Apich50

Carbohydrate fermentation patterns (APICH50; Biomérieux, Marcy-l’Étoile, France) were finally carried out for Lactobacillus to investigate the carbohydrate metabolism of interested isolates according to the manufacturer’s instructions. In this assay, API 50 CHL Medium is a ready-to-use medium which allows the fermentation of the 49 carbohydrates on the API 50 CH strip to be studied. Briefly, a suspension is made in the API 50 CHL Medium with each L. kunkeei colony to be tested and each tube of the strip is then inoculated with the suspension. During incubation, the carbohydrates are fermented to acids which produce a decrease in the pH, detected by the change in color of the indicator. The results make up the biochemical profile of the strain.

The procedures for obtaining LGG for colony counts in stool and capsules were as follows. 4.5 mL of sterile phosphate buffered saline was added to either 0.5 grams of stool or 0.5 grams of capsule contents and diluted to 10⁻⁶. 100 µL dilutions of 10⁻¹ to 10⁻⁶ were plated in duplicate onto Lactobacillus Selection (LBS) Agar (BBL Sparks, MD) which is selective for isolation and enumeration of lactobacilli [43] . LBS plates were incubated at 37°C in an anaerobic chamber (5% CO₂, 10% H₂, 85% N₂) for 48 hours. Typical white, creamy LGG colonies with a distinct buttery smell [44] were easily distinguishable from other lactobacilli on LBS agar. These colonies were counted in duplicate and the average result was reported as CFU/g of stool or capsule. Representative colonies were gram stained and LGG was preliminarily identified if there were gram positive rods in a palisade arrangement versus other Lactobacilli such as L plantarum, L fermentum, L para para caseii and non-LGG L rhamnosus. Isolates were analyzed by APIZYM (Biomerieux, Durham, NC) that distinguishes between Lactobacillus species based on enzymatic reactions and API CH-50 (Biomerieux, Durham, NC) that differentiates between the species based on carbohydrate reactions. Lactobacillus para casei and non-LGG strains of L rhamnosus were distinguished from LGG based on fermentation.