RP-HPLC analysis of furosine and acid-soluble β-lactoglobulin to assess the heat load of extended shelf life milk samples in Austria

Abstract

-Recent trends to extend the shelf life of pasteurized milk, without the negative flavour normally associated with ultra-high-temperature (UHT) milk, have resulted in the development of extended shelf life (ESL) milk. Acid-soluble β-lactoglobulin (β-Lg) and furosine contents were chosen as relevant indicators for heat load of ESL milk products. RP-HPLC methods were developed using the same column (Symmetry 300™, Waters), which enabled the separation of whey proteins within 22 min; furosine was analysed using ion-pair RP-HPLC within 8 min. Electrophoresis was used as a high-throughput and cost-effective screening method to assess the impact of thermal processes on milk and to distinguish different categories of heat-treated milk samples. Liquid milk samples (n = 128; including 7 raw, 33 pasteurized, 71 ESL, and 17 UHT milk samples) were obtained from retail outlets in Austria and analysed. Only 45% of the analysed samples designated as ESL milk showed furosine contents < 40 mg·100 g −1 protein as well as acid-soluble β-Lg contents > 1800 mg·L −1 milk, which had been discussed as threshold levels for ESL milk. A further 55% of the analysed ESL milk samples had low acid-soluble β-Lg (< 500 mg·L −1 ) and high furosine contents (> 40 mg·100 g −1 protein), levels comparable to the excessive heat load of UHT milk. Thus, there is an urgent need for an EU regulation to define legal limits for the tolerable heat load of ESL milk as soon as possible.extended shelf life milk / β-lactoglobulin / furosine / RP-HPLC / electrophoresis Article published by EDP Sciences Résumé -Analyse de la furosine et de la β-lactoglobuline soluble à pH acide par RP-HPLC pour évaluer la charge thermique de laits de longue conservation en Autriche. La tendance actuelle visant à allonger la durée de conservation du lait pasteurisé, sans avoir les défauts de flaveur habituellement associés au lait UHT, a conduit au développement du lait de longue conservation ESL. Les concentrations en β-lactoglobuline soluble à pH acide (β-Lg) et en furosine ont été choisies comme indicateurs pertinents de la charge thermique des produits laitiers ESL. Des méthodes RP-HPLC ont été développées avec la même colonne (Symmetry 300™, Waters), qui a permis de séparer les protéines sériques en 22 min ; la furosine était analysée par RP-HPLC en paire d'ions en 8 min. L'électrophorèse a été utilisée comme méthode économique de criblage à haut débit pour évaluer l'impact des traitements thermiques sur le lait et pour distinguer différentes catégories d'échantillons de lait traité thermiquement. Les échantillons de lait liquide (n = 128 ; comprenant 7 échantillons de lait cru, 33 pasteurisé, 71 ESL et 17 UHT) ont été prélevés dans des points de vente en Autriche et analysés pour leur teneur en β-lactoglobuline et furosine. Seulement 45 % des échantillons analysés désignés comme lait ESL présentaient des teneurs en furosine inférieures à 40 mg·100 g −1 de protéine et des teneurs en β-lactoglobuline soluble à pH acide supérieures à 1800 mg·L, valeurs qui ont été disc...

Pilot-scale purification of α-lactalbumin from enriched whey protein concentrate by anion-exchange chromatography and ultrafiltration

“…4a) as any remaining casein and other whey proteins had been denatured during heat treatment in the production of αWPC and were removed by sedimentation upon adjusting the pH to 4.6. Mayer et al (2010) and Tolkach et al (2005) applying a similar method to treat the initial whey protein solution also achieved removal of bovine serum albumin (BSA) and immunoglobulin. The minor whey proteins, especially BSA and immunoglobulin, like β-Lg, both contain free thiol groups, and irreversible denaturation of these already starts at 60°C (Vermeer and Norde 2000;Boye et al 1996).…”

High-throughput quantitation of bovine milk proteins and discrimination of commercial milk types by external cavity-quantum cascade laser spectroscopy and chemometrics

“…Reversed phasehigh performance liquid chromatography (RP-HPLC) analysis of α-LA and β-LG Sample preparation for the determination of α-LA and β-LG as well as RP-HPLC analysis were carried out according to the corresponding IDF/ISO standard 40 and as recently described in the literature. 8,11 Briefly, caseins and denatured whey proteins were precipitated at pH 4.6 by dropwise addition of HCl (2 mol L −1 ). Acid whey containing the acid-soluble whey proteins was separated by centrifugation and diluted (1 : 10, or 1 : 5 in the case of UHT milk) with sodium phosphate buffer solution (0.1 mol L −1 , pH 6.7).…”

“…10 The International Dairy Federation (IDF) suggests a minimum β-LG content of 2.6 g L −1 for pasteurized milk, 2.0 g L −1 for high-pasteurized (ESL) milk, and 0.05 g L −1 for UHT milk. 11 For liquid ESL milk, these limits are not obligatory, in contrast to pasteurized/UHT milk. In Austria, however, since July 2018 a minimum β-LG content of 1.8 g L −1 has been introduced for ESL milk in order to minimize the actual heat load of this upcoming milk type.…”

“…Nowadays, the traditional Kjeldahl method for determination of organic nitrogen in food and beverages continues to be the employed standard analytical technique for quantification of total protein content in milk, even though it is a fairly labour-intensive wet-chemical approach with low throughput. For quantitation of individual milk proteins including Cas, α-LA and β-LG, diverse techniques based on chromatographic [11][12][13] and electrophoretic 13 methods can be employed, all of them involving time-consuming, wet-chemical sample preparation steps. Mid-infrared (IR) spectroscopy has been applied for rapid and non-destructive analysis of quality and composition of dairy products due to its high-throughput capacity, simplicity and low cost.…”

From DNA to Emerging Technologies: Innovative Quality Markers

“…Although there are no maximum limits, excessive treatments give rise to some molecules (reaction by-products) which are useful markers to estimate thermal damages. Furosine, lactulose, and galactosyl-β-pyranone are among the most suitable molecules investigated to evaluate the severity of the heat treatment [12]. Similarly, these markers may also detect frauds such as the addition of reconstituting powder milk to UHT milk, analyzing the furosine/ lactulose ratio [13].…”