Pulsed electric field pre-treatment for drying of living bacteria

Bacteria are commonly used in food industry as starter cultures or probiotic supplements. Production of these bacterial food ingredients requires growth of the bacteria followed by storage until they are used as fermentation starter or ingested as probiotic. To prolong the shelf life of these bacteria, drying processes such as freeze drying and spray drying are used to dry these ingredients into a powder. A drawback of drying is that the viability of the dried bacterial cultures decrease along the process chain. Therefore, many studies focus on improving the survival of these cultures during drying and subsequent storage. Often the bacteria are dried in a carrier matrix consisting of carbohydrates and/or proteins to increase their survival. Some studies also suggest that increased intracellular concentrations of protective solutes such as for example trehalose can also result in better survival after processing. Trehalose is a disaccharide that is accumulated intracellularly by several organisms, e.g. bacteria, yeasts and plants, under desiccation stress. Intracellular trehalose concentrations can also be increased by a mechanical method, i.e. pulsed electric field treatment. Pulsed electric field (PEF) treatment is the application of short high voltage pulses to a product between two electrodes which can result in permeabilization of the cellular membrane. PEF pre-treatment leading to increased intracellular trehalose concentrations has been shown to enhance robustness of mammalian cells towards processing, such as cryopreservation. The aim of this thesis was to develop a PEF pre-treatment leading to increased intracellular protective solutes concentrations in bacteria in order to enhance survival during drying and subsequent powder storage. This aim was divided in two parts. First, to develop a PEF pre-treatment leading to increased intracellular trehalose concentrations, while maintaining culture viability. And secondly, to evaluate whether this PEF pre-treatment results in enhanced robustness during drying and subsequent powder storage.

In chapter 2 we obtained the proof of principle that PEF can be used to increase intracellular trehalose concentrations in our model bacterium Lactobacillus plantarum WCFS1. The effect of various electric field strengths was evaluated on survival and intracellular trehalose contents. Increased intracellular trehalose concentrations in L. plantarum WCFS1 were found when applying two pulses of 100 μs at electric field strengths of 7.5, 10 or 12.5 kV/cm. In particular, at 7.5 kV/cm both an increased intracellular trehalose concentrations and a high survival was found. Subsequently, the fluorescent stain propidium iodide (PI) was used to study the effect of PEF treatment on the permeabilization of the cells. The stain was added before and after PEF treatment to evaluate whether the cellular membrane was permeabilized and whether the permeabilization was reversible or irreversible. It appeared that at 7.5 kV/cm approximately 23% of the cellular membrane was permeabilized, of which approximately half was reversibly permeabilized for PI. In chapter 3, this investigation was extended. Here, a second staining method with two impermeable stains was added; namely addition of PI before PEF treatment and addition of SYTOX Green after PEF treatment. The first method resulted in reversible permeabilized fractions up to 14% of the cell population, while the second method indicated these would be up to ~40% of the population with the same PEF conditions. This difference shows that the choice of fluorescent staining technique influenced the conclusions drawn.

The second aim of this thesis was to evaluate whether a PEF pre-treatment, leading to increased intracellular trehalose concentrations, can increase robustness of bacteria during drying and subsequent powder storage. Therefore, L. plantarum cultures were PEF treated in trehalose electroporation medium and subsequently freeze dried in the same solution in chapter 4. Contrary to our hypothesis, the survival after freeze drying and subsequent powder storage did not differ between the PEF pre-treated and control cultures. Upon further studying the intracellular trehalose concentrations during the different steps of the freeze drying process we found that intracellular trehalose concentrations also increased after freezing the cells. Therefore, there was no difference in intracellular trehalose concentrations between the PEF pre-treated and control cultures anymore, which could explain the lack of difference in survival after drying and storage. Similar experiments were also performed with lactose electroporation medium. In these experiments increased intracellular lactose concentrations in L. plantarum WCFS1 were not observed after freezing, but only after freeze drying and reconstitution of the powder. This difference between intracellular trehalose and lactose accumulation indicates that potentially also biological processes play a role in the observed effects. Finally, also after spray drying increased intracellular trehalose and lactose concentrations were found upon drying in these carbohydrates.

While in chapter 4 L. plantarum WCFS1 cultures were PEF treated and subsequently dried in the same medium, in chapter 5 the cultures were PEF treated in trehalose electroporation medium and subsequently spray dried in various carrier matrices, namely trehalose, reconstituted skim milk (RSM), maltodextrin DE19 and whey protein. Interestingly, survival of L. plantarum WCFS1 greatly increased upon applying PEF treatment when drying in RSM. A similar increase was not observed for the other carrier matrices. Also during powder storage higher survival of PEF pre-treated cultures was found compared to control cultures without PEF when RSM was used as a carrier matrix. Furthermore, not only PEF pre-treatment in trehalose electroporation medium resulted in this increases survival during spray drying in RSM, also PEF treatment in lactose electroporation medium. Analysis of intracellular trehalose and lactose concentrations before and after PEF treatment revealed that the increased robustness could not only be related to these intracellular concentrations.

The type of carrier matrix during drying influences not only the added value of a PEF pre-treatment, it also affects bacterial survival during spray drying. In chapter 6 the effect of different carrier matrices on survival of L. plantarum WCFS1 during drying has been investigated by a single droplet drying approach. More specifically, particle morphology development during single droplet drying was linked to bacterial survival. Three types of particle morphologies were observed, namely dense and smooth particles, smooth and hollow particles and dented particles. Survival of L. plantarum WCFS1 was highest in carrier matrices resulting in dense and smooth particles, followed by hollow particles and lowest in dented particles. This observed relation could be explained by a combination of glass transition and diffusion dynamics in the drying droplets.

In chapter 7 the implementation of the developed PEF pre-treatment in an industrial drying process is discussed. PEF pre-treatment to enhance bacterial survival can lead to an energy reduction for drying of bacterial cultures and/or it may make a transition from the more energy and time consuming freeze drying process to spray drying possible. In addition, not only the pulse parameters described in chapter 2 and 3 can influence the outcome of a PEF treatment, also large effects of temperature and the addition of a membrane fluidizing component such as ethanol were observed.

Overall, the approach described in this thesis provides a first step towards using pulsed electric field pre-treatment, leading to increased intracellular protective solutes concentrations, to enhance bacterial robustness during drying and subsequent powder storage. Knowledge is gained on the role of intracellular protective solutes during drying of bacteria, as well as on the effect of various carrier matrices. These insights can be used to develop and optimize drying processes aimed at high bacterial culture viability.