N.F. Olson

Proteolysis of bovine caseins by cathepsin D: Preliminary observations and comparison with chymosin

Proteolysis of αs1-, as2, β- and κ-caseins by bovine cathepsin D (E.C. 3.4.23.5), an indigenous acid proteinase in milk, was studied by reversed-phase HPLC and urea-PAGE. The results indicate that cathepsin D hydrolyzed all casein fractions and was more active on αs1- than on β-casein; αs1-casein was optimally hydrolyzed at pH 4.0. Proteolysis of β-casein was more sensitive to inhibition by NaCl than was αs1-casein. Comparison of the proteolysis of individual caseins with that by chymosin (E.C. 3.4.23.4) showed that the hydrolysis of αs1-casein by the two enzymes was very similar, but the specificities on gas2-casein differed. The initial stage of β-casein hydrolysis by cathepsin D was similar to that by chymosin and HPLC profiles showed a number of peptides in common. Although cathepsin D hydrolyzed κ-casein slowly in solution and the HPLC profiles of hydrolysates were similar to those produced by chymosin, cathepsin D showed poor milk clotting ability.

Proteolytic specificity of plasmin on bovine αs1‐Casein

Abstract The proteolytic specificity of plasmin (fibrinolysin, E. C. 3.4.21.7, from bovine plasma) on bovine αs1‐casein was determined in solution in 50 mM ammonium bicarbonate buffer, pH 8.4, at 37°C. Peptides, isolated by reverse‐phase high performance liquid chromatography on a C18 column or by electroblotting from urea‐polyacrylamide gel electrophoretograms, were identified from their amino acid sequence and mass. The principal plasmin cleavage sites were found at Arg22‐Phe23, Arg90‐Tyr91, Lys102‐Lys103, Lys103‐Tyr104, Lys105‐Val106, Lys124‐Glu125 and Arg151‐Gln152. The initial cleavage sites and the order of production of small (pH 4.6‐soluble) peptides suggest that αs1‐casein was cleaved initially towards the centre of the molecule.

Use of immobilized lactase in milk systems

Immobilization of lactase for continuous hydrolysis of lactose in milk systems offers considerable potential for the improvement of fluid dairy products. The hydrolyzed product, which contains glucose and galactose, may possess improved functional and nutritional properties. In particular, the usage of cheese whey, a by-product of cheese manufacturing, may be expanded greatly with the development of immobilized lactase technology. Although lactases occur rather widely in nature, only microbial enzymes are of commercial value. The various approaches to lactase insolubilization have been reviewed. Additionally, pertinent factors in operating an immobilized lactase reactor system have been discussed. Commercial lactose reactors are being used currently for industrial production of low-lactose skimmed milk and appear to have economic potential for processing of cheese whey.

Use of a papain superpolymer to elucidate the structure of bovine casein micelles

Abstract Micellar and soluble casein samples were treated with papain which had been cross-linked with glutaraldehyde to form a superpolymer. Unhydrolyzed portions of casein samples were fractionated on polyacrylamide gels. The stained gels were scanned with a densitometer. In both casein samples, the amount of unhydrolyzed κ-, β-, and α s- caseins decreased gradually as the reaction proceeded. The percentage of hydrolysis after 60 min of reaction was 69% in micellar-casein and 78% in soluble casein. However, none of the three casein fractions was hydrolyzed completely. Unhydrolyzed casein in both samples had approximately the same composition throughout the entire reaction. These results suggest that κ-casein does not have a specific location in the casein micelle and that the three major casein fractions are distributed unformly throughout the micelle.

The coagulation of milk with immobilized enzymes: A critical review

Abstract The milk coagulation process is described including current understanding of the mechanism and kinetics involved with emphasis on aspects which impact on coagulation with immobilized enzymes. From the studies of coagulation with immobilized enzymes reported to date there is little evidence to suggest that immobilized enzymes can be used to coagulate milk.

Applications of Confocal Microscopy to Fat Globule Structure in Cheese

The development of cheese texture depends upon a number of different variables, for example, the relative proportions of chemical constituents (fat, water, protein) and the degree of proteolytic degradation of the casein matrix (Everett and Jameson, 1993). Fat exists enmeshed in the casein matrix within cheese and curd, and may influence texture through the size of the globules and also by the nature of the proteinaceous stabilizing species adsorbed at the fat globule/water interface (van Vliet and Dentener-Kikkert, 1982; Xiong and Kinsella, 1991). In the present study, the effect of protein type at the fat/serum interface on size distribution of fat particles in cheese was investigated by manufacturing cheese that contained different proteins at the fat globule/water interface. This was achieved by homogenization of cream prior to cheesemaking, or emulsification of milk fat with isolated native milk proteins.

Autolysis of lactic acid bacteria: Impact on flavour development in cheese

Abstract Early autolysis of cheese related microorganisms in cheese should potentially release the intracellular enzymes into the cheese matrix. Such an event should lead to a more rapid development of flavour in cheese. The autolytic properties of several cheese related bacteria as well as the different factors affecting the process are discussed. Enzyme release during cell autolysis is also considered.

Cell-wall associated peptide hydrolase and esterase activities in several cheese-related bacteria

Cell-wall associated proteinases, peptidases and esterases of Lactobacillus helveticus, Pediococcus sp., Leuconostoc mesenteroides ssp. mesenteroides, Brevibacterium linens, Propionibacterium acidipropionici and Bifidobacterium infantis were assayed. Proteolytic activity was measured using C14-labelled casein. Aminopeptidase, dipeptidase and esterase activities were measured using chromogenic substrates. All the cheese related bacteria tested exhibited cell-wall proteinase activities with highest being detected in Leuconostoc mesenteroides ssp. mesenteroides, Pediococcus sp. and Brevibacterium linens. Lactobacillus helveticus and Brevibacterium linens exhibited greater cell-wall esterase and aminopeptidase activities, but little of the total activities of these enzymes were associated with the cell envelope. Greater properties of total cellular dipeptidase activities were associated with the cell envelope, with Lactobacillus helveticus and Brevibacterium linens exhibiting highest specific activities. The optimum pH of the crude cell-wall proteinase of different strains ranged from pH 5·5 to 8·0 while the optimum temperature ranged from 20 to 40°C. The impact of proteinase inhibitors tested differed between species.

MICROENCAPSULATION OF MULTI-ENZYME SYSTEMS

ABSTRACT Microcapsules were formed by emulsifying an aqueous solution into milkfat and extruding the emulsion into cool water or skimmilk. Efficiency of encapsulation of the aqueous phase depends upon the types, proportions and amounts of emulsifiers used, the volume of encapsulated solution and the temperature of the dispersion liquid. Cell-free extracts of Streptococcus lactis subsp. diacetilactis were encapsulated with appropriate substrate and cofactors and incorporated into low-fat cheese. Total diacetyl and acetoin production was eight times greater in cheese containing the encapsulated system as compared to cheese containing unencapsulated systems. KEYWORDS Microencapsulation, encapsulation, enzyme encapsulation, cheese, cheese ripening, cheese flavor, cheese ripening acceleration.