Comparison of analytical and predictive methods for water,protein, fat,sugar, and gross energy in marine mammal milk

Mammalian milks may differ greatly in composition from cow milk, and these differences may affect the performance of analytical methods. High-fat, high-protein milks with a preponderance of oligosaccharides, such as those produced by many marine mammals, present a particular challenge. We compared the performance of several methods against reference procedures using Weddell seal (Leptonychotes weddellii) milk of highly varied composition (by reference methods: 27–63% water, 24–62% fat, 8–12% crude protein, 0.5–1.8% sugar). A microdrying step preparatory to carbon-hydrogen-nitrogen (CHN) gas analysis slightly underestimated water content and had a higher repeatability relative standard deviation (RSD_r) than did reference oven drying at 100°C. Compared with a reference macro-Kjeldahl protein procedure, the CHN (or Dumas) combustion method had a somewhat higher RSD_r (1.56 vs. 0.60%) but correlation between methods was high (0.992), means were not different (CHN: 17.2 ± 0.46% dry matter basis; Kjeldahl 17.3 ± 0.49% dry matter basis), there were no significant proportional or constant errors, and predictive performance was high. A carbon stoichiometric procedure based on CHN analysis failed to adequately predict fat (reference: Röse-Gottlieb method) or total sugar (reference: phenol-sulfuric acid method). Gross energy content, calculated from energetic factors and results from reference methods for fat, protein, and total sugar, accurately predicted gross energy as measured by bomb calorimetry. We conclude that the CHN (Dumas) combustion method and calculation of gross energy are acceptable analytical approaches for marine mammal milk, but fat and sugar require separate analysis by appropriate analytic methods and cannot be adequately estimated by carbon stoichiometry. Some other alternative methods—low-temperature drying for water determination; Bradford, Lowry, and biuret methods for protein; the Folch and the Bligh and Dyer methods for fat; and enzymatic and reducing sugar methods for total sugar—appear likely to produce substantial error in marine mammal milks. It is important that alternative analytical methods be properly validated against a reference method before being used, especially for mammalian milks that differ greatly from cow milk in analyte characteristics and concentrations.