2.4. Effect of pH, enzyme concentration, and temperature onmilk clottingIn order to study the influence of the pH used forcynarase extraction, stigmas (15 g) from dried flowers ofC. scolymus L. were homogenized in 50 mL of differentbuffers, and centrifuged at 50000 g for 30 min. Thesupernatants were used to study their clotting properties.The buffers employed were 50 mm sodium citrate for pHvalues 3, 4 and 5; 50 mm sodium phosphate for pH 6.5 and7.0; 50 mm Tris/HCl for pH 8. For each pH, three enzymeextracts were made. To study the effect of the milk pH onthe rennet’s clotting capacity, the pH was adjusted at 35 Cby the gradual addition of 0:1m lactic acid/NaOH duringrapid stirring.Standard solutions of each concentration were preparedby diluting the enzyme extract in 50 mm citrate bufferpH 4 (extraction buffer) to maintain the dilution effectresulting from adding the enzyme to the milk samples.Temperature was controlled using a Haake D1 waterbath (Haake Inc., Berlin, Germany), measuring the waterbath and milk temperatures before and after adding theextract.
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