Characterization of Volatile Compound Profiling of Germinated

Brown Rice Revealed by Headspace Solid-phase Micro Extraction

Coupled to Gas Chromatography Mass Spectrometry

Qiang Xia 1, Liping Wang

1, Peng Huang

1, Yunfei Li

1

1 Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong

University, Shanghai 200240, China

Abstract Due to impressive health promoting effects, germinated brown rice is an increasingly popular

functional food. As a wholegrain, odor and flavor characteristics largely influence consumer perception of

cereal based products, such as GBR. However, there is no quantitative information available for volatile

compounds contained in GBR. This work examined the profiling of volatile component in two representative

varieties of pre-germinated brown rice, JZDG (Oryza sativa L. ssp. Indica) and CMSG (O. sativa ssp.

Japonica), using headspace solid-phase micro extraction coupled to gas chromatography mass spectrometry

(HS-SPME-GC/MS). The results showed that significant difference in the volatile compounds was not only

present in the relative abundance of individual components, but also in the varieties of the identified

compounds between JZDG and CMSG. A total of 36 volatile compounds were detected in cooked GBR.

Aldehydes and alkanes were the major chemical categories identified. Among these compounds, hexanal,

pentanal and 2-pentyl-Furan showed relatively high concentration. Cyclotetradecane,

2-methyl-1-Penten-3-one, 4-Decanone, 6-methyl-5-Hepten-2-one 2-n-Heptylfuran,

3,3,5-trimethyl-Cyclohexene, 4-Methyl-1,5-Heptadiene, (E,E)-2,4-Decadienal, 2-Carene were only present in

JZDG, while some compounds were only detected in CMSG, such as 4-methyl-(Z)-2-Pentene,

2-Methoxy-4-vinylphenol and 2,6,10-Trimethyl-dodecane. Characterizing aroma compound profiles of GBR

is considered as the first step to understand flavor formation, and thus orientedly modify and design the

product flavor.

Keywords: HS-SPME-GC/MS, GBR, volatile compounds, profiles.

1. Introduction

Rice (Oryza sativa L.) is the staple food for more than half of people around the world. Rice cultivars

can be classified into two major categories, including the ecotype indica and japonica, which are

characterized by long and short grains, respectively [1]. Sensory attributes, such as texture and flavor, are

important parameters that largely affect consumer acceptance for cereal based products, particularly

bran-rich wholegrain foods [2]. Volatile compounds of different rice cultivars have been profiled by several

studies in recent years [3], where aroma active and characteristic marker compounds have been identified.

Aldehydes and alcohols were the major compound classes, and 2-acetyl-1-pyrroline (2-AP) together with

sulfur volatiles, including 2-acetyl-2-thiazoline, dimethyl sulphide, 3-methyl-2-butene-1-thiol,

2-methyl-3-furanthiol, dimethyl trisulphide, and methional, showed high odor activity widely present in

aromatic rice cultivars; moreover, 2-(E)-octenal, acetyl-1-pyrroline/1-octen-3-ol, and

3-methyl-1-butanol/2-methyl-1-butanol were identified as aging markers [3].

The above-mentioned investigation focused on the volatile compounds of polished rice. Due to the

impulse by impressively nutritional and health attributes, the consumption of brown rice (BR), particularly

germinated brown rice (GBR), has been encouraged to be increased for their proportion consumed in dietary

Corresponding author. Tel. +86-21-34206918; fax: +86-21-34206918.

E-mail address: yfli@sjtu.edu.cn.

International Proceedings of Chemical, Biological and Environmental Engineering, Vol.95 (2016)

DOI:10.7763/IPCBEE.2016.V95.11

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foods as wholegrain foods in many countries. However, little information has been found concerning the

aroma profiling of GBR, which is greatly necessary because aroma and flavor of GBR is considered as an

important parameter influencing consumers acceptance [2]. Volatiles in GBR are likely to show large

difference from polished rice because GBR possesses pericarp and seed coat which may regulate the release

of volatile molecules from the core of grains. On the other hand, some volatile components may be produced

or reduced during germination due to metabolic activities. In this way, it is necessary to identify and quantify

the volatile compounds contained in GBR.

Therefore, the aim of the current study was to characterize the profile of volatile organic compounds in

GBR. Volatile compounds were identified and semi-quantified by headspace solid-phase micro extraction

coupled to gas chromatography mass spectrometry (HS-SPME-GC/MS).

2. Materials and Methods

2.1. Materials and Sample Preparation

Two representative rice grains, DHX (Oryza sativa L. subsp. Indica) and SCM (O. sativa L. subsp.

Japonica), were obtained from a supermarket in Shanghai. Rice grains were dehulled by industry to produce

brown rice (BR). The grains were sealed and stored at 4oC prior to use. Germination procedures referred to

the previously documented method [4]. Germination was undertaken in an incubator (RGX-260B, Hualian

Med., China) for 36 hour at dark to finally obtain pre-germinated JZDG and CMSG with a 0.5-1.0 mm

sprout length.

2.2. HS-SPME Sampling

GBR grains were boiled for 30 min and no white spots leaching were observed [5]. The cooked rice was

placed in a 20-ml autosampler vial with a screw cap and the internal standard (IS), n-heptadecane, was added.

An automated headspace sampling system using a Divinylbenzene/Carboxen/Polydimethylsiloxane

(DVB/CAR/PDMS) 50/30 μm fiber (2 cm, Supelco, PA, USA) was selected to extract and concentrate aroma

compounds. The samples were equilibrated for 15 min at 50 oC and extracted by HS-SPME for 30 min. The

absorbed volatiles by the fiber were then desorbed into the injector port of GC for 4 min at 260 oC, which

was finally subjected to the chromatographic column for separation.

2.3. GC/MS Analysis

The identification and quantification of minor volatile compounds were performed on an Agilent

GC7890A coupled to 5975C mass spectrometry detector (MSD) operating in electron impact (EI) mode at 70

eV. An apolar fused silica capillary column (HP-5, J&W Scientific; 30 m × 0.25 mm 0.25 μm film thickness)

was employed. Carrier gas was high-purity helium (99.999%) at flow rate of 1 mL/min. The injector

temperature was 260 oC with splitless mode. The oven temperature was maintained at 40°C for 5 min, then

programmed from 40 to 250 °C at the rate of 5 oC/min and held at 250

oC for 5 min. Transfer line

temperature and quadrupole temperature were 200 oC and 150

oC, respectively, and ion source temperature

was set at 230 oC. Scan range was m/z 20–400 at scan rate of 1.000 amu/s.

Volatile compounds were firstly identified by the comparison of actual mass spectra with the published

authentic spectra database in the GC-MS libraries (NIST2011), and the compounds with similarity index

above 800 were selected. These compounds were further confirmed according to their RI if available.

2.4. Data Analysis

The results were the averages of three duplicates of experiments. To examine the significant difference

of volatile compounds between different cultivars, Students’t-test was carried out using SPSS software

(version 19.0, SPSS Inc., Chicago, IL, USA). Samples with P value less than 0.05 were considered as

statistical difference.

3. Results and Discussion

Thirty-six volatile compounds originating from cooked GBR grains were identified and semi-quantified

by HS-SPME-GC/MS. With respect to representative total ion current chromatograms shown in Fig. 1,

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obvious differences between JZDG and CMSG were not only present in the relative abundance of individual

volatiles and total contents of volatiles, but also found in the varieties of volatiles identified.

Fig. 1: Representative GC–MS chromatograms of volatile compounds from JZDG (A) and CMSG (B) by using

HS-SPME

Table 1 summarized the detected compounds, which could be classified into aldehydes (10),

hydrocarbons (10), ketones (4), aromatic compounds (4), furans (4) and other components (4). With regard

to the relative proportion of main chemical classes of volatiles (Fig. 2), the statistical difference (P<0.05)

between two GBR cultivars was observed. The relative proportion of aldehydes in CMSG was significantly

higher than that in JZDG, whereas JZDG showed greater relative proportion of hydrocarbons and furans than

CMSG. No significant difference (P>0.05) was observed for the relative abundance of aromatic compounds,

ketones and others group. However, due to a greatly lower response of IS and higher response of volatile

components (Fig. 1), CMSG had greatly higher relative concentration of the detected compounds than JZDG.

Fig. 2: Relative proportion of the primary classes of volatile compounds identified from the ecotype indica (JZDG) and

japonica (CMSG) cultivar. Lower-case letters labeled on the top of columns means significant difference (P<0.05).

In terms of their relative concentrations and varieties, aldehydes were the major group identified in the

GBR samples. A total of nine aldehydes were detected, with the levels ranging from not found to 802.29

μg/kg. The major aldehydes with high concentration included hexanal, heptanal, pentanal, nonanal, decanal,

and octanal, which are also the commonly found in different cultivars of polished rice or BR [6], [7],

suggesting that GBR retains the qualitatively similar flavor profiles even after germination by soaking in

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water for a certain duration. It was worth noting that aldehyde compounds often give grass and fresh odor at

a sufficiently low concentration in the samples, but high levels of these compounds lead to the sniffing of

rancidity. In addition to its low odor thresholds, the aldehydes significantly contributed to the overall flavor

of GBR. Particularly, as the major component of rice volatiles [8], hexanal had the highest concentration in

CMSG, being 802.29 μg/kg, while JZDG showed 58.08 μg/kg at averages. Hexanal is partially formed

during the ripening of the cereals, and it is also produced via the oxidation of free fatty acids (FFA) by

lipoxygenase and hydroperoxide lyase during the storage [9]. In addition, 3-methyl-Butanal and

(E)-2-Octenal were detected in both GBR and 2-methyl-Butanal was not present in CMSG.

Table 1: Volatile components identified and semi-quantified by HS-SPME-GC/MS in two typical germinated brown

rice

Peak no. RT Aroma compounds CAS no. Relative contents (μg.kg-1)

JZDG CMSG

a1 3.04 3-Methyl-butanal 000590-86-3 0.82a 11.53b

a2 3.16 2-Methyl-butanal 000096-17-3 0.43 n.d.A

a3 3.67 Pentanal 000110-62-3 8.31a B 120.2b

a4 35.64 Heptanal 000111-71-7 11.2a 90.97b

a5 10.54 Hexanal 000066-25-1 58.08a 802.29b

a6 6.67 (E)-2-Octenal 002548-87-0 0.66 17.24

a7 16.19 Nonanal 000124-19-6 20.8a 139.71b

a8 17.73 Decanal 000112-31-2 1.45a 14.34b

a9 20.87 Octanal 000124-13-0 15.88a 87.85b

a10 15.96 (E,E)-2,4-Decadienal 025152-84-5 0.37 n.d.

a11 28.82 Pentadecane 000629-62-9 2.23 11.9

a12 31.21 Hexadecane 000544-76-3 4.3 12.76

a13 33.58 Octadecane 000593-45-3 0.84 n.d.

a14 14.30 Decane 000124-18-5 11.14 19.45

a15 14.22 Undecane 001120-21-4 2.81 22.19

a16 17.60 Dodecane 000112-40-3 1.02 7.45

a17 20.71 Tridecane 000629-50-5 0.85 4.67

a18 23.59 Tetradecane 000629-59-4 2.05a 19.02b

a19 26.28 2,6,10-Trimethyl-dodecane 003891-98-3 n.d. 6.73

a20 27.82 Cyclotetradecane 000295-17-0 0.31 n.d.

a21 28.62 2-Methyl-1-penten-3-one 025044-01-3 1.72 n.d.

a22 5.58 4-Decanone 000624-16-8 0.44 n.d.

a23 18.44 6-Methyl-5-hepten-2-one 000110-93-0 1.2 n.d.

a24 13.63 6,10,14-Trimethyl-2-pentadecanone 000502-69-2 2.61 7.72

a25 36.48 Ethylbenzene 000100-41-4 1.42 10.54

a26 8.90 Toluene 000108-88-3 1.55 16.91

a27 5.46 Benzaldehyde 000100-52-7 3.91a 68.86b

a28 12.73 2-Methoxy-4-vinylphenol (4-Vinylguaiacol) 007786-61-0 n.d. 10.75

a29 23.84 2-Methyl-furan 000534-22-5 9.63 n.d.

a30 2.47 Tetrahydrofuran 000109-99-9 3.96 29.88

a31 2.71 2-Pentyl-furan 003777-69-3 21.01a 279.58b

a32 13.83 2-n-Heptylfuran 003777-71-7 0.23 n.d.

a33 20.45 3,3,5-trimethyl-Cyclohexene 000503-45-7 1.33 n.d.

a34 15.20 4-Methyl-1,5-Heptadiene 000998-94-7 0.36 n.d.

a35 17.26 2-Carene 000554-61-0 0.46 n.d.

a36 24.84 4-Methyl-(Z)-2-pentene 000691-38-3 n.d. 5.79 A n.d. denotes not detected;

B Different lower-case letter behind the data indicates statistical difference (P<0.05).

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The most abundant varieties of hydrocarbons in GBR were detected, which was possibly related to the

polarity of chromatographic column used. Overall, alkanes and alkenes possessed much less total levels in

both GBR cultivars compared with the relative contents of aldehydes. Most of hydrocarbons usually have

little effects on the aroma attributes. These compounds are byproducts of lipid oxidation, so the relative

abundance of hydrocarbons can reflect the extent of lipid oxidation [10]. Hexane, decane, tetradecane,

pentadecane and hexadecane were reported to be present in polished rice with and without fragrance [3], [11],

[12]. More abundant alkanes were found in GBR, which could attribute to the higher activity of enzymes

associated with the oxidation of fatty acids in comparison with polished rice. The previous study has

observed that in BR samples hydrocarbon was the most abundant classes, of which over 25 were detected by

GC/MS using separate polar (CP-WAX) and apolar (DB-5) capillary columns with same dimension [13].

Among four ketones detected in GBR, 2-methyl-1-penten-3-one, 4-decanone, and

6-methyl-5-hepten-2-one were not present in CMSG and 6,10,14-trimethyl-2-pentadecanone was found in

both GBR. Benzaldehyde showed relatively high contents among the aromatic compounds detected. The

relative abundance of 2-pentylfuran was up to 279.58 μg/kg, showing high concentration similar to rice when

compared with other volatiles contained in individual samples [7]. Simultaneously, 2-pentyl-furan,

benzaldyhyde and 2-Methoxy-4-vinylphenol contributes to the unique roasted and nutty aroma [14]. These

compounds can be formed by different pathways. For example, 2-pentyl-furan, 2-methyl-furan,

2-methyl-butanal and 3-methyl-butanal can be produced via Maillard reaction and Strecker degradation

reaction [15].

4. Conclusion

In summary, the main volatile compounds were identified and semi-quantified by HS-SPME combined

with GC/MS. The results demonstrated that aldehydes and hydrocarbons were the major volatile compounds

of GBR, and the profiling of aroma components contained was likely to show some difference from the

polished rice and brown rice, for which further investigation is needed using more representative samples to

obtain a comprehensive understanding of volatiles fingerprinting and how aroma compounds evolve with the

processing .

5. Acknowledgements

This investigation was financially supported by the Project of Prepared Food Research and

Industrialization in City Nutrition Catering (No.2014BAD04B08).

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