Revealing the biochemical profile and antioxidant activity of kodo millet ( Paspalum scrobiculatum L.

Kodo millet ( Paspalum scrobiculatum L.) is a traditional millet crop extensively cultivated in Asia, particularly India and Africa. The crop is grown in adverse environmental conditions and resilient to biotic and abiotic stresses, and the grains are prized for their higher nutritional value than major cereals. In the present study, biochemical components and antioxidant activity were examined in grains of four kodo millet cultivars and compared with rice. Results showed that four kodo millet cultivars had higher amylose content (14.8-29.66%), protein (7.1-9.7%), polyphenol (446.29-553.07 mg GAE/100 g), and elevated antioxidant activity (44.29-87.5 inhibition %) than rice cultivars. Also, we obtained a strong positive correlation between starch, amylose, protein content, antioxidant activity and total phenolic content (TPC). Notably, among the kodo millet cultivars, ATL 1 and CO 3 exhibited high amylose, protein, antioxidants, and phenolic content levels. Collectively, the information obtained in our study provides useful information to improve the targeted traits in kodo millet breeding programs.


INTRODUCTION
Kodo millet (Paspalum scrobiculatum L.) is recognized as a tetraploid with the chromosome of 2n=4x=40.This lesserknown small millet, belonging to the Poaceae family, is renowned for its ability to grow in adverse environmental conditions with less input.The crop is extensively cultivated in India, Pakistan, Thailand, Indonesia, Vietnam and Western Africa (Thakur et al., 2020).Kodo millet holds significance as a staple grain for inhabitants of the Deccan plateau region of India, as well as in specific areas of Maharashtra, Odisha, West Bengal, the Himalayas, Uttar Pradesh, and Rajasthan.This resilient grain boasts a carbohydrate content ranging from 60% to 70%, fats-1.4%,minerals-2.6%,iron-25.86-39.60ppm, making it a valuable source of energy (Girish et al., 2014).Additionally, kodo millet is rich in protein, with levels averaging around 7% to 13%, enhancing its nutritional value (Deshpande et al., 2015).Moreover, this hardy millet contains essential minerals such as iron, calcium, and phosphorus, contributing to its potential in addressing micronutrient deficiencies.Its ability to thrive in diverse agro-climatic conditions and adapt to varying soil types makes it an invaluable asset for crop improvement efforts in the face of climate change (Saxena et al., 2018).Additionally, it boasts heightened forage value, with its leaves serving as a palatable feed for goats and sheep.Compared to the other minor millets, kodo millet distinguishes itself by possessing superior phosphorus, phenols, and radical scavenging activity and decreased anticipated glycemic index in the grains than cereals (Hegde & Chandra, 2005;Neelam et al., 2013).Lecithin, a neurotransmitter, is abundant in kodo millet leaves, while its grains are employed in the production of gluten-free commodities such as biscuits, noodles, and laddus, which are gaining popularity in eateries for their health benefits especially for diabetes patients (Deshpande et al., 2015).On the other hand, for breeding and the management of germplasm, knowledge of the genetic variation among accessions or cultivars and the diversity of the available germplasm is important.Both farmers and plant breeders can use it to support their breeding efforts.Therefore, identification of starch and protein rich germplasm is necessary for kodo millet nutritional breeding programs.
In light of this context, the current study's objectives were to investigate the biochemical components and antioxidant activity in kodo millet cultivars and compare them to rice.The data from our study can help kodo millet breeding programs.

Plant Genetic Materials
Kodo millet cultivars viz., 'CO 3',' ATL 1','TNAU 86','CO 43' and 'Seeraga Samba' seeds were obtained from Center of Excellence for Millets, Tamil Nadu Agricultural University, Athiyandal, Tiruvannamalai, India and Paddy breeding station, Tamil Nadu Agricultural University, Coimbatore, India.The salient features of the kodo millet cultivars are given in Table 1.The grains of kodo millet and rice cultivars were well-dried and powdered prior to the analysis.500 mg of powdered sample was used for the estimation of biochemical traits.

Determination of Starch, Amylose, and Amylopectin
The estimation of starch was carried out utilizing the anthrone reagent method at the absorbance of 630 nm (DuBois et al., 1956).The carbohydrate in the sample (μg/mL) was estimated from the standard graph equation y = 0.0069x -0.0174 with an R² =0.99, with the standard as glucose.The glucose value was converted to starch by applying a conversion factor of 0.91 (MacRae, 1971).The total starch content was quantified as a percentage within a weight of 100 g of seeds (g/100 g of seed).Amylose content was determined using the iodine complex colorimetric method (McCready et al., 1950).This technique leverages the deposition of the iodine complex on the helical strands of amylose, inducing the manifestation of a blue coloration, which is subsequently measured using a spectrophotometer at 600 nm.The amylose content of the sample (μg/mL) was estimated from the reference standard graph equation y = 0.0014x -0.0131, R² =0.98, with potato amylose as standard.The quantification of total amylose content was expressed as a percentage within a weight of 100 g of seeds (g/100 g of seed).The quantification of amylopectin was achieved by calculating the difference between the quantities of total starch and amylose (Nikitha & Natarajan, 2020).

Total Protein and Phenolics Content
Protein content was quantified using Lowry's method and measured the absorbance at 660 nm (Lowry et al., 1951).The sample's protein content (μg/mL) was estimated from the reference standard graph, equation y = 0.0004x + 0.0061, R² value = 0.99, and standard as Bovine serum albumin.The total phenolic content (TPC) was quantified on the sample extracts using the Folin-Ciocalteau reagent, and absorbance was measured at 725 nm (Chandrasekara & Shahidi, 2010).Gallic acid was used as the reference standard.The TPC of samples was estimated from the regression equation y = 0.0014x -0.0136, R² = 0.9959 with standard as gallic acid.The concentration of phenols present within the sample was subsequently expressed in terms of milligrams of Gallic Acid Equivalents (GAE) per 100 g of seed sample (mg GAE/100 g of seed).

DPPH Assay
Within the phenolic fraction of kodo millet grains, antioxidant activity was evaluated by assessing radical scavenging activity (DPPH inhibition %) employing the DPPH method (Blois, 1958).The absorbance was measured at 517 nm using a UV-Vis spectrophotometer.The DDPH inhibition % was calculated from the formula, Percentage Inhibition = [(Abs blank -Abs sample )/Abs blank ] × 100, where, Abs blank is the absorbance of the blank and Abs sample is the absorbance of the sample.

Statistical Analysis
All the experiments were replicated in triplicates, and the results were reported as a mean±standard deviation.Duncan's new Multiple Range Test (DMRT) was performed for biochemical analysis.The significance level was p< 0.05 (de Mendiburu, 2019).Correlation analysis was performed to find the positive and negative correlating traits.All statistical analysis was performed in R software version 4.2.3 (Team, 2023).

RESULTS
The statistical analysis reflected significant genetic variation among the kodo millet genotypes at P = 0.01 or 0.05.The four genotypes were analyzed for various biochemical traits and revealed that genotypes were significantly different (Table 2).

Starch, Amylose, and Amylopectin
The percentage yields of starch obtained for the four distinct kodo millet cultivars, CO 3, ATL 1, TNAU 86, and RK 390-25, alongside the control samples (Table 2).The starch % for all the cultivars ranged from 43.17% to 59.68% within the kodo millet and from 51.69% to 95.86% within the rice cultivars.There was a significant difference between the percentage of highest starch and the lowest starch accumulating types within the kodo millet four cultivars.CO 3 starch % was on par with ATL 1, and TNAU 86 was on par with RK 390-25.Conversely, in the rice cultivars, the highest starch content was attained by the ADT 36 cultivar (Figure 1a).The amylose content within grains is pivotal in both industrial applications and health-related benefits.Among the considered cultivars, the amylose percentages ranged from 14.8% to 29.6% in kodo millet.Notably, a significant difference between kodo millet cultivars was recorded.The highest amylose content was identified in the ATL 1 cultivar, measuring at 29.66%, followed by CO 3 cultivar (28.01%) (Figure 1a).The determination of amylopectin content, calculated as the difference between total starch and amylose content, observed an overall lower percentage of amylopectin within the kodo millet cultivars.Moreover, the amylose content observed in kodo millet was comparable to the amylose-rich rice cultivars such as CO 43 and ADT 36 (Figure 1a).This underscores the potential of kodo millet as a source of amylose for industrial applications and its parallelism with certain rice cultivars known for their elevated amylose content.A higher ratio of amylose to amylopectin signifies a lower estimated Glycemic Index (eGI) associated with health benefits.Kodo millet displayed a notably high amylose-to-amylopectin ratio compared to rice, indicating its potential for a lower eGI and its consequent positive impact on health.Amylose: amylopectin ratio exhibited a significant difference between CO 3, ATL 1, and other cultivars within the kodo millet.This distinction further accentuates the potential of these specific kodo millet cultivars, suggesting their capacity to offer a particularly low eGI, which can contribute to healthier dietary choices.

Protein and Total Phenolic Contents
Across the four cultivars of kodo millet, the protein content ranged from 7.1-9.7%,with significant differences.In comparison, the protein content of kodo millet was higher than the considered rice cultivars (3.69% to 5.26%).Notably, the highest protein content value (9.7 g/100 g) was recorded for the ATL 1 cultivar within the kodo millet (Figure 1b).These findings underline the variability in protein content between kodo millet and rice cultivars, with the ATL 1 cultivar standing out for its particularly high protein content.This variation in protein content holds implications for their nutritional profiles and potential applications in various dietary and food contexts.The TPC exhibited a range of 446.29-553.07mg GAE/100 g, with significant differences between rice cultivars (Table 2).Among the kodo millet cultivars, the highest TPC content was observed in the ATL 1 cultivar (553.07 mg GAE/100 g), while the CO 3 cultivar recorded the lowest TPC content at 446.29 mg GAE/100 g (Figure 1c).This variation in phenolic content is significant for potential health benefits such as control for diabetes and cardiovascular diseases.

DPPH-Radical Scavenging Activity
The antioxidant activity of the phenolic fraction was evaluated using the DPPH inhibition percentage and resulted in significant differences within kodo millet cultivars.The DPPH inhibition % ranged from a low percentage of 44.29% to a high of 87.5%.Notably, the highest antioxidant activity was observed in the ATL 1 (87.5%) and CO 3 (80.98%)cultivars (Figure 1d).In contrast, the TNAU 86 cultivar displayed the lowest DPPH inhibition at 44.29%.Comparatively, kodo millet had higher antioxidant potential than rice cultivars and reported a significant difference.Such diversity in antioxidant activity emphasizes the potential health-promoting attributes of specific millet cultivars in their ability to counteract oxidative stress.

Correlation Analysis
A strong positive correlation was observed between the starch and amylose content and the levels of protein, TPC, and antioxidant activity (>0.8).A negative correlation between amylopectin and other traits has been observed (<1).Positive correlation also existed between antioxidant potential and TPC.This confirms that kodo millet has more antioxidant potential with high amylose and protein content (Figure 2).

DISCUSSION
Physiochemical analysis of the kodo millet cultivars for various biochemical traits compared to rice helped identify the potential benefits of kodo millet cultivars (Hariprasanna, 2017).The mean starch and amylose content values observed in rice checks were similar to the previous findings (Pandarinathan, 2019;Naik et al., 2023).In the context of kodo millet, the starch content observed in the cultivars ATL 1 and CO 3 cultivars aligns with those reported in the same cultivars of kodo millet (Nirmalakumari et al., 2022).The amylose content was higher than amylopectin in kodo millet cultivars, similar findings in barnyard millet genotypes were also reported (Dimri & Singh, 2022).While previous studies reported amylose content in kodo millet to be around 19.6% (Zhu, 2014) and 13.99% (Bangar et al., 2021).The higher amylose content in ATL 1 and CO 3 could be attributed to smaller granules and referred to as "B-type granules".Starch with B-type granules is resistant to digestion, thus indirectly conferring an antidiabetic nature (Yang et al., 2019;Sangwongchai et al., 2023).In pearl millet, it is reported that high amylose content can contribute to the presence of non-waxy-type starch (Suma & Urooj, 2015).Recent studies reported other physiochemical properties of kodo millet, such as swelling power, viscosity, and solubility.High amylose starches have good pasting behavior with higher density, higher gelatinization temperature, retrogradation, film-forming properties, and, more importantly, resistant starch formation prone to less digestibility (Garhwal et al., 2022).Its economic significance is emphasized because the stored starch in kodo millet emerges as a resistant starch type (Kaimal et al., 2021).Additionally, the amylopectin content was comparatively lower than amylose in kodo millet, highlighting the distinctive composition of these two starch components within the grain.Also, high amylose-to-amylopectin ratio plays a pivotal role in contributing to a lower Glycemic Index (GI) through the mechanism of slow digestion (Verma et al., 2020;Al-Atbi et al., 2023;Goudar et al., 2023).
The protein content in rice was similar to the results of previous studies (Jaiganesh et al., 2019;Parkavi et al., 2020).The current study reported that the protein content within kodo millet is notably higher than in rice cultivars (Yankah et al., 2020;Girish et al., 2014).The protein content of kodo millet observed in ATL 1, CO 3, and TNAU 86 was similar to the previous findings (Nirmalakumari et al., 2022).The analyzed cultivars have higher protein content than the other kodo millet cultivars (Deshpande et al., 2015;Rajput et al., 2019).This divergence between different cultivars in protein content emphasizes the influence of grain type and assessment techniques on the recorded values.The TPC content in kodo millet was similar to the study conducted with different kodo millet cultivars (Goudar et al., 2023).Predominantly, millet's seed coat contains a significant concentration of polyphenols, primarily phenolic acids, and flavonoids (Shobana et al., 2009).Polyphenols in millets offer multiple health benefits, encompassing glucose regulation and mineral absorption.Thus, confirming that millets consumption is beneficial for individuals with diabetes (Ren et al., 2016;Wang et al., 2022).Hydroxyl groups of polyphenols form hydrogen bonds with starch molecules exhibit a change in physiochemical properties which results in the slower digestibility of the starch (Chi et al., 2017;Ngo et al., 2022).It is worth noting that polyphenols, saponins, and tannins, categorized as antinutrients, are recognized to impede mineral availability.However, these substances undergo catabolism during germination, leading to improved mineral accessibility (Grewal & Jood, 2006).
In the current study, the DPPH inhibition % of kodo millet was greater than that of the rice cultivars, indicating a higher antioxidant potential.Also, DPPH inhibition percentages aligned with previous studies in kodo millet (Goudar et al., 2023).This high DPPH inhibition capacity contributes to the neutralization of reactive oxygen species (ROS) produced in the body as a byproduct of metabolism.As a consequence, the antioxidant activity serves to counteract the potentially harmful effects of ROS, which are implicated in various health issues (Chandrasekara & Shahidi, 2011).The notable antioxidant potential holds implications for several health benefits, including diabetes management through enhanced insulin sensitivity, the inhibition of cancer cell growth, and the promotion of cardiovascular health emphasizing the positive impact of antioxidant activity on diverse aspects of health and well-being (Ofosu et al., 2020).This trend aligns with similar findings of previous studies, such as the positive correlation between TPC and antioxidant activity (Aryal et al., 2019;Goudar et al., 2023).The current study's results underscore that kodo millet is a highly commendable alternative to rice due to its elevated amylose content and other health benefits.Upon comparing the various millet cultivars, it becomes apparent that ATL 1 boasts consistently high values across all conducted biochemical tests.CO 3 exhibited higher levels of starch, amylose, and protein when compared to the remaining cultivars, falling second to ATL 1.Meanwhile, RK 390-25 demonstrated elevated levels of TPC, placing it on par with the starch, amylose and protein content of TNAU 86.This comprehensive analysis of the cultivars highlights the distinct attributes and potential benefits associated with each.

CONCLUSION
The extensive findings of this study highlight significant differences between several kodo millet types in terms of their starch, amylose, TPC, protein content, and antioxidant properties.The elevated amylose with antioxidant activities and grain phenolics can exert various protective mechanisms against oxidative stress.Among the observed characteristics, ATL 1 emerged as having the highest amylose, protein, TPC, and antioxidant activity, followed by CO 3, RK 390-25, and TNAU 86.These attributes collectively position kodo millet as a valuable food option for individuals seeking a low glycemic index diet, particularly those managing diabetes.The information from our study can be utilized to improve breeding programs for kodo millet that target particular traits.

Table 2 : Biochemical estimation of physiological properties of kodo millet cultivars with comparison to rice
Note: Values represent Means±Standard Error in terms of g/100g seed weight.Means in the given column followed by same letter(a, b, c, d)are not significantly different from each other (P>0.05,DMRT) Louis et al.