Study On The Enzymatic Production Of Xylooligosaccharides From Apple Pomace

Study on functional oligosaccharides, which has the physical characteristics such as the promotion of Bifidobacterium and not being absorbed by human bodies, has become one of the most popular topics in the field of health food research and development. Xylooligosachhardies (XOs), is oligosaccharide mixture containing 2-7 xylose of linear or branched chains which is connected by (3-(1-4) glycosidic bond. Xylobiose (X2) and Xylotriose(X3), are two major elements presented in XOs. Except for the physiological function of proliferating the bifidobacteria, resisting saprodontia, boosting immunity and lowering blood-pressure and blood-lipids, XOs have some unique qualities such as, low-dosage of usage, good stability, etc. Apple pomace is the main by-product of apple juice industry. Xylan accounts for 25.5% of dry apple pomace. If apple xylan used fully, not only the pollution of apple pomace can be solved, but also the comprehensive utilization of apple can be improved, which can increase the economic and social benefits, and is more advantageous to the development of apple industry. Currently, the main raw materials for the production of XOs is corncob. Production of XOs by enzymatic hydrolysis of apple pomace has not been reported, thus we study the enzymatic hydrolysis of apple pomace and the separation and purification of XOs.First, apple pomace xylan was extracted by alkaline hydrogen peroxide method. Then, apple xylan was enzymatic hydrolyzed by xylanase. Based on the study of the impacts of enzyme concentration, pH value, substrate concentration, temperature, time to the production of XOs, we used orthogonal experiment to get the optimum technological conditions for enzymatic hydrolysis of xylan, with the content of reducing sugar (RS), soluble total sugar (STS) and the average degree of polymerization (DP) of the hydrolyzate as index. After purification of XOs by active carbon-diatomite chromatography, we got light-yellow amorphous crystal XOs. The main research results are shown as follows:1. Use alkaline hydrogen peroxide to extract apple xylan, do orthogonal experiment on the factors as follows:the concentration of hydrogen peroxide solution(H2O2), pH value, reaction time and reaction temperature. The optimum technological condition: H2O2 3.0%, pH 12.5,50℃, for 8 h. Under such condition, the yield of apple xylan was more than 85%.2. Apple xylan was hydrolyzed by xylanase. Single factor analysis was carried out before orthogonal experiment. The content of reducing sugar (RS) was analyzed by 3,5-dinitro salicylic acid (DNS). Single factor analysis shown that:the content of RS increased with the increase of the reaction time from 1 h to 12 h; the content of RS increased with the increase of the substrate concentration, which reached up to 50.87 mg when the concentration is 4.08%, while the hydrolysis ratio was on the decrease, so we selected 4.08% for optimum substrate concentration and did not select it as as a factor in orthogonal experiment; when enzyme concentration was 1500 U, soluble total sugar (STS) and RS were 129.85 mg and 63.42 mg, respectively, and the average degree of polymerization (DP) of the hydrolyzate was 2.05; at pH 5.0, the enzyme activity reach maximum, the average DP of the hydrolyzate was 2.09, and the main hydrolyzate was X3; at 40℃, both RS and STS nearly reached maximum, that were 63.40 mg and 134.03 mg, respectively, then decrease quickly after 50℃. The optimum conditions for enzymatic hydrolysis of xylan in orthogonal experiment was: enzyme concentration 1500 U, pH 5.0, 40℃, reaction time 8 h. The concentration of RS and STS were 6.42 mg/ml and 13.28 mg/ml, respectively, and average DP of the hydrolyzate was 2.07 under such condition.3. Use active carbon-diatomite chromatography column for the purification of XOs. Desorption of salts, monosaccharide and other impurities by deionized water (DW) before desorption of XOs by 30% ethanol. XOs can not be washed out until the eluant reached 175 ml. At 225 ml, the concentration of XOs reached maximum (16.79 mg/ml), and remain unchanged to 250 ml, and then began to fall. XOs was completely washed out at 375 ml.4. Thin-layer chromatography (TLC) of XOs showed that the main enzymatic hydrolyzate of apple xylan were X3 and X2, and the content of the X3 was large than X2. After separation, monosaccharides can be removed completely, and XOs be purified. Recycle ethanol by rotating evaporation. At last, we got a light-yellow amorphous crystal XOs when it was dried by vacuum freeze-drying.