Gabriela Zárate, Silvia González, and Adriana Pérez Chaia
The genus Propionibacterium consists of two principal groups, cutaneous and classical or dairy. Cutaneous species are predominant members of the microbial population of human skin and have also been isolated from the feces of humans and other vertebrate animals. They are often considered opportunistic organisms and have been occasionally associated with infections in humans (1).
Dairy propionibacteria are microorganisms extensively used in the industry for manufacture of Swiss-type cheeses and biological production of propionic acid and vitamin B12 (2). They can be isolated from soil, vegetables, silage, raw milk, and dairy products such as kefir and different cheeses with "eyes."
In the last decade, several studies have demonstrated probiotic properties for members of the genus Propionibacterium (3,4). The effects claimed are based on the production of bacteriocins (5), vitamins, stimulation of growth of other colonic bacteria like bifidobacteria (6), beneficial modification of the composition and metabolic activities of the intestinal microflora (3,4), immunomodulation (7), and antimu-tagenic activity (8).
It is thought that to produce many of these health benefits, the probiotic microorganisms must be able to survive the transit through the hostile conditions of the gastrointestinal tract (GIT) and remain at high levels in the intestine, avoiding removal by peristaltic contractions of the gut. In this sense, microorganisms with a short generation time or the ability to adhere to the intestinal mucosa will survive for prolonged periods in the body of the host (9). Therefore, two desirable properties for probiotic microorganisms are (1) resistance to gastric acidity, bile, and pancreatic enzymes; and (2) adhesion ability to mucosal surfaces.
Dairy bacteria are traditionally not considered to persist as normal inhabitants of the human intestinal tract. Therefore, survival under GIT conditions and adherence are important properties to be considered, and tests to study them would be useful tools.
In the present chapter we describe the methods used in our laboratory to assess survival, metabolic activity, and adhesion of dairy propionibacteria to intestinal epithelial cells after gastrointestinal digestion (10-13).
The techniques used are described under the following headings:
1. Gastrointestinal digestion in vitro: as a screening procedure for selection of acid- and bile-tolerant strains. The method considers both stresses in a sequential way instead of either of these alone, as in conventional testing. Microbial counts and |3-galactosidase activity are determined to evaluate damage produced by the digestion (10).
2. Adhesion to intestinal epithelial cells in vitro: as a useful tool to study the potential of strains to persist long enough in the intestine to exert beneficial effects (11,12).
3. Intestinal transit rate: to assess the time of permanence of the bacteria in each section of the GIT. This is important to determine the optimum sampling time in assays of adhesion to the mucosa. Spores of Bacillus stearothermophilus are commonly used as transit markers to determine the fate of bacteria in the bulk of the digestive tract (10).
4. Adhesion to the intestinal mucosa in vivo: as a useful tool to study the adhesion ability of selected strains in an animal model (11).
5. fi-galactosidase assay: most of the industrial strains of Propionibacterium have the ability to metabolize lactose. A simple assay of |3-galactosidase activity allows one to estimate the metabolic activity of the strains after gastrointestinal digestion, both in vitro and in vivo. Additional information about the ability of strains to improve lactose digestion within the intestine can be obtained (10,13-15).
1. de Man-Rogosa-Sharpe (MRS) broth (16): 10 g/L peptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 1.0 g/L Tween-80, 2 g/L K2HPO4, 5 g/L sodium acetate, 2 g/L ammonium citrate, 0.2 g/L MgSO4-7H2O, 0.05 g/L MnSO4-4H2O, pH 6.5. Autoclave at 121°C for 15 min and store at room temperature. Dehydrated MRS can be obtained from Difco (Detroit, MI).
2. YEL agar (sodium lactate agar): 10 g/L tryptone, 10 g/L yeast extract, 0.5 g/L L-cysteine, 0.5 g/L Tween-80, 0.25 g/L K2HPO4, 0.05 g/L MnSO4, 14 g/L sodium lactate, 15 g/L agar, pH 6.5. Autoclave at 121°C for 15 min and store at room temperature. Tryptone and yeast extract were obtained from Difco, components were from Sigma (St. Louis, MO).
3. Lactose broth: The same composition as YEL without sodium lactate. Autoclave at 121°C for 15 min and then supplement with filter-sterilized lactose for a final concentration of 1%.
4. Lactose agar: Lactose broth with 1.5% agar.
5. BHI broth: 10.0 g/L brain-heart infusion, 8.5 g/L casein peptone, 8.5 g/L meat peptone, 2.5 g/L glucose, 2.5 g/L Na2HPO4, 5.0 g/L sodium chloride, pH 7.4. Autoclave at 121°C for 15 min and store prepared media at 2-8°C protected from light.
6. Plate count agar: 5 g/L casein peptone, 2.5 g/L yeast extract, 1 g/L glucose, 14 g/L agar, pH 7.0. Autoclave at 121°C for 15 min and store at room temperature.
7. Milk-yeast extract: 10 g/L skim milk, 10 g/L glucose, 5 g/L yeast extract. pH 6.0. Autoclave for 20 min at 115°C (3/4 atm) and store at refrigeration temperature.
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