Plants are used to produce many secondary metabolites that are too difficult, expensive or impossible to make by chemical synthesis. Conventional cultivation of plants is of course subject to vagaries of weather, pests and availability of land; hence, the interest in highly controlled culture of plant cells and hairy roots in bioreactors as methods of producing various products. This project focussed on production of blue and red colors of Lavandula augustifolia and Beta vulgaris, respectively. Callus and suspension cell culture were successfully produced from L. augustifolia after extensive trials, but hairy roots could not be generated from this species. In contrast, a successful protocol was developed for consistently producing hairy roots from B. vulgaris, but calli could not be generated from this species. Effects of medium composition on growth of L. augustifolia calli and freely suspended cells and production of the blue pigment by the latter, were investigated. Optimal production of callus occurred in full-strength Murashige and Skoog (MS) medium supplemented with 2 mg/l of indole-3-acetic acid (IAA) and 1 mg/l of kinetin. Stable suspension cultures could be produced and maintained in full-strength MS medium supplemented with 1 mg/l each of IAA and kinetin. In suspension culture in full-strength MS medium, the following hormone combinations were tested: (1) 1 mg/l each of indole-3-acetic acid (IAA) and kinetin; (2) 2 mg/l of IAA and 1 mg/l of kinetin; (3) 2 mg/l of IAA and 1 mg/l of benzyl amino purine (BAP); and (4) 2 mg/l each of IAA and BAP. Combination (3) maximized cell growth, but the highest cell-specific production of the blue pigment was seen in combination (2), although pigment production occurred at all hormone combinations. The medium formulation that gave the best production of the pigment in shake flasks was scaled up to a 2 L aerated stirred tank bioreactor, but both the biomass and pigment productivities were reduced in the bioreactor apparently due to the high shear stress generated by the Rushton turbine impeller. Compared to suspension cultures of L. augustifolia, the hairy root cultures of B. vulgaris grew extremely rapidly. Hairy roots also produced large amounts of the red pigments. Growth of hairy roots was influenced by the composition of the medium. Although the full strength MS medium better promoted biomass growth compared to the half-strength MS medium, the final concentration of the biomass and the pigment were nearly the same in both media. Attempts were made to enhance production by using various hormones (i.e. naphthalene acetic acid, BAP, IAA added individually at a concentration of 0.5 mg/l), but none of the hormones proved useful. BAP adversely affected the growth of hairy roots. In summary, production of pigments by suspension culture of L. augustifolia and hairy root culture of B. vulgaris, is technically possible, but requires substantial further optimization for enhancing productivity than has been possible in this project. iii

The present study was aimed at extending our knowledge of the antioxidative properties of the milk protein products, whey protein isolate (WPI) and sodium caseinate (NaCas), in oil-in-water (O/W) emulsions rich in polyunsaturated fatty acids (PUFAs). In particular, the objective was to contribute to our understanding of the compositional and processing factors that influence the oxidative stability of protein-stabilised O/W emulsions. Linoleic acid (approximately 60 %) was used as the lipid for the oil phase (10.6 %). The emulsion samples were usually incubated at 50 °C to accelerate lipid oxidation. Lipid oxidation indicators were lipid hydroperoxides and headspace hexanal, determined by solid phase microextraction (SPME) combined with gas chromatography (GC). WPI- or NaCas-stabilised emulsions were prepared using a wide range of protein concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 7.0 or 10.0 %) at two droplet sizes (d32 = 0.31 and 0.65 µm). In general, higher lipid oxidation levels were found for the larger droplet size. Increasing protein concentration led to a decrease in the lipid oxidation rate. The greatest decrease in lipid hydroperoxide levels (values after 4 h) occurred at up to 4.0 % protein concentration. The greatest decrease in hexanal levels (values after 24 h) occurred at up to 4.0 % protein concentration in WPI emulsions (0.31 µm). The hexanal levels were more independent of the protein concentration in the other emulsion types. The hexanal level decreased at protein concentrations > 4.0 % in NaCas emulsions (0.31 and 0.65 µm) and at protein concentrations > 7.0 % in WPI emulsions (0.65 µm). The difference between lipid hydroperoxide generation in emulsions with small and large droplet sizes decreased with increasing protein concentration. This effect was more pronounced in NaCas emulsions. In general, NaCas was a better inhibitor of lipid oxidation than WPI, but WPI appeared to be the better antioxidant at some droplet size/protein concentration combinations. The protein in the continuous phase, i.e. the unadsorbed protein, played an important role in lipid oxidation. In principal, the lipid hydroperoxide and hexanal levels showed the same development over the continuous phase protein concentration as over the protein concentration in WPI and NaCas emulsions (d32 = 0.31 µm). A low NaCas level in the continuous phase already led to a relatively low hexanal level, whereas a higher WPI level was required. When NaCas solution was added to a WPI emulsion or WPI solution was added to a NaCas emulsion, a synergistic antioxidative effect was observed. The high molecular weight fractions (molecular weight = 12000-14000) of WPI and NaCas contained pro-oxidative metal ions that contributed to lipid oxidation in the emulsions. An enrichment of NaCas emulsions with the low molecular weight fraction of NaCas (with a molecular weight = 12000-14000) notably inhibited lipid oxidation. An enrichment of WPI emulsions with the low molecular weight fraction of WPI (with a molecular weight = 12000-14000) also seemed to inhibit lipid oxidation, but the effect was not significant. The protein solutions were enriched with these fractions before emulsion preparation. Pure WPI solution or mixed WPI/NaCas (1:1, weight/weight) solution with 1.12 or 2.24 % protein concentration was heated at 84 °C for up to 40 min, cooled and then used to prepare emulsions. Lipid oxidation was generally not affected by the heat treatment or the degree of whey protein denaturation. However, at the lower WPI concentration, more hexanal was produced for the longer heating times (20, 30 and 40 min) and this appeared to be connected with the physical instability of the emulsions. Greater oxidative stability was found at the higher protein concentration and when the proteins were mixed, pointing to a possible synergistic antioxidative effect of WPI and NaCas. The addition of the free radical source 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) greatly increased the oxygen uptake and the generation of lipid hydroperoxides in the emulsions. The oxidative stability increased with increasing protein concentration (1.0, 4.0 and 7.0 %). NaCas had a greater antioxidative effect than WPI. The inhibition of oxygen uptake appeared to be largely influenced by the free-radical-scavenging activity of the system, determined by the protein type and the protein concentration, as the radicals were produced linearly over time and oxygen was consumed linearly over time. It can therefore be concluded that free-radical-scavenging activity represents a major antioxidative mechanism of the milk proteins. Oxygen was consumed much faster in emulsions than in protein solutions when the same level of AAPH was incorporated. In a WPI (1.0 % protein) emulsion, much lower levels of protein hydroperoxides than of lipid hydroperoxides developed. This pointed to a much greater reactivity of linoleic acid than of the milk proteins with oxygen. In contrast, the exposure of WPI to oxidising linoleic acid in an emulsion (1.0 % protein) or to AAPH in aqueous solution led to oxidative damage of the whey proteins, indicated by the loss of amino acids. The loss of specific amino acids was different for proteins in the continuous phase or cream phase of an emulsion or in WPI solution. The present study confirms the antioxidative potential of WPI and NaCas and gives new insights into their functionality as oxidative stabilisers in O/W emulsions.

Reducing the production cost of industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to a complete lack of genetic tools and techniques available for the manipulation of this promising bacterium. This thesis encompasses the development of fundamental genetic tools and techniques that will permit extensive genetic and metabolic engineering of C. pasteurianum. We initiated our genetic work with the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency up to 7.5 × 104 transformants µg-1 DNA, an increase of approximately three orders of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection. Following development of a gene transfer methodology, we next aimed to sequence the entire genome of C. pasteurianum. Using a hybrid approach involving 454 pyrosequencing, Illumina dye sequencing, and single molecule real-time sequencing platforms, we obtained a near-complete genome sequence comprised of 12 contigs, 4,420,100 bp, and 4,056 candidate protein-coding genes with a GC content of 30.0%. No extrachromosomal elements were detected. We provide an overview of the genes and pathways involved in the organism’s central fermentative metabolism. We used our developed electrotransformation procedure to investigate the use of established clostridial group II intron biology for constructing chromosomal gene knockout mutants of C. pasteurianum. Through methylome analysis of C. pasteurianum genome sequencing data and transformation assays of various vector deletion constructs, we identified a new Type I restriction-modification system that inhibits transfer of vectors harboring group II intron gene knockout machinery. We designated the new restriction system CpaAII and proposed a recognition sequence of 5’-AAGNNNNNCTCC-3’. Overcoming restriction by CpaAII, in addition to low intron retrohoming efficiency, allowed the isolation of a gene knockout mutant of C. pasteurianum with a disrupted CpaAI Type II restriction system. The resulting mutant strain should be efficienty transformed with plasmid DNA lacking M.FnuDII methylation. Lastly, we investigated the use of plasmid-based gene overexpression and chromosomal gene downregulation to alter gene expression in C. pasteurianum. Using a β-galactosidase reporter gene, we characterized promoters corresponding to the ferredoxin and thiolase genes of C. pasteurianum and show that both promoters permitted high-level, constitutive gene expression. The thiolase promoter was then utilized to drive transcription of an antisense RNA molecule possessing complementarity to mRNA of our β-galactosidase reporter gene. Our antisense RNA system demonstrated 52-58% downregulation of plasmid encoded β-galactosidase activity throughout the duration of growth. In an attempt to perturb the central fermentative metabolism of C. pasteurianum and enhance butanol titers, we prepared several antisense RNA constructs for downregulation of 1,3-propanediol, butyrate, and hydrogen production pathways. The resulting downregulation strains are expected to exhibit drastically altered central fermentative metabolism and product distribution. Taken together, we have demonstrated that C. pasteurianum is amendable to genetic manipulation through the development of methods for plasmid DNA transfer and gene overexpression, knockdown, and knockout. Further, our genome sequence should provide valuable nucleotide sequence information for the application of our genetic tools. Thus, the genome sequence, electrotransformation method, and associated genetic tools and techniques reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.

Plants are used to produce many secondary metabolites that are too difficult, expensive or impossible to make by chemical synthesis. Conventional cultivation of plants is of course subject to vagaries of weather, pests and availability of land; hence, the interest in highly controlled culture of plant cells and hairy roots in bioreactors as methods of producing various products. This project focussed on production of blue and red colors of Lavandula augustifolia and Beta vulgaris, respectively. Callus and suspension cell culture were successfully produced from L. augustifolia after extensive trials, but hairy roots could not be generated from this species. In contrast, a successful protocol was developed for consistently producing hairy roots from B. vulgaris, but calli could not be generated from this species. Effects of medium composition on growth of L. augustifolia calli and freely suspended cells and production of the blue pigment by the latter, were investigated. Optimal production of callus occurred in full-strength Murashige and Skoog (MS) medium supplemented with 2 mg/l of indole-3-acetic acid (IAA) and 1 mg/l of kinetin. Stable suspension cultures could be produced and maintained in full-strength MS medium supplemented with 1 mg/l each of IAA and kinetin. In suspension culture in full-strength MS medium, the following hormone combinations were tested: (1) 1 mg/l each of indole-3-acetic acid (IAA) and kinetin; (2) 2 mg/l of IAA and 1 mg/l of kinetin; (3) 2 mg/l of IAA and 1 mg/l of benzyl amino purine (BAP); and (4) 2 mg/l each of IAA and BAP. Combination (3) maximized cell growth, but the highest cell-specific production of the blue pigment was seen in combination (2), although pigment production occurred at all hormone combinations. The medium formulation that gave the best production of the pigment in shake flasks was scaled up to a 2 L aerated stirred tank bioreactor, but both the biomass and pigment productivities were reduced in the bioreactor apparently due to the high shear stress generated by the Rushton turbine impeller. Compared to suspension cultures of L. augustifolia, the hairy root cultures of B. vulgaris grew extremely rapidly. Hairy roots also produced large amounts of the red pigments. Growth of hairy roots was influenced by the composition of the medium. Although the full strength MS medium better promoted biomass growth compared to the half-strength MS medium, the final concentration of the biomass and the pigment were nearly the same in both media. Attempts were made to enhance production by using various hormones (i.e. naphthalene acetic acid, BAP, IAA added individually at a concentration of 0.5 mg/l), but none of the hormones proved useful. BAP adversely affected the growth of hairy roots. In summary, production of pigments by suspension culture of L. augustifolia and hairy root culture of B. vulgaris, is technically possible, but requires substantial further optimization for enhancing productivity than has been possible in this project. iii

This study investigated pressure-induced starch gelatinisation in water and milk suspensions. A rheological method, termed ‘pasting curves’, provided an objective and analytical means to determine the degree of pressure-induced starch gelatinisation. In addition, a polarised light microscope was used to observe birefringence of the starch granules and the degree of starch swelling was measured. The preliminary investigation into pressure-induced gelatinisation of six different starches showed that potato starch was the most pressure resistant and was not gelatinised after a pressure treatment of 600 MPa for 30 min at 20 °C. Waxy rice, waxy corn and tapioca starches showed complete gelatinisation after the same treatment while normal rice and normal corn starches were only partially gelatinised despite the disappearance of birefringence. Based on the preliminary study, two starches (normal and waxy rice starches) were selected for more detailed studies. The effects of treatment conditions (pressure, temperature and duration) on the gelatinisation were investigated with these selected starches. The degree of gelatinisation was dependent on the type of starch and the treatment conditions. The results also indicated that different combinations of the treatment conditions (e.g. high treatment pressure for a short time and low treatment pressure for a longer time) could result in the same degree of gelatinisation. Both starch types exhibited sigmoidal-shaped pressure-induced gelatinisation curves and there was a linear correlation between the degree of swelling and the apparent viscosity of the starch suspension. After treatments at =500 MPa for 30 min at 20 °C, both starches lost all birefringence although the apparent viscosity and the degree of swelling of normal rice starch did not increase to the same extent as observed in waxy rice starch. Pressure-induced gelatinisation of starch was retarded when starch was suspended in skim milk. This was attributed to the effect of soluble milk minerals and lactose present in the milk whereas milk proteins (casein and whey) did not affect the degree of gelatinisation at the levels present in 10% total solids skim milk. The presence of soluble milk and/or lactose may lead to less effective plasticising of starch chains by the suspension medium. Interactions between milk components and starch molecules may also play a role in retarding gelatinisation by reducing the mobility of starch chains. The functionality of starch in a dairy application was tested using acid milk gels as a model system. Skim milk with added starch (waxy rice or potato starch) was either pressure treated (500 MPa, 20°C, 30 min) or heat treated (80°C, 30 min) and subsequently acidified to form acid milk gels. The addition of waxy rice starch resulted in firmer acid milk gels, and increasing the amount of starch caused an increase in the firmness of both pressure-treated and heat-treated samples. However, pressure-treated samples with added potato starch did not show significant changes in the firmness whereas the heat-treated counterparts showed a marked increase in the firmness as the level of potato starch increased. The difference between the effects of the two different starches can be explained by the extent of starch gelatinisation in skim milk. Starch granules absorb water during gelatinisation whether induced by pressure or heat which effectively increases milk protein concentration in the aqueous phase to form a denser protein gel network on acidification. The firmness of acid milk gels can be increased by adjusting the pH at pressure or heat treatment to higher than the natural pH of milk. The effect of pH at pressure or heat treatment and addition of starch on the acid milk gel firmness was additive and independent of each other up to a starch addition level of 1%. This study provided an insight into pressure-induced gelatinisation of starch by showing gelatinisation properties of starches of different botanical origins and the effects of the treatment conditions (treatment pressure, treatment temperature and duration) on the degree of gelatinisation. Furthermore, the results from the pressure treatments of starch in dairy-based suspensions showed that pressure-induced gelatinisation was affected by other components in the system. These results demonstrate the importance of understanding the gelatinisation properties of starch in complicated food systems in which a number of other components are present. In terms of the application of starch in dairy systems, when starch was added to milk and gelatinised by pressure treatment, the acid milk gel produced by subsequent acidification was firmer than the acid milk gel made from skim milk alone.

The present study was aimed at extending our knowledge of the antioxidative properties of the milk protein products, whey protein isolate (WPI) and sodium caseinate (NaCas), in oil-in-water (O/W) emulsions rich in polyunsaturated fatty acids (PUFAs). In particular, the objective was to contribute to our understanding of the compositional and processing factors that influence the oxidative stability of protein-stabilised O/W emulsions. Linoleic acid (approximately 60 %) was used as the lipid for the oil phase (10.6 %). The emulsion samples were usually incubated at 50 °C to accelerate lipid oxidation. Lipid oxidation indicators were lipid hydroperoxides and headspace hexanal, determined by solid phase microextraction (SPME) combined with gas chromatography (GC). WPI- or NaCas-stabilised emulsions were prepared using a wide range of protein concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 7.0 or 10.0 %) at two droplet sizes (d32 = 0.31 and 0.65 µm). In general, higher lipid oxidation levels were found for the larger droplet size. Increasing protein concentration led to a decrease in the lipid oxidation rate. The greatest decrease in lipid hydroperoxide levels (values after 4 h) occurred at up to 4.0 % protein concentration. The greatest decrease in hexanal levels (values after 24 h) occurred at up to 4.0 % protein concentration in WPI emulsions (0.31 µm). The hexanal levels were more independent of the protein concentration in the other emulsion types. The hexanal level decreased at protein concentrations > 4.0 % in NaCas emulsions (0.31 and 0.65 µm) and at protein concentrations > 7.0 % in WPI emulsions (0.65 µm). The difference between lipid hydroperoxide generation in emulsions with small and large droplet sizes decreased with increasing protein concentration. This effect was more pronounced in NaCas emulsions. In general, NaCas was a better inhibitor of lipid oxidation than WPI, but WPI appeared to be the better antioxidant at some droplet size/protein concentration combinations. The protein in the continuous phase, i.e. the unadsorbed protein, played an important role in lipid oxidation. In principal, the lipid hydroperoxide and hexanal levels showed the same development over the continuous phase protein concentration as over the protein concentration in WPI and NaCas emulsions (d32 = 0.31 µm). A low NaCas level in the continuous phase already led to a relatively low hexanal level, whereas a higher WPI level was required. When NaCas solution was added to a WPI emulsion or WPI solution was added to a NaCas emulsion, a synergistic antioxidative effect was observed. The high molecular weight fractions (molecular weight = 12000-14000) of WPI and NaCas contained pro-oxidative metal ions that contributed to lipid oxidation in the emulsions. An enrichment of NaCas emulsions with the low molecular weight fraction of NaCas (with a molecular weight = 12000-14000) notably inhibited lipid oxidation. An enrichment of WPI emulsions with the low molecular weight fraction of WPI (with a molecular weight = 12000-14000) also seemed to inhibit lipid oxidation, but the effect was not significant. The protein solutions were enriched with these fractions before emulsion preparation. Pure WPI solution or mixed WPI/NaCas (1:1, weight/weight) solution with 1.12 or 2.24 % protein concentration was heated at 84 °C for up to 40 min, cooled and then used to prepare emulsions. Lipid oxidation was generally not affected by the heat treatment or the degree of whey protein denaturation. However, at the lower WPI concentration, more hexanal was produced for the longer heating times (20, 30 and 40 min) and this appeared to be connected with the physical instability of the emulsions. Greater oxidative stability was found at the higher protein concentration and when the proteins were mixed, pointing to a possible synergistic antioxidative effect of WPI and NaCas. The addition of the free radical source 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) greatly increased the oxygen uptake and the generation of lipid hydroperoxides in the emulsions. The oxidative stability increased with increasing protein concentration (1.0, 4.0 and 7.0 %). NaCas had a greater antioxidative effect than WPI. The inhibition of oxygen uptake appeared to be largely influenced by the free-radical-scavenging activity of the system, determined by the protein type and the protein concentration, as the radicals were produced linearly over time and oxygen was consumed linearly over time. It can therefore be concluded that free-radical-scavenging activity represents a major antioxidative mechanism of the milk proteins. Oxygen was consumed much faster in emulsions than in protein solutions when the same level of AAPH was incorporated. In a WPI (1.0 % protein) emulsion, much lower levels of protein hydroperoxides than of lipid hydroperoxides developed. This pointed to a much greater reactivity of linoleic acid than of the milk proteins with oxygen. In contrast, the exposure of WPI to oxidising linoleic acid in an emulsion (1.0 % protein) or to AAPH in aqueous solution led to oxidative damage of the whey proteins, indicated by the loss of amino acids. The loss of specific amino acids was different for proteins in the continuous phase or cream phase of an emulsion or in WPI solution. The present study confirms the antioxidative potential of WPI and NaCas and gives new insights into their functionality as oxidative stabilisers in O/W emulsions.

The present study was aimed at extending our knowledge of the antioxidative properties of the milk protein products, whey protein isolate (WPI) and sodium caseinate (NaCas), in oil-in-water (O/W) emulsions rich in polyunsaturated fatty acids (PUFAs). In particular, the objective was to contribute to our understanding of the compositional and processing factors that influence the oxidative stability of protein-stabilised O/W emulsions. Linoleic acid (approximately 60 %) was used as the lipid for the oil phase (10.6 %). The emulsion samples were usually incubated at 50 °C to accelerate lipid oxidation. Lipid oxidation indicators were lipid hydroperoxides and headspace hexanal, determined by solid phase microextraction (SPME) combined with gas chromatography (GC). WPI- or NaCas-stabilised emulsions were prepared using a wide range of protein concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 7.0 or 10.0 %) at two droplet sizes (d32 = 0.31 and 0.65 µm). In general, higher lipid oxidation levels were found for the larger droplet size. Increasing protein concentration led to a decrease in the lipid oxidation rate. The greatest decrease in lipid hydroperoxide levels (values after 4 h) occurred at up to 4.0 % protein concentration. The greatest decrease in hexanal levels (values after 24 h) occurred at up to 4.0 % protein concentration in WPI emulsions (0.31 µm). The hexanal levels were more independent of the protein concentration in the other emulsion types. The hexanal level decreased at protein concentrations > 4.0 % in NaCas emulsions (0.31 and 0.65 µm) and at protein concentrations > 7.0 % in WPI emulsions (0.65 µm). The difference between lipid hydroperoxide generation in emulsions with small and large droplet sizes decreased with increasing protein concentration. This effect was more pronounced in NaCas emulsions. In general, NaCas was a better inhibitor of lipid oxidation than WPI, but WPI appeared to be the better antioxidant at some droplet size/protein concentration combinations. The protein in the continuous phase, i.e. the unadsorbed protein, played an important role in lipid oxidation. In principal, the lipid hydroperoxide and hexanal levels showed the same development over the continuous phase protein concentration as over the protein concentration in WPI and NaCas emulsions (d32 = 0.31 µm). A low NaCas level in the continuous phase already led to a relatively low hexanal level, whereas a higher WPI level was required. When NaCas solution was added to a WPI emulsion or WPI solution was added to a NaCas emulsion, a synergistic antioxidative effect was observed. The high molecular weight fractions (molecular weight = 12000-14000) of WPI and NaCas contained pro-oxidative metal ions that contributed to lipid oxidation in the emulsions. An enrichment of NaCas emulsions with the low molecular weight fraction of NaCas (with a molecular weight = 12000-14000) notably inhibited lipid oxidation. An enrichment of WPI emulsions with the low molecular weight fraction of WPI (with a molecular weight = 12000-14000) also seemed to inhibit lipid oxidation, but the effect was not significant. The protein solutions were enriched with these fractions before emulsion preparation. Pure WPI solution or mixed WPI/NaCas (1:1, weight/weight) solution with 1.12 or 2.24 % protein concentration was heated at 84 °C for up to 40 min, cooled and then used to prepare emulsions. Lipid oxidation was generally not affected by the heat treatment or the degree of whey protein denaturation. However, at the lower WPI concentration, more hexanal was produced for the longer heating times (20, 30 and 40 min) and this appeared to be connected with the physical instability of the emulsions. Greater oxidative stability was found at the higher protein concentration and when the proteins were mixed, pointing to a possible synergistic antioxidative effect of WPI and NaCas. The addition of the free radical source 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) greatly increased the oxygen uptake and the generation of lipid hydroperoxides in the emulsions. The oxidative stability increased with increasing protein concentration (1.0, 4.0 and 7.0 %). NaCas had a greater antioxidative effect than WPI. The inhibition of oxygen uptake appeared to be largely influenced by the free-radical-scavenging activity of the system, determined by the protein type and the protein concentration, as the radicals were produced linearly over time and oxygen was consumed linearly over time. It can therefore be concluded that free-radical-scavenging activity represents a major antioxidative mechanism of the milk proteins. Oxygen was consumed much faster in emulsions than in protein solutions when the same level of AAPH was incorporated. In a WPI (1.0 % protein) emulsion, much lower levels of protein hydroperoxides than of lipid hydroperoxides developed. This pointed to a much greater reactivity of linoleic acid than of the milk proteins with oxygen. In contrast, the exposure of WPI to oxidising linoleic acid in an emulsion (1.0 % protein) or to AAPH in aqueous solution led to oxidative damage of the whey proteins, indicated by the loss of amino acids. The loss of specific amino acids was different for proteins in the continuous phase or cream phase of an emulsion or in WPI solution. The present study confirms the antioxidative potential of WPI and NaCas and gives new insights into their functionality as oxidative stabilisers in O/W emulsions.

The present study was aimed at extending our knowledge of the antioxidative properties of the milk protein products, whey protein isolate (WPI) and sodium caseinate (NaCas), in oil-in-water (O/W) emulsions rich in polyunsaturated fatty acids (PUFAs). In particular, the objective was to contribute to our understanding of the compositional and processing factors that influence the oxidative stability of protein-stabilised O/W emulsions. Linoleic acid (approximately 60 %) was used as the lipid for the oil phase (10.6 %). The emulsion samples were usually incubated at 50 °C to accelerate lipid oxidation. Lipid oxidation indicators were lipid hydroperoxides and headspace hexanal, determined by solid phase microextraction (SPME) combined with gas chromatography (GC). WPI- or NaCas-stabilised emulsions were prepared using a wide range of protein concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 7.0 or 10.0 %) at two droplet sizes (d32 = 0.31 and 0.65 µm). In general, higher lipid oxidation levels were found for the larger droplet size. Increasing protein concentration led to a decrease in the lipid oxidation rate. The greatest decrease in lipid hydroperoxide levels (values after 4 h) occurred at up to 4.0 % protein concentration. The greatest decrease in hexanal levels (values after 24 h) occurred at up to 4.0 % protein concentration in WPI emulsions (0.31 µm). The hexanal levels were more independent of the protein concentration in the other emulsion types. The hexanal level decreased at protein concentrations > 4.0 % in NaCas emulsions (0.31 and 0.65 µm) and at protein concentrations > 7.0 % in WPI emulsions (0.65 µm). The difference between lipid hydroperoxide generation in emulsions with small and large droplet sizes decreased with increasing protein concentration. This effect was more pronounced in NaCas emulsions. In general, NaCas was a better inhibitor of lipid oxidation than WPI, but WPI appeared to be the better antioxidant at some droplet size/protein concentration combinations. The protein in the continuous phase, i.e. the unadsorbed protein, played an important role in lipid oxidation. In principal, the lipid hydroperoxide and hexanal levels showed the same development over the continuous phase protein concentration as over the protein concentration in WPI and NaCas emulsions (d32 = 0.31 µm). A low NaCas level in the continuous phase already led to a relatively low hexanal level, whereas a higher WPI level was required. When NaCas solution was added to a WPI emulsion or WPI solution was added to a NaCas emulsion, a synergistic antioxidative effect was observed. The high molecular weight fractions (molecular weight = 12000-14000) of WPI and NaCas contained pro-oxidative metal ions that contributed to lipid oxidation in the emulsions. An enrichment of NaCas emulsions with the low molecular weight fraction of NaCas (with a molecular weight = 12000-14000) notably inhibited lipid oxidation. An enrichment of WPI emulsions with the low molecular weight fraction of WPI (with a molecular weight = 12000-14000) also seemed to inhibit lipid oxidation, but the effect was not significant. The protein solutions were enriched with these fractions before emulsion preparation. Pure WPI solution or mixed WPI/NaCas (1:1, weight/weight) solution with 1.12 or 2.24 % protein concentration was heated at 84 °C for up to 40 min, cooled and then used to prepare emulsions. Lipid oxidation was generally not affected by the heat treatment or the degree of whey protein denaturation. However, at the lower WPI concentration, more hexanal was produced for the longer heating times (20, 30 and 40 min) and this appeared to be connected with the physical instability of the emulsions. Greater oxidative stability was found at the higher protein concentration and when the proteins were mixed, pointing to a possible synergistic antioxidative effect of WPI and NaCas. The addition of the free radical source 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) greatly increased the oxygen uptake and the generation of lipid hydroperoxides in the emulsions. The oxidative stability increased with increasing protein concentration (1.0, 4.0 and 7.0 %). NaCas had a greater antioxidative effect than WPI. The inhibition of oxygen uptake appeared to be largely influenced by the free-radical-scavenging activity of the system, determined by the protein type and the protein concentration, as the radicals were produced linearly over time and oxygen was consumed linearly over time. It can therefore be concluded that free-radical-scavenging activity represents a major antioxidative mechanism of the milk proteins. Oxygen was consumed much faster in emulsions than in protein solutions when the same level of AAPH was incorporated. In a WPI (1.0 % protein) emulsion, much lower levels of protein hydroperoxides than of lipid hydroperoxides developed. This pointed to a much greater reactivity of linoleic acid than of the milk proteins with oxygen. In contrast, the exposure of WPI to oxidising linoleic acid in an emulsion (1.0 % protein) or to AAPH in aqueous solution led to oxidative damage of the whey proteins, indicated by the loss of amino acids. The loss of specific amino acids was different for proteins in the continuous phase or cream phase of an emulsion or in WPI solution. The present study confirms the antioxidative potential of WPI and NaCas and gives new insights into their functionality as oxidative stabilisers in O/W emulsions.

Richmond Ltd. has recently undergone a change in strategy, away from the traditional commodity based meat industry, towards the modern food business. To do this, opportunities to add value to their current product range must be identified. This involves the conversion of traditionally low value commodity based products into products that demand a premium. An example of this is converting muscles that are currently used for grinding meat into a further processed convenience food (i.e. ready meals). Another method is to add further value to premium products by making them more appealing to consumers (i.e. nutritional information on labels). This work details investigations into the functional properties of selected beef forequarter muscles (low value commodity products) and the nutritional properties of selected export lamb products (premium products). The functional properties of a number of beef forequarter muscles were measured to identify which had the best potential for further processing applications with respect to ready meals. The functional properties of tenderness, cook loss and shrinkage were measured for the Latissimus Dorsi, Pectorialis Profundus (Point End Brisket), Infraspinatus (Cross Cut Blade), Triceps Brachi Longhead (Main muscle in Bolar Shoulder Clod), Supraspinatus (Chuck Tender), Serratus Ventralis and Triceps Brachi Medialhead (Muscle in Bolar Shoulder Clod. From the tests conducted the Infraspinatus and the Triceps Brachi Longhead have been identified as having the best functional properties with respect to further processing for ready meal applications. As well as conducting tests to identify the forequarter muscles with the best potential for further processing applications, investigations were carried out to identify cooking regimes that would optimise the functional properties. This work confirmed that there are three major chemical reactions, which determine the resultant functional properties of cooked meat. They are the denaturation and aggregation of the myofibrillar proteins and the denaturation and solubilisation of connective tissue (collagen). At around 50°C myosin (45% to 50% of the myofibrillar proteins) denatures, which results in a substantial increase in cook loss and reduction in water holding capacity. At around 60°C collagen (main connective tissue protein) denatures, which results in a substantial increase in tenderness and increase in cook loss. This is because as the collagen denatures it loses it mechanical strength (increase in tenderness) and can no longer support its own structure, and causes it to contract. This contraction causes fluid within the meat and cook loss caused by the denaturation of myosin to be expelled from the meat by compressive forces (squeezed out). At around 70°C actomyosin (22% of the myofibrillar proteins) denatures. This results in a substantial increase in the cook loss and firming of the meat. The increase in cook loss or decrease in water holding capacity that occurs with myofibrillar protein denaturation is due to the fact that when these proteins denature and aggregate their ability to bind water is greatly reduced. From the results of the cooking regime trials it is recommended that for functional property considerations that during the cooking of further processed meat products (i.e. ready meal applications) a meat temperature of 62°C should be aimed for, for the slowest heating region during cooking (usually the centre). This is because it has been identified that a cooking temperature of 65°C should not be exceeded otherwise detrimental effects can occur to the functional properties of the cooked meat. For health concerns a 7D bacterial death reduction has to be achieved. This means that for a cooking temperature of 62°C the meat has to be held at this temperature for at least 5 minutes. Therefore the total cooking time would be the time needed to heat all the meat to 62°C plus 5 minutes to ensure a safe product. The heating or cooking system employed should also ensure that a minimal amount of the meat is heated above 65°C. This can be easily achieved by minimising the external cooking temperature, but long cooking times will result. An industrial cooking process will be a compromise between the cost associated with longer residence time and product functionality. As mentioned earlier another way to add value is to supply nutritional information for selected cuts. Consequentially one of the objectives of this project was to provide some nutritional information for selected meat cuts. Though the primary objective of this part of the project was to develop a method for producing the needed information, so that Richmond N.Z. Ltd. can develop further information on an as needs basis. The nutritional characteristics of a number of export lamb cuts from the saddle region has also been investigated and a method devised to allow further characterisation of other cuts. The method involves breaking down a standard cut into its constituent components (e.g. Frenched rack consists of loin eye, fat cap, intercostals and fatty tissue). The constituent components are tested for their nutritional properties. The frenched rack nutritional properties are calculated from the nutritional properties of the constituents components and the yield data (percentage of each constituent component within a frenched rack) for frenched racks. This method allowed the identification of the main sources of variation for nutritional characteristics. These differences were found to be caused by the lean to fat ratio, not nutritional differences in lean tissue from the same region of lamb (i.e. loin eye and tenderloin very similar nutritionally). The difference in lean to fat ration also accounts for the variation between grades (i.e. PX grade lamb cuts have a higher fat content than YX grade lamb cuts due to PX grade cuts having a higher percentage fat tissue in their cuts). The cuts characterised were the shortloin section (whole section or chop), rack section (whole section or chop), 75mm racks frenched 25mm, boneless loin and tenderloin for both PX and YX grade lamb. The method will be applicable to other regions of lamb (i.e. hindquarter and forequarter) for which nutritional information already exists, but for which yielding data will have to be collected. The method would also be applicable to other species such as beef and venison, but both nutritional data for constituent components and yielding data would have to be collected.

Richmond Ltd. has recently undergone a change in strategy, away from the traditional commodity based meat industry, towards the modern food business. To do this, opportunities to add value to their current product range must be identified. This involves the conversion of traditionally low value commodity based products into products that demand a premium. An example of this is converting muscles that are currently used for grinding meat into a further processed convenience food (i.e. ready meals). Another method is to add further value to premium products by making them more appealing to consumers (i.e. nutritional information on labels). This work details investigations into the functional properties of selected beef forequarter muscles (low value commodity products) and the nutritional properties of selected export lamb products (premium products). The functional properties of a number of beef forequarter muscles were measured to identify which had the best potential for further processing applications with respect to ready meals. The functional properties of tenderness, cook loss and shrinkage were measured for the Latissimus Dorsi, Pectorialis Profundus (Point End Brisket), Infraspinatus (Cross Cut Blade), Triceps Brachi Longhead (Main muscle in Bolar Shoulder Clod), Supraspinatus (Chuck Tender), Serratus Ventralis and Triceps Brachi Medialhead (Muscle in Bolar Shoulder Clod. From the tests conducted the Infraspinatus and the Triceps Brachi Longhead have been identified as having the best functional properties with respect to further processing for ready meal applications. As well as conducting tests to identify the forequarter muscles with the best potential for further processing applications, investigations were carried out to identify cooking regimes that would optimise the functional properties. This work confirmed that there are three major chemical reactions, which determine the resultant functional properties of cooked meat. They are the denaturation and aggregation of the myofibrillar proteins and the denaturation and solubilisation of connective tissue (collagen). At around 50°C myosin (45% to 50% of the myofibrillar proteins) denatures, which results in a substantial increase in cook loss and reduction in water holding capacity. At around 60°C collagen (main connective tissue protein) denatures, which results in a substantial increase in tenderness and increase in cook loss. This is because as the collagen denatures it loses it mechanical strength (increase in tenderness) and can no longer support its own structure, and causes it to contract. This contraction causes fluid within the meat and cook loss caused by the denaturation of myosin to be expelled from the meat by compressive forces (squeezed out). At around 70°C actomyosin (22% of the myofibrillar proteins) denatures. This results in a substantial increase in the cook loss and firming of the meat. The increase in cook loss or decrease in water holding capacity that occurs with myofibrillar protein denaturation is due to the fact that when these proteins denature and aggregate their ability to bind water is greatly reduced. From the results of the cooking regime trials it is recommended that for functional property considerations that during the cooking of further processed meat products (i.e. ready meal applications) a meat temperature of 62°C should be aimed for, for the slowest heating region during cooking (usually the centre). This is because it has been identified that a cooking temperature of 65°C should not be exceeded otherwise detrimental effects can occur to the functional properties of the cooked meat. For health concerns a 7D bacterial death reduction has to be achieved. This means that for a cooking temperature of 62°C the meat has to be held at this temperature for at least 5 minutes. Therefore the total cooking time would be the time needed to heat all the meat to 62°C plus 5 minutes to ensure a safe product. The heating or cooking system employed should also ensure that a minimal amount of the meat is heated above 65°C. This can be easily achieved by minimising the external cooking temperature, but long cooking times will result. An industrial cooking process will be a compromise between the cost associated with longer residence time and product functionality. As mentioned earlier another way to add value is to supply nutritional information for selected cuts. Consequentially one of the objectives of this project was to provide some nutritional information for selected meat cuts. Though the primary objective of this part of the project was to develop a method for producing the needed information, so that Richmond N.Z. Ltd. can develop further information on an as needs basis. The nutritional characteristics of a number of export lamb cuts from the saddle region has also been investigated and a method devised to allow further characterisation of other cuts. The method involves breaking down a standard cut into its constituent components (e.g. Frenched rack consists of loin eye, fat cap, intercostals and fatty tissue). The constituent components are tested for their nutritional properties. The frenched rack nutritional properties are calculated from the nutritional properties of the constituents components and the yield data (percentage of each constituent component within a frenched rack) for frenched racks. This method allowed the identification of the main sources of variation for nutritional characteristics. These differences were found to be caused by the lean to fat ratio, not nutritional differences in lean tissue from the same region of lamb (i.e. loin eye and tenderloin very similar nutritionally). The difference in lean to fat ration also accounts for the variation between grades (i.e. PX grade lamb cuts have a higher fat content than YX grade lamb cuts due to PX grade cuts having a higher percentage fat tissue in their cuts). The cuts characterised were the shortloin section (whole section or chop), rack section (whole section or chop), 75mm racks frenched 25mm, boneless loin and tenderloin for both PX and YX grade lamb. The method will be applicable to other regions of lamb (i.e. hindquarter and forequarter) for which nutritional information already exists, but for which yielding data will have to be collected. The method would also be applicable to other species such as beef and venison, but both nutritional data for constituent components and yielding data would have to be collected.