| Bacteriophages (or phages) are viruses that invade bacteria cells and, in the case of lytic phages, multiply rapidly till the complete lysis of bacteria. Phage therapies have unique advantages compared with common antibiotics by their host-specificity, exponential growth, low side effects and their ubiquitous and plentiful nature. Therefore, bacteriophages have been suggested as possible alternatives to chemical antibiotics.Recent studies indicate that phages can be used effectively to control and treat bacterial infections in animals, but one problem is that the viability of orally administered phages may be rapidly reduced under acidic conditions of the stomach and in the presence of gastric acid, enzymes, and other digestive compounds such as bile. Without protection phages might not survive gastric passage and thus not be infective in the intestine. Therefore, it is highly necessary to develop an effective delivery system to protect orally administered phages from the harsh gastrointestinal environment enroute to the infection site in the intestine. One possible way of protection of phages is by encapsulating them in microspheres of pH-dependent polymers.Here, we reported a process for the microencapsulation of Salmonella bacteriophage Felix 01 using natural polysaccharides such as alginate and chitosan. The results showed that the phage was efficiently entrapped in the alginate gel matrix and the microsphere formation process had no detrimental effect on the viability of phage. The mean encapsulation efficiency of phage in Chitosan-Alginate microspheres was 93.3%. In vitro studies were used to determine the effects of simulated gastric fluid (SGF) and bile salts on the viability of free and encapsulated phages. Free phages Felix O1 were found extremely sensitive to acidic environment and were not detectable after 5 min incubation in SGF when the pH value was below 3.7. The viable count of microencapsulated phages decreased only 2.58 log10PFU/g during 1 h exposure to SGF with pepsin at pH 2.4. After 3 h of incubation in 1 and 2% bile salt solutions, the free phage count decreased by 1.29 and 1.67 log10PFU/g respectively, while the viability of encapsulated phages was fully maintained. Phages were completely released from microspheres upon exposure to simulated intestinal fluid (SIF, pH 6.8) within 6 hours. The encapsulated phages in wet microspheres retained full viability when stored at 4℃during the testing period (6 weeks). The microencapsulated phages in dried form had a 0.90 log10PFU/g reduction after 6 weeks storage at 4℃, whereas a 1.20 log10PFU/g reduction occurs after 6 weeks at 22℃. The results demonstrate that dried encapsulated phage stored at 4℃shows better stability than phage kept at room temperature.In order to develop an improved microsphere delivery system with enhanced acid resistance for oral delivery of phage. CaCO3 microparticles and whey protein were co-encapsulated with phage K into alginate microspheres and tested for its efficacy in improving the viability of phage under in vitro acidic conditions. Free phage was completely destroyed when exposed to SGF of pH 2.5. By adding CaCO3 as an antacid excipient to the alginate microspheres, the stability of encapsulated phage K in SGF was largely improved, with only a 0.17 log10PFU/g reduction after 2 h exposure to SGF at pH 2.5, but the release was delayed in SIF, the cumulative release was only about 68% after 12 h. Besides, a combination of alginate and whey protein also led to a better protection for phage K in SGF, the phage titer decreased only by 0.53 log10PFU/g after 2 h exposure to SGF at pH 2.5. In additon, the Alginate/Whey microspheres showed a rapid release profile, and an almost complete release of phage was achieved after 3 h incubation in SIF. The curve fitting data indicated that the phage release mechanism followed non-fickian diffusion pattern, which controlled by erosion of the matrix or enzymatic degradation of the hydrogel network. A number of protective agents including trehalose, sucrose, skim milk, and maltodextrin were tested and found to increase the viability of encapsulated phage K when subjected to drying. The protective effects varied with the type and concentration of each incorporated additives.In vivo, different phage-loaded microspheres were tested in chickens and mice to determine the release profile and gastrointestinal distribution of the encapsulated phage in the digestive tracts. The results showed that there was only a small portion of released phage was detected in chickens’ intestine after 1-4 h of administration of Chitosan-Alginate and Alginate/CaCO3 microspheres (<4 Log10PFU/g), whereas the Alginate/Whey microspheres showed a complete release of phage in both chick and mouse intestine. The free and Alginate/Whey encapsulated phage Felix 01 were then administered to chickens experimentally colonized with S. Typhimurium. Treatment with encapsulated phage resulted in a 1.34 and 1.27 log10CFU/g reduction of cecal Salmonella counts at 12 h and 24 h respectively after challenge as compared with untreated controls. But no significant reduction of cecal Salmonella counts was observed in the chickens treated by free phage. The results indicated that microencapsulation of phage Felix 01 into Alginate/Whey microspheres significantly enhanced its efficacy in reducing Salmonella colonization in chickens.The human adenocarcinoma derived Caco-2 cells were used as an in vitro model to investigate the killing activity of phage Felix 01 against intracellular Salmonella. The results showed that S. Typhimurium can invade and survive inside the Caco-2 cell monolayers. Phage Felix O1 has no lytic activity against intracellular Salmonella, but can inhibit the invasion of Caco-2 cells by the bacteria. The results indicated that the invasion and intracellular survival of Salmonella would be a limiting factor for the antimicrobial activity of bacteriophage.In conclusion, an oral microencapsulated form of bacteriophages were successfully prepared in this study, besides, the gastrointestinal stabilities, release profiles and antimicrobial activities of the microencapsulated phages were evaluated through both in vitro and in vivo experiments. The results demonstrated that microencapsulation can protect phages against gastric juice and bile, and also facilitate oral delivery of a high enough dose of phages to the intestine. Therefore, the current encapsulation approach would lay the foundation for the therapeutic use of phages to control bacterial infections in animals. |
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