Desenvolvimento de matriz de imobilização de lipase utilizando gelatina de diferentes blooms adicionada de plastificantes hidrofílicos

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico. Programa de Pós-Graduação em Engenharia QuímicaO emprego de enzimas imobilizadas vem aumentando devido às vantagens que as mesmas oferecem como, por exemplo, a possibilidade de reúso. A gelatina é uma proteína solúvel em água, de baixo valor agregado, que tem a capacidade de formar gel e seu uso associado com plastificantes vem sendo estudado no desenvolvimento de biofilmes. Neste trabalho foi realizado um estudo de imobilização de lipase comercial em gelatinas de diferentes valores de Bloom adicionadas de plastificantes hidrofílicos. Foram testadas amplas faixas de concentrações para as gelatinas e baixas concentrações dos plastificantes glicerol e manitol buscando avaliar a influência do valor de Bloom, bem como dos plastificantes na eficiência de imobilização. As gelatinas foram caracterizadas quanto am parâmetros físico-químicos, perfil de aminoácidos e interações dos géis formados com a água. Após a imobilização, avaliou-se a migração para as diversas combinações de gelatina e plastificante, sendo avaliados os plastificantes individualmente, obtidos os rendimentos de imobilização e realizadas microscopias das matrizes. A estabilidade de imobilização em relação a parâmetros operacionais foi verificada para posterior estudo do comportamento na à hidrólise de óleo de oliva para a lipase livre e imobilizada e o reúso da lipase imobilizada. Os resultados indicaram a necessidade de reticulação dos géis com glutaraldeído devido à alta solubilidade em água e razão de inchamento, justificada pelo perfil de aminoácidos que confirma a solubilidade da gelatina. O manitol apresentou maior eficiência na imobilização da lipase, com estruturas mais porosas e de poros mais uniformes. Estas estruturas também sofreram influência da concentração de gelatina, onde maiores concentrações desta associadas com concentrações intermediárias de plastificante proporcionaram matrizes com maior rendimento de imobilização. Através das análises de Difração de Raios-X, pode-se perceber uma estrutura semicristalina para as matrizes de imobilização, sendo que a maioria apresentou o pico correspondente às triplas hélices formadas durante a etapa de resfriamento do gel de gelatina, com maior intensidade para a matriz E8-280-M (correspondente ao experimento 8, com gelatina de 280 Bloom e manitol), levando a maior estabilidade à temperatura. A estabilidade de imobilização foi afetada por valores de pH, temperatura e agitação, sendo somente as matrizes E8-280-M e E9/10-280-M (correspondentes aos experimentos 9 e 10, com gelatina de 280 Bloom e manitol) mais estáveis. Nas reações de hidrólise de óleo de oliva, a atividade enzimática para a lipase livre e imobilizada foi estudada em relação à temperatura, pH e tempo, sendo 42 ºC a melhor temperatura para a lipase livre e imobilizada. Para a lipase livre, o pH ótimo foi 7,0, para a lipase imobilizada em E8-280-M o pH ótimo de atividade ficou na faixa de 5,5 - 7,5 e para a lipase imobilizada em E9/10-280-M, o pH ótimo ficou entre 6,5 e 7,5. Ao longo do tempo, avaliado até 720 minutos, tanto a lipase livre como a imobilizada apresentaram perdas de atividade enzimática. Foram possíveis 10 ciclos de reúso, sendo que até o 5º ciclo a lipase imobilizada em E8-280-M permaneceu com 77,66 % da atividade inicial e para a lipase imobilizada em E9/10-280-M, em 63,09 % da atividade inicial. A maior retenção de atividade na etapa de manutenção foi para a lipase imobilizada a 25 ºC na matriz E8-280-M com 71,76 % de atividade relativa após 30 dias de armazenamento. Foi possível a hidrólise de diferentes substratos com a lipase livre e imobilizada nas matrizes E8-280-M e E9/10-280-M, sendo o maior percentual de hidrólise obtido para a primeira matriz de imobilização para o óleo de canola.The use of immobilized enzymes is increasing due to the advantages that they offer such as the possibility of reuse. Gelatin is a water-soluble protein, low value, which has the ability to form gel and its association with plasticizers has been studied in the development of biofilms. In this paper we present a study of lipase immobilization of commercial gelatins of different values of hydrophilic plasticizers Bloom added. Were tested wide ranges of concentrations for the gelatin and low concentrations of the plasticizers glycerol and mannitol by assessing the influence of the Bloom value, as well as plasticizers in the efficiency of immobilization. The gels were characterized am physico-chemical parameters, amino acid profile and interactions of the gels formed with water. After immobilization, the migration was evaluated for various combinations of gelatin and a plasticizer, the plasticizer being evaluated individually, obtained yields of immobilization and performed microscopy of arrays. The stability of immobilization with respect to operating parameters was observed for further study of the behavior in the hydrolysis of olive oil for free and immobilized lipase and the reuse of immobilized lipase. The results indicated the necessity of crosslinking of the gels with glutaraldehyde due to high water solubility and rate of swelling, justified by the profile of amino acids confirming the solubility of the gelatine. Mannitol had a higher efficiency in the immobilization of lipases, with structures more porous and more uniform pores. These structures also influenced by the concentration of gelatin, where higher concentrations associated with this intermediate concentrations of plasticizer matrices provided with a greater yield of immobilization. By analysis of X-ray diffraction, it can be noticed a semicrystalline structure to immobilization matrices, and most showed a peak corresponding to triple helices formed during the cooling step the gelatin gel with greater intensity for E8-array 280-M (8 corresponding to the experiment with 280 Bloom gelatin and mannitol), leading to better temperature stability. The stability of restraint is affected by pH, temperature and agitation, with only the dies E8-280-M and E9/10-280-M (trials corresponding to 9:10, with a 280 Bloom gelatin and mannitol) more stable . In the hydrolysis of olive oil, the enzymatic activity for the free and immobilized lipase was studied in relation to temperature, pH and time, and 42 °C. The optimum temperature for free and immobilized lipase. For free lipase, the optimum pH was 7.0 for the immobilized lipase at E8-280-M the optimum pH of activity was in the range 5.5 to 7.5 and for immobilized lipase in E9/10-280 -M, the pH optimum was between 6.5 and 7.5. Over time, measured up to 720 minutes, both free and immobilized lipase showed loss of enzyme activity. 10 cycles were possible reuse, and by the 5th cycle, the immobilized lipase at E8-280- M remained with 77.66% of initial activity and the lipase immobilized on E9/10-280-M in 63.09% the initial activity. The higher retention of activity in the upkeep for the immobilized lipase was 25 ° C in the matrix E8-280-M with 71.76% of relative activity after 30 days of storage. Could the hydrolysis of different substrates with free and immobilized lipase matrices in E8-280-M and E9/10-280-M, the largest percentage of hydrolysis obtained for the first immobilization matrix for the canola oil

Extracts of Leaves of Ficus auriculata Lour.: Antioxidant, Antimicrobial and Phytotoxic Activity

Ficus auriculata Lour. is a tree of the genus Ficus. The aim of this study was to obtain the total phenolic compounds; and to determine the antioxidant (DPPH), antimicrobial (diffusion disc and Minimal Inhibitory Concentration) and phytotoxic activity (in cucumber - Cucumis sativus L.) of the extracts. Five extraction methods were used to obtain extracts of young and mature leaves of F. auriculata: water/ethanol (M1), water/ethanol/ultrasound (M2), water/ethanol/cellulase complex (M3), water (M4) and water/cellulase complex (M5). The phenolic content for extracts of young leaves was of 30.22 ± 2.99 mg GAE.g-1 dry sample (M1), 35.22 ± 0.53 mg GAE.g-1 dry sample (M2) and 28.90 ± 0.57 mg GAE.g-1 dry sample (M3) and for the mature leaves of 24.42 ± 0.04 mg GAE.g-1 dry sample (M2) and 17.13 ± 4.69 mg GAE.g-1 dry sample (M1). For the antioxidant activity, the lowest values of IC50 (or higher antioxidant activity) occurred for the extracts of young and mature leaves obtained by the M2. The extracts were able to inhibit all the evaluated bacteria, presenting MICs in the range of 21.60–90.32 µg.ml-1 for the Escherichia coli, 21.60-188.85 µg.ml-1 for the Salmonella enteritidis, 64.22-188.85 µg.ml-1 for the Staphylococcus aureus and 76.31-87.82 µg.ml-1 for the Listeria monocytogenes. The extracts of young and adult leaves presented herbicide potential, occurring suppression of the growth of cucumber plants. The use of the extract obtained in M3 led to the death of the plants. Extracts of F. auriculata presented antioxidant and antimicrobial activities, which agree with previous studies linking phenolic compounds to these properties

Extracts of Leaves of Ficus auriculata Lour.: Antioxidant, Antimicrobial and Phytotoxic Activity

Ficus auriculata Lour. is a tree of the genus Ficus. The aim of this study was to obtain the total phenolic compounds; and to determine the antioxidant (DPPH), antimicrobial (diffusion disc and Minimal Inhibitory Concentration) and phytotoxic activity (in cucumber - Cucumis sativus L.) of the extracts. Five extraction methods were used to obtain extracts of young and mature leaves of F. auriculata: water/ethanol (M1), water/ethanol/ultrasound (M2), water/ethanol/cellulase complex (M3), water (M4) and water/cellulase complex (M5). The phenolic content for extracts of young leaves was of 30.22 ± 2.99 mg GAE.g-1 dry sample (M1), 35.22 ± 0.53 mg GAE.g-1 dry sample (M2) and 28.90 ± 0.57 mg GAE.g-1 dry sample (M3) and for the mature leaves of 24.42 ± 0.04 mg GAE.g-1 dry sample (M2) and 17.13 ± 4.69 mg GAE.g-1 dry sample (M1). For the antioxidant activity, the lowest values of IC50 (or higher antioxidant activity) occurred for the extracts of young and mature leaves obtained by the M2. The extracts were able to inhibit all the evaluated bacteria, presenting MICs in the range of 21.60–90.32 µg.ml-1 for the Escherichia coli, 21.60-188.85 µg.ml-1 for the Salmonella enteritidis, 64.22-188.85 µg.ml-1 for the Staphylococcus aureus and 76.31-87.82 µg.ml-1 for the Listeria monocytogenes. The extracts of young and adult leaves presented herbicide potential, occurring suppression of the growth of cucumber plants. The use of the extract obtained in M3 led to the death of the plants. Extracts of F. auriculata presented antioxidant and antimicrobial activities, which agree with previous studies linking phenolic compounds to these properties

Extracts of branches and fruits of Ficus auriculata Lour: antioxidant, antimicrobial and phytotoxic activities

The present study aimed to obtain the phenolic compounds from Ficus auriculata Lour fruits and branches and to determine the antioxidant and antimicrobial activities and phytotoxicity. For this purpose, five extraction methodologies were used, containing water and ethanol as solvents, low-frequency ultrasound and a cellulases complex. The antioxidant activity was determined by eliminating the DPPH radical. Antimicrobial activity was determined against Gram-positive and Gram-negative bacteria, through disk diffusion and minimum inhibitory concentration. The extracts were also used for tests of phytotoxicity in cucumber (Cucumis sativus L.). The contents of total phenolic compounds showed up to 16.16 mg GAE·g-1 and 22.56 GAE·g-1 for the extracts obtained from the branches and by aqueous and enzymatic extraction. The highest antioxidant activity was found in the extract of the branches obtained with the mixture of ethanol and water and the use of ultrasound, with an IC50 value of 190.57 µg.mL-1. Extracts from branches and fruits showed antimicrobial activity against all tested microorganisms, with inhibition halos of up to 17 mm and minimum inhibitory concentrations of 25.5 μg.mL-1 to 122.25 μg.mL-1. The extracts showed phytotoxicity against cucumber seeds, with a 30% reduction in growth compared to the control. The extracts from the branches and fruits of F. auriculata have biological potential in relation to the tested activities and the difference in these was demonstrated according to the form of extraction

Extracts of branches and fruits of Ficus auriculata Lour: antioxidant, antimicrobial and phytotoxic activities

The present study aimed to obtain the phenolic compounds from Ficus auriculata Lour fruits and branches and to determine the antioxidant and antimicrobial activities and phytotoxicity. For this purpose, five extraction methodologies were used, containing water and ethanol as solvents, low-frequency ultrasound and a cellulases complex. The antioxidant activity was determined by eliminating the DPPH radical. Antimicrobial activity was determined against Gram-positive and Gram-negative bacteria, through disk diffusion and minimum inhibitory concentration. The extracts were also used for tests of phytotoxicity in cucumber (Cucumis sativus L.). The contents of total phenolic compounds showed up to 16.16 mg GAE·g-1 and 22.56 GAE·g-1 for the extracts obtained from the branches and by aqueous and enzymatic extraction. The highest antioxidant activity was found in the extract of the branches obtained with the mixture of ethanol and water and the use of ultrasound, with an IC50 value of 190.57 µg.mL-1. Extracts from branches and fruits showed antimicrobial activity against all tested microorganisms, with inhibition halos of up to 17 mm and minimum inhibitory concentrations of 25.5 μg.mL-1 to 122.25 μg.mL-1. The extracts showed phytotoxicity against cucumber seeds, with a 30% reduction in growth compared to the control. The extracts from the branches and fruits of F. auriculata have biological potential in relation to the tested activities and the difference in these was demonstrated according to the form of extraction

DESENVOLVIMENTO DE COBERTURA À BASE DE COLÁGENO PARCIALMENTE HIDROLISADO, MANITOL E ANTIMICROBIANOS APLICADA EM MORANGOS

O presente artigo apresenta o estudo experimental do desenvolvimento de coberturas à base de colágeno parcialmente hidrolisado e manitol (utilizado como plastificante) e posterior incorporação de antimicrobianos com o intuito de aumentar o tempo de vida útil de morangos. Testaram-se nove combinações de colágeno parcialmente hidrolisado e manitol, mediante planejamento experimental, buscando-se os efeitos dessas variáveis em relação à perda de massa dos frutos. A cobertura mais eficiente para a redução da perda de massa do fruto continha 7,5% de colágeno e 2,5% de manitol. Após definição da melhor formulação testou-se a ação dos antimicrobianos sorbato de potássio e do composto comercial, denominado neste trabalho como extrato de pomelo. Obteve-se aumento da vida útil dos morangos usando cobertura à base de colágeno e manitol com antimicrobianos incorporados, os quais alcançaram vida-de-prateleira de 12 dias

Biscuit Residue in the Nutrition of Laying Hens: Effects on Animal Health, Performance and Egg Quality

Background: Corn and soybean meal are common ingredients used in poultry feed in order to supply the demand for energy and protein, respectively. Also, these ingredients directly influence the final price of the diets, and consequently, the final cost of production. A major problem is associated to the oscillation of these grains in some months of the year. Therefore, there is a need to search for nutritionally and economically viable alternatives to mitigate this problem. The aim was to evaluate whether the addition of biscuit residue could partially replace the use of corn in the feed of laying hens in order to obtain economic viability without impairment on production, egg quality and animal health.Materials, Methods & Results:  The experiment was completely randomized and each repetition was considered one experimental unity. One hundred Hy-line brown 48 week-old chickens were allocated in cages and divided into five groups with four repetitions each, allocating five animals per cage. The group T0 was composed by animals that received the basal diet, i.e. without biscuit residue. The other groups were composed by increasing levels of biscuit residue to substitute corn i.e., group T7 (7% of biscuit residue), T14 (14% of biscuit residue), T21 (21% of biscuit residue) and T28 (28% of biscuit residue). Productive performance of the animals, egg quality, as well as analysis of blood parameters related to lipid metabolism, carbohydrates and proteins of birds were measured at the beginning of the experiment (day 0) and at the end of each cycle (days 21, 42 and 63 of experiment). Among the performance variables, only feed conversion was altered by biscuit residue, i.e., the feed conversion was lower in the groups that received the residue when compared to the control. The replacement of corn by biscuit residue did not affect laying rate (P > 0.05). Regarding egg quality, a less intense coloration was observed in the eggs of the treatments that received biscuit residue compared to the control group (P < 0.05), but without difference between treatments. Seric levels of uric acid were higher in the treatments that received biscuit residue in the days 21 and 42 compared to the control group, effect not seen in the third cycle (day 63). The cost ($) of feed reduced as the levels of inclusion of the biscuit residue in the diet were increased, with the control diet costing$ 0.244/kg, while 28% of biscuit inclusion lower its cost to $0.189/kg (22.3$ 0.524, 0.415, 0.441, 0.397 and 0.332 for 0, 7, 14, 21 and 28% inclusion, respectively.Discussion: Residues of biscuit is widely used to feed swine, with positive results as an ingredient to attend the demand for energy. Even though the Brazilian Tables for Poultry and Swine include biscuit residue as a possible ingredient, there is a lack of scientific research in laying hens. These study was verified that the inclusion of biscuit residue (18.936%) is feasible, since it favors the productive performance of the animals, without adverse effects on their health; in addition, the inclusion of biscuit residue reduces dietary costs, as the cost of egg production. Whereas animal feed accounts for a large part of the production cost, the reduction in feed costs without impairment on animal productivity, is a key factor in animal farming. At certain times of the year, food can account for 80% of the production costs and consequently it influences the final price of the product in the market. In our study, we observed a significant reduction on total costs of diets that received biscuit residue, which occurred because a part of the energetic portion of the diet was supplied by the biscuit residue, and not by corn, which influenced the final price.Keywords: egg quality, biscuit residue, economic viability, nutrition

Atividade antimicrobiana do urucum, ácido fosfórico e Biomax D® contra Listeria monocytogenes em salsicha / Antimicrobial activity of urucum, phosphoric acidand and Biomax D® against Listeria monocytogenes in sausage

Os produtos cárneos são frequentemente identificados como responsáveis por surtos e casos de listeriose. Compostos naturais como, extratos de plantas e frutos podem apresentar atividade antimicrobiana contra Listeria monocytogenes.  O presente estudo teve como objetivo avaliar a atividade antilisterial do urucum e ácido fosfórico, compostos utilizados na etapa de tingimento da salsicha, bem como do Biomax D® (extrato de pomelo e ácido ascórbico). Inicialmente, avaliou-se atividade antimicrobiana in vitro do urucum, ácido fosfórico e Biomax D® contra L. monocytogenes. Os compostos demostraram atividade antilisterial, no entanto, quando incorporado em alimentos esta ação antimicrobiana pode ser influenciada pelos componentes da matriz alimentar.  Para isto, salsichas foram contaminadas com diferentes concentrações de L. monocytogens e submetidas aos  tratamentos: T1 -  2,0% de urucum; T2 – 1,5% de ácido fosfórico; T3 – 2,0% de urucum e 1,5% de ácido fosfórico; T4 – 5,0% de Biomax D®; T5 – 5,0% de Biomax D® e  2,0% de urucum; T6 – 5,0% de Biomax D® e 1,5% de ácido fosfórico; T7 – 5,0% de Biomax D®, 2,0% de urucum e 1,5% de ácido de ácido fosfórico e armazenadas a 4 °C por 10 dias. Nos tempos zero, 5 e 10 dias, as salsicha foram submetidas as contagens de Listeria spp. em ágar Oxford e por PCR confirmou-sea espécie L. monocytogenes. Verificou-se que o urucum, o ácido fosfórico e o Biomax D® quando aplicados individualmente ou em conjunto apresentam atividade antilisterial em salsichas. A etapa de tingimento da salsicha pode contribuir para o controle deste micro-organismo. Além disso, o Biomax D® é um antimicrobiano natural, com potencial de utilização em salsicha

AVALIAÇÃO DE PARÂMETROS OPERACIONAIS DE UM SISTEMA DE LODOS ATIVADOS UTILIZADO NO TRATAMENTO DE EFLUENTE LACTEO

An activated sludge system operates satisfactorily in the removal of organic load and factors such as dissolved oxygen concentration, pH, and turbidity should be monitored. Parte superior do formulário. The purpose of this study was to verify possible oscillations of these parameters. The monitoring was conducted for 43 days in May and June of 2014.  Samples were collected on three points determined in the reactor and the analyzes were performed in triplicate. For pH, the values obtained were in the range 6.8 to 7.8. For dissolved oxygen, the value oscillated from 1 to 10 mg/L, but with more observations to 1 mg/L, which indicates that the process is operating efficiently, but for the days of higher concentration of dissolved oxygen, was observed the occurrence of tumid sludge, with a reduction of filamentous bacteria. For turbidity, it was found that for the three points of samples the profiles were similar, as one being activated sludge, occurs the complete mixture. During monitoring, the treatment system was operated without significant disruption thus ensuring the quality of the treated effluent.Para que o sistema de lodos ativados opere de maneira satisfatória na remoção de carga orgânica, fatores como concentração de oxigênio dissolvido, pH, e turbidez devem ser monitorados. O presente estudo teve como objetivo verificar possíveis oscilações desses parâmetros. O monitoramento foi realizado durante 43 dias durante a estação outono-inverno. As coletas das amostras foram realizadas em três pontos determinados no reator e as análises foram realizadas em triplicata. Para o parâmetro pH, os valores encontrados dentro da faixa de 6,8 a 7,8 evidenciou uma correlação com o oxigênio dissolvido reduzindo a sua solubilidade, resultando em uma boa condição de nitrificação reduzindo os nutrientes presentes na biomassa. No que se refere ao parâmetro oxigênio dissolvido, observou-se uma variação ocasionada pelo fenômeno de lodo intumescido, reduzindo as bactérias filamentosas, elevando o nível do oxigênio dissolvido. Para o parâmetro turbidez, verificou-se que para as 3 amostras analisadas os perfis foram semelhantes, pois sendo um sistema de lodos ativados ocorre à mistura completa, correlacionado ao pH e oxigênio dissolvido para realizar a biodegradação

HIDRÓLISE ENZIMÁTICA DE PROTEÍNAS DE EFLUENTE SINTÉTICO: ESTUDO DAS CONDIÇÕES OPERACIONAIS

The high content of protein present in wastewaters of slaughterhouse and dairy industries may lead to a low rate of biodegradation in conventional biological treatment systems. The use of hydrolytic enzymes as adjuvants in treatment systems may be an alternative to increasing the efficiency of biodegradation since the enzymes hydrolyze the substrate, making the substrate more easily assimilable to the microorganisms. The objective of this study was optimizing the best conditions of pH, temperature and concentration of papain in the hydrolysis of synthetic wastewater. Were used synthetic wastewater with the protein fraction simulated using bovine serum albumin (BSA) and gelatin. Were obtained different profiles of protein concentration decay for the BSA and the gelatin with the enzyme tested, papain, the most efficient hydrolysis of BSA. The hydrolysis conditions for simulated wastewater with BSA were pH 5-6 and temperature of 36°C, for all concentrations of enzyme. For the simulated wastewater with gelatin, all variables tested presented significant effect (p<0.05), with pH optimum between 5- 6, temperature of 30°C and concentration of enzyme of 0.1%.O alto conteúdo de proteína presente em efluentes de frigoríficos e de laticínios pode levar a uma baixa taxa de biodegradação nos sistemas de tratamento biológico convencionais. O uso de enzimas hidrolíticas como coadjuvantes nestes processos de tratamento pode ser uma alternativa para o aumento da eficiência da biodegradação, pois estas, ao hidrolisarem o substrato, tornam este mais facilmente assimilável para os micro-organismos. O objetivo deste trabalho foi estudar melhores condições de pH, temperatura e concentração de papaína na hidrólise de efluente sintético. Foram utilizados efluentes sintéticos, sendo a fração proteica simulada com o uso de albumina de soro bovina (BSA) e gelatina. Foram obtidos diferentes perfis de decaimento da concentração de proteína para a BSA e para a gelatina, sendo a enzima testada, a papaína, mais eficiente na hidrólise da BSA. As condições de hidrólise para o efluente simulado com BSA foram pH 5,0 e 6,0 e temperatura de 36ºC, para todas as concentrações de enzima testadas. Para o efluente simulado com a gelatina, todas as variáveis testadas exerceram efeito significativo (p<0,05), com ótimo de pH entre 5,0 e 6,0, temperatura de 30ºC e concentração de enzima de 0,1 %