Increased Survival of Lactococcus lactis Strains Subjected to Freeze-Drying after Cultivation in an Acid Medium: Involvement of Membrane Fluidity

Research background. Freeze-drying is the most widely used dehydration process in the food industry for the stabilization of bacteria. Studies have shown the effectiveness of an acid pre-stress in increasing the resistance of lactic acid bacteria strains to freeze-drying. Adaptation of bacteria to an acid stress is based on maintaining the properties of the plasma membrane. Indeed, the fatty acid composition of lactic acid bacteria membrane is often changed after an acid pre-stress. However, few studies have measured membrane fluidity after an acid stress realized during lactic acid bacteria strains cultivation. Experimental approach. In order to use two pH profiles, the strains Lactococcus lactis NCDO 712 and NZ9000 were cultivated in two media, without any pH control. The two pH profiles obtained were representative of initial media composition, media buffering properties, and strains metabolism. Absorbance at 600 nm and pH were measured during bacterial cultivation. Then, the two strains were freeze-dried and their survival rates determined. Membrane fluidity was evaluated by fluorescence anisotropy measurements using a spectrofluorometer. Results and conclusions. Cultivation under a more acidic condition significantly increased both strains survival to freeze-drying (p<0.05, ANOVA). Moreover, in both strains of L. lactis, a more acidic condition during cultivation significantly increased membrane fluidity (p<0.05, ANOVA). Our results revealed that cultivation in such condition, fluidifies the membrane and allows a better survival to freeze-drying for the two strains of L. lactis. A more fluid membrane can facilitate membrane deformation and lateral reorganization of membrane components, critical for the maintenance of cellular integrity during dehydration and rehydration. Novelty and scientific contribution: A better understanding of the involvement of membrane properties, especially of membrane fluidity, in bacterial resistance to dehydration is provided in this study.

Zeta potential of Escherichia coli DH5α grown in different growth media

Proteins and metabolites typically adsorb to bacterial cell surface through a variety of mechanisms such as van der Waals attraction and electrostatic interactions, and forms a layer of nonspecifically adsorbed ions and molecules on the cell surface. Thus, the bacterial cell surface charge comprised the contribution from the cell wall as well as layers of nonspecifically adsorbed ions and molecules on the cell surface. This is the cell surface charge perceived by other bacterial cells in the growth medium. Given that different growth medium comprises different ensemble of proteins and metabolites that could adsorb onto the cell surface of bacteria, it is important to examine the effect of growth in different medium on the cell surface characteristics of bacterial cells using zeta potential as the proxy parameter. Defined at the shear plane, zeta potential provides a comprehensive view of the cell surface charge that include the nonspecifically adsorbed ions and molecules on the cell surface and the intrinsic electric charges in the cell wall. Using Escherichia coli DH5α (ATCC 53868) as model organism, experiments performed with deionized water as wash and resuspension buffer revealed that the zeta potential-pH profiles of cells grown in LB Lennox, and LB Lennox with 2 g/L glucose overlapped each other over the entire pH range from 2 to 9. This suggested that there was little physiological adaptation of the cell envelope of cells grown in LB Lennox supplemented with 2 g/L glucose, which indicated that the medium could be used for increasing biomass yield without affecting cell surface characteristics. Similarly, the zeta potential-pH profiles of E. coli DH5α grown in LB Lennox and LB Lennox buffered with 89 mM potassium hydrogen phosphate buffer also overlapped each other, which highlighted that the buffered medium did not elicit physiological adaptation of the cell envelope. However, supplementation of the LB Lennox (buffered) medium with 6 g/L glucose resulted in a more negatively charged zeta potential-pH profile in the pH range from 4 to 12 compared to that during growth in LB Lennox. Growth of E. coli DH5α in other media such as Tryptic Soy Broth (TSB), formulated medium, and formulated medium with 6 g/L glucose also resulted in more negatively charged zeta potential-pH profiles compared to that during growth in LB Lennox medium. However, the point-of-zero-charge (pHzpc) of cells grown in TSB and formulated medium were the same as that of cells grown in LB Lennox medium. Collectively, physiological adaptation to growth in different media as well as different ensemble of proteins and metabolites nonspecifically adsorbed to the cell surface would generally result in different zeta potential-pH profiles of bacteria cultivated in growth media of different compositions. Understanding the cell surface charge characteristics of E. coli DH5α grown in different media would thus help unveil the mysteries of cell-cell interactions in the medium.

Zeta potential of Escherichia coli DH5α grown in different growth media

Proteins and metabolites typically adsorb to bacterial cell surface through a variety of mechanisms such as van der Waals attraction and electrostatic interactions, and forms a layer of nonspecifically adsorbed ions and molecules on the cell surface. Thus, the bacterial cell surface charge comprised the contribution from the cell wall as well as layers of nonspecifically adsorbed ions and molecules on the cell surface. This is the cell surface charge perceived by other bacterial cells in the growth medium. Given that different growth medium comprises different ensemble of proteins and metabolites that could adsorb onto the cell surface of bacteria, it is important to examine the effect of growth in different medium on the cell surface characteristics of bacterial cells using zeta potential as the proxy parameter. Defined at the shear plane, zeta potential provides a comprehensive view of the cell surface charge that include the nonspecifically adsorbed ions and molecules on the cell surface and the intrinsic electric charges in the cell wall. Using Escherichia coli DH5α (ATCC 53868) as model organism, experiments performed with deionized water as wash and resuspension buffer revealed that the zeta potential-pH profiles of cells grown in LB Lennox, and LB Lennox with 2 g/L glucose overlapped each other over the entire pH range from 2 to 9. This suggested that there was little physiological adaptation of the cell envelope of cells grown in LB Lennox supplemented with 2 g/L glucose, which indicated that the medium could be used for increasing biomass yield without affecting cell surface characteristics. Similarly, the zeta potential-pH profiles of E. coli DH5α grown in LB Lennox and LB Lennox buffered with 89 mM potassium hydrogen phosphate buffer also overlapped each other, which highlighted that the buffered medium did not elicit physiological adaptation of the cell envelope. However, supplementation of the LB Lennox (buffered) medium with 6 g/L glucose resulted in a more negatively charged zeta potential-pH profile in the pH range from 4 to 12 compared to that during growth in LB Lennox. Growth of E. coli DH5α in other media such as Tryptic Soy Broth (TSB), formulated medium, and formulated medium with 6 g/L glucose also resulted in more negatively charged zeta potential-pH profiles compared to that during growth in LB Lennox medium. However, the point-of-zero-charge (pHzpc) of cells grown in TSB and formulated medium were the same as that of cells grown in LB Lennox medium. Collectively, physiological adaptation to growth in different media as well as different ensemble of proteins and metabolites nonspecifically adsorbed to the cell surface would generally result in different zeta potential-pH profiles of bacteria cultivated in growth media of different compositions. Understanding the cell surface charge characteristics of E. coli DH5α grown in different media would thus help unveil the mysteries of cell-cell interactions in the medium.

Surface charge characteristics of Bacillus subtilis NRS-762 cells

Bacterial cell surface carries an electrical charge due to the myriad functional groups present, as well as assortment of ions and molecules nonspecifically adsorbed to the cell surface. Thus, solution in contact with the bacterial cell surface play a critical role in influencing the overall surface charge characteristics through conferring nonspecifically adsorbed ions and molecules. Various wash buffers are commonly used in removing nonspecifically adsorbed ions and molecules for revealing the real surface charge of the bacterium. Using electrophoretic mobility measurement of zeta potential, this study attempted to understand the surface charge characteristics of Bacillus subtilis NRS-762 (ATCC 8473) with the help of three wash buffers: deionized (DI) water, 0.1M sodium nitrate, and 9 g/L sodium chloride. Experiment results revealed that B. subtilis NRS-762 was negatively charged over the entire pH range from 1.5 to 12. Specifically, with deionized water as wash buffer, the point-of-zero-charge (pHzpc) was at pH 1.5, which indicated that large amount of negatively charged functional groups were present on the cell surface. Comparison between the zeta potential-pH profiles of B. subtilis NRS-762 cultivated at 30 oC and 37 oC revealed that the profile for growth at 37 oC was more negatively charged over the entire pH range compared to that for growth at 30 oC. This highlighted that physiological adaptation might had occurred on the cell surface for coping with growth at a higher temperature. Zeta potential-pH profiles obtained revealed that DI water could not remove significant quantities of the nonspecifically adsorbed ions and molecules. On the other hand, the zeta potential-pH profiles of cells washed with 0.1M sodium nitrate and 9 g/L sodium chloride overlapped each other substantially, and were more negatively charged over the pH range from 2 to 11, compared to that of cells washed with DI water. This revealed substantial removal of nonspecifically adsorbed ions and molecules with the use of 0.1M sodium nitrate (0.1M ionic strength) and 9 g/L sodium chloride (0.15M ionic strength), which helped reveal the actual surface charge of B. subtilis NRS-762 cells. Collectively, actual surface charge of B. subtilis NRS-762 was masked by nonspecifically adsorbed ions and molecules, which could be removed by 0.1M sodium nitrate and 9 g/L sodium chloride wash buffer. Thus, in the case of B. subtilis NRS-762, 0.1M ionic strength wash buffer was the threshold at which there was complete removal of nonspecifically adsorbed ions and molecules from the cell surface.