93:, κ) that account for nearly 80% of bovine milk proteins and that form soluble aggregates are known as "casein micelles" in which κ-casein molecules stabilize the structure. There are several models that account for the spatial conformation of casein in the micelles. One of them proposes that the micellar nucleus is formed by several submicelles, the periphery consisting of microvillosities of κ-casein Another model suggests that the nucleus is formed by casein-interlinked fibrils. Finally, the most recent model proposes a double link among the caseins for gelling to take place. All 3 models consider micelles as colloidal particles formed by casein aggregates wrapped up in soluble κ-casein molecules. Milk-clotting proteases act on the soluble portion, κ-casein, thus originating an unstable micellar state that results in clot formation.
219:) by κ-casein labeled with the fluorochrome fluorescein isothiocyanate (FITC) to yield the fluorescein thiocarbamoyl (FTC) derivative. This variation allows quantification of the κ-casein molecules degraded in a more precise and specific way, detecting only those enzymes able to degrade such molecules. The method described by Twining (1984), however, was designed to detect the proteolytic activity of a considerably larger variety of enzymes. FTC-κ-casein allows the detection of different types of proteases at levels when no milk clotting is yet apparent, demonstrating its higher sensitivity over currently used assay procedures. Therefore, the method may find application as an indicator during the purification or characterization of new milk-clotting enzymes.
67:
171:
pH or high temperature. Consequently, this may lead to confusing and irreproducible results, particularly when the enzymes have low activity. At the same time, the classical method is not specific enough, in terms of setting the precise onset of milk gelation, such that the determination of the enzymatic units involved becomes difficult and unclear. Furthermore, although it has been reported that κ-casein hydrolysis follows typical
192:
102:
170:
The conventional way of quantifying a given milk-clotting enzyme employs milk as the substrate and determines the time elapsed before the appearance of milk clots. However, milk clotting may take place without the participation of enzymes because of variations in physicochemical factors, such as low
214:
FTC-κ-casein method affords accurate and precise determinations of κ-caseinolytic degradation, the first step in the milk-clotting process. This method is the result of a modification to the one described by S.S. Twining (1984). The main modification was substituting the substrate previously used
178:
To overcome this, several alternative methods have been proposed, such as the determination of halo diameter in agar-gelified milk, colorimetric measurement, or determination of the rate of degradation of casein previously labeled with either a radioactive tracer or a
161:
pattern, the limiting step in milk-clotting being the degradation rate of κ-casein. The kinetic pattern of the second step of the milk-clotting process is influenced by the cooperative nature of micellar flocculation, whereas the
166:
properties of the gel formed depend on the type of action of the proteases, the type of milk, and the patterns of casein proteolysis. The overall process is influenced by several different factors, such as pH or temperature.
130:. There are also several milk-clotting proteases that, being able to cleave the Phe105-Met106 bond in the κ-casein molecule, also cleave other peptide bonds in other caseins, such as those produced by
48:(GMP). GMP is responsible for an increased efficiency of digestion, prevention of neonate hypersensitivity to ingested proteins, and inhibition of gastric pathogens. The human
1074:"Partial Identification of Water-Soluble Peptides Released at Early Stages of Proteolysis in Sterilized Ovine Cheese-Like Systems: Influence of Type of Coagulant and Starter"
561:
Carlson, Alfred; Hill, Charles G; Olson, Norman F. (1987). "Kinetics of milk coagulation: I. The kinetics of kappa casein hydrolysis in the presence of enzyme deactivation".
598:
Carlson, Alfred; Hill, Charles G.; Olson, Norman F. (1987). "Kinetics of milk coagulation: II. Kinetics of the secondary phase: Micelle flocculation".
124:
industry. However, there are milk-clotting proteases able to cleave other peptide bonds in the κ-casein chain, such as the endothiapepsin produced by
771:
Holt, C. (1992). "Structure and
Stability of Bovine Casein Micelles". In Anfinsen, C.B.; Richards, Frederic M.; Edsall, John T.; et al. (eds.).
1148:"Gelation Mechanism of Milk as Influenced by Temperature and pH; Studied by the Use of Transglutaminase Cross-Linked Casein Micelles"
86:
1239:
871:
788:
806:
Horne, David S. (1998). "Casein
Interactions: Casting Light on the Black Boxes, the Structure in Dairy Products".
854:
Kobayashi, Hideyuki (2004). "Polyporopepsin". In
Barrett, Alan J.; Woessner, J. Fred; Rawlings, Neil D. (eds.).
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183:
compound. All these methods use casein as the substrate to quantify proteolytic or milk-clotting activities.
829:"Studies on Milk Proteins. II. Colorimetric Determination of the Partial Hydrolysis of the Proteins in Milk"
120:
the peptide bond in Phe105-Met106 of κ- casein and is considered to be the most efficient protease for the
697:
Drøhse, Helle B.; Foltmann, Bent (1989). "Specificity of milk-clotting enzymes towards bovine κ-casein".
883:"Three-Dimensional Molecular Modeling of Bovine Caseins: A Refined, Energy-Minimized κ-Casein Structure"
200:
1117:
Twining, Sally S. (1984). "Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes".
1181:
Walstra, Pieter (1979). "The voluminosity of bovine casein micelles and some of its implications".
728:"Effect of pH on the Gelation Properties of Skim Milk Gels Made from Plant Coagulants and Chymosin"
528:"Fluorescein Thiocarbamoyl-Kappa-Casein Assay for the Specific Testing of Milk-Clotting Proteases"
1234:
Fluorescein
Thiocarbamoyl-Kappa-Casein Assay for the Specific Testing of Milk-Clotting Proteases
988:"Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422"
1224:
1266:
211:) derivative. This substrate is used to determinate the milk clotting activity of proteases.
953:"Invited Review: Perspectives on the Basis of the Rheology and Texture Properties of Cheese"
136:
or even bovine chymosin. This allows the manufacture of different cheeses with a variety of
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Rao, Mala B.; Tanksale, Aparna M.; Ghatge, Mohini S.; Deshpande, Vasanti V. (1998).
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637:"A Rapid Method for Measuring Protease Activity in Milk Using Radiolabeled Casein"
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699:
Biochimica et
Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
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Poza, M.; Sieiro, C.; Carreira, L.; Barros-Velázquez, J.; Villa, T. G. (2003).
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kinetics, it is difficult to determine with the classic milk-clotting assay.
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involved in several important physiological processes. Chymosin (found in
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672:"Casein Micelles as Colloids: Surface Structures and Stabilities"
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41:
33:
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Vasbinder, A.J.; Rollema, H.S.; Bot, A.; de Kruif, C.G. (2003).
431:
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1031:"Molecular and Biotechnological Aspects of Microbial Proteases"
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82:
37:
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Esteves, C.L.C.; Lucey, J.A.; Wang, T.; Pires, E.M.V. (2003).
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Ageitos, J.M.; Vallejo, J.A.; Poza, M.; Villa, T.G. (2006).
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The milk-clotting process consists of three main phases:
918:"Formation and Physical Properties of Milk Protein Gels"
397:
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Kumosinski, T.F.; Brown, E.M.; Farrell, H.M. (1993).
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Journal of
Industrial Microbiology and Biotechnology
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951:Lucey, J.A.; Johnson, M.E.; Horne, D.S. (2003).
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105:In red/blue Phe105-Met106 bond of κ- casein
1035:Microbiology and Molecular Biology Reviews
207:) to yield the fluorescein thiocarbamoyl (
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773:Advances in Protein Chemistry Volume 43
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199:Κ-casein labeled with the fluorochrome
44:(para kappa-casein) and water-soluble
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474:
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256:Kumosinski, Brown & Farrell 1993
40:) splits K-casein into an insoluble
1072:Silva, S.V.; Malcata, F.X. (2005).
70:Molecular surface model of K-Casein
13:
864:10.1016/B978-0-12-079611-3.50035-5
147:Enzymatic degradation of κ-casein.
14:
1278:
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1165:10.3168/jds.S0022-0302(03)73741-2
1091:10.3168/jds.S0022-0302(05)72870-8
970:10.3168/jds.S0022-0302(03)73869-7
935:10.3168/jds.S0022-0302(02)74078-2
900:10.3168/jds.S0022-0302(93)77586-4
846:10.3168/jds.S0022-0302(47)92412-0
775:. Vol. 43. pp. 63–151.
745:10.3168/jds.S0022-0302(03)73850-8
689:10.3168/jds.S0022-0302(98)75865-5
654:10.3168/jds.S0022-0302(87)80218-7
545:10.3168/jds.S0022-0302(06)72418-3
600:Biotechnology and Bioengineering
563:Biotechnology and Bioengineering
96:
856:Handbook of Proteolytic Enzymes
448:Carlson, Hill & Olson 1987b
436:Carlson, Hill & Olson 1987a
271:Lucey, Johnson & Horne 2003
1241:Biotechnology and Microbiology
1047:10.1128/MMBR.62.3.597-635.1998
228:
157:Each step follows a different
1:
820:10.1016/S0958-6946(98)00040-5
781:10.1016/S0065-3233(08)60554-9
519:
140:and organoleptic properties.
1131:10.1016/0003-2697(84)90553-0
711:10.1016/0167-4838(89)90039-3
61:
7:
808:International Dairy Journal
10:
1283:
377:Drøhse & Foltmann 1989
236:"Kappa casein (IPR000117)"
201:fluorescein isothiocyanate
195:Fluorescein isothiocyanate
1195:10.1017/S0022029900017234
1183:Journal of Dairy Research
1005:10.1007/s10295-003-0100-y
1152:Journal of Dairy Science
1078:Journal of Dairy Science
957:Journal of Dairy Science
922:Journal of Dairy Science
887:Journal of Dairy Science
833:Journal of Dairy Science
732:Journal of Dairy Science
676:Journal of Dairy Science
670:Dalgleish, D.G. (1998).
641:Journal of Dairy Science
532:Journal of Dairy Science
409:Silva & Malcata 2005
222:
1119:Analytical Biochemistry
635:Christen, G.L. (1987).
196:
150:Micellar flocculation.
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16:Mammalian milk protein
1262:Laboratory techniques
612:10.1002/bit.260290508
575:10.1002/bit.260290507
353:Vasbinder et al. 2003
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1225:InterPro: IPR000117
916:Lucey, J.A. (2002).
112:(EC 3.4.23.4) is an
827:Hull, M.E. (1947).
511:Ageitos et al. 2006
392:Esteves et al. 2003
127:Endothia parasitica
858:. pp. 111–5.
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187:FTC-Κ-casein assay
133:Cynara cardunculus
116:that specifically
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873:978-0-12-079611-3
790:978-0-12-034243-3
114:aspartic protease
46:glycomacropeptide
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1125:(1): 30–4.
164:rheological
138:rheological
1251:Categories
755:10316/3878
520:References
336:Horne 1998
312:Lucey 2002
118:hydrolyzes
1211:222355860
475:Hull 1947
324:Holt 1992
62:Structure
28:mammalian
1257:Proteins
1174:12778566
1110:15905424
1022:23067478
1014:14634834
979:14507008
944:11913691
764:12939079
628:44397261
620:18576490
591:38359395
583:18576489
554:16960051
240:InterPro
110:Chymosin
20:Κ-casein
1139:6442109
1065:9729602
909:8227653
799:1442324
719:2495817
663:3117854
159:kinetic
75:Caseins
42:peptide
34:protein
26:, is a
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38:rennet
1207:S2CID
1056:98927
1018:S2CID
624:S2CID
587:S2CID
223:Notes
22:, or
1199:PMID
1170:PMID
1135:PMID
1106:PMID
1061:PMID
1010:PMID
975:PMID
940:PMID
905:PMID
868:ISBN
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715:PMID
659:PMID
616:PMID
579:PMID
550:PMID
205:FITC
55:CSN3
50:gene
31:milk
1191:doi
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1051:PMC
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684:doi
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209:FTC
87:αS2
83:αS1
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