4 Change of biomass concentrations in gel beads co-entrapping () and subsp. mixed-strain starters in the effluent of a continuous reactor were obtained using this technology, and very high productivity resulted from the high cell density retained in the immobilized cell reactor (7, 8). However, a large cross-contamination of beads, initially entrapping pure cultures, was observed during continuous cultures over long fermentation occasions of 6 to 8 8 weeks in supplemented whey permeate (7, 8) or in milk (14). A theoretical model of cell release from cavities located near the gel bead surfaces has been recently proposed to explain this cross-contamination phenomenon (6). To experimentally validate this hypothesis and to identify factors responsible for this cross-contamination phenomenon, a method for specifically detecting the different strains in beads is needed. Ractopamine HCl A model system with a probiotic strain (subsp. biovar diacetylactis) as the competitive strain was chosen for this study. Bifidobacteria are increasingly used in fermented dairy products in combination with LAB strains because of their perceived importance in human health (9). Single (13) and dual Ractopamine HCl (1) labeling with green fluorescent protein has been reported to detect free Ractopamine HCl LAB cells and gram-negative bacteria in mixed free-cell culture, respectively. Fluorescent polyclonal antibodies were used to specifically detect genetic variants of in Ractopamine HCl mixed free-cell culture, using a direct and indirect fluorescence labeling method with fluorescein isothiocyanate (FITC) as a differential cell detection strategy (3). and coimmobilized in gel beads were detected separately using a two-step fluorescent-labeling method with FITC-labeled anti-rabbit antibody (4). Hence, green fluorescent colonies of either or were observed with this strategy. Dual immunofluorescent labeling has never been reported for the simultaneous and specific detection of probiotic and LAB cultures coimmobilized in gel beads. The subsp. biovar diacetylactis strain (Rhone Poulenc, Brampton, Ontario, Canada) was produced at 30C in M17 broth (Difco Laboratories, Detroit, Mich.) supplemented with 1% (wt/vol) lactose. The ATCC 15707 strain (Rosell Institute Inc., Montreal, Quebec, Canada) was cultivated at 37C in MRS broth (Rosell Institute Inc.) supplemented with 0.5 g of cysteine per liter, 0.2 g of Na2CO3 per liter, and 0.1 g of CaCl2 per liter (12). Polyclonal antibodies against both strains were raised in rabbits using cell wall suspensions as immunogens. Cross-reactivities of anti-antibody on subsp. biovar diacetylactis and anti-subsp. biovar diacetylactis antibody on were removed using a cross-adsorption protocol. All Rabbit Polyclonal to ARRB1 operations were carried out at 4C. Anti-antibody used at a final concentration of 5 g/ml was mixed with 10 ml of an subsp. biovar diacetylactis cell suspension (1010 CFU/ml) made up of protease inhibitors for 24 h in a rotary shaker at 4 rpm. The pH was adjusted to 7.5 0.1 with 1 N NaOH before adsorption. After adsorption, free immunoglobulin G (IgG) was recovered on a protein A/G column (Pierce, Rockford, Ill.), dialyzed against phosphate-buffered saline (PBS), and concentrated to 2 mg/ml using centricon (Millipore, Bedford, Mass.). The same technique was used for eliminating anti-subsp. biovar diacetylactis IgG cross-reacting with cells. The specificities of purified IgG (before and after adsorption) were determined by dot blot immunoassay on nitrocellulose membranes (Micron Separation Inc., Westboro, Mass.) using peroxidase-labeled antibodies (5). Two fluorescent dyes, ALEXA 488 and ALEXA 568, were used to label the adsorption-purified anti-and anti-subsp. biovar diacetylactis antibodies, respectively, using an ALEXA protein labeling kit (Molecular Probes, Inc., Eugene, Oreg.), according to the manufacturer’s instructions. The ALEXA 488-labeled anti-IgG and the ALEXA 568-labeled anti-subsp. biovar diacetylactis IgG have excitation maxima at 488 and 568 nm, respectively, and emission maxima at 517 and 603 nm, respectively (2). The immobilization procedure for -carrageenan and locust bean gum gel beads (2.75 Ractopamine HCl and 0.25% [wt/wt], respectively) was based on a two-phase dispersion technique (7) modified as follows. A 1% (vol/vol) mixed inoculum made of 90% (vol/vol) and 10% (vol/vol) subsp. biovar diacetylactis with cultures standardized at an absorbance of 0.5 at 550 nm, was used to favor the growth of the less competitive strain. Beads immobilizing real cultures of and subsp. biovar diacetylactis strains were also prepared using the same procedure but with an absorbance-standardized inoculum of 2% (vol/vol) in the polymer answer. All operations were then carried out with 0.1 M KCl to keep the bead structure. Beads coentrapping and subsp. biovar diacetylactis strains were incubated in supplemented MRS medium during six successive pH-controlled batch cultures for 16, 12, 8, 6, 4, and 4 h at 37C in a 500-ml bioreactor (BioFlo model C30; New Brunswick Scientific Co., Edison, N.J.), with CO2 injections in the headspace. Beads entrapping real cultures were incubated separately for only two successive fermentations of 16 and 8 h in appropriate medium. The bioreactor was inoculated with 20% (vol/vol) gel beads, pH was kept at 6 by addition of 6 M NH4OH, and mixing was set at 200 rpm. Beads coentrapping and subsp. biovar diacetylactis strains were.