A comparative study on comprehensive nutritional profiling of indigenous non-bio-fortified and bio-fortified varieties and bio-fortified hybrids of pearl millets

Abstract

Seven indigenous pearl millet varieties, including non-bio-fortified (HC-10 & HC-20) and bio-fortified (Dhanashakti) and bio-fortified hybrids, viz., AHB-1200, HHB-299, HHB-311, and RHB-233, were studied in the present work. There was not any significant difference observed in the crucial anti-nutrients content, i.e., phytate (24.88–32.56 mg/g), tannin (3.07–4.35 mg/g), and oxalate (0.33–0.43 mg/g). Phytochemical content and antioxidant activity showed significantly high (p < 0.05) TPC and FRAP, TFC, and DPPH radical scavenging activity in the HHB 299 and Dhanashakti, respectively. Quantitative analysis of polyphenols by HPLC (first report on these varieties) revealed that HHB-299 has the highest amount of gallic acid. Fatty acid profiling by GC-FID showed that Dhanashakti, AHB-1200, and HHB-299 have rich monounsaturated fatty acid (MUFA) and polyunsaturated fatty acids (PUFA). Mineral analysis by ICP-OES showed high iron (87.79 and 84.26 mg/kg) and zinc (55.05 and 52.43 mg/kg) content in the HHB-311 and Dhanashakti, respectively. Results of the present study would help facilitate the formulation of various processed functional food products (RTC/RTE) that are currently not reported/unavailable.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13197-022-05452-x.

Introduction

Millets are one of the most essential crops consumed throughout the world, primarily in the semi-arid and arid regions of Asia (China and India) and Africa. These are the first cereals grains used domestically by humans, having a round shape, small seeds, and belong to the Poaceae family. Millets are ranked as the sixth essential crop that feeds 1/3rd of the world population (Samuel and Peerkhan 2020; Singh et al. 2017). Millets possess a rich quantity of essential fatty acids, dietary fiber, fats, protein, etc., although they contain a good quantity of major micronutrients such as iron, calcium, magnesium, zinc, and potassium (Saini et al. 2021). Recently, millets gained much researchers' interest due to the presence of essential nutrients (carbohydrates, proteins, major micronutrients, i.e., Fe and Zn, vitamin B) and bioactive ingredients, which have promising health-boosting attributes. Furthermore, millet is also explored for its therapeutic potential as a nutraceutical towards chronic ailments, including diabetes, cancer, cardiovascular diseases, and obesity (Majid and Priyadarshini 2020).

Pearl millet (Pennisetum glaucum) is one of the most important cereals, widely cultivated and well recognized as future food crops that confirm people's nutritional security. In India, PM production has improved constantly with high-yielding hybrid varieties. Due to the immense nutritional level of dietary fiber, micronutrients, and bioactive components, whole PM-based natural food products demand has also increased (Gong et al. 2018). PM is also reported to contain high lipids compared to other grains such as sorghum and maize. They also contain a rich amount of essential fatty acids like linoleic acid (39–45%) and oleic acid (21–27%). Literature evidence has concluded that PM has rich amount of phenolic components than other food sources such as rye, barley, wheat, sorghum, maize, and oats (Tomar et al. 2021). PM grains show a rich concentration of bioactive components (phenolics and flavonoids) with immense antioxidative attributes. Several methods, such as HFRSA, TAC, FRAP ABTS, and DPPH, have been used to evaluate the antioxidative potential of PM grains (Salar and Purewal 2017).

Cereals biofortification is an excellent approach to alleviate malnutrition conditions using genetic tools to improve the nutritional value of food crops. To overcome the concerns of micronutrient deficiency (mainly Fe and Zn), the All India Coordinated Research Project on Pearl Millet (AICRP-PM) started the biofortification trial to yield micronutrient-rich crops in 2014. Initially, ICRISAT, with the support of harvest plus, released a biofortified variety of pearl millet with large Fe and Zn. Dhanashakti (Zn-43 and Fe-81 ppm) is the first high-Fe pearl millet released in 2013. After this, high iron and zinc-containing biofortified pearl millet varieties/hybrids, i.e., AHB-1200, AHB 1269, HHB-299, RHB-234, RHB-233, and HHB-311, were released between 2018 and 2020. One meal of pearl millet high-Fe biofortified variety can fulfill up to 50–100% of the daily iron requirement and help mitigate the iron deficiency in the population (children, men, and women) (Satyavathi et al. 2021).

Despite its rich nutritional value in terms of minerals, it contains anti-nutritional factors such as phytate, which profoundly decreases their bioavailability. Phytate is one of the critical anti-nutrients that possesses chelating properties and chiefly reduces the bioavailability of micronutrients such as zinc, copper, and iron. Moreover, other anti-nutritional factors, such as oxalate, tannins, etc., have been reported to reduce the bioavailability of the minerals in the food (Kaushik et al. 2018; Samtiya et al. 2020). PM grains have rich nutritional value, but comprehensive data of the selected varieties of PM is still lacking.

Further, there is a dearth of literature about comprehensive nutritional profiling for these bio-fortified PM hybrids and bio-fortified PM variety, Dhanashakti. These bio-fortified hybrids and bio-fortified varieties have been developed for the rich iron and zinc content, but still, their other nutritional and anti-nutritional content has not been discussed in detail in the previously published studies. Thus, the present study was conducted to assess the proximate composition, anti-nutrients (Tannin, oxalate, and phytate), minerals (Fe, Zn, Ca, Mg, Mn, Na, and K), phytochemical content (Total flavonoids and total phenolic compound), antioxidant activity (DPPH and FRAP), Fatty acids profiling, and quantitative estimation of polyphenols present in the seven pearl millet varieties being majorly cultivated in India to help develop various functional food products from the bio-fortified hybrid/varieties having the better nutritional profiling and least possible anti-nutritional factors.

Material and methods

Chemicals/reagents

All the reagents and chemicals used in this research study were molecular/analytical grade. Phytic acid, Gallic acid, 6-hydroxy-2,5,7,8 tetramethylchroman-2-carboxylic Acid (Trolox), 2,4,6-tri (2-pyridyl)-s-triazine (TPTZ), Quercetin, Diphenyl-1-picrylhydrazyl (DPPH) were procured from Sigma-aldrich Inc., USA. Methanol, Hydrochloric acid, Ferric chloride, Aluminium chloride, Sodium bicarbonate, Potassium acetate, Petroleum ether, Folin-Ciocalteu's reagent, Sulphuric acid, Sodium hydroxide, Acetone were procured from the Hi-Media Laboratories Pvt. Ltd., Mumbai. Supelco FAME Mixture (Supelco CRM No.47885), n-Hexane (HPLC Grade) (CAS No.110–54-3), Toluene (HPLC Grade) (CAS No. 108-88-3), Boron Trifluoride (7% in Methanol), Anhydrous sodium sulfate (AR Grade) (CAS No.7757-82-6), Grade-1 Reagent grade water as per IS 1070. Nitric acid, and Perchloric acid were procured from Qualigens (ThermoFisher Scientific, Mumbai, India). Tannic acid was procured from the Sisco Research Laboratories Pvt. Ltd., Mumbai, India.

Pearl millet varieties procurement and processing

Total seven varieties of Pearl millet (PM), i.e., Non-biofortified (HC-10 & HC-20) & biofortified (Dhanashakti), and bio-fortified hybrids, viz., AHB-1200, HHB-299, HHB-311, and RHB-233 were used in this study. Four PM varieties (HC-10, HC-20, HHB-311, HHB-299) were procured from the Bajra Section, CCS Haryana Agricultural University, Hisar, India. Other three PM Varieties (Dhanashakti, AHB-1200, RHB-233) were procured from the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, Telangana, India. Each variety was collected in 2–2.5 kg of weight. All raw PM varieties were appropriately cleaned and dried in sunlight in 4–5 h up to 8–10 percent moisture content. Further, all seven samples were ground in a hammer mill into fine flour and stored in air-tight containers till further analysis.

Proximate analysis of raw PM varieties flour

Raw PM flour was analyzed for protein, moisture, ash, crude fiber content by following standard procedures of the Association of Official Analytical Chemists (AOAC 2005). Protein content (N × 6.25) was estimated using Kjeldahl apparatus (Gerhardt, Analytical Systems, Germany), and crude fat content was estimated by standard Soxhlet method using automated Soxhlet equipment (Pelican Equipments, Chennai, India). Moisture content was measured after drying the flour samples at 105 °C for 16 h and repeated till weight was constant. Ash content was calculated by ashing the flour samples for 4–5 h using a muffle furnace at 550 °C. The crude fiber of samples was estimated after digesting with 1.25% sulfuric acid and subsequently with 1.25% sodium hydroxide solution. Total carbohydrate was calculated according to formula: Total carbohydrate = [100 – (crude protein + crude fat + crude fiber + moisture + ash)].

Estimation of phytochemical content and antioxidant activity

Ultra-sonic assisted extraction (UAE) and conventional extraction (CE)

PM samples were extracted using Ge et al. (2021) method with some modifications, using 80% of methanol (methanol:water) as a solvent. Different concentrations of methanol (nil to absolute) were first experimented to finalize 80% methanol as a preferred solvent for the extraction of PM (data not shown). In the UAE method, 1 g of flour sample was mixed in 10 mL of methanol and ultrasonicated for 30 min using an ultrasonic water bath (MRC, Israel) operated at 40 kHz at 30 °C. The supernatant was collected, and the residue was subjected to ultrasonication in the same manner two more time. All the three supernatants were pooled and centrifuged at 8000 rpm for 15 min followed by filtering through a syringe filter 0.45 µm and stored in the amber falcon tubes at −20 °C till further analysis.

In the CE method, 1 g flour sample was mixed in 10 mL methanol and kept in a shaker incubator for 30 min at 150 rpm at 30 °C (Innova 42, New Brunswick Scientific, USA). The supernatant was collected and re-shaked in the same conditions twice, and afterward same steps were followed as discussed in the UAE method.

Phytochemical content

Total phenolic content (TPC) and total flavonoid content (TFC)

TPC and TFC were estimated according to Singhal et al. (2021) with some modifications. Samples absorbance was measured at 765 nm using a multimode microplate reader with a cuvette port (Spectramax M2e system, Molecular Devices, USA). Gallic acid was used as standard with a range of 0–50 µg/mL to measure the TPC concentration, and results were expressed as mg of gallic acid equivalent (GAE)/g of flour.

For the estimation of TFC, sample extract (500 µL) was mixed with 1.5 mL methanol, followed by 100 µL of aluminum chloride (10% w/v), 100 μL 1 M potassium acetate, and 2.8 mL distilled water. Samples were incubated in dark conditions for 30 min at room temperature. The absorbance of the sample's solution was measured at 415 nm using a microplate reader with a cuvette port. Quercetin was used as standard with a range of 0 to 80 µg/mL, and results were calculated as mg of quercetin equivalent (QE)/g of flour.

Antioxidant activity

Ferric reducing antioxidant power (FRAP) activity

The FRAP assay was estimated according to the method described by Kumar et al. (2021) with minor modifications. The absorbance of the sample was measured at 593 nm using the cuvette port of the spectrophotometer. Trolox was used as a standard with a range (5–80 µM), and results were discussed in mM Trolox equivalent (TE)/g of flour.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity (RSA)

Free radical scavenging activity of samples was measured by DPPH assay according to Kumar et al. (2020) and Verma et al. (2021) method with modifications. Sample absorbance was measured at 517 nm against a blank (methanol only). The same amount of DPPH and methanol mixture was used as a control. All the analyses were measured in triplicates. RSA ability was calculated by:

$$Radicalscavengingability\left(RSA\%\right)=\left[{\text{A}}_1-\left({\text{A}}_{\text{D}}-{\text{A}}_{\text{S}}\right)\right]/{\text{A}}_1\times 100$$

Note: A₁ is control absorbance; A_D is DPPH solution absorbance with the sample; A_S is sample extract absorbance without DPPH.

Anti-nutritional factors

Phytic acid (PA)

The phytic acid content of the raw PM flour samples was estimated using the method described by Ahmed et al. (2020) with minor changes. The absorbance of the sample was calculated at 500 nm using a multimode microplate reader with a cuvette port. A standard curve of phytic acid (dissolved in 0.2 N HCl) was used to estimate PA content, and the results are discussed as PA in mg/gm of flour.

Tannin content

The tannin content of raw PM flour was estimated according to Owheruo et al. (2019) method with minor modifications. The absorbance of the sample was measured at 725 nm using a multimode microplate reader with a cuvette port. Tannic acid was used as a standard with a range (5–50 µg), and results were discussed as mg of tannic acid/gm of flour.

Oxalate content

The oxalate content of raw PM flour was estimated according to Ijarotimi et al. (2018) method. About 1 gm of flour sample was added into 100 ml of conical flask followed by 75 ml of H₂SO₄ (3 M). The sample solution was stirred for 1 h with a magnetic stirrer at 30 °C and filtered using filter paper (Whatman No 1). Exactly 25 ml of clear filtrate was taken and titrated hot (90 °C) against 0.1 M KMnO₄ solution till faint pink color appeared for at least 30 s. Oxalate was calculated using the below formula:

$$\text{Oxalate}\left(\text{mg}/\text{g}\right)={\text{V}}_{\text{T}}\times 0.9004$$

Note: V_T = Titre volume (mL).

Polyphenol profiling

Extraction of polyphenols

For the quantification of polyphenols, 3 g of raw PM flour was extracted in 10 mL methanol and ultrasonicated for 30 min using an ultrasonic water bath (MRC, Israel) operated at 40 kHz at 30 °C. The supernatant was collected, and the residue was re-extracted in the same manner twice. All the three supernatants were pooled and centrifuged at 8000 rpm for 15 min. The supernatant thus obtained was then filtered through a nylon syringe filter 0.45 µm and was stored in the amber-colored falcon tubes and used for phenolics profiling analysis on the same day.

HPLC analysis of polyphenols

Polyphenols were quantified by high-performance liquid chromatography (HPLC) with photodiode-array (PDA) detector (Agilent 1260 Infinity II, Agilent Technologies, USA) as per the Seal (2016) method with some modifications. Polyphenolic compounds in samples and standards were estimated on a reverse-phase C18 column (Spherisorb, 250 × 4.6 mm, 5 μm). HPLC column was controlled at 40° C thermostatically. The total run time of the separation protocol was 32 min, 1.0 mL/min of flow rate, and 10 μL injection volume was used. After testing several gradient settings and acetic acid concentrations (1, 0.5, and 0.1%), the following mobile phase conditions were used for the separation: solvent A (0.1% aqueous acetic acid) and solvent B (acetonitrile). The gradient increased at 10 min from 20 to 80% solvent B and was maintained for 10 min. Solvent B was then decreased to 20% at 27 min and held for 5 min. Individual components were identified by injecting the standard solutions at 10 μg/mL based on their UV spectrum. Caffeic and quercetin were detected at 320 nm and Gallic at 300 nm. Individual polyphenols were quantified by the area comparison corresponding to their dilutions in mg/100 g.

Fatty acid profiling

Fatty acids of raw PM flour samples were measured by Gas chromatography (GC) having Supelco SP-2560 fused silica capillary column (Agilent technology, 7890B) as fatty acids methyl esters (FAMEs) (AOAC 2002, Official Method 996.06). Two–three mL of petroleum ether was added to the extracted fat and fat was dissolved completely. Reconstituted fat was transferred into a Pyrex culture tube with a teflon screw cap (16 mm × 120 mm). The solvent was evaporated entirely under a gentle stream of nitrogen at room temperature. After that, 2 mL of 7% Boron trifluoride was added in methanol followed by addition of 1 mL toluene. Then, the pyrex tube was incubated at 100 °C for 45 min in a hot air oven. At every 10 min, tube was shaken gently during incubation. Vial was allowed to cool down to room temperature (20–25 °C), and then 5 mL of distilled water, 2 mL hexane, and approximately 1.0 g sodium sulphate were added. The vial was capped and shaken for 5 min in a vortex mixer. Consequently, layers were allowed to separate, and then carefully upper hexane layer was pipetted. Finally, the hexane layer was filtered through a 0.22 µm PTFE filter into an amber coloured GC vial to be used for the further analysis. Data are reported as the percentage of fatty acids.

Instrument programming

Operating conditions: Injector temperature—250 °C; Detector temperature—280 °C; Initial temperature—100 °C (held for 4 min). The temperature was then increased by 4 °C/min up to 240 °C and held for 25 min.

Minerals composition determination

Micronutrients in the PM varieties were estimated according to Wheal et al. (2011) with modifications. Digestion of samples was carried out by using a diacid (Nitric (HNO₃) and perchloric (HClO₄) acid) mixture. After 3–4 h of digestion, samples were collected when HNO₃ white fumes ceased, and collected samples were diluted with the milli-Q type I ultrapure water up to 50 mL. Total minerals (Fe, Zn, Ca, Mg, Mn, K, and Na) were analysed using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) (Optima 7000DV, PerkinElmer, USA).

Statistics

All measurements were carried out in triplicates, and data were analysed using GraphPad Prism (GraphPad Software version 5.01, San Diego, CA, USA). The results were represented as mean ± SD (standard deviation). One-way and two-way analysis of variance (ANOVA) was used for statistical analysis of data followed by Tukey post hoc test to separate the mean (p ≤ 0.05), which were considered statistically significant.

Results and discussion

Proximate analysis of raw PM varieties flour

The proximate composition of all the seven PM varieties is presented in Table 1. Our results showed that carbohydrate and moisture content of PM varieties was in the range of 66.98 ± 0.51 to 74.40 ± 0.28% and 6.13 ± 0.22 to 9.54 ± 0.15%, respectively. Dhanashakti variety had the highest carbohydrate content among all the seven varieties. The crude fiber and ash content of our samples was in the range 2.58 ± 0.02 to 1.606 ± 0.02% and 1.77 ± 0.21 to 3.08 ± 0.60%, respectively. Our results are in parallel to that of Kumari et al. (2018), who reported 70.77 to 67.90% carbohydrate in the 11different local, popular and new hybrid of PM varieties of Indian origin (such as PC 1201, HHB 203, Proagro 9444, HHB 67, Pioneer 86M86, etc.). Protein content was lowest in HHB-299 and was highest in the HC-10 variety. Fat content was highest in the AHB-1200 hybrid among all seven varieties. Siroha and co-workers have shown that different Indian pearl millet varieties have fiber, ash, protein, and fat content in the range 2.9–3.8%, 1.65–1.90%, 9.7–11.3%, and 5.1–7.2%, respectively, corroborating our results (Siroha et al. 2016). Findings suggested that PM is a rich source of protein and could be used to mitigate protein malnutrition by making different ready-to-cook/eat (RTC/RTE) products.

Table 1.

Proximate composition of non-bio-fortified and bio-fortified pearl millet cultivars

PM Varieties	Carbohydrate (%)	Protein %	Ash %	Fat %	Moisture %	Fiber %
HC-20	70.68 ± 0.42a	9.60 ± 0.20ac	2.28 ± 0.40ab	6.76 ± 0.56ab	8.46 ± 0.01af	2.19 ± 0.005a
HC-10	66.98 ± 0.51b	10.46 ± 0.09b	2.60 ± 0.69ab	8.62 ± 0.32b	9.54 ± 0.15b	1.78 ± 0.03b
HHB-299	73.37 ± 0.82c	9.38 ± 0.08a	2.05 ± 0.65ab	5.09 ± 0.28a	8.10 ± 0.22ae	1.99 ± 0.03c
HHB-311	68.46 ± 1.12ab	10.20 ± 0.13bdc	2.11 ± 0.20ab	7.81 ± 1.29b	9.19 ± 0.12bf	2.21 ± 0.01a
AHB-1200	67.21 ± 1.51db	9.53 ± 0.17ac	3.08 ± 0.59b	10.67 ± 1.06c	7.10 ± 0.17cde	2.40 ± 0.01d
RHB-233	69.88 ± 0.67a	10.03 ± 0.13dc	1.76 ± 0.20a	9.81 ± 0.38bc	6.12 ± 0.22d	2.51 ± 0.02e
Dhanashakti	74.40 ± 0.28c	9.82 ± 0.087c	1.78 ± 0.02a	5.17 ± 0.52a	7.21 ± 0.33e	1.60 ± 0.01f

Data is expressed as mean ± SD, n = 3. Mean values bearing different superscript letters (a, b, c, d) are significantly different at p < 0.05 in Tukey’s multiple comparison post-hoc test

Values are in dry weight basis

Phytochemical content

TPC and TFC

Figure 1a and b shows the comparative analysis of TPC and TFC for CE and UAE treatments. Results showed that TPC and TFC content in UAE was higher than that of CE in the studied PM varieties. TPC and TFC content of our samples for UAE treatment were in the range 2.58–3.25 mg GAE/g and 0.49–0.62 mg QE/g, respectively. Siroha et al. (2016) reported similar results for TPC; the study reported 3.13–2.39 mg of GAE/g of PM samples. Another study by El Hag et al. (2002) reported that raw flour of different PM cultivars had 3.04 mg/g and 4.44 mg/gm of total polyphenols content. There was a non-significant difference (p > 0.05) in the TPC between CE and UAE treatments for HHB-311 and AHB-1200 hybrids. Highest TPC and TFC content was found in the HHB-299 hybrid and Dhanashakti variety, respectively, for both the treatments (CE and UAE). The TFC results in the present study are in agreement with Owheruo and co-workers, who reported 0.91 mg/g in raw PM samples (Owheruo et al. 2019). Similar results were reported by Pushparaj and Urooj (2014) with 0.27 and 0.21 mg/g of TFC in two different varieties (Maharashtra Rabi Bajra and Kalukombu) of raw pearl millet.

Fig. 1.

Phytochemical content and antioxidant activity of methanolic extracts of non-bio-fortified and bio-fortified pearl millet cultivars using CE and UAE. Data is expressed as mean ± SD, n = 3. Mean values bearing *(p < 0.05); **(p < 0.01); ***(p < 0.001) are significantly different in Tukey’s multiple comparison post-hoc test

Antioxidant activity

Ferric reducing antioxidant power (FRAP) activity

FRAP assay is an easy and fast method to evaluate the antioxidant potential of different food components. Results of the FRAP assay are presented in Fig. 1c. In this assay color (straw) of FRAP is reduced to blue color by electron donor bioactive component in acidic environments. Our results showed that the FRAP value of CE and UAE treated samples was 14.70–21.11 mM of TE/g and 17.72 to 19.28 mM of TE/g of flour, respectively. The highest FRAP activity, i.e., 21.11- and 19.28-mM TE/g, was shown by the HHB-299 hybrid (CE treatment) and HC-10 variety (UAE treatment), respectively. Salar and Purewal (2017) reported FRAP activity ranging from 2.56 to 4.75 mM Fe²⁺/g of sample in twelve indigenous varieties of pearl millet (such as HHB-226, HHB-197, HC-20, PUSA-605, etc). Moreover, they reported a non-significant difference in the FRAP potential of nine PM varieties. Researchers have reported that different processing methods (i.e., roasting, germination, etc.) did not significantly change the FRAP activity (Pushparaj and Urooj 2014).

2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging (RSA) ability

The results of the DPPH assay are presented in Fig. 1d. RSA of CE extracted PM samples was 39.55–57.71%; whereas, UAE samples showed 45.10–64.00% RSA. A decent quantity of RSA was shown by samples extracted with the UAE method, which may be due to the higher leaching of polyphenols by UAE treatment owing to effective breakage of the cell wall of the grain. Minimum RSA was found in the RHB-233 variety for CE and UAE extracts. Our results are well supported by the findings of Salar and Purewal (2017), who reported DPPH inhibition activity of the twelve indigenous varieties of pearl millet (Indian origin) (such as HHB-226, HHB-197, HC-20, PUSA-605, etc.) in between 22.71 and 54.90% RSA. Another study has reported 49.95% DPPH RSA in the raw pearl millet samples (Owheruo et al. 2019).

Anti-nutritional factors

Phytic acid (PA)

Phytic acid has the property to chelate cations like iron, zinc, calcium, and magnesium, ultimately reducing their bioavailability; it is considered as one of the most effective and critical anti-nutrients in foods, causing mineral deficiency in human and animal foods (Samtiya et al. 2020). Figure 2a shows the PA content of seven PM varieties used in this study. The PA content of the studied varieties was found to range from 24.88 ± 7.16 to 32.56 ± 1.29 mg/g of flour samples. Results clearly showed that there was not any significant difference (p < 0.05) between the PA content of all seven varieties. The highest PA content was observed in the HC-20 variety, whereas the lowest PA was found in the HC-10. The PA content of different pearl millet has been reported to be 17.72 mg/g (Owheruo et al. 2019), 9.9 mg/g (Pushparaj and Urooj 2014) and 10.76 mg/g (El Hag et al. 2002). Our results showed a high amount of PA content, possibly due to the different agro-climatic conditions, different primary processing methods, and quantification protocol.

Fig. 2.

Anti-nutritional factors in non-bio-fortified and bio-fortified pearl millet cultivars. Data is expressed as mean ± SD, n = 3. Mean values bearing different superscript letters (a, b, c, d) are significantly different at p < 0.05 in Tukey’s multiple comparison post-hoc test

Tannin content

Tannin is a type of anti-nutrient that mainly affects the digestibility of proteins and decreases essential amino acids (EAAs) by forming tannin–protein complexes between the carbonyl group of proteins and hydroxyl group tannins (Samtiya et al. 2020). Figure 2b represents the tannin content of PM varieties used in the present study. Our results showed that tannin content in the PM ranged from 3.07 ± 0.03 to 4.35 ± 0.64 mg TA/g of flour. Results showed no significant (p < 0.05) difference noticed in the tannin content of all seven varieties. The highest tannin content was found in the AHB-1200, whereas the lowest was measured in the HC-10 variety. Our results are in agreement with Singh and co-workers, who reported 4.59 and 3.01 mg/g of tannin in the pearl millet and finger millet varieties, respectively (Singh et al. 2017). Other studies have found tannin content to be between 2.3 and 2.1 mg/g in the indigenous pearl millet (Maharashtra Rabi Bajra and Kalukombu) and 1.64 mg/g in finger millet (Nigeria), respectively (Pushparaj and Urooj 2014; Owheruo et al. 2019). Again, the probable reason for high tannin content in our studies varieties and hybrids might be attributed to differences in agro-climatic conditions, different primary processing methods, and quantification protocols.

Oxalate content

Oxalate is one of the critical anti-nutrients that hinder calcium's bioavailability mainly by making complex. Figure 2c represents the oxalate content of PM varieties explored in the present study. PM flour contained oxalate ranging from 0.33 ± 0.01 to 0.43 ± 0.01 mg/g of flour. The highest oxalate content was found in the AHB-1200 biofortified hybrid, whereas the lowest was in the HC-20. Our results are in parallel with Suma and Urooj (2014) and Amalraj and Pius (2015), who reported 0.32 to 0.36 mg/g and 0.2 mg/g oxalate in their pearl millet samples, respectively. Among all varieties studied, oxalate content was significantly different (p < 0.05) only between AHB-1200 and HC-20.

Anti-nutrients mainly hinder the bioavailability of minerals (such as iron, zinc, calcium, etc.); our study shows a non-significant difference in the anti-nutrients content in almost all the varieties/hybrids studied. So, we will select varieties based on their minerals, fatty acids, and phenolic content to further make a suitable functional food.

Quantification of phenolics

Pearl millet contains a diverse and ample quantity of phenolic compounds. Among some polyphenols quantified in this study, three phenolic compounds have been found to be present in the higher quantity in the seven PM varieties. Supplementary file S1 represents PM varieties' HPLC chromatograms of phenolics standard and one representative sample (HHB-299), and gallic acid, caffeic acid, and quercetin content of all varieties. The gallic acid, caffeic acid, and quercetin content ranged from 89.81 to 130.09 mg/100 g, 1.37 to 4.20 mg/100 g, and 41.85 to 57.39 mg/100 g, respectively. Biofortified hybrid, HHB-299 contained the highest amount of gallic acid, whereas the lowest was found in the RHB-233 hybrid. HC-10 had the lowest quantity of caffeic acid. AHB-1200 has the highest level of quercetin, while RHB-233 has the lowest quercetin content. Despite our best efforts with the literature search, we could not find a study to compare these results. This study is the first to quantify and report different types of polyphenols in the selected bio-fortified hybrids and bio-fortified variety, Dhanashakti. The results suggested that the HHB-299 could be an excellent candidate to formulate various RTE/RTC based functional food products, and the RHB-233 hybrid should not be the choice for the bio-fortified PM-based functional foods.

Fatty acid profiling

Our study results showed that PM varieties have suitable lipid concentrations. Saturated fatty acids like palmitic acid and stearic acid and polyunsaturated fatty acids like linoleic acid are the primary fatty acids creating the main portion of PM oil (Jukanti et al. 2016). In the present study, seven different fatty acids were identified in the PM lipid/fat: palmitic acid, oleic acid, stearic acid, linoleic acid, α-linolenic acid, arachidonic acid, and behenic acid. Table 2 represents the data of these seven fatty acids in percentage. Figure 3 shows the chromatograms of the three varieties (AHB-1200, RHB-233 and Dhanashakti) having rich essential fatty acid content. Supplementary file S1 represents the retention time (min) data for fatty acids of all varieties. The range of palmitic acid, oleic acid, stearic acid, linoleic acid, and α-linolenic acid were 18.75–20.89%, 26.31–31.48%, 3.63–5.80%, 40.81–44.74%, and 1.88–2.22%, respectively. The other two fatty acids, arachidonic acid and behenic acid ranged from 0.89 to 1.18% and 0.25 to 0.34%, respectively. HHB-299 hybrid showed the highest oleic acid (31.48%), and HC-10 showed the highest palmitic acid (20.89%), whereas the highest linoleic acid (44.74%) and α-linolenic acid (2.22%) were found in AHB-1200 and RHB-233 varieties, respectively. The study suggested that Dhanashakti, AHB-1200, RHB-233, and HHB-311 have a rich amount of PUFAs (linoleic acid and α-linolenic acid), and HHB-299 has a rich source of oleic acid, making them suitable for consumption by growth retarded individuals, mainly children. Linoleic acid and α-linolenic acid are two essential fatty acids that cannot be synthesized by human de novo, so these must be taken from food. A recent study by Tomar et al. (2021) have also reported fatty acids present in the indigenous PM variety which is in concurrence with our results. The lipids of PM are the immense source of fat-soluble vitamins such as vitamins K, E, D, and A and a rich source of essential fatty acids. Tomar et al. (2021) reported that PM varieties mainly contains nearly 25% of saturated fatty acids (SFAs), almost 50% of PUFA, and about 25% monounsaturated fatty acids (MUFA). Furthermore, oleic acid alone has been reported to make up approximately 25% of MUFA and linoleic acid make up nearly 45% of the PUFA (Jukanti et al. 2016). Oleic acid can lower the hydrolysis of starches eventually playing a vital function of hypoglycaemic ability in the PM. Hence, the presence of rich oleic acid content in the PM lipids may play a significant role in the diet of people with hypoglycaemic concerns (Annor et al. 2015).

Table 2.

Fatty acids profiling of non-bio-fortified and bio-fortified pearl millet cultivars by GC-FID

	Saturated fatty acids				MUFA	PUFA
PM Varieties	Palmitic acid (%)	Stearic acid (%)	Arachidonic Acid (%)	Behenic acid (%)	Oleic acid (%)	Linoleic acid (%)	α-Linolenic acid (%)
HC-20	19.39	5.80	1.11	0.29	27.92	43.38	2.11
HC-10	20.89	5.39	1.18	0.34	29.44	40.81	1.95
HHB-299	18.75	5.42	1.13	0.32	31.48	41.03	1.88
HHB-311	19.66	5.02	1.10	0.31	28.79	43.09	2.04
AHB-1200	20.63	3.63	0.89	0.29	27.41	44.74	2.14
RHB-233	20.53	4.37	0.94	0.28	26.31	44.67	2.22
Dhanashakti	20.03	4.14	0.93	0.25	29.24	43.39	2.01

MUFA: Monounsaturated fatty acid; PUFA: Polyunsaturated fatty acid

Values are presented as % of fatty acids in total % of lipids

Fig. 3.

Chromatograms of fatty acid analysis. a RHB-233, b AHB-1200 and c Dhanashakti

Our result is in concurrence with Slama and co-workers, who reported that pearl millet oil had a good amount of essential fatty acids, linoleic acid (47.5% ω-6 fatty acid18:2) and linolenic acid (2.15% ω-3 fatty acid18:3) (Slama et al. 2020). It has also been reported that a diet rich in polyunsaturated acids (PUFA) can provide ample benefits such as regulation of immune functions, maintaining blood cholesterol levels, and enhancing the high-density lipoproteins' fluidity. Consequently, rich PUFA fractions of the diet may be vital for the active transport molecules, enzymes functions, receptors, and structure and functions of several essential membrane proteins (Yaqoob 2002; Mehmood et al. 2008). Similarly, another study by Jellum and Powell (1971) has supported our fatty acids profiling results wherein different pearl millet varieties were shown to have 16.7–25.0% palmitic acid, 1.8–8.0% stearic acid, 40.3–51.7% linoleic acid.

Mineral composition

Pearl Millet has rich composition of minerals such as calcium, phosphorus, magnesium, manganese, potassium, zinc, and iron. Moreover, these minerals play several crucial functions, such as minerals acting as cofactors for many enzymes, modulating/controlling cell signaling, metabolic pathways, and immune functions in the human body (Krishnan and Meera 2018; Gharibzahedi and Jafari 2017). Our study results showed that PM varieties have favorable minerals composition. Seven micronutrients (i.e., Fe, Zn, Mg, Mn, Na, K, and Ca) were quantified in the present study. Table 3 represents the data of micronutrients in mg/kg of pearl millet. Results showed that in the selected varieties of PM, the Fe, Zn, Ca, Mg, Na, K and Mn ranged from 47.10 to 87.79, 38.37–55.05, 404–919.1, 1035–1716, 574.9–911.6, 2757–5205 and 6.69–12.45 mg/kg, respectively. The co-relation coefficient (r²) value for the Fe and Zn standards were 0.998 and 0.999, respectively. Dhanashakti variety had the highest Mg, Ca, K, and Mn; whereas HHB-299 contained the highest sodium (Na). Highest Fe and Zn content were found in the bio-fortified variety (Dhanashakti) and bio-fortified hybrids (AHB-1200, HHB-299, HHB-311, and RHB-233) compared to the indigenous varieties (HC-10 and HC-20). In the present study, high Fe and Zn content was recorded in the HHB-311 and Dhanashakti, i.e., (87.79 and 55.05 mg/kg) and (84.26 and 52.43 mg/kg), respectively. Our results are in agreement with the recent study by Tomar and co-workers, who have recorded iron (65.33–84.22 ppm), zinc (28.71–58.64 ppm), Ca (234.62–405.59 ppm), Mg (1635.23–2386.71 ppm), K (2495.57–4994.72 ppm), and Mn (8.21–12.57 ppm) in the different clusters of indigenous pearl millet germplasm (Tomar et al. 2021). Calcium and magnesium are vital minerals, and both have significant role in the bone formation, neuronal activities, and regulation of the internal cell environment. However, sodium controls the transmission of neural signalling, pH, contraction of muscle as well as regulation of the cellular fluid quantity. Our results evince that the bio-fortified hybrid, HHB-311, could be considered to formulate various iron-rich functional food products. These products could help tackle the crucial anaemia problem in the country by simply supplementing/adopting this hybrid and taking care of its anti-nutritional factors, especially the phytate.

Table 3.

Minerals composition of non-bio-fortified and bio-fortified pearl millet cultivars by ICP-OES

PM Varieties	Iron (mg/kg)	Zinc (mg/kg)	Calcium (mg/kg)	Magnesium (mg/kg)	Sodium (mg/kg)	Potassium (mg/kg)	Manganese (mg/kg)
HC-20	47.10	52.80	434.1	1330	749.9	3712	11.10
HC-10	54.88	38.37	464	1223	767.4	3183	6.69
HHB-299	77.71	50.76	436	1462	911.6	4210	8.02
HHB-311	87.79	55.05	564	1035	606	2757	9.78
AHB-1200	67.53	51.77	692.7	1419	574.9	4147	11.18
RHB-233	73.54	52.47	404	1704	874.7	3642	11.79
Dhanashakti	84.26	52.43	919.1	1716	793	5205	12.45

Conclusion

The present study showed that the Indian PM non-biofortified varieties, biofortified hybrids, and biofortified variety are rich in polyphenols, antioxidants, micronutrients (especially iron and zinc), and fatty acids (mainly essential); however, they also contain a significant amount of anti-nutritional factors (i.e., phytate, tannins, and oxalate). Biofortified hybrid, HHB-299, showed the highest TPC and FRAP, while biofortified variety, Dhanashakti, showed the highest TFC. Dhanashakti has a rich amount of carbohydrates and possesses a notably high concentration of omega-6 fatty acid, linoleic acid, and the omega-9 fatty acid, oleic acid. HHB-299 variety contained the highest amount of gallic acid and oleic acid among all the varieties. AHB-1200 has the highest amount of linoleic acid and quercetin among all the varieties tested. Dhanashakti variety had the highest Mg, Ca, K, and Mn; whereas, HHB-299 contained the highest sodium content. In the present study, high iron (87.79 and 84.26 mg/kg) and zinc (55.05 and 52.43 mg/kg) content were recorded in the HHB-311 and Dhanashakti, respectively. A non-significant difference was found in all the varieties' tannin and phytic acid content. Comparative data of our study suggested that the HHB-299, HHB-311, and Dhanashakti have rich nutritional profiles among the biofortified variety and bio-fortified hybrids of the pearl millet. Due to their rich fatty acid composition and nutritional attributes, these three varieties should be the choice to formulate various processed functional food products.

Supplementary Information

Below is the link to the electronic supplementary material.

Author’s Contributions

MS: Investigation, Formal analysis, Data curation, Validation, Writing—original draft. GAC: Methodology, Resources, Writing—review & editing. TD: Conceptualization, Resources, Methodology, Data curation, Writing—review & editing. RS: Resources, Formal analysis, Writing—review & editing. AK: Formal analysis, Validation, Writing—review & editing. AK: Data curation, Writing—review & editing. PCB: Conceptualization, Resources, Methodology, Data curation, Project administration, Supervision, Writing—review & editing.

Funding

The author are thankful to the Haryana State Council for Science Innovation and Technology India, for funding this research work and the project (HSCSIT/R&D/2021/461).

Availability of data and material

Not applicable.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors declare no conflicts of interest.

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.