Mechanistic study of the differences in lactic acid bacteria resistance to freeze- or spray-drying and storage.

Lactobacillus delbrueckii subsp. bulgaricus and Lactiplantibacillus plantarum are two lactic acid bacteria (LAB) widely used in the food industry. The objective of this work was to assess the resistance of these bacteria to freeze- and spray-drying and study the mechanisms involved in their loss of activity. The culturability and acidifying activity were measured to determine the specific acidifying activity, while membrane integrity was studied by flow cytometry. The glass transitions temperature and the water activity of the dried bacterial suspensions were also determined. Fourier transform infrared (FTIR) micro-spectroscopy was used to study the biochemical composition of cells in an aqueous environment. All experiments were performed after freezing, drying and storage at 4, 23 and 37 °C. The results showed that Lb. bulgaricus CFL1 was sensitive to osmotic, mechanical, and thermal stresses, while Lpb. plantarum WCFS1 tolerated better the first two types of stress but was more sensitive to thermal stress. Moreover, FTIR results suggested that the sensitivity of Lb. bulgaricus CFL1 to freeze-drying could be attributed to membrane and cell wall degradation, whereas changes in nucleic acids and proteins would be responsible of heat inactivation of both strains associated with spray-drying. According to the activation energy values (47-85 kJ/mol), the functionality loss during storage is a chemically limited reaction. Still, the physical properties of the glassy matrix played a fundamental role in the rates of loss of activity and showed that a glass transition temperature 40 °C above the storage temperature is needed to reach good preservation during storage. KEY POINTS: • Specific FTIR bands are proposed as markers of osmotic, mechanic and thermal stress • Lb. bulgaricus CFL1 was sensitive to all three stresses, Lpb. plantarum WCFS1 to thermal stress only • Activation energy revealed chemically limited reactions ruled the activity loss in storage.

Insights into the mechanisms of L. salivarius CECT5713 resistance to freeze-dried storage.

Ligilactobacillus salivarius is a lactic acid bacterium exhibiting several health benefits. However, it is sensitive to freeze-drying and storage in the dried state, thus limiting its commercial exploitation. Our objective was to identify markers of cell resistance by applying multiscale characterization to L. salivarius CECT5713 cell populations exhibiting different resistance to freeze-dried storage. Cells were produced under two different sets of production conditions differing in the culture parameters (temperature, neutralizing solution, and harvesting time) and the protective formulation composition. The culturability, membrane integrity, and cell biochemical composition assessed by Fourier transform infrared (FTIR) micro-spectroscopy were evaluated after freezing, freeze-drying, and subsequent storage at 37 °C. Membrane properties (fatty acid composition, membrane fluidity, and phospholipid organization), as well as matrix physical properties (glass transition temperature and water activity), were determined. The most resistant cells to freeze-dried storage exhibited the highest cyclic fatty acid content and the most rigid membrane. Freeze-drying and storage induced damage to membrane integrity, proteins, nucleic acids, and constituents of the peptidoglycan cell wall. From the FTIR spectra analysis, we propose the minimization of the variations of the 1058 and 1714 cm-1 vibration bands (that arise mainly from symmetric C-O-C stretching and CO stretching, respectively) induced by the freeze-drying process as a marker of storage stability. We confirmed that a matrix with a glass transition temperature at least 50 °C higher than the storage temperature is crucial for L. salivarius CECT5713 storage stability. In addition, this work explored promising FTIR methods for a better understanding of the protection mechanisms involved.

A rapid method for the assessment of the vitality of microorganisms using flow cytometry.

Cultivability, viability, and vitality make it possible to characterize the behavior of a cellular population. Vitality was assessed using the kinetic parameters of specific metabolisms depending on whether the strains were used, for example, for the acidification of lactic acid bacteria or for CO2 production in fermenting yeasts. However, these methods are time-consuming. We developed a cytometric descriptor based on the energy-dependent extrusion of carboxyfluorescein from cells, subsequent to carboxyfluorescein diacetate staining, and compared it to the measurements of metabolic activities of various bacteria and yeasts. For all of the microorganisms tested, the cytometric descriptor ΔFI15 was well correlated with the results of the metabolic measurements and, moreover, has the advantage of being easier and faster to use than metabolic methods. It can be very useful for evaluating the vitality of the starters before inoculation in industrial processes.

Environmental conditions during glycerol bioconversion affect 3-hydroxypropionic acid bioproduction by Limosilactobacillus reuteri DSM 17938.

3-Hydroxypropionic acid (3-HP) is a platform molecule whose biological production was carried out by the bacterium Limosilactobacillus reuteri according to a two-step process: first, a growth phase in batch mode on glucose, then a glycerol bioconversion into 3-HP in fed-batch mode. With the objective of improving 3-HP bioproduction, this study aimed at defining the operating conditions during the bioconversion phase that increases the bioproduction performance. A central composite rotatable design allowed testing various pH levels and specific glycerol feeding rates. By establishing response surfaces, optimal conditions have been identified that were different depending on the considered output variable (final 3-HP quantity, 3-HP production yield and production rate). Of them, 3-HP final quantity and 3-HP production yield were maximized at pH 6.0 and at specific glycerol feeding rates of 60 and 55 mggly  gCDW -1  h-1 , respectively. The specific 3-HP production rate was the highest at the upper limit of the specific substrate feeding rate (80 mggly  gCDW -1  h-1 ) but was not affected by the pH. An additional experiment was carried out at pH 6.0 and a specific glycerol feeding rate of 80 mggly  gCDW -1  h-1 to validate the previous observations. In conclusion, the results showed a significant improvement of 3-HP concentration by 13%, of specific production rate by 34% and of 3-HP volumetric productivity by 39%, as compared to the initial values.

Physiological and biochemical responses of Yarrowia lipolytica to dehydration induced by air-drying and freezing.

Organisms that can withstand anhydrobiosis possess the unique ability to temporarily and reversibly suspend their metabolism for the periods when they live in a dehydrated state. However, the mechanisms underlying the cell's ability to tolerate dehydration are far from being fully understood. The objective of this study was to highlight, for the first time, the cellular damage to Yarrowia lipolytica as a result of dehydration induced by drying/rehydration and freezing/thawing. Cellular response was evaluated through cell cultivability determined by plate counts, esterase activity and membrane integrity assessed by flow cytometry, and the biochemical composition of cells as determined by FT-IR spectroscopy. The effects of the harvesting time (in the log or stationary phase) and of the addition of a protective molecule, trehalose, were investigated. All freshly harvested cells exhibited esterase activity and no alteration of membrane integrity. Cells freshly harvested in the stationary phase presented spectral contributions suggesting lower nucleic acid content and thicker cell walls, as well as longer lipid chains than cells harvested in the log phase. Moreover, it was found that drying/rehydration induced cell plasma membrane permeabilization, loss of esterase activity with concomitant protein denaturation, wall damage and oxidation of nucleic acids. Plasma membrane permeabilization and loss of esterase activity could be reduced by harvesting in the stationary phase and/or with trehalose addition. Protein denaturation and wall damage could be reduced by harvesting in the stationary phase. In addition, it was shown that measurements of loss of membrane integrity and preservation of esterase activity were suitable indicators of loss and preservation of cultivability, respectively. Conversely, no clear effect of freezing/thawing could be observed, probably because of the favorable operating conditions applied. These results give insights into Y. lipolytica mechanisms of cellular response to dehydration and provide a basis to better understand its ability to tolerate anhydrobiosis.

Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage.

Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.