Extraction & Purification, Characteristics Of Konjac PPO And It's Effects On Rheological Property Of Konjac Polysaccharides

Konjac belongs to Amorphophallus Blume Maxim konjac genus, which is widely grown in the tropical and semitropical area in Asia and Africa. Konjac powder is the main rough product after starch is wiped off from the cracked konjac and studies on the konjac mostly start with the konjac glucomannan in konjac powder. In order to whiten the product, protecting color is necessary in the process of konjac powder. Physical method and inhibitor aimed at the PPO in fresh konjac are used to eliminate enzyme activity to protect color. SO2 can blanch product and eliminate enzyme activity, which is used to protect the color of konjac powder. However, the residue of SO2 has negative effect on the finished product, and SO2 is negative to human beings. So studies into PPO in the konjac help to find better ways to protect color. Though studis on PPO is of 60-year history, there haven't any reports of studies into the PPO in konjac. In this paper, fresh konjac was studied. And the PPO in konjac was extracted, purified and determined through modern separation techniques and analysis methods by first studied. At the same time enzymological properties and structure of PPO were studied and the effects of PPO on the polysaccharides rheology were also analyzed. The results are as follows:1. Extraction, purification and purity identification of PPO in konjacPPO was extracted from fresh konjac and was precipitated with (NH4)2SO4 of different saturations. Thereby the extraction wih the saturation of 70% was confirmed.Based on the fibrinous configuration characteristic (observed through atomic force microscope)of konjac PPO, series Sephadex G gel were used to separate PPO, after the primary purification. Results showed that several peaks could obtained on Sephadex G-75 column, which was a good way to purify PPO. And it was better to use DEAE-Cellulose-52 as pretreatment of PPO, not only through size exclusion chromatography .Two PPO isoenzymes were found in konjac and one of it was obtained in this paper, themolecular weight of which was 45K.2. Discussion on the structure of PPOThe observation through atomic force microscope showed that the konjac PPO was fibrinous,so that gel could not be chosen only according to PPO's molecular weight.AA analysis indicated that Glu, Arg, Asp and Ser were of high amount in konjac PPO. It probably concerned with Anti-parallel B-blotting and N-terminal leader sequences formed bymulti-B-chains of function domain of C-terminal.X-ray analysis showed that the konjac PPO obtained through the above method was partly crystallized, which might be aroused by the structural change of PPO during the extraction.CD spectra showed that the percentage of PPO secondary structure in optimum pH is: Helix 34.8%. Beta 28.2%, Turn 7.0%, Random 30%. It's secondary structure changed when substrate was puted into, the percentage of Helix reduced and Beta raised, it showed that the active domain existed in Helix; The percentage of konjac PPO secondary structure changed along with pH, the percentage of Helix reduced while Beta raised when solution pH was beyond the optimum. 3. Effects of PPO and enzymological properties on the polysaccharides rheologyKonjac PPO was of highest enzyme activity when pH was 6-7 and temperature was 30°C.And the enzyme activity increased as the concentration of enzyme and substrate increased, however the increasing trend was becoming tempered until getting the highest activity. Phytic acid, Na2SO3, Vc and EDTA could all restrain the PPO activity, with Na2SO3 of the highest effect and EDTA the lowest.Konjac PPO could influence both the apparent coefficient of viscosity of konjac powder and hyperfine powder. The reason might be that PPO oxidized polyphenol and broke the polysaccharide-polyphenol conjunct. All the coefficient of viscosity of rough enzyme group and rough enzyme&polyphenol group decreased notably, which could be resulted by the konjac glucomannan in the rough enzyme.Three methods to extract polyphenol showed that high concentration of ethanol could result in the loss of polyphenol. The reason was that polyphenol was precipitated with polysaccharide when high concentration ethanol was added, and at mean time the too low concentration of ethanol could also decrease the relative content of total hydroxybenzene. To extract polyphenol under the condition of fresh konjac/anhydrous ethanol = 1:10, blanch and 70 °C could get more polyphenol.