Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1994-08-10
1998-12-08
Eisenschenk, Frank C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 435 7021, 435346, 4241391, 4241411, 4241721, 530327, 530328, 530329, 530330, 5303871, 5303879, 5303881, G01N 3353, A61K 3808, C12N 512, C07K 1618
Patent
active
058467327
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a process which makes it possible to evaluate the degree of proteolytic denaturation of milk and milk products.
Proteolysis phenomena and, in particular, those due to bacterial proteases are responsible for a reduction in the quality of milk products.
The bacteria responsible for these phenomena are essentially Gram-negative bacteria, and especially Pseudomonas. These are psychrotrophic bacteria which are capable of developing at low temperature; the storage of milk at 4.degree. C. favours the development of these bacteria at the expense of other species.
The action of proteases from psychrotrophic bacteria has been studied essentially with respect to casein. It seems that the fraction which is most rapidly degraded is K-casein; .beta.-casein is also substantially attacked, and, to a lesser degree, .alpha.s1-casein is also degraded. Proteolysis of K-casein by psychrotrophic bacteria proteases releases a peptide of about sixty amino acids (for example 64 amino acids for cow's milk), caseinomacropeptide (CMP), also called glycomacropeptide (GMP), and corresponding to the C-terminal end of K-casein.
The protease activity of psychrotrophic bacteria is thermoresistant and is stable to UHT sterilisation treatments. The persistence of this activity in UHT products reduces the shelf life of these products and their organoleptic qualities. It is therefore desirable to have a test which makes it possible to evaluate the proteolysis (or the proteolytic potential) due to psychrotrophic bacteria in milk and milk products.
Such a test, when performed on milk on arrival at the dairy factory, will make it possible to determine the treatments and processing operations which are most suitable for the quality of the milk received. It would thus be possible to avoid certain manufacturing faults (jellification of UHT milks, bitter flavours, rancidity, decrease in cheese yields).
Furthermore, such a test would also be very useful for cheese manufacturing. Indeed, proteolytic enzymes from psychrotrophic bacteria definitely play a role in the maturing of cheese. Determination of the potential proteolytic activity before manufacturing would make it possible to better control the maturing by varying the ripening conditions.
Several techniques have consequently been proposed with the aim of evaluating proteolytic activity in milk, and in particular that due to psychrotrophic bacteria.
It has for example been proposed to directly evaluate the psychrotrophic flora responsible for proteolysis. This method, which involves techniques for culturing and enumerating bacteria, has all the disadvantages relating thereto; it is necessary to have a relatively large amount of specific equipment and a specialised staff and, on the other hand, the results are obtained only after the period required for the growth of the bacterial cultures. Recently, rapid indirect techniques (bioluminescence, impedimetry, and the like) have been proposed. However, they require specific equipment, are more or less suitable for heterogeneous cultures such as milk and milk products, and in practice are hardly used in the dairy industry.
It has also been proposed to directly assay these proteases; these assays are carried out either on the basis of the enzymatic activity, or directly by immunochemical quantification of the proteases.
Assay of the enzymatic activity is carried out for example by determining hydrolysis of natural or artificial substrates, releasing labelled products (coloured, fluorescent or radioactive). By way of example, the mentioned in which the substrate is collagen denatured by covalent bonding with a blue dye. This technique makes it possible to detect proteases produced by about 2.times.10.sup.6 psychrotrophs/ml.
Immunochemical methods have been proposed, for example, for proteinase P1 49, 382-387!. Assay of this proteinase by the ELISA technique makes it possible to detect the equivalent of about 10.sup.7 psychrotrophs/ml.
Other techniques involve the measurement of amino acids or non-protein nitrogen which are release
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Collin Jean-Claude
Perrod Jean-Louis
Eisenschenk Frank C.
Rabin Evelyn
Sanofi Diagnostics Pasteur
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