**5. Aroma extraction, identification and quantification methods**

The extraction process is influenced by various criteria viz., type of matrix, volatility of the analyte, concentration of constituents in the sample and extraction conditions; therefore, efficient methods of extraction is needed [14]. However, so far there is no single method that will prove ideal for aroma extraction, identification, and quantification of rice. Although, several traditional and modern methods are available for extraction and isolation of rice aroma chemicals which are coupled with analytical methods (**Figure 4**).

**Figure 4.** *Extraction and isolation methods of rice aroma.*

#### **5.1 Isolation and extraction methods**

Several extraction methods are available for extraction of VACs (volatile aromatic compounds) in rice. While considering the extraction efficiency, it differs dramatically for each method. Based on that, the selection of extraction technique can efficiently extract volatile compounds viz., alcohols, aldehydes, hydrocarbons, carboxylic acids, esters, furans, ketones, N2-containing, phenols and terpenes [94]. Most of the volatile compounds are insoluble in water so conventional extraction methods need non polar solvent as a medium.

The isolation techniques such as vacuum SDE apparatus; PTM, SDE, SEfbDI, SPME, SFE, HAS, and HSSE have their own distinguished characteristic feature. For extraction and isolation of 2-AP concentration from rice sample a desired efficient technique is a prerequisite. From the above methods, simultaneous distillation extraction (SDE), solid-phase microextraction (SFME) and supercritical fluid extraction (SFE) are the most widely used method for extraction of volatile compounds.

#### *5.1.1 Simultaneous distillation extraction (SDE)*

Simultaneous distillation extraction is a combination of vapor distillation and solvent extraction method for extraction of VACs [95]. SDE is also known as the Likens–Nickerson steam distillation, it is one of the most popular method for rice aroma chemical analysis. Solvent extract is the final product of these method. Many researchers have used this method for extraction of 2AP volatile compounds and it showed to be the most effective approach for a quantitative evaluation of 2AP. A laborious concentration step is still needed for traditional SDE procedure. Therefore, a modified version of so called micro-SDE device was proposed to overcome the problem [96].

The main advantage of this method is only a small amount of solvent is used for extraction and it also shortens the extraction time and improves the extraction efficiency. The solvents, such as: hexane, dichloromethane (DCM) and n-pentane, can be used and the amount of solvent required for SDE has been dramatically reduced compared to that of the conventional LLE. However, the major disadvantage of this method is the atmospheric pressure SDE was also obvious. Due to its high temperature, there is a possible occurrences of undesirable ester hydrolysis, Maillard reaction and sugar degradation [95].

#### *5.1.2 Solid-phase microextraction (SPME)*

This method was first introduced in early 1990s by Arthur and Pawliszyn [97]. Solid-phase microextraction is a newly emerging extraction technique for extraction of rice aroma compared with other method. It was applied in both laboratories as well as on-site [98]. Because of its persistence over other method of extraction, many results have been reported on 2AP aroma compound from rice grains [99].

Solid-phase microextraction has been used for the extraction of volatiles due to certain advantages, such as low-cost, simple, solvent-free, rapid and time-saving technique when compared with SDE method. This method can eliminate contamination, prolong the fiber lifetime and lead to reproducible results [98]. The chemistry of volatiles is decided with the help of desorption and adsorption behavior. If such extracted analytes are varied in their polarities that requires a different chemistry of SPME fiber [99].

*Rice Aroma: Biochemical, Genetics and Molecular Aspects and Its Extraction… DOI: http://dx.doi.org/10.5772/intechopen.98913*

#### *5.1.3 Supercritical fluid extraction (SFE)*

Supercritical fluid extraction is a separation and extraction process and it uses the supercritical fluids (SCFs) as the extraction solvent. It is a type of solvent which is clean and pure. The supercritical fluid is considered as 'Green Chemistry', because it is less toxic in compression compared to the organic solvents. The carbon dioxide (CO2) is an extensively used SCF; sometimes it is modified by ethanol or methanol as such co-solvents [100].

Nowadays, Supercritical fluid extraction (SFE) is widely used for extraction of volatile compounds from rice and also in other plant samples such as vegetables, fruits and so on [101].

It is a quick technique and it can recover the majority of the VACs [102]. Within 10–60 minutes the whole process would be completed and it produces the pure extract by releasing the pressure. One of the main disadvantage of SFE is less effective, than solvent extraction [101].

### **5.2 Identification and quantification methods**

The identification and quantification of volatile aromatic compounds from various types of rice is a tedious process. The research on aroma in rice is conducted by the researchers, scientists and industry groups for more than four centuries but still now it is not possible to identify all aroma compounds presented in rice. To determine the sensory quality of foods, need more concentration/efforts towards the application of modern and recently developed technologies.

There are several analytical methods for identification and quantification of rice aroma *viz.,* GC–MS, GC–MS-FID, GC–MS-AFID, GC–MS-FTD, GC–MS-SIM, Capillary GC–MS, GC-O, GC-FID, GC-PFPD, GC-TOF-MS, GLC, GLC-Capillary or GLC-Packed column. From the above methods, Gas Chromatography–Mass Spectrometry (GC–MS), Gas chromatography-olfactometry (GC-O), Gas Chromatography-Flame Ionization Detector (GC-FID) these three methods are widely used for identification and quantification of aroma in rice sample.

#### *5.2.1 Gas chromatography-mass spectrometry (GC-MS)*

Gas Chromatography–Mass Spectrometry (GC–MS) method was the most common instrumental analysis method for rice aroma analysis and effective method for analyzing volatiles, and widely used for qualitative and quantitative analysis of volatiles in rice [103]. In this method, the volatile compounds present in rice are determined and separated by GC and then identified by GC–MS [12]. This method can separate VACs having a molecular weight of less than 1,000 Dalton [104]. To date, the identification and quantification of volatile components from rice depends on advanced technologies and improved GC with multidimensional use. The qualitative and quantitative analysis of VACs is proved to be very sensitive in this method. The performance of MS is based on generated charged particle (ions) from molecules of analyses.

#### *5.2.2 Gas chromatography-olfactometry (GC-O)*

Gas chromatography-olfactometry (GC-O) is considered as one of the most advanced analytical method for the identification and quantification of VACs in sample matrix of rice. In GC-olfactometry (GC-O) system, human nose was applied to detect the odor intensity of volatiles. Two detectors which perceived the odor-active

compounds (hexanal, longifolene, 2-methoxyphenol and so on) eluted from the chromatographic column [105]. Although this method was very much useful for identification of aroma-active compounds from food samples. However, it is not suitable method for quantitative and qualitative analysis of VACs. As a result, the GC–O analytical method is not only an instrumental but also a sensorial analysis [106].

#### *5.2.3 Gas chromatography-flame ionization detector (GC-FID)*

Gas Chromatography-Flame Ionization Detector is the combination of FID with GC. This method is considered as very effective and crucial GC method because of its excellency [97]. It enables the separation, identification, and quantification of volatile compounds with their existing levels of concentrations from different food sample [98]. By comparing the retention time (RT) in GC-FID, the identification of volatile aromatic compounds of rice is completed and the retention times are converted into system-independent constant known as Kovatx retention index [99].

### **6. Factors affecting rice aroma**

#### **6.1 Genetic factors**

The genes controlling aroma was found to be a highly heritable and also relatively complex in nature. In chromosome 8 the main candidate gene was *fgr*/*badh2*/*Os2AP* homologous to betaine aldehyde dehydrogenase (BADH), whereas many other genes were also reported [103]. Deletion of 8 base pair in exon 7 or deletion of 7 base pair in exon 2 of BADH2 gene on chromosome 8 results in a loss of function of BADH2, which catalyzes the oxidation of 4-aminobutanal to 4-aminobutanoic acid. It was reported that 4-aminobutanal existed in solution equilibrium with its cyclic form 1-pyrroline which was a precursor for 2AP [107].

According to [88], the gene *fgr*/*badh2*/*Os2AP* was not the only aromatic gene in rice. The aromatic compound 2AP was identified in a number of rice varieties not carrying the 8-bp deletion. The aromatic landraces in Japan consists of six clades, none of which had the 8-bp deletion in exon 7 of BADH2 and also Japanese aromatic and non-aromatic landraces were found genetically different [108]. About 84 Subsp. indica rice landraces were investigated with respect to 8-bp deletion in BADH2 gene [109]. The results showed that aroma traits were genetically controlled by recessive monogenes, independent of cytoplasmic genes, however, aroma was also studied as a quantitative trait, and many genes were included in the expression [110].

#### **6.2 Planting and harvesting factors**

The maximum down regulation of BADH2 gene was reported in temperature of 25°C, highest 2AP content and excellent phenotypic aroma score, indicating the function of temperature on regulating phenotypic expression of aroma and final rice aroma quality. BADH2 gene expression is influenced by the temperature, phenotypic aroma score and 2AP content were investigated in three different temperatures (ambient or 28.29 ± 0.91°C, 25°C and 20°C) [2].

There is a significant positive effect on 2AP content in rice grains (Meixiangzhan and Nongxiang 18) by the application of manganese, which esults in probably improvement of enzyme activities involved in 2AP formation. Higher total soil nitrogen plays a major role in producing rice aroma. During flowering stage, it was found that Si contents in leaves were positively related with 2AP contents.

*Rice Aroma: Biochemical, Genetics and Molecular Aspects and Its Extraction… DOI: http://dx.doi.org/10.5772/intechopen.98913*

Thus, indicating that Si application to some amount will improve 2AP contents in grains [111].

An increase of 2AP content in grains with salinity was observed for three improved aromatic rice varieties and salinity was thought to have a positive effect on rice aroma quality [112]. NaCl stress enhanced aroma production in Tulaipanji, Radhunipagal and Gobindobhog rice varieties while weaken that in Kalonunia [113]. Shading treatments during grain filling significantly increased 2AP content in both Yuxiangyouzhan and Nongxiang rice varieties, and had a selective effect on the metabolism of other volatiles [114].
