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Master of Agribusiness / Department of Agricultural Economics / Keith D. Harris / Food processing facilities are faced with many challenges in ensuring that the food supply is safe for consumption. Listeria monocytogenes is a food pathogen that has been linked to ready-to-eat foods, including tree nuts. Listeria monocytogenes is part of the ubiquitous microorganism genus, Listeria. The most likely cause of Listeria contamination in food is post-processing contamination. The purpose of this research is to identify and examine possible solutions a nut processing facility might employ to mitigate a food safety risk. The outcome of this research helps to establish the most financially viable method a processing facility may implement to address and mitigate an established risk given defined premise construction and constraints. The research objective is to identify a solution, implement a course of action, and establish safeguards to prevent recurrence of the issue. Factoring in facility specific variables as well as industry data and relevant analyses, the research conducted concludes with recommended actions for the facility to make, including a combination of structural design changes coupled with extensive chemical sanitation techniques.

Ogi is a fermented food made from maize, sorghum or millet which serves as complementary food for infants and breakfast for adults in Nigeria, West Africa. This study characterized the microbial diversity of maize and sorghum grains and ogi produced by their natural fermentation in an attempt to understand the roles of the key microbial species and the impact of the population dynamics and selected species on changes in nutritional composition and aroma notes of ogi during fermentation. A combined approach of culture dependent and culture independent methods of analysis was applied to investigate the microbial community of grains and ogi from two different sources. Microbial diversity and viable populations varied with the source of the grain. Bacterial and fungal genera identified with the partial 16S rRNA and 26S rRNA sequence analysis respectively in maize and sorghum were Bacillus, Enterobacter, Micrococcus, Kytococcus, Pantoea, Staphylococcus, Amycolatopsis, Methanoculleus, Aspergillus, Penicillium, Eupenicillium, Acremonium, Schizosaccharomyces, Meyerozyma, Hyphopichia, and Pichia in maize grains; Enterococcus, Enterobacter, Pantoea, Bifidobacterium, Aspergillus, Cladosporum, and Penicillium in maize ogi; Enterococcus, Enterobacter, Pantoea, Aeribacillus, Cyanobacterium, Acinetobacter, Fusarium and Trametes in sorghum grains; and Pediococcus, Lactobacillus, Enterococcus, Bacillus, Cladosporum and Penicillium in sorghum ogi. Similar species were observed in both sources of maize while those of sorghum differed slightly. Predominant microbes included species of Enterobacteriaceae and moulds. Acetic acid bacteria were not identified as part of the diverse community. Following the predominance of moulds during the natural fermentation, preliminary screening was performed by PCR using specific biosynthetic gene primers to test whether they are the mycotoxin producing species. None of the genes tested were detected by PCR thus they may not be the toxin producing species. Starch, non-starch polysaccharide (NSP), phytate and phenolic compounds were determined in the grains and respective ogi to ascertain the levels of these nutritionally important components in the naturally fermented ogi and the impact of the varying microbial populations on the fate of these compounds during fermentation. In the grains, the average starch and NSP contents in each case were 80.35 g/100g and 9.40g/100g in maize and 93.12 g/100g and 8.14 g/100g in sorghum. Out of the total in grain the average percentage recovery of starch and NSP respectively in the ogi showed 63% and 42% in maize and 58% and 27% in sorghum. Maize showed good starch and fibre (NSP) retention than sorghum after fermentation. To further understand the types and levels of polymers in NSP hydrolysis in ogi fermentation, HPLC analysis of the hydrolysed extract was performed. Glucose was entirely present in maize and sorghum ogi which represents the beta-D-glucans while arabinose and xylose (in maize only), mostly lost with the pomace, signify the arabinoxylans. Overall variations in the microbial populations of sorghum seemed causal to the difference in starch and NSP recoveries. Phytate was assessed based on release of total phosphorus in the samples by enzymatic and chemical methods. Recovery of phytate in the naturally fermented ogi ranged from 18-25% in maize and 40-48% in sorghum suggesting greater phytase activity and more nutrient bioavailability in maize ogi than in the sorghum. Greater activity in maize reflects the presence of phytate hydrolysing species such as Aspergillus in the grain. Total phenolic content (TPC) was assessed by Folin-Ciocalteu colorimetric method after direct extraction of samples by saponification. TPC in the original grains ranged from 410–437 mg GAE/100g in maize and 221–247 mg GAE/100g in sorghum. Due to the nutritional significance, the amount of phenolics that are either freely soluble or are covalently bound to the food matrix were assessed. Soluble phenolics in ogi ranged from 16-38% in maize and 32-49% in sorghum based on the total soluble fraction in the original grain. In all cases loss of soluble phenolics with the waste waters accounted for 12-25% and 31-39% with the pomace. Only the LAB population seemed to correlate with the release of phenolics in the natural fermentation. Given the higher value of soluble phenolics, naturally fermented sorghum ogi appeared to have higher antioxidant potential than the maize ogi. Furthermore an attempt was made to ascertain whether the use of selected microbes would improve the antioxidant properties and aroma of ogi while minimizing the incidence of pathogens due to chance inoculation. Thus the impact of selected LAB (Pediococcus pentosaceus) and fungi (T. hirsuta and A. zeae previously shown to have phytase activity) on changes in phytate, phenolics and aroma of ogi was assessed following a parallel experiment to the previous study but using autoclaved grains. Five fermentation treatments of the pure and co-cultures were investigated. Cell populations in all culture fermentations varied and reached the average maximum of log 6-9 cfu/ml. Changes in the distribution of bound and soluble phenolics were observed showing esterase activity. Leaching of phenolics was evident in all cases but was higher in the sorghum fermentations. Higher levels of soluble phenolics were recovered in pure culture fermented ogi using T. hirsuta or P. pentosaceus than in the natural fermentation having 76% and 45% of the original soluble fraction in maize and sorghum respectively. This suggests greater antioxidant potentials than the naturally fermented ogi. Pure culture fermentations using T. hirsuta and co-culture of P. pentosaceus with A. zeae reduced phytate by 97% and 96% in maize and sorghum ogi respectively showing greater phytase activity and more nutrient bioavailabilty in the ogi than in the natural fermentation. The aroma profile of ogi was analysed by solid-phase microextraction and gas chromatography-mass spectrophotometry (SPME GC-MS). Ethyl acetate, butyl acetate and ethyl hexanoate were observed as the key active aroma components in ogi. The ester, methyl thiobutanoate was found to be unique to the naturally fermented ogi suggesting that it may have been generated by species other than the selected starter organisms. Overall in both natural and starter culture fermentations, maize ogi showed high relative abundance of volatile components suggesting good substrate compatibility and utilization during fermentation. Thus compounds with high threshold values may be significant in the aroma notes of maize ogi. P. pentosaceus and T. hirsuta in pure and in co-culture fermentations produced ogi with aroma notes mostly related to the naturally fermented product. In conclusion the diversity and levels of the initial microflora and the structural composition of grain could be major factors contributing to the nutritional compositional changes in ogi fermentation.

This thesis concentrates on gaining a fundamental understanding of the crystallisation, phase behaviour, and melting in relatively simple fat/oil blends. This is the first reported study of hot stage microscopy (HSM) experiments on tripalmitin (PPP)/triolein (OOO), 1,3-dipalmitoyl-2-oleoylglycerol (POP)/OOO, and cocoa butter (CB)/hazelnut oil (HZ) systems. The HSM technique allows the visualisation of the initial crystallisation, polymorphic transformations, and melting of fat crystals; melting points can also help identify polymorphic form. Supporting experiments were also performed using differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), and X-ray diffraction (XRD). In PPP-OOO samples, HSM visualised for the first time a melt-mediated transformation from β' to β across a small liquid gap between the untransformed (β') and transformed (β) material. This behaviour was not seen with pure PPP. Melting points obtained by HSM for the PPP/OOO system were above those predicted by the Hildebrand equation, but this is attributed to the non-equilibration of concentration gradients within the system. This was evidenced by the fact that a rapid cooling rate (to produce a finer microstructure with smaller crystals, and hence reduce diffusion distances) combined with a slower remelt rate enabled samples to melt close to ideal. Indeed, final melting points obtained via HSM were consistently higher than DSC results across all systems; convection and the more three-dimensional system in DSC (as compared with the two-dimensional HSM system) may have aided melting. The POP/OOO system displayed complex remelting behaviour, especially when warmed at a relatively slow rate; this also resulted in a higher production of β. Liquid oil content was shown to not only be important in aiding transformation of lower forms to β, but also reduced the number of polymorphs observed upon remelting, as compared with pure fat samples. Liquid oil content was also shown to be crucial for transformation to β in the CB/HZ experiments; very few β crystals were seen in pure CB samples. Experiments carried out on DSC for both CB and CB-HZ cooling at 1°C/min or faster produced both α and β' crystals, but for CB-HZ this also then led to some transformation from α to β. The β polymorph was not observed when only β' was formed at slower cooling rates. A curious result was that the effect was stronger the longer the samples were held at 0°C before rewarming, with the α becoming more resilient against transforming to β' and instead transforming directly to β. More extensive transformation to β occurred if the sample was held for 30 min at 18°C or 22°C during the rewarming step. As well as showing differences in melting temperatures, the HSM and DSC results also did not always match with respect to polymorphic form. Small quantities of β crystals were seen in HSM samples that were not always seen in thermograms of equivalent DSC samples. This either highlights the limitations of DSC or suggests that polymorphic behaviour in the more fluid DSC system differs to that in HSM, or both. Growth rate analysis of PPP (in OOO) showed that both reduced supersaturation and supercooling can be correlated with the growth rates of β' and β. Growth rates of β that occurred via the melting of β' were well correlated with driving forces that took into account that the concentration of PPP in the liquid gap between β' and β (from which the β crystal was growing) was limited by the solubility of β' at the sample temperature. Whilst temperature is often seen as a key driver in governing polymorphic transformation, part of the temperature effect may be an indirect effect via the extra amount of liquid content at higher temperatures. The ability of oil to aid transformation to β may be relevant to food systems where this higher polymorphic form is the preferred type, such as chocolate. Potential applications could include producing novel fat blends with relatively stable fat network structures at a lower overall saturate level, or developing blends with bloom inhibiting properties.

There is demand from the food industry for novel savoury seasonings based on low-cost food ingredients, sourced from within the EU. A nucleotide and protein rich spray dried powder which was derived from a Fusarium venenatum fermenter waste stream and food-grade Alphitobius diaperinus with potential as a savoury flavour enhancer was evaluated. Enzymatic digestion of these two raw materials as a source of flavour precursors was evaluated. Serial enzyme combinations, enzyme dosages, sequence of enzyme application, pH, temperature and length of digestion for the liberation of amino acids and nucleotides were optimised for the liberation of taste active compounds. For amino acids, free glutamine (GLN) and glutamic acid (GLU) could be enhanced using a combination of peptidases on both raw materials. Digesting a spray dried powder derived from the fermenter waste stream of Fusarium venenatum with exopeptidase (1% Flavourzyme TM), resulted in an improved yield of GLN (from 0.1 mg/g to 28.9 mg/g powder) and GLU (from 1.71 mg/g to 5.98 mg/g powder). For milled Alphitobius diaperinus, mixed use of exopeptidase (1% Flavourzyme TM) and endopeptidase (2% Alcalase 1.4-fold increased yield of GLU (17.5 mg/g powder) and 1.7-fold increased yield of GLN (1.2 mg/g) as best production was obtained. For nucleotides, digestions of the waste stream with a yeast lytic enzyme (YL-TLTM) followed by a nuclease (RP-1GTM) resulted in the highest 5’-guanosine monophosphate (GMP) and 5’-adenosine monophosphate (AMP) production. Specifically, a 2% and 0.05% treatment by YL-TLTM and RP-1GTM respectively was shown to be optimal, followed by a 0.05% DeamizymeTM treatment for the conversion of AMP to 5’-Inosinic acid (IMP) of 38 mg/g. For the solid digestion of Alphitobius diaperinus, being treated with a nuclease tretment (2% RP-1GTM) followed by a 0.2% DeamizymeTM treatment for the conversion of AMP to IMP, resulted in the highest GMP yield, a 3.5-fold increased (2.6mg/g), and 7.8-fold increased IMP (4.7mg/g) production.

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