USE OF PEA PROTEIN-POLYSACCHARIDE MICROCAPSULES AS A DELIVERY SYSTEM FOR PROBIOTIC BACTERIA; TESTING UNDER SIMULATED GASTRIC CONDITIONS AND IN ANIMAL MODEL SYSTEMS
Pea protein-polysaccharide microcapsules (2.0 % (w/v) of pea protein isolate (PPI), mixed with either sodium alginate, iota-carrageenan or gellan gum (0.5 % (w/v)), containing Bifidobacterium adolescentis were tested in a series of in vitro survival experiments to evaluate the ability of microcapsules to protect the bacterium under simulated stomach conditions, as well as their ability to release the encapsulated bacteria under simulated conditions of the lower gut. All tested capsule formulations provided significant protection for bifidobacteria relative to non-encapsulated bacteria. PPI-alginate and PPI-iota-carrageenan microcapsules released 70-79 % of encapsulated bacteria, with higher cell numbers being released from freeze-dried capsules. The number of released cells from PPI-gellan gum microcapsules was ~ 26-30 % lower. A rat feeding study was conducted with the test bacterium encapsulated in PPI-alginate. Bifidobacterium adolescentis-specific PCR and qPCR analyses confirmed the presence of DNA from this species in animal fecal samples, but only during the period of capsule intake. Using a mixture of two lactobacilli (a combination of Lactobacillus rhamnosus strain R0011 and Lactobacillus helveticus strain R0052) known to relieve Citrobacter rodentium colitis in mice, we assessed the impact of immobilization of these cells (PPI-alginate) using this animal disease model. The main objective was to determine whether PPI-alginate encapsulation matrix interfered with the ability of probiotic bacteria to reduce the symptoms of colitis. Animals were fed rodent AIN93G diets (control) or AIN93G diets supplemented with either freeze dried or microencapsulated bacterial cells. A second control consisting of PPI-alginate microcapsules only was also fed to mice. Half of the animals in each treatment group were infected with C. rodentium. Daily monitoring of disease symptoms, analyses of histopathological changes in intestinal tissues, cytokine expression levels, and bacterial densities in fecal samples were assessed. Additionally, 16S rRNA gene sequencing of the mucosal microbial communities in the distal colon was conducted. Infection of mice with C. rodentium led to a marked progression of infectious colitis, as evidenced from symptomatic and histopathological data, changes in cytokine expression levels, and alteration of composition of mucosa-associated bacterial communities. Probiotic administration affected disease markers in the cecum but not in the colon, but had no significant impact on cytokine profiles in infected animals. Based on cytokine expression analyses and histopathological data, it was evident that encapsulation contributed to the development of inflammation and worsened symptoms in this experimental animal model. In general, the results of this study suggest that even though the tested encapsulation materials are generally recognized as safe, they may in fact contribute to the development of an inflammatory response in certain animal disease models when administered in the capsule form. In the last study, PPI–alginate microcapsules with or without a chitosan coating and containing Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0052 were produced by extrusion and tested for survivability during storage and under in vitro gastrointestinal conditions. Storage at 4°C had no negative impact on capsule protective ability in both types of capsules. PPI-alginate microcapsules with chitosan containing showed much higher bacterial survival counts than microcapsules without extra coating during challenge tests even after the storage period.
probiotics, Citrobacter rodentium, colitis, pea protein, alginate, iota-carrageenan, high throughput sequencing, animal model
Doctor of Philosophy (Ph.D.)
Food and Bioproduct Sciences