Nutritional diversity and food potential of indigenous pigmented rice landraces from Koraput regions of Eastern Ghats

Compressive nutritional, nutraceuticals and mineral profiling was carried out in eight diverse pigmented rice landraces originated from Koraput and compared them with improved variety (IR 64). The proximate compositions such as moisture content varied from 8.23 to 11.65 g 100 g−1, ash 0.68–1.46 g 100 g−1, fat 1.07–2.23 g 100 g−1, protein 7.00–9.63 g 100 g−1, carbohydrate 76.37–80.66 g 100 g−1, fiber 0.11–1.69 g 100 g−1 and energy 346.3–362.11 kcal 100 g−1 in the studied rice lines. These landraces are rich in phenol, flavonoid, and antioxidant concentrations and varied from 3.0 to 9.0 mg g−1, 0.150 to 0.950 mg 100 g−1, and 10.8 to 40.20%, respectively. Principal component analysis explained 47.2% of the overall variation and reflected huge difference between explored genotypes. The heritability and genetic advance varied from 30.22–99.90% and 2.5–111.5%, respectively. In compared to improved IR 64 variety, rich in energy content was recorded in Paradhan, Bhatamali and Haladiganthi indicated its nutritional superiority. Further, exceptional rich in phenol, flavonoid, vitamin C, vitamin E and antioxidant capacity was recorded in Kalachudi, Bedagurumukhi and Kandulakanthi, which may create opportunities for its large-scale commercialization and cultivation. These nutrition rich landraces also hold great potential for future crop improvement programs aimed at enhancing quality.


Introduction
Rice is considered as staple food resources of majority of human population worldwide especially in Asia [1].In most underdeveloped nations where, white rice is the main staple meal, malnutrition and chronic diseases are common.On the other hand, pigmented rice variants are an alternative nutritious diet with strong antioxidant and other nutraceutical compounds, which have a substantial positive impact on human health [2,3].The pigmented rice is special-coloured landraces (Black, Purple, Reddish brown etc.) because of the presence of anthocyanin pigment [4,5].Now-a-days, cultivation of pigmented rice has become increasingly interest due to its rich in bioactive compound and health benefits [6][7][8].In addition, it has nutraceutical value showing beneficial effect against cardiovascular disease, obesity, cancer etc. [5,7,9].Despite its economic and nutritional significance, there is currently a shortage of sufficiently characterised germplasm for the breeding and selection of pigmented rice landraces with improved features.The dearth of knowledge on nutritional parameters had significantly contributed to the genetic erosion of rice.Therefore, proper phenotyping of grain quality traits in pigmented rice germplasm is important for crop improvement.
Earlier many studies highlighted the nutritional and quality assessment in pigmented rice from Thailand, China and Sri Lanka [10], pigmented rice landraces of Nigeria [11], red rice germplasm from Bangladesh [5], pigmented rice lines from Northeast India [12][13][14] and coloured rice germplasms of South India [1,8].However, genetic variation on nutritional compositions of pigmented rice lines from Eastern Ghats particularly from Koraput region is meager.
Koraput region of Eastern Ghats of India is rich in rice diversity and is considered the secondary center of origin of rice [15].The Food and Agriculture Organization recently designated this area as a global agricultural heritage site [12].Numerous indigenous pigmented rice landraces can be grown in this area by the tribal people [16].These are important resources in tribal societies due to their unique qualities, including medical, gastronomic, and cultural significance [15,16].However, in recent years, the diversity of these regionally adapted varieties established by the farmers over generations has been superseded by the development and dissemination of semi-dwarf high yielding rice types [12].According to Edwina et al. [17], these landraces have valuable genes that can help them endure un favourable environmental conditions.In order to provide food security by lowering social hunger, it is necessary to unearth the hidden genetic potential of neglected indigenous pigmented rice landraces.However, there is a lack of phenotypic knowledge regarding the quality traits and nutritional value of these pigmented rice landraces.Therefore, the objective of the study was to investigate the genetic variation in nutritional and nutraceutical compositions of pigmented rice landraces from Koraput and to assess the heritability and genetic advances, which will aid in future breeding programs.The study will also create opportunities for its large-scale commercialization and cultivation and will boost the local economy of tribal people.

Rice sample
A set of 8 pigmented rice landraces (originated from Koraput region of Eastern Ghats) collected from MSSRF, Koraput (Fig. 1).The non-pigmented rice (IR64) was collected from ICAR-NRRI, Cuttack.Table 1 provides information on all rice accessions, including ecotype, maturity period, and distinctive characteristics.Morphological variations of rice grains and kernel are shown in Fig. 2. The mature seeds were dehusked by using palm husker.The rice kernels were pulverized into powder form (2 mm particle size) by using grinder and stored in airtight containers for further quality trait analysis.

Grain morphological parameters
The five randomly chosen grains were measured in mm and classified as per IRRI [18].Based on the length to width ratio, grains are classified into three categories: medium (2.1 to 3.0 mm), large (2.0 mm), and long (> 3.0 mm) [19].

Moisture content
The oven drying method of AOAC 934.06 [20] was used to estimate the total moisture content of the rice sample.A petri plate (W1) containing 2 g of rice sample was dried in oven (at 100 °C for 24 h).Further, petri plate was placed for 30 min in a desiccator to cool completely before being weighed (W2).The moisture content was determined by applying the following formula: where W1 is the initial weight of the petri plate with sample, W2 is the final weight of the petri plate with sample after drying.

of the sample taken
× 100  Ash was appeared as greyish white colour which indicates all the organic matters of the sample was completely oxidized.The crucible was desiccated and reweighed (W3).The ash value was determined according to AOAC 923.03 [20] formula and expressed in percentage.

Fat content
The fat content of the indigenous rice varieties was determined utilizing soxhlet extraction method AOAC 920.39 [20].The solvent beaker of 100 ml was dried at 60 °C for 15 min.The blank solvent beaker weight was taken (W1) and filled with 80 ml of petroleum ether (40-60 °C).The extraction thimbles containing 2 g of rice powder were placed in solvent beaker.The soxhlet apparatus was assembled and allowed to reflux for 120 min.After extraction the thimbles were removed carefully.Then Ash content (%) = Difference in weight of ash Weight of the sample taken × 100 Fig. 2 Morphological variation of kernels of different pigmented rice landraces of Koraput solvent beaker was dried at 60 °C for 30 min till the petroleum ether was evaporated.After drying it was cooled in a desiccators and weight of solvent flask containing the fat was measured (W2).The fat content was calculated as per following formula: where W1: Beaker weight before extraction and W2: Beaker weight after extraction.

Fiber content
The acid and alkali digestion protocol of AOAC 2017.16 [20] was used to determine the crude fiber content of rice sample.Rice powder (2 g) was kept in a conical flask (500 ml) and 150 mL of H 2 SO 4 (1.25%) added to it and further the solution was boiled (for 30 min).The content was filtered and residue was collected.1.25% NaOH (150 mL) was added to the residue and allowed to boil for 30 min.The precipitate was rinsed with HCl (10%) and ethanol twice.The content was allowed to dry before collecting the residue in a pre-weighed crucible and dried overnight at 105 °C in a hot air oven.Further the sample was cooled in desiccators and the residues weighed (W1).The dried residue was then heated in a muffle furnace (at 600 °C, for 90 min.)before being cooled in desiccators and weighed again (W2).The Crude fiber (%) was determined applying following equation: Weight loss on ignition = W1-W2.

Protein content
The Kjeldahl method for nitrogen analysis was used to quantify the crude protein content.Rice sample of 200 mg was taken in the Kjeldhal flask and equal weight of salt mixture (467 g K 2 SO 4 with 28 g CuSO 4 5H 2 O and 4.7 mg metallic Selenium) was added.Thereafter, 3 mL of conc.H 2 SO 4 was added and left it overnight for digestion.The completion of sample's digestion was marked by formation of clear solution.The distillation process was carried out for 5 min each followed by titration with 0.01N HCl until changing of colour.The protein content was calculated as per the equation of AOAC 2016.014[20].
Crude protein (%) = 6.25 × % N where, S is the sample titration reading; N: normality of HCl; B: blank titration reading; D: dilution of the sample after digestion; V: the volume taken for distillation and 0.014 is the mili equivalent weight of nitrogen.

Carbohydrate content
Carbohydrate content of rice sample was calculated by Rachkeeree et al. [21]

Total Energy content
Total energy content was calculated by following equation of Rachkeeree et al. [21]

Amylose content
Using the method described by Sadasivam and Manickam [22], the amylose content was measured.Powdered rice (100 mg) was extracted with ethanol (1 mL) and 1N NaOH (5 mL) and incubated overnight.The mixture was centrifuged Total carbohydrate = 100 − (% moisture + % protein + % crude fat + % crude ash + % crude fibre) Total energy = (4 x g protein sample) + (9 x g fat sample) + (4 x g carbohydrate sample) 1 3 for 5 min at 6000 rpm.The supernatant was collected in a 50 ml conical flask and volume was made up with distilled water.The diluted extract of 2.5 mL was taken and three drops of phenolphthalein (0.1%) was added to it, then 0.1 N HCl was add on drop wise to this mixture until the solution starts to disappear its pink colour.The iodine reagent of one ml was mixed with the content and volume was maintained by addition of distilled water up to 50 mL.The colour change was analysed in a spectrophotometer at 590 nm.The amylose content was expressed as mg g −1 dwt.by using amylose as standard.

Vitamin C content
Vitamin C content was estimated using the 2, 4-dinitrophenylhydrazine-thiourea-copper sulphate (DTC) reagent following the method of Omaye et al. [23].Powdered rice sample of 1 g was extracted with 10% TCA (4.0 mL) and centrifuged at 3500 rpm for 20 min.0.5 mL of supernatant was mixed with 0.1 mL DTC reagent and further incubated at 37 °C (3 h).
After incubation chilled 65% H 2 SO 4 (0.75 mL) was added and further, incubated for an additional 30 min at room temp.The colour intensity was measured at 520 nm in spectrophotometer against a blank containing 0.5 mL of 10% TCA.Values were expressed as mg ascorbic acid 100 g −1 dwt.using known concentration of ascorbic acid (10-50 µg) as standard.

Vitamin E content
Vitamin E (α-tocopherol) content was estimated according to the method of Baker et al. [24].Powdered sample of 1.0 g was extracted with petroleum ether (2 mL) and ethanol (1.6 mL) and the reaction was generated in the dark after addition of 0.2 mL of 2, 2'-dipyridyl solution (0.2% in ethanol).The mixture was allowed to incubate for 5 min., in dark condition.The deep red colour was developed.After incubation, it was diluted with distilled water (4.0 mL) and mixed gently.The colour intensity of the aqueous layer was read at 520 nm in spectrophotometer against a blank containing only reagent.The amount of vitamin E value was expressed as mg 100 g −1 on dwt basis by taking standard as α-tocopherol in the range of 10-100 μg.

Phenol content
Phenol content was determined using the method of Sadasivam and Manickam [22].0.1 g of rice sample were refluxed in 80% methanol, evaporated, and 10 mL of the crude extract were produced.One ml of extract, 1 mL of Folin-Ciocalteu's reagent (2:1), and 2 mL of 20% Na 2 CO 3 were combined to make an aliquot, which was then incubated for two minutes in a water bath at 90 to 95 °C.The absorbance was measured at 650 nm.The phenol was estimated by taking different concentration of gallic acid as a standard.The total phenol content was expressed as mg g −1 dwt.sample.

Flavonoid content
According to Chang et al. [25], the total flavonoid content of the rice sample was calculated.Powdered rice sample of 0.5 g were soaked in ethanol (80%) for 10-15 h then centrifuged at 4000 rpm (for 30 min).The extract (0.5 mL) was diluted to 2 mL with methanol.To this 0.1 mL AlCl 3 (0.1 M), 0.1 mL potassium acetate (0.1 M) and distilled water (2.5 mL) was mixed well and incubate for 30 min.The absorbance was taken in spectrophotometer at 415 nm and the flavonoid value was presented as mg flavonoid 100 g −1 dwt.by taking different concentration of quercetin as a standard.

Antioxidant capacity
The total antioxidant capacity of the rice sample was estimated as per procedure of Prieto et al. [26].The powdered rice samples (50 mg) were extracted with 70% ethanol (25 mL) by vortex mixing.300 µL of rice extract was added with reagent solution (3 mL) (0.6 M H 2 SO 4 , 28 mM sodium dihydrophosphate dihydrate and 4 mM ammonium hepta-molybdate tetrahydrate).Then the absorbance was measured at 695 nm against blank after complete cooling.The percentage of antioxidant activity was calculated by following equation.

Determination of Zn and Fe composition
Zn and Fe content of the rice sample were estimated by following the methods of AOAC [20].A rice powder weighing 1 g was placed in a volumetric flask with a total volume of 100 mL.Then, 10 mL of the acid mixture HNO 3 :HClO 4 (9:4) was added to the flask.The contents were thoroughly mixed by swirling and the flask was placed in a digestion chamber.After completion of digestion, cooled the solution and volume was made up to 100 mL with distilled water and then filtered through filter paper (Whatman No.1).The digested sample was analyzed for mineral content using an atomic absorption spectrophotometer (AAS) (Perkin Elmer AAS, AAnalyst-200).
The AAS was filled with Zn and Fe EDL lamps using an air-acetylene flame at suitable detection wavelengths: Zn (213.86 nm) and Fe (248.32 nm).The accuracy of the metal analysis was assessed by analyzing certified reference materials.Linearity range and detection limits were ascertained using a Zn and Fe standards diluted up to 5 μg/mL.The accuracy and precision of the instrument was calibrated with standards with every 10 samples.The Zn and Fe content was recorded in parts per million (ppm) and later converted to micrograms per gram (µg g −1 ).

Data analysis
The parameters reported in all the tables are mean values of triplicate determinations (n = 3).Genetic variability parameters such as phenotypic coefficient of variance (PCV), genotypic coefficient of variation (GCV) and environmental coefficient of variation (ECV) were determined according to Burton and Devane [27].Further, genetic advance (GA) and genetic advance as mean% (GAM) were calculated.asper Johnson et al. [28].The CROPSTAT program was used for analysis of variance of studied parameters.The Fisher's least significant difference (LSD) was used for comparison of means.Principal component analysis (PCA) and cluster analysis were analyzed using PAST-3 software.

Grain morphological traits in pigmented rice landraces
The grain morphological parameters are significantly varied in pigmented rice landraces of Koraput (Table 2).The Grain length and Kernel length of pigmented rice lines are significantly smaller than that of high yielding rice IR64.The kernel length varied from 5.3 (Bedagurumukhi) to 6.7 (IR64).Based on the results these pigmented rice grains are short and medium grains rice.The grain length/width ratio and kernel Length/ width ratio were varied from 2.362 to 4.087 and 1.828 to 3.089 respectively.

Proximate composition in pigmented rice landraces
The studied pigmented rice landraces of Koraput have significantly different proximate components from one another (Table 3).Moisture content of the rice sample ranged from 8.23 to 11.65% with a mean value of 10.05%.The highest moisture content was found in Kalachudi and our results consistent with the moisture levels that have already been reported for several types of rice [29].A moisture percentage of under 12-14% is preferred to preserve rice's quality and for long-term storage [3].The ash contents ranged from 0.68 to 1.46% with an average value of 1.12%, Paradhan reported having the most ash content, followed by Bhatamali and Tikichudi.The protein contents range from 7.00 to 9.63 g 100 g −1 , with a mean value of 7.927 g 100 g −1 , respectively.Kandulakanthi was found to have the highest protein content, followed by Paradhan and Tikichudi compared to other landraces.These pigmented rice landraces of Koraput had higher protein content than rice landraces from Sri Lanka, Thailand, or China [10].The fat content of the pigmented rice landraces was varied from 1.07 to 2.23 g 100 g −1 and Kalachudi showed more fat content.These rice grains are lower fat content than that of south Indian coloured rice [8] and grains of China and Sri Lankan rice landraces [10].In the studied pigmented rice landraces carbohydrate content varied from 76.37 to 80.66 g 100 g −1 with an average value of 78.08 g 100 g −1 .The rice-eating population with type Antioxidant activity (%) = (Asample − Ablank) (Aascorbic acid − Ablank) × 100 II diabetes mellitus could be encouraged to use local rice types with reduced carbohydrate content [30].Fiber content in rice sample varied from 0.11 to 1.69 g 100 g −1 reported in the present study.Highest fiber was recorded in Paradhan followed by Malimankada and Bedagurumukhi.The examined rice landraces' total energy values ranged from 346 kcal 100 g −1 (Kalachudi) to 362 kcal 100 g −1 (Paradhan).Energy content of the current study was lower than the traditional rice reported by Verma and Srivastav [31] (367 kcal 100 g −1 ) and greater than the energy content of rice from Arunachal previously reported by Longvah et al. [1] (343 kcal 100 g −1 ).Native landraces Paradhan, Bhatamali and Haladiganthi showed exceptionally high energy content demonstrated their better nutritional status to other varieties, and these landraces may serve as dependable food security crops for the area's tribal populations.One of the key factors affecting the quality is the amylose.Paradhan had the highest amylose content, followed by Kalachudi and Haldiganthi in our study.According to Prasad et al. [32], high amylose kinds of rice could be favored for diabetes prevention and better management because they also have low glycemic load and glycemic index.

Nutraceutical compounds in pigmented rice landraces
The nutraceutical compositions, including phenol, flavonoid, and antioxidant capacity were shown in Fig. 3. Across the examined rice landraces, the phenol, flavonoid, and antioxidant capacity varied from 3.0 to 9.0 mg g −1 , 0.150 to 0.950 mg 100 g −1 and 10.8 to 40.20%, respectively.Among the studied rice landraces Kalachudi recorded maximum phenol content and Bedagurumukhi recorded high flavonoid content and antioxidant % was recorded highest in Kandulakanthi.The studied rice landraces are rich in vitamin C (1.6 to 20.16 mg 100 g −1 ) and Vitamin E (1.35 to 7.9 mg 100 g −1 ) (Fig. 4).In compared to the other genotypes, Kalachudi and Haldiganthi had the highest levels of vitamin C, whereas Kandulakanthi and Kalachudi had the highest levels of vitamin E recorded.According to Priya et al. [8], phenols and flavonoids are both recognized as helpful antioxidants that are especially adept at scavenging free radicals.These indigenous rice landraces demonstrate the effectiveness of its traditional uses and have several health advantages, including antidiabetic properties, anticancer properties, and a propensity to lower the risk of cardiovascular disorders [8,9].

Mineral compositions in pigmented rice landraces
The pigmented rice landraces exhibit high content of Iron and Zinc content (Fig. 5).The value of Fe content was varied from 3.8 to 6.4 µg g −1 and this value higher that of earlier reported mean iron content in Arunachal rice landraces (0.99 mg 100 g −1 ) by Longvah et al. [1].In the present study pigmented rice landraces Kalachudi, Malikankada and   Tikichudi recorded significantly higher Iron content than other.The Zn content of studied rice landraces varied from 0.783 to 1.005 µg g −1 .Zinc content was maximum in Kalachudi followed by Kandulakanthi and Tikichudi.Average zinc concentration was lower than the earlier value reported in Indian rice landraces (1.91 µg g −1 ) by Nirosha et al. [33].

Relationship among grain quality parameters
Multiple correlation analysis was used to determine the relationships between the grain quality parameters in the studied rice landraces (Table 4).Energy content was positively correlated with Fat (r = 0.48*), protein (r = 0.32*) and carbohydrates (r = 0.61*) whereas, inversely correlated with moisture (r = − 0.91**).Carbohydrate content was a negative correlation with moisture (r = − 0.74**) and protein (r = − 0.43*), but positively associated with Antioxidant.
Numerous earlier studies have also revealed a strong negative relationship between rice's carbohydrates, moisture, and protein [34].

Genetic variability findings of quality parameters
Genetic variability parameters in studied landraces are shown in Table 5. Results showed PCV was greater than GCV and ECV in all the traits and lower differences was reported in this study.This suggests that for the phenotypic expression of these traits and the afore mentioned traits, environment has the least influence and a significant contribution is genotypic variation [35].The traits with the highest PCV and GCV were phenol, flavonoid, vitamin c, Vitamin E, ash and fiber.This suggests that there is significant heterogeneity for these traits, based on which a useful selection of rice landraces may be made [36].These results are consistent with the report of Sivasankar et al. [37], and Sameera et al. [38] in different rice landraces.The broad sense of heritability and genetic advance of means for studied parameters were varied from 30.22% to 99.90%, and 2.5 to 111.5% respectively in the studied grain quality traits.High GAM and heritability was observed in traits like phenol, flavonoid, vitamin c, Vitamin E, ash and fiber, which proposed to using these characteristics in breeding programme and can be considered as selection criteria in rice breeding [39].

Principal component analysis
Principal component analysis was used to investigate genetic variations in grain quality parameters (Fig. 6).PCA is beneficial for pinpointing the key features that most significantly contribute to phenotypic variability and for revealing patterns of variation [36].The first two axes account for 47.2% of the overall variation and reflected huge difference between explored genotypes (Fig. 6).Two native rice landraces, Kalachudi and Kandulakanthi are grouped in one quarter with the high yielding IR64 variety in the biplot and are effectively distinguished from another by moisture and protein content.However, Tikichudi was present in the second quarter and distinguished from the others by characteristics like phenol, vitamins C and E, iron and zinc.On the basis of energy, fibre, amylose and ash content three genotypes (Paradhan, Matimankada, and Haldiganthi) present in the III quarter.Based on antioxidants and carbohydrate levels, the genotypes Bedagurumukhi and Bhatamali were distinguished from one another.there were no duplication genotypes and revealed a sizable genetic variance in the nutritional characteristic.Such variation of nutritional and nutraceutical traits among the studied rice landraces might be related to their genetic origin and geographical sources where they are grown.These pigmented rice landraces have been cultivated for several generations and continue to serve as a crucial food source for the indigenous people of the region.The degree of genetic variants among these landraces could be useful for germplasm conservation and global implications.The current study will also beneficial for breeders for creating breeding strategies for rice quality improvement and its economic use.

Conclusion
The study provides the baseline information on nutritional profiling of diverse pigmented rice landraces originated from Koraput.The pigmented rice landraces of Koraput showed adequate variability of nutritional parameters.These landraces should be preserved and sustainably used for future crop enhancement programs.The rich in energy content in landraces Paradhan, Bhatamali and Haladiganthi indicated its nutritional superiority than other.Further, exceptional rich in phenol, flavonoid and antioxidant capacity was observed in Kalachudi followed Bedagurumukhi and Kandulakanthi which also showed adequate vitamin C and vitamin E. Based on the results pigmented rice landraces Kalachudi, Malikankada and Tikichudi recorded significantly higher Iron and Zinc content compared to other genotypes.These nutrition rich pigmented landraces hold great potential for future crop improvement programs aimed to enhance the quality.Necessary steps should be taken for conserve these valuable genetic resources and production of this nutritious pigmented rice can boost the local economy of tribal people.

Fig. 1
Fig. 1 Map showing the geographical location of collection sites of pigmented rice germplasms used for the study

Fig. 3
Fig. 3 Phenol, flavonoid and antioxidant content in pigmented rice landraces of Koraput

Fig. 4
Fig. 4 Vitamin C and Vitamin E content in pigmented rice landraces of Koraput

Fig. 5
Fig. 5 Fe and Zn content in rice landraces of Koraput

Table 1
Details of rice genotypes from Koraput used for the study along with the land ecotype, length of maturity, seed and kernel colour LL lowland; ML medium land; UL upland

Table 2
Grain morphological parameters in studied rice landraces Values are mean ± standard deviation (SD) (n = 3).Values with different superscript letters in the same column are significantly different at p < 0.05.GL Grain length, GW grain width, G L/W ratio Grain Length/width, Kernel L Kernel length, Kernel W Kernel width, Kernel L/W ratio Kernel Length/width ratio, 100 Gwt 100 Grain weight

Table 3
Variation of Proximate parameters in pigmented rice landraces Values are mean ± standard deviation (SD) (n = 3).Values with different superscript letters in the same column are significantly different at p < 0.05.*: significance at P < 0.05; **: significance

Table 4
Relationship of different traits in pigmented rice genotypes of Koraput *