The pectin methylesterase inhibitor(PMEI)gene,cloned from the kiwi fruit,was linked to the expression vector pPICzαA through double digestion using EcoRI/XbaI.It was then transferred into Pichia pastoris GS115 by electro-transformation to obtain the positive strain GS115/pPICzαA-PMEI.After the PMEI expressed by GS115/pPICzαA-PMEI was concentrated and purified,it was applied in fruit wine fermentation.The results show that the recombinant P.pastoris expression strain GS115/pPICzαA-PMEI was successfully constructed,and the PMEI expression level was 35.38 mg/L.When PMEI was applied in fruit wine fermentation,the methanol in orange wine decreased by 47.54%from 189.75～99.55 mg/L.Similarly,the methanol content of apple wine dropped by 21.52%from 118.15～91.20 mg/L,whereas the methanol content of grape wine did not change significantly and remained below 20.00 mg/L.These results indicate that PMEI has a significant methanol reduction effect on oranges that are rich in pectin and endogenous pectinase.The minimum inhibitory concentration of PMEI for optimal orange wine fermentation was 8.84 mg/L and optimum temperature was 18℃.Under these conditions,the methanol content of orange wine reduced by 66.40%,to 89.10 mg/L.These findings provide a novel technique for producing low-methanol fruit wine by applying PMEI.
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