Production Of Mixed Enzymes With Apple Pomace In Solid State Fermentation

China is one of the largest apple productions in the world, and about 20% of apple are used to produce fruit juice, which can produce about one million tons apple pomace. At present, a small amount of apple pomace are used for fuel, fertilizer and processing, and the most are abandoned, which are not only waste resources but also seriously polluted the environment. Therefore, the development and utilization of apple pomace, feed resources development has become an important issue. This paper mainly studied in apple pomace as raw material which are produced mixed enzymes by solid state fermentatio, and the main results of cellulase, xylanase and pectinase activity are as follows:l. The main component of the apple pomace were analyzed.Apple pomace is composed with 96.2% fruit peel, fruit seed 3.1%, and stem 0.7%. Pomace contains nitrogen-free extract 61.5%, 12.3% of total sugar, 6.2% crude fat, crude protein content of 5.6%, sugar 11.4%, 0.5% fructose, sucrose 0.5%, 0.52% total acid, oleic acid 31.8%, Asia 33.5% oleic acid, sodium 454.72 mg / kg, K 0.436%.2. Three kinds of optimization of assay conditionsApple pomace fermentation can produce complex enzyme, so we should explore how to determine cellulose enzyme, xylanase, pectinase activity, that is to say, the best determination of enzyme activity of three wavelength scans, DNS plus, enzyme substrate, reaction time, reaction temperature, boiling time for the single factor analysis, the final determination of conditions to determine the various parameters: wavelength of three kinds of activity is 480 nm, measuring temperature is 40 o C, pH 5.0, and enzyme substrate volume is 2 ml, and choose blank sample to terminate. determination of cellulose substrate concentration is 10 g / l, enzymatic reaction time is 10 min; xylan substrate concentration is 10 g / l, reaction time is 15 min; pectinase optimum substrate concentration is 0.8 g / l, and reaction time is 15 min.3. Determination of solid-state fermentation mediumFermentation period is 48 h, the ratio of water are determined to be 1:1.2, from second day of the fermentation on, daily replenishment water is 1.0 ml, timely replenishment of the enzyme activity increased to a certain extent. Single factor and orthogonal tests are to determine influence of different nitrogen sources on solid-state fermentation of apple pomace, and to determine the solid medium for fermentation of apple pomace: 2.0% ammonium sulfate, potassium hydrogen phosphate 0.1%, 2.5% urea, marc: bran = 4:1.4. the selection of species combinationWe can use solid flat culture medium to choose the species which can develop thriving, and we can determine the activities of the three enzymes to choose the higher activities. Fungi which are chosed should be validated wheather they can growth harmonious with each other with line on the solid flat culture medium. Through experimentation, we get the best combination which including C-2, C-6, Asp-1. After fermentation with the mixed species, there enzyme activites are higher than single ones. Maybe the synergy effect is the best explanation for that.5. Through the fermentation situantion control of the combination species, we can get species proportion with the higher enzyme activity.We use orthogonal experiment to optimize experimental condition , including ferment temperature, O flux, inoculate time and volume of species No 2 and No3.6. Investigation of enzyme charatersThe optimum temperature of cellulase is 70 o C, the optimum temperature of xylanase is 55 o C, pectinase optimum temperature is 45 o C. The enzyme hot stability: cellulase has a good hot stability, which can be residued 80% enzyme at 60 o C for 30min. If temperature is higher than 60 o C, enzyme activity would be distinctly decreased. Xylanase is lessened a little at 40 o C, and it would residue 76.75% enzyme activity at 50 o C, 17.35% left at 60 o C. The optimum temperature of pectinase is 70 o C, pectinase would be left 70% from 30 o C to 60 o C.The optimum pH of cellulase is 4.6, xylanase and pectinasen is 4.4 and 5.0 respectively. Relative enzyme activity is above 60% at pH 4.2-5.4, so three enzymes are acid enzymes, and they can keep their activities for a long time at acid situation.The enzyme pH stability: pectinase has a good hot stability at pH 4-5, and it would residue 90% enzyme activity at pH 3-6. Xylanase is stable relatively at pH 4.5, and it would residue above 85% enzyme activity at pH 3.5-6.0, and it is also applied at other pH. Cellulase is more stability than xylanse, and cellulase is little losen at pH 3.0-8.0, which is residued more than 90%.