A novel ultra-high performance liquid chromatography method for the rapid determination of β-lactoglobulin as heat load indicator in commercial milk samples

Highlights

• The application of UHPLC for the determination of β-lactoglobulin in milk is proposed.

• The level of acid-soluble β-lactoglobulin was used to assess the applied heat load.

• Commercial liquid milk samples produced with different technologies were analyzed.

• β-Lactoglobulin amounts varied greatly within extended shelf life (ESL) milk samples.

Abstract

The level of undenatured acid-soluble β-lactoglobulin can be used as an indicator to assess the heat load applied to liquid milk, thus further allowing the discrimination between milk originating from different thermal production processes. In this work, a new UHPLC method for the rapid determination of bovine β-lactoglobulin in 1.8 min only (total runtime 3 min) is presented using simple UV detection at 205 nm. Separation selectivity for possibly co-eluting other major whey proteins (bovine serum albumin, lactoferrin, α-lactalbumin, immunoglobulin G) was verified, and the method validated for the analysis of liquid milk samples regarding linearity (20–560 μg/mL, R² > 0.99), instrumentation precision (RSDs < 2.8%), limits of detection and quantification (7 and 23 mg/L milk), repeatability of sample work-up (RSDs ≤ 2.6%) and method recovery (103%). In total, 71 commercial liquid milk samples produced using different preservation techniques (e.g., thermal or mechanical treatment), hence featuring different applied heat loads, were profiled for their intrinsic undenatured acid-soluble β-lactoglobulin levels. As expected, pasteurized milk showed the highest concentrations clearly above 3000 mg/L due to pasteurization being the mildest thermal treatment, while in contrast, ultra-high temperature heated milk featured the lowest amounts (<200 mg/L). For extended shelf life (ESL) milk, quite diverse levels were determined ranging from ∼100 up to 4000 mg/L, thus clearly illustrating variable applied heat loads and impacts on the “nativeness” of milk essentially due to the fact that the production technologies used for ESL milk may differ significantly, and are currently not regulated in the EU.

Introduction

In recent years, extended shelf life (ESL) milk, intended for closing the gap between traditionally pasteurized and ultra-high temperature treated (UHT) milk, has gained widespread acceptance in many countries, e.g., Austria and Germany [1], [2]. This new category of liquid milk fulfills the customer's requirements for more convenience due to a longer storage ability (21–24 days under chilled conditions) compared to pasteurized milk (10 days), simultaneously maintaining the fresh quality and non-cooked flavor [2], [3]. Nevertheless, the term “ESL” and its production processes are not legally defined within the European Union. Currently, different technological processes are available to produce ESL milk, including thermal processes (e.g., HTST – high temperature short time – ESL milk, using direct or indirect heat treatment and short heating times at temperatures above 120 °C) or a combination of non-thermal/mechanical and “milder” thermal processes (e.g., microfiltration followed by conventional pasteurization) [2], [4].

As more intensive heat treatment will always lead to stronger organoleptic and nutritional changes, the measurement of specific components extrinsic or intrinsic to milk (time temperature integrators, TTIs) offer the possibility for quantitative estimation of the heat load impact without the knowledge of the previous thermal history [5], [6]. Type-I TTI reactions include the denaturation, degradation or inactivation processes of heat labile components such as enzymes, vitamins and whey proteins like β-lactoglobulin (β-Lg) and are most suitable to indicate low-heat treatments. In contrast, type-II TTI reactions describe the formation of new substances that are (almost) not present in milk; these indicators are more effective for the assessment of high-temperature processes (e.g., hydroxy-methylfurfural, lactulose or furosine) [1], [7].

β-Lactoglobulin is the most abundant whey protein in milk with an average relative percentage of 55% of whey proteins (that accounts for ∼20% of total protein content). Denaturation occurs either with increasing temperatures above 60 °C, due to high calcium concentrations or at pH values above 8.6 [8], [9]. Thus, the remaining concentration of undenatured acid-soluble β-Lg is suitable to function as TTI and to distinguish between pasteurized milk and UHT milk. The International Dairy Federation (IDF) proposed a minimum content of 2600 mg β-Lg/L for pasteurized, 2000 mg/L for high-pasteurized, and 50 mg/L for UHT milk, respectively [1]. However, these recommendations are not obligatory and are rather of general, more informative nature. Similarly, the Austrian food codex currently (since 2011) suggests a threshold level of minimum 1800 mg β-Lg/L for liquid ESL milk, however, a transition period till 2018 is granted if indirect heating processes for ESL milk production are still in use [10].

Analytical determination methods include gel electrophoresis [1], capillary electrophoresis [11], immunochemical methods [12] or liquid chromatography [13], [14], whereas reversed-phase HPLC in combination with simple UV detection is proposed by the IDF as the method-of-choice to determine undenatured acid-soluble β-Lg in liquid milk [15]. Additionally, reversed-phase HPLC/UV methods have already been used to evaluate the heat-induced denaturation/changes in the profile of major bovine whey proteins including the application on different thermally treated liquid milk samples [16], [17].

Besides such “conventional” HPLC methods, also ultra-high performance/pressure liquid chromatography (UHPLC) and MS detection was used to determine the major bovine whey proteins (α-lactalbumin, β-Lg genetic variants A + B) in infant formulae offering the possibility for enhanced analysis speed (net separation time under 6 min) and lower detection limits [18]. Thus, given the continuous demand for faster chromatographic methods and the possibilities offered by the sub-2 μm particle/UHPLC approach to increase LC performance, the major objective of this study was to adapt the IDF HPLC method onto UHPLC conditions in order to facilitate the rapid, high-throughput LC analysis of β-Lg in liquid milk, to estimate the applied heat load, and to differentiate between different thermally treated milk samples.