Studies On Pepsin Inhibitor From Coriolus Versicolor By Submerged Fermentation

The research mainly focused on the following aspects about pepsin protease inhibitor from Coriolus versicolor by submerged fermentation, including fermentation conditions, extracting, purification, the basic properties and application .Compared the biomass of different fungi by submerged fermentation, the mycelium dry weight of Coriolus versicolor is the most heavy 12.97g/L, about 2.56 times of Gantheram Lucidum; Compared the inhibition rate of extracting effect on pepsin, Coriolus versicolor is still the best one; Compared the inhibition rate of Coriolus versicolor broth by pre-and post cell disruption effect on pepsin, after cell disruption the inhibition rate improved from1.09 IU/mL to 23.9 IU/mL, it was suggested that the pepsin inhibitor was the intracellular production.The main influencing factors were determined as operational volume, inoculum size and initial pH by using single-factor experimental method.As potato for culture medium, the Orthogonal experiment and single- factor tested for the best culture conditions indicated that fermentation time is 120 h, fermentation temperature is 28℃, rotate speed is 180 r/min, initial pH is 6.2, operational volume is 70 mL/250 mL, inoculum size is 12%; The inhibitory avtivity was improved to 56.5 IU/mL,increasing by 2.08 times.Pepsin inhibitor produced in liquid state fermentation by Coriolus versicolor was pretreated by ultrasonic cell disruption, centrifugation,decolorized by macroporous resin, and then isolated by DEAE-52, Mono Q ion-exchange chromatography and UltroGelACA-54 gel-filtration(be named CVPPI), the purificating times was 38.7. It was identified to be a protein and showed a single subunit by the methods of UltroGelACA-54 gel-filtration and SDS-PAGE, the relative molecular weight was 22.31 kDa.CVPPI was consisted of protein, amino acid composition date indicated that it was rich in acidic amino acid (aspartic acid and glutamic acid) , reaching to 37.1% and 12.4%; Experimentation indicated that the inhibitor was not proteinase;The best reacting time was 1 h; The inhibitor showed a remarkable heat stability, it retained its initial activity even at up to 100℃for 1 h; The pH stability range of it was about 3.0～7.0.By investigating the interaction between CVPPI and variety of proteinases, it is indicated that the inhibitor was more specific against aspartic acid proteinase (such as pepsin and yeast proteinase A) than other proteinases; The concentration required for 50% inhibition (IC50) to pepsin and PrA were 26.26μg/mL and 152.4μg/mL; The dissociation constants (Ki) for the two were 0.876μmol/L and 2.77μmol/L; The dissociation constants (Ki) for the two were 0.996μmol/L and 3.10μmol/L; Kinetic studies (Line weaver-Burk method) showed CVPPI was a kind of noncompetitive and anti-compeititve inhibitor to pepsin while non-competitive inhibitor to PrA; The combined mode of CVPPI and pepsin was: it maybe formed hydrogen bonds in the active groups of the pepsin and blocked binding sites of the pepsin and substrate.A kind of aspartic acid protease inhibitor (be named PAPI) was isolated by DEAE-52 ion-exchange chromatography and UltroGelACA-54 gel-filtration from potato juice, the relative molecular weight was 18.9 kDa. By experimentation indicating their molecular weight, reacting mechanism, composition and inhibition were all different, so that CVPPI was different from PAPI.By applying, its best reacting time to No.1 and 2 proteinases was 0.5 h. The ratios about the two proteinases to CVPPI were not linearity, as the inhibitionate reaching to 50%, the ratios were 10:1 and 4:1. Maybe CVPPI could improve the rheologic characteristics of wheat flour, there would be applying potential in food addiitive industry.