Screening of biopolymeric matrices for phage encapsulation against enterohemorrhagic E. Coli

DINI C; ISLAN GA; DE URRAZA PJ; CASTRO GR

Curitiba

Congreso; 4º Congreso Internacional de Bioprocesos en Industria de Alimentos; 2010

SOCIEDADE BRASILEIRA DE CIÊNCIA E TECNOLOGIA DE ALIMENTOS

Enterohemorrhagic E. coli (EHEC) infection is the leading cause of uremic hemoliticsyndrome with a high rate of mortality worldwide among children. The use of phage therapyis becoming an efficient biocontrol strategy to avoid or reduce EHEC transmision from bovineintestine to meat during slaugther. Orally dosed of animals seems to be the most practicalway of supplying phages. But an important obstacle could be the loss of viability of thephages across the extreme acidic conditions of the stomach. In a previous work an EHEClytic phage was selected as candidate for phage therapy in cattle (Dini et al.). However,this phage did not resist in-vitro pHs below 4. Phage encapsulation is an alternative toexplore to overcome this disadvantage . Carbohydrate biopolymers are in general non-toxic, biodegradable, tailorable, and gel-forming molecules that are regularly used in foods,pharmaceutical, cosmetics and others. That biopolymers could bind proteins (Triggeredrelease of proteins from emulsan?alginate beads, Guillermo R. Castro). In that way,proteases from the gastrointestinal lumen can be adsorb into the pectin or alginatemicrospheres, clearing the way for phages.The aim of the present work is to develop a biomatrix for microencapsulation of theEHEC liytic phage ?CA933P? to make it more suitable for use in oral delivery. Thisbiomatrix could be a protection barrier against harsh environmental conditions suchas stomach acidity or intestinal proteases.Microencapsulated phages viability was evaluated in-vitro on high acid conditions(pH 1.5-1.6) at 25 and 37ºC on different biopolymeric matrices. Pectin and alginatewere tested as matrices for phage protection against simulated gastric media.The effect of oleic acid and an emulsifier to the formulations, and coating sphereswith Guar Gum and High Methoxyl Pectin (HMP) on the protective capacitywere evaluated. Emulsified pectin and coating alginate beads with HMP showedthe highest protection after 30 min incubation at pH 1,60 (simulated stomachenvironment) with surviving bacteriophage titers of 106 and 5105 UFPs/mlrespectively at 25°C and 4.105 and 5.105 UFPs/ml at 37°C.It was also determined the effect of ionic strength and phosphate presence in thefacility of phage release. Incubation of spheres encapsulating phage in PectinEmulsion, containing 7,3E+06 UFPs/ml, was done at 37ºC 30 min in NaCl 1,2Msolution, reaching a final phage title of 1,96E+06, wich indicates preservationof phage viability. Microspheres in presence of PBS solution pH=7,40 at 37°C,showed a controlled release profile, releasing bacteriophage in 4 hours with a titer of4,75E+05 UFPs/ml.For the presented results, the development of a biopolymeric matrix consistingof Pectin Emulsion with oleic acid, provide acidity protection with the betterencapsulation rate and controlled release of phages. All these make this systemmore suitable for oral delivery in cows, and could provide protection and stability tophages against the gastrointestinal environment.