Microsoft Word - 3-31-38-PJAEC-15092022-460-C-Revised Galley Proof-19-05-2023-3.docx Cross Mark ISSN-1996-918X Pak. J. Anal. Environ. Chem. Vol. 24, No. 1 (2023) 31 – 38 http://doi.org/10.21743/pjaec/2023.06.03 Aroma Component Analysis Using HS/SPME-FID Gas Chromatograph in Basmati Rice Varieties of Punjab, Pakistan Farah Shamim*, Mohsin Ali Raza, Syed Sultan Ali, Samina Sarfraz and Misbah Riaz Rice Research Institute, Kala Shah Kaku, Punjab-39018, Pakistan. *Corresponding Author Email: farah_tirmazi@yahoo.com Received 15September 2022, Revised 16February2023, Accepted 20February 2023 -------------------------------------------------------------------------------------------------------------------------------------------- Abstract Aroma is a promising quality factor for rice grain that impacts consumer acceptability. The principal volatile compound that adds Basmati rice fragrance is 2-acetyl-1-pyrroline (2AP). Milled White rice of 04 promising varieties i.e., Super Basmati, Basmati-515, Basmati 2000and Basmati 370 were evaluated for volatile compounds by gas chromatography (GC) coupled with Solid Phase Micro Extraction unit (SPME) using Flame Ionizing Detector (FID). Six volatile compounds (nonanal, decanal, and alcohols such as benzyl alcohol, indole) were identified in the tested varieties, among them 2-AP is only present in aromatic rice varieties. This study confirmed the occurrence of 2-AP in all studied varieties with highest concentration in Super Basmati followed by Basmati-515, Basmati 2000 and Basmati 370. Keywords: Basmati rice, 2-acetyl-1-pyrroline, Aroma, Pakistan, Gas Chromatograph, SPME. -------------------------------------------------------------------------------------------------------------------------------------------- Introduction Fragrant/ aromatic rice varieties demand has amplified in this decade worldwide. Scent is supreme feature in rice grain, especially where consumer approval is taken a benchmark. Currently, buyer are more conscious of the eminence of the rice consumed [1,2]. Recent world consumption data and scientific studies showed that in European countries demand for aromatic rice varieties increased, particularly Basmati due to emergent attention in Eastern ethnic cuisine. Premium quality Basmati rice only originated in Pakistan and India, and jasmine rice grown in Thailand get good market price. The tropical countries have their own scented rice varieties that are popularly consumed locally besides basmati [3, 4]. Compounds responsible for aroma in rice have been under study since previous 33 years. Several compounds were recognized in cooked and un-cooked rice. Preliminary rice selection decisions were made by eating individual grains or breathing the aroma of leaf tissue or grains either heating in water or retorting with KOH. This practice is not quantifiable and it allows classification of progeny only as scented, moderately scented, and non-scented. Presence of 2-AP has been an indicator of aroma in the selection of fragrant rice lines [5]. In 1965, ammonia, hydrogen sulfide along with acetaldehyde were found in the volatiles of heated rice. The biochemical Pak. J. Anal. Environ. Chem. Vol. 24, No. 1 (2023) 32 nature of the aroma complex remained unidentified till 1982, once Ron Buttery with his team of scientists from the US Department of Agriculture (Albany, CA) successfully recognized 2-acetyl-1-pyrroline (2AP) as the prime compound apt for the pleasing aroma in rice [6]. 2-AP [IUPAC tag: 5-acetyl-3,4- dihydro-2H-pyrrole, density 1090 kgm−3, molecular weight 0.111145 kg mol−1] has minute odor verge (0.1 µgkg−1) so nominally detected by the human nose. It contributes popcorn like aroma in rice. In non-scented rice varieties, it is present at negligible levels reportedly 10 times lower than aromatic rice [4, 7, 8]. Rice aroma is interaction of numerous complex volatile compounds affected by pre- and post-harvest operations. The rapid advancement in the instrumentation and sampling methods for the isolation have made it possible to analyze compounds even at trivial concentrations (ppb levels). Extraction methods can be divided into traditional and modern. Traditional methods include purge and trap, steam distillation–solvent extraction, direct distillation and solvent extraction. Recent methods contain static headspace (SHS) followed by separation of aroma compounds using gas chromatography (GC) with flame ionization detector (FID) or mass spectroscopy (MS) and headspace–solid phase micro extraction (HS-SPME) [9, 10]. SPME is newest effective aroma analytical equipment combines sampling, extraction unified with liquid or gas chromatography. It has excellent repeatability and detection as compared to previous methods [11, 12]. Newly harvested aromatic rice varieties have robust smell but later successive operations i.e., milling, drying and storage decreases aroma. Lesser the milling degree, the greater the aroma absorbed in the milled rice [13]. Rice aroma component analysis is important to understand the aromatic nature of rice. Pakistan has quite a lot of traditional basmati rice cultivars retaining the fragrance. Nevertheless, their volatile profiles and the extent of impulsive compounds have thus far studied. This study may help to identity the variety and interpret the cooking quality of rice to relate it to further analysis for future rice breeding. Methods and Materials Rice varieties (Super basmati, Basmati-515, Basmati-2000, Basmati-370) were grown at experimental farm area of Rice Research Institute, Kala Shah Kaku, Punjab, Pakistan under standard agronomic conditions. Paddy samples were harvested at 20% moisture content, dried to 11% moisture, de- hulled, polished with Lab polisher (SATAKE TRG058, Japan) from Milling Lab to obtain white rice [14]. Freshly milled white rice samples were grinded using UDY Cyclone mill to make fine powder. 4g of flour sample was immediately placed in 20mL glass vial. Prior analysis empty vials were heated in Hot air oven at 150oC for 1 h to remove any unintended compound. After placing rice flour, vials were then instantly sealed with silicon septum/PTFE and crimp cap made with aluminum. Vial was well shaken at ambient temperature prior injection in GC [15]. The volatile compounds of rice were recognized by their different retention times from standard. Following standards were acquired from authentic manufacturer. i.e., Nonanal (Sigma Aldrich, USA), Decanal (Sigma Aldrich, USA), Vanillin (DAEJUNG, Korea), Benzyl Alcohol (Sigma Aldrich, USA), 2-acetylpyroline (Sigma Aldrich, USA), Indole (Supelco, Spain). Different dilutions of standards (0.1, 1.0, 2.0, 3.0, 4.0, 5.0 ppm and 10 ppm) were made in HPLC grade Toluene (Sigma Aldrich, USA). Pak. J. Anal. Environ. Chem. Vol. 24, No. 1 (2023) 33 Separation of volatiles mixture was achieved by Gas Chromatograph (Master GC with complete automation, DANI Instruments S.P.A, Italy) united by Flame Ionization Detector (FID), capillary column DN-WAX (30m×0.25mm×0.25µm) was used. Nitrogen gas used as carrier with flow rate of 1 mL/min. Initially 40°C column temperature was employed and then heated to 165°C @ 10°C/ min and then elevated to 250°C @ 15°C/min for 3min. The SPME fiber (57341- U) Supelco, Bellefonte, PA, USA) was desorbed in GC injector for 5 min having 250°C temperature in splitless mode. GC oven programme was hold for 1 min at 50°C to reach 100°C @ 4°C/min in increments then ramped to 240°C @ 50°C/min with ultimate hold of 2min. Identification of 2-AP and other selected volatiles was done by comparing the sample peak with standard solution peak. The vials were incubated in the auto sampler oven at 80°C, after 20 min the SPME fiber assembly was exposed to the headspace overhead the surface of the solid sample for 30 min at the same temperature. Extraction temperature set at 80°C, pre-incubation time at 10 min with adsorption period at 10 min was used for complete recovery. Area computation was used as extent of the comparing quantity during optimization. Calibration graph for quantification of volatile compounds viz. 2-AP, decanal, nonanal, vanillin, benzyl alcohol and indole was plotted. Volatile compounds were calculated by the fraction values got from the chromatogram. Chromatogram values were subtracted from the blank solvent reading. Clarity chromatographic station software was used in this study. Results and Discussion The concentration of aromatic compounds evaluated by GC analysis varied among tested aromatic rice varieties. Identification of individual volatile constituents were based on comparison with standard. Six volatile compounds were separated from all tested varieties. The chromatograms are presented as Fig.1. a, b, c, d. Component detail with data is summarized in Table 1-4, as peak number, name of volatile compounds (Benzyl alcohol, Vanillin, Nonanal, Decanal, Indole and 2-AP) retention time (12.8-19.5 min), percentage peak area (548.38 mV.s) and concentration (ppm) of the identified compounds. Aroma in rice may be created by assimilated expression of many volatile compounds. The predominant compounds identified in this study were Benzyl alcohol, Vanillin, Nonanal, Decanal, Indole and 2-AP respectively in different range as discussed below. Nonanal in food and feed is responsible for nutty flavor. Its concentration was highest in Super Basmati (14.06ppm) followed by Basmati 370 (12.52ppm) and Basmati 515. Indole exhibits musty,jasmine like flavor in food and feed.The highest concentration of Indole was present in Super Basmati and Basmati-370. Minimal traces of indole were detected in Basmati 2000. Though benzyl alcohol, responsible for the sweet floralodour, was identified in all varieties ranging 5.66 to10.67 ppm. 2-AP is the principal aromatic compound responsible for Basmati aroma, which was recognized in all studied varieties. The highest 2-APlevel was detected in Super Basmati, followed by Basmati 370 as depicted by Fig. 1. a, b, c, d. It is apparent from Table 5, that Super Basmati contained highest quantity of all volatiles except indole and vanillin. Basmati -515 is leading aromatic cultivar of Punjab, Pakistan exhibiting notable content of 2-AP. Likewise Basmati-370, an old variety still grown in Pakistan, showed aroma volatiles in prominent quantity. Basmati-2000 contained all the scented contents with the exception of Indole content which might be a varietal character. Pak. J. Anal. Environ. Chem. Vol. 24, No. 1 (2023) 34 Figure 1. a, b, c, d. Chromatogram of Basmati-515, Super Basmati, Basmati-370, Basmati-2000 (a) (b) (c) (d) Pak. J. Anal. Environ. Chem. Vol. 24, No. 1 (2023) 35 Table 1. Basmati-515 aroma component analysis. Retention Time (min) Area (m V.s) Concentration (ppm) Peak type Compound name 1 12.835 20.962 12.33 Ordnr Nonanal 2 14.688 82.167 5.33 Ordnr Decanal 3 16.576 60.325 9.25 Ordnr Benzyl Alcohol 4 16.875 1.549 0.46 Ordnr 2AP 5 18.619 62.848 9.9 Ordnr Indole 6 19.528 38.052 18.4 Ordnr Vanillin Total 265.902 45.67 - - Table 2. Super basmati aroma component analysis. Retention Time (min) Area (m V.s) Concentration (ppm) Peak type Compound name 1 12.827 56.378 14.06 Ordnr Nonanal 2 14.691 194.241 14.26 Ordnr Decanal 3 16.576 150.125 16.04 Ordnr Benzyl Alcohol 4 16.875 2.204 0.68 Ordnr 2AP 5 18.619 183.386 18.46 Ordnr Indole 6 19.536 34.573 11.67 Ordnr Vanillin Total 620.907 75.17 - - Table 3. Basmati 370 aroma component analysis. Retention Time (min) Area (m V.s) Concentration (ppm) Peak type Compound name 1 12.827 112.504 12.52 Ordnr Nonanal 2 14.691 404.245 17.92 Ordnr Decanal 3 16.576 301.525 10.67 Ordnr Benzyl Alcohol 4 16.875 37.305 0.59 Ordnr 2AP 5 18.619 305.783 19.27 Ordnr Indole 6 19.517 59.244 21.71 Ordnr Vanillin Total 1240.608 132.14 - - Table4. Basmati 2000 aroma component analysis. Retention Time (min) Area (m V.s) Concentration (ppm) Peak type Compound name 1 12.824 27.839 12.33 Ordnr Nonanal 2 14.693 11.077 5.33 Ordnr Decanal 3 16.6 3.723 9.25 Ordnr Benzyl Alcohol 4 16.84 3.816 0.46 Ordnr 2AP 5 18.619 0.987 9.9 Ordnr Indole 6 19.523 18.69 18.4 Ordnr Vanillin Total 66.144 28.75 Pak. J. Anal. Environ. Chem. Vol. 24, No. 1 (2023) 36 Table 5. Aroma component analysis of basmati varieties of punjab, Pakistan. Sr. No. Compound name Basmati 515 Super Basmati Basmati 2000 Basmati 370 1 Nonanal 12.38b 14.06a 11.60c 12.52b 2 Decanal 5.33d 14.26b 11.37c 17.92a 3 Benzyl alcohol 9.25c 16.04a 5.66d 10.67b 4 2-AP 0.46c 0.68a 0.43c 0.59b 5 Indole 9.90b 18.46a 0.00d 19.27c 6 Vanillin 18.40b 11.67d 14.69c 21.17a Results are in line with research findings of many Indian scientists. Bryantetal. 2011 [16] documented hexanal (0.787 ppm), decanal (0.065 ppm), nonanal (0.242 ppm), benzyl alcohol (0.052 ppm), guaiacol (0.033 ppm) and vanillin (0.324 ppm) in Indian Basmati 370. Scented cultivars Basmati, Della Khao Dawk and Mali were evaluated for flavor quantification and 2-AP concentration in these varieties varied from 0.05 to 0.34 ppm [17]. In another study, [15] Jezussek et al. 2011 reported 0.122 to 0.411 ppm 2-AP concentration in 11 basmati varieties of India. Furthermore, 0.214 ppm was reported in Basmati 370 collected from market of Punjab, India in the same study. In a latest study, 152 volatile compounds were separated among the tested 12 rice cultivars using solvent extraction technique [18]. Besides about 65 volatiles identified in rice bran previously [19]. Another study [15] revealed that Basmati grown in parts of Karnataka state and Maharashtra excel in 2AP (0.122 to 0.411 ppm) on other area’s Basmati. In another study slight 2AP contents were reported in black rice [20]. The majority of the volatile (heterocyclic) are prevalent in freshly harvested rice than stored [21]. The present study confirmed the prior findings that the aroma strength in rice is affected by variety selection and growing conditions mainly. Aromatic rice cultivars cannot adequately characterize just by 2-AP because other distinctive odors (e.g., earthy, nutty, roasty and green) are also demonstrating the expression [9, 22, 23]. In scented rice genotypes, 2AP can be spotted in all parts of the plant excluding roots [6] whereas in non- aromatic rice, 2AP is also found at a much lower concentration (0.0015 mg/kg) that cannot be professed easily [13]. Conclusion Aroma profile analysis and quantification in Pakistani rice varieties was accomplished first time regarding the cultivars which was not reported earlier. We can apply it as targeted breeding approach in aromatic rice variety development as it is time- consuming and laborious to cultivate new varieties without aroma profiling. 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