Accelerated Oxidation Test of Freeze-Dried Pseudomonas Fluorescens BTP1, BB2 and PI9 Strains
The Asia Journal of Applied Microbiology
Pseudomonas fluorescens strains are very susceptible to damage during freeze-drying and storage. It would be useful to have longer lasting viable powders. Membrane oxidation is one of the factors responsible for damages suffered by P. fluorescens strains during storage. Singlet oxygen ( 1 O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. We exposed freeze-dried powders of three strains P. fluorescens (BB2, BTP1 and PI9)
... (BB2, BTP1 and PI9) to singlet oxygen produced by photosensitisation of Methylene Blue/ light during 2 hours. We observe a significant loss in survival rate, a pronounced oxidation of polyunsaturated fatty acids, protein oxidation (carbonyl groups) and protein insolubilisation. This study enables better predictions of types of packaging suitable for freeze-dried powders of P. fluorescens. Contribution/ Originality This study is one of very few studies which have investigated on Pseudomonas fluorescens strains. This study has objectives to develop a method for to stabilise and storage freeze-dried bacteria gram negative which is very sensitive to all form of drying. useful to have longer-lasting cultures  . However, freeze-drying also renders the powder vulnerable to Reactive Oxygen Species (ROS) attack during storage [2, 3] . Lipids are major targets during oxidative stress. Free radicals can directly attack polyunsaturated fatty acids in membranes and initiate lipid peroxidation  . These highly reactive free radicals can oxidize lipids, proteins and DNA, contributing to a variety of important types of cellular damage. In biological membranes, lipid peroxidation is frequently a consequence of free radical attack [4, 5] . A primary effect of lipid peroxidation is a decrease in membrane fluidity, which alters membrane properties and can disrupt membrane-bound proteins significantly. This effect acts as an amplifier in which more radicals are formed and polyunsaturated fatty acids are degraded into a variety of products. Some of these, such as aldehydes, are very reactive and can in turn damage other molecules such as proteins [4, 6] . The main protein modifications observed are: loss of catalytic activity, amino acid modifications, carbonyl group formation, decreases in thermal stability, changes in viscosity, changes in fluorescence, fragmentation, formation of protein-protein crosslinks, formation of disulfide bridges, and increased susceptibility to proteolysis  . Subsequent to the finding that some amino acid residues (including lysine, arginine, proline and threonine) can be oxidized to carbonyl derivatives  , several methods to detect the carbonyl content of proteins were developed and used to measure protein damage  . The aim of this work is to determine the damage caused by ROS on freeze-dried powders of three P. fluorescens strains. To do this, freeze-dried P. fluorescens powders were exposed to singlet oxygen produced by the action of light on Methylene Blue (MB/ light). In this paper, we examined overall viability as well as specific oxidative damage to fatty acids, proteins and DNA in freeze-dried P. fluorescens powders after exposure to Methylene Blue/light. MATERIALS AND METHODS Organisms and Cultivation The strain used in our study is P. fluorescens BTP1 from the Wallon Center of Industrial Biology laboratory (CWBI), P. fluorescens BB2 and PI9 from Faculty of Agro-Vets and Biological ,Saad Dahlab of Blida University Algeria  . The strain was grown in a 20 L bioreactor (Biolafitte) containing 16 L of 863 medium for 20 hours and then concentrated 20-fold by centrifugation (Sorval RC 12 BP) at 4700 rpm. Afterward, the pellets were dried in a freeze-drier (LOUW KOELTECHNIEK BVBA) using a standard program in which the temperature was gradually increased from -25°C to 25°C at 0.9 mbar pressure during 48 hours . Photosensitisation Samples were exposed to 40 µM of Methylene Blue (MB) in the presence of light. Methylene Blue produces singlet oxygen when exposed to an incandescent bulb . Water Activity (aw) and Survival Rate The water activity (aw) of freeze-dried samples was measured at 25°C using a water activity meter (Gbx-meter, Paris, France). Standard salt solutions (Gbx) of known water activity were In a previous work  P. fluorescens were produced in a 100 L bioreactor, with or without the addition of cryoprotector (2% glycerol) and freeze-dried. The storage stability of the freeze-dried samples was then studied by monitoring several parameters such as: loss in viability, protein insolubilisation and changes in membrane structure (precisely C18:2 and C18:3). To be able to improve long term storage of bacteria requires knowledge of the phenomena such as oxidation (lipid, proteins and DNA) responsible for sample degradation over time  . We know that 1 O2 can be generated in cells, such as under conditions of oxidative stress, decomposition of lipid peroxides, or by spontaneous dismutation of superoxide  . In our study, three strains of P.fluorescens were used: a psychrophilic strain, P.fluorescens BTP1, from the Walloon Center of Industrial Biology (CWBI) and thermophilic , BB2 and PI9, sent from Algeria to Saad Dahlab of Blida University [2, 9] . After freeze-drying, the powders were conditioned in glass tubes for an accelerated oxidation test. The accelerated oxidation test was monitored by losses in cell viability, changes in carbonyl groups, protein concentration, DNA oxidation and lipid oxidation (by profiling fatty acids) during a 90 minute exposure of the powders to Methylene Blue/ light. The activity water (aw) of all powders was measured before exposure and samples were taken every 30 minutes during exposure for analysis. The water activity of all powders was high; e.g., 0.33 for the BTP1 strain 0.29 for the PI9 strain and 0.39 for the BB2 strain. The percentage of survivors is expressed by logarithmic values of the surviving fractions after 90 minutes exposure. We observe that the PI9 strain shows a higher survival rate after exposure than the BTP1 and BB2 strains. The decrease in survival rate observed after 30 minutes exposure to Methylene Blue/light is more pronounced for the BB2 strain (25%) compared to BTP1 and PI9 (60%), but after 60 minutes exposure the decrease is more pronounced with all strains. This shows that the singlet oxygen produced has a considerable impact on the viability of the freeze-dried powders. Lipids are also affected by this exposure; as with survival rates, levels of polyunsaturated fatty acids (C18:2 and C18:3) also decrease. This decrease is more pronounced with the BB2 strain, which has a greater value of water activity (Figure 2 ). Of all the strains, P.fluorescens BTP1 from the Walloon Center of Bio-Industry showed a lower percentage of polyunsaturated fatty acids (C18:2 and C18:3)  than BB2 and PI9 from the Algerian Republic. Lipid oxidation of membrane fatty acids has been deemed responsible for cell death during storage [11, 17] . The main relative percentages from all the strains before or after exposure are presented in Fig. 2 . This decrease shows that our three strains are very sensitive to Methylene Blue /light exposure. Protein oxidation mediated by reactive oxygen species is accompanied by the conversion of proline, lysine, arginine and histidine residues into carbonyl derivatives, thereby providing a convenient assay for oxidative modification  . As shown in (Figure 3 and Figure 4 ) protein oxidation and protein insolubilisation were significantly increased in three strains during exposure to Methylene Blue /light. However, the BB2 strain showed significantly higher levels of carbonyl groups and protein concentration compared to the BTP1 and PI9 strains during this exposure. Oxidative damage to membranes results in increased membrane fluidity, compromised integrity, and inactivation of membrane-bound receptors and enzymes  . The decrease in protein concentration shows that proteins are being altered and insolubilized by the oxygen singlet produced by exposure to Methylene Blue/ light. Protein denatured is also pronounced after 30 minutes exposure, reflected in the decreased concentrations of soluble proteins. This decrease is more pronounced for the BB2 strain but it is still constant after 30 minutes for the BTP1 and PI9 strains. Carbonyl groups increase more for BB2 strain after a 30 minute exposure, whereas most of the increase for BTP1 and PI9 began after 60 minutes. This shows that the BB2 strain is the first strain to undergo protein oxidation, and in fact BB2 has a high water activity. CONCLUSION The results presented here provide experimental support for the hypothesis that Oxygen is one key element responsible for the mortality of Pseudomonas fluorescens during storage of the freeze-dried powder. These results indicate that water activity and lipid peroxidation represent important intermediary steps in the process of oxygen radical-induced genetic damage during storage of the powder. The differences observed in survival rate, fatty acids composition, proteins and carbonyl content for the three strains are due in nature of strain (BTP1 is mesophilic, BB2 and PI9 are thermophilic), because thermophilic strains have a great polyunsaturated fatty acids content. This test allows us to understand the mechanisms responsible for loss of viability in freeze-dried powders during storage. Our results show that exposure of powders to singlet oxygen produced by photosensitisation with Methylene Blue /light (MB/light), leads to membrane oxidation. This oxidation is responsible for the loss in viability during storage of freeze-dried bacteria powders. To reduce oxygen damage during storage, the protective compounds must be used before freeze-drying and powder packed in a bag impervious to light and with oxygen.