Vitamin D3 formation in milk by UV treatment - Novel insights into a rediscovered process.

Vitamin D3 is essential for several functions in the human body and the demand is usually covered by natural reactions in skin with UV radiation delivered by the sun. But living beyond a latitude of 35° can lead to a lack of sufficient exposition to the deciding wavelength. Here, many countries fortify their milk prophylactically with artificial vitamin D3. However, the precursor molecule of vitamin D3 (7-deydrocholesterol) is already naturally located in the milk fat globule membrane. Thus, this study deals with the transformation of the naturally occurring 7-dehydrocholesterol into vitamin D3 through UV treatment of the milk - a mechanism that was observed a century ago only indirectly. Different parameters such as temperature (10 - 50°C), fluid flow regimen (turbulent vs. laminar thin film, i.e., 0.6 mm) and wavelength (254, 280 and 313 nm) were investigated in this study for their efficiencies. The UV dose of each experiment was measured with chemical actinometry delivering the actually applied dose reaching the milk. Thus, the connection between applied UV dose and generated vitamin D3 content in the milk measured quantitively with LC-MS/MS was evaluated here that both were not possible a hundred years ago. The experimental results revealed that temperature generally promotes the vitamin D3 formation at 254 nm. Further, a turbulent flow is not as efficiently treated as a laminar thin film flow that is as narrow as 0.6 mm. As expected from absorbance spectra of the precursor molecule 7-dehydrocholesterol, 280 nm turned out to be the most efficient wavelength, followed by intermediate success through irradiation with 254 nm and almost no effect by 313 nm. Generally, it was shown that vitamin D3 concentration of milk was easily increased by UV treatment with today's technologies and that adjustment of certain physical parameters have a significant effect on the efficiency.

Differences in binding behavior of (-)-epigallocatechin gallate to ß-lactoglobulin heterodimers (AB) compared to homodimers (A) and (B).

The lipocalin ß-lactoglobulin (ß-LG) exists in different natural genetic variants--of which ß-LG A and B are predominant in bovine milk. At physiological conditions the protein dimerizes--building homodimers of ß-LG A and ß-LG B and heterodimers of ß-LG AB. Although ß-LG is one of the most intensely characterized lipocalins, the interaction behavior of ligands with hetero- and homodimers of ß-LG is largely unknown. The present findings revealed significant differences for hetero- and homodimers regarding ligand binding capacity as tested with a model ligand (i.e. surface binding (-)-epigallocatechin gallate (EGCG)). These findings were confirmed using FT-IR, where the addition of EGCG influenced the ß-sheet backbone of homodimer A and B with significantly higher intensity compared to heterodimer AB. Further, shape analysis by SAXS revealed oligomerization of both types of dimers upon addition of EGCG; however, homodimer A and B produced significantly larger aggregates compared to the heterodimer AB. In summary, the present study revealed that EGCG showed significantly different interaction reactivity (binding sites, aggregation size and conformational changes) to the hetero and homodimers of ß-LG in the order ß-LG A > B > AB. The results suggest that conformational differences between homodimers and heterodimers strongly influence the EGCG binding ability. This may also occur with other polyphenols and ligands of ß-LG and gives not only important information for ß-LG binding studies, but may also apply for polymorphisms of other self-aggregating lipocalins.

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.

Unraveling the effects of low protein-phenol binding affinity on the structural properties of beta-lactoglobulin.

Non-covalent interactions of phenolics with proteins cannot always be readily identified, often leading to contradictory results described in the literature. This results in uncertainties as to what extent phenolics can be added to protein solutions (for example for bioactivity studies) without affecting the protein structure. Here, we clarify which tea phenolics (epigallocatechin gallate (EGCG), epicatechin and gallic acid) interact with the whey protein ß-lactoglobulin by combining various state-of-the-art-methods. STD-NMR revealed that all rings of EGCG can interact with native ß-lactoglobulin, indicating multidentate binding, as confirmed by the small angle X-ray scattering experiments. For epicatechin, unspecific interactions were found only at higher protein:epicatechin molar ratios and only with 1H NMR shift perturbation and FTIR. For gallic acid, none of the methods found evidence for an interaction with ß-lactoglobulin. Thus, gallic acid and epicatechin can be added to native BLG, for example as antioxidants without causing modification within wide concentration ranges.

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.

Uridine as a non-toxic actinometer for UV-C treatment: influence of temperature and concentration.

UV-C treatment is an effective method to inactivate microorganisms and therefore gets increasingly more attention in food industry, especially for liquid products. To test and monitor different UV-C reactor designs, a photochemical actinometer is required that gives reliable UV-C dose values and is non-toxic allowing frequent control of the production chain. Here, a variable concentrated aqueous uridine solution is tested as a photochemical actinometer. Uridine reacts at 262 nm by photohydration to a single photoproduct not absorbing any light. A concentration dependent quantum yield (Ф) was quantified in the range of 0.2-3.0 mM uridine. Results show that uridine is as accurate as the commonly accepted iodide/iodate actinometry, but not as precise. Especially at higher concentrations a higher number of measurements becomes necessary. Further, a temperature correction is presented for 10 °C > ϑ > 30 °C. Taking these results into account, uridine can certainly be considered as a non-toxic dosimeter for UV-C systems.

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.

Nucleoprotein-derived and unbound ribonucleosides: bioactivity and potential applications.

Naturally occurring and chemically modified ribonucleosides have interesting bioactive effects. Dietary ribonucleosides are ingested mainly as nucleoproteins and are converted in the course of intestinal digestion to monomeric compounds. Different bioactive effects of dietary ribonucleosides have been described, including the ability to enhance gut growth and maturation and to increase iron absorption. Cytochemical studies with human cells showed that several ribonucleosides can induce apoptosis in human cells, and therefore may be potentially anticancerogenic compounds. Even if suboptimal concentrations of single bioactive nucleo compounds are available from food, the total content of different bioactive ribonucleosides may reach physiologically effective concentrations in vivo where intestinal cells may represent the main target sites of a selective apoptotic activity. Modified ribonucleosides serve as valuable pathobiochemical marker molecules for cancer. Chemically modified ribonucleosides have already found interesting applications as pharmaceutically active compounds in the treatment of different illnesses including AIDS. Regarding therapeutic and pharmaceutical aspects, further studies are required to evaluate the bioactive efficacy of indigenous ribonucleosides. The findings demonstrate the great variety of potential applications of ribonucleosides, e.g. in functional foods as well as pharmaceutical preparations.