Elsevier

LWT

Volume 47, Issue 2, July 2012, Pages 407-412
LWT

The pH-dependent thermal and storage stability of glycosylated caseinomacropeptide

https://doi.org/10.1016/j.lwt.2012.02.001Get rights and content

Abstract

Bioactive properties of bovine glycosylated caseinomacropeptide (gCMP) like antibacterial effects are reported to be closely associated with their structure in terms of attached glycans. Technological properties are also influenced by the carbohydrate side chains. However, during product manufacturing gCMP can be modified due to processing. Processing conditions, which influence the degree of glycosylation of gCMP lead to alterations of bioactivity and techno-functional properties of gCMP and accordingly gCMP-containing products. Hence, gCMP was studied for its glycan stability during heat treatment and storage under different pH values. Process stability (preservation of native protein structure in terms of attached glycans) was analysed by quantifying the release of the terminal carbohydrate, N-acetylneuraminic acid (Neu5Ac), from gCMP. The results clearly showed that the thermal stability of gCMP is strongly influenced by pH. When the pH was decreased from 7 to 2, reduced stability was found even at low heating temperatures. Minimal destabilisation effects were found at neutral pH. Similar observations were found during storage of gCMP. Neu5Ac was released after six days of storage, with a maximum release of 30% at pH 2. Acidic pH conditions were responsible for the hydrolysis of the glycans from the peptide backbone during heat treatment and storage.

Highlights

► GCMP possesses interesting functional properties related to attached glycans. ► Stability of gCMP during heat treatment and storage was studied. ► Process stability is strongly influenced by pH. ► High stability at neutral pH.

Introduction

Bovine caseinomacropeptide (CMP), the hydrophilic fragment f(106–169) of κ-casein released by the action of chymosin possesses interesting nutritional and technological properties such as antibacterial and antiadhesive effects as well as foaming and emulsifying properties (Janer et al., 2004, Kawasaki et al., 1992, Kreuss, Krause et al., 2009, Kreuss, Strixner et al., 2009, Marshall, 1991). Therefore, it is a potential ingredient for food applications such as infant formula and/or as prebiotics in adult nutrition (Fox, 2001, Korhonen, 2002, Steijns, 2001). However, CMP shows a high degree of heterogeneity due to the genetic variations of κ-casein and mainly due to post-translational glycosylation, since all sites for O-glycosylation of κ-casein are found in the C-terminal domain f(106–169) (Holland et al., 2005, Saito and Itoh, 1992). Glycosylation of CMP differs in the numbers of occupied glycosylation sites, and further due to variability in size and structural composition of each of those glycans, which may be composed of N-acetyl galactosamine (GalNAc), galactose (Gal) and N-acetylneuraminic acid (Neu5Ac) (Holland et al., 2006, Kawasaki et al., 1992, Saito and Itoh, 1992).

Since the attached glycans affect physicochemical properties like the net charge, conformation, solubility, in addition to protecting the protein from proteolytic degradation (Kundra and Kornfeld, 1999, Parodi, 2000, Sinclair and Elliott, 2005, Sola and Griebenow, 2009), differences in functionality occur between the glycosylated (gCMP) and non-glycosylated (aCMP) fractions of CMP. While aCMP possesses stronger emulsifying activity and foaming properties (Kreuss, Krause et al., 2009, Kreuss, Strixner et al., 2009), biofunctional effects are reported to be strongly mediated by the attached carbohydrates in gCMP (Byrne et al., 2007, Kawasaki et al., 1993, Kawasaki et al., 1992, Lemeste et al., 1990). Pathogenic organisms and toxins bind to cell surface carbohydrates to gain access to the mucosal membrane (Arnold et al., 2007, Newburg, 1999, Varki, 1993). Oligosaccharide sequences on soluble glycoconjugates such as gCMP can serve as cognate receptors for the encounter of rotavirus (Varki, 1993), cholera toxin (Kawasaki et al., 1992) or Escherichia coli strains (Parkkinen, Finne, Achtman, Vaisanen, & Korhonen, 1983). Upon binding to these glycoconjugates microorganisms, parasites or toxins are swept out leaving the mucosal cell untouched (Wang & Brand-Miller, 2003). The monosaccharide Neu5Ac is highly involved in this activity due to its location at the terminus of an oligosaccharide chain (Byrne et al., 2007, Schauer, 2000, Wang and Brand-Miller, 2003). Based on its high negative charge, Neu5Ac furthermore affects the technological properties of glycoproteins to a great extent (Byrne et al., 2007, Schauer, 1982, Varki, 1993).

Functional properties of proteins or peptides such as CMP are widely utilised in the dairy industry and other foods (Fox, 2001, Korhonen, 2002, Walstra, 2003). However, CMP can be subjected to modifications during product manufacturing. On the on hand, this concerns processing steps, which are necessary during the isolation of CMP from bovine milk and during subsequent applications of CMP. It should be noted that bovine milk already undergoes several processing prior to CMP isolation. On the other hand, processing during the manufacture of dairy and other CMP-containing food products may alter it.

Thermal treatment is a fundamental operation, which is applied to improve food safety, sensory qualities and shelf life. Furthermore, it is well known that heat treatment of milk and milk protein solutions affect the functional properties of native proteins. Hence, milk proteins can be subjected to modification such as conformational changes due to denaturation, intermolecular reactions or Maillard reaction (Fenaille et al., 2006). In addition, unfavourable conditions are created by a low pH, promoting the hydrolysis of saccharides (Klewicki, 2007). As intrinsically disordered peptide without a secondary or tertiary structure, CMP is less vulnerable to denaturation. However, the cleavage of glycosidic linkages of gCMP, resulting to a loss of carbohydrate side chains, is likely to occur during processing. In that case gCMP would be altered, resulting in the acquisition of properties comparable to aCMP. This affects biological and technological properties, and therefore would also affect properties of food products containing gCMP.

However, the impact of manufacturing such as heat treatment and variations of environmental factors on the stability of gCMP is not yet well known. In order to preserve and utilize the original functionality of gCMP for nutritional purposes in consumer products, the native glycoprotein structure including the carbohydrate chains, is required (Abe et al., 1991, Arnold et al., 2007, Sola and Griebenow, 2009). Moreover, to manufacture consistent products, providing desired characteristics, it is essential to know the impact of manufacturing, process parameters and storage conditions on gCMP stability, especially when CMP would be deliberately used as biofunctional ingredient.

The aim of the present study was therefore to determine the effect of heat treatment on the stability of carbohydrate side chains of gCMP. Since it is desirable to have ingredients or foodstuffs that are stable over time, storage stability was additionally studied. Due to its important role within several biological functions and its terminal position within the glycoconjugate, Neu5Ac was used as an indicator molecule for the release of carbohydrates from gCMP during processing. Total and non-protein-bound Neu5Ac content of the samples was determined in order to quantify the stability of gCMP. The impact of heat treatment on the pH-dependent stability of gCMP was determined at conventional processing conditions applied to raw milk, and dependent on the heating time. The storage stability was studied with respect to the pH- and storage temperature dependence.

Section snippets

Sample preparation

Caseinomacropeptide (CMP) was obtained from Arla Foods Ingredients, Viby J, Denmark (Lacprodan CGMP-10). Protein content is 857 mg g−1, and content of major protein fractions are as follows: aCMP 369 mg g−1, gCMP 454 mg g−1, α-Lactalbumin 60 mg g−1, β-Lactoglobulin 117 mg g−1 (Kreuss & Kulozik, 2009). CMP solutions of 10 mg mL−1 were prepared by suspending CMP-powder in double distilled water and solutions were stored at 4 °C over night prior to usage. The pH value of the solution was adjusted

Conventional processing conditions

Heat stability of gCMP was measured by quantitatively assessing the destruction (total Neu5Ac, Fig. 1a) and release of Neu5Ac (non-protein-bound Neu5Ac, Fig. 1b) and was studied at conventional processing conditions applied to raw milk (Table 1).

As shown in Fig. 1a, the total Neu5Ac amount of pH-adjusted CMP solutions at heating conditions below 100 °C (low-temperature long-time, LTLT; high-temperature short-time I, HTST I; high-temperature short-time II, HTST II) and at extended-shelf-life

Conclusion

The present study describes the stability of gCMP during thermal treatment and storage. No detailed analysis of the stability of glycosylated CMP regarding the attached glycans during thermal processing and storage has been reported so far. Release of Neu5Ac was found in this study, when gCMP is processed at acidic environmental conditions. Depending on the heating temperature and time, up to 72% of the total Neu5Ac starting content was found to be unbound to gCMP at pH 2 (120 °C, 90 s).

Acknowledgements

The authors are grateful to the German Federal Ministry for Education and Research (BMBF), for financial support through the research project BioChangePLUS 031632A.

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