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 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.

Accelerostat study in conventional and microfluidic bioreactors to assess the key role of residual glucose in the dimorphic transition of Yarrowia lipolytica in response to environmental stimuli.

Yarrowia lipolytica, with a diverse array of biotechnological applications, is able to grow as ovoid yeasts or filamentous hyphae depending on environmental conditions. This study has explored the relationship between residual glucose levels and dimorphism in Y. lipolytica. Under pH stress conditions, the morphological and physiological characteristics of the yeast were examined during well-controlled accelerostat cultures using both a 1 L-laboratory scale and a 1 mL-microfluidic bioreactor. The accelerostat mode, via a smooth increase of dilution rate (D), enabled the cell growth rate to increase gradually up to the cell wash-out (D ≥µmax of the strain), which was accompanied by a progressive increase in residual glucose concentration. The results showed that Y. lipolytica maintained an ovoid morphology when residual glucose concentration was below a threshold value of around 0.35-0.37 mg L-1. Transitions towards more elongated forms were triggered at this threshold and progressively intensified with the increase in residual glucose levels. The effect of cAMP on the dimorphic transition was assessed by the exogenous addition of cAMP and the quantification of its intracellular levels during the accelerostat. cAMP has been reported to be an important mediator of environmental stimuli that inhibit filamentous growth in Y. lipolytica by activating the cAMP-PKA regulatory pathway. It was confirmed that the exogenous addition of cAMP inhibited the mycelial morphology of Y. lipolytica, even with glucose concentrations exceeding the threshold level. The results suggest that dimorphic responses in Y. lipolytica are regulated by sugar signaling pathways, most likely via the cAMP-PKA dependent pathway.

A Guide for Ex Vivo Handling and Storage of Stool Samples Intended for Fecal Microbiota Transplantation.

Owing to the growing recognition of the gut microbiota as a main partner of human health, we are expecting that the number of indications for fecal microbiota transplantation (FMT) will increase. Thus, there is an urgent need for standardization of the entire process of fecal transplant production. This study provides a complete standardized procedure to prepare and store live and ready-to-use transplants that meet the standard requirements of good practices to applied use in pharmaceutical industry. We show that, if time before transformation to transplants would exceed 24 hours, fresh samples should not be exposed to temperatures above 20 °C, and refrigeration at 4 °C can be a safe solution. Oxygen-free atmosphere was not necessary and simply removing air above collected samples was sufficient to preserve viability. Transplants prepared in maltodextrin-trehalose solutions, stored in a -80 °C standard freezer and then rapidly thawed at 37 °C, retained the best revivification potential as  proven by 16S rRNA profiles, metabolomic fingerprints, and flow cytometry assays over a 3-month observation period. Maltodextrin-trehalose containing cryoprotectants were also efficient in preserving viability of lyophilized transplants, either in their crude or purified form, an option that can be attractive for fecal transplant biobanking and oral formulation.

Subcellular membrane fluidity of Lactobacillus delbrueckii subsp. bulgaricus under cold and osmotic stress.

Cryopreservation of lactic acid bacteria may lead to undesirable cell death and functionality losses. The membrane is the first target for cell injury and plays a key role in bacterial cryotolerance. This work aimed at investigating at a subcellular resolution the membrane fluidity of two populations of Lactobacillus delbrueckii subsp. bulgaricus when subjected to cold and osmotic stresses associated to freezing. Cells were cultivated at 42 °C in mild whey medium, and they were exposed to sucrose solutions of different osmolarities (300 and 1800 mOsm L-1) after harvest. Synchrotron fluorescence microscopy was used to measure membrane fluidity of cells labeled with the cytoplasmic membrane probe 1-[4 (trimethylamino) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Images were acquired at 25 and 0 °C, and more than a thousand cells were individually analyzed. Results revealed that a bacterial population characterized by high membrane fluidity and a homogeneous distribution of fluidity values appeared to be positively related to freeze-thaw resistance. Furthermore, rigid domains with different anisotropy values were observed and the occurrence of these domains was more important in the freeze-sensitive bacterial population. The freeze-sensitive cells exhibited a broadening of existing highly rigid lipid domains with osmotic stress. The enlargement of domains might be ascribed to the interaction of sucrose with membrane phospholipids, leading to membrane disorganization and cell degradation.

Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation.

During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms.

Understanding the cryotolerance of lactic acid bacteria using combined synchrotron infrared and fluorescence microscopies.

Freezing is widely used for preserving different types of cells. Frozen concentrates of lactic acid bacteria (LAB) are extensively used for manufacturing food, probiotic products and for green chemistry and medical applications. However, the freezing and thawing processes cause cell injuries that result in significant cell death. Producing homogeneous bacterial populations with high cryotolerance remains a real challenge. Our objective was to investigate the biochemical and physiological changes in a LAB model at the cell scale following fermentation and freezing in order to identify cellular biomarkers of cryotolerance. Infrared spectra of individual bacteria produced by applying different fermentation and freezing conditions were acquired using synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy to achieve sub-cellular spatial resolution. Fluorescent microscopy was concomitantly assessed, thus making possible to simultaneously analyse the biochemistry and physiological state of a single cell for the first time. Principal component analysis was used to evaluate changes in cell composition, with particular focus on lipids, proteins and polysaccharides. SR-FTIR results indicated that before freezing, freeze-resistant cells grown in a rich medium presented a high content of CH3 groups from lipid chains, of cell proteins in an α-helix secondary structure and of charged polymers such as teichoic and lipoteichoic acids that constitute the Gram-positive bacterial wall. Moreover, SR-FTIR microspectroscopy made it possible to reveal cell heterogeneity within the cluster of resistant cells, which was ascribed to the diversity of potential substrates in the growth medium. Freezing and thawing processes induced losses of membrane integrity and cell viability in more than 90% of the freeze-sensitive bacterial population. These damages leading to cell death were ascribed to biochemical modification of cell membrane phospholipids, in particular a rigidification of the cytoplasmic membrane following freezing. Furthermore the freeze-resistant cells remained viable after freezing and thawing but a modification of protein secondary structure was detected by SR-FTIR analysis. These results highlighted the potential application of bimodal analysis by SR-FTIR and fluorescence microscopy to increase our knowledge about mechanisms related to cell damage.

Freeze-drying of lactic acid bacteria.

Lactic acid bacteria are of great importance for the food and biotechnology industry. They are widely used as starters for manufacturing food (e.g., yogurt, cheese, fermented meats, and vegetables) and probiotic products, as well as for green chemistry applications. Freeze-drying or lyophilization is a convenient method for preservation of bacteria. By reducing water activity to values below 0.2, it allows long-term storage and low-cost distribution at suprazero temperatures, while minimizing losses in viability and functionality. Stabilization of bacteria via freeze-drying starts with the addition of a protectant solution to the bacterial suspension. Freeze-drying includes three steps, namely, (1) freezing of the concentrated and protected cell suspension, (2) primary drying to remove ice by sublimation, and (3) secondary drying to remove unfrozen water by desorption. In this chapter we describe a method for freeze-drying of lactic acid bacteria at a pilot scale, thus allowing control of the process parameters for maximal survival and functionality recovery.