Optimization of β-Mannanase production from Bacillus subtilis H and its application for the formulation of fish feed containing palm kernel cake.

Palm kernel cake (PKC) has been largely produced in Malaysia but consider as a non cost-effective feed ingredients because of several constraint such as high fiber (mannan) content. Fish, a monogastric animal is lacking of capability to digest fiber in higher percentage. Hence, PKC is not suitable for monogastrics animal as feed without any pretreatment or supplementation. The thesis researched on the β-mannanase production and the addition of this enzyme as supplement in feed formulation containing PKC for red tilapia hybrid (Oreochromis sp.). The study started with the selection of β-mannanase producing microorganism from the fermented PKC was conducted. The optimal temperature and pH for purified β-mannanase activity produced by the Bacillus subtilis H was at 50oC and pH 8.0; respectively. The crude enzyme showed stability over a broad range of pH 4.5-10.0 and displayed relatively high activity on elements such as Mg, K, Mn, Na, Cu and Ca that has been reported present in PKC. These properties make the β-mannanase more useful in the animal feed and detergent industries. The response surface methodology (RSM) was used for the optimization experiments. The effect of medium composition and culture conditions to enhance the production of enzyme, in shake flask and fermenter; respectively was then studied. The highest β-mannanase production in shake flasks, had demonstrated the used of relatively low cost ingredients of 53.8 g/l PKC, 2.0 g/l yeast extract, 2.5 g/l (NH4)2SO4 and 4.0 g/l NH4NO3. However the production of enzyme was far much lower in fermenter as compared to shake flask experiment. Supplementation of protectant during freeze drying process for preserving of protein activity was also conducted. Skim milk showed the largest effect in preserving the β-mannanase activity when exposed to all tested environment or conditions. It can be concluded that the empirical model developed through RSM in terms of effective medium composition, culture condition and protectant addition during freeze drying were found adequate to describe the optimal values for all experiments. β-Mannanase can degrade mannan structure in PKC however it is considered as a costly approach and a significant reduction in cost will be important for their commercial use. The optimal condition for enzymatic saccharification of PKC was then studied. The evaluation on the influence of β-mannanase supplementation to the fish feed containing PKC on red tilapia was then conducted. During the growth experiment, the final body weight, weight gain and feed conversion ratio were significantly better in tilapia fed diet with enzyme supplementation. The inclusion of enzyme could also reduced the released of ammonia to the cultured environment. In conclusion, supplementation β-mannanase in the diet containing 40% of PKC could improved the growth performance, energy and nutrient digestibility of tilapia, thus would increased the nutritional value of PKC as feed ingredient.