Process and formulation parameters influencing the survival of Saccharomyces cerevisiae during spray drying and tableting

Abstract

Probiotic microorganisms provide health benefits to the patient when administered in a viable form and in sufficient doses. To ensure this, dry dosage forms are preferred, with tablets in particular being favored due to several advantages. However, the microorganisms must first be dried as gently as possible. Here, the model organism Saccharomyces cerevisiae was dried by spray drying. Various additives were tested for their ability to improve yeast cell survival during drying. In addition, the influence of various process parameters such as inlet temperature, outlet temperature, spray rate, spray pressure and nozzle diameter was investigated. It was possible to dry the yeast cells in such a way that a substantial proportion of living microorganisms was recovered after reconstitution. Systematic variation of formulation and process parameters showed that the use of protective additives is essential and that the outlet temperature determines the survival rate. The subsequent compression of the spray-dried yeast reduced viability and survival could hardly be improved by the addition of excipients, but the tabletability of spray-dried yeast protectant particles was quite good. For the first time, loss of viability during compaction of spray-dried microorganisms was correlated with the specific densification, allowing a deeper understanding of the mechanism of cell inactivation during tableting.

Introduction

The production of tablets containing viable microorganisms is of particular interest for the effective administration of probiotic microorganisms (Vorländer et al., 2020). These microorganisms provide health benefits to the patient through a variety of mechanisms, including competition with pathogenic microorganisms for adhesion sites or nutrients and thus displacement of pathogens, additional beneficial catabolic or anabolic metabolic pathways, immunomodulatory functions, and the expression and secretion of specific antimicrobial bacteriocins or comparable inhibitory substances (Bermudez-Brito et al., 2012, Oelschlaeger, 2010). Due to their high specificity, probiotic microorganisms are therefore sometimes considered as a promising possibility to reduce the use of antibiotics, especially with regard to the increasing spread of antibiotic resistance (Broeckx et al., 2016).

However, to achieve the desired benefits of the probiotic microorganisms, they must reach their site of action in sufficient doses and in a viable form (Joint FAO/WHO Working Group, 2002). Therefore, the production of appropriate dosage forms is an important challenge. In general, liquid dosage forms have limited storage stability and frozen products are expensive to handle (Santivarangkna, 2016). Thus, dry dosage forms are usually preferred, which are characterized by improved shelf life, although the drying step itself is problematic here due to thermal stress and dehydration (Broeckx et al., 2016). Spray drying is a promising process for life-sustaining drying of microorganisms (Santivarangkna et al., 2007). The cell suspension to be dried is sprayed directly into hot drying air with temperatures up to 200 °C (Broeckx et al., 2016). Due to the large surface area of the millions of small droplets formed, both energy and mass transfer are accelerated, enabling very rapid drying (Santivarangkna et al., 2007). Towards the end of the drying process, when a crust has already formed and evaporation is slowing down, the droplets/particles reach the highest temperature as the effect of evaporative cooling is reduced (Santos et al., 2018). When microorganisms are spray dried, it is assumed that their thermal inactivation occurs at this time (Broeckx et al., 2016, Peighambardoust et al., 2011). The influence of individual spray drying parameters on the survival of probiotic microorganisms has been investigated using among others different strains of lactobacilli and bifidobacteria. Mostly, a variation of the outlet temperature by variation of the inlet temperature (Ananta et al., 2005, Fávaro-Trindade and Grosso, 2002) or by variation of the feed rate (Bielecka and Majkowska, 2000, Gardiner et al., 2000, Golowczyc et al., 2010, Romano et al., 2014, Wang et al., 2004) was studied, but also the influence of the atomization pressure (Riveros et al., 2009) or the drying gas (air vs. nitrogen) has been considered (Ghandi et al., 2012). In principle, the process parameters for spray drying should be chosen to minimize stresses (shear, heat, osmolality, residual moisture, oxidation).

Even with the most gentle process parameters, dehydration of living microorganisms is critical because water molecules are essential for maintaining various biological structures (Ananta et al., 2005, Crowe et al., 1987, Oliver et al., 1998). However, some substances are able to help stabilize biological structures and thus mitigate the negative effects of desiccation. Broeckx et al. reviewed numerous possible additives from various publications, including different sugars, especially disaccharides, sugar alcohols, other carbohydrates, amino acids and proteins (Broeckx et al., 2016). Maintaining the integrity of the cell membrane is critical for cell survival. Normally, the membrane is in a lamellar fluid phase, but upon dehydration, it can change to a gel phase with increased interactions between phospholipids. Therefore, the reverse phase transition during reconstitution may be associated with packing defects causing lethal leakage of cellular components (Crowe et al., 1988). The critical phase transition of the cell membrane can be prevented by the addition of protective substances, especially sucrose and trehalose, for which different mechanisms have been postulated: vitrification, water replacement and preferential exclusion / hydration (Belton and Gil, 1994, Broeckx et al., 2016, Cordone et al., 2007, Wolkers and Oldenhof, 2021). The structure and function of required proteins is maintained by the same mechanisms (Leslie et al., 1995).

For accurate dosage and easy administration of the dried microorganisms, further processing into tablets is appropriate. The first published studies on the tableting of viable microorganisms were performed dozens of years ago. However, further studies are needed to elucidate the associated mechanisms of microbial damage. Especially for spray-dried microorganisms, studies on tableting are scarce (Byl et al., 2020), and findings on compression of lyophilizates or granules are not necessarily transferable due to the different product structures. However, it is clear that with increasing compression stress, the stress on the cells increases and viability decreases (Ayorinde et al., 2011, Blair et al., 1991, Chan and Zhang, 2002, Fassihi and Parker, 1987, Muller et al., 2014, Plumpton et al., 1986a, Plumpton et al., 1986b, Poulin et al., 2011, Stadler and Viernstein, 2001, Vorländer et al., 2023a, Vorländer et al., 2023c, Vorländer et al., 2023b, Vorländer et al., 2020). The extent of the decrease depends on various aspects, such as the deformation behavior of the formulation (Ayorinde et al., 2011, Blair et al., 1991, Byl et al., 2019, Fassihi and Parker, 1987, Plumpton et al., 1986a). Despite the challenge of maintaining viability during compression, the processing of probiotic microorganisms into tablets is a target, for example in the context of probiotic oral-pharyngeal administration, where lozenges are more suitable than loose powders or capsules, which are also significantly more expensive to produce.

In the present work, the yeast Saccharomyces cerevisiae is used as a model organism. This is closely related to the probiotic yeast Saccharomyces cerevisiae subsp. boulardii (Edwards-Ingram et al., 2007), so it can be assumed that the relationships and mechanisms identified in this work also apply to the probiotic yeast. Yeast cells are dried by spray drying, and cell damage associated with dehydration and heat is reduced by the use of protective additives. In addition, the influence of process parameters such as spray pressure, nozzle diameter, inlet temperature, and drying gas flow rate on microorganism survival is considered.

The spray-dried product is then tableted, either directly or after the addition of various dry binders in different proportions, and the physico-mechanical and microbiological tablet properties are analyzed. Structural parameters of the tablets are used to derive stress mechanisms and to explain formulation influences. In contrast to bacteria, yeast cells as eukaryotes possess more complex membrane structures, a nucleus with chromosomes, and a variety of organelles such as mitochondria or the endoplasmic reticulum, and are typically larger than bacterial cells. Therefore, yeast cells are thought to have a lower tolerance, particularly to shear stress during compression, and this sensitivity makes them more suitable than bacteria for elucidating mechanisms of damage during tableting. This deeper mechanistic understanding will provide the basis for a future rational formulation of probiotic microorganisms into tablets.