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. 2021 Aug 7;26(16):4796. doi: 10.3390/molecules26164796

Characterization of Aroma Compounds in Cooked Sorghum Using Comprehensive Two-Dimensional Gas Chromatography-Time-of-Flight Mass Spectrometry and Gas Chromatography-Olfactometry-Mass Spectrometry

Shuang Chen 1,*, Li Wang 2, Derang Ni 2, Lin Lin 2, Heyu Wang 2, Yan Xu 1
Editor: Domenico Montesano
PMCID: PMC8401407  PMID: 34443387

Abstract

Sorghum is the major raw material for the production of Chinese Baijiu (Chinese liquor) and has a great effect on the flavor of Baijiu. Volatiles in cooked glutinous and non-glutinous sorghum samples were extracted using solid-phase microextraction (SPME) and analyzed via comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and gas chromatography-olfactometry/mass spectrometry (GC-O/MS). A total of 145 volatile compounds and 52 potent odorant compounds were identified from both sorghum types according to the retention index, MS, aroma, and standards. Based on their aroma features, the compounds were grouped into eight general categories, and the intensities of each aroma group were summed. Moreover, most of the compounds detected in the cooked sorghums were also detected in commercial Chinese Baijiu, indicating that the aroma compounds produced during the sorghum cooking process have a direct and significant influence on the final flavor quality of Baijiu.

Keywords: cooked sorghum, aroma compounds, SPME, GC×GC-TOFMS, GC-O/MS

1. Introduction

Sorghum one of the oldest crops, has been widely grown in the semiarid regions of the world [1]. It is the fifth most important cereal crop in the world and has been widely used in feed and food industries globally. However, sorghum is the most commonly used raw material for the production of Chinese Baijiu (Chinese liquor) [2]. Chinese Baijiu, also known as sorghum spirit, is a traditional Chinese distilled spirit and the most consumed spirit in the world (over 7 billion liters annually) [3]. Baijiu is usually distilled from fermented sorghum, and the demand for sorghum exceeds 4 million tons per year [3]. The production of Baijiu involves a typical repeated-batch fermentation and distillation process [4]. Fermented sorghum is usually mixed with soaked fresh sorghum to undergo solid-state distillation. Alcohol distillation and cooking of the fresh sorghum occur simultaneously. The aroma compounds generated during the cooking process are directly infused into the freshly produced Baijiu [3]. Therefore, the aroma compounds of cooked sorghum can greatly impact the final flavor quality of the Baijiu [5]. Depending on its amylose content, sorghum can be classified as non-glutinous or glutinous [5]. Both types of sorghums are widely used for Baijiu production, with the overall flavor characteristics of the Baijiu produced being significantly different [3].

As a pleasurable alcoholic beverage, the quality of Baijiu is highly dependent on its aroma quality, with the presence of volatile compounds being vital to its aroma characteristics. As far as Baijiu manufacturing is concerned, a significant amount of research on the aroma of Baijiu has already been carried out [6,7]. More than 300 aroma-active compounds have been identified in Baijiu, comprising numerous chemical classes, including esters, alcohols, fatty acids, pyrazines, and terpenes [8]. These aroma-active compounds may be derived from the raw materials, fermentation, distillation and aging processes used during production [9]. Since sorghum is the major raw material in the production of Chinese Baijiu, a few authors have identified the volatile compounds of sorghum. Using simultaneous distillation-extraction (SDE) and headspace-solid-phase microextraction (SPME) with gas chromatography-mass spectrometry (GC-MS), Lian et al. [10] identified 108 volatile compounds in sorghum.

However, not all of these compounds are aroma-active compounds. Gas chromatography-olfactometry (GC-O) is the most widely used analytical method for evaluating which volatile compounds are also important odorants in foods. Wu et al. [11] were the first to study the aroma-active compounds of cooked sorghum by GC-O/MS, tentatively identifying a total of 26 aroma-active compounds through comparison with the NIST retention index database. However, the aroma-active compounds that are responsible for the aroma profiles of different types of sorghum have not yet been clearly elucidated.

Therefore, the aims of this work were to: (I) develop an in-vial cooking method to capture and enrich the volatile compounds of cooked sorghum; (II) characterize the overall volatile compound profile by means of GC×GC-TOFMS; (III) characterize the aroma-active compounds of cooked sorghum by GC-O/MS; and (IV) compare and identify differences in the aroma profiles of glutinous and non-glutinous sorghum. The results of this study could provide a foundation for understanding the aroma role of sorghum as an ingredient in foods and beverages, such as Baijiu.

2. Results and Discussion

2.1. Optimization of the Aroma Extraction Method for Cooked Sorghum

Solid-phase microextraction (SPME) is widely used for the analysis of aroma compounds due to its relatively simple sample preparation and fully automated operation [12]. In this study, SPME was used to analyze the aroma compounds in cooked sorghum. Before analysis, the SPME conditions were optimized. Firstly, four SPME fibers with different coatings, PDMS, PDMS/DVB, CAR/PDMS, and DVB/CAR/PDMS were evaluated using GC-MS. Among them, the DVB/CAR/PDMS fiber, with a bipolar coating, had the highest extraction yield of aroma compounds. As a result, this fiber was selected to further optimize the extraction temperature and time of the SPME procedure. GC-MS analysis revealed that the volatile compound content of cooked sorghum increases significantly (p < 0.05) with increases in the extraction temperature (40–80 °C) (Figure 1A) and time (30–120 min) (Figure 1B). Therefore, 80 °C (upper temperature limit of the instrument) and 120 min were determined to be the optimal conditions for aroma extraction.

Figure 1.

Figure 1

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(A) extraction temperatures, and (B) extraction times of solid-phase microextraction (SPME) with corresponding gas chromatography-mass spectrometry (GC-MS) peak areas. Different letters indicate significant differences in values (p < 0.5).

2.2. Analysis of Volatiles in Cooked Sorghum Samples by Using GC×GC-TOFMS

Figure 2 shows the bubble plot obtained by GC×GC-TOFMS analysis of the volatile components of two cooked sorghum samples. Tentative compound identification was performed by firstly comparing database mass spectra with experimental ones, where database matches were considered with a spectral similarity of higher than 80%, and secondly, comparing between calculated and literature retention values, where a difference value within 50 is desirable. Thirdly, the positive identification of 98 compounds (~67% of the total number) was performed with commercially available pure standard compounds injected for qualitative analysis. Finally, a total of 145 volatile compounds were retained in this study, among which aldehydes were present in the highest number (34), followed by alcohol (25), ketones (19), esters (16), terpenes (14), acids (14), heterocyclics (12), sulfides (4), aromatics (3), pyrazines (2), and lactones (2) (Table 1).

Figure 2.

Figure 2

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The 2D bubble plot of glutinous and non-glutinous sorghum samples.

Table 1.

Comparison of volatile compounds detected in cooked glutinous and non-glutinous sorghum by HS-SPME-GC×GC-TOFMS.

Class Number of Compounds
Glutinous Non-Glutinous Total
Phenol 2 3 3
Pyrazines 1 2 2
Lactones 2 2 2
Sulfur compounds 3 4 4
Acids 11 12 14
Heterocyclics 10 12 12
Esters 13 8 16
Ketones 12 15 19
Terpenes 14 14 14
Alcohols 19 18 25
Aldehydes 25 32 34
Total 112 122 145
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As shown in Figure 3, aldehydes were the most abundant class of volatile compounds in the cooked sorghum samples and both saturated and unsaturated linear chain compounds were representative of this class [5]. Aldehydes usually derive from lipid oxidation, and these results could be related to the larger amount of lipids present in the bran layers. Saturated fatty aldehydes which are aromatics, have a greater contribution to the aroma of a cereal product because of their low odor threshold values (OAVs), and provide almond, malt, pungent (pentanal), grassy, green and fatty (hexanal), fatty, citrus and rancid (heptanal), fatty, soapy and green (octanal), fatty, citrus and green (nonanal) characteristics. (E)-2-enal is an important kind of aldehyde, there are (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal and (E)-2-decenal, respectively. Most of them show green, fruity, and fatty odors and the odor characters change with increases of the carbon chain length (Table 2). (E)-2-enals were not detected in the free aroma of original sorghum, but were detected in various aroma types of Baijiu, indicating that sorghum cooking can promote the introduction of fatty aldehydes into liquor, thus improving the flavor quality of baijiu.

Figure 3.

Figure 3

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Comparison of identification compounds in cooked glutinous and non-glutinous sorghum obtained by HS-SPME-GC×GC-TOFMS.

Table 2.

Volatile compounds identified in cooked glutinous and non-glutinous sorghums by HS-SPME-GC×GC-TOFMS.

Compounds Peak Area RI Aroma c Identification d
Glutinous Sorghum Non-Glutinous Sorghum Cla-RI a Lit-RI b
Aldehydes
Acetaldehyde 9,675,368 ± 350,920 2,416,216 ± 104,048 709 718 pungent, ether, fresh RI, MS, STD
Butanal 903,728 ± 59,123 22,033,094 ± 215,879 875 854 pungent, green RI, MS, Tent
2-Propenal 95,002 ± 8637 164,626 ± 10,770 832 838 almond, cherry RI, MS, Tent
2-Methyl-2-propenal 53,945 ± 4904 95,014 ± 6216 887 893 wild, hyacinth, foliage RI, MS, STD
2-Methyl-butanal 989,169 ± 64,712 34,559,663 ± 61,488 904 910 malt RI, MS, STD
3-Methyl-butanal 2,194,192 ± 79,582 2,973,227 ± 45,278 917 916 cocoa, almond RI, MS, STD
Pentanal 923,054 ± 60,387 100,296,059 ± 69,876 985 976 almond, malt, pungent RI, MS, Tent
2-Butenal 162,609 ± 6776 842,320 ± 113,039 1056 1042 floral RI, MS, Tent
Hexanal 2,355,706 ± 85,440 6,868,006 ± 104,589 1095 1083 grass, tallow, fatty RI, MS, STD
(E)-2-Pentenal - 804,648 ± 655,501 1125 1147 strawberry, fruity RI, MS, STD
Heptanal 105,864 ± 4411 475,576 ± 63,822 1189 1178 fatty, citrus, rancid RI, MS, Tent
3-Methyl-2-butenal 89,259 ± 8115 101,051 ± 6611 1212 1215 sweet fruity RI, MS, Tent
2-Hexenal 197,246 ± 8219 1,106,558 ± 14,849 1207 1216 apple, green RI, MS, STD
Octanal 1,390,299 ± 50,426 2,519,793 ± 38,373 1310 1319 fatty, soapy, green RI, MS, STD
(E)-2-Heptenal 590,306 ± 38,618 - 1333 1320 soapy, fatty, almond RI, MS, STD
(E)-2-Octenal 3,337,651 ± 121,055 2,579,505 ± 39,282 1443 1436 green RI, MS, STD
Nonanal 11,917,467 ± 432,240 15,915,159 ± 685,342 1403 1422 fatty, citrus, green RI, MS, STD
2-Octenal - 14,342,203 ± 116,837 1443 1445 fatty green herbal RI, MS, Tent
Decanal 1,631,776 ± 59,184 2,340,543 ± 35,643 1506 1506 soapy, orange peel RI, MS, STD
Nonenal - 11,040,264 ± 89,938 1440 1453 green cucumber RI, MS, STD
(E)-2-Nonenal 829,816 ± 54,287 - 1520 1535 cucumber, fatty, green RI, MS, STD
Benzaldehyde 3,571,972 ± 129,554 15,600,161 ± 23,756 1549 1524 almond, burnt, sugar RI, MS, STD
(E, Z)-2,6-Nonadienal - 1,781,208 ± 14,510 1597 1591 cucumber, wax, green RI, MS, STD
Undecanal - 2,441,022 ± 19,885 1611 1609 oil, pungent, sweet RI, MS, STD
2,4-Octadienal - 832,551 ± 678,232 1575 1585 green, cucumber RI, MS, STD
(E)-2-Decenal - 1,776,196 ± 14,469 1650 1641 tallow, waxy, fatty RI, MS, STD
Phenylacetaldehyde 16,153,454 ± 585,877 26,877,978 ± 11,574 1658 1650 honey RI, MS, STD
Dodecanal - 1,731,008 ± 141,015 1715 1709 soapy, citrus, green RI, MS, Tent
(E, E)-2,4-Nonadienal 729,557 ± 47,728 48,460,352 ± 474,815 1713 1686 fatty, tropical fruity RI, MS, STD
(E)-2-Undecenal 510,997 ± 3430 5,568,899 ± 56,396 1719 1736 soapy, fatty, green RI, MS, STD
(E, E)-2,4-Decadienal 466,460 ± 30,516 40,596,386 ± 397,762 1778 1783 fried, wax, fatty RI, MS, STD
2,4-Dimethyl-benzaldehyde 96,234 ± 4010 158,918 ± 21,327 1748 1710 cherry, almond, vanilla RI, MS, Tent
2-Methyl-undecanal 384,132 ± 3806 357,289 ± 18,627 1609 1636 waxy, fatty, metallic RI, MS, Tent
1H-Pyrrole-2-carboxaldehyde - 179,875 ± 46,534 2046 2059 musty, beefy, coffee RI, MS, Tent
Alcohols
2-Propanol - 2,823,662 ± 23,002 921 950 musty, woody RI, MS, Tent
2-Methyl-butanol - 284,448 ± 12,249 1200 1214 wine, onion RI, MS, STD
Pentanol 4,122,806 ± 149,532 - 1261 1275 fruity RI, MS, STD
3-methyl-3-butenol 87,123 ± 3630 111,599 ± 14,977 1136 1118 sweet fruity RI, MS, STD
2-Heptanol 4,272,658 ± 54,967 - 1316 1280 mushroom RI, MS, STD
(Z)-2-Pentenol 99,697 ± 4154 115,061 ± 15,441 1310 1327 green, metallic RI, MS, STD
Hexanol 16,714,881 ± 60,239 5,390,684 ± 22,135 1356 1350 resin, flower, green RI, MS, STD
3-Hexenol - 621,386 ± 506,208 1393 1398 green leafy RI, MS, Tent
1-Octen-3-ol 8,675,498 ± 314,656 6,647,311 ± 26,248 1442 1456 mushroom RI, MS, STD
Heptanol 1,929,513 ± 69,983 431,169 ± 6566 1440 1421 green RI, MS, STD
2-Ethyl-hexanol - 983,534 ± 42,353 1490 1481 rose, green RI, MS, Tent
(E)-2-Heptenol 133,566 ± 5565 - 1531 1546 pungent, fatty RI, MS, Tent
2-Nonanol 980,889 ± 64,171 8,535,137 ± 862,710 1555 1532 cucumber RI, MS, Tent
Octanol 3,985,051 ± 144,536 1,399,177 ± 21,308 1565 1572 moss, nut, mushroom RI, MS, STD
(Z)-3-Octenol 45,855 ± 4169 - 1566 1563 melon, earthy RI, MS, Tent
4-Methoxy-benzenemethanol - 31,493 ± 1356 1161 1178 flower, cherry, vanilla RI, MS, Tent
(E)-2-Octenol 723,534 ± 47,334 - 1616 1645 mushroom RI, MS, Tent
4-Methyl-pentanol 75,541 ± 6868 - 1334 1316 nutty RI, MS, Tent
Nonanol 1,639,336 ± 59,458 728,439 ± 11,093 1663 1651 fatty, green RI, MS, STD
Decanol 194,979 ± 8124 267,183 ± 35,856 1761 1784 fatty RI, MS, Tent
2-Undecanol 153,446 ± 6394 291,288 ± 39,091 1734 1717 fresh wax RI, MS, Tent
1-Hepten-3-ol - 299,299 ± 43,822 1346 1331 sweet, green RI, MS, STD
Benzenemethanol 544,118 ± 35,597 102,365,753 ± 297,758 1885 1902 sweet, flower RI, MS, STD
Phenylethyl alcohol 4,612,402 ± 167,289 6,865,240 ± 295,633 1900 1931 honey, spice, rose RI, MS, STD
2-Phenoxy-ethanol 48,375 ± 4398 - 2126 2142 rose, cinnamyl RI, MS, Tent
Ketones
2-Propanone - 493,389 ± 1936 791 816 solvent, pear RI, MS, Tent
2-Pentanone - 2,038,749 ± 16,608 963 1003 ether, fruity RI, MS, Tent
2,3-Butanedione 940,134 ± 34,098 - 965 977 butter, sweet, creamy RI, MS, STD
2,3-Pentanedione 99,279 ± 9026 - 1086 1062 sweet, butter, creamy RI, MS, Tent
2-Heptanone - 6,160,141 ± 18,317 1213 1191 soap RI, MS, Tent
3-Octanone - 4,162,680 ± 91,099 1226 1252 soap, gasoline RI, MS, Tent
2-Octanone 2,021,178 ± 73,307 4,559,765 ± 69,438 1261 1309 herb, butter, milk RI, MS, STD
3-Hydroxy-2-butanone 192,650 ± 8027 - 1269 1277 butter, cream RI, MS, STD
6-Methyl-5-hepten-2-one 1,476,409 ± 53,549 3,481,086 ± 53,012 1315 1342 pepper, mushroom RI, MS, Tent
2,3-Octanedione 4,153,568 ± 61,383 3,576,519 ± 5446 1325 1330 cream, sweet RI, MS, STD
2-Nonanone 73,548 ± 6686 - 1390 1385 sweet, coconut RI, MS, STD
3-Octen-2-one 1,637,944 ± 59,408 2,551,762 ± 38,860 1391 1408 nut, crushed bug RI, MS, Tent
3-Penten-2-one - 325,033 ± 264,786 1159 1120 fruity, phenolic RI, MS, Tent
2-Decanone - 2,296,398 ± 1,870,745 1466 1480 flower, fatty RI, MS, Tent
3-Nonen-2-one 403,732 ± 26,413 33,365,624 ± 691,571 1493 1520 berry RI, MS, Tent
3,5-Octadien-2-one - 638,633 ± 52,025 1538 1563 green grassy RI, MS, Tent
2-Undecanone 1,521,845 ± 55,197 603,365 ± 9189 1571 1592 orange, fresh, green RI, MS, STD
Acetophenone 5,212,317 ± 189,048 3,985,709 ± 17,163 1667 1655 floral RI, MS, STD
4-(5-Methyl-2-furanyl)-2-butanone 44,108 ± 4010 242,127 ± 15,840 1769 1745 bitter, roasted RI, MS, STD
Esters
Ethyl formate 2,930,691 ± 10,629 4,730,110 ± 72,032 806 842 pungent RI, MS, STD
Acetic acid, ethyl ester 2,141,451 ± 77,669 2,424,633 ± 36,924 864 902 fruity, sweet RI, MS, STD
3-Methylbutanoic acid, ethyl ester 339,055 ± 33,600 - 1078 1067 fruity RI, MS, STD
Pentanoic acid, ethyl ester - 1,340,571 ± 10,920 1112 1159 yeast, fruity RI, MS, STD
Hexanoic acid, methyl ester 151,637 ± 6318 - 1189 1183 fruity, fresh, sweet RI, MS, Tent
Hexanoic acid, ethyl ester 2,006,843 ± 72,787 1,214,227 ± 18,491 1199 1240 apple, sweet RI, MS, STD
Acetic acid, hexyl ester 262,775 ± 26,041 - 1265 1277 herb, fruity, sweet RI, MS, STD
2-Hydroxy- propanoic acid, ethyl ester 6,528,852 ± 23,678 7,666,361 ± 33,013 1321 1345 fruity RI, MS, Tent
Trans-2-octenoic acid, ethyl ester 70,963 ± 6451 - 1534 1540 fruity, pear, plum RI, MS, Tent
Octanoic acid, ethyl ester 115,957 ± 4832 - 1413 1435 fruity, wine RI, MS, STD
Heptanoic acid, methyl ester 101,821 ± 4243 - 1283 1292 sweet fruity, green RI, MS, Tent
Hexanoic acid, pentyl ester 21,086 ± 1917 - 1513 1501 green fruity RI, MS, STD
Hexanoic acid, hexyl ester 87,149 ± 7923 - 1585 1599 apple peel, peach RI, MS, STD
Benzoic acid, methyl ester - 1,546,633 ± 12,599 1612 1637 prune, lettuce, herb RI, MS, Tent
Benzoic acid, 1-methylethyl ester - 8,085,255 ± 65,659 1637 1639 sweet, fruity, floral RI, MS, Tent
Hexadecanoic acid, ethyl ester 635,304 ± 41,562 161,126,361 ± 157,871 2228 2240 waxy, fruity RI, MS, Tent
Terpenes
α-Terpinene 383,922 ± 38,047 276,282 ± 14,404 1162 1172 citrus-like, floral RI, MS, STD
α-Limonene 273,432 ± 27,097 161,047 ± 8396 1193 1200 citrus-like RI, MS, STD
γ-Terpinene 116,456 ± 4853 172,494 ± 2314 1230 1238 citrus-like, lemon RI, MS, STD
Terpinolene 154,506 ± 6438 240,321 ± 3225 1306 1280 citrus-like, floral RI, MS, STD
β-Elemene 168,909 ± 7038 343,075 ± 46,041 1598 1586 fruity, sweet RI, MS, STD
(-)-α-Cedrene 180,884 ± 7537 367,397 ± 49,305 1568 1571 cypress RI, MS, STD
Thujopsene 255,330 ± 25,303 150,385 ± 7840 1600 1606 cypress RI, MS, STD
β-Terpineol 108,746 ± 4531 161,074 ± 21,616 1632 1616 clove RI, MS, STD
Linalool 80,301 ± 7300 130,720 ± 8552 1552 1535 flower, wood RI, MS, STD
β-Damascenone 402,071 ± 3984 366,180 ± 19,090 1832 1827 sweet, honey RI, MS, STD
Geranyl acetone 578,093 ± 37,819 14,052,357 ± 37,684 1861 1862 fruity RI, MS, STD
(E)-β-ionone 334,172 ± 33,116 267,967 ± 13,970 1952 1953 violet RI, MS, STD
D-nerolidol 343,040 ± 33,995 231,477 ± 12,068 2272 2290 mild floral RI, MS, STD
(E)-Nerolidol 299,822 ± 29,712 387,590 ± 20,206 2040 2054 rose, apple RI, MS, STD
Acids
Acetic acid 18,084,293 ± 655,907 23,545,032 ± 1,013,901 1415 1398 acid, sour RI, MS, STD
2-Methyl-propanoic acid 90,203 ± 3759 - 1565 1524 rancid, butter, cheese RI, MS, STD
Butanoic acid 805,495 ± 8566 507,462 ± 6012 1625 1628 acid, sweaty RI, MS, STD
3-Methyl-butanoic acid - 593,181 ± 25,544 1660 1648 sweat, acid, rancid RI, MS, STD
Pentanoic acid 656,331 ± 42,938 - 1737 1733 acidic, cheesy RI, MS, STD
Hexanoic acid 32,242,500 ± 1,169,417 15,491,525 ± 667,099 1850 1857 fatty, sweat, cheese RI, MS, STD
Heptanoic acid 8,631,813 ± 313,071 1,850,130 ± 79,671 2134 2142 rancid, sour, cheesy RI, MS, STD
Octanoic acid 11,893,186 ± 431,359 3,281,620 ± 141,314 1472 1462 sweat, cheese RI, MS, STD
Nonanoic acid 11,290,020 ± 409,483 3,026,817 ± 130,342 2164 2180 green, fatty RI, MS, STD
Decanoic acid 1,549,490 ± 56,199 1,115,042 ± 16,981 2253 2264 rancid, fatty RI, MS, STD
Hydrocinnamic acid - 27,120 ± 1168 2603 2633 sweet, fatty, rose RI, MS, STD
Hexadecanoic acid - 38,378,443 ± 165,266 2886 2876 waxy, creamy fatty RI, MS, Tent
Benzoic acid 1,480,735 ± 53,706 3,555,504 ± 54,145 2410 2404 urine RI, MS, STD
Tetradecanoic acid 2,554,084 ± 92,635 4,176,313 ± 63,599 2716 2734 fatty, soapy, coconut RI, MS, Tent
Heterocyclics
2-Methylfuran 286,945 ± 28,436 1,597,938 ± 83,305 848 875 chocolate RI, MS, Tent
2,5-Dimethyl-furan 195,451 ± 8144 462,775 ± 62,104 934 958 chemical, meaty, gravy RI, MS, STD
2-Vinylfuran - 3,956,245 ± 32,229 1059 1085 coffee RI, MS, STD
2-Pentylfuran 30,803,201 ± 11,172 17,918,860 ± 771,626 1209 1241 green bean, butter RI, MS, STD
Furfural 1,337,092 ± 48,496 16,802,768 ± 25,588 1459 1486 almond RI, MS, STD
2-Acetyl-5-methylfuran 88,602 ± 8055 402,960 ± 26,362 1612 1608 musty, nutty, coconut RI, MS, STD
2-Furanmethanol 96,955 ± 4040 419,643 ± 56,316 1645 1666 burnt RI, MS, STD
1-(5-Methyl-2-furanyl)-propanone - 1,748,396 ± 14,243 1671 1686 green, hazelnut-like RI, MS, Tent
2-Pentanoylfuran 176,476 ± 7353 106,465 ± 14,288 1750 1747 sweet caramel RI, MS, Tent
2 (5H)-Furanone 106,282 ± 4429 234,854 ± 31,517 1761 1787 buttery RI, MS, STD
2-Hexanoyl furan 312,315 ± 30,950 183,949 ± 9590 1858 1872 sweet, fruity, green RI, MS, Tent
2-Acetylpyrrole 1,648,279 ± 59,782 137,960 ± 2101 1987 2002 popcorn-like RI, MS, STD
Sulfur compounds
Methanethiol 283,822 ± 28,127 289,490 ± 15,092 602 643 sulfur, gasoline, garlic RI, MS, STD
Dimethyl disulfide 316,357 ± 31,351 891,894 ± 46,497 1059 1078 onion, cabbage, putrid RI, MS, STD
Dimethyl trisulfide 338,026 ± 33,498 763,285 ± 39,792 1353 1400 sulfur, fish, cabbage RI, MS, STD
2-Pentyl-thiophene - 667,400 ± 54,360 1422 1460 sweet, fruity RI, MS, Tent
Phenols
4-Ethyl-2-methoxy-phenol - 318,607 ± 25,955 2019 2048 smoky, clove RI, MS, STD
2-Methoxy-4-vinylphenol 7,974,386 ± 28,922 647,661 ± 27,890 2214 2213 smoky, clove RI, MS, STD
Vanillin 127,696 ± 5321 736,429 ± 98,828 2551 2530 sweet RI, MS, STD
Pyrazines
2,6-Dimethyl-pyrazine - 4,684,846 ± 38,164 1335 1308 roasted nut, cocoa RI, MS, STD
Tetramethyl-pyrazine 59,882 ± 5444 1,142,923 ± 74,771 1472 1462 nutty, coffee, burnt RI, MS, STD
Lactones
γ-Octalactone 3,815,443 ± 13,838 284,300 ± 4330 1941 1924 coconut milk RI, MS, STD
γ-Nonalactone 7,974,386 ± 28,922 647,661 ± 27,890 2053 2044 coconut milk RI, MS, STD
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a Cal RI: calculated linear retention indices. b Lit RI: literature linear retention indices obtained from the NIST library (https://webbook.nist.gov/chemistry/, (accessed on 7 Auguest 2021)). c The aroma characteristic of the compound itself. d Identification: tentative identification (Tent.) based on retention indices (RI) and mass spectra (MS), positive identification based on retention times of authentic standards (STD).

Alcohols are usually produced by the decomposition of the secondary hydroperoxides of fatty acids. Pentanol, hexanol, heptanol and octanol are saturated fatty alcohols, mostly showing grass, flower and fruity odors. 1-octene-3-ol, also known as mushroom alcohol, has a typical mushroom smell, and the threshold in the air is only 1 μg/L [5].

Ketone compounds formed by the autoxidation of fatty acids were additional major volatile components in the cooked sorghum. Among the various ketones identified in the current study, most of them present butter, cream and fruity odors.

In total, 16 esters were identified in cooked sorghum, and they usually contributed to desirable odors, like fruity, floral and sweety.

Terpenes are a kind of natural hydrocarbon that are widely found in plants. In the current study a total of 14 terpenoid compounds were detected, including mono- and poly-terpene hydrocarbons, alcohols, carbonyls and esters.

A total of 14 acid compounds were identified in this study, of these identified acids, the homologous series of straight-chain monocarboxylic fatty acids from C4–C10, C14, and C16 were all detected, which contributed to acidic, rancid and cheesy odors.

Some heterocycles were also detected in the samples, including furan, furanone and pyrrole. 2-Pentylfuran exists in the cooking process of sorghum and it is derived from lipid peroxidation. This compound has a typical butter odor at a dilute concentration, but at higher concentrations, it has the less pleasant aroma characteristic of soybeans [13]. Furfural (sweet, woody and almond) which has been identified as an aroma-active compound in some cereals, was only detected in cooked samples.

2.3. Analysis of Aroma-Active Volatiles in Cooked Sorghums by Using GC-O/MS

The volatile compounds of the cooked glutinous and non-glutinous sorghum samples were directly extracted using the optimized headspace-SPME method and analyzed by GC-O/MS. The analysis results showed that 51 and 48 aroma-active volatiles were detected in glutinous and non-glutinous sorghum, respectively (Table 3). Most of the aroma compounds detected in the cooked sorghums have also previously been identified in Chinese Baijiu [6,7,14,15], indicating that the aroma compounds in sorghum contribute to the overall flavor of Baijiu. The 52 different identified aroma volatiles included 14 aldehydes, 13 terpenes, 8 acids, 5 ketones, 5 alcohols, 5 heterocyclic compounds, 1 sulfur-containing and 1 aromatic compound. Numerically, the aldehyde group comprised 27% of the total aroma-active volatiles in the cooked sorghum samples, followed by terpene (25%), acid (14%), ketone (10%), alcohol (10%), heterocyclic (10%), aromatic (2%), and sulphur-containing (2%) compounds. The aroma intensity of (E)-2-heptenal, nonanal, (E)-2-octenal, benzaldehyde, (E)-2-undecenal, (E, E)-2,4-decadienal, α-limonene, (-)-α-Cedrene, thujopsene, β-elemene, β-damascenone, geranyl acetone, 2,3-octanedione, acetophenone, 2-pentylfuran, 2-acetylpyrrole and dimethyl disulfide, present significant differences between the two samples (p < 0.05). (E)-2-undecenal was undetected in glutinous sorghum, and (E, E)-2,4-decadienal, α-limonene, thujopsene, and β-elemene were undetected in non-glutinous sorghum (Figure 4).

Table 3.

Aroma compounds in cooked glutinous and non-glutinous sorghum.

No. a Compound Aroma Descriptor Aroma Intensity b Significant c LRI d Identification
Glutinous Sorghum Non-Glutinous Sorghum
Aldehydes
1 2-Methylbutanal Green, malty 3.25 ± 0.27 3.08 ± 0.20 ns 912 MS, RI, Aroma, STD
2 3-Methylbutanal Green, malty 4.08 ± 0.49 4.17 ± 0.25 ns 925 MS, RI, Aroma, STD
4 Hexanal Grassy, green 2.92 ± 0.38 2.58 ± 0.37 ns 1084 MS, RI, Aroma, STD
11 Octanal Fatty, soapy, orange 2.83 ± 0.41 3.17 ± 0.25 ns 1289 MS, RI, Aroma, STD
13 (E)-2-Heptenal Fatty 2.83 ± 0.61 2.00 ± 0.45 * 1315 MS, RI, Aroma, STD
15 Nonanal Green 4.33 ± 0.52 2.50 ± 0.20 *** 1396 MS, RI, Aroma, STD
17 (E)-2-octenal Green 4.58 ± 0.38 4.00 ± 0.45 * 1435 MS, RI, Aroma, STD
22 Decanal Fatty/waxy 2.50 ± 0.45 2.08 ± 0.51 ns 1506 MS, RI, Aroma, STD
23 Benzaldehyde Almond 5.00 ± 0.00 4.33 ± 0.25 ** 1541 MS, RI, Aroma, STD
30 (E)-2-decenal Green 1.00 ± 0.71 0.83 ± 0.45 ns 1656 MS, RI, Aroma, STD
31 Phenylacetaldehyde Rose 1.50 ± 0.45 2.00 ± 0.38 ns 1675 MS, RI, Aroma, STD
33 (E, E)-2,4-Nonadienal Green, fatty 2.83 ± 0.61 3.08 ± 0.45 ns 1711 MS, RI, Aroma, STD
37 (E)-2-Undecenal Soapy, metallic - 0.92 ± 0.32 *** 1736 MS, RI, Aroma, STD
38 (E, E)-2,4-Decadienal Fatty, vegetable 1.75 ± 0.27 - *** 1820 MS, RI, Aroma, STD
Terpenes
5 α-Terpinene Citrus-like, floral 1.83 ± 0.61 1.50 ± 0.38 ns 1185 MS, RI, Aroma, STD
6 Terpinolene Citrus-like, floral 1.17 ± 0.61 1.00 ± 0.32 ns 1200 MS, RI, Aroma, STD
7 α-Limonene Citrus-like 1.25 ± 0.69 - ** 1270 MS, RI, Aroma, STD
9 γ-Terpinene Citrus-like, lemon 2.25 ± 0.52 2.00 ± 0.41 ns 1273 MS, RI, Aroma, STD
24 (-)-α-Cedrene Cypress 5.00 ± 0.00 3.17 ± 0.40 *** 1574 MS, RI, Aroma, STD
25 Thujopsene Cypress 1.50 ± 0.45 - *** 1620 MS, RI, Aroma, STD
28 β-Terpineol Clove 0.92 ± 0.49 1.33 ± 0.38 ns 1626 MS, RI, Aroma, STD
36 β-Elemene Fruity, sweet 1.83 ± 0.41 - *** 1598 MS, RI, Aroma, STD
39 β-Damascenone Sweet, honey 5.00 ± 0.00 4.08 ± 0.45 ** 1833 MS, RI, Aroma, STD
42 (E)-β-Ionone Violet 2.25 ± 0.52 1.75 ± 0.38 ns 1952 MS, RI, Aroma, STD
45 Geranyl acetone Fruity 5.00 ± 0.00 4.25 ± 0.25 *** 1856 MS, RI, Aroma, STD
46 (E)-Nerolidol Rose, apple 1.50 ± 0.45 1.92 ± 0.37 ns 2015 MS, RI, Aroma, STD
52 Vanillin Sweet 1.83 ± 0.61 2.17 ± 0.51 ns 2542 MS, RI, Aroma, STD
Acids
20 Acetic acid Acid 0.83 ± 0.52 1.50 ± 0.49 ns 1455 MS, RI, Aroma, STD
29 Butanoic acid Sweaty, acid 2.17 ± 0.41 2.67 ± 0.66 ns 1657 MS, RI, Aroma, STD
34 3-Methyl-butanoic acid Acid 2.25 ± 0.52 2.50 ± 0.55 ns 1687 MS, RI, Aroma, STD
35 Pentanoic acid Sweaty 2.25 ± 0.27 2.58 ± 0.45 ns 1754 MS, RI, Aroma, STD
40 Hexanoic acid Sweaty, cheese 5.00 ± 0.00 5.00 ± 0.00 ns 1844 MS, RI, Aroma, STD
43 Heptanoic acid Sweaty 1.67 ± 0.26 2.17 ± 0.68 ns 1915 MS, RI, Aroma, STD
49 Octanoic acid Grease 1.75 ± 0.61 2.08 ± 0.68 ns 2060 MS, RI, Aroma, STD
51 Decanoic acid Grease 3.17 ± 0.68 3.50 ± 0.45 ns 2284 MS, RI, Aroma, STD
Ketones
10 2-Octanone Milk 1.58 ± 0.49 1.83 ± 0.58 ns 1282 MS, RI, Aroma, STD
12 2, 3-Octanedione Cream, sweet 3.33 ± 0.41 2.67 ± 0.20 ** 1368 MS, RI, Aroma, STD
14 2-Nonanone Sweet, coconut 1.17 ± 0.41 1.00 ± 0.45 ns 1388 MS, RI, Aroma, STD
27 2-Undecanone Orange 1.17 ± 0.61 1.58 ± 0.45 ns 1596 MS, RI, Aroma, STD
32 Acetophenone Floral 4.00 ± 0.45 3.08 ± 0.51 * 1668 MS, RI, Aroma, STD
Alcohols
16 Hexanol Floral 3.50 ± 0.71 3.00 ± 0.38 ns 1376 MS, RI, Aroma, STD
18 1-Octen-3-ol Mushroom 3.33 ± 0.52 3.50 ± 0.38 ns 1440 MS, RI, Aroma, STD
19 Heptanol Green 0.92 ± 0.49 1.17 ± 0.38 ns 1450 MS, RI, Aroma, STD
26 Octanol Fruity 2.50 ± 0.63 2.75 ± 0.51 ns 1575 MS, RI, Aroma, STD
41 Phenethyl alcohol Honey 3.50 ± 0.45 4.17 ± 0.60 ns 1942 MS, RI, Aroma, STD
Heterocyclics
8 2-Pentylfuran Grass, bean 2.92 ± 0.58 2.00 ± 0.38 * 1218 MS, RI, Aroma, STD
21 Furfural Almond 1.17 ± 0.61 1.00 ± 0.58 ns 1464 MS, RI, Aroma, STD
44 2-Acetylpyrrole Popcorn-like 0.75 ± 0.42 1.83 ± 0.45 ** 1957 MS, RI, Aroma, STD
47 γ-Octalactone Coconut milk 3.50 ± 0.63 3.00 ± 0.74 ns 1886 MS, RI, Aroma, STD
48 γ-Nonalactone Coconut milk 2.92 ± 0.58 2.50 ± 0.38 ns 2059 MS, RI, Aroma, STD
Sulphur
3 Dimethyl disulfide Onion 2.00 ± 0.32 1.17 ± 0.38 ** 1078 MS, RI, Aroma, STD
Aromatic
50 2-Methoxy-4-vinylphenol Smoky, clove-like 2.25 ± 0.52 2.50 ± 0.63 ns 2200 MS, RI, Aroma, STD
Open in a new tab

a Numbers represent the aroma compounds listed in Figure 4; b The aroma intensities of odorant compounds were represented as the mean value of sextuplicate samples ± standard deviation; c Symbols indicates a significant aroma intensity difference between two sorghum samples. Significance: “***”, p < 0.001 (very highly significant), “**”, p < 0.01 (highly significant) “*”, p < 0.05 (significant); “ns”, p > 0.05 (not significant). d LRI: linear retention indices.

Figure 4.

Figure 4

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(A) Gas chromatography-mass spectrometry (GC-MS) and (B) gas chromatography-olfactory (GC-O) chromatograms of cooked glutinous sorghum. Numbers correspond to the compounds listed in Table 3.

To estimate the contributions that these aroma-active volatiles had on the overall aroma impression, the 52 compounds were grouped into eight general aroma categories: sweet/fruity, green, floral, fatty, volatile acids, citrus-like, roasty/nutty, and miscellaneous (sulphury/smoky/mushroom). To compare the differences in the aroma intensities of each group between the cooked glutinous and non-glutinous sorghum samples, the intensities of each group were summed (Table 4).

Table 4.

Summed aroma intensities of each aroma group present in the cooked glutinous and non-glutinous sorghum samples.

Aroma Impression a Volatile Compounds Summed Aroma Intensities b Significance c
Glutinous Non-Glutinous
sweet/fruity 2-octanone, 2,3-octanedione, 2-nonanone, octanol, β-elemene, β-damascenone, phenethyl alcohol, geranyl acetone, γ-octalactone, γ-nonalactone, vanillin 32.17 ± 2.32 28.42 ± 1.48 *
green 2-methylbutanal, 3-methylbutanal, hexanal, 2-pentylfuran, nonanal, (E)-2-octenal, 1-heptanol, (-)-α-cedrene, thujopsene, (E)-2-decenal, (E, E)-2,4-nonadienal 33.33 ± 1.75 26.58 ± 1.51 ***
floral hexanol, β-terpineol, phenylacetaldehyde, acetophenone, (E)-β-ionone, (E)-nerolidol 13.67 ± 1.67 13.08 ± 1.51 ns
fatty octanal, (E)-2-heptenal, decanal, (E)-2-undecenal, (E, E)-2,4-decadienal, octanoic acid, decanoic acid 14.83 ± 0.75 13.75 ± 1.80 ns
volatile acids acetic acid, butanoic acid, 3-methyl-butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid 14.17 ± 0.75 16.42 ± 1.06 **
citrus-like α-terpinene, (E)-β-terpinolene, α-limonene, γ-terpinene, 2-undecanone 7.67 ± 0.55 6.08 ± 0.53 ***
roasty/nutty furfural, benzaldehyde, 2-acetylpyrrole 6.92 ± 0.61 7.17 ± 0.69 ns
miscellaneous (sulphury/smoky/mushroom) dimethyl disulfide, 1-octen-3-ol, 2-methoxy-4-vinylphenol 7.58 ± 0.79 7.17 ± 0.47 ns
Open in a new tab

a The aroma characteristics; b The summed aroma intensities of odorant compounds were represented as the mean value of sextuplicate samples ± standard deviation; c Symbols indicate a significant aroma intensity difference between two sorghum samples. Significance: “***”, p < 0.001 (very highly significant); “**”, p < 0.01 (highly significant) “*”, p < 0.05 (significant); “ns”, p > 0.05 (not significant).

2.3.1. Sweet/Fruity

Sweet/fruity was found to be the major aroma character in both types of cooked sorghums, with the glutinous sorghum containing 12% more sweet/fruity aroma than the non-glutinous sorghum. Four types of compounds (terpenes, heterocyclics, alcohols, and ketones) provided the sweet/fruity aroma note in the cooked sorghums. Among the four terpenes (elemene, β-damascenone, geranyl acetone, and vanillin), β-damascenone and geranyl acetone were significant contributors to the overall aroma of the cooked sorghums. During the GC-O/MS analysis, although no obvious peak for β-damascenone was observed, an aroma of rich honey sweetness was sniffed at 26.84 min, with an RI of 1833 (Figure 4), and the compound was identified using its retention time and mass ions. β-Damascenone is an ionone isomer belonging to the isoprenoid class of compounds. These compounds often exude the aroma of lemon at low concentrations, while at high concentrations the aromas of apple, rose, and honey are expressed [16]. According to the literature, β-damascenone is an important aroma contributor to Chinese Baijiu [6], having extremely low aroma threshold values in air (0.002–0.004 ng/L) and water (0.00075–0.002 μg/L) [17]. β-Damascenone and geranyl acetone are both bound aroma compounds of sorghum, with geranyl acetone also being an important aroma compound in the soy sauce aroma type Baijiu [6]. Vanillin is another common aroma compound in Chinese Baijiu [18]. It has a sweet vanilla aroma feature with a low aroma threshold of 26 μg/L in chinese rice wine [17,19]. The two sweet/fruity heterocyclic compounds (γ-octalactone and γ-nonalactone) contributed a coconut aroma to the cooked sorghums. Both compounds are γ-lactones, a significant class of aroma lactones that mostly have coconut, sweet, and peach aromas [20]. γ-Octalactone and γ-nonalactone also have relatively low aroma thresholds, 7 and 30–65 μg/L, respectively. Additionally, two alcohols and two ketones contributed to the sweet/fruity aromas of the cooked sorghums. Among these, phenethyl alcohol was a significant aroma contributor having an aroma intensity of 3.50 and 4.17 in glutinous and non-glutinous sorghum, respectively (Table 3).

2.3.2. Green

Green was another significant aroma note in the cooked sorghums. In this study, 11 green aroma compounds were detected, having total aroma intensities 20% higher in glutinous versus non-glutinous sorghum (Table 4). Seven of the eleven compounds were aldehydes. Saturated aldehydes and (E)-2-enal unsaturated aldehydes often demonstrate grass and malty aromas [14,17]. Nonanal and 3-methylbutanal (saturated aldehydes) and (E)-2-octenal (an unsaturated aldehyde), were evaluated as having strong aroma intensities in both cooked sorghums. Lipids in sorghum are susceptible to oxidization, and the heat imposed during sorghum distillation may accelerate the oxidation process [21], possibly contributing to the presence of aldehyde compounds in the cooked sorghum samples. (E)-2-Enal aldehydes have not been detected in uncooked sorghum [10,11], while they have been detected in a variety of Chinese Baijiu [14], thus further substantiating that the distillation (cooking) process of sorghum may improve the release of bound aroma compounds and these compounds influence the final flavor quality of Baijiu. (-)-α-Cedrene, a terpene, showed the strongest green aroma note (5.0) in glutinous sorghum (Table 3). As (-)-α-Cedrene is also an aroma compound in the soy sauce aroma type Baijiu, this compound may strongly contribute to the overall aroma of Baijiu [22].

2.3.3. Floral

Six compounds were detected as having floral aromas in the cooked sorghums. The summed aroma intensities of these compounds for the two types of sorghum were similar (Table 4). Of the compounds primarily responsible for the floral aromas of the cooked sorghums, acetophenone had the highest relative floral aroma intensity, 4.00 and 3.08 in glutinous and non-glutinous sorghum, respectively.

2.3.4. Fatty

Seven compounds were detected as having fatty aromas. Compounds with long carbon chains, such as octanal and decanoic acid, often demonstrate fatty aromas. However, with decreases in the carbon chain length, compounds may demonstrate different aroma notes, and their aroma thresholds may also decrease [17].

2.3.5. Volatile Acids

Organic acids are important aroma compounds in many traditional fermented foods [23]. Six compounds having acids aromas were detected, and their summed aroma intensities were 14% lower in glutinous versus non-glutinous sorghum (Table 4). Among these, hexanoic acid was evaluated as having sweaty and cheese aromas, with aroma intensities of 5.00 for both types of cooked sorghums (Table 3).

2.3.6. Citrus-Like

The citrus-like aroma is a unique feature of the aroma profile of cooked sorghums. Four terpenes and one ketone were detected with citrus-like aromas. Among these, γ-terpinene showed a relatively strong aroma intensity of 2.00 both in glutinous and non-glutinous sorghum (Table 3).

2.3.7. Roasty/Nutty

Three compounds were detected as having roasty/nutty aromas. Benzaldehyde demonstrated strong almond aroma intensities of 5.00 and 4.33 in glutinous and non-glutinous sorghum, respectively (Table 3).

2.3.8. Miscellaneous

Besides the typical aroma notes of cooked sorghum, 1-octen-3-ol (mushroom aroma), dimethyl disulfide (onion aroma), and 2-methoxy-4-vinylphenol (smoky aroma), were also detected and contributed to the overall aromas of the cooked sorghums (Table 3).

Based on the ANOVA, the summed intensity of four aroma categories: sweet/fruity, green, volatile acids and citrus-like, present significant differences between glutinous and non-glutinous sorghum samples (Table 4).

3. Materials and Methods

3.1. Reagents and Chemicals

Two sorghum cultivars (glutinous and non-glutinous) produced in Guizhou Province in 2018, were provided by a liquor producing enterprise. The aroma standards were purchased from Sigma Aldrich (Shanghai) Trade Co. Ltd. All compounds were of chromatographic purity, and the purity was >95%. The n-alkane (C7–C30) for the linear retention index (RI) determination was obtained from Sigma-Aldrich Co., Ltd. (Shanghai, China). Ultrapure water was obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA).

3.2. Sorghum Cooking

Sorghum samples were first moistened by adding 300 g of sorghum to 400 mL of distilled water in a 1 L beaker. The mixture was then sealed with foil and heated in an oven at 75 °C overnight [24]. Then, 5 g of the moist sorghum and 1 mL of distilled water were added to a 20 mL headspace glass vial that was purged with nitrogen gas and loosely sealed with a Teflon-coated septum screw cap. Next, the vial was placed in a boiling water bath (100 °C) for 1 h, and directly subjected to headspace-solid-phase microextraction.

3.3. Optimization of Headspace-Solid-Phase Microextraction Sampling

Four SPME fiber types, polydimethylsiloxane (PDMS), PDMS/divinylbenzene (DVB), carboxen (CAR)/PDMS, and DVB/CAR/PDMS, were evaluated for their capabilities at extracting and concentrating the headspace volatiles of cooked sorghum. Extraction temperatures (40, 50, 60, 70, and 80 °C) and times (30, 60, 90, 120, and 150 min) were evaluated to determine the optimal conditions for extracting the volatiles using each type of SPME fiber. After optimization, the DVB/CAR/PDMS SPME fiber was inserted into the headspace vial of the sorghum sample and left for 120 min at 80 °C. Subsequently, the fiber was inserted into the injection port of the GC-MS for 5 min at 250 °C for thermal desorption of the flavor volatiles.

3.4. HS-SPME-GC×GC-TOFMS Instrumentation

Following a method presented in the literature [25], experiments were performed on a LECO Pegasus® 4D GC×GC-TOFMS system (LECO Corp., St. Joseph, MI, USA). The column configurations for GC×GC analysis consisted of two columns which were coupled in tandem. The 1D column was a 60 m × 0.25 mm × 0.25 µm DB-FFAP (Agilent Technologies, Palo Alto, CA, USA), and the 2D column was a 1.5 m × 0.25 mm × 0.25 µm Rxi-17Sil MS secondary column (Restek, Bellefonte, PA, USA). Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. The injector temperature was set at 250 °C. The initial oven temperature was held at 45 °C for 3 min, and then ramped at a rate of 4 °C/min to 150 °C, and held for 2 min, reaching 200 °C at 6 °C/min and 230 °C at 10 °C/min for 20 min. The secondary oven was set at a 5 °C offset, relative to the primary oven. The modulator was set at a 20 °C offset, relative to the primary oven, and a modulation period of 4 s was used. The TOFMS was operated at an acquisition rate of 100 spectra/s and scanned from 50 m/z to 350 m/z.

The detector voltage was set to 1350 V, and the electron energy was −70 V.

3.5. Gas Chromatography-Olfactometry/Mass Spectrometry (GC-O/MS) Analysis

Gas chromatography was performed using an Agilent 6890N GC equipped with an Agilent 5975 mass selective detector (Agilent Technologies, Santa Clara, CA) and olfactometer (ODP 2, Gerstel GmbH and Co. KG, Mulheim, Germany). Samples were separated and evaluated using a polar DB-FFAP column (60 m × 0.25 mm i.d, 0.25 μm film thickness, Agilent Technologies, Inc., Santa Clara, CA). The oven temperature was programmed to 40 °C for 2 min, then increased at a rate of 4 °C/min to 230 °C, and maintained there for 10 min. The carrier gas used was helium, at a flow rate of 2.0 mL/min. A split ratio of 1:1 was used for injection.

Half of the carrier flow was diverted to the olfactory port, and mixed with warm, humid air for sniffing. The sniffing port temperature was set to 250 °C. Three trained panelists (two females and one male) received two months of training on GC-O involving, at a minimum, 30 aroma-active reference compounds at concentrations that were 10 times their odor thresholds in air. During GC operation, each panelist placed his/her nose near the olfactory port to record the retention times, intensity values, and aroma descriptions of the samples. Odor intensities were evaluated using a 6-point intensity scale from 0 to 5, with “0” = no, “3” = moderate, and “5” = extreme intensity. Each panelist conducted the GC-O evaluation four times. The final intensity value of each aroma was an average of the aroma intensities obtained from all of the panelists.

The other half of the carrier flow was diverted to the mass spectrometer. The ion source temperature was set to 230 °C, electron impact ionization was applied in positive ion mode (70 eV), and the mass range scanned was m/z = 25~300.

3.6. Identification of Aroma Compounds

Identifications were made through comparisons of the aromas, mass spectra, and linear retention indices (LRIs) with reference/standard compounds. Aroma determinations were made by comparing the odor descriptions of the compounds analyzed by GC-O to Flavornet and Flavor DB (citations). Mass determinations were made by comparing the mass spectra of the compounds to the NIST/EPA/NIH mass spectral library (NIST 14, version 2.0) (NIST, Gaithersburg, MD). LRI analyses involved comparisons of the aroma LRIs with those of reference compounds. The LRIs of the odorants were calculated from the linear retention times of n-alkanes (C5–C30) using a DB-FFAP column according to a modified Kovats method [26]. All of the aroma-active compounds detected by GC-O were also confirmed through the use of standard compounds.

3.7. Statistical Analysis

The analysis of variance (ANOVA) was performed using Microsoft Excel 2013 software. Significant differences were estimated as “***”, p < 0.001 (very highly significant); “**”, p < 0.01 (highly significant); “*”, p < 0.05 (significant); “ns”, p > 0.05 (not significant).

4. Conclusions

In this study, an SPME method for the extraction of aroma compounds from cooked sorghums was developed. Using this method, a total of 145 volatile compounds and 52 aroma-active compounds were detected in cooked glutinous and non-glutinous sorghum by GC×GC-TOFMS and GC-O/MS respectively. These compounds were further identified through comparisons with reference/standard compounds. The aroma features and intensities of these compounds were evaluated by three well-trained panelists using olfactory detection. The aroma intensity of (E)-2-heptenal, nonanal, (E)-2-octenal, benzaldehyde, (E)-2-undecenal, (E, E)-2,4-decadienal, α-limonene, (-)-α-Cedrene, thujopsene, β-elemene, β-damascenone, geranyl acetone, 2,3-octanedione, acetophenone, 2-pentylfuran, 2-acetylpyrrole and dimethyl disulfide, present significant differences between the two samples (p < 0.05). (E)-2-undecenal was undetected in glutinous sorghum, and (E, E)-2,4-decadienal, α-limonene, thujopsene, and β-elemene were undetected in non-glutinous sorghum. Based on their aroma features, the compounds were grouped into eight general aroma categories, and the aroma intensities of each aroma group were totaled. The summed intensity of four aroma categories: sweet/fruity, green, volatile acids and citrus-like, present significant differences between glutinous and non-glutinous sorghum samples (p < 0.05). Glutinous sorghum contained 12%, 20% and 21% more sweet/fruity, green and citrus-like total intensities, respectively than non-glutinous sorghum; and contained 14% less acid total intensity, emphasizing the aromatic differences between the two cooked sorghums. In addition, most of the compounds detected in the cooked sorghums were also detected in commercial Baijiu, indicating that the aroma compounds produced during sorghum distillation (cooking) have direct and significant influences on the final flavor quality of Baijiu.

Author Contributions

S.C., Conceptualization, Methodology, Validation, Analysis, Writing, Review & Editing, project administration; L.W., Supervision & Review; D.N., Validation, Analysis; L.L., Methodology, Analysis; H.W., Methodology, Validation, Analysis; Y.X., Supervision & Review. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key R&D Program of China (NO. 2018YFC1604100), the China Postdoctoral Science Foundation (NO. 2018M631971), National First-class Discipline Program of Light Industry Technology and Engineering (LITE2018-12), and 111 Program of Introducing Talents (NO. 111-2-06).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from the authors.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

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