Impact of spray-drying on the pili of Lactobacillus rhamnosus GG.

The preservation of the viability of microorganisms in probiotic formulations is the most important parameter ensuring the adequate concentration of live microorganisms at the time of administration. The formulation and processing techniques used to produce these probiotic formulations can influence the preservation of the microbial viability. However, it is also required that the bacteria maintain their key probiotic capacities during processing, formulation and shelf life. In this study, we investigated the impact of spray-drying on different cell wall properties of the model probiotic strain Lactobacillus rhamnosus GG, including its adherence to intestinal epithelial cells. The dltD gene knock-out mutant, L. rhamnosus GG CMPG5540, displaying modified cell wall lipoteichoic acids, showed significantly increased colony-forming units after spray-drying and subsequent storage under standard conditions compared to wild-type L. rhamnosus GG. In contrast, disruption of the biosynthesis of exopolysaccharides or pili expression did not impact survival. However, spray-drying did significantly affect the adherence capacity of L. rhamnosus GG. Scanning electron microscopy confirmed that the pili, key surface factors for adherence to intestinal cells and mucus, were sheared off during the spray-drying process. These data thus highlight that both the functionality and viability of probiotics should be assessed during the spray-drying process and subsequent storage.

Ultrasound-assisted extraction optimization and validation of an HPLC-DAD method for the quantification of polyphenols in leaf extracts of Cecropia species.

Cecropia species are traditionally used in Latin American folk medicine and are available as food supplements with little information warranting their quality. The optimum conditions for the extraction of chlorogenic acid (CA), total flavonoids (TF) and flavonolignans (FL) from leaves of Cecropia species were determined using a fractional factorial design (FFD) and a central composite design (CCD). A reversed-phase high-performance liquid chromatographic method coupled to a diode array detector (HPLC-DAD) was validated for the quantification of CA, TF and FL, following the ICH guidelines. Quantitative and Principal Component Analysis (PCA) was also performed. The extraction-optimization methodology enabled us developing an appropriate extraction process with a time-efficient execution of experiments. The experimental values agreed with those predicted, thus indicating suitability of the proposed model. The validation parameters for all chemical markers of the quantification method were satisfactory. The results revealed that the method had excellent selectivity, linearity, precision (repeatability and intermediate precision were below than 2 and 5%, respectively) and accuracy (98-102%). The limits of detection and quantification were at nanogram per milliliter (ng/mL) level. In conclusion, the simultaneous quantification of chemical markers using the proposed method is an appropriate approach for species discrimination and quality evaluation of Cecropia sp.

Enhancing the viability of Lactobacillus rhamnosus GG after spray drying and during storage.

Increasing knowledge about the human microbiome has led to a growing awareness of the potential of applying probiotics to improve our health. The pharmaceutical industry shows an emerging interest in pharmaceutical formulations containing these beneficial microbes, the so-called pharmabiotics. An important manufacturing step is the drying of the probiotics, as this can increase the stability and shelf life of the finished pharmabiotic product. Unfortunately, drying also puts stress on microbial cells, thus causing a decrease in viability. We aimed to examine the effect of different drying media and protective excipients on the viability of the prototype probiotic strain Lactobacillus rhamnosus GG after spray drying and during subsequent storage for 28 weeks. The presence of phosphates in the drying medium showed to have a superior protective effect, especially during long-term storage at room temperature. Addition of lactose or trehalose resulted in significantly improved survival rates after drying as well as during long-term storage for the tested excipients. Both disaccharides are characterized by a high glass transition temperature. Maltodextrin showed less protective capacities compared to lactose and trehalose in all tested conditions. The usage of mannitol or dextran resulted in sticky powders and low yields, so further testing was not possible. In addition to optimizing the viability, future research will also explore the functionality of cellular probiotic components after spray drying in order to safeguard the probiotic activity of the formulated pharmabiotics.

Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics.

The increasing knowledge about the human microbiome leads to the awareness of how important probiotics can be for our health. Although further substantiation is required, it appears that several pathologies could be treated or prevented by the administration of pharmaceutical formulations containing such live health-beneficial bacteria. These pharmabiotics need to provide their effects until the end of shelf life, which can be optimally achieved by drying them before further formulation. However, drying processes, including spray-, freeze-, vacuum- and fluidized bed drying, induce stress on probiotics, thus decreasing their viability. Several protection strategies can be envisaged to enhance their viability, including addition of protective agents, controlling the process parameters and prestressing the probiotics prior to drying. Moreover, probiotic viability needs to be maintained during long-term storage. Overall, lower storage temperature and low moisture content result in good survival rates. Attention should also be given to the rehydration conditions of the dried probiotics, as this can exert an important effect on their revival. By describing not only the characteristics, but also the viability results obtained by the most relevant drying techniques in the probiotic industry, we hope to facilitate the deliberate choice of drying process and protection strategy for specific probiotic and pharmabiotic applications.

Evaluation of a newly developed HPMC ophthalmic insert with sustained release properties as a carrier for thermolabile therapeutics.

A novel drug delivery system (DDS) with sustained release properties was developed to allow ocular protein delivery. The DDS developed is aimed at overcoming stability issues during preparation such as denaturation of proteins caused by shear forces applied or due to elevated temperatures and air entrapment potentially causing oxidation of the molecule. The rod-shaped HPMC inserts were loaded with lysozyme and several HPMC types were studied and compared. An aqueous colloidal HPMC solution (hydrogel) was prepared and subsequently dried at 150°C to dehydrate the polymer solution. This partially dehydrated polymer cylinder was loaded with an aqueous glycerol/lysozyme solution at 2°C. A 2(4) full factorial design was set up to evaluate the effect of the different preparation parameters on water uptake and release properties. As a result, four out of sixteen formulations revealed homogenous distribution for lysozyme in both duplicates. The change in water uptake over time was dependent on the type of HPMC polymer used but not between the chosen HPMC percentages. After 240min, 50% of lysozyme loaded was released depending on the chosen formulation. Lysozyme molecules exhibit slower release from a K100M matrix compared to E10M inserts, albeit the overall effect is relatively limited.