Abstract

The rapid routine method currently used to monitor the ability of milk to coagulate does not provide information on the primary clotting phase. The purpose of this work was to assess if the principal critical events related to the primary clotting phase could be detected by NIR and by other methods based on the interaction of light and material (fluorescence spectroscopy, tristimulus colorimetry) that provide a quick response. Six different coagulation tests were carried out. Reconstituted skim milk powder was used as standard substrate and the same liquid calf rennet solution was added. Milk NIR spectra were collected at 35°C using an InfrAlyzer 500 (Bran+Luebbe). Spectra were taken every 70 seconds, from rennet addition up to about 10 minutes after clotting time. Data were processed by IDAS-PC software to find the most discriminant wavelengths in highlighting spectral differences. For fluorescence tests, the primary clotting phase was monitored collecting samples at different times and stopping the enzymatic reaction at 0°C in an ice-bath. Spectrofluorimetric titrations were carried out by using ANS as hydrophobic probe and a Luminescence Spectrometer LS-B. Protein Surface Hydrophobycity (PSH) values were calculated. The ANS relative distribution was also monitored after ultracentrifugation at 0°C. In this case ANS was added to milk before rennet addition. Colorimetric assays were carried out by using a tristimulus colorimetry and data collected at the same time as NIR spectra. Differences in luminosity values were calculated. The clotting time was calculated using both a Formagraph Instrument and by visual observation of coagulation. NIR data show two critical events during the primary clotting phase, confirmed by the other techniques used. The first appears a few minutes before clotting. The second, in accordance with the visual observation of coagulation, detects the clotting point, before Formagraph in corrispondence of micellar aggregation. Interesting results are obtained by plotting selected NIR 2nd derivative absorbance values against time; this establishes a trend of coagulation, which is also detectedby the other techniques. The interrelationship among the different techniques is discussed and spectral data are interpreted in terms of water bonds and/or different amounts of free water due to the formation of new products and/or protein structure rearrangements.

© 1998 NIR Publications

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