Proof of concept: real-time viability and metabolic profiling of probiotics with isothermal microcalorimetry

Abstract

Isothermal microcalorimetry (IMC) is a potent analytical method for the real-time assessment of microbial metabolic activity, which serves as an indicator of microbial viability. This approach is highly relevant to the fields of probiotics and Live Biotherapeutic Products (LBPs), offering insights into microbial viability and growth kinetics. One important characteristic of IMC is its ability to measure microbial metabolic activity separately from cellular enumeration. This is particularly useful in situations where continuous tracking of bacterial activity is challenging. The focus on metabolic activity significantly benefits both probiotic research and industrial microbiology applications. IMC's versatility in handling different media matrices allows for the implementation of viability assessments under conditions that mirror those found in various industrial environments or biological models. In our study, we provide a proof of concept for the application of IMC in determining viability and growth dynamics and their correlation with bacterial count in probiotic organisms. Our findings reinforce the potential of IMC as a key method for process enhancement and accurate strain characterization within the probiotic sector. This supports the broader objective of refining the systematic approach and methods used during the development process, thereby providing detailed insights into probiotics and LBPs.

Keywords: beneficial microbes; isothermal microcalorimetry; metabolic activity; probiotics; real-time; viability assessment; viability enumeration.

FIGURE 1

Isothermal microcalorimetry (biocalorimetry) principle. Isothermal incubation of a microbial culture in a vial positioned above a heat sensor on a heat sink. As metabolically active cells release energy, a byproduct of metabolic pathway reactions, the generated heat disperses over the heat sensor toward the heat sink. The heat sensor detects and converts these minute heat changes into an analog signal in μW. Heat flow is continuously measured every 2 s, enabling real-time monitoring of the sample’s activity.

FIGURE 2

Robustness and reproducibility of isothermal microcalorimetric measurements using the calScreener™. Thermograms of two independent experiments on different days of L. plantarum started from independent batches of commercial probiotic product. Shading of each curve shows the SD of the 32 individual replicates per experiment.

FIGURE 3

Serial dilution of L. plantarum to build a standard curve. (A) Thermograms of serial dilution of overnight culture of L. plantarum in MRS broth. Initial inoculum sizes are depicted above the thermograms. (B) For standard curve, the time to peak (TTP) of each dilution from the thermogram was determined and values were plotted against CFU/ml of respective dilution as confirmed by plate count. Relationship between TTP and CFU was described in a linear regression model.

FIGURE 4

Correlation of TTP and CFU is universal independent from medium type. Metabolic activity of serial dilution of an overnight culture of L. plantarum in 1:1 MRS and oat drink. Dilution factors are indicated above thermograms.

FIGURE 5

Reproducibility of standard curve under different pre-culture and incubation conditions of L. plantarum. Step-wise dilutions of L. plantarum (10⁹ CFU/ml) overnight culture in MRS or a one-to-one MRS medium-oat drink mixture or step-wise dilution of reconstituted powder of commercial freeze-dried formulation of L. plantarum in MRS. Measurement by IMC and confirmation of initial cell number by plate counting. For the standard curves, TTP and CFU values were plotted on x–y axes.

FIGURE 6

Real-time metabolic activity measurements to complement acidification and cell count data. Three growth medium formulations, medium A, B and C, were evaluated using acidification curves, isothermal microcalorimetry (plotted in panels A,C) and time point measurements using flow cytometry (shown in panels B,D). Two different strains, L. rhamnosus and L. fermentum, were used for the experiments. IMC data are presented as heat flow in μW, acidification curves depict pH values and for flow cytometry fluorescent units (FU) with total FU (TFU) and alive FU (AFU) are displayed.