The extraction of nitrogenous compounds from cod (Cadus morhua) offal by enzymic hydrolysis with papain, the membrane separation of the peptides in the soluble fraction and their freeze and membrane concentration were investigated with the aim of obtaining a high yield of functional products. The following aspects were covered: kinetics of the hydrolysis and of the enzyme inactivation, separation of insoluble solids and yield of nitrogen under different operating conditions (water to fish offal ratio, temperature and particle size of the raw material). Three of the processing options -the hydrolyses, without and with added water, and the water extraction of the minced fish offal followed by hydrolysis of the residue- gave nitrogen yields of 51%, 62% and 69% of the total nitrogen in the fish offal, and required water: fish offal ratios of 0,1 and 1.86 respectively. The peptides in the hydrolysate supernatants were not amenable to membrane separation but those in the water extract supernatant could be split by pH precipitation and ultrafiltration. Eight functional properties of the potential products were evaluated. The hydrolysate supernatants lacked most of them except for high solubility but their low ash content and the particular molecular weight distribution of their peptides may be useful in special feeding diets. The pH precipitate and the concentrate from the ultrafiltration of the supernatant from the pH precipitation exhibited all the functional properties tested to some extent. The precipitate exhibited low solubility and high buffering capacity, high viscosity and good foaming properties. The concentrate had good solubility, gel strength and emulsifying properties.

No description available.

Mechanically separated turkey meat (MSTM) is one of the cheapest sources of protein; however its use for production of further-processed poultry products is limited due to undesirable composition. pH-shifting extraction was applied to overcome the problems associated with MSTM. In the first study the effect of acid pH-shifting extraction with the aid of citric acid and calcium ions on lipids and heme pigments removal from MSTM was investigated. The maximum removal of total, neutral and polar lipids was achieved with addition of 4, 6 and 2 mmol/L of citric acid, respectively. Addition of 6 or 8 mmol/L of citric acid was the most efficient for total heme pigments removal. In the second and third studies chemical, functional and rheological properties of proteins isolated from MSTM were investigated as influenced by different (2.5, 3.5, 10.5 and 11.5) extraction pH. Gel-forming ability was found the highest for pH 3.5 extracted protein. / Food Science and Technology

Doctor of Philosophy / Department of Biological & Agricultural Engineering / Lisa R. Wilken / Microalgae are a potential feedstock for renewable and sustainable bioproducts and energy but there are significant scientific and engineering challenges to address before widespread acceptance of this platform. In particular, biorefining microalgae serves to maximize biomass valorization and minimize waste to improve process economics. The overall goal of this dissertation was the development of a biological-based microalgae biorefinery to enhance the economic feasibility of Chlamydomonas reinhardtii as a source of multiple products including native proteins and lipids. Specific objectives included accumulating biomass enriched in target biomolecules and determining processing strategies that eliminated the need to dry biomass, employed mild conditions to maintain extractability and quality, and minimized application of petroleum-derived and toxic solvents during extraction. The microalgae biorefinery developed included biomolecule accumulation, biomass harvesting, and targeted enzymatic degradation of the cell wall and organelles for release of native proteins and lipids. Biomass was cultivated, and kinetic studies indicated that 48 h nitrogen deprivation was adequate for protein and lipid accumulation. Four lytic enzymes were screened for their ability to permeate the C. reinhardtii cell wall and the C. reinhardtii-produced enzyme, autolysin, led to >85% cell disruption. TEM imaging confirmed cell disruption and retention of lipid droplets in organelle remnants indicating that protein, lipids, and starch could be distinctly partitioned and recovered. A design of experiments optimization study determined that incubation of disrupted biomass at pH 12 for 4 h at 45°C resulted in up to 65% of total protein released from disrupted biomass followed by 40-50% protein recovery with isoelectric precipitation. The cell disruption and protein extraction steps were subsequently integrated to minimize unit operations, processing time, and energy inputs. Secondary application of trypsin led to release of ~73% of total lipids (enriched in triacylglycerols) from the disrupted biomass. Characterization by thin layer chromatography and GC-FID of released lipids revealed similar profiles of enzymatically released lipids as compared to those released by conventional extraction procedures. Finally, the composition of released lipids indicated favorable combustion behavior, high oxidation stability, and suitability as biodiesel. The developed biological-based biorefinery is a promising step towards adoption of microalgae as a source of bioproducts to provide energy and food to meet the needs of a growing population. The second focus of the work was mitigation strategies for isolation of critical impurities (or potential co-products) while processing microalgae and plant hosts. Specific emphasis was placed on evaluating the impact of proteases, polysaccharides, phenolic compounds and pigments, phytic acid, and host cell proteins on the processing of microalgae and other plant hosts for extraction, recovery, and purification of therapeutic proteins. This review served as evaluation of the broader implications of application of the biorefinery to transgenic microalgae and other plants.

Proteins in catfish by-products were extracted by two methods: Alkaline extraction (AE) and salt extraction (SE). Properties of the fish protein isolate (FPI) were measured by protein yield and content, moisture content, SDS-PAGE protein patterns, color and texture profile, and compared with commercial surimi products. Our results showed that catfish frame had higher protein yield and color similarity with commercial products than the head; AE-FPI had higher yield and gel strength than SE-FPI; SDS-PAGE protein patterns of FPI from catfish frame by SE method was comparable with commercial surimi products. Based on the results, further optimization of the recovery yield of protein extracted by alkaline extraction method, and effect of microbial transglutaminase (MTGase) on gelation properties under various concentration and reaction time was investigated. Results indicate that the protein yield reached up to 60%, and the addition of MTGase in protein isolate effectively improved the gel forming ability.

The main objective of this research was to examine uses for distillers dried grains with solubles (DDGS), a coproduct of ethanol production plant, in the fiber-reinforced plastic composites industry. Initially the effort intended to take advantage of the DDGS components, using chemical reactions, to produce coupling agents to improve the physical properties of the composite. Four different chemicals plus water were used to convert proteins into soluble amino acids. The results were not as expected, and appeared to show an early pyrolysis of DDGS components. This may be due to regeneration of proteins when pH of solutions is neutralized. Procedures were then investigated to utilize DDGS for different markets. Considering that oils and proteins of DDGS can thermally decompose, it seemed important to separate the major components and work with DDGS fiber alone. A procedure to extract oil from DDGS using ethanol and then to hydrolyze proteins with ethanol diluted with water, acid and sodium sulfite, was developed. The resulting DDGS fiber or residual material, with a low content of oil and proteins, was used as filler in a propylene matrix with a lubricant and coupling agent to make natural fiber plastic composites (NFPC). Composites containing wood flour (WPC) were prepared simultaneously with those of DDGS fiber to compare tensile properties and fracture surfaces of the specimens by scanning electron microscope (SEM). This study demonstrates that DDGS fiber can replace wood fiber as a filler in NFPC.

Oilseeds may be processed to yield a number of potentially valuable compounds and fractions including oil, protein and small molecules. However, energy costs associated with industrial processing of oilseeds can be significant. For example, processes that use water to dissolve and separate materials are burdened with the costs associated with concentrating value-added products from dilute solutions. The ethanol industry produces large amounts of an aqueous solution called thin stillage that has little value and is used in animal feed. Thin stillage contains some of the necessary salts used in protein extraction but has a low pH. Protein extraction and protein isolate production is commonly conducted at higher pH. Waste alkali from biodiesel production has a high pH and can be used to adjust the pH of thin stillage to improve its ability to extract protein from oilseed meal. By combining the properties of the waste products of both the ethanol and the biodiesel industries, a complementary process is possible that may have greater economic potential than current practices in industry.<p> In this study, processes for protein extraction from mustard (<i>Brassica juncea</i> (L.) Czern.) meal using thin stillage from ethanol production and glycerol from biodiesel production were studied. The osmotic potential of thin stillage used in this research was lower than that of water, whereas both the density and the viscosity were higher. The pH was typically 3.7-3.8, and the total Kjeldahl nitrogen was approximately 0.080.10 %, w/w. Organic compounds identified in thin stillage were isopropanol, ethanol, lactic acid, 1,3-propanediol, acetic acid, succinic acid, glycerophosphorylcholine, betaine, glycerol and phenethyl alcohol. In addition, yeasts, bacteria and fungi were also found. Moreover, the salt types and their concentrations in thin stillage were predictable. The salt types present in thin stillage were CaCl2, NaCl, K2SO4, NaNO3, Mg(OH)2, Na2SO4 and KOH. A model thin stillage synthesized for the purposes of this research had components and chemical and physical properties comparable to those of thin stillage from ethanol production. Protein was extracted from ground, defatted meal using thin stillage at different pHs and salt concentrations. The results showed that pH and salt content affected protein extraction efficiency. However, no differences were found in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability or amino acid composition of protein extracted with thin stillage, model thin stillage or sodium chloride solution. Moreover, extracted protein did not display significant hydrolysis. The results from peptide sequencing showed that napin and cruciferin were the most prevalent proteins in the extracted fractions. When increasing the scale of the extraction, the efficiency of protein extraction and the percentage of protein in the extracted protein were decreased. Protein recovery achieved with the complementary protocol was higher than that reported for a published protocol. Allyl isothiocyanate was found in protein extracts.

Oilseeds may be processed to yield a number of potentially valuable compounds and fractions including oil, protein and small molecules. However, energy costs associated with industrial processing of oilseeds can be significant. For example, processes that use water to dissolve and separate materials are burdened with the costs associated with concentrating value-added products from dilute solutions. The ethanol industry produces large amounts of an aqueous solution called thin stillage that has little value and is used in animal feed. Thin stillage contains some of the necessary salts used in protein extraction but has a low pH. Protein extraction and protein isolate production is commonly conducted at higher pH. Waste alkali from biodiesel production has a high pH and can be used to adjust the pH of thin stillage to improve its ability to extract protein from oilseed meal. By combining the properties of the waste products of both the ethanol and the biodiesel industries, a complementary process is possible that may have greater economic potential than current practices in industry.<p> In this study, processes for protein extraction from mustard (<i>Brassica juncea</i> (L.) Czern.) meal using thin stillage from ethanol production and glycerol from biodiesel production were studied. The osmotic potential of thin stillage used in this research was lower than that of water, whereas both the density and the viscosity were higher. The pH was typically 3.7-3.8, and the total Kjeldahl nitrogen was approximately 0.080.10 %, w/w. Organic compounds identified in thin stillage were isopropanol, ethanol, lactic acid, 1,3-propanediol, acetic acid, succinic acid, glycerophosphorylcholine, betaine, glycerol and phenethyl alcohol. In addition, yeasts, bacteria and fungi were also found. Moreover, the salt types and their concentrations in thin stillage were predictable. The salt types present in thin stillage were CaCl2, NaCl, K2SO4, NaNO3, Mg(OH)2, Na2SO4 and KOH. A model thin stillage synthesized for the purposes of this research had components and chemical and physical properties comparable to those of thin stillage from ethanol production. Protein was extracted from ground, defatted meal using thin stillage at different pHs and salt concentrations. The results showed that pH and salt content affected protein extraction efficiency. However, no differences were found in the efficiency of extraction, SDS-PAGE profile, digestibility, lysine availability or amino acid composition of protein extracted with thin stillage, model thin stillage or sodium chloride solution. Moreover, extracted protein did not display significant hydrolysis. The results from peptide sequencing showed that napin and cruciferin were the most prevalent proteins in the extracted fractions. When increasing the scale of the extraction, the efficiency of protein extraction and the percentage of protein in the extracted protein were decreased. Protein recovery achieved with the complementary protocol was higher than that reported for a published protocol. Allyl isothiocyanate was found in protein extracts.

Orientador: Marco Aurelio Zezzi Arruda / Tese ( doutorado) - Universidade EStadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-12T12:53:25Z (GMT). No. of bitstreams: 1 Magalhaes_CristianaSchmidtde_D.pdf: 4986578 bytes, checksum: d903003de832394edae53527286bdab8 (MD5) Previous issue date: 2008 / Resumo: Este trabalho de Tese apresenta os resultados da avaliação de onze procedimentos de extração de proteínas nas plantas medicinais ginkgo biloba (Ginkgo biloba L.) e castanha da Índia (Aesculus hippocastanum) e no fitoterápico Espirulina (Spirulina maxima). Os procedimentos variaram desde a simples agitação até àqueles onde se somavam várias etapas, tais como agitação, maceração, sonicação e centrifugação. A avaliação foi feita em termos da comparação da concentração de proteínas totais extraídas por meio de cada procedimento, utilizando-se o método de Bradford e de Kjeldhal. Os procedimentos contendo mais etapas se mostraram mais eficientes na extração de proteínas. Também foi feito o mapa protéico da castanha da Índia (Aesculus hippocastanum) e avaliada a influência dos procedimentos de extração de proteínas no perfil protéico da referida amostra. Para isso foi feita a separação das proteínas presentes nos extratos protéicos utilizando-se eletroforese SDS-PAGE. Esta separação (para as proteínas desnaturadas e sob condição não-redutora) permitiu identificar, em termos de massa molar, quais proteínas compunham os extratos protéicos, onde se verificou uma banda mais expressiva (MM = 33,2 ± 0,9 kDa), independentemente do procedimento de extração usado, fato que foi relacionado à mobilidade da proteína. Quando a separação ocorria sob condições redutoras, a banda mais expressiva apresentava MM = 23,5 ± 0,5 kDa. Com a finalidade de se fazer uma investigação mais detalhada, as proteínas da castanha da Índia que foram extraídas pelo procedimento de maceração e centrifugação, foram também separadas por eletroforese bidimensional, na qual houve o desdobramento da banda mais expressiva em pelo menos nove bandas protéicas. Estas bandas foram decompostas tripticamente e foram analisadas por espectrometria de massas, com a obtenção de várias seqüências peptídicas que se repetiam em várias bandas diferentes. Estes resultados sugerem que estas proteínas se tratam de isoformas. Finalmente, foi proposta a identificação de quais íons metálicos estariam ligados às proteínas, o que foi possível por meio do mapeamento das bandas protéicas utilizando-se a fluorescência de raios-X com radiação Síncrotron. Foram identificados 4 íons metálicos, os quais foram investigados quantitativamente. As bandas separadas por SDS-PAGE e por 2D-PAGE foram decompostas por radiação microonda e a determinação das concentrações dos íons metálicos foi obtida por ETAAS e FAAS. Para as bandas separadas por 2D-PAGE observou-se uma interessante distribuição não homogênea dos íons metálicos, sugerindo que algumas se trataram de metaloproteínas, enquanto outras não. Assim, estas proteínas podem ser de origem citosólica e de armazenamento, e que, embora sejam apenas coadjuvantes na ação medicinal da castanha, participariam ativamente nos processos germinativos e metabólicos da planta. / Abstract: This work presents the evaluation of eleven protein extraction procedures using ginkgo biloba (Ginkgo biloba L.), horse chestnut (Aesculus hippocastanum) medicinal plants, and also using the phytomedicine Spirulina (Spirulina maxima). The procedures varied since the agitation only until the addition of several steps, such as agitation, maceration, sonication, and centrifugation. This evaluation was made by comparing the total extracted proteins through Bradford and Kjeldhal methods. The more efficient protein extraction procedures were those whose steps were added. The influence of protein extraction procedures was also evaluated in the protein map of horse chestnut (Aesculus hippocastanum). The proteins present in extracts were separated by SDS-PAGE. This separation (for denatured and non-reduced proteins) allowed identifying the more expressive protein band (MM = 33.2 ± 0.9 kDa) independently of the used procedure. When the separation was carried out under reduced and denatured conditions, the more expressive protein band had MM = 23.5 ± 0.5 kDa. This difference in MM was attributed to protein mobility. The horse chestnut proteins extracted by maceration and centrifugation were also separated by twodimensional electrophoresis in order to obtain a more detailed protein profile. Through this separation, the more expressive band was folded in, at least, nine protein bands. These bands were triptically decomposed and analysed by mass spectrometry. Several peptide sequences that repeated in different protein bands were obtained. These results suggested to deal of isoforms. Finally, the identification of metallic ions bonded to proteins, using Sincrotron radiation- X-ray fluorescence was also proposed. Four metallic ions were identified, which were quantitaively investigated. The protein bands separated by SDS-PAGE and by 2D-PAGE were decomposed using microwave radiation, and metallic ion concentrations were determined by ETAAS and FAAS techniques. For 2D-PAGE bands an interesting non-homogeneous metallic ion distribution was observed, suggesting that some proteins can be metalloproteins or metal-binding proteins. Therefore, these studied proteins probably present a citosolic origin and storing function. However, they may be coadjuvants at medicinal action, and much probably participate in germinating and metabolic processes. / Doutorado / Quimica Analitica / Doutor em Ciências

Orientador: José Antônio Dell’Aqua Junior / Resumo: O estudo proteômico é utilizado como ferramenta na reprodução animal como auxílio para compreesão da fisiologia das células. Neste sentido esta técnica é empregada em espermatozoides na tentativa de elucidar os processos biológicos e assim determinar os mecanismos moleculares envolvidos na separação do sexo. Assim, o objetivo deste estudo foi descrever as proteínas das células espermáticas de bovinos em diferentes aspectos. No estudo 1, investigou-se a influência do tampão de extração, associado ou não ao método mecânico (flash-frozen), e da concentração celular sobre a quantidade de proteínas extraídas de espermatozoides de bovinos. Foram utilizados como tampões TRIS contendo Nonidet P-40 (NP), RIPA modificado (RP) e uréia/tiuréia/CHAPS (UT), e as concentrações de 2, 4, 6, 8 e 10 x 106 espermatozoides/mL em grupos submetidos ou não ao flash-frozen. As concentrações de proteína total foram quantificadas e gel de eletroforese SDS-1D foi confeccionado. O tratamento UT recuperou maior concentração de proteínas, porém no RP as proteínas apresentaram melhor resolução no gel de eletroforese. A concentração protéica aumentou de acordo com a concentração de células no NP e UT. A influência do flash-frozen variou de acordo com o tratamento. No estudo 2, o objetivo foi traçar o perfil proteico de células espermáticas sexadas (X e Y) de bovinos. Foram utilizadas amostras comerciais de espermatozoides sexados X (n = 6) e Y (n = 6). As proteínas foram solubilizadas, submetidas a espectrom... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The proteomic study is used as a tool in animal reproduction as an aid to understanding of the cells physiology. In this regard, this technique is used in spermatozoa in an attempt to elucidate the biological processes and thus determine the molecular mechanisms involved in the sex separation. Therefore the aim of this study was to describe the bovine sperm cell proteins in different aspects. In study 1, the influence of the extraction buffer, associated or not to mechanical method (flash-frozen), and cellular concentration on the amount of proteins extracted from bovine spermatozoa were investigated. TRIS buffers containing Nonidet P-40 (NP), modified RIPA (RP) and urea/thiourea/CHAPS (UT) were used as well as concentrations of 2, 4, 6, 8 and 10 x 106 spermatozoa/mL in groups submitted or not to flash-frozen. Total protein concentrations were quantified and SDS-1D gel electrophoresis was prepared. The UT treatment recovered a higher concentration of proteins, but in RP the proteins showed better resolution in electrophoresis gel. The protein concentration increased according to the concentration of cells in the NP and UT. The influence of flash-frozen varied according to the treatment. In study 2, the objective was to map the protein profile of sexed sperm cells (X and Y) of cattle. Commercial samples of sexed spermatozoa X (n = 6) and Y (n = 6) were used. The proteins were solubilized, submitted to mass spectrometry SWATH analisys. 459 proteins common to the groups were ide... (Complete abstract click electronic access below) / Doutor