The Effects of Cellular Membrane Damage on the Long-Term Storage and Adhesion of Probiotic Bacteria in Caco-2 Cell Line.

Adhesion is one of the main factors responsible for the probiotic properties of bacteria in the human gut. Membrane proteins affected by cellular damage are one of the key aspects determining adhesion. Fluid-bed-dried preparations containing probiotic bacteria were analyzed in terms of their stability (temperature of glass transition) and shelf life in different conditions (modified atmosphere, refrigeration). Imaging flow cytometry was utilized to determine four subpopulations of cells based on their physiological and morphological properties. Lastly, adhesion was measured in bacteria cultured in optimal conditions and treated with heat shock. The results show that the subpopulations with no or low levels of cell membrane damage exhibit the ability to adhere to Caco-2 cells. The temperature of protein denaturation in bacteria was recorded as being between 65 °C and 70 °C. The highest glass transition temperature (Tg) value for hydroxypropyl methylcellulose (used as a coating substance) was measured at 152.6 °C. Drying and coating can be utilized as a sufficient treatment, allowing a long shelf-life (up to 12 months). It is, however, worth noting that technological processing, especially with high temperatures, may decrease the probiotic value of the preparation by damaging the bacterial cells.

Imaging Flow Cytometry Demonstrates Physiological and Morphological Diversity within Treated Probiotic Bacteria Groups.

Probiotic bacteria can be introduced to stresses during the culturing phase as an alternative to the use of protectants and coating substances during drying. Accurate enumeration of the bacterial count in a probiotic formulation can be provided using imaging flow cytometry (IFC). IFC overcomes the weak points of conventional, commonly used flow cytometry by combining its statistical power with the imaging content of microscopy in one system. Traditional flow cytometers only collect the fluorescence signal intensities, while IFC provides many more steps as it correlates the data on the measured parameters of fluorescence light with digitally processed images of the analyzed cells. As an alternative to standard methods (plate cell counts and traditional flow cytometry) IFC provides additional insight into the physiology and morphology of the cell. The use of complementary dyes (RedoxSensorTM Green and propidium iodide) allows for the designation of groups based on their metabolic activity and membrane damage. Additionally, cell sorting is incorporated to assess each group in terms of growth on different media (MRS-Agar and MRS broth). Results show that the groups with intermediate metabolic activity and some degree of cellular damage correspond with the description of viable but nonculturable cells.

Current Trends in the Production of Probiotic Formulations.

Preparations containing probiotic strains of bacteria have a beneficial effect on human and animal health. The benefits of probiotics translate into an increased interest in techniques for the preservation of microorganisms. This review compares different drying methods and their improvements, with specific reference to processing conditions, microorganisms, and protective substances. It also highlights some factors that may influence the quality and stability of the final probiotic preparations, including thermal, osmotic, oxidative, and acidic stresses, as well as dehydration and shear forces. Processing and storage result in the loss of viability and stability in probiotic formulations. Herein, the addition of protective substances, the optimization of process parameters, and the adaptation of cells to stress factors before drying are described as countermeasures to these challenges. The latest trends and developments in the fields of drying technologies and probiotic production are also discussed. These developments include novel application methods, controlled release, the use of food matrices, and the use of analytical methods to determine the viability of probiotic bacteria.