Utilization Of Orange Peels: Multienzyme Production In Solid-state Fermentation

The orange peels contain abundant pectin, cellulose, soluble sugar and minerals (mainly Ca, K, P, and Si), so they can be used as the substrates for microbial growth. China has the largest planting area of citrus and amount of orange peels and pulps are produced as waste. So it's important to make use of them. Enzymes, such as cullulase, protease, xylanase and pectinase, have a wide application in area of feed and food industry. The work described in this paper was aimed at studying the utilization of orange wastes as substrates for multienzyme production by fungal strains. The work mainly included strain screening, strain mutation, optimization of fermentation conditions and composition of culture media, and properties of enzymes. The result of this study was reported below:1. Screening and mutation of strains for multienzyme productionAspergillus usaanii, Aspergillus nigerI and Aspergillus nigerII were obtained from a lot of fungal strains, which were tested of their abilities of multienzyme production by growing on orange peels mediaImproved PDA culture media, which contained 2% CMC-Na and 0.5% dextrose, was chosen for screening plating media. The correlation coefficient of hydrolytic diameter /colony diameter and CMCase activity, hydrolytic diameter /colony diameter and acid protease activity, CMCase activity and acid protease activity were 0.770(**), 0.780 (**) and 0.870(**), respectively. The culture media was proved to be effective.A.usaanii was chosen for further study of mutation by Y -ray radiation and low energy N⁺ ions beam implantation. The effects of death rate of the spores and the dose of ions implantation were studied in the work. Primary study of mechanism of mutation by ions beam implantation was carried out. The result showed that: every plate spread with 0.2mL spores suspension of 10⁵ spores/mL was suitable for mutation work. The doses of 50×2.6×10¹³N⁺/cm².s and 150×2.6×10¹³N⁺/cm².s were proved to be the most effective. A mutant A.usaanii-CF1 was finally obtained with genetic stability. Acid protease, CMCase and xylanase of the mutant were improved by 62.7%, 48.6% and 44.3%, respectively.2. Optimization of culture conditionsThe initial moisture content of culture media significantly affected the multienzyme productivity of A.usaanii-CFl in solid-state fermentation. The initialmoisture content of the highest enzyme productivity for each enzyme component was different. The highest enzymes activities were obtained with the initial moisture content of a range of 50% to 55% for acid protease, 55% for cellulase, a range of 60% to 65% for xylanase. The initial moisture content of 55% to 60% was chosen for further study.Growth temperature had significant effects on enzymes production. Maximum enzyme production could be obtained at 28 °C. It was found that initial culture pH had no significant effects on multienzyme production. The natural pH was used in the subsequent study.Enzymes production time course was investigated in this study. CMCase, FPA, 3 -glucosidase, xylanase and acid protease reached high levels of enzyme activities at 54-72h, 66-72h,72-84h, 60-66h and 60-66h, respectively. In consideration of all the enzyme components, the culture time was chosen at 60 to 72h finally.We couldn't find significant effects of the inoculation volume on multienzyme productivities. But an inoculation volume of less than 3% would delay cell growth and enzyme production with an increasing possibility of infection. It was suggested we chose an inoculation volume of 5% or more. 3. Optimization of culture mediaThe multienzyme productivities could be improved by adding some bran except xylanase.The amount of defatted soybean had significant effects on multienzyme production of A.usaanii-CFl. Acid protease couldn't be detected with low cellulase and xylanase productivity when there was no defatted soybean in culture media. The effects of the ratio of orange peels and defatted soybean on enzyme production were investigated in the study. When the ratio was 8:2, CMCase, FPA and xylanase got the highest enzymes activities. Acid protease and 3 -glucosidase got the highest activities at a ratio of 7:3 and 6:4. A ratio of 8:2 to 7:3 of orange peels and defatted soybean was chosen for further study.Nitrogen sources had significant effects on enzymes production. NttfNC^ (NH4) 2SO4 and CO(NH)2 were proved to have positive effects on multienzymes production. CO(NH)2 was proved to be the most effective and potential nitrogen source for enzyme production.Inorganic mineral salts had different effects on multienzyme production. CaCb could improve cellulase production while it had restraining effects on xylanase...