(W0930-05-28) Enhancing Oral Delivery of Alginate-Encapsulated Probiotics: The Novel and Versatile Role of Casein as an Excipient

Purpose: Encapsulating probiotics in lyophilized alginate beads is commonly used for controlled release during oral delivery and to shield against external environmental effects. However, this method compensates cell viability during manufacturing, storage, and gastrointestinal transit: During manufacturing, the lyophilization process can expose the payload to harsh conditions which increase the risk of viability loss. Additionally, during storage, residual moisture can lead to further viability loss. The payload is also vulnerable to gastric acid-induced viability loss as alginate matrix is acid-permeable [1]. To combat these degrading events, we introduce casein - a key protein in milk, that serves multiple functions as a novel excipient. Acting as a lyoprotectant, an agent that reduces water absorption from the environment, and a local buffering agent, this protein-based formulation strategy offers a comprehensive solution for preserving cell viability from manufacturing through to patient use. To evaluate its effectiveness, we compared the lyoprotection efficacy, water absorption profiles, and pH-buffering capabilities in simulated gastric fluid, among three chitosan-alginate encapsulation systems: alginate-chitosan (alg-chi), alginate—chitosan-trehalose (alg-chi-tre), and alginate-chitosan-casein (alg-chi-cas).

Methods: Solutions of alg (1.2% sodium alginate, wt%), alg-tre (1.2% sodium alginate, 6% trehalose, w/v), and alg-cas (1.2% sodium alginate, 6% casein, w/v) were prepared for further use. Probiotic yeast strain (FZY012-G7-E1) was harvested from yeast-peptone-dextrose (YPD) media after 22 hours of culture at 30°C and collected to mix with alg, alg-tre, or alg-cas solution. The mixture was extruded through a #16 needle dropwise to the solution of 0.2M CaCl2 with a dropping height of 2 cm. The resulting beads was stirred for 1 hour in the CaCl2 for hardening, and transferred to a solution of 0.5% chitosan (pH=5) and stirred for another 1 hour to enable chitosan deposition onto alginate beads [2], resulting in three different encapsulation systems: alg-chi, alg-chi-tre, and alg-chi-cas. (Figure 1) The products were collected and aliquoted to glass vials for lyophilization, with the process consisting of a freezing stage of 180 minutes at -50°C, followed by a transition to primary drying with the ramping rate of 1°C/min to -36°C under 60 mTorr. Primary drying continues for 1440 mins before moving to secondary drying with the ramping rate of 0.1 °C/min to 30°C under 75 mTorr and kept for 360 mins. Cell viability was assessed by calculating the percentage of colony-forming units (CFUs) within the entire cell population, as determined by optical density measurements at 600 nm. PH-buffering capability of alg-chi, alg-chi-tre, and alg-chi-cas beads was conducted in fast-state simulated gastric fluid (FaSSGF, pH=1.63). Measurements were taken with a Thermo Fisher Orion Lab Star PH111 pH/mV bench meter, which was equipped with a 9810BN micro pH electrode. Water-absorption measurements were conducted by placing the beads in a stability chamber (25C, 60%RH). The beads were sampled at various time points and their water activity was measured using a LabMaster Neo water activity meter.

Results: To incorporate buffering agents into alginate beads, various agents were added to the alginate-yeast mixture prior to crosslinking in a CaCl2 solution (Figure 1). These agents included four inorganic salts (phosphate, citrate, tartrate, and acetate) and one protein (6% casein, w/v). The outcomes of bead formation are recorded in Table 1. It was noted that the use of traditional inorganic anions consistently resulted in the failure of bead formation, likely due to the high tendency of calcium ions to precipitate with these anions. In contrast, the incorporation of casein into the alginate successfully resulted in bead formation. Comparative analysis of post-lyophilization survivability among different bead formulations revealed that trehalose provided a significantly higher protective effect (2.68%), while casein offered a marginally better protection (0.20%) compared to the negative control (alg-chi, 0.05%) (Figure 2a). In assessments of pH-buffering capability, alg-chi-cas exhibited a longer time before the pH decrease relative to alg-chi and alg-chi-tre, suggesting that casein enhanced the protection from acid permeation (Figure 2b). The cell survivability during a 1-hour gastric simulation also demonstrated that casein enabled a higher survival rate, consistent with its superior buffering capability (Figure 2c). Furthermore, the water-absorption curves showed that incorporating casein into alginate beads resulted in slower water absorption compared to beads made of alginate alone, thereby facilitating better moisture control in subsequent manufacturing and packaging processes (Figure 2d).

Conclusion: We demonstrated the superiority of casein as a novel-category excipient to support alginate-based oral delivery of probiotics, acting as a protectant to improve cell survivability upon lyophilization, reducing potential water absorption during downstream product process to enable better on-shelf stability, and serving as a pH-buffering agent to enable successful bead formation where traditional buffers inhibit bead formation. These features enabled the utilization of casein as a novel formulation strategy to encompass various challenges associated with cell viability loss during manufacturing, storage, and gastric transit. This underscores the potential of casein as a next-generation ingredient in pharmaceutical formulations and highlights the need for further research into proteins as an innovative category of excipients.

References: [1] Fangfang Nan, Jie Wu, Feng Qi, Yan Liu, To Ngai, Guanghui Ma, Uniform chitosan-coated alginate particles as emulsifiers for preparation of stable Pickering emulsions with stimulus dependence, Colloids and Surfaces A: Physicochemical and Engineering Aspects, Volume 456, 2014, Pages 246-252.
[2] Wang X, Gao S, Yun S, Zhang M, Peng L, Li Y, Zhou Y. Microencapsulating Alginate-Based Polymers for Probiotics Delivery Systems and Their Application. Pharmaceuticals (Basel). 2022 May 23;15(5):644.

Acknowledgements: This work is funded by R01AI148357-01 (NIAID).


Figure 1. Workflow of the preparation of alg-chi-excipient beads.


Table 1. Incorporating inorganic salts often results in failed bead formation, whereas proteins and sugars do not exhibit this issue.


Figure 2. Comparative analyses of different beads formulations. (a) Although casein served as a lyoprotectant to enhance post-lyophilization survivability, its effectiveness was not as pronounced as that of trehalose. (b) Alg-Chi-Cas showed better pH-buffering capability in simulated gastric fluid (c) Alg-Chi-Cas enhanced survivability following exposure to simulated gastric conditions. (d) Alg-Chi-Cas demonstrated reduced hygroscopicity.