The West African soft cheese (wara) is traditionally produced using latex extracts of the leaves of Calotropis procera. Even though no death has been apparently traced to it, the extract is said to be poisonous causing death by paralysis of the heart. This project, therefore, aims at obtaining an alternative (in this case lactic acid bacteria protease) to the latex extract of C. procera in order to rule out possible poisoning effect. The physiological characteristics of such alternative protease was studied and compared to that of the extracts from C. procera. Seven lactic acid bacteria species were isolated from samples of the West African soft cheese (wara) obtained from local retailers. They were identified as Streptococcus lactis , Streptococcus pyogenes, Lactobacillus acidophilus, Lactobacillus brevis, Leuconostoc mesenteroides, Lactococcus lactis and Streptococcus faecalis. The isolates were subjected to growth and protease production studies. Optimization of protease production was carried out on three selected isolates - Streptococcus lactis, Lactobacillus acidophilus and Lactococcus lactis. Enzymes obtained were further characterized and separated using molecular exclusion procedures. Results obtained were compared to those of different extracts of C. procera. It was observed that Streptococcus lactis had the highest (4.25 units/ml) protease production at 28°C after 72 hours with peptone and casein as nitrogen sources in the presence of glucose as carbon source. The ethanolic extract of Calotropis procera had the highest protease activity (12.60 units/ml) at 5% substrate concentration. Lactococcus lactis and C. procera extracts were inhibited (0.00 units/ml) at 0.2M and 0.3M concentrations EDTA respectively thus suggesting the presence of a metallic protease. Na2+ ion was seen to enhance protease activity in all the extracts. Comparative results show that Streptococcus lactis protease had a Km of 0.42 mg/ml for casein at a Vmax of 2.0; Lactobacillus acidophilus protease had a Km of 0.77 mg/ml for casein at a Vmax of 2.50; Lactococcus lactis protease had a Km of 3.3 mg/ml for casein at a Vmax of 0.87; The crude extract of Calotropis procera had a Km of 5.0 mg/ml for casein at a Vmax of 1.54; the ethanolic extract of C. procera had a Km of 2.44 mg/ml for casein at a Vmax of 8.77 while the methanolic extract of C. procera had a Km of 5.0 mg/ml for casein at a Vmax of 4.17. In conclusion, the lactic acid bacteria protease from Streptococcus lactis was found to fare better physiologically, while the protease from the ethanolic extract of C. procera faired better amongst the plant extracts.
Brewery Wastes Reuse for Protease Production by Lactic Acid Bacteria Fermentation
