Theses and dissertations (Applied Sciences)
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Item Profiling and antibiotic resistance of lactic acid bacteria isolated from commercial aMasi samples(2020-03) Pillay, Yovani; Ijabadeniyi, Oluwatosin Ademola; Swalaha, Feroz MahomedaMasi is traditionally fermented milk that constitutes part of the South African heritage and is regarded as a supplementary staple food. Its inclusion into the South African Food Based Dietary Guidelines has led to the encouraged consumption of this product. Given the fact that aMasi is a rich source of lactic acid bacteria (LAB), such bacteria are of economic importance to the food, feed and pharmaceutical industries. The main concern regarding food safety is ability to acquire and disseminate antibiotic-resistant genes. Although LAB bility of resistance genes to human and animal opportunistic and pathogenic bacteria which could make treatment of bacterial infections more complex to treat in the future. Numerous reports globally, have documented antibiotic resistance among LAB isolated from commercial dairy and pharmaceutical products over the last decade. Therefore, the aim of this study was to determine if LAB isolated from commercial aMasi samples harbour antibiotic-resistant genes. To achieve this aim, the total bacterial population and LAB population of 10 aMasi samples were surveyed using culture-dependent techniques and the proportional prevalence of LAB to the total bacterial population were determined by using a 100% stacked-column. In all 10 samples, LAB was the predominating population ranging from 87.44% to 99.77%. A total of 30 LAB isolates were characterised after isolation and sequencing of 16S rDNA of these isolates showed that LAB were Leuconostoc pseudomesenteroides and Leuconostoc mesenteroides with two isolates being identified as Lactococcus lactis CP028160.1. The relationship between the growth of LAB and selected physicochemical properties (pH, titratable acidity, water activity (aw), moisture content, fat content and estimation of reducing sugars (lactose)) were determined using principal component analysis (PCA) and classification and regression tree (CART) to illustrate the likelihood of LAB present in aMasi samples based on LAB count and pH. From the PCA results, approximately 75.25% of variances in the data were retained by the first three principal components (PCs). The first principal component (PC1) had accounted for the highest total variance of 33.16%. PC1 increased with an increase in lactic acid % and aw, whilst it negatively correlated with LAB count, moisture % and lactose (mg/25ml lactose·H2O). The results showed an increase in LAB count with an increase in moisture % and lactose (mg/25ml lactose·H2O) whilst, LAB count had decreased with an increase in lactic acid % and aw. Moreover, pH and fat % had no effect on PC1, high LAB counts were observed for samples 6 and 7 whist low LAB counts were observed for samples 9 and 10. On the other hand, PC2 had accounted for approximately 27.53% of the total variance. PC2 increased with an increase in fat % and lactose (mg/25ml lactose·H2O), whilst it negatively correlated with LAB count and pH. It was observed that the growth of LAB had increased with an increase in pH, whilst it decreased with an increase in fat % and lactose (mg/25ml lactose·H2O). Moreover, lactic acid %, aw and moisture % had no effect on PC2. High LAB counts were observed for samples 7 and 8 and low LAB counts were observed for samples 2 and 4. Nine out of the 30 LAB isolates were selected due to these isolates having a different GenBank Accession number and were subjected to antibiotic susceptibility testing using the disc diffusion method against a total of 11 antibiotics. Most of the LAB isolates exhibited multiple resistance towards some of the most commonly used antibiotics as well as last-resort antibiotics. All the isolates showed high levels of resistance towards vancomycin, colistin sulphate, fosfomycin and pipemidic acid except for Lactococcus lactis CP028160.1 which was susceptible to vancomycin. All isolates were susceptible to tetracycline and erythromycin whilst eight out of nine isolates were susceptible to chloramphenicol with seven out of nine isolates being susceptible to ampicillin. Furthermore, the isolates had displayed intermediate resistance mainly towards kanamycin and streptomycin. The present study showed that multiple antibiotic resistance is prevalent in different species of starter culture strains, which may pose a food safety concern. LAB that exhibit phenotypic resistance to antibiotics should also be evaluated on a molecular level to monitor their resistance. The presence of such a variety of expressed AR genes in probiotic isolates is a worrying trend. The impact of the interactions of these bacteria with pathogenic strains and their transfer of these AR genes is yet to be assessed. Furthermore, antibiotic sensitivity is an important criterion in the safety assessment for the evaluation of food-grade and potential food-grade LAB.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.Item Incidence of mycotoxigenic fungi during processing and storage of bambara groundnut (Vigna subterranea) composite flour(2019) Olagunju, Omotola Folake; Ijabadeniyi, Oluwatosin Ademola; Mchunu, Nokuthula PeaceFungal contamination of food commodities is a global food security challenge that impacts negatively on the health of consumers. Mycotoxins are produced as secondary metabolites by some pathogenic fungi and may contaminate agricultural products while on the field or during harvesting and storage. Processing operations and storage conditions of temperature and relative humidity have marked effect on the ability of fungal pathogens to grow and produce mycotoxins in agricultural food commodities. The consumption of mycotoxin- contaminated foods, even at low doses over a prolonged period of time, may have deleterious effects on health of consumers. Bambara groundnut (Vigna subterranea (L.) Verdc) is an African legume gaining wide acceptance in various food applications due to its favourable nutritional composition, especially the high protein content. In several parts of Africa, it is used as a supplement in cereal-based foods, especially in weaning food for infants and young children. Bambara groundnut grows near or under the soil, which serves as inoculum of pathogenic fungi. Very little information is presently available on fungal and mycotoxin contamination of Bambara groundnut from Southern Africa. Hence, its safety for consumption from a mycological standpoint requires further studies. To establish the profiling of fungal contaminants in food commodities consumed in Durban, South Africa, 110 samples of regularly consumed food samples which included rice (23), spices (38), maize and maize-derived products (32), and Bambara groundnut (17) were randomly collected over a period of five months from retail stores and open markets. The food samples were screened for fungal contamination using conventional and molecular methods. Fungal isolates were characterized following DNA extraction, polymerase chain reaction and sequencing. Using a modified QuEChERS method, the detection and quantification of mycotoxins in Bambara groundnut was performed via Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), and isolation and detection of the causative pathogen was carried out. The effect of processing operations of milling, a combination of roasting and milling, and spontaneous fermentation on the survival of the natural fungal population of Bambara groundnut, and aflatoxin production under simulated tropical conditions of storage was further studied. Processed Bambara groundnut flour samples were stored at temperature of 30±1 °C and 85±2% relative humidity for 30 days, vi and samples withdrawn at 5-day intervals for analyses, i.e., fungal counts, aflatoxin accumulation and changes in water activity during storage. Following the detection of aflatoxins in Bambara groundnut flour and the isolation of aflatoxigenic Aspergillus flavus in the seed, the effect of milling, roasting and milling, or lactic acid bacteria fermentation on the survival, growth and aflatoxin production of A. flavus in Bambara groundnut flour was studied. Irradiated seeds of Bambara groundnut were artificially inoculated with a 3-strain cocktail of A. flavus (2 x 106 spores/mL) and processed by milling, roasting at 140 °C for 20 min and milling. Slurries of irradiated Bambara groundnut flour were also inoculated with A. flavus spores and 1 x 108 CFU/mL inoculum of Lactobacillus fermentum or Lactobacillus plantarum. All inoculated samples were incubated at 25 °C for 96 h, samples withdrawn every 24 h were analyzed for viable A. flavus counts, changes in water activity during incubation, and aflatoxin production using Enzyme- linked Immunosorbent Assay (ELISA). Bambara groundnut flour samples fermented with lactic acid bacteria were further analyzed for pH, total titratable acidity, and viable lactic acid bacteria counts over the incubation period. The degradation of aflatoxin (AF) B1 by both lactic acid bacteria was also studied. Slurries of irradiated Bambara groundnut flour were spiked with 5 µg/kg of aflatoxin B1 (AFB1) and the percentage reduction over the incubation period was determined using HPLC. The survival, growth and aflatoxin production of A. flavus in Bambara groundnut and maize- composite flours as affected by milling, roasting and milling or lactic acid bacteria fermentation during storage was also studied. Processed and irradiated Bambara groundnut flour, maize flour and maize-bambara composite flour (70:30) were inoculated with 2 x 107 spores/ml of A. flavus and stored for up to 10 weeks at a temperature of 25±2 °C and relative humidity of 75±2%. Samples were withdrawn weekly and analyzed for viable populations of A. flavus, concentrations of aflatoxins B1, B2, G1 and G2, changes in pH and water activity over the storage period. The colonization of Bambara groundnut by A. flavus and the effects of fungal infection on the seed coat, storage cells and tissue structures were also studied. Irradiated Bambara groundnut seeds were artificially inoculated with spore suspension of aflatoxigenic A. flavus (2 x 106 spores/mL) and stored at a temperature of 25±2 °C and relative humidity of 75±2% for 14 days. Samples were withdrawn at 24 h intervals for 4 days, then at 7 and 14 days and examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Various fungal genera were isolated from the food samples under study with Aspergillus (52.5%) and Penicillium (31.8%) as the dominant genera. All the 110 food samples were contaminated with more than one fungal species. A. flavus and other Aspergilli, Penicillium citrinum and Fusarium oxysporum were isolated from Bambara groundnut seeds. Aflatoxigenic A. flavus was isolated from Bambara groundnut seed, with a co-occurrence of Aflatoxin (AF) B1 (0.13–6.90 µg/kg), AFB2 (0.14–2.90 µg/kg), AFG1 (1.38–4.60 µg/kg), and AFG2 (0.15–1.00 µg/kg) in the flour. The fungal counts of the samples during storage significantly (p≤0.05) increased, irrespective of the processing method from 6.3 Log10 CFU/g in Bambara groundnut flour to 6.55 Log10 CFU/g in fermented Bambara groundnut flour. Aflatoxin concentration was affected markedly by the processing methods in Bambara groundnut flour (0.13 µg/kg) and fermented Bambara groundnut flour (0.43 µg/kg), aflatoxin was not detected in roasted Bambara groundnut flour. The survival and growth of A. flavus was also markedly affected by lactic acid bacteria fermentation and roasting during incubation. Within 24 h of fermentation with L. fermentum, significant (p≤0.05) changes were recorded in viable population of A. flavus (6.30‒5.59 Log10 CFU/mL), lactic acid bacteria count (8.54‒13.03 Log10 CFU/mL), pH (6.19‒4.12), total titratable acidity (0.77‒1.87%) and a reduction by 89.2% in aflatoxin B1 concentration. Similar significant changes were recorded in Bambara groundnut flour fermented with L. plantarum. Aspergillus flavus in the artificially contaminated seeds were completely eliminated by roasting. Aflatoxin production was not detected in Bambara groundnut flour samples over the incubation period. During storage for 10 weeks, the population of A. flavus significantly (p≤0.05) decreased in roasted Bambara groundnut flour from 7.18 to 2.00 Log10 CFU/g. Similar significant (p≤0.05) decrease in A. flavus viable counts was recorded in fermented Bambara groundnut flour from 6.72 to 2.67 Log10 CFU/g, however after 7 weeks of storage and beyond, A. flavus was not detected. Significant (p≤0.05) decrease in aflatoxin B1 (0.36‒0.26 µg/kg) and aflatoxin G1 (0.15‒0.07 µg/kg) accumulation was also recorded in roasted Bambara groundnut flour. While A. flavus viable population significantly (p≤0.05) decreased in maize-Bambara composite flour from 6.90 to 6.72 Log10 CFU/g, aflatoxin B1 accumulation significantly (p≤0.05) increased from 1.17 to 2.05 µg/kg. Microscopy studies showed that the seed coat of Bambara groundnut was rapidly colonized by A. flavus within 24 h of inoculation. The infection of internal tissues of the cotyledon was through the ruptured seed coat, resulting in a disruption of the cellular architecture. Cell wall collapse, development of cavities in parenchymatous cells and ruptured storage cells resulted from A. flavus infection of the seed. This study reports a high prevalence of fungal contamination in some food commodities consumed in Durban, South Africa. The isolation of live mycotoxin-producing fungi from the food commodities necessitates the need for regular routine checks to ensure the mycological safety of agricultural products offered for sale to consumers. The detection of aflatoxigenic A. flavus and aflatoxins in Bambara groundnut flour at levels above the maximum tolerable limits raises health concerns on its utilization in food applications, and in supplementary feeding for infants and young children. Although roasting was effective in degradation of aflatoxins in Bambara groundnut seeds, elimination of fungal contaminants was not achievable which resulted in continued production of aflatoxin during storage. Fermentation using L. fermentum or L. plantarum is effective in eliminating A. flavus and degrading AFB1 in Bambara groundnut flour. Compositing Bambara groundnut with maize increased aflatoxin production in the flour. It is therefore necessary to implement legislation for aflatoxins in Bambara groundnut, and develop effective management practices during planting, harvesting and storage that will mitigate A. flavus infection in Bambara groundnut.