Submitted in fulfillment of the academic requirement for the degree Doctor of Philosophy (Ph.D.) in Food Science and Technology, Durban University of Technology, Durban, South Africa, 2017. / Bambara groundnut (Vigna subterranea) is an indigenous underutilised leguminous crop to Africa. It is a good source of protein and carbohydrate including starch. Bambara groundnut is a traditional crop grown mainly for subsistence in Southern Africa. Bambara groundnut has the advantage of being drought tolerant and can thrive in hot temperatures and poor soil conditions. Therefore, it has great potential as an alternative crop to soya bean and peanuts for cultivation and utilisation. Bambara groundnut starch can potentially be used for various industrial applications. However, native starches are not suitable for most industrial applications, hence the need for modification. Bambara groundnut starch has been previously modified using physical and chemical modification methods. Natural alternatives such as the use of lipids are being sought to modify starches due to the associated risk with chemically modified starch. In this research, Bambara groundnut starch was modified with lipids to improve functional properties, utilisation and application. Specifically, the physicochemical properties of native Bambara groundnut starch obtained from five Bambara groundnut genotypes and three landraces (maroon, brown and cream) were determined. Bambara groundnut starch was modified with lipids (palmitic acid, stearic acid, oleic acid, linoleic acid and lysophosphatidylcholine) and the physicochemical properties of the modified starch were investigated. Further, the influence of high-pressure homogenization on complexation of Bambara groundnut starch with lipids was assessed in comparison with maize and potato starches. Lastly, an application of modified Bambara groundnut starch in biofilm production was also studied. Bambara groundnut landraces generally showed higher amylose contents (approx. 33%) than the genotypes (approx. 28%). Differences were observed in the crystalline patterns of these starches. Bambara groundnut genotypes exhibited the C-type-crystallinity, while the landraces showed the unusual A-type pattern. In terms of functionality, landrace starches showed better swelling than the genotypes. Subsequent studies on modification used maroon Bambara groundnut starch since the amylose content was higher than other landraces and there was a consistent supply of the grains during the period of the study. Generally, Bambara groundnut starch showed higher complexing ability with all the lipids than maize and potato reference samples. These differences in complexing ability among the starches could be due to the variation in amylose contents (Bambara groundnut starch: 31.5%, maize: 22.5% and potato: 24.6%). Fatty acids complexed better with Bambara groundnut starch than lysophosphatidylcholine, which could be due to the structural differences in comparison with the lysophosphatidylcholine molecule. The number of fatty acid in the glycerol backbone and the additional steric hindrance of the polar phosphatidic acid group in the lysophosphatidylcholine may have reduced its complexing ability. Among the fatty acids, palmitic acid complexed better than stearic and the unsaturated fatty acids, possibly due to its short chain length compared to other fatty acids. Bambara groundnut starch showed reduced peak and setback viscosities in the presence of stearic acid, linoleic acid and lysophosphatidylcholine, suggesting the formation of V-amylose complex. Bambara groundnut starch pasted with lipids displayed reduced gelling ability compared to their unmodified counterparts. XRD studies of freeze-dried paste revealed peaks at 2Ѳ = 7.4, 12.9 and 19.9o confirming the formation V-amylose complexes in Bambara groundnut starch. Modification of Bambara groundnut starch with lipids resulted in reduced digestibility. High-pressure homogenization significantly increased the complexing ability of Bambara groundnut starch with lipids. Homogenized Bambara groundnut starch-lipid complexes generally exhibited higher complex index than their unhomogenized counterparts. The higher complexing ability could be attributed to the effect of high-pressure which may have enhanced greater dispersion of lipids in the starch-water system. X-ray diffraction studies also revealed the formation of higher complexes as shown by high intensities at peaks (2Ѳ= 7.4, 12.9 and 19.9o) corresponding to V-amylose complexes. Bambara groundnut starch-lipid complexes displayed significantly higher melting temperatures (95.74-103.82oC) compared to native uncomplexed starch (77.32oC). Homogenized Bambara groundnut starch complexes were non-gelling while the unhomogenized types produced weak gels, with G′ ˃ G″ in the range of 0.1- 10 Hz. Complexation of Bambara groundnut starch with lipids using high-pressure homogenization may be employed in the production of modified starch with non-gelling properties and higher thermal stability suitable for certain industrial application, such as fat replacers in mayonnaise, frozen foods and desserts for a better mouth feel. The physicochemical and mechanical properties of biofilm prepared from Bambara groundnut starch modified with stearic acid at varying concentrations of 0, 2, 4, 6, 7 or 10% were further studied. By SEM, Bambara groundnut starch films containing stearic acid (˃ 2%) showed a progressively rough surface compared to those with 2% stearic acid and the control. The addition of 2% stearic acid to Bambara groundnut starch film reduced water vapour permeability by approximately 17%. However, mechanical properties of starch films were generally negatively affected by stearic acid. Bambara groundnut starch film may be modified with 2% stearic acid for improved water vapour permeability and thermal stability with minimal effect on tensile strength. / D

The objective of the research was to carry out the biogeneration of lipophenols by enzymatic esterification of tricaprylin and caprylic acid with catechin and catechol in a model hexane system. Commercial lipases, including Lipase N from Rhizopus niveus, Lipozyme IM from Mucor miehei and Novozym 435 from Candida antarctica were used throughout this study. The effects of reaction time, incubation temperatures and agitation speeds on enzymatic hydrolytic activity were investigated to determine the optimal conditions for biocatalysis. The optimal temperatures for biocatalysis were determined to be 37.5°C for Lipase N, and 55°C for Lipozyme IM and Novozym 435; the optimum agitation speed was 100 rpm. Using Lipase N, maximum hydrolysis of 1.66 mumol free fatty acids/mL was obtained after 1.5 days of incubation, while with Lipozyme IM, maximum hydrolysis of 8.1 and 8.5 mumol free fatty acids/mL was obtained after 1 and 4 days, respectively. With Novozym 435, the highest hydrolysis of 4.0 and 6.1 mumol free fatty acids/mL were found after 2 and 9 days, respectively.

No description available.

Plant-based proteins have gained popularity because of their high nutritional value and more sustainable alternative to animal-based proteins. Soybean and chickpea are two widely consumed plant-based proteins, whereas tempeh is a popular plant-based fermented whole food product that is rich in protein. With the increase in the development of plant-based food products, there is little research into how plant proteins affect gut microbiota characteristics and metabolites. Therefore, there is a need to understand the underlying mechanisms surrounding the consumption of these foods. The purpose of this study was to investigate the health benefits of soybean, chickpea, and their tempeh counterparts’ consumption as whole foods on the gut microbiota and metabolites. Our results showed that soybean tempeh significantly increased the abundance of beneficial probiotic bacteria such as Roseburia and Ruminiclostridium 5 in the gut microbiota of mice. Additionally, soybean tempeh and soybean significantly increased Muribaculaeceae abundance, known to increase SCFA production in the colon. Lachnospiraceae NKA136 was significantly increased in soybean tempeh, soybean, and chickpea groups which may allow these foods to be used as a way of probiotic restoration. Our results showed that all dietary supplementation groups had significantly altered metabolic profiles compared to the control group. The soybean tempeh group had higher levels of peroxide (vitamin B6), myoinositol, and tetrahydrobiopterin while the chickpea tempeh group had higher levels of metabolites such as 3 hydroxyanthranilic acid. The soybean group had higher levels of metabolites such as 3-hydroxytryptophan (Oxitriptan) whereas the chickpea protein group had higher levels of metabolites such as 3-hydroxyanthranilic acid and oxitriptan. In conclusion, our study suggests that different plant-based foods can have distinct effects on gut microbiota and metabolic profiles in mice. These findings may have implications for human health and warrant further investigation into the effects of plant protein consumption on human metabolism.

Absorption bands responding to changes in fat, protein, and lactose concentrations in milk were determined. The effects of milk fat variation and lipolysis on the infrared spectrum were studied. Absorbances from 1283 to 1100 cm-1 correlated with fat, protein, and lactose concentration and showed a low response to milk fat variation and lipolysis. A Fourier transform infrared spectrometer equipped with an attenuated total internal reflectance cell was calibrated using these absorption band s, partial least squares statistics, and milk samples from herds in Minnesota. When the fat, protein, and lactose concentrations in these samples were predicted, the standard deviations of difference (reference - infrared) were .22, .06, and .02% . When the fat, protein, and lactose concentrations in a separate set of samples from herds in California were predicted, the standard deviations of difference were 1.23, .10, and .07%. Substitution of a 15 μm pathlength transmission cell for the attenuated total internal reflectance cell changed the standard deviations of difference to .07, .11, and .06% in the calibration (Minnesota) samples and .09, .10, and .16% in the validation (California) samples. Infrared spectroscopy was used to measure whey powder in an adulterated sample of nonfat dry milk. Mixtures of nonfat dry milk containing whey powder at various concentrations were analyzed using absorption bands between 1400 and 1200 cm-1 in the infrared spectrum. There was a strong correlation (r > .99) between predicted and measured concentrations of whey powder in adulterated samples. Accuracy was not affected by processing conditions , source of nonfat dry milk, and origin of whey powder. A rapid method for detecting soybean oil in process cheese was developed. The infrared spectrum of each sample was collected using an accessory designed for analysis of solid samples. A linear relationship fit (= .98) when the ratio of absorbance at 2957 and 2852 cm-1 was plotted versus percent adulteration.

Whey permeate was anaerobically digested in laboratory scale upflow anaerobic sludge blanket reactors. Nine hydraulic retention times between 5 and 0.2 days were examined with a fixed influent concentration of 10.6 ± 0.2 g COD/L. Chemical oxygen demand removal efficiency ranged from 99.0 to 18.9% and maximum production rate of methane gas was 2.67 L/L/day at a hydraulic loading rate of 12.97 kg COD/m3/day. About 70% of the chemical oxygen demand removed was converted to methane. Both the nonlinear least square method with 95% confidence interval and linear regression were used to evaluate kinetic coefficients. The maximum substrate utilization rate, k, and half saturation coefficient, KL, were determined to be 1.269 ± 0.163 Kg COD/kg VSS/day and 1.000 ± 0.179 kg COD/kg VSS/day. The yield coefficient, Y, and biomass decay rate coefficient, Kd, were also determined to be 0.160 ± 0.012 kg VSS/kg COD and 0.027 ± 0.004 day-1, respectively.

Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq) / Ao longo dos anos e de inúmeras pesquisas na área biotecnológica os biossurfactantes começaram a ser sintetizados por uma gama de microrganismos que, atuando sobre uma fonte de carbono, e auxiliado por uma fonte de nitrogênio e minerais, produzem compostos com características superiores quando comparados aos surfactantes. O uso destes tem sido ampliado, porém o custo produtivo dos biossurfactantes ainda é alto quando comparado aos surfactantes sintéticos. Nesse sentido, pesquisas que contribuam para a melhoria do bioprocesso de produção dos biosurfactantes se fazem necessárias. Neste trabalho, as ferramentas quimiométricas de planejamento experimental multivariado foram de fundamental importância para acelerar e otimizar as condições experimentais. O planejamento Doehlert auxiliou na otimização do tipo do ácido e suas melhores condições experimentais para a hidrólise do melaço de cana-de-açúcar. Os resultados apontaram que o ácido sulfúrico 10% com um tempo de reação de 60 minutos proporciona um aumento na concentração de açúcares redutores. A etapa de clarificação do melaço hidrolisado (MH) foi otimizada através de planejamento fatorial 22. Os clareamentos utilizando subacetato de chumbo e ultrafiltração foram considerados na etapa de produção do biosurfactante. Ao longo do bioprocesso observou-se a formação crescente do biosurfactante e uma redução na quantidade de açúcares redutores disponíveis. Uma avaliação estatística mostrou diferença significativa no início do bioprocesso realizado a partir do MH clarificado com subacetato de chumbo e a partir do MH clarificado por ultrafiltração. Entretanto, a diferença não é estatisticamente significativa, no nível de 99%, ao final da bioreação. A PCA, aplicada com a fusão dos dados de espectroscopia NIR e resultados laboratoriais para biomassa, produto, proteína e açúcar redutor, ao longo da bioreação, permitiu explorar de forma mais objetiva o bioprocesso. Foi possível inferir sobre a formação de biosurfactante até o tempo de 108 horas de bioreação quando ocorre uma estabilização no bioprocesso. Além disso, até o tempo de 60 horas de bioreação as variáveis que apresentam maior influência no bioprocesso são o açúcar redutor e as absorções na região do NIR correspondente ao segundo overtone das ligações CH, CH2, CH3 e ArOH entre 1200 – 1400 nm e, ao primeiro overtone da ligação ArCH em torno de 1600 nm. Após 72 horas de bioprocesso as variáveis que mais influenciam a bioreação são a biomassa e o produto. Tais variáveis estão correlacionadas com as regiões do NIR entre 900 -1100 nm (absorção do terceiro overtone das ligações CH, CH2, CH3, ArOH, ROH, H2O, RNH2 e ArCH) e 1400 -1500 nm (absorções do segundo overtone das ligações CH, CH2, CH3, CONHR, ArOH, ROH, H2O, RNH2 e CONH2). O borbulhamento realizado em 3 etapas aumentou a concentração de produto e apresentou diferença estatística significativa, no nível de 95%, no teste de Tukey. / Over the years and numerous researches in the biotechnology area biosurfactants began to be synthesized by a range of microorganisms that, acting on a carbon source, and aided by a source of nitrogen and minerals, produce compounds with superior characteristics when compared to synthetic surfactants. Its uses have been expanded, but the production cost of biosurfactants is still high when compared to synthetic surfactants. In this sense, research that contributes to the improvement of bioprocess production of biosurfactants is necessary. In this work, the chemometric tools of multivariate experimental design were of fundamental importance to optimize the experimental conditions. The Doehlert experimental design contributed in the optimization of the acid type and its experimental conditions for the hydrolysis of sugarcane molasses. The results indicated that 10% sulfuric acid with a reaction time of 60 minutes provides an increase in the concentration of reducing sugars. The clarification step of hydrolyzed molasses (MH) was optimized through factorial design 22. Clarification using lead subacetate and ultrafiltration were considered in the biosurfactant production stage. Throughout the bioprocess the growing formation of the biosurfactant and a reduction in the amount of reducing sugars were observed. A statistical evaluation showed a significant difference at the beginning of the bioprocess made from MH clarified with lead subacetate and from MH clarified by ultrafiltration. However, the difference is not statistically significant, at the 99% level, at the end of the bioreaction. PCA, applied to data fusion of NIR spectroscopy and laboratory results for biomass, product, protein and reducing sugar, allowed to explore the bioprocess in a more objective way. It was possible to infer about the formation of biosurfactant until the time of 108 hours of bioreaction when a stabilization occurs in the bioprocess. Moreover, until 60 hours of bioreaction, the variables that have the significant influence on the bioprocess are the reducing sugar and absorptions in the NIR region corresponding to the second overtone of the CH, CH2, CH3, and ArOH between 1200-1400 nm, and to the first overtone of the ArCH around 1600 nm. After 72 hours of bioprocess, the variables that most influence bioreaction are biomass and the product. These variables are correlated with NIR regions between 900-1100 nm (absorption of the third overtone of CH, CH2, CH3, ArOH, ROH, H2O, RNH2 and ArCH) and 1400-1500 nm (absorptions of the second overtone of CH bonds CH2, CH3, CONHR, ArOH, ROH, H2O, RNH2 and CONH2). The bubbling performed in 3 steps increased the product concentration and presented a statistically significant difference, at the 95% level, in the Tukey test.

Submitted in fulfillment of the academic requirement for a degree in Doctor of Philosophy (Ph.D.) in Food Science and Technology, Durban University of Technology, Durban, South Africa, 2017. / There has been a global increase in fresh produce consumption, due to its attendant nutritional a nd health benefits. On the other hand, increase in the outbreak of diseases, accompanied with health and economic implications, have been traced to this deve lopment. A good number of pathogenic contaminants along the food chain have been identified as causative agent s with Listeria monocytogenes identified as one of such. Among other control strategies, the use of bacteriophage, was recommended as a palliative measure. Furthermore, the a ppli cation of artificial neural networks (ANN) in food safety remains an emerging concept in risk assessment study. Therefore, the aim of this research is to investigate the effect of bacteriophage or phage control and artificial neural network prediction in the inactivation of L. monocytogenes ATCC 7644 on fresh produce. Fresh-cut tomato and carrot were artificially inoculated with L. monocytogenes (108 CFU/ml) and subjected to antimicrobial treatment of Listex P100 bacteriophage (108 PFU/ml), sucrose monolaurate (SML at 100, 250 and 400 ppm), with chlorine (sodium hypochlorite at 200 ppm) used as control. Also, application of ANN to predict the risk effect of antimicrobial treatments of bacteriophage, sucrose monolaurate and chlorine was evaluated on the fresh-cut produce. Mathematical models were developed using a linear regression and sigmoid (hyperbolic and logistic) activation function-(120). Data sets were trained using Back propagation ANN, containing one hidden layer with four hidden neurons. Furthermore, carbon utilization profile of phage-treated L. monocytogenes using phenotypic micro array method was evaluated. In the first phase, susceptibility of L. monocytogenes subjected to certain stress-adapted conditions (acid,-adapted AA, chlorine-adapted CA, heat-adapted HA) and non-adapted-NA to phage treatment inoculated on the fresh-cut produce stored for 10 days at 4, 10 and 25oC was evaluated. The second phase investigated the combination of bacteriophage and sucrose monolaurate (using chlorine at 200 ppm as control) to inhibit the L. monocytogenes growth on the fresh-cut produce stored for 6 days at 4, 10 and 25oC. Physicochemical properties (pH, titratable acid-TTA, total soluble solids-TSS, and colour values-CIE L* a* b*) of the fresh produce after treatment were evaluated. In the third phase, ANN as a predictive tool was used to evaluate the risk involved in the relationship among the initial bacterial load, fresh-produce type, antimicrobial concentration and residual bacteria. In the final phase, 100 µL of phage-treated L. monocytogenes was introduced into a 96-micro well plate impregnated with a tetrazolium dye. The Carbon utilization profile was evaluated at intervals of 4 hours for 48 hours using a biolog micro station. Generally, L. monocytogenes grew on both fresh-cut produce and the storage temperature did not adversely affect the lytic ability of the phage treatment. Antimicrobial treatment of phage and sucrose monolaurate had minimal variations on the physicochemical properties of both fresh-cut samples. All stress-adapted and non-adapted L. monocytogenes were (p ≤ 0.05) susceptible to bacteriophage control. Phage treatment reduced non-adapted, acid adapted, chlorine-adapted, and heat-adapted L. monocytogenes population by 0.57, 0.81, 0.86 and 0.95 log CFU/ml in fresh-cut tomato, and 2.26, 2.41, 2.49 and 2.54 log CFU/ml in fresh cut carrot respectively. Furthermore, the additive effect of SML at 100 and 250 ppm had no significant effect on phage lysis. However, combination of phage with SML at 400 ppm significantly (p ≤ 0.05) resulted in 1 and 3 fold reductions in tomato and carrot respectively. Control treatment with chlorine resulted in 1-2 log reductions on both fresh produce. Algorithm data set trained using ANN gave 100% accuracy. Prediction with logistic activation function showed the highest positive correlation relationship between predicted and observed values with ~ 0.99 R2-value and MSE of 0.0831. Carbon utilization profile showed hexose and pentose sugars-ribose, glucose, fructose and sugars were maximally utilized while oligosaccharide sugars of sucrose, cellobiose and gentiobiose were similarly observed to be utilized. Notably, utilization of glucose-6-phosphate which determines L. monocytogenes pathogenicity was not very pronounced in the carbon profile. Bacteriophage application in the inactivation of L. monocytogenes contamination of fresh produce provides a safe means of control. Its perceived limitation however, can be overcome by combining with other antimicrobials. Similarly, the use of artificial neural networks prediction, remains an improved approach to harness the potential risk that could occur through this method. / D

Hierarchical systems that integrate nano- and macroscale structural elements can offer enhanced stability over traditional immobilization methods. Microparticles were synthesized using interfacial assembly of lipase with (CLMP-N) and without (CLMP) nanoparticles into a crosslinked polymeric core, to determine the impact of the highly ordered system on lipase stability in extreme environments. Kinetic analysis revealed the macrostructure significantly increases the turnover rate (kcat) following immobilization. The macrostructure also stabilized lipase at neutral and basic pH values, while the nanoparticles influenced stability under acidic pH conditions. A greener solvent, choline chloride and urea, was applied to produce sugar ester surfactants. Microparticles exhibited decreases in the turnover rate (kcat) and catalytic efficiency (kcat/Km) following exposure, but retained over 60% and 20% activity after exposure at 50 ºC and 60 ºC, respectively. CLMP and CLMP-N outperformed the commercially available lipase per unit protein in the production of sugar esters. The utilization of greener solvent systems with hierarchical immobilized enzyme systems has the potential to improve processing efficiency and sustainability for the production of value-added agricultural products.

In addition to proving an excellent source of nutrients, eggs are used in the food, cosmetic, and biotechnology industries for their rheological and bioactive properties. Much of the potential for the added value is in individual components of the egg, rather than the whole egg. At low speed centrifugation, yolk separates into two distinct fractions—granules and plasma. It is becoming increasingly popular in the industry to remove the plasma fraction of the egg yolk to use for its livetins, particularly immunoglobulin Y, leaving behind a granule by-product (“yellow cake”). Previous research has shown potential added-value from the granule fraction, especially from its phosvitin and phospholipids. Granules are protein aggregates with complexes of phosvitin and high density lipoproteins linked by phosphocalcic bridges. In their native form, the proteins are mostly insoluble, however previous studies have shown the links can be broken by alterations in pH, ionic strength, and mechanical treatments. This thesis project seeks to find potential uses for the egg yolk by product after the removal of the livetin fraction by means of further fractionation with mechanical treatment (filtration). Two variables were tested to extract more proteins from the yellow cake. Salt was added to 10% solids solution of yellow cake in water before filtration at four different NaCl levels: 0%, .05%, 1%, and 2.5%. Additionally pH was tested at four different levels: 4.6, 4.8, 5.0, 5.2. The samples were also tested for antibacterial properties against Escherichia v coli with a minimum inhibitory concentration assay (MIC). Analysis with BCA showed salt concentration had a significant effect on the yield of protein. The highest concentration of salt tested, 2.5%, had the highest protein yield. Additionally, SDS PAGE showed 2.5% salt had the most unique protein bands. This could be to the disruption of the phosphocalcic links between the phosvitin and HDL by NaCl, allowing the protein to solubilize. pH did not have a significant effect on the yield or types of proteins in the range tested in this experiment. There is no conclusive evidence of antibacterial properties against E. coli from the protein extract. The MIC assay had growth show up in all wells with the protein extract, however there was a visible decrease in turbidity with higher concentration of the protein extract. This could mean that the protein extract does have some antibacterial properties, but needs testing at higher concentrations or with isolated proteins/peptides. The SDS-PAGE revealed bands showing phosvitin present, which has known antibacterial properties. Overall, improvements to the methods for further protein extraction from egg yolk by-products will help lead the industry to finding novel uses and product applications.