Insights into lactic acid bacteria cryoresistance using FTIR microspectroscopy.

Freezing is widely used for bacterial cell preservation. However, resistance to freezing can greatly vary depending on bacterial species or growth conditions. Our study aims at identifying cellular markers of cryoresistance based on the comparison of three lactic acid bacteria (LAB) exhibiting different tolerance to freezing: Carnobacterium maltaromaticum CNCM I-3298, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, and Lactobacillus delbrueckii subsp. bulgaricus CFL1. A thorough characterization of their cytoplasmic membrane properties was carried out by measuring their fatty acid composition, membrane fluidity, and lipid phase transition upon cooling from 50 to -50 °C. Vitrification temperatures of the intra- and extra-cellular compartments were also quantified by differential scanning calorimetry. Additionally, the cell biochemical characterization was carried out using a recently developed Fourier transform infrared (FTIR) micro-spectroscopic approach allowing the analysis of live bacteria in an aqueous environment. The multivariate analysis of the FTIR spectra of fresh and thawed cells enabled the discrimination of the three bacteria according to their lipid, protein, and cell wall peptidoglycan components. It also revealed freezing-induced modifications of these three cellular components and an increase in bacteria heterogeneity for the two strains of L. bulgaricus, the freeze-sensitive bacteria. No cellular damage was observed for C. maltaromaticum, the freeze-resistant bacteria. Comparison of the results obtained from the different analytical methods confirmed previously reported cryoresistance markers and suggested new ones, such as changes in the absorbance of specific infrared spectral bands. FTIR microspectroscopy could be used as a rapid and non-invasive technique to evaluate the freeze-sensitivity of LAB.

Dynamic Modeling of Carnobacterium maltaromaticum CNCM I-3298 Growth and Metabolite Production and Model-Based Process Optimization.

Carnobacterium maltaromaticum is a species of lactic acid bacteria found in dairy, meat, and fish, with technological properties useful in food biopreservation and flavor development. In more recent years, it has also proven to be a key element of biological time-temperature integrators for tracking temperature variations experienced by perishable foods along the cold-chain. A dynamic model for the growth of C. maltaromaticum CNCM I-3298 and production of four metabolites (formic acid, acetic acid, lactic acid, and ethanol) from trehalose in batch culture was developed using the reaction scheme formalism. The dependence of the specific growth and production rates as well as the product inhibition parameters on the operating conditions were described by the response surface method. The parameters of the model were calibrated from eight experiments, covering a broad spectrum of culture conditions (temperatures between 20 and 37 °C; pH between 6.0 and 9.5). The model was validated against another set of eight independent experiments performed under different conditions selected in the same range. The model correctly predicted the growth kinetics of C. maltaromaticum CNCM I-3298 as well as the dynamics of the carbon source conversion, with a mean relative error of 10% for biomass and 14% for trehalose and the metabolites. The paper illustrates that the proposed model is a valuable tool for optimizing the culture of C. maltaromaticum CNCM I-3298 by determining operating conditions that favor the production of biomass or selected metabolites. Model-based optimization may thus reduce the number of experiments and substantially speed up the process development, with potential applications in food technology for producing starters and improving the yield and productivity of the fermentation of sugars into metabolites of industrial interest.

Freeze-Drying of Lactic Acid Bacteria: A Stepwise Approach for Developing a Freeze-Drying Protocol Based on Physical Properties.

Freeze-drying or lyophilization has become a reference process for preserving lactic acid bacteria. The development of stable freeze-dried lactic acid bacteria (LAB) requires maintaining the biological activity of the cells and the macroscopic porous structure while increasing the efficiency of the manufacturing process. Physical properties of protective solutions, such as glass transition and collapse temperatures, are key elements not only for process optimization but also for the stability of freeze-dried LAB. This chapter provides a stepwise approach for developing a protective formulation for the long-term preservation of LAB and an efficient freeze-drying process. Methods for determining glass transition and collapse temperatures of protective solutions and cell suspensions, as well as water activity and water content of freeze-dried products, are described.