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Efficacy of pea protein isolate-alginate encapsulation on viability of Lactobacillus reuteri in the porcine gastrointestinal tract
The selection and application of probiotic bacteria is limited by the challenge of maintaining their viability in the digestive tract. Here, we hypothesized that a pea protein isolate (PPI)-alginate (AL) matrix (PPC) can improve probiotic viability during transit of the porcine gastrointestinal tract using an antibiotic resistant L. reuteri strain as a marker.
Lactobacillus reuteri ATCC 53608 was selected on de Man, Rogosa and Sharpe (MRS) media containing rifampin (400 µg/mL) and streptomycin (4000 µg/mL). Early stationary phase antibiotic resistant L. reuteri (LRR) cultures were centrifuged, washed and either resuspended in 1 volume of 15.0% skim milk (LRR-M) or encapsulated in a 4.0% PPI:0.6% AL aqueous solution followed by extrusion and crosslinking in 5.0% CaCl2 +1.0% Tween 80 (w/w) solution (LRR-PPC). Bacteria (LRR-M and LRR-PPC) were freeze dried and stored at -80 °C until use. Weaned piglets (age= 27d, n=24) were divided into three treatments supplemented with either LRR-PPC or LRR-M at a level of 106 cfu/g feed. The third treatment served as an unsupplemented control group. Experimental diets were prepared with a fresh aliquot of frozen probiotic each morning on experimental days 1-3. Fecal samples were collected on days 0, 2, 3 and 4. All piglets were killed on day 4 and intestinal contents were collected from the stomach, duodenum, jejunum, ileum, cecum, proximal colon and distal colon. To enumerate LRR, samples were plated on MRS agar containing 200 µg/mL rifampin and 2000 µg/mL streptomycin.
No LRR was enumerated in feces collected on day 0. On experimental day 4, fecal shedding of LRR from LRR-PPC treatment (5.92±0.10 log cfu/g feces) was significantly (P<0.001) higher than the LRR-M group (5.13±0.10 log cfu/g feces). Recovery of LRR in digesta varied according to treatment and location. For the LRR-M group, higher counts (P<0.001) were observed in the stomach and duodenum compared to LRR-PPC, while counts in the LRR-PPC group were significantly (P<0.001) higher than LRR-M in the proximal and distal colon. Homogenization of stomach contents increased viable LRR counts such that there was no significant difference between LRR-PPC (4.90±0.22 log cfu/g content) and LRR-M (4.82±0.20 log cfu/g content).
The pattern of abundance of L. reuteri in the digestive tract showed that PPC disintegrated and released the probiotic in the distal small intestine permitting increased colonization in distal locations and abundance in feces. Pea protein isolate-alginate encapsulation may be effective in improving probiotic viability in the gastrointestinal tract and permit an increase in the range of bacterial species candidates for probiotic application.
Keywords: encapsulation, porcine gastrointestinal tract, Lactobacillus reuteri