 16
18	5°C	16 ± 2	27 ± 5	33 ± 3	34 ± 3	24 ± 2	31 ± 2	27 ± 2	35 ± 3	49 ± 8	13 ± 2	23 ± 3	23 ± 7
	40°C	68 ± 3	97 ± 14	83 ± 16	83 ± 4	94 ± 11	77 ± 7	51 ± 12	54 ± 12	68 ± 11	52 ± 12	54 ± 10	49 ± 5
19	5°C	10 ± 2	13 ± 1	14 ± 2	14 ± 0	14 ± 2	15 ± 1	10 ± 0	13 ± 2	15 ± 2	7.4 ± 1.3	16 ± 4	12 ± 4
	40°C	10 ± 0	7.6 ± 0.4	7.7 ± 0.8	7.7 ± 0.6	9.4 ± 1.4	8.2 ± 1.0	4.9 ± 1.3	7.6 ± 1.5	5.1 ± 0.8	10 ± 4	9.8 ± 1.3	8.0 ± 1.3
20	5°C	2.3 ± 1.0	5.0 ± 0.9	3.8 ± 0.4	5.7 ± 1.2	4.0 ± 0.6	2.8 ± 0.9	2.7 ± 0.8	9.1 ± 5.9	2.7 ± 1.0	0.8 ± 0.0	1.9 ± 1.3	3.5 ± 2.8
	40°C	3.9 ± 0.4	13 ± 0	12 ± 3	13 ± 1	10 ± 2	9.7 ± 1.4	5.9 ± 2.5	8.3 ± 2.8	4.3 ± 1.3	1.5 ± 0.0	3.1 ± 0.5	3.9 ± 0.9
21	5°C	12 ± 2	13 ± 2	20 ± 1	14 ± 1	15 ± 1	14 ± 1	8.2 ± 1.9	16 ± 2	13 ± 3	5.6 ± 1.4	10 ± 2	11 ± 2
	40°C	44 ± 5	40 ± 11	47 ± 10	47 ± 11	51 ± 16	41 ± 10	21 ± 4	34 ± 7	22 ± 2	27 ± 4	27 ± 3	26 ± 3
22	5°C	28 ± 4	112 ± 20	120 ± 6	127 ± 5	91 ± 7	89 ± 8	63 ± 19	74 ± 11	77 ± 23	22 ± 3	65 ± 11	101 ± 64
	40°C	86 ± 5	160 ± 15	179 ± 31	202 ± 12	166 ± 17	145 ± 15	84 ± 12	111 ± 26	63 ± 12	53 ± 25	78 ± 18	86 ± 9
23	5°C	9.4 ± 1.5	10 ± 1	14 ± 1	9.7 ± 0.9	13 ± 1	13 ± 1	5.7 ± 0.9	8.7 ± 0.8	10 ± 2	8.2 ± 1.6	10 ± 3	11 ± 2
	40°C	24 ± 2	21 ± 6	17 ± 10	20 ± 6	27 ± 4	22 ± 2	12 ± 2	18 ± 4	24 ± 4	16 ± 3	20 ± 3	18 ± 2
24	5°C	18 ± 4	23 ± 7	24 ± 1	24 ± 3	28 ± 10	22 ± 8	13 ± 3	24 ± 9	43 ± 13	15 ± 2	22 ± 9	22 ± 3
	40°C	26 ± 7	59 ± 12	41 ± 9	65 ± 5	46 ± 6	49 ± 5	43 ± 13	42 ± 9	47 ± 12	23 ± 5	29 ± 12	27 ± 8
25	5°C	5.3 ± 0.9	4.8 ± 1.6	7.1 ± 1.5	8.0 ± 2.8	6.9 ± 0.9	4.9 ± 3.0	2.6 ± 0.4	4.0 ± 1.9	4.4 ± 0.4	3.5 ± 1.3	4.4 ± 0.2	3.4 ± 1.0
	40°C	20 ± 1	29 ± 3	24 ± 7	27 ± 1	27 ± 4	22 ± 2	13 ± 5	19 ± 7	19 ± 6	16 ± 3.5	12 ± 1	10 ± 1
26	5°C	23 ± 5	32 ± 8	27 ± 3	41 ± 1	32 ± 5	32 ± 2	24 ± 5	41 ± 4	74 ± 13	19 ± 4	28 ± 5	29 ± 14
	40°C	64 ± 2	98 ± 6	97 ± 19	99 ± 5	100 ± 15	89 ± 8	54 ± 14	89 ± 23	78 ± 15	54 ± 10	53 ± 7	62 ± 8
27	5°C	20 ± 3	92 ± 16	161 ± 10	122 ± 12	73 ± 8	88 ± 9	77 ± 12	77 ± 7	137 ± 26	19 ± 3	44 ± 15	57 ± 9
	40°C	177 ± 18	249 ± 30	325 ± 67	301 ± 13	322 ± 21	254 ± 27	122 ± 20	207 ± 41	174 ± 29	127 ± 15	203 ± 28	180 ± 20
28	5°C	38 ± 3	141 ± 22	115 ± 6	161 ± 12	95 ± 13	101 ± 12	113 ± 17	98 ± 22	108 ± 19	30 ± 4	59 ± 7	91 ± 42
	40°C	138 ± 13	362 ± 31	378 ± 81	470 ± 24	401 ± 54	346 ± 31	212 ± 25	252 ± 53	157 ± 27	85 ± 11	137 ± 14	168 ± 10
29	5°C	140 ± 14	202 ± 28	218 ± 19	188 ± 8	193 ± 19	206 ± 11	79 ± 10	239 ± 16	124 ± 14	66 ± 12	113 ± 27	142 ± 56
	40°C	270 ± 21	430 ± 52	438 ± 88	458 ± 19	434 ± 35	369 ± 39	224 ± 55	272 ± 49	300 ± 37	139 ± 27	202 ± 21	205 ± 27
30	5°C	3.0 ± 0.3	2.7 ± 0.4	2.9 ± 0.4	2.4 ± 0.3	2.9 ± 0.1	3.2 ± 0.5	ND	3.0 ± 0.3	3.9 ± 0.3	1.8 ± 0.4	2.6 ± 0.7	2.8 ± 1.4
	40°C	8.1 ± 1.1	9.6 ± 1.2	11 ± 1.6	9.7 ± 0.4	11 ± 1	8.0 ± 0.8	4.7 ± 0.8	8.3 ± 1.2	11 ± 1	5.3 ± 1.1	6.3 ± 0.7	6.6 ± 0.5
31	5°C	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
	40°C	12 ± 0	29 ± 2	26 ± 6	32 ± 2	27 ± 5	22 ± 3	7.6 ± 3.3	18 ± 9	15 ± 7	5.7 ± 2.7	3.7 ± 0.5	4.8 ± 0.8
32	5°C	18 ± 5	22 ± 11	23 ± 18	20 ± 7	18 ± 5	21 ± 2	28 ± 21	27 ± 11	24 ± 14	22 ± 10	15 ± 8	13 ± 6
	40°C	28 ± 6	35 ± 13	20 ± 7	36 ± 18	23 ± 5	29 ± 6	31 ± 28	28 ± 13	28 ± 11	27 ± 7	21 ± 13	19 ± 12
33	5°C	19 ± 6	18 ± 3	14 ± 3	16 ± 2	17 ± 3	29 ± 12	22 ± 12	18 ± 4	33 ± 19	14 ± 2	20 ± 12	21 ± 8
	40°C	61 ± 5	76 ± 7	39 ± 12	64 ± 5	57 ± 2	90 ± 18	63 ± 10	46 ± 9	76 ± 14	45 ± 14	46 ± 8	41 ± 9
34	5°C	88 ± 31	41 ± 25	99 ± 40	72 ± 32	76 ± 8	38 ± 4	27 ± 14	42 ± 10	25 ± 8	33 ± 4	30 ± 9	39 ± 3
	40°C	202 ± 10	55 ± 3	62 ± 13	56 ± 17	89 ± 11	95 ± 16	37 ± 22	54 ± 15	38 ± 11	89 ± 15	84 ± 20	57 ± 4
35	5°C	57 ± 5	366 ± 64	294 ± 25	460 ± 50	273 ± 38	208 ± 30	288 ± 66	178 ± 38	266 ± 80	62 ± 9	112 ± 45	216 ± 65
	40°C	108 ± 20	151 ± 17	140 ± 22	197 ± 10	140 ± 8	141 ± 19	122 ± 8	112 ± 21	995 ± 17	85 ± 14	143 ± 48	125 ± 24
36	5°C	2.8 ± 0.1	9.4 ± 1.6	6.9 ± 1.0	8.6 ± 0.9	5.8 ± 0.9	6.7 ± 0.8	9.1 ± 2.0	6.7 ± 2.3	7.7 ± 1.6	1.3 ± 0.2	2.4 ± 0.7	4.2 ± 2.1
	40°C	25 ± 4	84 ± 8	67 ± 15	102 ± 3	67 ± 8	62 ± 7	48 ± 6	44 ± 10	49 ± 8	19 ± 3	28 ± 4	28 ± 4
37	5°C	102 ± 11	173 ± 34	363 ± 41	382 ± 36	272 ± 31	301 ± 38	179 ± 39	229 ± 55	362 ± 114	162 ± 29	241 ± 149	256 ± 31
	40°C	192 ± 35	131 ± 16	165 ± 51	205 ± 10	207 ± 13	183 ± 28	74 ± 8	113 ± 21	144 ± 31	151 ± 52	242 ± 46	168 ± 37
38	5°C	81 ± 9	140 ± 23	134 ± 18	175 ± 16	130 ± 22	121 ± 15	128 ± 21	122 ± 10	175 ± 34	72 ± 9	100 ± 34	110 ± 27
	40°C	87 ± 13	164 ± 15	130 ± 25	178 ± 9	139 ± 15	144 ± 17	100 ± 7	104 ± 20	85 ± 11	68 ± 7	96 ± 21	93 ± 7
39	5°C	65 ± 6	59 ± 5	63 ± 6	59 ± 5	61 ± 9	70 ± 16	60 ± 16	72 ± 22	93 ± 28	43 ± 4	100 ± 14	78 ± 39
	40°C	351 ± 28	185 ± 14	290 ± 47	232 ± 14	225 ± 9	236 ± 31	163 ± 16	304 ± 50	198 ± 32	324 ± 49	342 ± 84	259 ± 44
40	5°C	3.6 ± 0.5	3.6 ± 0.9	4.5 ± 0.9	4.3 ± 0.2	4.3 ± 0.6	4.7 ± 1.5	1.8 ± 0.2	4.8 ± 0.7	6.1 ± 1.0	2.5 ± 0.4	4.0 ± 0.9	4.5 ± 1.8
	40°C	7.8 ± 1.2	13 ± 3	16 ± 2	14 ± 1	15 ± 1	10 ± 1	7.6 ± 0.7	11 ± 1	13 ± 1	6.7 ± 1.4	9.1 ± 1.0	9.4 ± 1.1
41	5°C	139 ± 15	221 ± 35	571 ± 35	412 ± 43	320 ± 33	356 ± 34	281 ± 47	424 ± 20	606 ± 136	215 ± 25	376 ± 165	387 ± 82
	40°C	548 ± 88	338 ± 63	504 ± 69	442 ± 18	529 ± 50	450 ± 56	259 ± 30	458 ± 41	436 ± 42	429 ± 98	623 ± 123	478 ± 68
42	5°C	2.1 ± 0.1	2.4 ± 0.6	2.6 ± 0.5	2.6 ± 0.0	2.2 ± 0.4	3.1 ± 0.5	1.7 ± 0.3	2.4 ± 0.2	3.3 ± 1.4	1.4 ± 0.3	2.5 ± 0.3	1.9 ± 0.6
	40°C	6.0 ± 0.6	6.3 ± 0.4	5.0 ± 0.2	6.0 ± 0.5	6.1 ± 0.5	6.9 ± 1.2	3.6 ± 1.1	3.9 ± 1.5	4.2 ± 2.0	3.5 ± 1.3	3.6 ± 1.5	3.7 ± 0.4
43	5°C	4.5 ± 0.2	9.6 ± 1.5	8.8 ± 1.7	12 ± 1	7.8 ± 1.7	7.8 ± 1.3	9.8 ± 2.9	6.5 ± 0.4	10 ± 4	3.4 ± 0.7	4.7 ± 1.9	6.8 ± 1.8
	40°C	10 ± 1	30 ± 2	28 ± 5	42 ± 3	30 ± 3	27 ± 5	22 ± 2	20 ± 5	17 ± 5	6.9 ± 1.8	12 ± 2	14 ± 2
44	5°C	12 ± 2	13 ± 1	15 ± 3	14 ± 1	25 ± 2	17 ± 3	15 ± 4	15 ± 1	34 ± 11	13 ± 1	19 ± 8	15 ± 3
	40°C	53 ± 9	68 ± 3	44 ± 12	69 ± 4	88 ± 8	64 ± 9	41 ± 3	41 ± 5	87 ± 16	39 ± 14	46 ± 8	46 ± 8
45	5°C	7.8 ± 1.1	15 ± 3	12 ± 2	20 ± 2	12 ± 2	11 ± 1	19 ± 3	17 ± 3	35 ± 8	8.1 ± 0.9	9.2 ± 2.9	12 ± 4
	40°C	14 ± 2	56 ± 6	40 ± 6	52 ± 3	41 ± 4	38 ± 3	35 ± 6	50 ± 8	41 ± 2	10 ± 2	17 ± 11	31 ± 4
46	5°C	135 ± 12	86 ± 7	89 ± 10	57 ± 3	100 ± 7	102 ± 13	45 ± 14	65 ± 12	89 ± 21	85 ± 8	116 ± 44	77 ± 10
	40°C	133 ± 11	25 ± 0	ND	ND	ND	ND	30 ± 1	25 ± 4	49 ± 6	99 ± 19	87 ± 17	49 ± 4
47	5°C	0.5 ± 0.1	2.5 ± 0.2	1.8 ± 0.4	2.7 ± 0.4	1.5 ± 0.0	1.1 ± 0.0	ND	ND	ND	ND	ND	ND
	40°C	59 ± 7	1115 ± 90	677 ± 120	1187 ± 84	782 ± 147	591 ± 60	639 ± 43	617 ± 127	494 ± 82	60 ± 17	100 ± 10	241 ± 21
48	5°C	8.9 ± 1.1	7.1 ± 0.7	14 ± 3	9.9 ± 2.2	9.3 ± 2.7	9.5 ± 2.2	7.8 ± 1.6	8.8 ± 1.4	9.4 ± 1.6	8.7 ± 1.0	11 ± 5	9.8 ± 2.0
	40°C	33 ± 3	16 ± 4	22 ± 5	18 ± 5	22 ± 2	23 ± 3	22 ± 11	14 ± 1	25 ± 4	23 ± 6	27 ± 3	19 ± 1
49	5°C	31 ± 2	160 ± 37	169 ± 8	238 ± 25	92 ± 15	119 ± 19	269 ± 33	146 ± 43	305 ± 77	30 ± 3	62 ± 20	104 ± 44
	40°C	182 ± 35	551 ± 66	527 ± 119	748 ± 34	504 ± 55	513 ± 75	449 ± 51	479 ± 80	366 ± 59	128 ± 31	292 ± 75	331 ± 42
50	5°C	261 ± 11	3265 ± 470	2045 ± 110	3210 ± 191	1940 ± 256	1645 ± 211	3945 ± 111	1643 ± 300	2353 ± 533	318 ± 35	494 ± 325	1408 ± 299
	40°C	712 ± 136	3156 ± 256	2074 ± 636	2969 ± 119	2247 ± 183	2098 ± 224	2912 ± 156	2567 ± 2	2650 ± 237	670 ± 293	1676 ± 310	1940 ± 556
51	5°C	216 ± 24	278 ± 35	219 ± 8	248 ± 16	233 ± 30	241 ± 22	271 ± 47	263 ± 66	235 ± 72	154 ± 12	119 ± 42	141 ± 21
	40°C	806 ± 30	1858 ± 116	1344 ± 373	1766 ± 48	1512 ± 141	1515 ± 66	1179 ± 225	1196 ± 202	1014 ± 197	449 ± 132	539 ± 200	762 ± 285
52	5°C	13 ± 5	10 ± 1	17 ± 6	15 ± 5	14 ± 7	16 ± 9	12 ± 3	14 ± 5	16 ± 4	10 ± 4	15 ± 2	14 ± 5
	40°C	56 ± 7	36 ± 5	38 ± 13	41 ± 3	48 ± 4	49 ± 9	29 ± 3	39 ± 1	36 ± 2	41 ± 19	45 ± 7	38 ± 12
53	5°C	19 ± 4	34 ± 8	21 ± 4	25 ± 2	24 ± 2	34 ± 2	49 ± 12	30 ± 3	40 ± 7	14 ± 2	13 ± 7	17 ± 4
	40°C	55 ± 13	232 ± 20	137 ± 16	138 ± 19	151 ± 5	190 ± 11	171 ± 28	104 ± 13	111 ± 19	38 ± 16	49 ± 2	63 ± 20
54	5°C	8.9 ± 0.9	15 ± 4	16 ± 8	14 ± 1	10 ± 3	11 ± 1	16 ± 8	17 ± 4	20 ± 3	12 ± 5	10 ± 4	11 ± 3
	40°C	44 ± 9	88 ± 4	64 ± 21	86 ± 8	74 ± 5	62 ± 3	54 ± 7	75 ± 9	72 ± 13	38 ± 15	50 ± 8	51 ± 14
55	5°C	122 ± 8	89 ± 2	88 ± 18	90 ± 5	93 ± 15	105 ± 38	92 ± 29	92 ± 13	174 ± 66	120 ± 11	134 ± 97	127 ± 39
	40°C	190 ± 29	130 ± 16	87 ± 34	108 ± 12	139 ± 14	119 ± 17	98 ± 10	115 ± 8	193 ± 35	171 ± 80	165 ± 55	128 ± 58
56	5°C	6.1 ± 1.7	12 ± 2	4.0 ± 0.8	6.1 ± 0.9	9.4 ± 0.7	9.3 ± 0.7	14 ± 5	5.9 ± 2.3	6.8 ± 1.7	4.6 ± 3.7	4.0 ± 1.9	4.3 ± 1.1
	40°C	9.0 ± 1.7	26 ± 2	13 ± 4	22 ± 2	21 ± 1	16 ± 1	21 ± 5	16 ± 3	13 ± 3	6.3 ± 1.5	9.8 ± 2.0	10 ± 3

Peak area of quantified ion was expressed as mean ± standard deviations of four replications. ND means not detected.

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