Volatile Composition of Smoked and Non-Smoked Iranian Rice

In this work, the volatile profiles of smoked and non-smoked Iranian rice were identified, and their relative abundance was calculated and compared. Headspace solid-phase microextraction together with gas chromatography-mass spectrometry (SPME-GC-MS) were used to extract and identify the volatile compounds. The main groups of volatiles in Iranian rice were aldehydes, ketones, phenol derivatives, furans, linear hydrocarbons, esters and terpenes. The chemical family aldehydes was the most abundant one in the profile of non-smoked rice, while phenol derivatives and furans predominated in smoked samples. This study is the first one reporting comparative data of volatile compounds between smoked and non-smoked Iranian rice.


Introduction
Rice (Oryza sativa) is an essential food for the people of the world, being the second major crop after wheat [1]. Iran is also a relevant world rice producer and exporter [2]. The most important area of rice cultivation, with more than 80% of rice production, is the north part of Iran and includes the provinces of Mazandaran and Gilan, with 238,000 ha planted area [1][2][3]. Even the local rice varieties have low grain yields (averaging 2.5 to 3.5 tones/ha), and more than 80% of the total rice area in Iran is still under these traditional varieties because of their fragrance and other excellent quality traits [3,4].
After the harvest, some rice cultivars undergo a smoking process to enhance the flavor. Exposing bulk rice filled in cotton bags to the smoke obtained by burning special woods, such as those from beech, alder, oak, and fruit trees, for several hours is the traditional method for flavoring and preserving rice. Smoked rice consumption is common in many parts of Iran, especially in northern areas.
The fragrance and taste of the Iranian aromatic rice are the main reasons supporting the higher prices for this type of rice as compared to non-aromatic cultivars in Iran [3]. The compounds responsible for this high quality of the aromatic rice samples are the volatile compounds released from the grains [5]. They have been fully investigated, due to their important aspects for consumer preference and acceptance [6]. For instance, it has been demonstrated that 2-acetyl-1-pyrroline (2-AP) is a volatile compound with an attractive aroma, the sensory descriptors of which are: popcorn, pandan (Pandanus amaryllifolius), nutty, and white bread. Also, the 2-AP can be detected in non-aromatic rice, but at trace levels [6,7].
More than 200 volatiles have been identified in rice using the traditional methods which include static headspace, purge and trap, steam distillation-solvent extraction, and direct solvent extraction for Foods 2016, 5, 81 2 of 8 collection/concentration [8]. The separation is conducted by gas chromatography with flame ionization or a mass spectrometer [9]. Static headspace (SHS) is an effective technique for simple and rapid quantitative analysis of the aroma compound of rice, in which the sample headspace is automatically brought directly to the gas chromatograph (GC) [10]. However, Ezquerro et al. demonstrated that headspace solid-phase microextraction (HS-SPME) is more sensitive than SHS for the identification of volatile compounds from the packaged materials [11]. Also, Lin et al. investigated the volatile compounds of rice using SPME combined with gas chromatography and mass spectrometry (GC-MS); this method proved to have several advantages, including easy preparation, quick analysis, no use of organic solvents and low cost [12]. The extraction temperature and exposure time are the most important factors in these techniques [10,11].
Even though there are several studies about the volatile profiles of rice, there is scarce information on the aromatic components of smoked rice. Consequently, the purpose of this study was to isolate, identify, and compare the volatile profiles of smoked and non-smoked rice samples from different Iranian cultivars. Fifteen rice cultivars four of which were smoked, were evaluated by HS-SPME combined with GC-MS.

Experimental Material
Fifteen rice samples were cultivated in non-organic farms from various provinces of Iran and were harvested in summer of 2012. Most of the samples were grown in the north part of Iran (Table 1), and the number of samples to be harvested and analyzed was decided according to the rice productions of the different regions. Unfortunately, it was not possible to get similar numbers of samples from non-smoked and smoked rice, but the ratio of samples non-smoked:smoked (11:4) is quite representative of the one reaching the Iranian market.
The products were obtained by Department of Food Science and Technology of Ramin Agriculture and Natural Resources University, Iran. Samples were obtained in local markets, to fully reproduce the type of samples being marketed in Iran and available in the main Iranian cities. Samples were bought in triplicate, meaning samples from three different batches were obtained and used for this study. Just after collection, the rice samples were immediately air-mailed to the Universidad Miguel Hernández de Elche (Spain) where they were analyzed for their volatile profiles. Food smoking is an old process of flavoring, cooking, and preserving foods using wood smoke. Exposing cotton bags of rice (up to 50 kg) to the smoke of burning special forest woods, such as beech, alder, oak, and fruit-tree for several hours (4-5 h) is the traditional method of its flavoring and preserving in Iran and it is called in Persian "berenge doudi". The process is done in a wooden cottage it is quite similar in all areas of northern Iran. However, in future studies the effect of the different parameters involved in the smoking process will be studied, for instance rice variety, type of wood, time of smoking, etc.

Extraction Method
Headspace solid phase micro-extraction (HS-SPME) was used to isolate the volatile compounds from the rice samples. Approximately 5 g of ground rice and 10 mL of ultrapure water was settled in 50 mL vial with polypropylene cap and PTFE/silicone septa. Moreover, 0.75 g NaCl, 1 µL anethole (1 mg/L, used as internal standard), and a magnetic stirring bar were added to the vial. The vial was settled in a 40 • C water bath and kept for 15 min to reach an equilibrium between the rice sample mixed in water and the headspace. After this equilibration time was elapsed, a 50/30 µm DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) fiber (StableFlex/SS, 2 cm, and 24 Ga; Supelco, Sigma-Aldrich Co, Bellefonte, PA, USA) was introduced into the vial through a hole in the cap and exposed to the sample headspace for 50 min at 40 • C (simulating approximately the mouth temperature). After the extraction process, the fiber was quickly removed and injected in the port of gas chromatograph and kept for 3 min in the desorption port. This type of fiber was adequate for its high capacity to trap volatile chemicals, and provided excellent results in other matrixes, such as pistachios or pomegranate juice [15,16].

Chromatographic Analyses
The rice volatile compounds were analyzed and identified using a Shimadzu GC-17A gas chromatograph connected with a Shimadzu QP-5050A mass spectrometer detector (Shimadzu Corporation, Kyoto, Japan).
The GC-MS system consisted of a TRACSIL Meta.X5 (95% dimethylpolysiloxane and 5% diphenylpolysiloxane) column (60 m × 0.25 mm, 0.25 µm film thickness; Teknokroma S. Coop. C. Ltd, Barcelona, Spain). The carrier gas used to perform the analysis were helium, at a column flow rate of 0.6 mL·min −1 , and a total flow of 6.0 mL·min −1 in a split ratio of 1:6. The oven program was as follows: (a) 80 • C for 0 min; (b) increase of 3 • C·min −1 from 80 • C to 210 • C, and hold for 1 min; (c) increase of 25 • C·min −1 from 210 • C to 300 • C, and hold for 3 min. The injector and detector temperatures were 230 • C and 300 • C, respectively.
Three protocols were used to identify the rice volatile compounds: (1) retention indices and its correlation with those from the literature; (2) GC-MS retention times (authentic compounds); (3) mass spectra (authentic chemicals and NIST05 spectral library collection) [17]. Only fully identified compounds have been reported in this study.
The relative abundance of the volatile compounds (%) was performed on a gas chromatograph, Shimadzu 2010, with a flame ionization detector (FID). The column and chromatographic conditions were those previously reported for the GC-MS analysis. The injector temperature was 200 • C and nitrogen was used as carrier gas (1 mL·min −1 ). The relative abundance was obtained from electronic integration measurements using flame ionization detection (FID). Anethole (1000 mg·L −1 ) was added as internal standard at the beginning of the distillation procedure to simulate the behavior of volatile compounds; this chemical was used as internal standard after checking that it was absent in herbs, it separates well from other volatiles, it possesses similar FID and MS response factors to most of the volatiles in the aromatic herb essential oil, it is stable at high temperatures and does not react with water. This internal standard, anethole, was used to normalize the area of all the rice volatiles.
The volatile composition analysis was run in triplicate and results were expressed as a percentage of the total area represented by each one of the volatile compounds [16,18]. Thus, GC-MS was used for identification of the volatile compounds while GC-FID was used for establishing the relative abundance of the volatiles. This combination of GC-MS and GC-FID has been successfully used for the identification and relative abundance (%) or quantification (µg·L −1 or µg·kg −1 ) of volatile compounds in several matrices, such as aromatic herbs [18,19], wild bitter almonds (Amygdalus scoparia) [20], and Origanum majorana [21], among others.

Statistical Analysis
To compare the data two consecutive tests were performed: (i) one-way analysis of variance (ANOVA), and (ii) Tukey's multiple-range test. To be considered statistically significant the differences must be, of at least, at p < 0.05. All statistical analyses were performed using StatGraphics Plus 5.0 software (Manugistics, Inc., Rockville, MD, USA). Table 2 shows the 37 volatile compounds that have been identified by GC-MS. These compounds can be grouped into seven main chemical families: (i) aldehydes (10 compounds); (ii) ketones (four); (iii) phenol derivatives (11); (iv) furans (eight); (v) linear hydrocarbons (two); (vi) esters (one); and (vii) terpenes (one). Table 2 also shows the main sensory descriptors of each of the volatiles identified in the rice profile. From this list, it is important to highlight that several compounds have descriptors related to the wood fumes (smoked notes), such as furfural, 2-acetylfuran, 5-methyl-furfural, benzofuran, and guaiacol.

Volatile Compositions of Non-Smoked and Smoked Iranian Rice
The volatile profile of non-smoked rice was dominated by only five compounds: tetradecane (32.9%), hexanal (17.6%), benzaldehyde (14.0%), p-ethylguaiacol (13.7%), and nonanal (9.9%) ( Table 3); the concentrations of these compounds were higher than those in the non-smoked samples. Most of the compounds found in non-smoked rice samples were already found in California long-grain rice by Buttery et al. [23], as early as1988, and later confirmed in other studies [24,25]. In this way, Malekzadeh et al. also obtained similar results by concluding that the major compounds identified in Iranian rice were alkyl aldehydes [10].

Principal Component Analysis (PCA)
The PCA scores plot (Figure 1) successfully group the four smoked rice samples (R1, R5, R6, and R12). Besides, it can be observed that around these four samples, most of the compounds found belonged to two chemical families, phenol derivatives and furans; this result agrees quite well with the previous comments and results compiled in Table 4. The aldehyde and linear hydrocarbon groups were related to the 11 remaining samples of non-smoked rice and are located on the left side of the graph.

Conclusions
The volatile profiles of non-smoked rice were controlled by aldehydes (hexanal, benzaldehyde, nonanal, and decanal) and linear hydrocarbons (tetradecane), while those of smoked samples were dominated by phenol derivatives (guaiacol, phenol, 2-methoxy-4-methylphenol, etc.) and furans (furfural, and 5-methylfurfural). In the future, it will be necessary to conduct consumer studies to be able to establish correlations among the volatile compounds and the consumer preferences.

Conclusions
The volatile profiles of non-smoked rice were controlled by aldehydes (hexanal, benzaldehyde, nonanal, and decanal) and linear hydrocarbons (tetradecane), while those of smoked samples were dominated by phenol derivatives (guaiacol, phenol, 2-methoxy-4-methylphenol, etc.) and furans (furfural, and 5-methylfurfural). In the future, it will be necessary to conduct consumer studies to be able to establish correlations among the volatile compounds and the consumer preferences.

Conflicts of Interest:
The authors declare no conflict of interest.