الفهرس 
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Abstract
The present study was conducted to investigate the possible utilization of the environmentally polluting solid residue obtained after olive oil extraction, olive oil cake (OC), as a solid support under solid state fermentation for microbial production of the tanninolytic enzyme, tannase, with concomitant production and extraction of pharmaceutical bioactive compounds having antioxidant and anticancer activities and to improve the chemical composition of OC through fermentation which can allow it to be used as a composite or in animal feeding.
The studies showed the following:
1. A screening program was done for ten strains; bacterial strains (Bacillus amyloliquifaciens NRRL B-14393 and Bacillus subtilis NRRL B-4219), yeast strains (Kluyveromyces marxianus NRRL Y-8281, Kluyveromyces marxianus NRRL Y-7571, Saccharomyces cerevisiae NRRL Y-12632, Candida bambicola NRRL Y-17069 and Candida guilliermondii NRRL Y-2075) and fungal strains (Agaricus blazei, Ganoderma lucidum and Hericium erinaceus) to examine their ability to produce tannase using OC as a sole carbon source for different incubation periods. Of the ten strains tested, Kluyveromyces marxianus NRRL Y-8281 showed the highest tannase productivity using raw OC under SSF after 2 days of incubation.
2. The chemical composition of unfermented (UFOC) and fermented (FOC) olive cake was investigated and the results showed that the fermentation altered OC chemical composition so that the crude fiber was decreased by 8.56%, while crude protein, fat and carbohydrate contents were increased by 2.74%, 2.63% and 3.57%, respectively keeping the organic matter and ash content nearly unchanged.
3. Overproduction of tannase (1714.7 U/ gds) was achieved by optimizing fermentation conditions. The effect of incubation time (from 12 hours to 4 days) was studied and the 48th hour of incubation was chosen as the best incubation period. The effect of supplementation of different carbon (glucose, galactose, mannose, sucrose, lactose, fructose, sorbitol, mannitol, starch, tannic acid, gallic acid and methyl gallate) and nitrogen (urea, peptone, yeast extract, malt extract, ammonium nitrate, ammonium chloride and sodium nitrate) sources on tannase production was investigated. Neither carbon nor nitrogen sources used in this study demonstrated any stimulating effect on tannase production. The effect of initial pH of the medium (from 4 to 8) and incubation temperature (from 25 to 50°C) on enzyme production was evaluated. The optimum pH and temperature was found to be 6.0 and 45°C, respectively. Different inoculum sizes (5-30%) were tested. The highest enzyme activity was obtained using 20% inoculum size. The effect of moisture level (0-60%) on enzyme production was tested. The optimum moisture level was found to be 35% which represented the original moisture content of OC.
4. The crude enzyme preparation was obtained by conducting fermentation processes under the optimal culture conditions. The whole culture medium collected from several batches was used as a source of the crude enzyme.
5. Partial purification of crude K. marxianus tannase was carried out by fractional precipitation with ammonium sulfate. The precipitation with 60-80% was the most powerful precipitating agent that showed the highest specific activity (664.3 U/mg) reaching 15.68 purification fold with good recovery of enzyme activity (67.94%).
6. Further purification of the partially purified enzyme was carried out by gel filtration chromatography on Sephadex G-200 chromatographic column which led to obtaining tannase enzyme with 24.21 purification fold, compared to that of the culture filtrate, with 1026.12 U/mg specific activity and suitable recovery (64.6%).
7. The UV spectrum of tannase exhibited an absorbance maximum at 349 nm with no significant absorbance could be traced in the visible region indicating the absence of chromophore.
8. The physicochemical properties of pure K. marxianus NRRL Y-8281 tannase enzyme showed that:
- The molecular weight of the pure enzyme was found to be 66.62 x 103 and 65 x 103 Da as determined by gel filtration technique and SDS polyacrylamide gel electrophoresis, respectively.
- The pure enzyme exhibited maximum activity at pH 4.5 and 8.5 with 0.2M acetate buffer and tris-HCl buffer, respectively. For pH stability, at least 64.5% of tannase activity was maintained from pH 4.0 to 6.0 after incubation at 30C for 30 minutes.
- The optimum temperature was 35C for pure tannase and the activation energy was found to be 8.84 Kcal mol-1. The enzyme was stable up to 70˚C and retained 62.3% of its activity till 60 minutes of incubation.
- The rate of hydrolysis of tannic acid by pure K. marxianus NRRL Y-8281 tannase increased with increasing time till 10 minutes followed by a decrease in hydrolysis rate.
- A linear relationship was observed between the rate of the reaction and the concentration of tannase enzyme up to 300 µg/reaction mixture.
- The purified tannase was tested for substrate specificity. Tannic acid, methyl gallate and propyl gallate were good substrates for enzymatic activity. The enzyme was less active on epigallocatechin gallate.
- The Km value for K. marxianus tannase was 0.77 mM and Vmax was 263.16 µmole min-1 ml-1 using tannic acid as a substrate.
- The effect of metal ions (1mM) on purified tannase was evaluated. Ca++, Cd++, Cu++, Fe+++ and Li+ enhanced the enzyme activity. Moderate inhibitory effect on tannase activity was caused by Na+, Mg++, Co++, Ba++ and Zn++. While, K+ and Hg++ had higher inhibitory effects on tannase activity. The highest inhibitory effect on the enzyme activity was brought by Mn++.
9. HPLC analysis data indicated that the purified enzyme could carry out 24.65% tannic acid conversion within 30 minutes only with concomitant production of 784.55 µg gallic acid reflecting an increase in its concentration by 5.25 folds.
10. Phenolic compounds were recovered from both UFOC and FOC simultaneously with the evaluation of the antioxidant activity of the extracts using DPPH system. The effect of different conditions (extracting solvent, sample to solvent ratio, extraction time and extraction temperature) on both total phenolic extraction efficiency and in vitro antioxidant activity was estimated. Methanol showed both highest total phenolic extracting capacity and antioxidant activity among all tested solvents. Maximum total phenolic recovery (160.08 and 149.00 mgGAE/gds for UFOC and FOC, respectively) and antioxidant activity (96% and 97% for UFOC and FOC, respectively) were achieved by optimization of extraction parameters using a sample to solvent ratio of 1:10 at 50°C for two hours.
11. The effect of methanol extract concentration on total phenolic content and antioxidant activity by using both DPPH radical scavenging activity assay and β-carotene/linoleic acid system assay was evaluated. For both UFOC and FOC methanolic extracts, a concentration of 4 mg/ml expressed the highest antioxidant activity in DPPH system (96% and 97% for UFOC and FOC, respectively) and β-carotene / linoleic acid system (44.5% and 68.9% for UFOC and FOC, respectively), while the total phenolic content increased linearly with increasing the extract concentration.
12. HPLC analysis revealed that K. marxianus mediated fermentation of OC resulted in a sharp decrease in OC tannin content by 96.75% which was accompanied with 2.8 times increase in gallic acid concentration.
13. GC/MS analysis data revealed that major compounds identified in UFOC methanolic extract were methyl palmitate, methyl oleate and ethyl oleate. While, analysis of FOC methanolic extract confirmed the production of carvacrol, thymol, eugenol, caryophyllene oxide and methyl isopalmitate by fermentation.
14. In vitro anticancer activity of UFOC and FOC methanolic extracts was assessed against different human cancer cell lines. The results revealed that although both extracts did not show any effect against lung A549, cervix Hela cancer cell lines or normal HFB4 cells, they exerted anticancer effect close to the value of the doxorubicin drug against liver HepG2 and breast MCF-7. Moreover, both extracts showed moderate activity against prostate PC3 and colon HCT116 cell lines. However, the fermented extract was more potent than the unfermented one.
Fermentation of OC by K. marxianus via SSF does not only eliminate the environmental pollution resulting from its accumulation, but also presents a new eco-friendly valorization technique for the production of an industrially important enzyme, tannase, simultaneously with gallic acid production and opens new prospects for the generation of antioxidant and anticancer pharmaceuticals leaving the OC with altered chemical composition allowing its use as animal feed or compost.