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The use of multi angle laser light scattering on-line to size exclusion chromatography, analytical ultracentrifugation and viscosity measurements for the determination of molecular weights and conformations of polysaccharide systems is described. The use of several independent techniques for the characterization of polysaccharide systems was found to be essential due to the polydisperse nature of the materials. These techniques were used to investigate the changes in molecular size and conformation of guar gum due to irradiation. Molecular weights and viscosities were found to decrease significantly with increasing radiation dose. Conformational studies on the resulting series of homologous samples confirmed the random coil-like conformation of guar gum. Investigations on BSNdextran complexes, obtained by dry-heating at different molar ratios showed that complex formation (most likely by a Maillard-type reaction) only took place when a low molecular weight dextran fraction was used. The highest molecular weight complexes were obtained when the BSA ratio was high, suggesting that some form of association between individual complexes and/or BSA had taken place.

The work described in this thesis addresses two classes of mixed biopolymer systems: (a) starches and sodium caseinate ; (b) gelling seaweed polysaccharides (x-carrageenan - both with and without locust bean gum - agar and alginate) and proteins (gelatin and blood plasma proteins). The viscosity and swelling volume of a 1% potato starch paste in distilled, deionised water is markedly reduced in the presence of caseinate. Similar effects were seen with simple electrolytes suggesting that this occurs as a consequence of a non-specific ionic strength effect. In contrast a 4% maize starch paste in distilled, deionized water undergoes a viscosity and swelling volume increase in the presence of caseinate. However, when pasted in a 0.1M, pH 7.0 phosphate buffer caseinate addition has little effect on the viscosity of the fresh paste and at high concentrations appears to prevent retrogradation on ageing. It is suggested that in buffer caseinate prevents the leaching of starch polysaccharides from the swollen granule and therefore maintains amylose in the granular phase. This is attributable to the high ionic strength of the solvent, allowing caseinate and the starch polysaccharides to phase separate. In water the unfavourable entropy, change due to the uneven distribution of the counter-ions, prevents phase separation and results in an interpenetrating network. Studies on the large deformation stress relaxation behaviour and melting points of 2% carrageenan, 0.5% carrageenan/0.5% locust bean gum (LBG) and 2% agar gels in a variety of solvent media indicate that the inclusions of 0-20% gelatin and 0-5% bovine serum albumin (BSA) give different results depending on both protein and polysaccharide. The main points of this study show that agar/gelatin mixed gel undergoes a distinct phase inversion at 4-7% gelatin levels, which is not seen with the carrageenan gels containing gelatin. Even when 20% gelatin is incorporated into a 2% carrageenan gel the melting point of the gel is unaltered from that of carrageenan alone. In the presence of high levels of BSA the carrageenan/LBG gel undergoes a marked increase in melting point. Investigations using locust bean gums of variable protein content suggests a possible (LBG)protein-BSA interaction since the melting point increases with the LBG protein content. It is shown that carrageenan/LBG gels have clear regions when formed by autoclaving in the presence of blood plasma. This supports the idea of an association between the protein in the insoluble husk and the blood plasma proteins. The interaction mixtures of BSA with sodium alginate at the interface and in bulk solution have been studied through the techniques of microelectrophoresis and ultracentrifugation respectively, to further elucidate the association between denatured proteins above their isoelectric point and anionic polysaccharides. Both techniques clearly show that the macromolecules can associate electrostatically at pH's above the pI of the protein.

Deterioration of flavour quality during processing and storage is often brought about by oxidative processes. These typically involve oxygen or an active form of oxygen in effecting transformation of a wide range of volatile and non-volatile compounds, including key quality chemicals, flavour precursors and antioxidants. To investigate the nature of the chemical and biochemical change within vegetables and herbs, unblanched frozen parsley was selected as a suitable tissue. The chemical status of parsley during technological processing was determined using novel analytical protocols (SNCV A/SNCNV A) implemented as part of a unified strategy for the quantitative analysis of volatile and non-volatile species. The analysis utilized a single stabilized solution produced from plant tissue, under a regime which minimized isolation stress and artifact formation. On frozen storage (-10OC) the principal volatiles of parsley, myrcene, beta-phellandrene and menthatriene were extensively degraded to non-volatile products at differential rates. p-Cymenene and the tentatively assigned menthatriene diepoxide were formed as minor volatile oxidation products. Myristicin remained largely unchanged. Under similar frozen storage, chlorophyll 'a' displayed significant degradation with only minor amounts of chlorophyllide 'a', pheophytin 'a' and 13[superscript]2 hydroxychlorophyll 'a' formed. Ascorbic acid was extensively degraded in timescales preceding monoterpene and chlorophyll loss. Thermal blanching of parsley extensively prevented the degradation of the monoterpenes, suggesting that endogenous enzymes were responsible for the changes. Elimination of oxygen, in the absence of blanching, prevented volatile loss, confirming the requirement for oxygen. The hypothesis that peroxidase can operate in a co oxidative couple with the flavonoid, apigenin-7-glucoside and hydrogen peroxide, as proposed by Yamauchi (1985), was investigated to establish its potential role in the degradation of terpenoids and chlorophyll. In model experiments, using horseradish peroxidase, menthatriene and chlorophyll showed extensive degradation only when all components of the couple were present. In addition the requirement for oxygen was also established. Naringenin and umbelliferone have been shown to behave similarly to apigenin, as co-substrates for peroxidase. Lycopene, with some structural similarity to menthatriene, was also susceptible to co-oxidation. Polyphenol oxidase, proposed to operate in a similar fashion to peroxidase with mono- and di-phenols as substrates (Montedoro et al. 1995), in model experiments did not cause the degradation of chlorophyll. The co-oxidative role of lipoxygenase in parsley is believed to be of minor significance, however, it is likely to be responsible for the production of low levels of hexanal observed during thawing of frozen parsley. From this thesis it is concluded that the aroma and colour quality loss in frozen unblanched parsley probably results from the oxidative degradation of the unsaturated monoterpenes and chlorophyll 'a' respectively via an oxidative cascade initiated by the action of peroxidase.

Lipase 3 is a fungal lipase produced industrially for use as a dough-conditioning enzyme in bread making. Mutants of Lipase 3 were designed to improve enzyme specific activity and to prevent N-linked glycosylation, which was found to cause a drop in activity on industrial-scale production. These were based on three-dimensional model structures of Lipase 3 in `open' and `closed' conformational states derived from crystallographic data of fungal lipases sharing high sequence homology. Lipase variants were expressed and secreted by Pichiapastoris yeast and purified by anionexchange chromatography, which allowed the separation of two active isoforms. Analysis of the `glycosylation' mutants by SDS-PAGE and MALDI-TOF mass spectrometry implied that mutation of N-linked glycosylation sites prevented attachment of oligosaccharide groups to these sites. Mutants were characterised by measurement of specific activities with soluble and emulsified substrates and determination of kinetic constants with emulsified substrate. None of the `activity' mutants showed improved activity over wild type, and the significant drop in activity with emulsified substrate on mutation of a `lid' tryptophan residue indicated that this residue was required for interaction with long-chain triacylglycerol substrates. Specific activities and kinetic constants measured for the ‘glycosylation' mutants did not differ significantly from those of the wild type enzyme. Sigmoidal kinetic curves were observed for lipases expressed in Pichia pastoris and Aspergillus niger with emulsified substrate. Co-operativity was measured using the Hill plot and found to be positive. The possibility of a kinetic mechanism, rather than an allosteric mechanism (involving interactions between ligand binding sites), for cooperativity is discussed.

This thesis is a documented energy audit and long term study of energy and water reduction in a ghee factory. Global production of ghee exceeds 4 million tonnes annually. The factory in this study refines dairy products by non-traditional centrifugal separation and produces 99.9% pure, canned, crystallised Anhydrous Milk Fat (Ghee). Ghee is traditionally made by batch processing methods. The traditional method is less efficient, than centrifugal separation. An in depth systematic investigation was conducted of each item of major equipment including; ammonia refrigeration, a steam boiler, canning equipment, pumps, heat exchangers and compressed air were all fine-tuned. Continuous monitoring of electrical usage showed that not every initiative worked, others had pay back periods of less than a year. In 1994-95 energy consumption was 6,582GJ and in 2003-04 it was 5,552GJ down 16% for a similar output. A significant reduction in water usage was achieved by reducing the airflow in the refrigeration evaporative condensers to match the refrigeration load. Water usage has fallen 68% from18ML in 1994-95 to 5.78ML in 2003-04. The methods reported in this thesis could be applied to other industries, which have similar equipment, and other ghee manufacturers.

Hubbard Foods Limited of Auckland makes a variety of oats-based value-added products. In the preparation of a range of products at Hubbard Foods, technical staff has become aware of a bitterness problem that sporadically appears in toasted oats. Toasting involves dry heating to about 150°C resulting in the golden colour and flavour development necessary for range of products. Bitterness development has been described in the literature, but Hubbard staff is necessarily focussed on production issues, rather than on a sporadic problem seemingly outside the scope of production variables. The author of this thesis set out to identify the cause and suggest a remedy. Prior research with oats has shown that bitterness and associated off-flavours are linked to the accumulation of free fatty acids, their volatile oxidation products, and possibly amino acids and certain phenols. Oats are distinguished from related grains by their high relative fat content, about seven percent, and an associated very active lipase. The free fatty acids stem from the lipase activity that should be, but may not be, inactivated at source in Australia. This is achieved in the milling process by physical disruption and moist heating to a temperature at which the enzyme is denatured. However, residual lipase activity may adversely affect oats quality during time in storage and transit. A number of analytical methods for cereals were adapted to match the constraints of time and resources. These methods were for colour, moisture, peroxidase activity, p-anisidine, and fat and free fatty acids content, composition of fatty acids, total phenols, volatiles, and bitterness as perceived by an analytical sensory panel of four people. Determination of lipase activity is very expensive, so peroxidase activity is commercially used as an indicator. If the latter is inactive, the former will necessarily be also inactive. The designed methods were first applied to 17 oats lots passing through the Hubbard environment, where 14 were paired raw and toasted. The values of moisture, fat content, free fatty acids content and total phenols were within the normal limits expected for commercial lots of oats compared with the previous studies. Not much variation was observed among the 17 oats lots, with the exception of lot DWHE25. Lot DWHE25 was a faulty product, which had high moisture content, high free fatty acids content, and tasted very bitter. The results suggested that moisture content, free fatty acids and bitterness were usually correlated. In spite of the differences encountered and the clues provided by extremes, the data generated from Hubbard oats lots did not provide enough variation in quality to lead to a definitive chemical model of bitter flavour development. But perhaps crucially, it was found that most samples as received from Hubbard Foods were peroxidase-active which conflicted with the results reported on specification sheets prepared by the oats supplier. These specifications accompanied each lot delivered to Hubbard Foods. Therefore, the supplier’s method was examined and was found to be deficient in one critically important respect. Their method omitted the key reactant hydrogen peroxide. Therefore, it is possible that the lipase was active in many of the samples. Therefore, some experiments were conducted where raw oats, from Hubbard Foods and a supermarket, were treated with water additions and stored for a period to examine the effect of moisture content on the quality and flavour deterioration on subsequent storage. Water-treated oats were toasted to simulate a typical Hubbard process, yielding a total of 58 samples with carrying moisture contents. The data set was statistically analysed to identify the cause of bitterness and the means of its control. The free fatty acids content, volatile compounds particularly hexanal, and total phenols increased with moisture content and storage time. The correlations between chemical analysis and sensory test indicated that free fatty acids positively correlated with bitterness (r = 0.71), and hexanal was also positively correlated. Total phenols did not appear to correlate with bitterness. Oats lots with high peroxidase activity tended to have the poorest quality, strongly implicating residual lipase activity as the critical factor. There were no important interactions between water addition and toasting for most of the experiments. Therefore, it seems likely that the toasting procedures at Hubbard Foods are not responsible for bitterness formation. The cause(s) of bitterness is certainly at source, with a faulty peroxidase test strongly implicated.

Hubbard Foods Limited of Auckland makes a variety of oats-based value-added products. In the preparation of a range of products at Hubbard Foods, technical staff has become aware of a bitterness problem that sporadically appears in toasted oats. Toasting involves dry heating to about 150°C resulting in the golden colour and flavour development necessary for range of products. Bitterness development has been described in the literature, but Hubbard staff is necessarily focussed on production issues, rather than on a sporadic problem seemingly outside the scope of production variables. The author of this thesis set out to identify the cause and suggest a remedy. Prior research with oats has shown that bitterness and associated off-flavours are linked to the accumulation of free fatty acids, their volatile oxidation products, and possibly amino acids and certain phenols. Oats are distinguished from related grains by their high relative fat content, about seven percent, and an associated very active lipase. The free fatty acids stem from the lipase activity that should be, but may not be, inactivated at source in Australia. This is achieved in the milling process by physical disruption and moist heating to a temperature at which the enzyme is denatured. However, residual lipase activity may adversely affect oats quality during time in storage and transit. A number of analytical methods for cereals were adapted to match the constraints of time and resources. These methods were for colour, moisture, peroxidase activity, p-anisidine, and fat and free fatty acids content, composition of fatty acids, total phenols, volatiles, and bitterness as perceived by an analytical sensory panel of four people. Determination of lipase activity is very expensive, so peroxidase activity is commercially used as an indicator. If the latter is inactive, the former will necessarily be also inactive. The designed methods were first applied to 17 oats lots passing through the Hubbard environment, where 14 were paired raw and toasted. The values of moisture, fat content, free fatty acids content and total phenols were within the normal limits expected for commercial lots of oats compared with the previous studies. Not much variation was observed among the 17 oats lots, with the exception of lot DWHE25. Lot DWHE25 was a faulty product, which had high moisture content, high free fatty acids content, and tasted very bitter. The results suggested that moisture content, free fatty acids and bitterness were usually correlated. In spite of the differences encountered and the clues provided by extremes, the data generated from Hubbard oats lots did not provide enough variation in quality to lead to a definitive chemical model of bitter flavour development. But perhaps crucially, it was found that most samples as received from Hubbard Foods were peroxidase-active which conflicted with the results reported on specification sheets prepared by the oats supplier. These specifications accompanied each lot delivered to Hubbard Foods. Therefore, the supplier’s method was examined and was found to be deficient in one critically important respect. Their method omitted the key reactant hydrogen peroxide. Therefore, it is possible that the lipase was active in many of the samples. Therefore, some experiments were conducted where raw oats, from Hubbard Foods and a supermarket, were treated with water additions and stored for a period to examine the effect of moisture content on the quality and flavour deterioration on subsequent storage. Water-treated oats were toasted to simulate a typical Hubbard process, yielding a total of 58 samples with carrying moisture contents. The data set was statistically analysed to identify the cause of bitterness and the means of its control. The free fatty acids content, volatile compounds particularly hexanal, and total phenols increased with moisture content and storage time. The correlations between chemical analysis and sensory test indicated that free fatty acids positively correlated with bitterness (r = 0.71), and hexanal was also positively correlated. Total phenols did not appear to correlate with bitterness. Oats lots with high peroxidase activity tended to have the poorest quality, strongly implicating residual lipase activity as the critical factor. There were no important interactions between water addition and toasting for most of the experiments. Therefore, it seems likely that the toasting procedures at Hubbard Foods are not responsible for bitterness formation. The cause(s) of bitterness is certainly at source, with a faulty peroxidase test strongly implicated.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 79-86).

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