Recovery of hyaluronic acid using thermosensitive polymer-salt aqueous biphasic system with polymer recycling

Microbial fermentation emerges as a viable alternative to animal tissue-based extraction for industrial hyaluronic acid (HA) production. However, microbial HA requires rigorous separation and purification processes to meet high purity standards for cosmeceutical use. Aqueous biphasic system (ABS) presents a promising strategy for HA recovery due to its cost-effectiveness, high yield, purity and eco-friendly nature. Thermosensitive polymer, specifically ethylene oxide-propylene oxide (EOPO)-salt ABS, was proposed in the present study to recover microbial HA from Streptococcus zooepidemicus fermentation broth. The effects of molecular weight (MW) of EOPO, types of salt, phase composition, and the amount of crude extract on the efficiency of ABS for HA recovery were studied. Furthermore, the recovery efficiency of ABS formed using recycled EOPO was investigated. HA exhibited a preference for the salt-rich bottom phase of ABS because of its hydrophilic surface features. Highest partition coefficient of 11.41 ± 0.00 and yield of 93.42 % ± 0.00 % were achieved in ABS containing 14 % (w/w) EOPO₂₅₀₀, 12 % (w/w) sodium citrate at pH 8.2 and 15 % (w/w) crude extract. More than 70 % of EOPO₂₅₀₀ was recovered through the thermo-separation process, and the 1st recycling of EOPO₂₅₀₀ maintained a recovery yield of HA above 80 % in ABS. Moreover, Fourier transform infrared spectroscopy validated that HA retained its structure after the recovery process. In conclusion, this study demonstrates the effectiveness of EOPO-salt ABS for microbial HA recovery, achieving high recovery yield. The recycling of EOPO enhances the economic viability and environmental sustainability of the system. These findings pave the way for greener, scalable HA production practices while maintaining product integrity.