This study describes the characterisation and identification of two species of sulphate-reducing bacteria isolated from marine environments. The isolate coded Ind 1 was recovered from the heavily corroded hull of an oil storage vessel moored off the Indonesian coast. An isolate, referred to as Al 1, originated from a soured oil reservoir in Alaska. Observations using microscopy (light, scanning electron and atomic force) revealed that cells were Gram-negative, rod-shaped and very motile. Physiological characterisation, analysis of the fatty acid profiles and partial and full 16S rRNA sequencing demonstrated strong similarities between the two species and members of the Desulfovibrio genus. The position of the strains within phylogenetic trees showed Al 1 clustering closely with Desulfovibrio vietnamensis. Ind 1 revealed a high degree of similarity with both Desulfovibrio gigas and Desulfovibrio gabonensis and these three strains formed a separate cluster in the delta subdivision of the Proteobacteria. However, whole-cell protein profiles and Fourier-transform infrared spectroscopy studies showed that there is enough dissimilarity between the two isolates and the remaining species of the genus Desulfovibrio to consider Al 1 and Ind 1 as new separate species. Purification, physico-chemical and spectroscopic characterisation of the key enzymes involved in the sulphate metabolism was carried out for both isolates. Nuclear magnetic resonance and electron paramagnetic resonance studies revealed that the proteins of Al 1 and Ind 1 exhibited various features in common with their counterparts from other members of the genus Desulfovibrio. In particular, proteins from Ind 1 showed many similarities with the enzymes previously described for D. gigas. Based on the obtained results, the classification of Ind 1 as Desulfovibrio indonensiensis sp. nov. and Al 1 as Desulfovibrio alaskensis sp. nov. are proposed. The overall results highlight the complexity of the relationship between cell physiology and the organisms' environmental impact.

The focus of this research was to develop a plant-based microcapsule for flax oil by complex coacervation. Complex coacervation involves the electrostatic attraction between two polymers of opposing charges. Specifically, the research aimed to: a) identify the ideal biopolymer and solvent conditions required for complex coacervation involving pea protein isolate (PPI) and gum Arabic (GA); b) understand the functional behaviour of PPI-GA complexes as food and biomaterial ingredients; and c) develop methodologies for encapsulating flax oil within PPI-polysaccharide capsules. Complex coacervation between PPI-GA was found to be optimized at a biopolymer weight mixing ratio of 2:1 in the absence of salt. The functional behaviours of the optimized biopolymer mixture were then investigated as a function of pH (4.30-2.40) within a region dominated by complex coacervation. Emulsion stability was found to be greater for PPI-GA mixture systems relative to PPI alone at pH values between 3.10 and 4.00; emulsions produced under one-step emulsification exhibited higher stability compared to those of two-step emulsification at all pH values. Foam expansion was independent of both biopolymer content and pH, whereas foam stability improved for the mixed system between pH 3.10 and 4.00. The solubility minimum was broadened relative to PPI at more acidic pH values. These findings suggested that the admixture of PPI and GA under complexing conditions could represent a new food and/or biomaterial ingredient, and has potential as an encapsulating agent. Two encapsulation processes were employed in this research: high speed mixing (two-step emulsification) and low speed mixing (one-step emulsification). Flax oil capsules formed using the gelatin-GA mixture (as control) under high speed mixing exhibited low moisture content, water activity and surface oil, and afforded adequate protection against oxidation relative to free oil over a 25 d storage period. The PPI-GA mixture failed to produce acceptable capsules using either high or low speed mixing. In contrast, PPI-alginate capsules were produced and exhibited similar chemical properties as gelatin-GA capsules, except with lower encapsulated flax oil content (30% vs. 50% w/w). However, oxidative stability may adversely affected by the low speed mixing condition during encapsulation.

The focus of this research was to develop a plant-based microcapsule for flax oil by complex coacervation. Complex coacervation involves the electrostatic attraction between two polymers of opposing charges. Specifically, the research aimed to: a) identify the ideal biopolymer and solvent conditions required for complex coacervation involving pea protein isolate (PPI) and gum Arabic (GA); b) understand the functional behaviour of PPI-GA complexes as food and biomaterial ingredients; and c) develop methodologies for encapsulating flax oil within PPI-polysaccharide capsules. Complex coacervation between PPI-GA was found to be optimized at a biopolymer weight mixing ratio of 2:1 in the absence of salt. The functional behaviours of the optimized biopolymer mixture were then investigated as a function of pH (4.30-2.40) within a region dominated by complex coacervation. Emulsion stability was found to be greater for PPI-GA mixture systems relative to PPI alone at pH values between 3.10 and 4.00; emulsions produced under one-step emulsification exhibited higher stability compared to those of two-step emulsification at all pH values. Foam expansion was independent of both biopolymer content and pH, whereas foam stability improved for the mixed system between pH 3.10 and 4.00. The solubility minimum was broadened relative to PPI at more acidic pH values. These findings suggested that the admixture of PPI and GA under complexing conditions could represent a new food and/or biomaterial ingredient, and has potential as an encapsulating agent. Two encapsulation processes were employed in this research: high speed mixing (two-step emulsification) and low speed mixing (one-step emulsification). Flax oil capsules formed using the gelatin-GA mixture (as control) under high speed mixing exhibited low moisture content, water activity and surface oil, and afforded adequate protection against oxidation relative to free oil over a 25 d storage period. The PPI-GA mixture failed to produce acceptable capsules using either high or low speed mixing. In contrast, PPI-alginate capsules were produced and exhibited similar chemical properties as gelatin-GA capsules, except with lower encapsulated flax oil content (30% vs. 50% w/w). However, oxidative stability may adversely affected by the low speed mixing condition during encapsulation.

Poly(ethylene oxide) (PEO) is known for facilitating the electrospinning of biopolymer solutions, that are otherwise not electrospinnable. The objective of this study was to investigate the mechanism by which PEO enables the formation of whey protein isolate (WPI) electrospun fibers under different pH conditions. This investigation revealed that the addition of PEO increased the viscosity of WPI/PEO (10% w/w WPI; 0.4% w/w PEO) solutions. Difference in pH levels of the polymer solutions affected electrospinnability and fiber morphology. Acidic solutions resulted in smooth fibers (700 ± 105 nm) while neutral solutions produced spheres (2.0 ± 1.0 um) linked with ultrafine fibers (138 ± 32 nm). In comparison, alkaline solutions produced fibers (191 ± 38 nm) that were embedded with spindle-like beads (1.0 ± 0.5 um). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analyses revealed that the native globular configuration of WPI was not altered under neutral conditions. By contrast, the electrophoresis and spectrometry data indicated that WPI was denatured and hydrolyzed under acidic conditions, which facilitated the formation of smooth fibers. C13 nuclear magnetic resonance (NMR) and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopies showed that the increase random coil and a-helix secondary structures in WPI contributed to the formation of bead-less electrospun fibers. Also, C13 NMR analysis showed no evidence of chemical interaction between WPI and PEO. Scanning transmission electron microscopy coupled with energy dispersive X-rays (STEM-EDAX) revealed that WPI was uniformly distributed within WPI/PEO electrospun fibers. Observations by scanning electron microscopy (SEM) and field emission scanning electron microscopy (FESEM) indicated that fibers possessed a solid core. All these findings suggested that PEO enables the formation of WPI/PEO electrospun fibers by entanglement/entrapment/deposition. Preliminary studies were conducted on hydroxypropyl methyl cellulose (HPMC). In the absence of PEO, HPMC enabled the formation of WPI electrospun fibers under acidic conditions (124 ± 46 nm). FTIR analyses indicated that there was no interaction between HPMC and WPI, suggesting that HPMC aided in the electrospinning of WPI fibers, also by entanglement/entrapment/deposition. Hence, HPMC and PEO aid in the electrospinning of WPI fibers by entanglement/entrapment/deposition, which can be manipulated by alterations in the protein configuration and solution properties. / Natural Sciences and Engineering Research Council (NSERC) of Canada and the Dairy Farmers of Ontario (DFO)

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on October 30, 2007) Leaves 114-117 are blank. Includes bibliographical references.

Submitted by Milenna Moraes Figueiredo (milennasjn@gmail.com) on 2016-04-11T17:54:58Z No. of bitstreams: 1 irene recuperao das protenas provenientes de pescado utilizando o processo de variao de ph.pdf: 1216568 bytes, checksum: ff5b9e6719a564c8f0bc6e66a6239af3 (MD5) / Approved for entry into archive by cleuza maria medina dos santos (cleuzamai@yahoo.com.br) on 2016-04-29T20:10:03Z (GMT) No. of bitstreams: 1 irene recuperao das protenas provenientes de pescado utilizando o processo de variao de ph.pdf: 1216568 bytes, checksum: ff5b9e6719a564c8f0bc6e66a6239af3 (MD5) / Made available in DSpace on 2016-04-29T20:10:03Z (GMT). No. of bitstreams: 1 irene recuperao das protenas provenientes de pescado utilizando o processo de variao de ph.pdf: 1216568 bytes, checksum: ff5b9e6719a564c8f0bc6e66a6239af3 (MD5) Previous issue date: 2011 / As indústrias processadoras de pescado geram produtos assim como co-produtos de baixo valor comercial em grande quantidade, sendo considerável fonte de proteínas, fator este, que junto à busca por alimentos saudáveis, desperta o interesse por realizar estudos para aproveitar e agregar valor ao pescado de baixo valor comercial, assim como aos resíduos provenientes de sua industrialização para aplicação em produtos para consumo humano. O processo de extração química das proteínas baseia-se em dois mecanismos: solubilização ácida e/ou alcalina das proteínas e recuperação das mesmas no ponto isoelétrico. O objetivo deste trabalho foi avaliar o processo de variação de pH para recuperação das proteínas do pescado, comparando uma espécie gorda (anchoita) com outra magra (corvina), aplicando o processo de solubilização ácida e/ou alcalina e comparar-los com o surimi tradicional. Foi determinada a composição proximal (cinzas, umidade, proteínas e lipídeos); propriedades funcionais (capacidade de retenção de água, capacidade de retenção de óleo e solubilidade), pH, cor, força do gel e rendimento. O processo de solubilização ácida obteve-se maior rendimento para o concentrado de músculo de corvina (98,5%), o menor teor de umidade (71,2%), sendo a menor redução lipídica (33,7%) apresentada pelo concentrado protéico de resíduo de anchoita. Os isolados obtidos por este processo apresentaram teor protéico de até 96,0% (b.s.). A maior brancura foi atribuída ao concentrado de músculo de corvina e ao surimi de corvina. O gel do concentrado do músculo de corvina apresentou maior qualidade em relação ao surimi de corvina e aos demais concentrados protéicos. No processo alcalino, o concentrado protéico de resíduo de anchoita apresentou um rendimento de (35,7%) menor que o rendimento do concentrado de resíduo de corvina (63,7%). O teor protéico foi superior a 94,0% (b.s.) exceto para o concentrado de resíduo de anchoita que apresentou 72,9% (b.s.). A menor brancura foi apresentada pelo concentrado de resíduo de corvina (47,4) quando comparado com o concentrado do músculo de corvina (78,7). O gel do concentrado protéico de resíduo de corvina apresentou qualidade superior ao gel do surimi de corvina. Todos os concentrados protéicos apresentaram ausência de Salmonella sp. O maior valor de solubilidade foi encontrado em pH 11 e o menor em pH 5 para o isolado de músculo (1,5%) de corvina obtido pela solubilização ácida em relação com o valor de 46,3% para o isolado de resíduo de corvina obtido pela solubilização alcalina. A maior capacidade de retenção de óleo foi apresentada pelos isolados de resíduos de corvina e anchoita obtidos no processo de solubilização alcalina (8,3 mL/g proteína e 7,3 mL/g proteína respectivamente). A maior capacidade de retenção de água foi apresentada pelo surimi de corvina (22,0g H2O/g proteína), quando comparados todos os produtos obtidos. / Low commercial value products as well as their co-products are generated in high quantity by fish processing industries, being excellent protein source, factor which together the search for healthy food, interests to accomplish studies in order to profit and join value to fish with low commercial value, as well as also the wastes from its industrialization to the application in products to human intake. The chemistry extraction process of protein is based on two mechanisms: acid and/or alkaline protein solubilization and recovery of theirs at protein isoeletric point. The objective of this work was to evaluate the pH variation process in the recovery of fish proteins, comparing a fat specie (Argentine anchovy) with a thin specie (Whitemouth croaker), applying the acid and/or alkaline solubilization process, and comparing them with that of surimi croaker. It was determined the proximal composition (ash, moisture, proteins and lipids), functional properties (water holding capacity, retention capacity and solubility), pH, color, gel strength and yield. The acid solubilization process presented highest yield in the croaker muscle concentrate (98.5%), lowest moisture content (71.2%), while the lowest lipidic reduction (33.7%) was presented by the anchoita wastes protein concentrate. The isolate obtained by this process presented protein contents until 96.0% (dry basis). The highest whiteness was assigned to croaker muscle concentrate and croaker surimi. The croaker muscle concentrate gel showed the best quality in comparison with croaker surimi and the other protein concentrates. In the alkaline process, the croaker wastes protein concentrate showed less yield (35.7%) than croaker wastes concentrate yield (63.7%). The protein content was higher than 94.0% (d. b.), except for anchovy wastes concentrate that presented 72.9% (d. b.) for this component. The lowest whiteness was presented by croaker muscle concentrate (47.4) compared with the croaker muscle concentrate (78.7). The croaker wastes protein concentrate gel presented superior quality to croaker surimi gel. All the protein concentrate presented absence of Salmonella sp. The highest solubility value was found at pH 11 and the lowest value at pH 5 (1.4%) to the croaker muscle isolate obtained from acid solubilization, when compared with the value of 46.3% for croaker wastes isolate from alkaline solubilization. The highest oil retention capacity was presented by the croaker and anchovy wastes isolates from the process of alkaline solubilization (8.3 mL/g protein and 7.3 mL/g protein, respectively). The highest water retention capacity was showed by croaker surimi (22.0g H2O/g protein), when comparing all the products obtained.

The influence of pH (3.0, 5.0 and 7.0) and ionic strength (0, 50 and 100 mM NaCl) on the physicochemical and emulsifying properties of cruciferin-rich (CPI) and napin-rich (NPI) protein isolates were examined. Specifically, the surface characteristics (charge and hydrophobicity), solubility, interfacial tension and emulsifying activity (EAI) and stability (ESI) indices were measured. In the case of the cruciferin-rich protein isolate, surface charge was found to be negatively and positively charged at pHs above and below its isoelectric point (~4.6-4.8), respectively, ranging in potential from -33 mV at pH 8.0 to +33 mV at pH 3.0. In the presence of NaCl, the overall magnitude of charge became reduced at all pHs. In contrast, hydrophobicity, solubility and the ability for CPI to reduce interfacial tension all were found to be dependent upon both pH and NaCl concentration. Solubility was found to be lowest at pH 5.0 (~11%) and 7.0 (16%) for CPI without salt, but was significantly improved with the addition of NaCl (>80%). Interfacial tension was found to be lowest (10-11 mN/m) for pH 5.0 – 0 mM NaCl and pH 7.0 – 50/100 mM NaCl. Overall, the presence of salt reduced EAI with increasing levels of NaCl at pH 5.0 and 7.0, but not at pH 3.0. In contrast, ESI became reduced with the addition of NaCl (regardless of the concentration) from ~15.7 min at 0 mM NaCl to ~12 min with 50/100 mM NaCl, from ~14.7 min at 0 mM NaCl to ~11.5 min with 50/100 mM NaCl and from 15.1 min at 0 mM NaCl to ~11.7 min with 50/100 mM NaCl for pH 3.0, 5.0 and 7.0, respectively. ESI also was found to be unaffected by pH. In the case of a napin-rich protein isolate, surface charge for the NPI in the absence of NaCl ranged between ~ +10 mV to ~ -5 mV depending on the pH, becoming electrically neutral at pH 6.6. The addition of NaCl acted to reduce the surface charge on the NPI and caused a shift in its isoelectric point to pH 3.5 and 3.9 for the 50 and 100 mM NaCl levels, respectively. Overall, surface hydrophobicity for the NPI was reduced as the pH increased, whereas as NaCl levels were raised the hydrophobicity declined. In contrast, NPI solubility was found to be high (~93-100%) regardless of the solvent conditions. The ability of NPI to reduce interfacial tension was enhanced at higher pHs, however the effect of NaCl was pH dependent. Overall, EAI values were similar in magnitude at pH 3.0 and 5.0, and lower at pH 7.0. The effect of NaCl on EAI was similar at pH 3.0 and 7.0, where EAI at the 0 mM and 100 mM NaCl levels were similar in magnitude, but increased significantly at 50 mM NaCl. However, the EAI values at pH 5.0 were reduced as the level of NaCl increased. Overall, the stability of NPI-stabilized emulsions degraded rapidly and the addition of salt induced faster emulsion instability. In summary, CPI and NPI were very different in terms of their physicochemical properties. However, the emulsifying properties were similar in magnitude indicating that they had similar emulsifying potential under the solvent conditions examined.

Gelation properties of a salt extracted pea (Pisum sativum) protein isolate (PPIs) were evaluated with a goal of using this isolate as a meat extender. Microbial transglutaminase (MTG) was used to improve gelation of PPIs, muscle protein isolate (MPI) from chicken breast and the two combined. Gelation properties were evaluated using small amplitude oscillatory rheology and texture analysis. SDS-PAGE and differential scanning calorimetry were used to examine protein structure. Minimum gelation concentration for PPIs was 5%, lower than the 14% obtained for a commercial pea protein isolate (PPIc), possibly because the PPIc undergone denaturation whereas PPIs had not. Storage modulus (G') and loss modulus (G") increased with protein concentration and maximum gel strength for PPIs occurred at pH 4.0 in 0.3M NaCl. Higher or lower pH values affected protein charge and the potential for network formation. Higher salt concentrations resulted in increased denaturation temperatures, to a point where the proteins did not denature at the 95ºC temperature used for gel formation. When both heating and cooling rate were increased, gel strength decreased, though the cooling rate had a greater impact. Chaotropic salts enhanced gel strength, whereas non-chaotropic salts stabilized protein structure and decreased gel formation. Based on effects of guanidine hydrochloride, urea, propylene glycol, β-mercaptoethanol, dithiothreitol and N-ethylmaleimide, hydrophobic and electrostatic interaction and hydrogen bonds were involved in pea protein gel formation but disulfide bond contribution was minimal. Gels formed with MPI at concentrations as low as 0.5% and were strongest at 95ºC, higher than the ~ 65ºC normally used in meat processing. Good gels were formed at pH 6 with 0.6 to 1.2 M NaCl. Addition of MTG increased gel strength for PPIs, MPI, and a combination of the two. SDS-PAGE showed that bands in the 35~100kDa range became fainter with higher MTG levels but no new bands were found to provide direct evidence of interaction between muscle and pea proteins. Improved gel strength for the MPI/PPI mixture (3:1) containing MTG suggested that some crosslinking occurred. Higher heating temperatures and MTG addition led to the formation of MPI/PPI gel and demonstrated the potential for utilization of pea protein in muscle foods.

Gelation properties of a salt extracted pea (Pisum sativum) protein isolate (PPIs) were evaluated with a goal of using this isolate as a meat extender. Microbial transglutaminase (MTG) was used to improve gelation of PPIs, muscle protein isolate (MPI) from chicken breast and the two combined. Gelation properties were evaluated using small amplitude oscillatory rheology and texture analysis. SDS-PAGE and differential scanning calorimetry were used to examine protein structure. Minimum gelation concentration for PPIs was 5%, lower than the 14% obtained for a commercial pea protein isolate (PPIc), possibly because the PPIc undergone denaturation whereas PPIs had not. Storage modulus (G') and loss modulus (G") increased with protein concentration and maximum gel strength for PPIs occurred at pH 4.0 in 0.3M NaCl. Higher or lower pH values affected protein charge and the potential for network formation. Higher salt concentrations resulted in increased denaturation temperatures, to a point where the proteins did not denature at the 95ºC temperature used for gel formation. When both heating and cooling rate were increased, gel strength decreased, though the cooling rate had a greater impact. Chaotropic salts enhanced gel strength, whereas non-chaotropic salts stabilized protein structure and decreased gel formation. Based on effects of guanidine hydrochloride, urea, propylene glycol, β-mercaptoethanol, dithiothreitol and N-ethylmaleimide, hydrophobic and electrostatic interaction and hydrogen bonds were involved in pea protein gel formation but disulfide bond contribution was minimal. Gels formed with MPI at concentrations as low as 0.5% and were strongest at 95ºC, higher than the ~ 65ºC normally used in meat processing. Good gels were formed at pH 6 with 0.6 to 1.2 M NaCl. Addition of MTG increased gel strength for PPIs, MPI, and a combination of the two. SDS-PAGE showed that bands in the 35~100kDa range became fainter with higher MTG levels but no new bands were found to provide direct evidence of interaction between muscle and pea proteins. Improved gel strength for the MPI/PPI mixture (3:1) containing MTG suggested that some crosslinking occurred. Higher heating temperatures and MTG addition led to the formation of MPI/PPI gel and demonstrated the potential for utilization of pea protein in muscle foods.

Master of Science / Department of Food Science / Karen A. Schmidt / Milk proteins are excellent sources of sulfur-containing amino acids methionine and cysteine, in particular whey proteins. Cysteine is synthesized from methionine by γ-cystathionase. However, cysteine has to be included in the diets of certain subpopulations due to diminished γ-cystathionase activity. Cysteine, a heat- liable amino acid, may lose bioavailability during thermal processing. The objective of this research was to enhance cysteine content in yogurt while maintaining its quality. First, yogurt mixes were formulated to a total solids content of 12.5% with nonfat dry milk (NDM) (N) or a combination of NDM (10%) and whey protein isolate (WPI) (2.5%) (W), and processed at 70°C (20 min) (70) or 90°C (7 min) (90). Yogurt was prepared and maintained at 4oC for 60 days. Three replications were performed and data were analyzed using SAS®. The W mixes had 65%, 32% and 190% more cysteine, true protein and whey protein contents respectively, compared to N mixes prior to processing. However in day 1 yogurt, the highest cysteine content (398.3 mg/L) was found in the W70 yogurt and its gel quality was comparable to the N90 yogurt except for firmness. During a 60 day storage period the W70 and N90 were similar in gel quality except for firmness. Secondly, a hedonic test was done on the W70 (HC) and N90 (LC) yogurts which had been reformulated to contain sugar and vanillin. One replication was performed and data were analyzed using SAS®. The LC and HC yogurts did not vary in liking of flavor (6.1), aftertaste (6.1) and overall acceptability (6.3) corresponding to the words of “like slightly” when compared. However, the appearance of the LC yogurt was liked more than the HC yogurt (6.7 vs. 6.1) whereas the thickness of HC yogurt was liked more than the LC yogurt (6.4 vs. 5.8). These results suggest that addition of WPI along with lower process treatment resulted in yogurt with enhanced cysteine; however, further studies may be needed to optimize the WPI addition to improve the visual characteristics of the yogurt for consumer acceptance.