Biophysical characterization of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and osmotic stress and its relevance for cryopreservation

Abstract

Freezing lactic acid bacteria often leads to cell death and loss of technological properties. Our objective was to provide an in-depth characterization of the biophysical properties of the Lactobacillus delbrueckii subsp. bulgaricus membrane in relation to its freeze resistance. Freezing was represented as a combination of cold and osmotic stress. This work investigated the relative incidence of increasing sucrose concentrations coupled or not with subzero temperatures without ice nucleation on the biological and biophysical responses of two strains with different membrane fatty acid compositions and freeze resistances. Following exposure of bacterial cells to the highest sucrose concentration, the sensitive strain exhibited a survival rate of less than 10 % and 5 h of acidifying activity loss. Similar biological activity losses were observed upon freeze-thawing and after osmotic treatment for each strain thus highlighting osmotic stress as the main source of cryoinjury. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to membrane lipid organization characterized by FTIR spectroscopy. Both approaches made it possible to investigate the specific contributions of the membrane core and the bilayer external surface to cell degradation caused by cold and osmotic stress. Cold-induced membrane rigidification had no significant implication on bacterial freeze-thaw resistance. Interactions between extracellular sucrose and membrane phospholipid headgroups under osmotic stress were also observed. Such interactions were more evident in the sensitive strain and when increasing sucrose concentration, thus suggesting membrane permeabilization. The relevance of biophysical properties for elucidating mechanisms of cryoinjury and cryoprotection is discussed.

Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes

“…The concentration of sucrose was likely too low to induce a significant osmotic stress. Moderate osmotic stress was shown to occur at much higher concentration of sucrose (730 mM) for Lactobacillus delbrueckii (Meneghel et al ., ). In another study, growth inhibition of Staphylococcus aureus , Listeria monocytogenes , Cronobacter sakazakii , Enterococcus faecium , Escherichia coli and Salmonella Typhimurium was observed in growth medium supplemented with sucrose concentrations ranging from 1.1 to 1.8 M (Cebrian et al ., ).…”

Factors influencing the membrane fluidity and the impact on production of lactic acid bacteria starters

“…Besides, the phosphate symmetric stretching vibration band (vPO 2 − asym, around 1220 cm −1 ) has been employed for studying the interaction between phospholipid headgroups and the extracellular environment of LAB cells (Meneghel et al 2017b).…”

“…Moreover, research works on changes of membrane physical properties due to process conditions are also scarcely reported. To our knowledge, only two works have investigated simultaneously fatty acid composition, membrane phase transition, and fluidity in relation with the LAB resistance to process (Velly et al 2015;Meneghel et al 2017b).…”

“…The fermentation conditions and the resulting membrane lipid composition are commonly related to the resistance of LAB to stabilization processes. Even it is well admitted that membrane fluidity is governed by the fatty acid composition of the membrane, few works report direct assessment of membrane fluidity after fermentation (Velly et al 2015;Bouix and Ghorbal 2017) and following stabilization process (Schwab et al 2007;Passot et al 2014;Meneghel et al 2017b).…”

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus

“…Among various lactic acid bacteria used in starters, L. bulgaricus is extremely labile to various operations used to produce concentrated cultures (Wright and Klaenhammer, 1983a;Santos et al, 2014;Meneghel et al, 2017). Loss of viability at every stage-concentration, freezing, and lyophilization-occurs with L. bulgaricus, even when high populations are attained at the end of propagation.…”