Browsing by Subject "peptidase activity"

The literature review focused on the proteins and insoluble fibre, ?-(1->4)-galactan, of blue lupin seed and how they degrade during germination. The review also dealt with the food applications of lupins and the harmful substances of lupins: allergens and ?-galactosides. The object of the experimental study was to determine the peptidase activities in the blue lupin seeds at the different stages of germination, classify the peptidases in the seeds and investigate the changes occuring in the proteins during germination and fermentation. The percentage of the water-soluble protein in the seeds was also determined. Blue lupin seeds were soaked in water over night and were germinated in the dark (15 ° C, RH 100 %). Peptidase activities were determined spectrofotometrically using azo-casein as a substrate. Class-specific peptidase-inhibitors (Pepstatin A, PMSF, E-64 and O-FEN) were used for classification of peptidases. Lactobacillus brevis and Lactobacillus rhamnosus were used in the fermentations (35 °C, 24 h) as well as baking yeast. The changes which occured in the proteins during germination and fermentation were investigated by electrophoresis (SDS-PAGE). The Dumas method was used to determine the percentage of the water-soluble protein in the extracts composed of soaked and germinated (2 day) seeds. Peptidase activities increased until the second day of germination and then remained constant until the fourth day. Serine- and aspartic peptidases were identified by inhibitor tests but not cysteine peptidases, even though cysteine peptidases have been previously known to break down legume proteins during germination. The cysteine peptidase inhibitor, E-64, used in this study has been observed earlier to inhibit cysteine peptidases belonging to papain family but not to legumain family. Proteins degraded slightly when the germination continued 4 days, and the degradation continued further during the fermentations. Large polypeptides (MW 45–100 kDa) mainly degraded during germination and fermentation. In addition, 17 kDa polypeptides degraded during fermentation. Possible 20 kDa hydrolysis products also formed during fermentation. The degradation of proteins in fermentations was more efficient when seeds germinated for 2 days were used compared to ungerminated seeds. The content of the water-soluble protein in the soaked seeds and the seeds germinated for 2 days varied between 35–96 %, and the content increased markedly when the pH of the extraction solvent increased from 6 to between 7.5–9.0. Thus the alkalinity of the extraction solution changed the structure of the storage seed proteins to more water-soluble form. The peptidase activity of germinated blue lupin could be applied in varied fermentation processes. The storage proteins of blue lupin were extremely water-soluble, so this discovery could be utilised for manufacturing substitutes for dairy products.