Characterization and encapsulation of probiotic bacteria using a Pea-protein Alginate matrix

Abstract

Research was undertaken to examine different in vitro characteristics of probiotic bacteria, including Lactobacillus acidophilus ATCC® 11975™, Bifidobacterium infantis ATCC 15697D, Bifidobacterium catenulatum ATCC® 27675 and Bifidobacterium adolescentis ATCC® 15703™ in order to identify suitable strain(s) for encapsulation. Under simulated gastric conditions (pH 2.0), L. acidophilus was the most acid-tolerant strain (D-value 10.2 ± 0.8 min), and was able to survive for 30 min; whereas, the other tested probiotics underwent a rapid (within the first 5 min at pH 2.0) 4-5 log colony forming units (cfu)/mL loss in viability. All probiotics tested were able to survive 5 h exposure to 0.3% Oxgall bile at pH 5.8. The relative ranking of probiotic adherence to Caco-2 cells was determined to be: L. acidophilus > B. catenulatum > B. adolescentis > B. infantis, which correlated with 4.5 104, 3.1 103, 2.6 101, and 1.5 101 cfu/mL associated with Caco-2 cell monolayers, respectively. The most hydrophobic probiotics included L. acidophilus (46.5 ± 6.1%) and B. catenulatum (65.5 ± 5.2%); their hydrophobicity were positively correlated with auto-aggregation ability. Addition of divalent cations, EDTA, and bile salts were found to affect hydrophobicity as well; for example, 0.5 mM MgCl2 resulted in a 20% increase in cell surface hydrophobicity of L. acidophilus from baseline levels; whereas, the addition of 0.1 and 0.5% bile salts decreased L. acidophilus hydrophobicity from control levels by 60 and 90%, respectively. Cell free culture supernatant of L. acidophilus effectively inhibited the growth of Escherichia coli O157:H7, and Clostridium sordelli. Bactericidal activity of L. acidophilus cell-free supernatant (the lethal factor was determined to be both heat and trypsin-resistant) against Escherichia coli O157:H7 and Clostridium sordelli ATCC 9714 over 24 h resulted in reductions of 5.5 and 3.5 log cfu/mL, respectively. Further examination of probiotics revealed varying degrees of resistance to the iv antimicrobial agents ciprofloxacin (4 ìg/mL), naladixic acid (32 ìg/mL), kanamycin (64 ìg/mL) and sulfisoxazone (256 ìg/mL). Determination of carbon source utilization patterns indicated that B. catenulatum utilized a number of carbohydrates including -methyl-D-glucoside, D-xylose, D-cellobiose, and -D-lactose; whereas,L. acidophilus, B. infantis, and B. adolescentis utilized D-xylose. Lactobacillus acidophilus was ultimately selected for encapsulation in a 3 mm diameter pea protein-alginate matrix followed by in vitro challenge to simulated gastric conditions (pH 2.0). Encapsulation of L. acidophilus demonstrated a significant (P < 0.05) protective effect during the 2 h exposure to simulated acidic stomach conditions; within capsules, there was approximately 1 log cfu/mL loss in cell viability, whereas unprotected cells experienced > 6 log/mL loss in cell viability over the same period.