Fractionation of whey proteins from red deer (Cervus elaphus) milk and comparison with whey proteins from cow, sheep and goat milks
Introduction
Although milk production in the dairy industry in many countries is dominated by cow milk, other farmed animals, such as sheep and goats are increasingly used for milk production with products having interesting properties (Casper et al., 1999), including hypo-allergenicity compared to cow (El-Agamy, 2007). Milk production from these small ruminants is well established and most of this milk is processed into specialist cheese or low allergenicity milk products. Red deer and also elk are farmed for meat production in New Zealand and more recently dairy herds of red deer and elk have been established, creating potential for developing a novel dairy industry, and trial productions of cheese from these milk sources have been reported (Ashton, 2013). Cheese production results in the generation of significant quantities of whey as a by-product, which is often under-utilized in the dairy industry. Whey from non-bovine milk is usually disposed by land spreading (Casper et al., 1999).
Whey proteins have been recognized to have significant nutritional and health-promoting value and have been utilized in various products (see review by Hernandez-Ledesma et al., 2011). Whey protein hydrolysates are increasingly being recognized as an important source of bioactive peptides (Pihlanto-Leppala, 2000). A recent investigation of the in vitro production of peptide hydrolysates from red deer whole milk in comparison with that of cow milk demonstrated that red deer milk was more digestible and produced more peptides (Opatha-Vithana et al., 2012). Subsequently it has been shown that red deer milk hydrolysates generated peptides with significant immunomodulatory bioactive properties (Opatha-Vithana, 2012).
To our knowledge there has been only one previous report on red deer, which was limited by the technology available at the time and focused mainly on the most abundant whey protein beta-lactoglobulin (β-Lg) (McDougall and Stewart, 1976). While milk protein composition is reported to vary for several different species and differences in physical characteristics of the proteins are reported to be due to differences in amino acid sequence (Martin et al., 2003), literature on red deer whey proteins is very limited. Although a substantial literature on fractionation of milk proteins from mainly cow but also other species is available, many of the methods are more suitable for laboratory analytical purposes and are not necessarily readily scalable for industry. The analysis of milk proteins by FPLC using ion exchange chromatography on Mono Q and Mono S columns and using urea in the buffer (Andrews et al., 1985), or the use of RP-HPLC using trifluoroacetic acid/acetonitrile buffers (Bordin et al., 2001) are examples of this.
To compare red deer milk proteins with those of other ruminants we initially performed a fractionation of defatted whole milk proteins from red deer, cow, sheep and goat by RP-HPLC and 1D-SDS PAGE. In order to gain further insight into the characteristics of less abundant milk proteins, proteins with higher abundance such as caseins, β-Lg and alpha-lactalbumin (α-La) were depleted. Red deer, cow, sheep and goat sweet whey was subjected to ammonium sulfate fractionation, followed by anion exchange chromatography, which are industry scalable procedures, and the sub-fractionated whey proteins were analyzed by 1D- and 2D-PAGE. Ammonium sulfate fractionation of whey proteins has been reported to be useful for enrichment of the relatively highly abundant whey protein components (Brodbeck et al., 1967) and was used in this report to achieve depletion of these two proteins from the whey protein fraction to improve comparative visualization of other less abundant whey proteins. Anion exchange chromatography is reported to be generally useful to achieve fractionation of whey proteins and we elected to use a HiTrap-Q FF cartridge, as a relatively cost-effective and scalable ion exchange procedure, to further demonstrate differences of deer whey proteins compared to other species.
Section snippets
Materials
All chemicals were obtained from Sigma Aldrich New Zealand Ltd., Auckland, New Zealand, unless otherwise stated. 1D-PAGE electrophoresis materials were from Life Technologies, Auckland, New Zealand. 2D-PAGE electrophoresis materials and ion exchange chromatography materials were from GE Healthcare, Auckland, New Zealand. Bovine milk protein standards were purchased from Sigma Aldrich New Zealand Ltd., Auckland, New Zealand.
Milk samples
Unpasteurized milk samples were collected from red deer (Cervus elaphus)
Comparison of proteins in defatted whole milk from red deer with that from cow, sheep and goat by 1D-PAGE and RP-HPLC
1D-PAGE of defatted whole milk from red deer, cow, sheep and goat (Fig. 1) reveals differences in the red deer milk protein profile compared to that of cow, sheep and goat. The red deer caseins migrated with apparent lower Mr on 1D-PAGE compared to other species, which may be due to differences in amino acid sequence. The proportion of α-La relative to β-Lg in red deer milk appears to be different compared to other species. α-La of goat milk migrates quite differently compared to the other
Conclusion
Red deer milk caseins migrate on 1D-SDS PAGE with apparent higher mobility than cow, sheep and goat caseins, although the retention profile of red deer caseins on RP-HPLC was similar to other species. The relative proportion of red deer α-La and β-Lg was different from the other species based on 1D-PAGE and the mobility of these two proteins from red deer is also quite different. From analysis by 1D-PAGE, red deer whey proteins fractionate with ammonium sulfate in a similar manner to whey from
Conflict of interest
None declared.
Acknowledgments
We thank the Lincoln University deer farm, Lincoln, New Zealand, for supplying red deer whole milk, and local farm supply of cow, sheep and goat milk. M. Ha acknowledges receipt of a University of Otago PhD Scholarship.
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