Nutra-fragnance a sustainability boost of Odisha landraces

Introduction

Asia's role in the cultivation and production of aromatic rice varieties is significant due to its favourable climatic conditions, traditional agricultural practices and rich biodiversity (Smith, 2020) [1] . Regions like India, Thailand, and Vietnam, with their tropical and subtropical climates, provide an ideal environment for aromatic rice growth (Johnson, 2019) [2] . These climates offer the necessary warmth and humidity for varieties such as Basmati and Jasmine, which are in high global demand and are crucial export commodities that significantly contribute to the economies of rice-producing nations in Asia (Lee et al., 2021) [3] . India is a country of Asia, is recognized as a significant producer of diverse rice varieties, particularly aromatic rice land races, predominantly featuring small to medium grains. It is acknowledged as the origin center for cultivated rice, boasting extensive genetic diversity (De Candolle 1886; Vavilov 1992) [4,5] . These indigenous aromatic rice accessions are cultivated across nearly all Indian states, including Odisha. They are distinguished by their unique aroma, superior cooking and eating qualities and enhanced nutritional content compared to basmati rice alongside their adaptability to local environmental conditions. These varieties have naturally evolved over many years, resulting in high genetic diversity and distinct alleles for various agronomic traits (McCouch et al., 1997). Odisha, a state in eastern India, is known for its rich biodiversity, especially in rice landraces. These landraces are traditional varieties that have been cultivated and passed down through generations, often adapted to local conditions and possessing unique traits. The state has its own set of aromatic short grain rice, which is being cultivated in almost all districts in different agro-climatic zones. The aromatic rice is considered sacred among the people. These rice accessions hold enormous potential to be utilized as value added products, thereby providing higher economic return to small and marginal farmers and hold significant cultural and aesthetic value due to their distinctive aroma and superior grain quality. It is found from review the aromatic landraces are comparatively lesser yield value due to various agro-morphological characteristics like tall plant leads to high lodging, low response to nutrients, despite of this, the farmer of the land unhabituated to grow landraces in their own practice (Rao et al., 2006; Dixit et al., 2013) [7,6] . Aroma in rice is primarily attributed to the presence of 2-acetyl-1pyrroline (2AP), a volatile compound that gives rice its characteristic fragrance. The aroma is influenced by genetic factors, with certain genes controlling the synthesis of 2AP. In Odisha, aromatic rice landraces are often evaluated using sensory methods and molecular markers to identify genotypes with desirable aroma traits (Panda et al., 2020; Hossain et al., 2020) [9,8] . Proximate composition which includes moisture, protein, fat, fiber, and ash content and these components are essential for settling the nutritional quality and processing characteristics of rice. Landraces of Odisha have been studied for its proximate composition to understand their nutritional importance and potential for breeding programs. To understand the aroma and proximate composition of rice landraces is crucial for several reasons, like breeding programme on identifying and incorporating desirable traits into new rice varieties can enhance their market value and consumer acceptance (Chaudhary et al., 2001; Prodhan et al., 2020) [10,11] . On documenting the unique traits of landraces that helps in their conservation, sustainable use and in increasing economic value as aromatic rice often commands a premium price in the market making it economically beneficial for farmers. To satisfyl the demand of need, promoting the market, provide value added to farmers, this study of aroma with proximate composition in rice landraces of Odisha is itself a challenge to fill the gap and find the varieties of landraces on the mid-economic land like Odisha. This provides valuable insights into their genetic diversity, nutritional quality, and potential for improving rice varieties (Singh et al., 2005) [12] .

Methodology

Estimation of carbohydrate by Anthrone method

The powdered rice sample was taken in a boiling tube. It was hydrolyzed for 3 h with 5 ml of 2.5 N HCl. Cooled and neutralized by adding solid sodium carbonate followed by centrifugation. Then it was spectroscopically analysed against standard and the value was calculated.

Carbohydrate in 100 mg sample = ( mg of glucose/Volume of test sample ) × 100

Estimation of starch by Anthrone reagent

The milled rice was taken for estimation of starch by following Sadasivan and Manickam (1991). The rice sample was homogenized in hot ethanol (80%) followed by centrifugation Repeated washing was done with ethanol by testing with anthrone reagent followed addition of 5 ml of water and 6.5 ml of 52% perchloric acid. Then it was spectroscopically analysed against standard and the value was calculated. .

Estimation of protein by Lowry's method

Powered rice sample was taken by adding buffer against Bovine serum as standard. The test followed by 5 ml of alkaline copper solution, 0.5 ml of Folin-ciocalteau. The test was spectroscopically analysed.

Estimation of free fatty acids (Sadasivam and Manickam, 1992)

Powdered rice sample and absolute ethanol was taken for analysis followed by a few drops of phenolphthalein. The titration was done against 0.1N potassium hydroxide. Then it was calculated by,

Assessment of Crude fiber content (Sadasivam and Manickam, 1992)

Dried powdered sample was taken along with 200 ml of sulphuric acid followed by adding 200 ml of sodium hydroxide solution. Then it washed with 25 ml of boiling 1.25% H 2 SO 4 . Again, it was washed with three 50 ml parts of water and 25 ml of alcohol. The residues were removed and it was transferred to a washing dish (pre weighted dish W 1. ). The residue was dried for 2 h at 130 ± 2 °C. The dish was cooled in a desiccator and the weight was taken (W 2 ). It was then ignited for 30 minutes at 600 ± 15 °C. It was then cooled in a desiccator and reweight was taken (W 3 ). Fiber content was calculated using the following formula.

Assessment of mineral content (Iron and Zinc) -by wet digestion process using atomic absorption spectrophotometer.

Powdered rice sample was digested with 10 ml of diacids mixture (nitric acid and perchloric acid) at a ratio of 2:1 (v/v). The absorbance of the standard solution and the sample solution was taken by atomic absorption spectrophotometer at 560 nm and 510 nm respectively. Then the concentration value was plotted and noted down from graph on known to unknown value.

Biochemical identification, of scented compounds

Separation and analysis of volatile compounds were done using HS-SPME coupled with GC-MS (Varian 430-GC and 210-MS, Japan) with a factor four capillary column VF5-MS (30 m × 0.25 mm × 0.25 μm) of 5% diphenyl, 95% dimethylpolysiloxane. Research-grade helium (99.999%) was used as the carrier gas under a constant flow of 28.6 cm/s (1 mL/min). The SPME fiber was desorbed for 5 min in the GC injector with a temperature of 260 °C. The optimum performance of the SPME fiber was monitored after every ten extractions with the standard fiber maintained separately. The fiber showing comparable performance was continued in further analysis. The GC oven program was optimized for the separation of 2AP from the seed. Initially, the oven temperature was kept at 45 °C for 1 min and ramped to 55 °C at a rate of 5 °C/min, then increased to 120 °C at a rate of 9 °C/min, further ramped to 240 °C at a rate of 15 °C/min with a final hold of 1 min. The total GC cycle consisted of 19.

Statistical analysis

Statistical analysis and Principal component analysis (PCA) was conducted on the different traits using PAST-4.03 software to identify sources of variability.

Results and Discussion

Nutritional quality

Protein of rice is a major identifiable bio macronutrient that impact the nutritional quality Sompong et al. (2011) [13] due to its special amino acid composition though the amount is less in rice. The results find mean protein value 9.99% maximum in Kalajeera and 9.670% percentile of studied landraces are having good protein content which is close agreement to Resurrection et al., (1979) [14] . Protein content greater than 10% is mostly acceptable by food products and can be used to cure malnutrition Resurrection et al., (1979) [14] . The lower and similar result has been found by Masniawati et al. (2018) [15] , Juliano et al., (1985) [16] , Anjum et al., (2007) [17] , Chaudhary et al., (2001) [10] and Verma et al., (2017) [18] . The variation of protein content in rice may be due to environment, biotic and abiotic stresses, and application of fertilizer, growing areas, temperature and growth condition for synthesis and accumulation of protein in rice Buresova et al., (2010) [19] . Fat is another bio macronutrient representing the linoleic acid and other fatty acid without cholesterol Eggum et al., 1982 [20] . Low fat content is also a mark of aromatic short grain Singh et al., 1998 [21] . The taste of rice may differ due to high fat content and less starch. The results are in agreement with earlier findings (Verma & Srivastav, 2017 [18] ; Chen et al., 2004 [22] ; Cameron and Wang, 2005 [23] ; Rohman et al., 2014 [24] ; Saikia, 2012; Sompong et al., 2012). The fat in rice is mostly present at the aleurone layer of the kernel so, milling is the major cause of variation in getting it from storing nutrient of rice Wang et al., 2004 [25] . The variation of fat which is mostly unsaturated due to oxidation by atmospheric oxygen. Total carbohydrate comprises starch and cellulose. Total carbohydrate percentage ranged 64.281% of Kalajeera to 78% of Basumati-4, the result is in agreement with earlier finding of Verma et al., (2017) [18] . In total carbohydrate the starch comprising amylose and amylopectin. Rice starch is quick digest nutrient component than other starchy food Oko et al., 2012 [26] . The result shows Starch content ranges 66.9% of Gitanjali to 91.89% of Chinikamini. Similar result studied by Subudhi et al. (2013) [27] and Shayo et al.

(2006) [28] , whereas lower content has been reported by, Oko et al., (2010) [29] , Ebuchi et al. (2006) [30] , Yadav et al. (2007) [31] , Thomas et al., (2013) [32] , Verma & Srivastav, (2017) and Saikia et al., (2012) [33] . The lower starch content is the cause of no stickiness of rice whereas higher value is the cause of stickiness of rice after cooking Mbatchou et al. (2013) [34] . Crude fibre the important macronutrient biochemically reduces the blood cholesterol and decreases blood sugar which help in control of diabetics Yeager, (1998) [35] also release constipation by controlling bowel disorders FAO/WHO, (1998) [36] . The diet fibre directly enhances the bulk of faces that evacuate out-turn in gut (Mbatchou et al., 2013) [34] . It is studied that well refined rice has crude fiber percentage ranged from 0.5% to 1.0% (Oko et al., 2010) [29] . The present result of crude fiber percentage ranges from 0.46% (Bishnubhog) to 0.99% (Acharamati and Thakurbhog) that is little lower and close to an agreement to the standard value (Verma et [18,37,34] . The present finding of, fiber percentage are lower than the findings of many researchers [26,24,17,32,13] whereas it is higher than the finding of Saikia, (2012) and Diako et al. (2011) [38] . The digestibility of rice impacted by crude fibre. The digestibility will be lower in higher content of crude fiber in diet (WHO, 1985) [39] . The minerals are the bio micronutrients of rice effectively play its role in bio metabolic pathway. The Zinc ranges from 18.370 mg/kg to 23.390 mg/kg and Iron ranges from 6.58 mg/kg to 20 mg/kg. The result is in agreement with Juliano, (1980) [40] . Level of Zinc decreases by milling (Itani et al., 2004) [41] (Fig 1). The correlation (Table 1; Fig 2 ) shows protein is significantly and positively correlate with crude fiber (r = 0.300**), with starch (r = 0.203*) and with total carbohydrate (r = 0.261**). Crude fiber with Iron (r = 0.196*). Similarly Starch with Zinc (r = 0.230**). This signifies the various nutrient traits concentration are dependent among themselves. The PCA study (Table . 2) results Thakurbhog, Kalajeera-7, Pimpudibasa, Krusnabhog, Dubraj-2, Basumati, Jaygopal might be exploited for crop improvement, plant breeding and genetic conservation program. The present finding on nutrient traits correlation among Protein with crude fiber (r = 0.300*) and with carbohydrate (r = 0.261**) is in equivalent support of Meena et al. (2020) [42] who recorded protein was significantly correlate with amylose content. (A-Protein%, B-Crude fat%, C-Crude Fibre%, D-Starch%, E-Total Carbohydrate%, F-Iron(mg/kg), G-Zinc(mg/kg)) Scatter diagram of PCA on nutrient characters of different aromatic landraces were observed. The scatter diagram grouped the landraces into four quarters. In the first quarter (X, X) 42 genotypes such as sunsuniasunaphool, Kalajeera-5, Pimpudibasa, Tulashi, Kalamalliphula etc. were present based on nutrient characters such as crude fibre, Iron, Protein, and total carbohydrates. In the second quarter (-X, Y) 9 landraces (Desibasumati, Durgabhog, Basubhog, Ramachandrabhog, Bindli etc.) were present based on crude fat%. In the fourth quarter (X, -Y), 23 landraces (Krishnabhog, Basumati, Kanika, Heerakhandi, kalakati, Raktasiali etc..) were present based on nutrient character like Zinc and starch. In third quarter there was no differentiation of landraces based on nutrient characters. (Fig. 3).

Detection of scented compounds by GCMS analysis

In a detail analysis of 128 rice landraces using GCMS headspace techniques, a diverse array of scented compounds was identified (Fig. 4). The predominant chemical groups associated with the aroma encompassed alkanes, heterocycles, ethers, esters, ketones, and aliphatic amino acids. Notably, 2-Acetyl-1-pyroline (2AP), which imparts a characteristic popcorn aroma, was consistently present across all landraces, reinforcing its significance in rice aroma research. Along with this Ethane, 1-isothiocyanato-2-methoxy is notable for its fruity scent and is prominent in the Basumati landrace which demonstrated multiple peaks associated with this compound. The results justify on existing studies that have established the crucial role of 2AP in rice fragrance and it was established that concentration of 2AP is a strong determinant of the aromatic quality in rice varieties especially in traditional landraces such as Jasmine and Basmati. However, the detection of other new compounds like Dimethyl ether and Formic acid, 1methylethyl ester adds depth to the aromatic profile of the rice that suggests the potential unexplored avenues for enhancing aroma through breeding and processing. Focusing and finding of other studies like by Juliano et al. (1985) [16] and Koh et al. (2007) on the quantification and sensory evaluation of aromatic compounds, this study provides a more nuanced chemical profiling of the landraces. The comprehensive mass fragmentation patterns and retention times documented contribute valuable data for further genetic studies and breeding programs aimed at improving aromatic properties. The result finding of this particular research not only corroborates previous findings on key aroma compounds but also expands the known aromatic spectrum of rice, determining the complexity and diversity present in different landraces. Further studies could build on these findings to explore the relationship between specific compounds and sensory attributes, potentially guiding the development of rice varieties with enhanced aroma profiles for culinary applications.

Analysis of aromatic compounds

Rice that discharges fragnance on hulling and cooking (Jefferson, 1985) [43] is mostly due to aromatic compounds biosynthetically present in it. The aroma in aromatic rice is produced, accumulated and varied due to genetic (Panda et al., 2020) [9] , preharvesting conditions like environmental, abiotic stress, temperature, soil and cultural practices (Hossain et al., 2020) [8] . 2AP compound which is popcorn like smell, (Tanchotikul et al., 1991) [44] is mostly present in all studied aromatic short grain landraces. Similar result was found by (Hinge et al., 2016) [45] . This compound was detected as contributor of aromatic odour (Buttery et al., 1982) [46] . The quantification results minimum concentration 0.14 ppm in Dhusara and maximum 0.99 ppm in Thakurbhog. 2-acetyl-1-pyrolline concentration in Della (76.2 ppb), Basmati 370 (87.4 ppb) and in Jasmine (156.1 ppb) was also found. Buttery et al., 1983 [47] found the concentration of similar 2-acetyl-1pyroline in 10 aromatic rice which range from 0.09 ppm-0.006 ppm in milled rice and 0.2 ppm to 0.1 ppm. in brown rice. The surface layer is the major storage region of aromatic compound in grain of aromatic rice for which the raw milled or brown rice had 2-acetyl-1-pyrroline concentration ranged from 100ppb-200ppb and milled rice concentration range was from 6ppb-90ppb (Webber et al., 2011) [48] . The change in concentration of aromatic compound in scented rice may be due to temperature factor and late harvesting (Itani et al., 2004) [41] and cultural practice (Prodhan et al., 2020) [11] . Besides the major aromatic component 2AP, other scented compounds were also detected. These include Ethane, 1-isothiocyanato-2-methoxy (fruity smell), Dimethyl ether (sweet smell), Formic acid, 1-methylethyl ester (ethereal odour), Alanine (umami smell), and Boric acid trimethyl ester (sweet odour) (Fig 5: Table 3). The results surpass the findings of Hinge et al. (2016) [45] , who identified 88 scented compounds. The present analysis of the resulting scented compounds reveals a completely five new group of scented compounds found in the studied indigenous landraces of Odisha. The high protein contents are the cause of low aroma (Champagne et al., 2007; Champagne, 2008; Prodhan et al., 2020) [49,50,11] . So nutrient and aromatic compounds may biosynthetically correlate.

Quantification of 2AP

The concentration range, particularly the high value of 0.99 ppm in Thakurbhog and the lower value of 0.14 ppm in Dhusara, corroborates findings from other researchers, who often find substantial variability in 2AP levels among aromatic cultivars. For example, studies have shown that some landraces can exhibit concentrations exceeding 2 ppm, while others may fall below 0.1 ppm. Moreover, the coefficient of variation of 24.03% you reported indicates a moderate level of variability, which is consistent with findings reported in other studies, where the genetic diversity of rice landraces resulted in significant differences in aromatic compound profiles. The observation that 75% of the aromatic landraces had a concentration around 0.28 ppm reflects a trend noted in previous research, where a large proportion of aromatic rice varieties tend to cluster within specific ranges of 2AP concentration. The findings regarding 2-acetyl-1-pyrroline (2AP) concentration in scented rice landraces align with the body of research on aromatic compounds in rice. Studies conducted by various scientists have consistently identified 2AP as a significant contributor to the characteristic aroma of rice varieties. For instance, a notable study by Septiningsih et al. (2009) [51] reported similar variations in 2AP levels across different rice varieties, emphasizing the genetic basis for these differences.

Conclusion

The present research finding on the nutritional and aromatic qualities of various rice landraces, focusing the major macronutrients such as protein, fat and carbohydrates as well as micronutrients like zinc and iron. The results show that protein content varies among the landraces as Kalajeera having the highest at 9.99%. The average starch content ranges from 66.9% to 91.89% affecting the stickiness of cooked rice. Crude fiber levels are lower than some prior studies ranging from 0.46% to 0.99%. The result reveals a significant correlation between protein and other nutrients that indicates interdependence among these traits. The finding also identifies aromatic compounds using GCMS with 2-Acetyl-1-pyroline (2AP) being predominant. The concentration of 2AP varies significantly across landraces predominant its importance in the aroma profile of rice which provides an in-depth understanding of the nutritional composition and aromatic characteristics of rice landraces. The positive significant correlations identified suggest that certain nutrient traits can influence each other and they may be beneficial for breeding programs aimed at enhancing both nutritional and aromatic qualities in rice. The detection of a diverse range of aromatic compounds particularly 2AP underscores the genetic diversity and culinary potential of these landraces facilitating for further exploration into their breeding and processing for improved rice varieties.

Acknowledgement

Acknowledgement-The author is thankful to the P. G. Department of Botany, Utkal University, Vani Vihar, BBSR, Odisha and P. G. Department of Botany, Dhenkanal Autonomous College, Dhenkanal, Odisha, India for providing facilities for doing this research work.

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