Achieving delayed release of freeze-dried probiotic strains by extrusion, spheronization and fluid bed coating - evaluated using a three-step in vitro model

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Abstract

Intake of probiotics is associated with many health benefits, which has generated an interest in formulating viable probiotic supplements. The present study had two aims. The first aim was to achieve gastrointestinal protection and delayed release of viable probiotics by pelletizing and coating freeze-dried probiotic strains, using riboflavin as a marker for release. The second aim was to set up a dynamic three-step in vitro model simulating the conditions in the human gastric, duodenum/jejunum and ileum compartments using physiologically relevant media to evaluate delayed release of the formulations. To simulate lowered bile acid concentrations in the ileum area of the gastrointestinal tract, a novel method using the bile acid sequestrant cholestyramine to lower bile acid concentrations in the small intestinal medium to physiologically relevant levels was attempted. Granulation, extrusion and spheronization was used to develop pellets containing viable probiotics using freeze-dried Lactobacullus reuteri as a model strain. Fluid bed coating the pellets with the pH-sensitive polymers Eudragit S100 or Eudragit FS30D resulted in targeted release in the ileum step of the three-step in vitro model based on release of the marker riboflavin.

Introduction

In recent years, evidence of numerous health benefits associated with the intake of probiotics have been indicated and has generated great interest in the formulation of probiotic supplements and products (Vijaya Kumar et al., 2015). Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (Hill et al., 2014). Hence, in order to achieve the health benefits associated with probiotics, it is important to maintain viability during formulation and manufacturing processes, storage, and once ingested, during transit through the upper gastrointestinal tract (GIT). It can be a challenge to both formulate and to deliver viable probiotics to the GIT by the oral route (Ding and Shah, 2007). Many probiotic strains are susceptible to humidity, oxygen, low pH and bile acids and a daily dose around 108–1012 colony forming units (CFU) is needed, depending on the strain and clinical application (Guarner et al., 2012). The composition of the gut microbiota is stratified with microbial density and diversity increasing along the GIT (Guarner et al., 2012, Peterson et al., 2009). Thus, targeted delivery of specific probiotics to different areas in the GIT may increase their beneficial effects.

Many probiotic products on the market are sold in the form of dairy products or other food matrices including yoghurts, ice-creams and juices (Guarner et al., 2012, Vijaya Kumar et al., 2015). However, one of the major challenges is the stability of the probiotics during storage of the products and during transit through the upper GIT after consumption (Charteris et al., 1998). Alternative dosage forms include capsules and tablets containing probiotics in freeze-dried form, which can be modified to achieve gastric resistance (Guarner et al., 2012). Capsules and tablets are, however, not suitable for consumers with dysphagia or other swallowing difficulties including the paediatric and geriatric population (Schiele et al., 2013). Consequently, there is a need for alternative, easy-to-swallow dosage forms that provide the same protection for the probiotics as enteric coated capsules/tablets. For this purpose, multiparticulate dosage forms, including pellets, may be applied.

For the production of pellets extrusion-spheronization is a commonly used technique (Vervaet et al., 1995). The production method involves five steps; a granulation step where the dry powders, including the active ingredient and excipients are mixed, wetting of the mass by addition of granulation liquid, shaping of the wetted mass into long cylindrical rods by extrusion through a die, breaking up and rounding of the extrudates to form pellets by transferring them to a spinning plate in a spheronizer and finally drying of the pellets (Muley et al., 2016, Vervaet et al., 1995). Previous studies have demonstrated that pellets containing live probiotics could be produced by extrusion-spheronization. In these studies, the probiotics were applied either in the form of a suspension (Brachkova et al., 2009, De Barros et al., 2015) or dried bacterial pellet (Bajaj et al., 2010). Microcrystalline cellulose (MCC) is commonly used as an excipient for formulation of pellets using the extrusion-spheronization method, as wetted MCC has the rheological properties, cohesiveness and plasticity needed for the formation of uniform pellets (Dukić-Ott et al., 2009). Alginate can crosslink in the presences of Ca2+-ions and this mechanism may be exploited in the extrusion-spheronization process to obtain robust pellets as previously shown by De Barros et al. (2015).

Targeted delivery of probiotics to the lower GIT may improve their survival and efficacy and this may be achieved by film coating pellets with pH-sensitive polymers responding to the physiological pH changes in the GIT. Eudragit S100 (ES100) and Eudragit FS30D (EFS30D) are synthetic substituted copolymers dissolving at pH > 6.8–7 and hereby resist the low pH in the stomach and release in the more neutral conditons in the lower GIT (Nikam et al., 2011, Thakral et al., 2013).

It is a challenge to evaluate the targeted delivery of a dosage form to the lower GIT and for this purpose there is a need for physiologically relevant in vitro models mimicking the parameters, including pH levels, bile concentrations, and digestive enzyme activities found in the entire human GIT. In humans, the gallbladder releases bile into the duodenum which then passes through the small intestine before being actively reabsorbed in the ileum. This results in lower concentrations of bile acids in the ileum area of the GIT, which therefore needs to be accounted for in in vitro models simulating these intestinal areas (Reppas et al., 2015, Ridlon et al., 2006).

For easier quantification of the targeted delivery potential of a probiotic dosage form during in vitro evaluation a marker for release can be used. The criteria for such a marker is that is detectable and not degradable in the media used in the in vitro model.

On this basis, the current study aims at developing an easy-to-swallow formulation consisting of pellets where the probiotic strain is incorporated directly in its freeze-dried form, using a freeze-dried Lactobacillus reuteri (L. reuteri) strain as a model probiotic. Additionally, the study aims at developing a physiologically relevant three-step in vitro model simulating the conditions found in the human gastric, duodenum/jejunum and ileum compartments, in order to evaluate the release from the produced probiotic pellets (PPs). The aim for the simulation of the ileum step was to find a method to lower bile concentrations to physiologically relevant levels.

Section snippets

Materials

Freeze-dried L. reuteri LR92 was provided by Deerland Probiotics & Enzymes (Hundested, Denmark). MCC (Avicel PH-101), alginic acid, dextrin, riboflavin, bovine bile extract, gastric lipase (Rhizopus oryzae), pepsin, pancreatin (from porcine pancreas), maleic acid, peptone, MRS (De Man, Rogosa and Sharpe) agar, polysorbate 80, NaCl, CaCl2, 4-bromophenylboronic acid (4-BBBA) and bile acid assay kit were purchased from Merck (Darmstadt, Germany). Talcum was purchased from Fagron (Copenhagen,

Development of probiotic pellets

Initially pellets were produced using 10% probiotic powder, 37.5% MCC, 47.5% alginate, 4.5% dextrin, 0.5% riboflavin and a 20% (w/v) CaCl2 granulation solution. As this formulation resulted in uniform pellets the amount of probiotic powder was slowly increased while decreasing the amount of MCC. When reaching 40% probiotic powder the extrudates became slightly fragile and shredded. The MCC amount was then adjusted from 7.5% to 15% to optimize plasticity and cohesiveness of the wetted mass and

Conclusion

The PPs coated with the pH-sensitive polymers ES100 or EFS30D developed in this work showed gastrointestinal protection and targeted release based on release of the marker riboflavin. It was shown that the extrusion-spheronization method could be used to produce viable PPs. In addition a dynamic physiologically relevant in vitro model simulating the conditions in the fasted human gastric, PSI and ileum compartments was developed and it was possible to lower bile acid concentrations in a fast

CRediT authorship contribution statement

Natashia Mai Yde Jacobsen: Writing - original draft, Methodology, Investigation. Ibrahim Caglayan: Validation, Investigation. Aslihan Caglayan: Validation, Investigation. Daniel Bar-Shalom: Conceptualization, Writing - review & editing, Supervision, Funding acquisition. Anette Müllertz: Conceptualization, Writing - review & editing, Supervision, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Thanks to Deerland Probiotics & Enzymes for providing the probiotic strain used in this work.

Funding

This work was supported by the Innovation Fund Denmark, Grand solutions project “Stable Probiotic Powder Targeted Children and the Elderly” and Department of Pharmacy, University of Copenhagen.

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