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Metabolomics, Physicochemical Properties and Mycotoxin Reduction of Whole Grain Ting (a Southern African fermented food) Produced via Natural and Lactic acid bacteria (LAB) fermentation A Thesis submitted to the Faculty of Science, University of Johannesburg, South Africa In partial fulfilment of the requirement for the award of a Doctoral Degree in Food Technology By OLUWAFEMI AYODEJI ADEBO STUDENT NUMBER: 201477310 Supervisor : Dr. E. Kayitesi Co-supervisor: Prof. P. B. Njobeh October 2018 EXECUTIVE SUMMARY Drought and challenges related to climate change are some of the issues facing sub-Saharan Africa countries, with dire consequences on agriculture and food security. Due to this prevailing situation, drought and climate resistant crops like sorghum (Sorghum bicolor (L) Moench) can adequately contribute to food security. The versatility and importance of sorghum is well reflected in its use as a major food source for millions of people in sub-Saharan Africa. Recent interest in gluten free food products has equally positioned this crop as a potential substitute for wheat, with numerous studies attesting to this possibility. Although other food processing techniques for the transformation of these sorghum grain exists, fermentation is an important and dominant technique for processing sorghum into other food products particularly in the developing world. Porridge and gruels are commonly and usually consumed in developing countries. A form of this sorghum fermented food is ting, referred to as mabele, bogobe (when processed to hard porridge) or motogo (when processed to soft porridge). It is commonly consumed in South Africa, Botswana and other neighbouring Southern African countries. Although ting is conventionally processed using refined grains, the role of whole grain meals with regards to the provision of desirable health benefits is emphasized in recent literatures. Their composition yields better contents of bioactive, health promoting and beneficial compounds in subsequent products as compared to products from refined grains. The composition of fermented foods depends on such factors as fermentation time and temperature, meaning that changes and choice of these conditions would influence the quality of the final product. Such differences in fermentation conditions would thus affect ting quality, necessitating an optimization of these conditions. Accordingly, a response surface methodology (RSM) optimization technique was employed in this study to optimize fermentation conditions of ting from two whole grain sorghum types: high tannin sorghum (Avenger) and low tannin sorghum (Titan). The effect of fermentation time and temperature on whole grain-ting parameters such as pH, titratable acidity (TTA), total viable bacteria count (TBC), total lactic acid bacteria count (TLABC), total fungal and yeast count (TFYC), tannin content (TNC), total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activities (AA) was investigated. Scanning electron microscopy (SEM) was also used to investigate possible morphological changes in the samples during fermentation. i Results obtained showed that fermentation significantly (p 0.05) influenced all the parameters investigated in both high tannin (HT) and low tannin (LT) ting samples. Higher levels of pH, TTA, TBC, TLABC, TFYC were observed in the LT-ting samples, with lower amounts of TPC, TFC, TNC and AA when compared to HT-ting samples. More visible modifications in morphology were observed for the LT-ting with no considerable morphological changes noted in HT-ting samples. Statistical models generated and validated using different statistical indices [absolute average 2 deviation (AAD), bias factor (퐵푓), accuracy factor (퐴푓) and coefficient of determination (R )] suggested the validity of the models obtained. A multi response numerical optimization (MRNO) technique for production of whole grain (WG) ting with better quality indicated optimal conditions for WG-ting from the HT- and LT-sorghum type as 28 oC for 72 h and 34 oC for 24 h, respectively. Subsequent predicted values confirmed via experiments (in triplicates) showed relatively close experimental values with the predicted ones, further indicating the strength of the models obtained. The study further investigated the effect of natural (spontaneous) fermentation as compared with a controlled fermentation using two LAB strains namely Lactobacillus fermentum FUA 3165 and L. fermentum FUA 3321 on different ting quality parameters. Optimal fermentation conditions earlier obtained with the multi response numerical optimization were adopted for both the HT- and LT-sorghum types. Both pH and TTA values of naturally fermented WG-ting were low, while accelerated fermentation led to a rapid drop in pH and significantly (p 0.05) higher TTA values, after fermentation with LAB strains. Likewise, a significant (p 0.05) decrease in the TNC, TPC and TFC was observed in LAB-fermented WG-ting samples, suggesting an increased metabolism of phenolic compounds and possible production of compounds that contributed to higher AA. Quantification of some bioactive compounds using liquid chromatography tandem mass spectrometry (LC-MS/MS) showed that WG-ting from the HT-sorghum had higher composition of catechin, quercetin and gallic acid. A combination of the two strains used gave relatively significant (p 0.05) lower values for WG-ting parameters investigated, indicating a negative competitive action between the LAB strains. Mycotoxin contamination of food crops and persistence in derived food products have been of immense concern due to associated health and economic implications. An effective, safe and practicable way of reducing these mycotoxins in food through fermentation was explored in this study. Although the initial mycotoxin content of the whole grain sorghum types (HT- and LT-) ii was well below levels regulated in South Africa, fermentation was observed to significantly (p 0.05) reduce these contents. This was particularly more pronounced in LAB fermented WG-ting samples, with over 80% reduction in fumonisin B2 (FB2), T-2 toxin (T-2) and alpha-zearalenol (α- ZEA). A 98% reduction in fumonisin B1 (FB1) was also recorded with other mycotoxins equally reduced at varying rates. Observations made in this phase of this study correlate with those of the preceding paragraph, indicating the effective potential of these L. fermentum strains as starter cultures for fermentation. Adopting a metabolomics approach, an untargeted analysis of the metabolites in WG-ting was done using a gas chromatography high resolution time of flight mass spectrometry (GC-HRTOF- MS) system. The raw HT and LT-ting samples were included to understand changes in sorghum metabolome before the formation of WG-ting. Multivariate data analysis using principal component analysis (PCA) effectively separated the tested samples into different clusters, relative to fermentation types (natural and control) and sorghum types. Subsequent orthogonal partial least square discriminant analysis (OPLS-DA) on the dataset revealed significant metabolites contributing to differences observed in both HT- and LT-sorghum samples as groups of acids (methylene cyclopropanecarboxylic acid and 3,4-difluorobenzoic acid, 2,2,2-trichloroethyl), pesticide, phenol and fatty acid ester (FAEs), some which might contribute to reduced fungal load and attendant mycotoxin reduction in both samples as noted. Although OPLS-DA equally revealed variations (majorly reduction of metabolites) as a result of fermentation (natural fermentation, use of L. fermentum FUA 3165, L. fermentum FUA 3321 singly and in combination), different significant metabolites were identified as being responsible for these differences. Differences in raw HT-sorghum and derived ting were due to a phenol, ketone, pesticide, fatty acids and fatty acid esters, with more volatiles metabolites differentiating LT-ting from the raw LT-sorghum. A reduction of 4-chlorobenzonitrile (a pesticide) was however observed in HT-ting derived from fermentation with L. fermentum 3165, indicating the degradative ability of this strain, corresponding to results of mycotoxin reduction. Among the variations observed, only ethanone, 1-(2-hydroxyl-5-methylphenyl) (a ketone) was identified as the only significant (p ≤ 0.05) metabolite common to all the samples, an indication that this is an important volatile compound present in these samples. iii Addressing food security will not only be achieved by providing adequate nutrition, but also ensuring these food products contain functional components that can improve health and mitigate certain diseases among consumers. Fermentation, especially with L. fermentum strains