HPLC Methods with Different Detection Types Determine Whey Protein Content in Cheese


High performance liquid chromatography (HPLC) was paired with both diode array and fluorescence detection (DAD and FLD, respectively) to measure Edam cheese for its total and acid-soluble whey content.

A study published in 2023 in the journal LWT—Food Science and Technology brought together 11 researchers across Germany: from Technische Universität Berlin, the University of Hamburg, DMK Group in Bremen, and the Federal Research Institute of Nutrition and Food in Kiel, to collectively report on the quantitation of whey protein in cheese using high performance liquid chromatography (HPLC) with either ultraviolet (UV) or fluorescence detection (FLD) (1). The study generally refers to the former method as HPLC-UV but specifies that a diode array detector (DAD) was used as part of the experiment.

Edam cheese on a cutting board | Image Credit: © Picture Partners - stock.adobe.com

Edam cheese on a cutting board | Image Credit: © Picture Partners - stock.adobe.com

Because whey, a traditional cheese byproduct, has a high nutritional content, according to these authors, it is being increasingly used in foodstuffs like beverages and instant powders. But in Germany, the fortification of traditional cheese with whey proteins is not yet being permitted. So the researchers set out to detect both total and acid-soluble whey protein (TWP and ASWP, respectively) content already present in cheese, in this case a foil-ripened, Edam-type cheese made in laboratory.

The researchers produced nine standard cheeses and nine whey protein-enriched cheeses; of the whey protein-enriched cheeses, amounts of 10%, 20%, and 30% high-heat milk had been added to the pasteurized cheese milk, then tested three times after a ripening period of six weeks at 80% humidity (1). Throughout this ripening process, the authors said they monitored TWP and ASWP content to measure the influence of proteolysis on both native and denatured whey proteins (TWP representing the sum of these two protein types).

This study did not attempt to determine which HPLC method was superior between the diode array or fluorescence detector, but did report that both approaches showed that the individual whey proteins α-lactalbumin and β-lactoglobulin were not degraded during ripening (1). However, ASWP content was shown to increase by up to 25% during that time.

In addition, β-lactoglobulin levels in the whey protein-enriched cheese with 30% high-heat milk increased by a factor of 3.5 compared with the standard cheese in the experiment. One significant limitation in the analysis arose regarding whey protein quantitation in more mature cheese; this was prevented by the proteolytic effects of caseines, the authors said, as well as the complex cheese matrix (1). Primary casein breakdown is known in a culinary sense by a much more familiar word, curdling, and it is just one of the many biochemical processes known to occur during cheese ripening.

To combat those matrix-related influences, the research team proposed that in future analysis, a proteomic mass spectrometry system be utilized, in part to quantify marker peptides. Not only, they said, could this help ensure accurate determination of whey protein content in Edam-type cheese, but further fine-tuning of these approaches could one day allow for informative separations of all cheese types, from hard to semihard, to soft and cream (1).


(1) von Oesen, T.; Treblin, M.; Staudacher, A.; et al. Determination and Evaluation of Whey Protein Content in Matured Cheese via Liquid Chromatography. LWT—Food. Sci. Technol. 2023, 174, 114347. DOI: 10.1016/j.lwt.2022.114347

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