Physicochemical and Sensory Characterization of Whey Protein-Enriched Semihard Cheese.

In view of potential future changes of German food legislation with regard to cheese product quality parameters, this study aimed to evaluate the quality of whey protein-enriched semihard cheese (WPEC). Model WPEC was produced in a pilot plant and on an industrial scale by adding defined amounts of high-heat (HH) milk to the cheese milk and comprehensively analyzed during cheese processing. The dry matter, total protein, pure protein, fat, and sodium chloride content of six-week ripened cheese samples were not significantly different (p < 0.05) when the technologically necessary heating of the curd was adapted to the amount of HH milk. However, the ripening, firmness, and melting behavior of WPEC was different compared to cheese without HH milk. During ripening, no formation of whey protein peptides was observed, but differences in the amount of some bitter peptides deriving from the casein fraction were found. Sensory data suggested a slightly more bitter taste perception by the panelists for the WPEC. Further technological adjustments are recommended to obtain marketable WPEC.

Identification of Marker Peptides for the Whey Protein Quantification in Edam-Type Cheese.

Several technologies are available for incorporating whey proteins into a cheese matrix. However, there is no valid analytical method available to determine the whey protein content in matured cheese, to date. Consequently, the aim of the present study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of individual whey proteins based on specific marker peptides ('bottom-up' proteomic approach). Therefore, the whey protein-enriched model of the Edam-type cheese was produced in a pilot plant and on an industrial scale. Tryptic hydrolysis experiments were performed to evaluate the suitability of identified potential marker peptides (PMPs) for α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG). Based on the findings, α-LA and ß-LG appeared to be resistant to proteolytic degradation during six weeks of ripening and no influence on the PMP was observed. Good levels of linearity (R2 > 0.9714), repeatability (CVs < 5%), and recovery rate (80% to 120%) were determined for most PMPs. However, absolute quantification with external peptide and protein standards revealed differences in model cheese depending on the PMP, e.g., 0.50% ± 0.02% to 5.31% ± 0.25% for ß-LG. As protein spiking prior to hydrolysis revealed differing digestion behavior of whey proteins, further studies are required to enable valid quantification in various cheese types.

High-Performance Thin-Layer Chromatography-Immunostaining as a Technique for the Characterization of Whey Protein Enrichment in Edam Cheese.

Whey protein-enriched cheese can be produced by means of a high-temperature treatment of a part of the cheese milk. In this way, the nutritional quality of the resulting cheeses can be increased while resources are conserved. High-performance thin-layer chromatography-immunostaining (HPTLC-IS) using specific ß-lactoglobulin (ß-LG) antibodies was applied to study the implementation and stability of ß-LG in two different sample sets of whey protein-enriched Edam model cheeses, including industrial-scale ones. Two methods were compared for the extraction of the proteins/peptides from the cheese samples. By applying tryptic hydrolysis directly from a suspended cheese sample instead of a supernatant of a centrifuged suspension, a better yield was obtained for the extraction of ß-LG. When applying this method, it was found that selected epitopes in the tryptic ß-LG peptides remain stable over the ripening period of the cheese. For four of the tryptic ß-LG peptides detected by immunostaining, the amino acid sequence was identified using MALDI-TOF-MS/MS. One of the peptides identified was the semi-tryptic peptide VYVEELKPTP. A linear relationship was found between the content of this peptide in cheese and the proportion of high-heated milk in the cheese milk. ß-LG enrichment factors of 1.72 (n = 3, sample set I) and 1.33 ± 0.19 (n = 1, sample set II) were determined for the cheese samples containing 30% high-heated milk compared to the non-enriched samples. The relative ß-LG contents in the cheese samples with 30% high-heated milk were calculated to be 4.35% ± 0.39% (sample set I) and 9.11% ± 0.29% (sample set II) using a one-point calibration. It can be concluded that the HPTLC-IS method used is a suitable tool for the analysis of whey protein accumulation in cheese, being therefore potentially directly applicable on an industrial scale. For more accurate quantification of the whey protein content in cheese, an enhanced calibration curve needs to be applied.

Two-dimensional high-performance thin-layer chromatography for the characterization of milk peptide properties and a prediction of the retention behavior - a proof-of-principle study.

High-performance thin-layer chromatography (HPTLC) is a suitable method for the analysis of peptides and proteins due to a wide selection of stationary and mobile phases and various detection options. Especially, two-dimensional HPTLC (2D-HPTLC) enables a higher resolution compared to one-dimensional HPTLC in the separation of complex peptide mixtures. Similar to 2D electrophoresis, characteristic peptide patterns can be obtained, allowing a differentiation of ingredients based on varying protein origins. The aim of this study was to evaluate 2D-HPTLC with regard to its suitability for the characterization of proteins/peptides and to verify whether it is possible to predict the retention behavior of peptides based on their properties. As models, the five most abundant milk proteins α-lactalbumin, ß-lactoglobulin, α-, ß-, and κ-Casein were used. In order to determine the repeatability of the peptide separation by 2D-HPTLC, each tryptic protein hydrolyzate was separated eight times. The standard deviations of the retardation factors for the separated peptides varied between 1.0 and 11.1 mm for the x-coordinate and 0.5-7.3 mm for the y-coordinate. It was also shown that after the chromatographic separation, peptides of the individual protein hydrolyzates were located in specific areas on the HPTLC plate, so that a clustering could be obtained for the whey proteins' as well as the caseins' hydrolyzates. For establishing correlations between the properties of the peptides and their retardation factors, 51 of 85 selected peptides were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). On this basis, statistically significant correlations (α = 0.05) between the retardation factors of the peptides and their isoelectric points, as well as the percentage of anionic and non-polar amino acids in the peptides were established. Finally, it was investigated, whether the retardation factors for peptides can be predicted on the basis of a linear regression of the percentage of non-polar amino acids in a peptide. For this purpose, a mixture of artifical (synthetic) peptides (n = 14) was separated by 2D-HPTLC and the measured retardation factors were compared with the corresponding retardation factors calculated. Absolute deviations of 0.3-17.9 mm were obtained. In addition, the universal applicability of the method to other protein sources other than milk proteins (animal protein) was tested using a mixture of pea peptides (plant protein, n = 3) resulting in absolute deviations of 0.7-8.6 mm.

In Vitro Determination of Protein Conjugates in Human Cells by LC-ESI-MS/MS after Benzyl Isothiocyanate Exposure.

Glucosinolates and their breakdown products, especially isothiocyanates (ITCs), are hypothesized to exert a broad range of bioactivities. However, physiological mechanisms are not yet completely understood. In this study, formation of protein conjugates after incubation with benzyl isothiocyanate (BITC) was investigated in vitro. A survey of protein conjugates was done by determining BITC cysteine and lysine amino acid conjugates after protein digestion. Therefore, a liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated. Stability studies showed that cysteine conjugates are not stable under alkaline conditions, and lysine conjugates did not show any correlation to pH values, although stability increased at low temperatures. Lysine conjugates were the preferred form of protein conjugates, and longer BITC exposure times led to higher amounts. Knowledge about the reaction sites of ITCs in eukaryotic cells may help to understand the mode of action of ITCs leading to health promoting as well as toxicological effects in humans.

Determination of isothiocyanate-protein conjugates in milk and curd after adding garden cress (Lepidium sativum L.).

Isothiocyanates (ITC) play an important role in health promotion and cancer prevention due to their anti-bacterial, anti-inflammatory, and anti-cancerogenic properties. However, ITC are highly reactive so that a reaction with further food components is very likely. For example, a reaction of ITC with nucleophilic amino acid side chains of proteins such as cysteine and lysine can occur, reducing the bioavailability of indispensable amino acids and protein functions may be altered. Therefore, it is of great interest to investigate the fate of ITC in the food matrix. Accordingly, the aim of the present study was to investigate the interaction of milk proteins and the ITC benzyl isothiocyanate (BITC) and allyl isothiocyanate (AITC) forming dithiocarbamates and thioureas in milk and curd. After incubating milk and curd with pure ITC or ITC-containing garden cress (Lepidium sativum L.), proteins were isolated, digested, and analyzed via LC-ESI-MS/MS as amino acid derivatives ("conjugates"). Protein conjugates of AITC and BITC were detected in all samples investigated. Further, the acidic pH value in curd favored the formation of dithiocarbamates over the formation of thioureas. Slightly acidic or neutral conditions like in fresh milk favored the formation of thioureas. The investigations also indicated that AITC shows a higher reactivity and dithiocarbamates are formed preferably, whereas incubation with BITC lead to less protein conjugates and the ratio of thioureas and dithiocarbamates was more balanced. In addition, amino acid modifications were often analyzed with indirect methods like measuring the decline of the amino acid residues. In this study, the modified amino acids were analyzed directly leading to more reliable results concerning the amount of modification.