Detection of proteolysis in milk by four selected methods
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Date
2009
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Publisher
The University of Reading
Abstract
Sensitive methods that are currently used to monitor proteolysis are limited due to their
high cost and lack of standardisation for quality assurance in the various dairy
laboratories. In this study, four methods, trinitrobenzene sulphonic acid (TNBS), reverse
phase high pressure liquid chromatography (RP-HPLC), gel electrophoresis and
fluorescamine, were selected to determine their suitability for the detection of proteolysis
in milk by added trypsin, plasmin, Pseudomonas fluorescens NCIMB 701274 (414) and
Pseudomonas fluorescens NCIMB 702085 (416) and Bacillus licheniformis enzymes.
Raw, pasteurised and UHT milk used to study proteolysis were analysed by the four
methods. Pseudomonas fluorescens enzyme was extracted in this study since commercial
enzymes are not available. All four methods confirmed that Pseudomonas fluorescens
416 was more proteolytic than Pseudomonas fluorescens 414. Dialysis was effective in
the purification of Pseudomonas fluorescens (Ps. fl.) enzymes increasing the detection
limit for the fluorescamine method, which had a low upper detection limit. Prominent
peaks by RP-HPLC were shown to occur between 20-30 min for Pseudomonas
fluorescens but 20-25 min for Bacillus licheniformis. RP-HPLC confirmed a peptide peak
at 35 min in pH 4.6 soluble extract, which was absent in 6% TCA soluble extract, was
from plasmin. Casein breakdown profiles by gel electrophoresis confirmed preference for
0-casein degradation over a and K-caseins by both Ps. fl. and B. licheniformis.
Comparison of raw and pasteurised milk (72, 85 and 90°C for 15 s) revealed that
pasteurisation was insufficient to inactivate plasmin inhibitors. The pH 4.6 and 6% TCA
soluble extracts of UHT skim milk with added trypsin or plasmin showed high
correlations (R2 > 0.93) by the TNBS, fluorescamine and RP-HPLC methods, confirming
increased proteolysis during storage. Gel electrophoresis showed that breakdown products
from trypsin were similar to plasmin although the former caused more extensive
proteolysis than the latter due to higher enzyme activity, y-caseins, formed as a result of
P-casein degradation disappeared (1484 and 742 BAEE units of added trypsin on days 3
and 7) due to extensive proteolysis. This finding had not previously been reported. Milk
processed at high temperatures (110, 120, 130 and 142°C for 2 s) had lower proteolytic
activities than raw milk and milk heated at 85°C implying inactivation of plasmin at
temperatures of 110°C and above. This was observed in all the methods assessed.
The TNBS method was recommended on the basis of its accuracy, reliability, simplicity
and cost.
Description
PhD Thesis
Keywords
Milk