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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.
Economic Analysis of the Production Process of Probiotics Based on the Biological and Physiological Parameters of the Cells
Probiotic bacteria confer a range of health benefits and are a focus of a growing number of studies. One of the main issues is their stability during drying and storage, which is why techniques, such as fluid bed drying and coating or treatment with stress factors during culturing, are utilized. The methods of the evaluation of probiotic viability and quality are, however, lacking and we need a way of distinguishing between different subpopulations of probiotic bacteria. To address this issue, imaging flow cytometry (IFC) has been utilized to assess cells after simulated in vitro digestion of dried and coated preparations treated with pH stress and heat shock. Samples were analyzed fresh and after 12 months of storage using RedoxSensor green and propidium iodide dyes to assess metabolic activity and cell membrane integrity of the cells. The results were then used to design a drying process on an industrial scale and evaluate the economic factors in the SuperPro Designer v13 software. Based on the number of biologically active and beneficial cells obtained utilizing tested methods, the coating process and treatment with heat shock and pH stress have been the most effective and up to 10 times cheaper to produce than only by drying. Additionally, samples after 12 months of storage have shown an increase in the proportion of cells with intermediate metabolic activity and small amounts of cell membrane damage, which are still viable in probiotic products. This subpopulation of bacteria can still be considered live in probiotic products but is not necessarily effectively detected by pour plate counts.
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.