Production of violacein nanoparticles via sonication technique with the aid of surfactants as stabilizer

Abstract

Violacein, a violet pigment produced from Chromobacterium violaceum UTM5, has gained interest due to its biodegradability and pharmacological properties. However, its high production cost and limited solubility in water have become the major stumbling blocks for the pigment to be applied in different industries. In this study, liquid pineapple waste was used as an alternative inexpensive growth medium for bacteria cultivation instead of expensive synthetic nutrient broth, thus reducing the production cost of this pigment. The cultivation of C. violaceum in 50 L bioreactor gave a crude yield of 11846 ± 925 mg L-1, which was comparable to the yield obtained using commercial growth medium. The crude pigment was successfully extracted using ethyl acetate. The presence of violacein, the major active compound of the crude pigment, was confirmed using high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR) and ultraviolet-visible spectrophotometry (UV-Vis). Thermal gravimetric analysis was used to determine crystallinity and thermal degradation while Zetasizer analyzer was used to identify the isoelectric point, stability at various pHs, and particle size of violacein. Violacein nanoparticles were produced via sonication technique, with the aid of surfactants (Tween 80, Triton X-100, sodium dodecyl sulfate and dodecyltrimethylammonium bromide) as solubilizing and stabilizing agent, to address the violacein’s poor solubility in water. The violacein nanoparticles were characterized using UV-Vis spectrophotometry, FTIR, thermal analysis and Zetasizer analysis. Water soluble violacein nanoparticles were produced at surfactant concentration greater than its critical micelle concentration, as indicated by FTIR. Zetasizer analysis showed the smallest violacein nanoparticle, which was 131.5 ± 2.001 nm, with polydispersity index (PDI) of 0.180 ± 0.018, which indicated a monodispersed violacein nanoparticle distribution. The thermal analysis showed that violacein nanoparticles were in amorphous state and stable upon dispersion in water, with a zeta potential of -49.8 ± 3.49 mV. The violacein nanoparticles have better solubility than the crude violacein pigment. The solubilized violacein nanoparticles remained well-dispersed upon storage in 28 days at different temperatures. In addition, the violet color of the violacein nanoparticles was maintained at pH range of 3 to 11, temperatures of up to 60°C, and under dark condition, despite its nanoscale size. Higher degradation rate was observed at high temperature and upon light illumination, with k = 6.51 × 10-3 h-1, t1/2 = 148 h and k = 6.75 × 10-4 h-1, t1/2 = 1027 h, respectively, following the first-order kinetics. In conclusion, this study confirmed the feasibility of using liquid pineapple waste as cheap growth medium for cultivation of C. violaceum UTM5 in pilot scale (50-L bioreactor) while production of water-soluble violacein nanoparticles via sonication method with the aid of surfactants as stabilizers would increase its usefulness, especially in pharmaceutical industry.