Development of a system for carrying or release bioactive molecules based on a composite matrix of alginate and kefiran

Abstract

Resumo: Os biopolimeros sao polissacarideos encontrados na natureza que apresentam funcoes biologicas e uteis aplicacoes na industria farmaceutica e de alimentos. Neste trabalho forum empregados dois biopolimeros o alginato e o kefiran. O alginato e um polissacarideo lineal produzido industrialmente por algumas algas marrom (Macrocystis pyrifera a mais comum), alem pode se encontrar em algumas bacterias (Pseudomonas aeruginosa), estruturalmente esta composto pelo acidos ƒÀ -D-manuronico e ƒ¿- L .guluronico e e comumente utilizado na sintese de geis por gelacao ionotropica. Por outro lado, o Kefiran e um glucogalactano produzido pela bacteria Lactobacillus kefiranofaciens encontrada nos graos de kefir, apresenta capacidade de formar geis e tem a vantagem de possuir atividades biologicas como antimicrobiana, antioxidante entre outras, mas ainda nao e produzido industrialmente, por conseguinte, forum otimizadas as condicoes fisico . quimicas para a producao de kefiran, encontrando como as melhores, o uso de soro de leite suplementado com glicose ao 15% (p/v) como meio de fermentacao, 30‹C, 10 h e sem agitacao; o produto resultante foi analisado com cromatografia de exclusao de tamanho de alta pressao (HPSEC), cromatografia gasosa - espectrometria de massa (GC . MS), ressonancia magnetica nuclear (RMN), espectroscopia de infravermelho com transformada de Fourier (FTIR) e espectroscopia RAMAN e foi confirmado que o polimero produzido e o kefiran. Do mesmo modo as propriedades antimicrobianas do kefiran forum testadas contra microrganismos patogenos (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, e quatro cepas de Salmonella typhimurium), os resultados mostraram que o processo de otimizacao nao altera as propriedades biocidas do mesmo. Alias, um sistema para carregar ou liberar moleculas foi desenvolvido baseado numa matriz composta de alginato e kefiran, de tal forma que para o encapsulamento e liberacao controlada do ciprofloxacino microesferas de alginato e kefiran forum produzidas melhorando o efeito bactericida contra patogenos comuns. Contudo, a estrutura das microesferas foi analisada com microscopia eletronica de varredura (SEM) encontrando uma forma esferica com uma superficie lisa em comparacao com microesferas feitas so com alginato, logo a seguir a liberacao do ciprofloxacino in vitro foi avaliada encontrando uma liberacao do 3% e 5% num pH estomacal (pH 1.2) e 5% e 25%

Abstract: Biopolymers are polysaccharides found in nature with biological functions and promising useful applications in food and pharmaceutical industries. Two polysaccharides were used in this study alginate and kefiran. Alginate is a linear polysaccharide produced industrially by some brown algae (i.e., Macrocystis pyrifera, among others) and also found in some bacteria (i.e., Pseudomonas aeruginosa), is composed of ? -D-mannuronic and ?- L -guluronic acids and is commonly employed in gel synthesis by ionotropic gelation. Kefiran is a water-soluble glucogalactan produced by Lactobacillus kefiranofaciens present in kefir grains, is able to form gels with the advantage of displaying antimicrobial, antioxidant and others biological activities, but until now is not produced industrially. In that way, the physicochemical conditions for kefiran production were optimized, finding as the best fermentation medium whey supplemented with 15% (w/v) of glucose and the best physical conditions were 30°C, 10 h and without shaking. The resulting product was analyzed using High - Pressure Size Exclusion Chromatography (HPSEC), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), Fourier - transform infrared (FTIR) and Raman spectroscopy, the polymer, was characterized as kefiran. Antimicrobial properties of kefiran against microbial pathogen (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and four strains of Salmonella typhimurium) were tested; the results showed that the optimization process did not alter the biocidal properties of the polymer. On the other hand, a system for carrying or release bioactive molecules was developed based on a composite matrix of alginate and kefiran, in that way, kefiran-alginate gel microspheres were developed to encapsulate ciprofloxacin for antimicrobial controlled release and enhanced bactericidal effect against common pathogens. Scanning electron microscopy (SEM) analysis of the hybrid gel microspheres showed a spherical structure with a smoother surface compared to alginate gel matrices. In vitro release of ciprofloxacin from kefiran-alginate microspheres was less than 3.0% and 5.0% at pH 1.2 (stomach), and 5.0% and 25.0% at pH 7.4 (intestine) in 3 and 21 h, respectively. Fourier transform infrared spectroscopy (FTIR) of ciprofloxacin-kefiran showed the displacement of typical bands of ciprofloxacin and kefiran, suggesting a cooperative interaction by hydrogen bonds between both molecules. Additionally, the thermal analysis of ciprofloxacin-kefiran showed a protective effect of the biopolymer against ciprofloxacin degradation at high temperatures. Finally, antimicrobial assays of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhymurium, and Staphylococcus aureus demonstrated the synergic effect between ciprofloxacin and kefiran against the tested microorganisms.