Process Optimization Of Pectin Esterase Fermentation By Aspergillus Tubingensis And Pomelo Peel Pectin Extraction

Pectin esterase?PE?is highly specific for polygalacturonic acid methyl ester.This enzyme can remove the methyl group from pectin,and then the pectin's solubility is improved.Pectin esterase has been widely used in fruit and vegetable processing,wine making,textiles,tea drinks,paper making,pharmaceutical and cosmetics industries to improve product quality,especially in the low methoxyl pectin preparation.At present,some scholars have studied the fermentation technology of PE from Aspergillus niger,but the research on the fermentation technology of PE from Aspergillus tubingensis is relatively few.In this study,the fermentation technology of A.tubingensis CICC 2651 producing PE and the enzymatic properties was explored.And the de-esterification of pectin by PE was studied on the basis of acid extraction of pomelo pectin.The main results are as follows:First of all,the effects of medium composition and culture condition on PE production by A.tubingensis were studied by determining the yield of PE.The results showed that the optimum medium composition for PE producing was?w/v?:3%high-ester pectin,0.5%ammonium sulfate,0.04%Na₂SO₄,MgSO₄·7H₂O 0.04%,K₂HPO₄ 0.2%,FeSO₄·7H₂O0.005%,100 mL distilled water.The optimum fermentation conditions were as follows:initial pH 4.5,loading volume 40 m L/250 mL,glass beads addition 5,fermentation temperature 30°C.Under the optimum medium composition and culture condition,the activity of PE reached 1.53 U/m L,which was 1.78 times higher than that before optimization.Secondly,high purity PE was extracted from fermentation broth by precipitation with ammonium sulfate,DEAE-Sepharose Fast Flow ion exchange chromatography and Seohacryl S-100 gel chromatography,and then the enzymatic properties of PE were investigated in the study.The results showed that the optimal temperature for enzyme catalyzation was 50°C.And after incubation at 45°C and 50°C for 150 min,the residual enzyme activity of PE reached 62.5%and 36.5%of the initial acitivity of PE,respectively.As the temperature was higher than 45°C,the stability of PE was decreased.After incubation at 55°C and 60°C for 150 min,the residual enzyme acitivity of PE were less than 20%of the initial activity of PE.Besides,the optimum pH for PE reaction was 4.0 and this enzyme was stable within the pH range of 3.5–8.0 for 0.5 h.Cu²⁺and Ag⁺inhibited PE activity significantly when the metal ion's concentration was 1 mmol/L,while the effect of other metal ions on PE activity was insignificantly.When the metal ion concentration was 10mmol/L,Fe²⁺,Fe³⁺,Zn²⁺,Cu²⁺and Ag⁺had strong inhibitory effect on PE,and Ba²⁺,Ca²⁺,Mn²⁺,Mg²⁺had inhibitory effect on PE activity,while Na⁺and K⁺had no apparent effect.The Km and Vmax values of PE were found to be 15.2 mg/m L and 10.3?mol/?mL·min?using Lineweaver-Burk double reciprocal method.Finally,he effects of extraction temperature,extracting pH,extraction time,ratio of liquid to material on the yield of pomelo pectin by acid extraction and the type of decolorizer,decolorization time and temperature on the decolorizing effect were studied.The optimized conditions for pomelo pectin extraction and decolorization were as follows:extraction time90 min,extracting pH 1.5,the ratio of liquid to material 20,extraction temperature 100°C,2%addition amount of perlite as the decolorizer,decolorization temperature 70°C,decolorization time 20 min,pectin extract:ethanol 5:4.Under the optimal conditions,the yield of pectin reached 11.5%.Based on the previous process,the effects of PE dosage and processing time on the deesterification of pectin were studied.The results showed that the content of methoxy group was 37.3%and the yield was 12.3%,when the enzyme activity was 7.5 U and the enzyme treatment time was 50 min,respectively.