Theses and dissertations (Applied Sciences)
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Item Characterization and modification of Bambara groundnut globulin fractions for the enhancement of functional properties(2023-05) Alabi, Opeyemi Olaitan; Amonsou, Eric OscarThere is a growing interest in the utilization of leguminous grain proteins for food and industrial applications. Bambara groundnut is a xerophyte pulse grain and a potential source of protein that can replace soybean protein, a trusted and widely used food ingredient in the food industry. However, the use of Bambara groundnut proteins including the subunits (legumin and vicilin) is limited in food applications. The understanding of the composition of Bambara groundnut proteins at the subunit level is vital to unlocking their potential and facilitating utilization. In this study, Bambara groundnut globulin is characterized in terms of the structures, composition, and physicochemical properties at the subunit level, and then modified using atmospheric plasma and enzymatic hydrolysis. Bambara globulin consisted of about 70% vicilin, whilst legumin protein was found in relatively low quantity. Gel electrophoresis revealed three major protein bands in globulin similar to vicilin with predominant β-sheet structures. The presence of a disulfide bond was also revealed in legumin. Bambara globulin showed major vicilin (7S, Mw: 120 kDa) and minor legumin (11S, Mw: 410 kDa) components. Fluorescence and hydrophobicity data suggested a folded structure for the legumin fraction dominated by the helical secondary structure compared to the vicilin fraction. Bambara proteins contain an appreciable amount of methionine that is even higher than the FAO/WHO recommended value. Bambara vicilin had the highest amount of negatively and positively charged amino acids compared to globulin and legumin. This coincides with its high solubility profile (approximately 82% at pH 3.5). The least gelation concentration (LGC) significantly increased in the order of globulin (8%) < legumin (18%) < vicilin (20%) at pH 7. Bambara groundnut proteins formed weakly structured gels as indicated by the frequencydependent behaviours of both the storage (Gʹ) and loss (Gʺ) moduli with a difference of lesser than 1 log cycle. The highest Gʹ of vicilin gel indicated more firmness of the gel compared to the gel formed by globulin and legumin. The sol-gel transition temperatures increased in the order of globulin (40℃) < legumin (50℃) < vicilin (80℃). The Gʹ and Gʺ of globulin showed relatively low dependency on heating time beyond the gel point compared to legumin and vicilin subfractions, suggesting a more rapid establishment of its gel network during gelation. Vicilin gel consisted of a microporous structure with a small lath sheet-like structure compared to globulin and legumin. Emulsifying stability of the proteins significantly differed (p < 0.05) at pH 7. The foaming capacity of the vicilin fraction was significantly (p < 0.05) higher than that of the storage protein at pH 3, 7, and 9. Atmospheric cold plasma-activated water (PAW) and enzymatic modification of Bambara groundnut globulin were further assessed. The cold plasma treatment resulted in the loss of the helical structure of Bambara globulin. The plasma treatment increased the hydrophobicity of Bambara globulin indicating an unfolded structure that was also reflected in the observed redshift in fluorescence intensity. No major changes were observed in gel electrophoresis, protein surface charge, and solubility profiles, except for about a 20% reduction in the glutamic acid content of the amino acid profile. Bambara globulin had reduced emulsifying capacity after treatment with PAW. However, foaming capacities were significantly better and stable at up to 15 mg protein/mL. Hydrolysates produced from Bambara groundnut globulin and vicilin, respectively using a combination of pepsin and pancreatin were investigated for ACE and renin inhibitory activities. The hydrophobic amino acid residues in both globulin and vicilin hydrolysates are high, improving the entry of their peptides into target organs via hydrophobic associations. Surface hydrophobicity increased significantly (p<0.05) with an increase in peptide size from <1 to <3 kDa with that of vicilin hydrolysate and membrane fractions having the highest values. The low molecular weight peptide (<1 kDa) membrane fractions from globulin at 1 mg/mL exhibited significantly higher (p<0.05) invitro ACE inhibitory activities compared to vicilin hydrolysate and its fractions. However, higher molecular peptide fraction (<3 kDa) favoured renin inhibitory activity at the same concentration. Vicilin is the major protein fraction of Bambara groundnut globulin. Bambara groundnut globulin was stabilized by disulfide linkages from the legumin, a minor fraction of the storage protein. Bambara globulin and its subfractions formed a weakly structured gel with the dominance of an elastic structure. The dominancy of the β-sheet structure in vicilin protein and the high crosslink density of the vicilin gel could be related to the firmness of the vicilin gel. The variations in the gel points of Bambara globulin and the subfractions were linked to the differences in their amino acid and subunit composition, the thermal unfolding properties of the protein fractions, and the presence of disulfide linkages. Modification of Bambara groundnut globulin using cold plasma-activated water treatment and enzymatic hydrolysis, respectively increased the hydrophobicity of the protein and influenced the emulsifying and foaming properties and the invitro angiotensin-converting enzyme (ACE) and renin inhibitory activities. Therefore, Bambara groundnut globulin could be a potential functional ingredient in the food system. The low molecular weight peptide (<1 kDa and <3 kDa) membrane fractions from globulin have the potential to serve as functional bioactive peptides against hypertension.Item Quality and microbiological study of bambara groundnut fortified injera, a fermented flat bread(2020-04) Jula, Mellisa Nokulunga; Ijabadeniyi, Oluwatosin AdemolaCereal fermented products are popular in developing countries, especially in Asia and Africa, because of their unique taste and fulfilment. Throughout the years, they have played a vital part in bringing up infants as part of their weaning foods and contributing to the daily diet of many households. Food fortification and supplementation of cereal grains with inexpensive readily available legumes, which have higher protein content compared to cereals may lead to a potential decrease in protein-energy malnutrition. Underutilised and indigenous crops such as Bambara groundnut can be in incorporated into the fermentation of cereal fermented foods, such as injera. In this study, injera was prepared by substituting only 9% and 12% Bambara groundnut flour and comparing them with the traditionally fermented original control, which is injera made from only tef flour. The first part of the study was to identify and characterise the lactic acid bacteria (LAB) and yeast involved in the spontaneous fermentation of traditional tef-injera and the newly developed injera fortified with Bambara groundnut (which contains 12% Bambara groundnuts) at different fermentation intervals of 0, 24, 48, and 72 hour. A total of 70 LAB isolates and 30 yeast isolates were identified from both fermentations using rep-PCR fingerprinting followed by sequencing the 16S rRNA gene and the D1/D2 region of the 26S rRNA gene. Weissella confusa, Lc. lactis and Lb. curvatus predominated in both fermentations at different intervals of the fermentation. The second part of the study investigated the effectiveness of the isolated LAB starter cultures on the production of injera and injera fortified with Bambara groundnut after which their physicochemical properties were evaluated. There was a significant increase (p<0.05) in titratable acidity and a significant decrease in pH to below four within 24 hours; recorded for samples inoculated with LAB starter cultures when compared to samples fermented without inoculation. The third and fourth parts of the study investigated the proximate composition and storage stability of the injera samples. Injera fortified with 12% Bambara groundnut + LAB culture had a significantly high (p<0.05) protein of 23.21%, the lowest protein content being Tef injera at 7.35%. The protein digestibility of Tef injera increased with the addition of Bambara groundnut and LAB starter culture. The digestibility of protein increased from 40% for Tef injera to 80% for injera fortified with 12% Bambara flour + LAB culture. There was no significant increase (p >0.05) in the amino acid content after the addition of Bambara flour + LAB cultures; the amino acid concentrations were slightly lower than the standard concentration recommended by the Food and Agricultural Organisation/World Health Organisation for adults. Injera samples fortified with Bambara groundnut flour and inoculated with lactic acid starter cultures were stable with microbial counts ranging from 4.42 log cfu/g to 4.68 log cfu/g for TPC at 4 ̊C, yeast and mould, coliforms and aerobic spore formers were not detected in all the samples from day 0 to day three upon storage. Higher counts had been perceived at room temperature ranging from 4.60 log cfu/g to 7.53 log cfu/g for moulds and 4.90 log cfu/g to 9.26 cfu/g for TPC; coliforms were detected in one tef injera only ranging from 4.48 log cfu/g to 6.16 log cfu/g and no detection of aerobic spore formers in all samples. Refrigeration temperatures effectively maintained the microbiological quality of injera for three days. The nutritional quality, distinctively the protein content increased with the addition of Bambara groundnut flour and through the use of lactic acid bacteria as a starter culture This will potentially pave the way for the commercialisation of injera in the industry with the use of LAB starter culture to ensure a fast and continuous supply of fresh injera that is in high demand.