الفهرس 
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Abstract
The conversion of xylan into value added useful products by enzymatic process holds strong promise for the use of a variety of agro-industrial residues. The results obtained substantiated the potential of orange peels biomass for extraction of xylan (OPX) and subsequent enzymatic production of functional XOS. The characteristic properties of the applied crude hydrolyzing enzyme,
B. amyloliquifaciens xylanase, validated the efficiency of the enzyme for marketable XOS production without formation of undesirable xylose by-products. The optimal operating conditions for enzyme activity were compatible for industrial level process.
The multivariable technique RSM based on CCD was highly efficient for optimization of XOS yield. The method explicated the interactive effects among the enzymatic reaction variables and the significance of each variable on the overall production process. Furthermore, the statistical based method proved advantageous over one factor at a time optimization studies in terms of time and cost.
The achieved optimum OPX derived XOS yield (16.059 ±
0.19 mg/ml xylan) was almost equivalent to XOS produced by enzymatic hydrolysis of purified BWX (16.02 mg XOS/ml xylan). The results further supported the notion of OPX as more economical alternative xylan source compared to standard commercial xylan types.
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The low polymerization degree of OPX derived XOS (2-5) preferred for prebiotics as well as their high antioxidant capacity evaluated in terms of IC50 satisfied the requirement of nutraceutical industry. The absence of monosaccharides in OPX hydrolysate allowed for minimum purification steps and acceptable recovery yield. Furthermore, the purity levels of XOS separated fractions namely xylobiose (75%) and xylopentose (77%) fulfilled strict marketing criteria.
The biochemical and histopathological examination of liver and kidney suggested that XOS supplementation has therapeutic effect on CCl4 induced oxidative liver damage in a much effectual way than the protective approach. Meanwhile, the conferred protective effect of XOS was found to be remarkable against CCl4 induced oxidative kidney damage.
Recommendations
The continuous application of bio-based technologies sustains the expansion of global economy. from this perspective, XOS produced by enzymatic hydrolysis of OPX offers great scope to the nutraceutical and pharmaceutical industries in terms of cost and quality. Large scale production and final product development are recommended. The full potential health benefits of OPX derived XOS can be further established by additional in vivo studies.
The conversion of xylan into value added useful products by enzymatic process holds strong promise for exploitation of agro- industrial residues. Xylooligosaccharides (XOS) are sugar oligomers produced during the hydrolysis of xylan with multiple applications in pharmaceutical, nutraceutical and feed industries. Cheap xylan sources, instead of the hardwood xylan, are necessary for economic production of XOS.
The present study targeted the production of XOS by enzymatic hydrolysis of xylan extracted from orange peels (OP). Commercial beech wood xylan (BWX) was used for comparative analysis. Bacillus amyloliquifaciens NRRL B-14393 xylanase in its crude form was used for the hydrolysis process and its biochemical characteristics required for satisfactory bioprocess were investigated. Response surface methodology (RSM) based on central composite design (CCD) was applied to optimize the enzymatic reaction conditions related to the production cost. Identification, purification, separation and characterization studies on the produced OPX derived XOS were performed. Furthermore, the antioxidant activity of crude OP derived XOS and their correlated phenolic content were evaluated in vitro.
The major findings of the in vitro study are summarized as follows:
- The chemical composition analysis of OP wall constituents revealed significant presence of hemicellulose fraction (17.11 ±
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0.13 %) and low acid detergent lignin (3.10 ± 0.01 %) indicating the biomass suitable as potential xylan source.
- Simple dilute-alkali extraction of xylan from dried OP at laboratory scale proved effective in terms of xylan true recovery (10.00 ± 0.26 %, w/w), as well as hemicellulose relative recovery (58.44 ± 0.97 %, w/w).
- FTIR spectra of extracted xylan confirmed the presence of characteristic β-glycosidic linkages and β-D-1, 4-xylopyranose residues.
- The amorphous nature of OPX was evident from scanning electron microscope (SEM).
- The crude xylanase extract proved to be β-xylosidase-free.
- The crude xylanase was active over a broad pH range with a maximum activity at pH, 8 and retained more than 80% of its activity at pH range 5–7 upon incubation at 30 °C for 60 minutes.
- The highest xylanase activity was observed at 50 °C, the crude enzyme displayed an appreciable half-life up to 60˚C for a period of 90 minutes and was thermostable up to 70 °C for 30 minutes.
- Optimum XOS production from OPX xylan (16.06 ± 0.19 mg/ml xylan; 401.48 ± 4.75 mg/g xylan) was achieved at xylanase dose of 4.44 mg /g xylan, substrate concentration at 3 %, w/v and 5 h incubation time as deduced using RSM.
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- HPLC analysis indicated xylobiose (1.27 %) and xylopentose (98.72 %) as the main end products of OPX hydrolysis under optimum conditions.
- from economic stand point, reducing OPX concentration to 1.46%, w/v yielded 14.064 ± 0.19 mg/ml xylan (352.25 ± 4.75 mg XOS / g xylan) applying enzyme dose of 2.22 mg/ g xylan and 1 h incubation period. Xylobiose (0.75 %) and xylopentose (99.25
%) were identified as the main end products under these conditions.
- Optimum XOS production from BWX (16.02 ± 0.56 mg XOS/ml xylan; 400.45 mg XOS/g xylan) was achieved at xylanase dose of 1.702 mg /g xylan, substrate concentration at 1.08 %, w/v and 4.91 h incubation time using RSM.
- HPLC analysis indicated xylobiose (0.36 %) and xylopentose (99.52%) as the main end products of BWX hydrolysis under optimum conditions.
- Purification and separation steps of OPX derived XOS encompassed microfiltration followed by gel permeation chromatography. The recovery yield of the purified XOS mixture was 68.00%.
- The purity of separated XOS fractions identified as xylobiose and xylopentose were of 75 and 77%, respectively.
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- The UV-Vis scan of the eluted refined XOS mixture attested the lack of protein, nucleotides as well as the absence of un- hydrolyzed polysaccharide.
- Purified XOS (β glycosidic linkages) presence was confirmed through structural studies SEM, FT-IR, 1H and 13C NMR spectroscopy which are very useful techniques for the identification and characterization of the detailed structures of XOS.
- Morphological changes of refined XOS mixture, xylobiose and xylopentose surfaces signified by scanning electron microscope (SEM) confirmed the xylanolytic hydrolysis of OPX.
- Estimated IC50 value of XOS scavenging capacity against DPPH, ABTS•+ and hydroxyl free radicals were 1.10, 1.06 and 1.25 mg XOS, respectively.
- The ferrous ion-chelating activity of the XOS mixture against positive control EDTA showed IC50 values of 23.95µg/ml and 0.011mM, respectively.
- Reduction power of XOS mixture was dose dependent and increased steadily at higher concentrations.
- The total phenolic content of XOS mixture was 105 ± 0.75 mg GAEq/g XOS.
- Vanillin was identified by HPLC as the major bound phenolic acid in the XOS mixture (431.48 µg/g XOS) followed by catechin
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(193.93 µg/g XOS), coffeic acid (184.44 µg/g XOS), coumaric acid (173.81µg/g XOS) and rutin (82.34 µg/g XOS).
**The effectiveness of OPX derived XOS supplementation in treatment and prevention of CCl4 induced hepatic oxidative stress in rats were evaluated. Biochemical (serum ALT, AST, ALP, urea, creatinine, sodium and potassium levels besides liver concentration of MDA, NO, GSH content, SOD, GPx and catalase activities) and histopathological studies (liver and kidney tissues) were completed. Fifty adult male Albino rats weighing about 110- 130 g were divided into five groups each group contained 10 rats. group I (control) rats were left without any treatment and were given distilled water, group II (XOS received) rats were orally administrated with OPX derived XOS (700 mg / kg body weight) daily for 5 consecutive weeks, group III (CCl4 group) rats were intraperitoneally (IP) injected with a mixture of CCl4 in olive oil (1:1) at a single dose of (1 ml/kg body weight) to induce oxidative stress, group IV (CCl4 then XOS) rats were IP injected with a mixture of CCl4 in olive oil (1:1) at a single dose of 1 ml/kg body weight 24 h prior to daily oral administration of OPX derived XOS at a dose of 700 mg/kg body weight for 5 consecutive weeks and finally group V (XOS then CCl4) rats were orally administrated with OPX derived XOS at a dose of 700 mg/kg body weight daily for 5 consecutive weeks then IP injected with
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a mixture of CCl4 in olive oil (1:1) at a single dose of 1 ml/kg body weight.
The major findings of the in vivo study are summarized as follows:
- group III (CCl4 group) recorded significant increase (P< 0.001) in liver enzymes activities (serum ALT, AST and ALP), renal biomarkers (serum urea, creatinine, sodium and potassium) as well as non-enzymatic antioxidants markers (liver homogenate MDA and NO). Meanwhile, a significant decrease (P< 0.001) in the levels of GSH and antioxidant enzymes (CAT, GPx and SOD) activities in hepatic tissue were observed when compared to group I (control group).
- Based on biochemical and histopathological studies, group II (XOS received group) generally demonstrated non-significant differences (P>0.05) when compared to group I (control group).
- XOS supplementation to group IV (XOS post-treated) and group V (XOS protected group) significantly improved (P <0.001) the activities of serum ALT, AST and ALP when compared to group III (CCl4 group).
- In groups IV (XOS post-treated) and V (XOS protected groups) non- significant changes (P > 0.05) of serum urea, creatinine sodium and potassium concentrations were noted compared to group I (control group).
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- XOS supplementation significantly restored MDA levels in groups IV and V (XOS post-treated and XOS protected groups) to near the level of normal control group (P <0.05).
- The increase in NO levels in the tissue homogenate of rats allocated in IV group (XOS- post treated group) was moderately significant (P<0.01). While, in group V (XOS protected group) the increase was non-significant (P >0.05) compared to group I (control group).
- The restored levels of GSH in group IV (P <0.01) posed a higher positive activity towards the normal levels than that of group V (P <0.001).
- Animals allocated in experimental group IV (XOS post-treated group) showed non-significant differences (P >0.05) in SOD, CAT as well as GPx activities compared to group I (control group).
- Experimental group V (XOS protected group) presented non- significant decrease (P >0.05) in CAT activity and moderately significant decrease (P <0.01) in both SOD and GPx activities compared to group I (control group).
- Histopathological examination of group IV (XOS post-treated group) proved potent curative effect of XOS against CCl4 induced oxidative stress in liver and kidney.
- Histopathological abnormalities in the liver observed in group V (XOS protected group) suggested XOS supplementation has less
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distinct protective effect against CCl4 induced oxidative liver damage.
- XOS supplementation has a remarkable protective effect against renal injury as evidenced by complete restoration of kidney tissue to normal state in group V (XOS protected group).
The results suggested that XOS produced by enzymatic hydrolysis of OPX are applicable in food and pharmaceutical industries. The overall production process was economic and the produced OPX derived XOS proved highly marketable. Moreover, the produced OPX derived XOS have very effective renal protective effect and could be considered as potent therapeutic agent against hepato-renal chemically induced oxidative stress.