Kodo millet (Paspalum scrobiculatum) are the minor crops and have several nutritional components having benefits against health issues. The present study involves the proximate analysis of kodo millet, was evaluated providing the data that will help in the utilization of grains in food processing. The proximate analysis of the kodo millet recorded the moisture content of 6.0%, crude protein 6.61%, crude fibre 8.5%, crude fat 2.82%, crude ash 0.62% and nitrogen free extract (carbohydrates) 63.62%. The α-glucosidase inhibition of methanol extracts of kodo millet reported the 15.37% in methanol, 49.3% in acid methanol, 48.6% in acid methanol heated, 12% in aqueous and 50.26% in aqueous heated extracts. The above information helps in the nutritional value and inhibitory activity of the kodo millet in various solvents extraction which may provide the idea of preventing or managing the diabetes mellitus.
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Nutritional analysis and inhibition activity of kodo millet
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kodo millet
proximate analysis
α-glucosidase
diabetes mellitus
Kodo millet (Paspalum scrobiculatum) is widely distributed across the tropics and subtropics of the world. It is native cereal of India and consumed as healthy food in all over other parts of India especially in southern part of India (Hegde and Chandra, 2005). It has annual production of about 310,710 tonnes (Neelam et al, 2013). Kodo millet are closely resembles the rice and helps to use in weight loss. It is rich in phytochemicals which helps in preventing different lifestyle related diseases and also helps in reducing the joint pain (Deshpande et al, 2015). Kodo millet flour can be used for novel foods like idli, dosa, biscuits and bread (Kalpana and Koushikha 2013). Diabetes is the world’s fastest-growing metabolic disorder in recent years that are affecting the world population of various age group. This is due to the poor nutrition availability and living a sedentary life style. To control the diabetes in early stage involves the inhibition of the carbohydrate-hydrolyzing enzymes, α-amylase and α-glucosidase, in the digestive tract. Thus, we considered the study of the proximate composition analysis and α-glucosidase inhibition by the kodo millet extracts to understand the nutritional aspects of the grain which helps in the managing the metabolic disorders.
2.1 Materials
Kodo millet (Paspalum scrobiculatum) was obtained from the super market of Kolar and Tumkur, Karnataka, India. The sample was grinded to powder and the flour was sieved and stored in dry place for further analysis. All methods were carried out in accordance with relevant guidelines.
2.2 Chemicals and reagents
The chemicals like acids and inorganic chemicals were purchased from SD fine Chemicals of analytical grade, India. Methanol HPLC grade was obtained from Ranbaxy Chemicals, India. Glucose oxidase kit for amyloglucosidase inhibitory assay Erba glucose kit was procured from Transasia biomedicals ltd, India.
2.3 Proximate composition analysis of kodo millet
2.3.1 Estimation of moisture content
The moisture content of the sample was estimated by AOAC Method 930.15, 1990. 5 g of flour of sample was transferred to pre-weighed, clean and dry aluminium moisture cups (W1) and recorded the weight (W2). The samples containing aluminium moisture cups were transferred to hot air oven at 1050 C for 3 h and transfer to dessicator to cool. After drying transfer the dish to the dessicator to cool. Reweigh the dish and its dried sample (W3). Using the below formula moisture content of the sample was calculated.
2.3.2 Estimation of crude protein
Protein content of the sample was determined by the Kjeldahl method as modified by AOAC method 981.10, 1990 method. 0.5 g of the Sample was digested by heating with concentrated H2SO4 in a digestion block using (K2SO4 and CuSO4) catalyst. Samples were distilled with 10M NaOH. Boric acid (4%) was used to trap ammonia from the distillation and the distillate was titrated with 0.2 M NaOH using mixed indicator. Percent nitrogen was used to estimate percent protein concentration by means of a nitrogen-to-protein conversion factor 6.25. The blank reagent was also titrated similarly.
% Protein= (Vol. Acid – Vol. Blank) X 1.4007 X 0.2 N X6.25/g sample.
2.3.3 Estimation of crude fat
The crude fat in the sample was estimated by described in AOAC 920.39, 1990. 5 g flour sample (w) was subjected to drying and the dried sample was transferred to clean and dry extraction thimble, which was packed with glass wool to permit the free flow of solvent. A pre-weighed clean and dry heating flask (w1) was filled with petroleum ether fat was extracted at a condensation rate of 5 to 6 drops per second for 8 hours. After the extraction of fat, the ether was removed by vacuum evaporator and the residual ether was dried in oven at 100oC for 30 min. The flask was then cooled in vacuum desiccator for 2 hr. and recorded the weight (w2).
Percent crude fat was calculated as [(w2–w1)/ (w1–w)] x 100.
2.3.4 Estimation crude fibre
The estimation of crude fibre in kodo millet was performed by AOAC Method 978.10, 1990. A 2 g of the defatted sample was hydrolysed by boiling with 150 ml of 0.25 N H2SO4 for 30 min. The suspension was filtered, washed with hot distilled water and the residue was hydrolysed by boiling with 150 ml of 0.313 N NaOH. The crucible containing sample was dried in oven for 3 hr at103oC and the weight recorded (w1). The sample was then ignited in a Muffle furnace at 600oC for 3 hour and was held in an oven at 103oC for 1 hr and weight recorded (w2).
Percent crude fibre was calculated as [(w1-w2)/ (w)] x 100.
2.3.5 Estimation of ash
The crude ash was estimated by AOAC Method 942.05, 1990. The 3 g sample was dried in a hot air oven at 100oC for 5hr and further ignited in a muffle furnace for 3 h at 600oC. Cool the crucible in desiccators and weigh. At the end of the process the ash content will be in whitish grey colour
% Crude ash = (W2-W)/ (W1-W) X100
2.3.6 Determination of nitrogen free extract
Nitrogen Free Extract (NFE) or soluble carbohydrates of sample was determined by difference method. %NFE = 100 – (% moisture + % crude protein + % crude fat +% crude ash + % crude fibre.
2.4 Determination of α-glucosidase inhibitory activity
2.4.1 Preparation of extracts
The methanol and aqueous extracts have been used to determine the α-glucosidase inhibitory activity of kodo millet. The1g of the millet flour was extracted in methanol (10ml) for 72 hours at 30°C in rotary shaker. The extract was filtered and the filtrate centrifuged for 15 min and 10000 rpm at 4oC methanol extract was collected. To the above residue was treated with acidified methanol (1% HCL in methanol) for 2 h in a rotary shaker shaking at 80 rpm at 30°C. The extract was cleared by filtration and centrifugation as shown above and was dried to obtain the acid methanol extract. To the acid methanol heated extracts done as shown above but the sample was heated for 10 min in a boiling water bath prior to extraction. 1g of the sample was extracted in 10 ml of distilled water by boiling for 2 hours to get the aqueous extracts heated and without heating for aqueous extracts non-heated. The extract was filtered and centrifuged for 15 min and 10000 rpm at 4oC.
The determination of a-glucosidase activity of test solution was performed using the method (Mai et al, 2007) with slight modifications. A suitable solutions (200µL) were pre-incubated with α-glucosidase (200µL in PO4 buffer) for 15 min at 37°C. To the pre-incubated sample, a 1% of starch solution and the volume adjusted to 1 ml by the addition of required amount of phosphate buffer (0.1M, pH 6.8). The enzyme assay mixture was incubated for 3 minutes and the activity of enzyme was stopped by heating the tube in a boiling water bath for 3 minutes. The a-glucosidase inhibition was studied by analysing the release of glucose from starch using standard glucose oxidase/peroxidase enzyme reagent. The released glucose was analysed after 10 minutes of addition of GOD/POD reagent. The absorbance was measured at 510nm. The solution without a sample (inhibitor) used as a blank. Calculating percentage inhibition of α-glucosidase activity\(\)by the following formula using standard curve.
$$Inhibition of {\alpha }-\text{g}\text{l}\text{u}\text{c}\text{o}\text{s}\text{i}\text{d}\text{a}\text{s}\text{e}=\left(\right(OD of Control-OD of Sample\left)\right)/\left(OD of Control\right)\times 100$$
3.1 Determination of the proximate analysis
The results on the proximate composition of kodo millet (Paspalum scrobiculatum) is prescribed in the Table 1.The moisture content of kodo millet was found to be 6.0%. Our results matches with the earlier report by Neelam et al. 2013, of 7.3%. Kulkarni et al. 1992; Thilagavathi et al. 2015; REVATHI et al. 2016; Kumar et al. 2016; Sheela et al. 2018 reported the higher moisture content in kodo millet of 10.17%, 11.72%, 9.7%, 12.71% and 11.82% respectively. The present of crude protein in the kodo millet was found to be 6.61%. Various other investigations reported the values of 8.30% by Mitkal et al. 2021. Patil et al. 2021, reported the highest crude protein content in kodo millet of 13.80%. Our investigation showed the lower protein content in kodo millet compare to other previous literature, this is due to the different sample variety and analytical variations.
| Component | g/100 g |
|---|---|
| Moisture | 6.0 |
| Crude protein | 6.61 |
| Crude fibre | 8.5 |
| Crude fat | 2.82 |
| Ash | 0.62 |
| Carbohydrate | 63.62 |
The crude fat in kodo millet flour sample was estimated to 2.82%. our result showed the higher value than the previous literature reported by Mitkal, et al. 2021, as 1.30%, similar value observed by Neelam et al. 2013, (1.29%); Deshpande et al. 2015, (1.4%); Muthamilarasan et al. 2016, (1.4%). The crude fiber in the kodo millet was found to be 8.5%. Our results matches with earlier reports by Mitkal et al. 2021 and Neelam et al. 2013. Kulkarni et al. 1992, reported that highest crude fiber in kodo millet such as 6.33% than the little millet (5.73%), proso millet (5.51%) and barnyard millet (5.35%). The previous studies showed crude fiber of 9.0% by Muthamilarasan et al. 2016, 9.2% by Nithyashree et al. 2020, 9.6% by Sheela et al. 2018, 9.7% by Sharma et al. 2017, 9.95% by Thilagavathi et al. 2015. Our investigation showed the carbohydrate content present in the kodo millet was estimated as 75.45%. Similar results are reported by REVATHI et al. 2016 of 78.0% and Patil et al. 2021 reported 71.81%. Other investigations reported the total carbohydrate content in kodo millet as 63.62%, 65.3%, 65.50%, 65.9%, 68.2% and 67.4% by Thilagavathi et al. 2015; Muthamilarasan et al. 2016; Sheela et al. 2018; Rajkumar et al. 2019; Nithyashree et al. 2020 and Mounika et al. 2022. Hymavathi et al. 2020 reported the carbohydrate content present in dehulled kodo millet was 74.71g. The ash content was found to be 0.62%. Our report fall within the range of earlier reports by Geervani and Eggum 1989 and Hymavathi et al. 2020 in the dehusked kodo millet grains contains 1.04% and 0.83g ash content. The sprouted kodo millet showed the ash content of 3.70% [Panda et al. 2020].
3.2 Amyloglucosidase inhibitory activity of kodo millet
Our results recorded the promising inhibition activity of kodo millet. Figure 1 indicated the inhibition activity of Kodo millet of 15.37% in methanol, 49.3% in acid methanol, 48.6% in acid methanol heated extracts, whereas aqueous extracts heated reported the highest inhibition activity of 50.26% than the aqueous extracts non-heated (12%). Chiranthika et al. 2021, reported kodo millet showed the inhibitory activity of 45.5%. Goudar et al. 2023, reported the free and bound form of kodo millet showed the α-glucosidase inhibition activity of 92.6% and 34.49%. α-glucosidase inhibitors helps in the slowdown the carbohydrate digestion hence reduces the blood sugar level (Liu, 2012; Duodu and Awika 2019). Amyloglucosidase is an enzyme which helps in the breakdown the starch into glucose in the digestive system, resulting in the raise of blood glucose level. Hence, it becomes the chosen factor for controlling the diabetes mellitus.
The present investigation reveals the nutrition composition and inhibition activity of kodo millet which helps in the designing and developing the balanced diet to prevent the metabolic disorder.
Author Contribution
Gargi G P and Manohar shinde both prepared the text, table and graph
Data availability
All data generated or analysed during this study are included in this published article.
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No competing interests reported.