Research was undertaken to examine different <i>in vitro</i> characteristics of probiotic bacteria, including <i>Lactobacillus acidophilus</i> ATCC® 11975, <i>Bifidobacterium infantis</i> ATCC 15697D, <i>Bifidobacterium catenulatum</i> ATCC® 27675 and <i>Bifidobacterium adolescentis</i> ATCC® 15703 in order to identify suitable strain(s) for encapsulation. Under simulated gastric conditions (pH 2.0), <i>L. acidophilus</i> 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: <i>L. acidophilus</i> > <i>B. catenulatum</i> > <i>B. adolescentis</i> > <i>B. infantis</i>, 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 <i>L. acidophilus</i> (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 <i>L. acidophilus</i> from baseline levels; whereas, the addition of 0.1 and 0.5% bile salts decreased <i>L. acidophilus</i> hydrophobicity from control levels by 60 and 90%, respectively. Cell free culture supernatant of <i>L. acidophilus</i> effectively inhibited the growth of <i>Escherichia coli</i> O157:H7, and <i>Clostridium sordelli</i>. Bactericidal activity of <i>L. acidophilus</i> cell-free supernatant (the lethal factor was determined to be both heat and trypsin-resistant) against Escherichia coli O157:H7 and <i>Clostridium sordelli</i> 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 <i>B. catenulatum</i> utilized a number of carbohydrates including -methyl-D-glucoside, D-xylose, D-cellobiose, and -D-lactose; whereas,<i>L. acidophilus, B. infantis</i>, and <i>B. adolescentis</i> utilized D-xylose. <i>Lactobacillus acidophilus</i> was ultimately selected for encapsulation in a 3 mm diameter pea protein-alginate matrix followed by <i>in vitro</i> challenge to simulated gastric conditions (pH 2.0). Encapsulation of <i>L. acidophilus</i> 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.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-08282009-170854 |
| Date | 24 September 2009 |
| Creators | Kotikalapudi, Bhagya Lakshmi |
| Contributors | Korber, Darren R., Low, Nicholas H., Nickerson, Michael, Shand, Phyllis |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-08282009-170854/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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