DISCUSSION

It is well known that viruses are typically damaged irreversibly by exposure to low pH, organic solvents, dehydration, and heat ( 32 , 33 , 45 ). The current experiment showed that the viability of free phage Felix O1 was rapidly lost upon exposure to a simulated acidic gastric environment and, to a lesser extent, upon exposure to bile salts. Therefore, ensuring phage stability is the key consideration in the design of effective microencapsulation methods. Not only should microencapsulation processes employ physically mild conditions, but also the selected materials should be compatible with the phage and not compromise its biological activity. Moreover, when the objective is to deliver viable phage to the gut, the encapsulation materials should protect phage from acid and enzymes present in gastric fluid and dissolve or swell easily in a weakly alkaline intestinal medium. The current encapsulation process was performed in a mild aqueous-based environment, and our results show that these encapsulation and coating processes had no detrimental effects on phage viability. Similar to previous reports on encapsulation of probiotic bacteria in chitosan-alginate microspheres ( 24 ), a high phage loading efficiency was also found in this study (93.3%). When the sodium alginate solution containing phage was dropped into a gelation medium of calcium chloride, the droplets formed gel microspheres instantaneously, entrapping the phage in a three-dimensional network of ionically cross-linked alginate. Additionally, the encapsulated phage showed excellent stability during storage under wet conditions. Spermine-alginate and poly( dl -lactide-co-glycolide) have been previously used for rotavirus microencapsulation, but only approximately 14% and 30%, respectively, of the initial quantity of virus were entrapped within microspheres ( 29 , 39 ). Therefore, it appears that Ca-alginate matrices have a good compatibility with encapsulated phage Felix O1.

Gastric juice survival is a prerequisite when phage is administered orally. The nonencapsulated phage Felix O1 was extremely sensitive to low-pH conditions, which is similar to the pig stomach pH. This is in agreement with a previous report in which none of three Vibrio vulnificus phage strains was recovered from SGF within 2 min at pH 2.5 to 2.7 ( 23 ). However, phage λ appears to be more acid resistant and was stable in SM buffer of pH 3.0 during storage at ambient temperature for 24 h ( 21 ). Clearly, there was a substantial strain variation in response to acid, but variations in assay buffer and incubation conditions might also contribute to the differences in the acid tolerance of phage. The alginate gel network has the properties of shrinking in low pH and dissolving in higher pH ( 15 ), which permits both the protection of phage from adverse gastric conditions and the release of phage at desired sites (small intestine) in a viable form. In this study, the survival of phage encapsulated in chitosan-alginate microspheres was much higher than that of free phage in SGF, even in the lower-pH solution, although partial loss of viability was still evident. A previous study also showed that survival of calcium-alginate-immobilized bifidobacteria was increased compared to nonencapsulated bacteria upon exposure to simulated gastrointestinal conditions ( 26 ). The protection mechanism of phage by encapsulation is likely achieved by limiting the direct contact of phage with an acidic medium. Immediate exposure of naked free phage to a very low pH SGF decreases viability. Presumably, encapsulation in a gel network protects the phage by reducing the diffusion rate of the protons into the bead matrix. Tang et al. have shown that the diffusion rate of acids (0.1 M hydrochloric acid) is significantly lower in an alginate network than in water ( 42 ). In addition, the drying and chitosan coating of alginate microspheres resulted in a very compact structure with a significantly reduced pore size, as demonstrated by DeGroot and Neufeld ( 11 ) and Gåserød ( 14 ), retarding proton diffusion into alginate matrix. It is therefore possible that phage located in the core of the microsphere is subjected to a more moderate pH during the initial exposure. However, the partial loss of phage indicates that the microspheres cannot completely prevent the acids from diffusing into the microspheres. The fact that phage viability decreased with increasing exposure time to SGF supports this hypothesis. A previous study has shown that calcium alginate microspheres with a diameter below 100 μm were not effective in protecting bifidobacteria subjected to simulated gastric acid ( 19 ). Therefore, it is reasonable to expect that the protection effects could be further improved by increasing the size of the microspheres.

Gastric emptying rates are an important factor that should be considered for survival of phage Felix O1 passing through the stomach. Transit time through the stomach is highly variable and is affected by the dosage form and the particle size in the case of administration of a solid meal ( 9 ). Previous studies indicate that solutions and small pellets of less than 2 mm empty from the stomach rapidly ( 9 ). The rate of gastric emptying in pigs is somewhat slower than that found in humans, with the mean times for 50% gastric emptying for liquid and pellet systems of 1.4 to 2.2 h ( 10 ), while typical values for the emptying of such dosage forms in humans are 0.5 to 1.5 h ( 9 ). The size of the microspheres produced in this study and the fact that the encapsulated phage appears to be viable over a 1.5-h period at pH 2.4 suggest that a sufficient amount of phage should remain viable after passage through the stomach when administered along with feed. This is because the stomach pH is likely to be much higher than pH 2.4 after a meal due to the buffering effect of ingested food. For example, during a meal, gastric pH increases to an average value of 5.0 in humans ( 13 ). In addition, the ingested food constituents may provide protection for phage against extreme pH values, which has been demonstrated in a study involving bacteria ( 49 ). O'Flynn et al. ( 34 ) have previously reported that phage Felix O1 can survive exposure to gastric contents directly collected from porcine stomachs at pH 2.5 for up to 2 h; this disagreement is presumably due to the difference in assay medium used. It is expected that as soon as the phage leaves the stomach, the low pH surrounding the microspheres will be neutralized by intestinal juice, and the microspheres will be gradually disintegrated at this higher pH, causing release of phage from the microspheres. In this study, complete release of phage from the microspheres was observed within 6 h of incubation in SGF. The released phage would be distributed throughout the small intestine during transit and should be available to bind and infect the target pathogens.

Bile acid formed from cholesterol in the liver is one of the anionic surfactants secreted in the gastrointestinal tract. Although there are few reports on bile resistance of bacteriophage, phage Felix O1 appears to be less bile tolerant than the staphylococcal phage K (unpublished data) and three previously reported phage of V. vulnificus ( 23 ), which were found to be resistant to 1% and 2% bile without loss of viability for up to 3 h at 37°C. The difference between the reduction in titer of free phage and that of encapsulated phage was statistically significant ( P < 0.05). When microencapsulated phage was exposed to bile salts, the viability of phage was fully maintained. Chitosan is a polysaccharide with polycationic properties; its salts are able to interact with anionic compounds in solution. Chitosan could adsorb bile acids through the ion-exchange action that occurs when chitosan salts and bile acids form an insoluble complex on the chitosan-alginate membrane ( 24 , 30 ). This will hinder the diffusion of bile salts into the microsphere core and protect the encapsulated phage from interacting with the bile salts. In a previous study, Krasaekoopt et al. also reported that the chitosan-coated alginate beads provided better protection for probiotic bacteria in bile salt solution than either poly- l -lysine or alginate ( 24 ). Although exposure to bile resulted in some loss in viability of phage Felix O1 compared to gastric acid, bile does not appear to be a major concern for oral delivery of phage Felix O1.

In conclusion, microencapsulation of bacteriophage Felix O1 in alginate-chitosan microspheres significantly improves the survival of this phage under laboratory conditions designed to simulate the pig gastrointestinal tract. The current encapsulation approach could provide a possible delivery technology for improving the efficacy of bacteriophage in oral therapeutic applications. Future work is required to explore other surface-coating materials with characteristics that will allow for long-term storage of encapsulated phage and targeted delivery of phage to sites in the gastrointestinal tract and that will enhance the protection of bacteriophage under harsh acidic conditions. The enteric polymers would be an attractive choice for use since they have been demonstrated to provide effective protection for bacteria against in vitro simulated gastric conditions ( 5 , 35 ).

Acknowledgments

Footnotes

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