Directional Preparation Of Endoinulinase And Its Application In Production Of Fructooligosaccharide

Fructooligosaccharide(FOS)is a kind of functional oligosaccharide which composed of a linear chains of 1-4 fructose residues linked by ?-(2,1)bonds terminated by a sucrose molecule.Endoinulinase(EC 3.2.1.7),yields fructooligosaccharide by acting on the internal ?-(2,1)glycosidic bonds at random.It may have an significant value for industrial production of FOS using natural endoinulinase.In this paper,one strain with high inulinase activity was screened from rhizosphere soil where Jerusalem artichoke was planted in Qinghai province.In order to obtain endoinulinase with low exoinulinase activity,to realize the subsequent production of FOS by endoinulinase.On the basis of optimizing the enzyme-producing conditions,a method of absorption was applied to the directional separation of endoinulinase and exoinulinase.Furthermore,the production process of FOS using endoinulinase with low exoinulinase activity had alse been specific studied.The main results of the paper were as follows:1.A strain with high inulinase activity was obtained from the rhizosphere soil of Jerusalem artichoke grown in Qinghai Province using preliminary screening and secondary screening.The strain was identified as Aspergillus niger Z421 by colony morphology observation and ITS-rDNA sequence identification.The endoinulinase activity of Aspergillus niger Z421 was6.46 U/mL and exoinulinase activity was 9.04 U/mL,I/S ratio was 0.73.2.In order to increase endoinulinase activity and I/S ratio,the conditions for inulinase from Aspergillus niger Z421 was optimized as follows: inulin was 20 g/L and yeast powder was 10g/L,pH was natural,no inorganic salts or mental ions was added,rotational speed was 170 rpm and temperature was 30 oC.After 72 h of fermentation,the activity of endoinulinase from Aspergillus niger Z421 was up to 11.78 U/mL,which was 65.45 % higher than that before optimization,exoinulinase was 9.34 U/mL.I/S ratio was 1.26,which was 72.60 % higher than that before optimization.3.For the purpose of producing endoinulinase with low exoinulinase activity,a method of absorption was applied to the directional separation of endoinulinase and exoinulinase,reducing exoinulinase and increasing I/S ratio.The optimal separation conditions of inulinase was achieved by single-factor experiment,the concentration of Jerusalem artichoke residue was 10 %(w/v),pH was 5.0,natural inulinase was added and absorped at 4 oC and 80 rpm for 5 h.The recovery rate of endoinulinase was 67.92 % and the recovery rate of exoinulinase was 19.16 %.After separation,I/S ratio was increased from 1.26 to 4.02,and the increment of I/S ratio was219 %.4.Endoinulinase with low exoinulinase activity was used for the production of fructooligosaccharide by hydrolyzing inulin.The best conditions for enzyme hydrolysis were as follows: inulin was 40 g/L,temperature was 50 oC,the enzyme dosage was 20 U/g inulin.After10 h of hydrolysis,the content of FOS was 16.06 g/L and the yield of FOS was 40.16 %.5.Through the comparison of FOS content by hydrolysis using inulinase which was after separation or not,the concentration of FOS was increase from 6.31 g/L to 16.06 g/L,and the yield of FOS was also increased from 15.78 % to 40.16 %.Moreover,the increment of FOS yield reached up to 178 %.