Improving thermostability of bromelain via immobilization onto clay-carboxymethylcellulose composites for aquafeed application

Supplementation of bromelain, a protease from the pineapple plant, in feed can significantly improve the growth performance of aquatic animals. However, the instability of bromelain at high temperatures (≥ 100 ºC) during feed processing limits its applications in aquaculture. To improve thermal stability, the immobilization of bromelain onto bentonite-carboxymethylcellulose (CMC) composite through an ionotropic gelation technique was introduced in this study. The composites containing bentonite clay and CMC were prepared in different CMC to calcium ion (Ca2+) proportions of 1:10, 1:20, 1:30, 1:40, and 1:50 (w/w). High immobilization yield (> 95%) with loading capacity in a range of 0.8-0.9% was obtained from all conditions. Less than 1% of enzyme leakage was found after placing it in water for 30 min. Among the immobilized enzymes, composite with a CMC: Ca2+ mass ratio of 1:20 showed the highest enzyme activity at 0.16 ± 0.01 units/mg protein. After thermal treatment at 100 ºC for 10 min, the residual activity of free bromelain was found at 0.96 ± 0.19%, while that of the immobilized enzyme was 139 ± 20% from its original activity. The study showed that incorporating bentonite clay with CMC in the presence of calcium ions created a good synergetic effect to improve thermal stability with low leakage loss of immobilized bromelain. These results in a high potential for its wide utilization in aquafeed applications.