Plasminogen activators in bovine milk: Fractionation, assays, and concentration in milk, cheese curd, and milk protein products

Lilian Mirembe Were, Purdue University

Abstract

Plasmin exists initially in milk in its zymogen form, plasminogen (PG), hence the importance of plasminogen activators (PAs), which convert the zymogen to active plasmin (PL). There is inconsistency in the literature as to the relative amounts of tissue-type PA (t-PA) and urokinase-type (u-PA) in bovine milk. The research objectives were to: (1) Fractionate PAs using various published methods. (2) Determine effect of cheese making cook temperature and salt concentration on PL, PG, and PA activities. (3) Determine PG, PL, and PA activities in various commercial dairy products. The PAs were fractionated from bovine milk using sulfuric acid and buffer containing dimethylformamide (DMF), &egr;-amino-n-caproic acid (EACA), or sodium chloride (NaCl). Protein content was determined by the bicinchoninic acid method. Plasminogen activators were determined colorimetrically, and also were visualized using casein-plasminogen gels. Fractionation with DMF resulted in the lowest total solids but highest protein concentration compared to other treatments. Urokinase-PA level (10.38 IU/ml) was greater than that of t-PA (7.33 IU/ml). Most PAs in the NaCl-treatment samples were found in the supernatant fraction while the majority of PAs in EACA-treatment samples were in the pellet fraction. However, total PA levels in EACA- and NaCl-treatments were at least 40 times lower than in the DMF-treatment samples per ml of sample. The high PA levels of DMF-treatment samples were consistent with results of assays using specific substrates. Modification of the DMF fractionation method resulted in higher total PA level when EACA was incorporated, but lower levels with NaCl addition. The 55°C cooked curd exhibited the lowest enzyme levels, and uncooked curd had the highest levels at 0 and 5% NaCl. The effect of NaCl on enzyme levels was temperature dependent, with an increased level for the uncooked and 37°C cooked curds when NaCl was added. Acid casein had more PL compared to rennet casein. No difference in PL levels between sodium and calcium caseinates from two sources was observed, but sodium caseinate from one source had higher PL compared to calcium caseinate. Whey protein concentrates had a higher PL level compared to whey protein isolates.

Degree

Ph.D.

Advisors

Nielsen, Purdue University.

Subject Area

Food science

COinS