Accelerated storage testing of freeze-dried Pseudomonas fluorescens BTP1, BB2 and PI9 strains

Freeze-dried cultures of Pseudomonas fluorescens are used in agriculture and microbiological industry. However, P. fluorescens is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to increase culture viability during storage. The viability of freeze-dried P. fluorescens strains (BTP1, PI9 and BB2) was evaluated by using the Arrhenius model. This model was described by measuring the reaction rate constants (D or k) and temperature sensitivity of rate constant (z or Ea). The freeze-dried P. fluorescens strains were stored in glass tubes at 60, 37 and 4°C for 8 h, 28 days and two months, respectively. D value decreased or k increased with an increase of the storage temperature. By comparing their decimal reduction time (D), we observed that BB2 strain was more resistant than BTP1 and PI9 at 37 and 60°C. The activation energy of all P. fluorescens strains were not significantly different and thermal inactivation may occur by the same mechanism. Thus, it was possible to compare rate constants of survival for the freeze-dried P. fluorescens strains. These results will be useful to the development of improved reference materials and samples held in culture collections.Key words: Arrhenius model, accelerated storage testing (AST), freeze-drying, storage stability

Accelerated storage testing of freeze-drying Pseudomonas fluorescens BTP1, BB2 and PI9 strains

peer reviewedFreeze-dried cultures of P.fluorescens are used in agriculture and microbiological industry. However, P. fluorescens is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to increase culture viability during storage. The viability of freeze-dried P. fluorescens strains (BTP1, PI9 and BB2) was evaluated by using the Arrhenius model. This model was described by measuring of reaction rate constants (D or k) and temperature sensitivity of rate constant (z or Ea). The freeze-dried P.fluorescens strains was stored in glass tubes at 60°C, 37°C and 4°C for 8 h, 28 days and 2 months, respectively. D value decreased or k increased with an increase of the storage temperature. By comparing their decimal reduction time (D), we observed that BB2 strain was more resistant than BTP1 and PI9 at 37 °C and 60 °C. The activation energy of all P. fluorescens strains are not significantly different and thermal inactivation may occur by the same mechanism. Thus it was possible to compare rate constants of survival for the freeze-dried P. fluorescens strains. These results will be useful to the development of improved reference materials and samples held in culture collections.Conseil Alimentaire (CONSALIM

CONTRIBUTION A L’ETUDE DE LA RESISTANCE AU SECHAGE DE PSEUDOMONAS FLUORESCENS

The objective of this thesis is to study the resistance to drying of Pseudomonas fluorescens. Freeze-drying is the most suitable method for drying P. fluorescens. However, freeze-drying induced loss of cell viability. This loss of viability is mainly due to membrane rupture, temperature and oxidation of fatty acids, membrane proteins and glutathione. For this purpose, the use of protective compounds during freeze-drying has allowed us to obtain a powder having a high viability. We then studied the impact of these protective compounds, oxygen and storage temperature on the viability of P. fluorescens during storage. Analyses of fatty acids, proteins, glutathione and the study of membrane integrity during the various manufacturing processes and storage have established a link between the degree of oxidation and cell death. The results of flow cytometry showed that the freeze-drying longer affects the viability of P. fluorescens rather than storage. We have increased the yield of the production in bioreactor of P. fluorescens and time of culture was halved.Consali

Impact of drying on Pseudomonas fluorescens viability. A review

The drying Pseudomonas fluorescens makes more economical storage, transportation and marketing. It aims to stop and to stabilize all biological activities for an optimal storage, compatible with the conservation of maximum viability of microorganisms desired. The viability of bacteria after drying depends on the operating conditions of the latter. One of the most important criteria to consider during the drying of biologically active products is the quality of the final dried product. Freeze-drying is the most drying method used for Pseudomonas. But temperature changes it induced are not without consequence for the cells. They are responsible for cell damage (peroxidation of fatty acids) and genetic (proteins and DNA oxidation). However, use of protective compounds during freeze-drying and during storage increases significantly the rate of cell viability.Conseil Alimentaire (CONSALIM

Study of viability of Pseudomonas fluorescens BTP1 freeze-dried during storage at 4 and 20°C

The drying of bacteria remains a major alternative in order to keep them long term. After centrifugation, the bacterial pellet of Pseudomonas fluorescens BTP1 was divided in two fractions one with and one without cryoprotectants (2% glycerol and 5% maltodextrine) and freeze-dried. After freeze drying, powders were sealed in aluminium bag under vacuum and storage at 4 or 20°C. The storage stability of freeze-dried powders was studied by parameters such as loss of viability on the Plate Count Agar (PCA) (e.g. Concentration of Cells with glycerol (PG) at CFU/g before storage 109 and after 7 month, 108 at 4°C and 107 at 20°C) and evolution in membrane composition by measuring the ratio of unsaturated/saturated fatty acid. These ratios decrease in function of time (e.g. at 4°C the ratios of C18:3 and C18:2 by C16:0 decreases respectively of 0,013 to 0,001 and 0,05 to 0,03 after 60 days of storage). Viability (%) and concentration (CFU/g) of bacterial during storage at 4 or 20°C with aw = 0,32 was determined using a procedure published by (Kurtmann et al., 2009). In the present study, flow cytometric analysis was applied to evaluate the state in which are the cells at the end of storage time. Furthermore, we compared result the survival of bacteria as obtained by plate count with the flow cytometric analysis results.Conseil Alimentaire (CONSALIM

Optimisation de la production de Pseudomonas fluorescens BTP1 en fermenteur.

The optimisation of production and freeze-drying of P. fluorescens used as a bio-control agent was investigated. P. fluorescens BTP1 was produced in a bioreactor with different against-pressure value (0.1 and 0.3 bar for bioreactor 1 and 2 respectively) and cells were harvested during the stationary phase (2 h and 4 h for bioreactor 1 and 2 respectively). A mixture of protective compounds were tested for freeze-drying, and the highest result was found for glycerol and maltodextrine (26%) followed by glycerol, maltodextrine and ascorbic acid (18.9%) and glycerol with ascorbic acid (8.5%). We observed that the survival rate is better at 4°C than at room temperature and those powders with protective compounds have a survival rate greater than the powder without protective compounds during storage.Conseil Alimentaire (CONSALIM

Impact of Protective Compounds on the Viability, Physiological State and Lipid Degradation of FreezeDried Pseudomonas Fluorescens BTP1 during Storage

The drying of bacteria remains a major alternative in order to keep them long term. After centrifugation, the bacterial pellet of Pseudomonas fluorescensBTP1 was divided in two fractions one with protecting compounds (2% glycerol or 5% maltodextrine) and one without and freeze-dried. After freeze drying, powders were sealed in aluminium bag under vacuum and stored at 4 or 20°C. The parameters such as viability, the conductivity and the ratio of polyunsaturated fatty acids/saturated fatty acids were used to investigate the viability of freeze-dried powders during storage. For example cell concentration of powder with glycerol (PG) at CFU/g before storage is 4.109 and after 7 month 2.108 at 4°C and 3,5.107 at 20°C). The ratio of polyunsaturated fatty acids/saturated fatty acids decrease in function of time (e.g. at 4°C the ratios of C18:3 and C18:2 by C16:0 decreases respectively of 0,013 to 0,001 and 0,05 to 0,03 after 60 days of storage). In the present study, flow cytometric analysis was applied to evaluate the state in which the cells are at the end of storage time. We compared the survival results of bacteria obtained by plate count with the flow cytometric analysis results.Conseil Alimentaire (CONSALIM

Impact of glycerol and storage temperature on gluatathione concentration and physiological state of Pseudomonas fluorescens BTP1 freeze-dried

Pseudomonas fluorescens is commonly used as bio-fungicides in agriculture. For this use it requires formulations as either liquid or powder. Formulations have two advantages, storage and transport. Freeze-drying is a commonly used method to preserve bacteria. However, freeze-drying damages the cells, which results in loss of viability. Protective compounds are used to reduce loss of viability during process (freeze-drying and storage). In our study we used flow cytometry analysis to assess the physiological state in which cells are at the end of freeze-drying and Glutathione (GSH) was measured before and during storage.Conseil Alimentaire (CONSALIM

Effects of glycerol on Pseudomonas fluorescens BTP1 freeze-dried

The storage stability of freeze-dried powders was studied by parameters such as loss of viability on the Plate Count Agar (PCA). Powder with glycerol (PG) contains 8.4x1010cfu/g before storage 1.1x1010cfug after 3 months at 4°C and 6.0x108cfu/g after 3 months at 20°C. The concentration of soluble proteins (mg/g) decrease during storage at 4°C from 3.77 to 0.80 after 90 days; and the ratios of unsaturated to saturated fatty acids (C18:3/C16:0 and C18:2/C16:0) decrease respectively from 0.05 to 0.04 and 0.007 to 0.004 after 3 months at 4°C. This ratio characterises the membrane fluidity. Powder without glycerol (PS) contains 1.1x1010 cfu/g before storage and 1.4 x 108 cfu/g after 3 months at 4°C and 1.4 x 107 cfu/g after 3 months at 20°C. The concentration of soluble proteins (mg/g) decrease during storage at 4°C from 4.08 to 0.42 after 90 days, the glutathione concentration decrease during storage at 4°C from 2.2 to 1.4. The beneficial effect of glycerol on fatty acid composition during freezedrying is shown and the ratios of unsaturated to saturated fatty acids (C18:2/C16:0 and C18:3/C16:0) decrease respectively from 0.019 to 0.004 and 0.054 to 0.036 after 90 days storage at 4°C. Analysis by flow cytometry was used to assess the physiological state in which cells are at the end of freeze-drying. We found 13.5% live cells, 36.1% dead cells and 50.4% cells in an intermediate state for powder with glycerol (PG) after freeze-drying. These results shows that glycerol play an important role in Pseudomonas fluorescens BTP1 desiccation during freeze-drying, by maintaining a degree of viability after freeze-drying and during storage.Conseil Alimentaire (CONSALIM