Produção de polihidroxialcanoatos por escherichia coli recombinante

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia de Alimentos.Polihidroxialcanoatos (PHAs) são poliésteres sintetizados por várias bactérias que possuem características termoplásticas, biocompatíveis e biodegradáveis. Sua aplicação comercial é limitada pelo alto custo de produção. Do ponto de vista econômico, o substrato contribui mais significativamente com os custos de produção totais. Para reduzir tais custos, linhagens recombinantes e estratégias de cultivo vêm sendo desenvolvidas. Desta forma, estudaram-se linhagens recombinantes de Escherichia coli, DH10B e JM101, ancorando os genes para a biossíntese de PHA de Ralstonia eutropha (Alcaligenes eutrophus), utilizando fontes de carbono de baixo custo. Visando estabelecer as melhores condições de cultivo com relação à fonte de carbono (amido de milho hidrolisado e óleo de soja) e suplemento (soro de queijo), efetuou-se um planejamento experimental 23 nas concentrações de 0 e 5% para cada substrato ou suplemento. Para a linhagem DH10B o maior acúmulo de PHA deu-se no meio com o amido (53,21% da MCS) enquanto o maior crescimento celular (MCS) foi obtido em meio contendo amido, soro e óleo (4,60g.L-1). Para a linhagem JM101 o comportamento foi análogo, com maior acúmulo de PHA com amido (68,27% da MCS) e maior crescimento celular em amido, soro e óleo (2,42g.L-1). A combinação das respostas fornecidas pelos modelos estatísticos indicou como melhores, de acordo com os níveis propostos, os valores de 5% (m/v) de amido e 5% (v/v) de soro para ambas linhagens, enquanto para o óleo, os valores foram estabelecidos em 0% (v/v) para DH10B e 1,5% (v/v) para JM101. Com estes resultados desenvolveu-se um segundo planejamento experimental 25 visando-se testar as concentrações de inóculo (2 e 5% sobre uma Abs=1,2), IPTG e ácido acrílico (0 e 0,1mg.L-1), tempo (48 e 96h) e temperatura (30 e 37°C) em E. coli JM101, ancorando os genes para a biossíntese de PHA de R. eutropha, em meio mineral contendo 5% de amido e 5% de soro e 1,5% de óleo (obtidos pelo planejamento anterior), as melhores respostas foram: MCS de 3,5g.L-1, 75% de PHB, com o acúmulo de 2,5g.L-1 de PHB, quando inóculo foi fixado em 5% (Abs = 1,2), sem a adição de IPTG e ácido acrílico durante 96h a 37°C. IPTG não teve influência significativa sobre as respostas, ácido acrílico não atendeu ao objetivo de permitir a incorporação do monômero HHx ao polímero e a temperatura de 30°C favoreceu preferencialmente o acúmulo de PHB e 37°C o acúmulo de massa celular. A fim de promover a síntese de PHAs de cadeia média (PHAMCL), um terceiro estudo foi realizado em E. coli recombinante, linhagens DH10B e JM101, ancorando os genes para a biossíntese de PHA de Pseudomonas aeruginosa, utilizando fontes de carbono de baixo custo. Através de um planejamento experimental 24 avaliou-se a composição de amido de milho hidrolisado, soro de queijo e óleo de soja ao meio mineral e a adição de ácido acrílico como inibidor da b-oxidação para a síntese de PHAMCL. As melhores respostas experimentais obtidas para E. coli DH10 foram MCS de 0,95g.L-1, 20% de PHA, com o acúmulo de 0,2g.L-1 de PHA, em meio mineral contendo 5% de amido, 5% de soro e 5% de óleo, além de 0,1mg.L-1 de ácido acrílico, e para E. coli JM101 alcançou-se MCS de 0,85g.L-1, 3% de PHA, com o acúmulo de 0,015g.L-1 de PHA, em meio mineral contendo 5% de amido e 5% de soro e 1,5% de óleo. Com o mesmo objetivo, utilizaram-se diversos óleos vegetais (algodão, arroz, canola, dendê, girassol, milho, oliva, soja) para o acúmulo de PHAMCL por E. coli JM101, ancorando os genes para a biossíntese de PHA de P. aeruginosa em meio mineral contendo 5% de soro e 1% do respectivo óleo vegetal, sendo que o maior acúmulo de PHA deu-se em óleo de dendê (11,64%). Por fim, analisou-se a estabilidade dos plasmídios, que até 24h de cultivo mantiveram praticamente 100% de sua estabilidade

Development and characterisation of cooked inlaid sausages with fillet and mechanically separated meat of Nile tilapia (Oreochromis niloticus)

The limited consumption of fish is due to a limited availability of products based on this type of meat. Therefore, innovation and development of products that meet consumers’ preferences are essential. The aim of this work was to develop and characterize cooked inlaid sausages prepared with fillet and mechanically separated Nile tilapia (Oreochromis niloticus) meat (MSM). Three sausages types were considered, F1 (100% MSM), F2 (50% MSM, 50% fillet) and F3 (25% MSM, 75% fillet). Microbiological quality of the raw materials and products obtained were within the standard limits. F1 had the lowest protein (34.68%) and the highest lipid (2.88%) contents. F1 and F2 had the highest luminosity L*, which was related to the MSM composition, as the L* was higher as the percentage of MSM increased in the formulation. The shear strength was higher for F2 (18.55 N) and lower for F1 (6.43 N) and differed among sausages types. The F2 sausages presented a better acceptability regarding the sensory attributes evaluated, with acceptation indexes of 88.00, 85.55, and 84.66% for flavour, texture, and odour respectively. This finding indicates that this formulation would be very well accepted in the market if available for commercialisation

Development and characterization of multilayer films based on polyhydroxyalkanoates and hydrocolloids

New biodegradable polymeric materials have been developed in order to minimize the environmental impact caused bythe traditional packaging found in the market. Polyhydroxyalkanoates (PHAs) are biopolymers with important features such as biode-gradability and biocompatibility; however, the costs associated with PHAs production and the limited mechanical properties reducetheir application. Whey is a residual product from dairy industry and gelatin is a biopolymer with good processing characteristics. Inthis context, a filmogenic solution based on these two biopolymers was incorporated into pure PHA films to improve their optical,mechanical, and structural properties. The filmogenic solution was prepared from gelatin, cheese whey and PHAs, using glycerol asplasticizer agent. The multilayers films (gelatin concentrations at 3 and 5%, w/v) showed higher values for all properties when com-pared with the PHA standard film. In this sense, the addition of this solution was responsible for the improvement of the film prop-erties. The 5% gelatin multilayers films added of cheese whey and PHAs showed better results when compared with the multilayersfilms with gelatin at 3%. The film composed of gelatin 5% and cheese whey showed a water vapor permeability ranging from 0.45 gmm/m2/d/kPa, elongation of 2.18%, and opacity of 14.5%. However, the results of the morphological analysis showed that both filmspresented a homogeneous surface without cracks. Moreover, the results of the thermal analysis of both films indicate polymeric misci-bility. Thus, the choice of the best film will depend on its applicabilityPostprint (published version

Development and characterisation of cooked inlaid sausages with fillet and mechanically separated meat of Nile tilapia (Oreochromis niloticus)

The limited consumption of fish is due to a limited availability of products based on this type of meat. Therefore, innovation and development of products that meet consumers’ preferences are essential. The aim of this work was to develop and characterize cooked inlaid sausages prepared with fillet and mechanically separated Nile tilapia (Oreochromis niloticus) meat (MSM). Three sausages types were considered, F1 (100% MSM), F2 (50% MSM, 50% fillet) and F3 (25% MSM, 75% fillet). Microbiological quality of the raw materials and products obtained were within the standard limits. F1 had the lowest protein (34.68%) and the highest lipid (2.88%) contents. F1 and F2 had the highest luminosity L*, which was related to the MSM composition, as the L* was higher as the percentage of MSM increased in the formulation. The shear strength was higher for F2 (18.55 N) and lower for F1 (6.43 N) and differed among sausages types. The F2 sausages presented a better acceptability regarding the sensory attributes evaluated, with acceptation indexes of 88.00, 85.55, and 84.66% for flavour, texture, and odour respectively. This finding indicates that this formulation would be very well accepted in the market if available for commercialisation.Peer reviewe

Effect of treatments and washing cycles on the quality of Nile tilapia (Oreochromis niloticus) protein concentrate

The extraction of proteins from wastes reduces production costs and environmental pollution. The aim of this work was to evaluate the effect of two treatments involving decanting/sieving or centrifugation and the number of washing cycles on the quality of protein concentrate obtained from mechanically separated meat (MSM) of Nile tilapia (Oreochromis niloticus). Results were analyzed in terms of final yield and proximate composition (moisture, protein, fat and ash) after each washing cycle. Moisture did not vary statistically with the treatments and after the third washing cycle. However, the process involving centrifugation was more efficient for protein concentration because the final protein content increased 2.0 folds (79.82%, dry basis) and fat decreased 6.1 folds (8.29%, dry basis). After four washing cycles, it was obtained a protein concentrate with 79.82% protein, 8.29% lipid and 0.45% ash (dry basis), and 80.0% yield, using the centrifugation procedure. Visual whiteness was highly improved after four washing cycles using both processes. It was concluded that the centrifugation process with four washing cycles was the most appropriate method for producing protein concentrate from MSM of Nile tilapia

UV-B irradiation effect on microalgae performance in the remediation of effluent derived from the cigarette butt cleaning process

Funding Information: This research was funded by the Fondazione Cassa di Risparmio di Lucca, grant number FOCUS 2019/2021. Publisher Copyright: © 2022 by the authors.Abstract: In this study, the potential of ultraviolet B (UV-B) radiation to alleviate the effects of pollutants in cigarette butt wastewater (CBW) was investigated using different Chlorella sorokiniana strains (F4, R1 and LG1). Microalgae were treated with UV-B (1.7 W m−2 ) for 3 days prior to their exposure to CBW and then incubated for 4 days in the absence or presence of UV-B. UV-B-untreated microalgae were used as the control. Comparative physiological responses, including photosynthetic pigments and non-enzymatic antioxidants, as well as nicotine and nicotyrine removal, were evaluated in 7-day cultures. UV-B treatments did not negatively impact algal chlorophyll or carotenoid production. UV-B acclimation was strain-dependent, correlating with native environment adaptations and genetic constitutions. UV-B as a pretreatment had long-term positive effects on non-enzymatic antioxidant capacity. However, LG1 needed more time to readjust the pro-oxidant/antioxidant balance, as it was the most UV-B-sensitive. Phenolic compounds played an important role in the antioxidant system response to UV-B, while flavonoids did not contribute to the total antioxidant capacity. Although cross-resistance between UV-B and CBW was observed in F4 and R1, only R1 showed nicotine/nicotyrine catabolism induction due to UV-B. Overall, the results suggest that UV-B activates defense pathways associated with resistance or tolerance to nicotine and nicotyrine.In this study, the potential of ultraviolet B (UV-B) radiation to alleviate the effects of pollutants in cigarette butt wastewater (CBW) was investigated using different Chlorella sorokiniana strains (F4, R1 and LG1). Microalgae were treated with UV-B (1.7 W m−2) for 3 days prior to their exposure to CBW and then incubated for 4 days in the absence or presence of UV-B. UV-B-untreated microalgae were used as the control. Comparative physiological responses, including photosynthetic pigments and non-enzymatic antioxidants, as well as nicotine and nicotyrine removal, were evaluated in 7-day cultures. UV-B treatments did not negatively impact algal chlorophyll or carotenoid production. UV-B acclimation was strain-dependent, correlating with native environment adaptations and genetic constitutions. UV-B as a pretreatment had long-term positive effects on non-enzymatic antioxidant capacity. However, LG1 needed more time to readjust the pro-oxidant/antioxidant balance, as it was the most UV-B-sensitive. Phenolic compounds played an important role in the antioxidant system response to UV-B, while flavonoids did not contribute to the total antioxidant capacity. Although cross-resistance between UV-B and CBW was observed in F4 and R1, only R1 showed nicotine/nicotyrine catabolism induction due to UV-B. Overall, the results suggest that UV-B activates defense pathways associated with resistance or tolerance to nicotine and nicotyrine.Peer reviewe

INDÚSTRIAS PRODUTORAS DE BIODIESEL: DESTINAÇÃO CORRETA AOS EFLUENTES ATRAVÉS DE IMPLANTAÇÃO DE POLÍTICAS DE PRODUÇÃO MAIS LIMPA (P+L)

Among the current biofuels, biodiesel stands out for the increase in demand in recent years and this situation is a consequence of many advantages, mainly in relation to the environment. In every industrial process, there is the formation of by-products and residues that can become possible environmental contaminants, it would not be different in the production of biodiesel. Among the waste generated in this process, the effluents appear in a prominent place and this massive production ends up generating a large volume. Such effluents present in their composition glycerin, alcohol, traces of the catalyst among others, which prevent its disposal in rivers and soils. In order to carry out the treatment of effluents with the objective of inserting them within the current environmental legislation, producers have encountered many operational, economic and environmental problems. The objective was to present a possible means of mitigating the problem of the destination to be given to the effluent generated by the production of biodiesel, introducing the proposal of cleaner production policies and applying a solution of reuse to the effluents. Adding it to glycerin, which consists of a by-product of low commercial value generated in the production of biodiesel. It is necessary to highlight that the realization of this mixture causes an increase in the volume of glycerin. The results obtained throughout the research showed the feasibility, since there was a considerable decrease in the polluting factors involved in the analyzes.Entre los biocombustibles actuales, el biodiesel destaca por el aumento de la demanda en los últimos años y esta situación es consecuencia de muchas ventajas, especialmente en relación con el medio ambiente. En todos los procesos industriales, hay formación de productos y residuos que pueden convertirse en posibles contaminantes ambientales, no serían diferentes en la producción de biodiesel. Entre los residuos generados en este proceso, los efluentes aparecen en un lugar prominente y esta producción masiva termina generando un gran volumen. Estos efluentes presentes en su composición glicerina, alcohol, rastros del catalizador, entre otros, que impiden su eliminación en ríos y suelos. Con el fin de tratar a los efluentes con el fin de insertarlos dentro de la legislación ambiental vigente, los productores se han encontrado con muchos problemas operativos, económicos y ambientales. El objetivo era presentar un posible medio para mitigar el problema del destino que debía darse al efluente generado por la producción de biodiesel, introducir la propuesta de políticas de producción más limpias y aplicar una solución de reutilización a los efluentes. Añadiéndolo a la glicerina, que consiste en un subproducto de bajo valor comercial generado en la producción de biodiesel. Es necesario destacar que la realización de esta mezcla provoca un aumento en el volumen de glicerina, que termina generando un aumento en el valor financiero, un hecho beneficioso para las industrias,. Dentre os biocombustíveis atuais, o biodiesel destaca-se pelo aumento da demanda nos últimos anos e este quadro é consequência de muitas vantagens, principalmente em relação ao meio ambiente. Em todo processo industrial, há formação de subprodutos e resíduos que podem se tornar possíveis contaminantes ambientais, não seria diferente na produção de biodiesel. Dentre os resíduos gerados neste processo, os efluentes aparecem em lugar de destaque e essa produção massiva acaba gerando um grande volume. Tais efluentes apresentam em sua composição glicerina, álcool, traços do catalisador entre outros, que impedem seu descarte em rios e solos. Para a realização do tratamento dos efluentes com objetivo de inseri-los dentro das legislações ambientais vigentes, produtores tem encontrado muitos problemas operacionais, econômicos e ambientais. Objetivou-se apresentar um possível meio de atenuação do problema da destinação a ser dada ao efluente gerado pela produção de biodiesel, introduzindo a proposta de políticas de produção mais limpa e aplicando uma solução de reuso aos efluentes. Acrescentando-o à glicerina, que consiste em um subproduto de baixo valor comercial gerado na produção de biodiesel. É necessário destacar que a realização dessa mistura provoca um aumento no volume da glicerina, o que acaba por gerar um aumento no valor financeiro, fato benéfico para as indústrias, uma vez que o efluente representa apenas custos enquanto que a glicerina representa lucro. Os resultados obtidos ao longo da pesquisa mostraram a viabilidade, uma vez que houve um decréscimo considerável dos fatores poluentes envolvidos nas análises

Nile tilapia (Oreochromis niloticus) waste protein-based films

The demand for new materials and technologies that are able to minimize the environmental impact generated by the disposal of materials from fossil sources has increased significantly in the past years. As these materials normally include gelatin, a high cost ingredient, our aim was to find a more effective solution through the development of films based on protein isolate sourced from Nile tilapias. Thickness, water solubility, water vapor permeability (WVP), tensile strength, elongation, color attributes, and opacity were evaluated. Casting technique was used to obtain the films, using different combinations of protein isolate, gelatin, and plasticizers (glycerol or sorbitol) in an experimental design 23. The films showed good continuity and handling. Surface was free from bubbles and cracks. Key results observed: thickness from 0.04 to 0.09 mm, solubility in water from 4.11 to 28.72%, WVP from 7.12 to 16.36 g mm d-1 KPa-1 m-2, tensile strength from 12.43 to 155.57 MPa, and elongation from 2.14 to 125.33%. Gelatin protein films were promising from the point of view of mechanical properties, visual appearance and easy handling, as well as low WVP and low solubility in water. However, the replacement of gelatin in the formulations implicated in films with reduced tensile strengthsPostprint (published version