Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health effect on the host” [1]. One of the primary effect of probiotics in humans is their antipathogenic effects [[2], [3], [4]]. They are believed to produce antimicrobial compounds which includes acidic metabolites and bacteriocins that inhibit pathogens [5,6]. They are also believed to reduce intestinal permeability, adhere onto the cell surfaces of its hosts, preventing pathogenic bacteria from attaching to the same surfaces and compete for nutrients with pathogens [7,8]. Another important mechanism in their antipathogenic effect is their ability to stimulate host's immunity [9].

Demonstration of direct antagonism through inhibitory substance production of probiotics is often assessed for potential probiotic candidates to establish antipathogenic activity. This is routinely demonstrated through diffusion tests or co-culture assays where test microorganism is co-cultured with potential probiotic supernatants or the live probiotic. Co-culture assays with live microorganisms could be laborious, requiring continuous sampling, serial dilutions, and plate assays if the experimenter wants to determine the profile of growth. Another disadvantage of the experiment is that data could be far from reality because of the retrospective nature of data collection. Furthermore, when molecular methods are combined, the assays become more expensive, requiring other skills and expertise. More experimenters therefore prefer the diffusion method for routine determination of antipathogenic activity of probiotics [[10], [11], [12], [13], [14]]. The diffusion test has its own limitation, for instance, the dependence of the inhibitory metabolite to diffuse irrespective of size or potential interaction in a solid medium [15,16]. In our previous study, we compared isothermal microcalorimetry with the agar diffusion and broth culture assays for assessing the inhibitory activity of probiotic culture supernatants. The results demonstrated that isothermal microcalorimetry could detect inhibitory activity of neutralised probiotic culture supernatant which could not be detected with the traditional broth culture and the agar well diffusion methods [15].

The importance of co-culture assays with live microorganisms cannot be ignored. For instance, they help us understand specific interactions and patterns that occur amongst microorganisms, which has led to some breakthroughs and discoveries and enhanced our insights in microbial ecology. The current approach in these co-culture assays with live microorganisms as mentioned, could be time intensive, labour intensive, require special equipment or expensive when molecular methods are combined. We propose an isothermal microcalorimetric method for determining growth profile of mixed cultures of microorganisms. Briefly, isothermal microcalorimetry is a technique based on the principle of measurement of heat. It measures heat flow of physical, chemical and biological processes. It has been previously used in the study of bacterial growth kinetics [15,17]. The technique is simple and offers the opportunity to monitor the growth of live microorganisms in real time. However, its application is often limited to monoculture bacterial assays. Its potential in mixed bacterial assays, and in application to live probiotic assays have not been clearly understood yet.

In this work, the microcalorimeter was explored to study the growth of Lactobacillus acidophilus, Bifidobacterium lactis and Bifidobacterium bifidum probiotic species in mixed culture with Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli.