Research On The Separation, Purification And Function Of Total Flavone From Fermentation Fluid Of Citrus Hidetrimmings

Processing Research Group, Citrus Research Institute, CAAS, through ten years of continuous exploration and numerous tests, successfully developed a new kind of feed additive owning the intellectual property right and be called as fermentation feedstuff of citrus waste. This technology had approved the achievements authentication by Chongqing science commission in July 2005, and provided a good access to deal with the citrus peel, so it has important economic, social and environmental significance. Besides it is in line with national resource recycling and environmental policy, beneficial to the utilization of citrus and pollution control, and offer a new thread to the utilization of the other agricultural products. In 2006, with the substantial support from ministry of science and technology and Chongqing science commission, this technology successfully declared a national agricultural scientific and technological achievements into capital projects, "Pilotplanttest for the key technology of fermentation feedstuff of citrus waste" and a major project of Chongqing Municipal Science and Technology about Citrus, "Research on the key utilization technology and complete equipment of citrus waste in the three gorges reservoir", which marks the attention paied to the utilization of citrus waste by the country and province. In 2007, under combined efforts of Citrus Research Institute of CAAS,Chongqing University and Wanzhou factory of Huiyuan Group, research on the pilotplanttest and complete equipment for the fermentation feedstuff of citrus waste was successfully completed, a complete technological line and perfect technical parameters were formed, at the same time the fermentation feedstuff of citrus waste have passed the feeding experiment of liability company Chongqing Longsheng feed.This paper mainly studied the fermentation fluid produced during the fermentation of Citrus Hidetrimmings, analyzed the active ingredient of the fermentation, comparability studied the extraction process of total flavone from the fermentation, studied the purification and biological activity of total flavone. The results play the guiding role of utilization and dealing with the large number of fermentation fluid produced during the industrial production of fermentation feedstuff from citrus waste. 1. Through analyzing six fermentation samples from pilotplanttest and industrial production, we can see the composition of it. They are:crude protein 0.9825%; total flavone 3554.615 mg/kg; invert sugar1.5630g/100mL; reducing sugar 1.319g/100mL; sucrose 0.2317g/100mL; total sugar 1.5514 g/100mL; titratable acid 0.8016(to citric acid meter, g/100mL); soluble solids 9.0367Brix°; pH value 3.8680; Vc 4.5251 (mg/100mL); crude fat 0.0154%; dry matter 7.73%.2. Research on the extraction process of total flavone from the fermentation by ethanol bath. Through the orthogonal test, we can see the best conditions are:ethanol 70%, temperature 65℃, extraction time 70min, solid-to-liquid ratio 1:40. Under this condition, repeat the test four times, the total flavone of the fermentation is 17.281±0.15g/kg.3. The Ultrasonic-assisted extraction process of total flavone from fermentation fluid of citrus hidetrimmings technical parameters was optimized by means of quadratic general rotary unitize design. The regression equation of the four factors to the total flavone extraction rate (Y) was:Y=21.93771-0.1912x1-0.2417x2+0.11796x3-0.07152x4-0.43125x12-0.3573x22-0.51350x32-0.592 50x42-0.55606x1x2-0.09956x1x3+0.09956x1x4-0.1186x2x3-0.08994x2x4-0.04369x3X4According to the extreme value analysis of the model and the single experiment, we obtained the optimum extraction condition, they are:supersonic time 4min, solid-to-liquid ratio 1:40, ethanol 70%, supersonic power 70%. Under this condition, the total flavone extraction rate is 21.935±0.03g/kg.4. Researched on the extraction of total flavanone from fermentation fluid of citrus hide trimmings with the method of microwave cooperated which took alcohol as resolver. The optimum extraction conditions were obtained by the extreme value analysis of the model and the single experiment, the results as followed:microwave time:4min, solid-to-liquid ratio 1:40, ethanol 70%,microwave power 70%. Under these conditions, he total flavone extraction rate is 23.7375±0.07g/kg.5. By studying the different types of resin on the adsorption and desorption of the crude extracts of total flaconoid from fermentation liquid of citrus peel, we can see that the resin of AB-8 type was suitable for the purification of the crude extracts of total flaconoid from the fermentation liquid of citrus peel, which is high selectivity,fast adsorption rate,the large capacity of adsorption and easy desorption.The optimum purifying conditions were obtained using L25(56) orthogonal analysis method, the results as followed:solid-to-liquid ratio 1:14, liquid pH 5, adsorption time 25h, desorption concentration 70%, desorption solution pH 6, desorption time 3 h. Under these conditions, repeat the test, adsorption capacity is 17.19±0.16 mg/g. Desorption rate is 81.97±0.64%, total flavonoids is 70.46±1.07mg. 6. HPLC typical value:the peak time of purify from crude extract before the hydrolys is: 8.04min,9.78min,23.03min,25.15min,31.73min. We can see from the peak area, there are two substances whose peak time are 9.78min,8.04min. The main peak time of hydrolys is:7.84min, 8.73min,9.78min,10.773min,20.707min,24.50min,30.78min,32.32min. By constructing the before hydrolysis with after hydrolysis, we can see that the purify from crude extract have something in the form of aglycone. At the same time, we can see:under the hydrolysis conditions, the total flavonoids of purify from fermentation fluid of citrus hidetrimmings is not complete hydrolyze, its components' structure remains to be further studied.By useing standard products spectra and hesperidin, naringin marking rutin standard curve regression equation,we can see:in purify fermentation fluid of citrus hidetrimmings, hesperidin content is 55.91%, naringin matter rutin content is 12.57%, the total flavonoids content is 68.48% or more.7. The paper have studied the reducing power and nitrite scavenging effect on lipid peroxide, superoxide anions (O2-·), hydroxyl radical (·OH) and nitriteion of purify fermentation fluid from citrus hidetrimmings, Take Vc and hesperidin as a comparison, take EC50 (it is drug concentration when clearance rate come to 50%) as a reference index. The results showed that:the effect of extraction on the removal of lipid peroxides EC50 is about 4.2 times Vc,1.3 times hesperidin, showing the stronger effect on the removal of lipid peroxides; the effect of extraction on the removal of superoxide anions EC50 is about 9.7 times Vc,1.9 times hesperidin, showing the weaker effect on the removal of superoxide anions; the effect of extraction on the removal of hydroxyl radical EC50 is about 4.2 times Vc,1.8 times hesperidin, showing the stronger effect on the removal of hydroxyl radical. In conditions of same concentration, the crude extraction of citrus waste is weaker than Vc and hesperidin on residue reduction, but the test show that the crude extraction of citrus waste have some reducing power; the effect of extraction on the removal of nitrite EC50 is about 10.1 times Vc, 1.4 times hesperidin, showing the weaker effect on the removal of nitrite.