Arno C. Alting

Number of thiol groups rather than the size of the aggregates determines the hardness of cold set whey protein gels

Variation of protein concentration during heating resulted in the formation of protein aggregates with clearly different structural and chemical characteristics. Heating conditions were chosen such that differences in the degree of aggregation were excluded. Acid induced gelation of dispersions of these aggregates resulted in gels with clearly different hardness. Although gel hardness seemed to correlate with the different structural aggregate features as reported before in literature, the differences in hardness could for the most part be cancelled by blocking of the thiol groups. Application of thiol-blocked protein aggregates enabled us to make a distinction between the effect of structural- and chemical-properties of the aggregates. Formation of larger disulfide cross-linked protein structures paralleled the increase in gel hardness and dominated the effect of structural characteristics on mechanical properties of cold-set gels. In addition, the effect of the presence of native non-aggregated protein on the final gel properties can be excluded, since in our gel-experiments most protein (>95%) participated in the formation of a protein network. Therefore, we can conclude that the hardness of cold set whey protein gels is determined by the number of thiol groups rather than by the size of the aggregates or other structural features.

Texture of acid milk gels:formation of disulfide cross-links during acidification

Denaturation of whey proteins during pasteurization of milk results in the formation of whey protein aggregates and whey protein-coated casein micelles. After cooling a substantial number of thiol groups remains exposed. Formation of larger disulfide-linked protein structures during acidification at ambient temperature was demonstrated by analytical methods. The time-dependent formation of these structures attributed significantly to the mechanical properties of acid milk gels, resulting in gels with an increased storage modulus and hardness. Addition of the thiol-blocking agent N-ethylmaleimide prevented the formation of disulfide-linked structures. The mechanical properties are shown to be the result of the contribution of denatured whey proteins to the protein network as such and the additional formation of disulfide bonds. Surprisingly, these sulfhydryl group-disulfide bond interchange reactions take place at ambient temperature and under acidic conditions. Therefore, the disulfide cross-linking is highly relevant for textural properties of acid-milk products, like yoghurt.

Quantification of heat-induced casein /whey protein interactions in milk and its relation to gelation kinetics

This paper describes a quantitative study on the distribution of denatured whey proteins over whey protein aggregates and whey proteins associated with the casein micelles in heated milk. This was done by combination of enzymatic fractionation and capillary electrophoresis. More severe heat treatment at the natural pH of milk caused more denaturation, but the ratio of denatured whey proteins associated with the casein micelle and present in aggregates remained constant. The distribution is related to changed acid-induced gelation properties and textural properties of milk-derived products. We clearly demonstrated that the observed shift in gelation pH of heated milk is linearly correlated with the distribution of denatured whey proteins. Quantitative description will allow better control and tuning of the final gel properties of milk-derived products.

The role of starter peptidases in the initial proteolytic events leading to amino acids in Gouda cheese

Abstract The initial proteolytic steps in Gouda cheese leading to amino acid nitrogen (AN) have been found to reflect the essential action of the cell-bound, cell-envelope proteinase (CEP) of lactococci on the primary product of chymosin action on αs1-casein, fragment 1–23 (αs1-CN(f1–23)) as observed in vitro under cheese conditions. In the absence of CEP, αs1-CN(f1–23) accumulates in cheese and the production of AN falls. In such a cheese, only a slow conversion of αs1-CN(f1–23), catalysed by an intracellular endopeptidase, could be detected. In the presence of CEP, the early appearance in cheese of products of the action of this endopeptidase indicates significant early lysis of cells. The distinct specific actions of different types of CEP, characteristic of different lactococcal strains, on αs1-CN(f1–23) in vitro are also recognized in cheese and therefore CEP may be expected to direct further proteolysis to some extent. Increasing salt concentration in the cheese moisture does not influence initial αs1-casein conversion but inhibits the subsequent conversion of αs1-CN(f24–199), as well as AN production in cheese significantly. Dehydration of the rind additionally influences proteolysis negatively. The importance of initial αs1-casein conversion and degradation of αs1-CN(f1–23) for the development of flavour in cheese is discussed.

Purification and characterization of the mature, membrane-associated cell-envelope proteinase of Lactobacillus helveticus L89

Lactobacillus helveticus L89 possesses a cell-envelope proteinase (Lb-CEP) that is biochemically and genetically related to that of the lactococci (Lc-CEP). The in-situ proteinase is resistant to autoproteolysis and remains associated with the membrane during lysozyme treatment of cells and subsequent mechanical disruption of the treated cells. The proteinase was purified from isolated membranes by a procedure that preserves the complete in-situ proteinase (mature proteinase) assumed to be the N-terminally processed translation product including the membrane anchor: its monomer molecular mass is approximately 180 kDa. The purified enzyme appeared to be more stable towards heat than hitherto known related, but C-terminally truncated cell-envelope proteinases of lactobacilli and lactococci, which were released from the cells by autoproteolysis. On the basis of its specificity towards caseins, towards the αsl-casein-(1-23)-fragment and towards two differently charged chromophoric peptides, the proteinase was recognized as an (Lb-)CEPI/III mixed-type variant different from those identified so far among the lactococcal proteinases.

Conversion of αS1-casein-(24-199)-fragment and β-casein under cheese conditions by chymosin and starter peptidases

Summary The sequence of proteolytic cleavages characterizing the action of chymosin on the αauthor-casein-(24-199)-fragment (αauthor-I) and on β-casein in vitro under conditions as present in Gouda cheese, and the possible intervention by the lactococcal cell-envelope proteinase (CEP) in the subsequent chain of reactions in cheese, have been established. Primary cleavage sites with approximately the same susceptibility to chymosin are Leu 149 - Phe 150, Leu 156 - Asp 157 and Trp 164 - Tyr 165 in αauthor-I and Leu 192 - Tyr193 in β-casein. Two of the three main primary products of αauthor-I degradation, viz. fragments f24–149 and f150–164, are rapidly converted by chymosin to several, mostly small fragments. These fragments, together with the primary product f165–199, are considered to be substrates for starter peptidase action in cheese. As long as cell lysis is not significant in cheese, CEP seems to be mainly responsible for further degradation of these peptides. The phosphoserine-containing αauthor-casein fragment f24–74 and the bitter-tasting β-casein fragment f193–209 appear to be most resistant to both chymosin and CEP action.

Proteins as clean label ingredients in foods and beverages

Abstract: This chapter discusses the use of proteins as a source of clean label ingredients. The chapter first reviews the different classes of proteins and their basic functionalities, then discusses the development of specific protein ingredients for application as viscosifiers, antimicrobial agents and emulsifiers to stabilize foams and emulsions. Finally, the chapter gives an overview of future trends in the development of protein ingredients.

Application of whey proteins as coating ingredients

Technologies are described where activated whey proteins are used for encapsulating sensitive food materials. The mechanism, governing the solubility of whey protein films and coatings, on which these technologies are based was investigated.The building blocks for these films and coatings were aggregated whey proteins. Solubility was related to the continuity of the network of disulfide cross-links between the building blocks and the dynamics of rearrangements of disulfide bonds occurring via the socalled SH/S-S exchange reaction.By controlling either the accessibility of thiol groups or the accessibility of disulfide bonds within the whey protein aggregates, solubility of whey protein coatings and films could be tuned. These new insights will help to improve the properties of whey protein coatings promoting the applicability of whey proteins in films, encapsulates and coatings.The technologies have been intellectual property rights (IPR)-protected (1-5) by NIZO food research.

Engineering stability and specificity of the Lactococcus lactis SK11 proteinase

Engineering of the cell envelope-located serine proteinase of L. lactis strain SK11 was performed to alter its caseinolytic specificity and improve its stability. Mutant proteinases with both broader and narrower specificity than wild-type were obtained by alteration of residues in the substrate binding region. Stability is directly related to autoproteolysis, and can be altered by changing enzyme specificity. Stability was enhanced by 2°C by introduction of an extra Asp residue in the binding region. Deletion of surface loops or domains had various effects. Removal of a 151-residue domain had little effect on stability and specificity, while removal of a 14-residue loop led to complete loss of activity.