The large number of probiotic products now available makes the decision about which product to choose difficult both for the consumer and for the specialist providing dietary/nutritional advice. Data on the viability of the bacteria in these products, in the in-vivo situation, are therefore important. This study was designed to explore the comparative health and survival of probiotic species in various commercial formulations, using more realistic test systems that might allow further understanding of factors that must be controlled to give the greatest chance of delivery of live healthy bacteria to the lower gut.A total of eight commercially available probiotic preparations were selected for enumeration tests and in-vitro gastric tolerance tests. Tolerance assays were conducted in porcine gastric fluid (PGF) fed and fasted state (pH 3.4 ± 0.04), simulated gastric fluid (SGF, pH adjusted to 1.2 and 3.4) and fasted state simulated gastric fluid (FaSSGF, pH adjusted to 1.6 and 3.4). Isothermal microcalorimetry was also used to measure real-time growth of probiotics after exposure to simulated gastric fluid.Results from the enumeration tests indicated that recovery of viable organisms per dose is the same as or better than the stated label claims for liquid-based formulations but lower than the stated claim for freeze-dried products. Results from the in-vitro tolerance tests overall suggest that the PGF provided a harsher environment than the simulated systems at similar pH. In general, liquid-based products tested tended to give superior results in terms of survival compared with the freeze-dried products tested. Results from tests in the fed state in PGF suggested that food greatly affects viability. Microcalorimetric data showed that for some products probiotic species were able to grow following exposure to gastric fluid, suggesting that viable bacteria reach the gut in-vivo.

The aim of the study was to investigate the influence of some commercial probiotics on the growth of Clostridium difficile using the isothermal microcalorimeter, an instrument which can monitor the real time growth of bacteria. Commercial probiotic strains and products, Lactobacillus acidophilus LA-5 ® , Bifidobacterium lactis BB-12 ® , Probio 7 ® and Symprove tm were co-cultured with C. difficile in Brain Heart Infusion (BHI) broth supplemented with 0.1% (w/v) L-cysteine hydrochloride and 0.1% (w/v) sodium taurocholate and monitored in the microcalorimeter. Pseudomonas aeruginosa NCIMB 8628 was also co-cultured with C. difficile and studied. The results indicated inhibition of C. difficile by the probiotics. The inhibition of C. difficile was shown to be pH-dependent using neutralized and unmodified cell free supernatant (CFS) produced by the probiotic strains. However, concentrated CFS of the probiotics also inhibited the intestinal pathogen in a non pH-dependent manner, likely due to specific antimicrobial substances produced. The results also indicated that C. difficile growth was greatly influenced by the presence of sodium taurocholate and by the pH of the medium. A medium pH of between 6.45 and 6.9 demonstrated maximum growth of the organism in the microcalorimeter.

Cell free supernatants (CFS) obtained from probiotic species are routinely used to preliminary investigate the antimicrobial activity of potential probiotic isolates by the agar well diffusion and broth culture. Both methods have documented limitations. In this work, the potential of isothermal microcalorimetry (IMC), a technique based on the measurement of heat produced by growing bacteria was used to investigate the antimicrobial effects of two commercial probiotic species (Lactobacillus acidophilus LA-5 ® and Bifidobacterium lactis BB-12 ® ) and one reference strain (Bifidobacterium bifidum ATCC 11863) against Pseudomonas aeruginosa NCIMB 8628 using unmodified, neutralised and heat-treated CFS. P. aeruginosa was inhibited in growth by the unmodified CFS of all the species. No inhibitory activity was recorded for neutralised CFS of all the species using the agar well diffusion assay. Plate count during co-incubation of P. aeruginosa with the neutralised CFS of all the species showed no inhibition. However, IMC data showed significant inhibition with neutralised CFS obtained from the two Bifidobacterium species suggesting presence of other non-acidic inhibitory compounds in the CFS. The results in this work demonstrated that IMC has potential in probiotic bioassay as it has the capability to record in real-time and capture even subtle effects, which could be unnoticed with traditional assays.

17Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus 18and Enterococcus faecium, which are the component species of a 19 commercially available probiotic mixture (Symprove ® , P1), were grown in co-20 culture to determine whether they would inhibit each other in vitro using an 21 isothermal microcalorimeter (IMC). The growth profiles in the IMC were 22 characteristic and unique to each species while the growth profile of P1 was 23 most similar to that of L. plantarum, suggesting this is the dominant organism 24 in mixed-culture. Bacterial growth in the cell free supernatants (CFS) of the 25 probiotic species were also evaluated by IMC and viable counts determined at 26 the end of the incubation period. L. plantarum was found to be the most 27 effective species at inhibiting L. rhamnosus. Conversely, L. rhamnosus was 28 the most effective at limiting the growth of L. plantarum. Both L. plantarum 29 and L. rhamnosus were inhibitory toward L. acidophilus and E. faecium. E. 30 faecium was the least inhibitory towards all the other species. The study 31shows how complex, multi-species probiotic products can be analysed to 32 determine the predominant species, and so provides a route to formulation of 33 new products. 34