Development of polymeric resin for enhanced in-situ hyaluronic acid recovery and its subsequent hyaluronic acid/ silver nanoparticle composite synthesis

Hyaluronic acid (HA) is a nonsulfated polysaccharide used in various biomedical and cosmetic applications due to its biocompatibility and viscoelastic properties. Industrially, it is produced through streptococcal fermentations, but microbial fermentation methods face challenges like low oxygen transfer efficiency due to high broth viscosity, limiting HA yield. Additionally, physical and chemical synthesized silver nanoparticle (AgNP) involve harmful reductants and are costly, and less effective antimicrobial properties due to AgNP aggregation. To overcome these challenges, in-situ product recovery (ISPR) of HA using polymeric resins was investigated, aiming to enhance yield and recovery prior preparing HA/AgNP composites. Five adsorbent resins, Amberlite IRA 400 Cl, Amberlite IRA 900 Cl, Amberlite IRA410 Cl, Amberlite IRA 402 Cl, and Amberlite IRA 67 (IRA 67) were tested, IRA 67 showing the highest adsorption capacity and selectivity for HA. Kinetic studies revealed HA adsorption onto IRA 67 followed pseudo-second order kinetics (R2= 0.985), and Freundlich isotherm model fitted better than Langmuir with a R2 value of 0.989. The study of ISPR via 2-L stirred tank bioreactor of Streptococcus zooepidemicus HA-116 was investigated to elucidate the adsorption of HA onto IRA 67 via dispersed and internal column systems employing helical ribbon (HRI) and Rushton turbine impeller (RTI). The utilization of an RTI surpassed the HRI, resulting in a more efficient HA yield. The application of a dispersed IRA 67 with RTI improved the HA production compared to the conventional fermentation without resin addition by 1.37-fold. The HA production was further improved by 1.36-fold with an internal column packed with IRA 67 resin system (3.928 g/L) over that obtained with dispersed IRA 67 system. The cultivation with an internal column system shows the highest reduction of viscosity value, from 58.8 to 23.4 (mPa⋅s). Two purification methods were compared for samples from extractive fermentation: Clarified Broth (without cells) and Resin Eluent. Method 2, which skipped the precipitation step, achieved to remove > 99% of proteins and 59% of nucleic acid (NA) from the Clarified Broth samples with 71% HA recovery. Method 2 have removed 93% proteins and 78% NA while retaining 58% HA for the Resin Eluent samples. The purified HA was successfully characterized by Fourier transform infrared (FTIR), however, only the HA from the Clarified Broth sample met all of the standards set by the British Pharmacopeia. The HA/AgNP were synthesized using microalgae extracts and characterized. The best HA/AgNP, synthesized by Spirulina platensis (HA-SP-AgNP) with Zaverage size and polydispersity index (PDI) of 66.98 nm and 0.494, respectively, indicative of its stable characteristics. The HA-SP-AgNP demonstrated better PDI value and antimicrobial activity against S. aureus and E. coli compared to SP-AgNP. The improved biosynthesis of HA indicated that the extractive fermentation by ISPR is effective and able to facilitate the subsequent purification steps, whilst the use of HA in the preparation of HA/AgNP nanocomposite can provide eco-friendly and nanometric material which has potential for clinical application.

Text 124855 fulltext.pdf Download (1MB) Official URL or Download Paper: https://ethesis.upm.edu.my/id/eprint/18787

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