Fermentation of palm kernel expeller using local fungal enzymes for production potential prebiotics

The rapid expansion of livestock industry to meet the increasing demand for animal protein has stimulated the search for appropriate agro by-products as alternative feed resources. Among the available by-products, palm kernel expeller (PKE) has been identified to be of great potential, because large quantity of this by-product from the palm oil industry is being produced annually in Malaysia. Many attempts have been made to reduce the high non-starch polysaccharides (NSP) of PKE to a tolerable level for monogastric animal, particularly poultry diets. This includes the use of exogenous enzymes, which has been proven rather successful in reducing the NSP contents, but when the enzyme treated PKE was fed to the animal, results in term of animal performance was inconsistent. In addition, scanty research data suggested that PKE can be a potential source for prebiotic instead of only as a source of dietary energy for monogastric animals. Thus, to overcome the current over-dependent on the use of commercial enzymes, which are normally imported at high cost to hydrolyse the NSP, the primary objective of this thesis was to increase the production of prebiotic oligosaccharides from PKE by using enzyme produced by indigenous fungi isolated from PKE itself. Ten fungi were isolated from local PKE. From a preliminary screening, three of the ten isolates which had the highest cellulase, mannanase, and xylanase activity were selected and identified using molecular analysis of fungal internal transcribed spacer (ITS) region. One isolate showed similarities to Paecilomyces variotii, while the other two showed similarity to Aspergillus terreus. Enzymes extracted from each of the above strain were tested for their ability to degrade PKE, as measured by the total reducing sugar production at the end of the solid state fermentation (SSF). Enzymes produced from isolate A. terreus K1 were most efficient in degrading the NSP of PKE into soluble sugar was selected for subsequent optimization process. Response surface methodology (RSM) was employed to optimize critical factors (temperature, moisture, inoculum, and pH) that affect SSF. A four-factor and five- level central composite design (CCD) was employed. Using PKE as sole substrate, a maximum cellulase (18.05 U/g DM) was produced at 30°C, 61% moisture, pH 5.3, and 7.5 × 105 spores/g PKE while maximum mannanase activity (42.03 U/g DM) was obtained at 31°C, 61% moisture, pH 6.4, and 6% spores, whereas maximum xylanase (339.68 U/g DM) was obtained at 29°C, 70% moisture, pH 4.6, and 7.7 × 105 spores/g PKE. In order to maximize the co-production of all the three enzymes simultaneously, CCD with three responses was used. It was predicted that maximum endoglucanase, mannanase, and xylanase (17.37, 41.24, and 265.57 U/g DM, respectively) can be produced by fermenting PKE at 30.5°C, 62.7% moisture, pH 5.8, and 6.0 × 105 spores/g PKE. Verification of the predicted condition was conducted in triplicate, and the enzyme activities obtained (19.97, 44.12, and 262.01 U/g DM, respectively) were close to the predicted values. The effectiveness of enzyme treatment to improve the prebiotics potential of PKE was evaluated using rats as animal model. Data of HPLC analysis showed that monosaccharides content increased 3 folds in the PKEENZ (enzyme-treated PKE) and 4 folds in SPKEENZ (steam + enzyme treated PKE) treatments as compared to the untreated PKE. These increases were reflected by the higher mannanoligosaccharides in the PKEENZ and SPKEENZ samples (3.95 and 8.07 mg/g PKE, respectively) compared to untreated PKE-extract (1.79 mg/g PKE). In vitro study showed that the different PKE-extracts used in this study were able to support growth of three different strains of Lactobacillus sp.. However, their growth varied significantly among species and PKE-extracts used (P < 0.05), with L. brevis I 218 had the highest growth compared to the other two strains (L. salivarius I 24 and L. gallinarum I16), and its growth was the highest when incubated in the SPKEENZ extract. In order to confirm the results of the in vitro study, an in vivo study were conducted using Sprague-Dawley rats fed standard rodent diet supplemented with the different PKE extracts. Results of the in vivo study showed that all the PKE-extracts tested can support growth of beneficial bacteria (Lactobacillus and Bifidobacterium), however, only rats in the SPKEENZ treatment group had significantly higher population of Lactobacillus and Bifidobacterium and lower population of E. coli compared to the control group. In conclusion, this thesis revealed that enzyme produced from fungi isolated from local PKE can be used to hydrolyse the NSP of PKE into mono- and oligo-saccharides and provided in vitro and in vivo data to show that they possess prebiotic properties. Comparison between different treatment-methods of PKE showed that pre-treatment by steaming prior to enzymatic treatment could further enhance the beneficial effects of PKE-extract as prebiotics.

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