Exploring The Inactivation Of Probiotics Cells And The Injury On Cellular Membrane During Spray Drying

Probiotics has high application potential as health-promoting supplements,pharmaceuticals,fermentation starting cultures and biopreservatives.The activity of probiotics could be preserved by dehydration approaches such as spray drying or freeze drying.Spray drying is preferred in industrial mass production because the process is relatively facile,fast,and of high productivity.However,there are many stressful factors in the process,which could lead to the inactivation of probiotics cells after being dried.Such adverse environmental factors include heat stress from high temperature,osmotic stress caused by water loss,oxidative stress when cells are exposed to the oxygen in the air,increased intracellular p H,and increased intracellular salt concentration due to water loss and so on.Cells injuries caused by thermal and dehydration stresses are usually considered as major factors responsible for cell inactivation.These stresses could cause injuries on multiple cellular structures such as cell membrane,cell wall,DNA,RNA and ribosome,resulting in the loss of metabolic activity and reproduction capacity.Therefore,to maximize the survival of the bacteria after spray drying,it is of paramount importance to identify the main cellular structures involved in the deactivation of probiotic cells,and to explore the mechanism of each stress influencing the cell viability during spray drying.Such understandings will facilitate a rational design of protective carrier formulation.The present study selected cellular membrane as the representative cellular structure and studied its injury history during spray drying,aiming at exploring the relationship between cell inactivation and damages in cellular membrane.Changes in the viability of Lactobacillus rhamnosus GG(LGG)and injuries on cell membrane were experimentally investigated in three processes,viz.,heat treatment,single droplet drying and spray drying.In the first part of this paper,LGG cells were suspended in lactose and trehalose carriers,and changes in the cell viability were monitored under simulated spray drying conditions using the single droplet drying(SDD)technique.The results w ere compared to control experiments with heat treatment process to unveil the influence of dehydration stress on LGG viability.At environmental temperature of 90°C,the inactivation of LGG cells in the SDD process was initially observed between 61 and 65°C,approximately 10°C higher than in the heat treatment,indicating that the heat tolerance of the cells in the single droplet drying is notably better than in the heat treatment.The enhanced heat stability was attributed to the favorable temperature history stemming from evaporative cooling at the initial drying stage,in addition to the reduced water activity of the semi-dried particles.Cell survival at the later treatment stage was influenced by the carrier material.Trehalose demonstrated an outstanding thermoprotective effect in the heat treatment,but such effect was attenuated during drying,indicating the presence of other stresses at the later drying stage,which might cause a rapid loss of bacterial viability.To further the understandings on the effect of dehydration stress on LGG cells,the second part of this paper investigated the injuries of cell membrane during both the heat treatment and the single droplet drying.In the heat treatment,membrane damages reached the maximum level between 60 and 70 °C,but during the SDD,there were still viable LGG cells with intact cytoplasmic membrane at a similar temperature range.The enhanced stability of cellular membrane observed in the SDD process was attributed to two reasons.Firstly,the temperature variation rate in the SDD process was higher than that in the heat treatment,so that cells took shorter time to achieve a similar temperature range.Secondly,the diffusion of intracellular substances through the damaged membrane to the environment might be impaired by the high viscosity of semi-dried particles.The correlation between cell viability and membrane damages was evaluated by plotting the two terms as ordinate and abscissa,respectively.The results showed that at the initial stage,damages in the cell membrane damages were associated with the viability loss of part of the cell population.However,at the later stage,there could be other cellular structures getting denatured or deactivated,resulting in the rapid loss of cell viability.The results from the above two parts of the study indicated that dehydration stress might be able to counteract the adverse effect of heat stress to some extent at the early stage of drying,and damages on the cell membrane need to be minimized for the optimization of cell survival after drying.In the third part of the study,LGG cells were spray dried in five types of protective carriers(lactose,trehalose,reconstituted skim milk-RSM,WPI/trehalose,WPI/lactose)using a new type of spray dryer – Monodisperse Microfluidic-Jet Spray Dryer.The residual cell viability and the degree of cellular membrane damages were investigated at varied spray drying conditions.The results showed that drying at higher inlet and outlet temperatures led to a lower survival ratio of the bacteria.The highest survival ratio,49.3%,was achieved with the inlet temperature of 84 oC and outlet temperature of 53~54 oC in the RSM carrier.Adding WPI to either type of sugar at a ratio of 2:8 did not improve the survival of dried cells,when compared to the results of pure sugar carriers.Carrier material not only affected the survival of probiotics,but also influenced the residual moisture content of spray dried powders,which could be another factor influencing the cell survival.Da mages on cellular structures and functional properties showed a similar trend to the result of viability loss.At higher spray drying temperature,the cellular membrane of dried LGG cells was more permeable,and cells also exhibited a longer lag period in the growth curve.The severe membrane damages observed for all tested spray drying conditions could be an important factor responsible for the loss of cell viability.In future studies,the development of an efficient carrier formulation for protecting cellular membrane during drying would be helpful for minimize the loss of cell viability after spray drying.