Characterization of partially purified milk‐clotting enzyme from sunflower (Helianthus annuus) seeds

Abstract

This study was aimed to extract milk-clotting enzyme from sunflower seeds and to determine its potentiality for manufacturing white soft cheese from cows and goats milk. The seeds were blended and extracted using two types of buffers and milk-clotting and proteolytic activities were evaluated. The enzyme was partially purified using ammonium sulfate fractionation techniques. Results indicated that sunflower seeds extracted with 5% NaCl in 50 mmol/L acetate buffer, pH 5.0, had the highest milk-clotting activity (MCA) and lowest coagulation time compared to that extracted with only acetate buffer (pH 5.0). Ammonium sulfate at 30-50% saturation purified the enzyme to 4.3 folds with MCA of 241.0 U/mL and final enzyme yield of 10.9%. The partially purified enzyme was characterized by SDS-PAGE that showed two bands with molecular weight of 120 and 62 kDa. When compared with other plant enzymes, the partially purified sunflower enzyme was found to have higher milk-clotting activity and lower proteolytic activity. Also, both milk sources and enzyme types significantly affected the cheese yield and curd formation time. The cheese made from cow milk using sunflower enzyme had higher yield compared to that obtained using commercial rennet, whereas the opposite was observed when using goat milk. 734

CARACTERIZACIÒN PARCIAL DE UNA FRACCIÓN PROTEICA POTENCIALMENTE BIOACTIVA DE Solanum marginatum L. f.

“…Later, the plant was cut, lyophilized and, crushed in a food processor. Then, 10 g of crushed leaves were extracted with a sodium acetate buffer 50 mM and NaCl 5% under constant stirring (Nasr et al, 2016). Subsecuently, the proteins were separated by precipitation with ammonium sulfate (80% saturation index) for 24 hours at 4°C.…”

Elaboración de queso tipo panela con extracto de hoja de ortiga y su comparación de parámetros fisicoquimicos

“…The cheese control was elaborated following the procedure described by Talib et al (2009) with some modifications. The elaboration of the cheeses with the treatments of leaf extract (1.0% and 0.75%) was used the technique described by Nasr et al (2016) with some modifications, where milk was incubated to 90°C and was added calcium chloride and the leaf extract (1.0% and 0.75%), was agitated and when precipitation of the curd was obtained, sodium chloride was added. Later it was pressed, molded and packed for its conservation where 5 cheeses of each treatment were elaborated.…”

EXTRACTION, PARTIAL PURIFICATION AND SOME FACTORS AFFECTING MILK CLOTTING ACTIVITY OF A MILK COAGULANT PREPARED FROM SUNFLOWER (Helianthus annus) SEEDS

“…From these results, it could be seen that the level of NPN in the partially purified extract was somewhat higher than that of commercial rennet but it was lower than that of crude extract. This could be explained by the presence of partial proteases in the crude extract which might contribute to the excessive proteolysis (Barros et al, 2001;Nasr et al, 2016). Several investigator have showen that many plant coagulants had higher proteolytic activity compared with calf rennet (Abd El-Gelil and Zawahary, 2004; Chazarra et al, 2007;Ahmed et al, 2009;Elmazar et al, 2012;Abdel-Raouf et al, 2017).…”

“…The ratio of milk clotting to proteolytic activity is a valuable indicator of the protease efficiency to be used as a rennet substitute in cheese making (Arima et al, 1970;Ahmed et al, 2009;Nasr et al, 2016). Table 8 compare the milk clotting to proteolytic activity of both crude and partial purified extracts of sunflower seeds and compare them with that of commercial calf rennet.…”

“…Sunflower seeds protease from mass spectrometry analysis were found to hydrolyse K-casein at the Phe 105-Met 106 bond as chymosin. Meanwhile, several inrestigators have shown that sunflower seeds possess proteases with high clotting/proteolytic ratio which clotted milk readily without developing any undesirable bitterness or excessive proteolytic activity (Park et al, 2000;Darwish, 2016;Nasr et al, 2016).…”