On the influence of non-enzymatic crosslinking of caseins on the gel strength of yoghurt

Abstract

After storage of UHT milk at 37 degrees C resp. 50 degrees C, yoghurt was prepared. For a storage temperature of 37 degrees C, breaking strength of the yoghurt samples increased from 2.7 to 5.8 N with increasing storage duration of the UHT milk. A plateau is reached after 17 days of storage. This increase in breaking strength correlates with a significant increase in non-reducible casein oligomerization from 14% for fresh UHT milk to 25% measured using size exclusion chromatography under reducing and denaturing conditions and calculated as sum of predominantly formed dimers and trimers at the total casein fraction. At a storage temperature of 50 degrees C, a less increase in breaking strength from 2.7 to 4.6 N with a plateau after 17 days was observed while casein oligomerization increased to 63%. After acid hydrolysis, only lysinoalanine and histidinoalanine were detected in the caseinate samples via amino acid analysis. The quantified concentration of lysinoalanine and histidinoalanine could not explain the observed casein oligomerization. Thus, unknown crosslinked amino acids must have been formed during storage, inducing significant changes in the functional properties of milk proteins.

Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

“…Alkaline treatment, especially in combination with heating, transforms particular amino acid residues into reactive intermediates, which form cross-links by reactions with other functional groups. Typical products are lysinoalanine, histidinoalanine and lanthionine, which can be determined by chromatographic techniques, e.g., [43][44][45][46][47][48]. Severe heat treatment may even induce the formation of isopeptide bonds between lysine and glutamine (N-ε-(γ-glutamyl)-lysine) or asparagine (N-ε-(β-aspartyl)-lysine), however, these reactions are competed by the Maillard reaction and therefore restricted in the presence of reducing carbohydrates [23,42,49,50].…”

“…SEC column materials differ in composition and pore volume. The column most frequently used for studying cross-linked casein is Superdex 200, e.g., [30,44,47,48,54,100,115,131,[134][135][136][137][138][139][140][141][142][143][144][145][146][147][148], which is provided by GE Healthcare (Uppsala, Sweden). The column material is based on agarose/dextran with a ratio of pore volume to void volume of V i /V o = 1.7 and exhibits a fractionation range from 10 to 600 kg/mol for globular proteins [128].…”

“…For instance, the highest number of casein dimers in TGase-catalysed polymerisation was observed in moderately cross-linked samples, i.e., at high levels of residual monomers (>50%), indicating that dimers as well as smaller oligomers are good substrates for further cross-linking by TGase [32,78,146]. Moreover, the polymerisation degree (sometimes also referred to as oligomerisation degree) was calculated in several studies from size exclusion chromatograms by relating peak areas of cross-linked caseins to the entire sample area [30,32,44,47,48,53,78,[134][135][136][146][147][148]. Because reference casein samples often reveal a low level of polymerised casein (see Figures 9 and 10), the polymerisation degree after cross-linking was also expressed as the difference between cross-linked samples and reference [137][138][139]144,145].…”

“…From studies on non-enzymatic cross-linking it was concluded that [CLAA] min higher than experimental data (e.g., for lysinoalanine or histidinoalanine) implies the occurrence of unknown cross-links, and that lower [CLAA] min is an indicator for intramolecular cross-linking [44,45,48]. Higher [CLAA] min may also indicate underestimation by insufficient protein hydrolysis prior to amino acid analysis.…”

Kinetic description of heat‐induced cross‐linking reactions of whey protein‐free casein solutions

“…Gel-permeation chromatography was performed according to Walter (1995) and Lauber et al (2001), using a high-performance liquid chromatography system equipped with a pump P 900 and a detector A 900 together with a Superdex 200 HR 10/30 (71-7059-00) column ( € Akta design, Amersham Biosciences, Freiburg, Germany). The column was eluted at room temperature at a flow rate of 0.5 mL/min using a 0.1 M sodium phosphate buffer pH 6.8, containing 6 M urea, 0.1 M sodium chloride and 0.1% 3-((3-cholamidopropyl)-dimethyl ammonium)-1-propane-sulphonate (CHAPS).…”