Theses and dissertations (Applied Sciences)
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Item Anticancer and antioxidant potential of Amaranthus cruentus protein and its hydrolysates(2020) Ramkisson, Shanece; Mellem, John J.; Venter, SonjaTraditionally, amaranth has been acknowledged to possess vital pharmacological properties with anticancer peptides having been found in Amaranthus cultivars. However, limited knowledge is available over the use of pepsin and alcalase enzymes to form hydrolysates. Thus, this study was aimed at comparing the in vitro anticancer effect of Amaranthus cruentus (grain) protein isolate and hydrolysates using alcalase, trypsin and pepsin. The safety of hydrolysates was investigated using the Ames mutagenicity and Brine Shrimp Lethality assay. Protein hydrolysates were thereafter investigated for their antioxidant potential using the FRAP, ABTS and DPPH assays. Subsequently, the protein hydrolysates were tested for their anticancer and apoptotic potential. The MTT assay was conducted to evaluate the cytotoxic potential of the protein hydrolysates using the HEK 293 (non-cancerous), A549 (cancerous) and MCF-7 (cancerous) cell lines. After that, morphological alterations were examined using the acridine orange and ethidium bromide double stain. Following this, the Annexin V apoptotic detection kit was used to quantify apoptosis together with the Glomax Caspase 3/7 kit to detect changes in the cell cycle Results show A. cruentus isolate and hydrolysates had no mutagenic response against Salmonella typhimurium TA 98 and TA 100 strains. The tested samples did not induce any significant increase in the death percentage of Artemia spp. in comparison to potassium dichromate (control). DPPH assay revealed that the hydrolysed samples had an enhanced scavenging activity compared to the unhydrolyzed sample, with pepsin having the greatest IC50 of 23.06 µg/ml. Amaranthus cruentus isolate (IC50 17.57 µg/ml) was a greater scavenger of the Fe+ ions compared to the control glutathione (IC50 79.81 µg/ml). For ABTS, all hydrolysates had a greater antioxidant scavenging potential compared to the isolate. The MTT cytotoxicity assay revealed that the isolate produced a greater cytotoxic effect on the MCF-7 and A549 cell line when compared to the control (camptothecin). For the non-cancerous cell line (HEK 293), trypsin hydrolysate had the highest toxicity. Apoptotic results revealed that trypsin hydrolysate was the most effective compared to the isolate, which was confirmed from morphological and Caspase 3/7 results. It may be concluded from the findings of this research that hydrolysates from food protein isolates have the potential for use as possible anticancer therapeutics. However, more research needs to be conducted to determine the peptides responsible for anticancer activity as well as the possible mechanism of action.Item Composition and functional bioactive properties of bambara groundnut protein and hydrolysates(2016) Arise, Abimbola Kemisola; Amonsou, Eric Oscar; Ijabadeniyi, Oluwatosin AdemolaBambara groundnut (Vigna substerranea) is an indigenous legume of African origin which is currently experiencing a low level utilisation. It is tolerant to drought and can grow under poor soil conditions in which other lucrative crops such as groundnut cannot grow. Bambara is a good source of protein comparable to that of cowpea and slightly lower than soya bean. In order to assess the potential use of bambara protein as a functional ingredient in food systems and as an important ingredient for the formulation of therapeutic product, the knowledge of its protein composition, structure and functionality becomes important. The main goal of this thesis was to determine the composition and bioactive properties of bambara protein and its hydrolysates. Specifically, a comparative study was carried out on the protein content, yield and functional properties of protein concentrates prepared from three different bambara landraces using different extraction methods (Salt solubilisation and Acid precipitation). There was no significant difference in protein content, yield and functional properties of the landraces. However, the method of extraction had an influence on their physicochemical and functional properties. Acid precipitation produced bambara protein concentrates with high protein content and yield (79% and 52% respectively) when compared to salt solubilisation (protein content - 57% and yield - 25%). Protein concentrates prepared through salt solubilisation method exhibited better functional properties in terms of water absorption capacity, oil absorption capacity, foaming capacity, foaming stability and emulsion activities when compared to concentrates obtained through acid precipitation. Furthermore, the composition of bambara proteins produced through isoelectric precipitation was determined. SDS PAGE revealed four major bands; a broad band at 55 kDa which was analysed to be vicilin, two medium bands at 62 kDa and 80 kDa and a high molecular weight (HMW) protein at 141 kDa. Further investigation of bambara protein revealed vicilin (55 kDa) with two sub units as the major protein in bambara and this was also confirmed by the proteomic map. The proteomic map revealed acidic amino acids as the major protein of bambara which is characteristic of vicilin, the map also showed that there were differences in the number of spots across the landraces with 77 spots matching each other. Circular dichroism spectroscopy exhibited reductions in α-helix, and β-pleated sheet conformations as pH varies. In addition, the tertiary structures as observed from the near-UV CD spectra were also influenced by shifts in pH conditions. Differential scanning calorimetry thermograms showed two endothermic peaks at around 67 and 81oC respectively. These can be attributed to thermal denaturation of vicilin and the HMW protein. Subsequent studies used isolates from red bambara since the composition of the landraces were similar. Bambara protein isolate was subjected to enzymatic hydrolysis using three proteases (alcalase, pepsin and trypsin) to produce various bambara protein hydrolysates (BPHs). BPHs were investigated for antioxidant and antihypertensive activities. The in vitro structural and functional characteristics of bambara protein and its enzymatic protein hydrolysate revealed that bambara groundnut possessed antioxidant properties against a variety of physiologically relevant free radicals. High surface hydrophobicity and the molecular size of the peptide seem to be important for scavenging of hydroxyl radicals, ferric reducing power and metal chelation. BPHs and peptide fractions were able to scavenge DPPH radicals with greater affinity for smaller size. Less than 1 and 1-3 kDa pepsin fraction was able to scavenge DPPH radical more than glutathione, BPHs and its fractions scavenge ABTS•+ three folds than the isolate. Scavenging of superoxide radicals was generally weak except for 5-10 kDa peptide fractions. All BPHs inhibited linolenic acid oxidation with greater affinity for the lower molecular size peptide. BPHs showed potential antihypertensive properties because of the in vitro inhibition of activities of angiotensin converting enzyme (ACE) and renin inhibition. The molecular size had significant effect on the ACE inhibitory properties with low molecular weight peptide (<1 kDa) fractions exhibiting significantly higher (p<0.05) inhibitory activities. However, enzyme type had synergistic effects on renin inhibition with alcalase hydrolysate showing highest inhibition at 59% when compared to other hydrolysates and their membrane fractions. The fractions with <1 and 1-3 kDa peptides showed a higher potential as antihypertensive and antioxidant peptides. Based on this study, incorporation of bambara protein isolate as an ingredient may be useful for the manufacture of high quality food products. Likewise, the bambara protein hydrolysates, especially the <1 kDa and 1-3 kDa fraction represent a potential source of bioactive peptides in formulating functional foods and nutraceuticals.