L. casei strains 21/1, 12A, M36, UCD174, A2-362, 32G, T71499, CRF28, UW1, UW4, and ATCC 334 were transferred from -80C freezer stocks into MRS broth (Difco Laboratories, Detroit, MI) and grown overnight (16–18 h) at 37°C. Strains were inoculated at 1% (v/v) into a filter-sterilized chemically defined amino acid medium (CDAA) with 25 mM galactose and incubated 16–18 h at 37°C. The CDAA was comprised of 114 mg sodium acetate, 171 mg sodium citrate, 171 mg ammonium chloride, 343 mg potassium phosphate (monobasic), 343 mg potassium phosphate (dibasic), 114 mg magnesium sulfate tetrahydrate, 6 mg iron sulfate hexahydrate, 6 mg manganese sulfate tetrahydrate, 4 g sodium chloride, 228 mg L-phenylalanine, 455 mg L-tyrosine, 6 mg L-adenine, 6 mg L-guanine, 6 mg L-uracil, 6 mg L-xanthine, 351 mg DL-aspartate, 245 mg L-glutamate, 545 mg L-tryptophan, 443 mg L-alanine, 312 mg L-arginine, 746 mg L-asparagine, 857 mg L-cysteine, 816 mg L-glutamine, 341 mg glycine, 900 mg L-histidine, 923 mg L-isoleucine, 326 mg L-leucine, 428 mg L-lysine, 148 mg DL-methionine, 93 mg L-proline, 946 mg DL-serine, 404 mg DL-threonine, 651 mg L-valine, 24 mg L-cystine, 1 mL trace elements solution , 0.4 mL Tween 80, 0.4 mL Tween 20, 0.4 mL Tween 60, plus 10 mL RPMI 1640 vitamin solution (100X; added prior to experimentation), and pH adjusted to 5.5.
Cells were collected by centrifugation at 13,000 x g for 5 min at 4°C, then suspended in CDAA lacking carbohydrate. Samples of each strain were then added to a final absorbance at 595nm (A595) of 0.1 into 1 mL CDAA adjusted to pH 5.5 that contained 2 mM galactose as a growth booster plus one of the following substrates: 25 mM meso-erythritol, D-xylose, D-ribose, D-arabinose, D-adonitol, D-arabitol, D-xylitol, D-glucose, D-mannose, D-galactose, D-fructose, lactone, D-mannitol, D-galactitol, D-sorbitol, myo-inositol, D-glucosamine, n-acetyl D-glucosamine, sialic acid (Indofine Chemical Company, Inc., Hillsborough, NJ), lactulose, D-lactose, D-sucrose, D-turanose, D-maltose, D-cellobiose, D-trehalose, D-maltitol, D-lactitol, D-raffinose, or D-melezitose; or 4.5 mg/mL (which is equivalent to 25 mM glucose) of heparin, N-acetyl-D-galactosamine, fucose, panose, galactosamine, amylopectin, high methylated pectin, stachyose, pectin, arabinogalactan, rhamnose, inulin, mucin, or phytic acid; or 12.5 mg/mL isomaltose, galacturonic acid, polydextrose, glucuronic acid, amygdalin, maltotriose, pullulan, amylopectin, carboxymethyl cellulose, xylan, lignin, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, dextrin, or amylose. Unless noted, substrates were purchased from Sigma-Aldrich Co. (St. Louis, MO). Strains A2-362 and UCD174 were not included in these studies because they were unable to grow well in CDAA.
Inoculated mixtures were incubated at 37°C under static conditions. Aliquots (50 μl) were periodically collected over a 48 h period, placed in a 96-well microtiter plate, and A595 was determined using a 96 well plate-reader (Bio-Rad, Hercules, CA). Uninnoculated mixtures containing individual substrates were used as blanks for the plate reader. Carbohydrates that produced a turbid sample at time 0 (amylopectin, mucin, carboxymethyl cellulose, xylan, lignin, α-cyclodextrin, γ-cyclodextrin, β-cyclodextrin, dextrin, and amylose) were diluted in a 0.9% sterile saline solution and plated on MRS agar (Difco Laboratories) using the drop plate method . Plates were incubated at 37°C for 48 h prior to colony enumeration.
All growth experiments were performed in triplicate, and the ability to utilize a particular substrate was determined by two-tailed student’s t-test comparison (α = 0.05) between the mean A595 values from cells incubated in CDAA containing no carbohydrate versus CDAA containing the substrate of interest. A dendrogram showing the relationships between strains in regard to substrate utilization was created using the Ward method in hierarchical clustering (JMP version 9, SAS Institute Inc., Cary, NC).