BIOACTIVE PROPERTIES OF PIGEON PEA (Cajanus cajan) SEED PROTEIN-DERIVED PEPTIDES AND FUNCTIONAL CHARACTERIZATION OF THE SEED STARCH

Abstract

There has been an increasing demand, application and consumption in recent years, for functional foods and nutraceuticals to replace some synthetic drugs which have been shown to have attendant side effects. Pigeon pea seeds were dehulled and processed via alkaline solubilization to extract the protein and starch components. The protein isolate obtained was subjected to enzymatic hydrolysis using four food grade enzymes namely alcalase, pancreatin, pepsin, thermoase and the hydrolysates obtained were separated by membrane ultrafiltration into peptides with different molecular weight (MW) sizes (<1 to >10 kDa). The hydrolysate with the most potent inhibitory activity against Angiotensin I- Converting Enzyme (ACE) was further purified using Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) and nineteen fractions were obtained. The isolate, hydrolysate and peptides were evaluated for their in vitro antioxidant, antihypertensive and antidiabetic properties. The isolate and hydrolysates were also orally fed to spontaneously hypertensive rats (SHRs) for in vivo antihypertensive evaluation. Graded acetylation of the starch extract was achieved by the addition of different volumes of acetic anhydride (CH3CO)2O). The physicochemical, thermal properties and microstructural characteristics of the starches were evaluated. The food application of pigeon pea flour and starch was evaluated, the starch was used as stabilizer in yoghurt; the physicochemical and sensory attributes of the yoghurt was evaluated. Acha flour was substituted with pigeon pea flour to produce a functional snack, the nutritional, antidiabetic, antioxidant properties and consumer acceptability of the biscuit were also evaluated. Results revealed that enzymatic hydrolysis significantly (P<0.05) increased the antioxidant properties of pigeon pea hydrolysates compared to the activity of the protein isolate. Pancreatin hydrolysed pigeon pea protein (PPHPa) exhibited the highest activity for all the antioxidant properties evaluated. Also, peptides from membrane fractionation showed improved antioxidant activity, evidenced by the lower 50 % inhibition concentration (EC50) compared to those of the corresponding crude hydrolysates. Pepsin-pancreatin hydrolysed protein (PPHPp) exhibited the highest ACE inhibitory activity (EC50 = 0.24 mg/mL) while pancreatin hydrolysed protein (PPHPa) had the highest percentage inhibition against renin (14.28 %). High molecular weight peptides (>10 kDa) exhibited the highest ACE inhibitory activity with the exception of thermoase hydrolysed protein (PPHT) that showed highest ACE inhibition in 5-10 kDa peptide. Low molecular weight peptides (<1-3 kDa) of alcalase hydrolysed protein (PPHA) showed high ability to inhibit renin while 1-3, 3-5 and >10 kDa peptides of PPHPp equally showed significantly high ability to inhibit renin. The hydrolysates caused a significant reduction in systolic blood pressure (SBP) in SHRs within the first 2 h till 24 h after oral administration. Three fractions (F 9, 10, 11) from the RP-HPLC exhibited highest activity in both antioxidant and antihypertensive properties. Acetylated starch with degree of substitution (DS) 0.08, 0.09 and 0.14 was obtained. Bulk density, water and oil absorption capacity, dispersibility, swelling capacity and solubility increased with increase in DS while viscosity and gelatinization temperature reduced with increased DS. DS 0.08 showed the least retrogradation tendency (30.54 %) and glycemic index (51.81 %). Lightness and whiteness index of the acetylated starches were also significantly (P<0.05) higher than the native starch. Fourier Transform Infrared (FTIR) spectra of acetylated pigeon pea starch showed distinctive peaks at 1245, 1370 and 1732 cm-1, assigned to C-C, C-O and C=O respectively. The intensities of the peaks increased with increase in DS. The microstructural properties of pigeon pea starches showed both round and oval shaped granules with sizes ranging between 14 to 43 μm, PPAS3 exhibited granule porosity with some identations. Incorporation of acetylated starch into yoghurt reduced whey syneresis and the yoghurt compared favourably with market sample in terms of overall acceptability. Incorporation of pigeon pea flour into acha flour at ratio of 20:80 and 30:70 in biscuit production revealed an increase in the protein content of the biscuit with increase in substitution of acha flour with pigeon pea flour from 10.47 to 19.18 %. The Na/K of the formulated biscuit was less than 1.0. The biscuit contained high amount of essential amino acid which exceeded the Food and Agriculture Organization/World Health Organisation (FAO/WHO) standard. Acha-pigeon pea (70:30) biscuit (APC2) exhibited strong antioxidant and antidiabetic properties as well as possessed low glycemic index (47.95 %). High MW peptide fractions from PPHPa and PPHPp may serve as potential ingredients to formulate functional foods against cardiovascular diseases. Acetylated pigeon pea starch may be useful as stabilizer in yoghurt production. APC2 could serve as a potential functional snack for the management of hyperglycemia.