Study On Gingerbread Plum (Neocarya Macrophylla) Kernel Proteins: Nutritional Assessment, Functional Properties And Antioxidant Activities

Gingerbread plum (Neocarya macrophylla Parinari macrophylla Sabinea source of edible oilseed having various uses. Keeping in view the potential of gingerbreadplum kernel as an underutilized oilseed, this study was carried out to investigate thephysicochemical properties; assess the nutritional values based on amino acids; study thefunctional properties; evaluate the antioxidant activities upon hydrolysis and finally, purify andidentify antioxidant peptides from gingerbread plum kernel proteins. For the purpose, ahis hypothesis, various in-vitro experiments wereconducted.Firstly, the proximate chemical composition of gingerbread plum (Neocarya macrophylla)kernels, mineral, fatty acid and amino acid compositions were evaluated. The proximate analysisrevealed the following composition: moisture10.57and11.76%, ash4.43and7.49%, fat47.28and2.14%, crude protein20.37and56.72%, carbohydrates17.34%and21.89%for gingerbreadPlum kenel (GPK) and defatted gingerbread plum kernel flour (DGPKF) respectively. Oleic,linoleic and arachidonic acids were the major unsaturated fatty acids with47.15,19.10and17.64%respectively. Saturated fatty acids accounted for14.72%of total fatty acids. The mainsaturated fatty acids were palmitic and stearic, with minute amounts of arachidic. Magnesium,potassium and calcium were the predominant elements present in the kernels. Copper, iron andmanganese were also detected in appreciable amounts. Essential amino acids were above therecommended amount by FAO/WHO for humans.Secondly, the effect of drying (freeze and vacuum) methods on gingerbread plum kernelprotein isolate was studied. Gingerbread plum kernel protein isolate was extracted from defattedgingerbread plum kernel flour (DGPKF) by alkali solution along with isoelectric precipitation.The protein isolate was subjected to freeze or vacuum drying process. Freeze dried gingerbreadplum kernel protein isolate (FGPKPI) and vacuum dried gingerbread plum Kernel protein isolate(VGPKPI) were evaluated for their physicochemical and functional properties (protein solubility,water/oil binding capacity, emulsifying capacity, foaming capacity). Among physicochemicalparameters, the proximate composition, amino acid composition, minerals, differential scanning calorimetry (DSC), SDS-PAGE and color attributes were studied. Both FGPKPI and VGPKPIcontained over90%protein versus DGPKF (56.72%) used as raw material. The method ofdrying had significant effect (p<0.05) on the physicochemical characteristics of FGPKPI andVGPKPI except for amino acids composition. The functional properties were variable amongsamples. DGPKF had higher emulsifying, water holding and oil binding capacities comparedwith FGPKPI and VGPKPI. FGPKPI exhibited better emulsifying capacity and water holdingcapacity than VGPKPI. FGPKPI also showed comparable oil binding capacity and bulk densityto commercial soy protein isolate (SPI).The next step consisted to the evaluation of the antioxidant capacity of gingerbread plumkernel protein fraction (albumin, globulin and glutelin) hydrolysates (GPKPFHs). Gingerbreadplum kernel protein fractions were hydrolyzed through a combined action of two digestiveenzymes (pepsin and trypsin). The hydrolyzed fractions were subjected to antioxidant test viaseveral chemical assays: DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity,hydroxyl radical-scavenging activity, reducing power and metal chelating activity. Totalphenolic compounds, amino acid composition and molecular weight distribution were alsoevaluated. The glutelin fraction hydrolysate showed the strongest antioxidative activitythroughout the entire investigation:79.09,58.81,52.08%and140.99x102mg/mL GAE forDPPH, hydroxyl radical, chelating activity and total phenolics respectively. GPKPFHs possess amolecular weight ranging from300to4000Da and also showed much more high reducing-tocopherol indicating that, hydrolysatesderived from gingerbread plum kernel protein could be a new antioxidants source.The impact of hydrolysis on the nutritional, functional properties and antioxidant activities ofgingerbread plum kernel protein isolates (HGPKPIs) were also investigated. HGPKPIs wereprepared at different times (5-180min) using two food-grade proteases (pepsin and trypsin).The hydrolysis time showed a significant effect (p<0.05) on nutritional parameters such as aminoacid score, essential amino acid index, biological value and protein efficiency ratios. Emulsifyingactivity index and foaming capacity decreased as hydrolysis time increases. During the wholehydrolysis period of180min, the antioxidant activities (hydroxyl radical scavenging, ferrouschelating, reducing power and DPPH radical scavenging) of HGPKPIs increased as function ofincubation time. And, within all samples a large amount of small-sized peptides (500-3000Da)was observed with increasing hydrolysis time. The above results indicated that hydrolysis time positively affects nutritional and antioxidant parameters of HGPKPIs but had a negative impacton the functional properties studied.Finally, gingerbread plum kernel protein hydrolysate obtained from the previous step after180min of hydrolysis (GPKPI-180) and showing the strongest antioxidant activities wassubjected successively to membrane ultra-filtration, gel filtration chromatography, RP-HPLCand matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry(MALDI-TOF/TOF-MS) to purify and identify the active peptide in GPKPI-180. The antioxidantactivities were evaluated on the basis of DPPH radical scavenging, hydroxyl radical scavengingand Fe2+chelating ability. GPKPI-180exhibited great hydroxyl radical scavenging activity andDPPH radical scavenging activity as well as Fe2+chelating ability through the variouspurification steps, suggesting the presence of peptides with antioxidant activity. The activeantioxidant peptide in GPKPI-180was a pentapeptide and the sequence has been identified asLeu-Leu-Leu-Ala-His. The estimation of the physicochemical properties of the purified peptideindicated that it is composed of20%basic amino acid residues and80%hydrophobic residueswith an isoelectic point and net charges at neutral pH of pH7.85and0.1respectively.