| D-erythorbic acid is a novel biological antioxidant for food and used widely in food industry because of its safe and efficient characters. China is the largest EA produced country in the world. But at present the sold EA in market is produced by indirectly fermenting. It has complex workmanship and low product yield with high energy consumption. This is violated to the low carbon economy advocated by government. Direct fermentation method for producing D-EA has simple workmanship and low cost with energy consumption. Products safety and reliability can be guaranteed. So it has a wide application prospect in researching and developing straight process. The study aims to obtain a high yielding strain in producing EA and optimizing the fermenting conditions with some progress in the workmanship technology of producing EA by straight process.The results of experiment showed that the optimal condition for preparation of protoplast to Penicillium griseofulvum HL as the starting strain was incubation time of mycelium of 48h, osmotic stabilizer of 0.7 mol/L sodium chloride, enzyme combination of 0.5% snailase +0.5% cellulase, hydrolysis temperature of 30℃ and enzymolysis time of 3h with pH value of 6.0. Under this condition the yield of protoplast could reach 3.14×10’/mL.The optimal condition for regeneration of protoplast of Penicillium griseofulvum HL was on improved Czapek medium with osmotic stabilizer of 0.7mol/L sucrose. Under this condition the regeneration rate of the protoplast was 24.93%. The protoplasts of Penicillium griseofulvum HL were induced by ultraviolet ray, diethyl sulfate, ultraviolet ray and diethyl sulfate,ultraviolet ray and LiCl, a high-yield strain of erythorbic acid ZD4 was obtained by first screening of flat plate and second screening of shake flask. The yield of erythorbic acid was increased from the initial 1.08mg/mL to 5.28mg/mL. And it can descend stably after six generation.The aim of this study is comparing the characters between the original Penicillium griseofulvum HL and ZD4 with protoplast mutagenesis. Results show that HL and ZD4 are similar in all kinds of variation tendency. Make fermental cultivation for 24 hours and the yielding of EA Tends to be stable, The content of EA in HL fermentation liquor reaches 1.21mg/mL meanwhile 5.18mg/mL in ZD4 fermentation liquor.Optimize fermentation medium of ZD4 strain by using PB experiment-design, Steepest ascent design and central composite design. Obtained the best nutrient media components for ZD4 strain producing EA are rice liquid glucose 13.5Brix, carbamide 0.5%, corn steep liquor 1.2%, magnesium sulfate 0.005%, dipotassium phosphate 0.005%, monopotassium phosphate 0.005%, Zn2+0.014%,tween-801%. In this medium the EA yielding is 6.92mg/mL.By using single factor test, obtain the best fermentation conditions are:the age is 96h,initial pH 5.5, inoculum size is 3%, loaded liquid is 70mL/250mL, Table speed is 180rmp,28℃,120 hours, the yielding of EA promotes to 7.23mg/mL under the optimized culture condition with the best medium.This article researched the different influencing of the different Fe2+ concentration to ZD4 strain producing EA. Set different Fe2+ concentration in medium to make fermental cultivation and draw the growth curve by track recording the ZD4 strain’s dry weight. Observing colony characteristics and assaying the EA content. Add Fe2+ in the different fermentative stage to study Fe+ reversibility in restraining the ZD4 producing EA.The results show that the different Fe2+ concentration have inbibitional effect on the capacity of ZD4 producing EA and also the ZD4 growingand the effect is irreversible. |
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