Julia K. Keppler

Characterization of the covalent binding of allyl isothiocyanate to β-lactoglobulin by fluorescence quenching, equilibrium measurement, and mass spectrometry

Reversible binding of small compounds through hydrophobic interactions or hydrogen bonding to food proteins (e.g. milk proteins) is a thoroughly researched topic. In contrast, covalent interactions are not well characterized. Here, we report a rare form of positive-cooperativity-linear binding of allyl isothiocyanate with β-lactoglobulin, resulting in the cleavage of a disulfide bond of the protein. We compared three methods (i.e. fluorescence quenching, equilibrium dialysis, and headspace–water equilibrium) to characterize the binding kinetics and investigated the molecular binding by mass spectrometry. The methodologies used were found to be comparable and reproducible in the presence of high and low ligand concentrations for fluorescence quenching and equilibrium-based methods respectively.

Influence of fermentation on glucosinolates and glucobrassicin degradation products in sauerkraut.

A systematic investigation was carried out on the influence of fermentation on glucosinolates and their degradation products from fresh raw cabbage, throughout fermentation at 20 °C and storage at 4 °C. Glucosinolates were degraded dramatically between Day 2 and 5 of fermentation and by Day 7 there was no detectable amount of glucosinolates left. Fermentation led to formation of potential bioactive compounds ascorbigen (13.0 μmol/100 g FW) and indole-3-carbinol (4.52 μmol/100g FW) with their higher concentrations from Day 5 to Day 9. However, during storage indole-3-carbinol slowly degraded to 0.68 μmol/100 g FW, while ascorbigen was relatively stable from Week 4 until Week 8 at 6.78 μmol/100 g FW. In contrast, the content of indole-3-acetonitrile decreased rapidly during fermentation from 3.6 to 0.14 μmol/100 g FW. The results imply a maximum of health beneficial compounds after fermentation (7-9 days) in contrast to raw cabbage or stored sauerkraut.

Differences in heat stability and ligand binding among β-lactoglobulin genetic variants A, B and C using 1H NMR and fluorescence quenching

The structure of β-lactoglobulin (β-LG) is well characterized, but the exact location of binding sites for retinol and (-)-epigallocatechingallate (EGCG) is still a subject of controversy. Here we report that the genetic β-LG variants A, B and C have different numbers of binding sites for retinol (almost completely incorporated into the calyx), as well as for EGCG (exclusively bound on the surface), and β-LG A with the most binding sites for EGCG, which include Tyr(20), Phe(151) and His(59). Upon heat related unfolding, new unspecific binding sites emerge, which are comparable in number and affinity for retinol and for EGCG, and in the three genetic variants A, B and C. The findings of our study provide new insights into the use of β-LG as nanotransporter.

Enrichment of enzymatically mineralized gellan gum hydrogels with phlorotannin-rich Ecklonia cava extract Seanol® to endow antibacterial properties and promote mineralization

Hydrogels offer several advantages as biomaterials for bone regeneration, including ease of incorporation of soluble substances such as mineralization-promoting enzymes and antibacterial agents. Mineralization with calcium phosphate (CaP) increases bioactivity, while antibacterial activity reduces the risk of infection. Here, gellan gum (GG) hydrogels were enriched with alkaline phosphatase (ALP) and/or Seanol(®), a seaweed extract rich in phlorotannins (brown algae-derived polyphenols), to induce mineralization with CaP and increase antibacterial activity, respectively. The sample groups were unmineralized hydrogels, denoted as GG, GG/ALP, GG/Seanol and GG/Seanol/ALP, and hydrogels incubated in mineralization medium (0.1 M calcium glycerophosphate), denoted as GG/ALP_min, GG/Seanol_min and GG/Seanol/ALP_min. Seanol(®) enhanced mineralization with CaP and also increased compressive modulus. Seanol(®) and ALP interacted in a non-covalent manner. Release of Seanol(®) occurred in a burst phase and was impeded by ALP-mediated mineralization. Groups GG/Seanol and GG/ALP/Seanol exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus. GG/Seanol/ALP_min, but not GG/Seanol_min, retained some antibacterial activity. Eluates taken from groups GG/ALP_min, GG/Seanol_min and GG/ALP/Seanol_min displayed comparable cytotoxicity towards MG-63 osteoblast-like cells. These results suggest that enrichment of hydrogel biomaterials with phlorotannin-rich extracts is a promising strategy to increase mineralizability and antibacterial activity.

β-Lactoglobulin as nanotransporter – Part II: Characterization of the covalent protein modification by allicin and diallyl disulfide

The whey protein β-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified β-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified β-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to β-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein.

Validation of a two-step quality control approach for a large-scale human urine metabolomic study conducted in seven experimental batches with LC/QTOF-MS

After his study of food science at the Rheinische Friedrich-Wilhelms University of Bonn, Tobias J Demetrowitsch obtained his doctoral degree in the research field of metabolomics at the Christian-Albrechts-University of Kiel. The present paper is part of his doctoral thesis and describes an extended strategy to evaluate and verify complex or large-scale experiments and data sets. Large-scale studies result in high sample numbers, requiring the analysis of samples in different batches. So far, the verification of such LC-MS-based metabolomics studies is difficult. Common approaches have not provided a reliable validation procedure to date. This article shows a novel verification process for a large-scale human urine study (analyzed by a LC/QToF-MS system) using a two-step validation procedure. The first step comprises a targeted approach that aims to examine and exclude statistical outliers. The second step consists of a principle component analysis, with the aim of a tight cluster of all quality controls and a second for all volunteer samples. The applied study design provides a reliable two-step validation procedure for large-scale studies and additionally contains an inhouse verification procedure.

β-Lactoglobulin as nanotransporter for allicin: Sensory properties and applicability in food

The thiosulfinate allicin is a labile, bioactive compound of garlic. In order to enrich allicin in a functional food, a delivery system which stabilises the compound and masks its intense flavour is necessary. In the present study allicin was covalently bound to the whey protein β-lactoglobulin and the incorporation of this transporter in a food matrix was tested. The sensory properties of the pure functional ingredient as well as of an enriched beverage were characterised by quantitative descriptive analysis. The concentration of volatile compounds was analysed by headspace gas chromatography-mass spectrometry. The garlic-related organoleptic properties of garlic powder were significantly improved by the binding of allicin in combination with spray drying. After purification of the modified β-lactoglobulin the garlic taste and smell were barely perceptible. β-Lactoglobulin modified with allicin provided a stable functional ingredient that can be used to enrich a broad range of food products.

β-Lactoglobulin as nanotransporter--Part I: Binding of organosulfur compounds.

The binding reaction of allicin and diallyl disulfide with β-lactoglobulin and the influence of pH value and protein denaturation on this reaction have been examined in the present study. Regardless of the structural similarity of both the organosulfur compounds, their binding behavior was significantly different. Both ligands were covalently bound by the free thiol group of the protein, whereas the affinity for allicin was significantly higher. In addition, diallyl disulfide was non-covalently bound. The binding reaction of both ligands was very sensitive to the pH value during incubation. The optimal pH range was between pH 8.0 and 9.0. Protein denaturation increased the reaction rate and reduced the number of binding sites for allicin, whereas the number of non-covalent binding sites increased for diallyl disulfide. Based on these findings, it can be proposed that the covalent modification of β-lactoglobulin functions as a specific transporter stabilizing allicin or diallyl disulfide.

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

The lipocalin β‐lactoglobulin (β‐LG) exists in different natural genetic variants—of which β‐LG A and B are predominant in bovine milk. At physiological conditions the protein dimerizes—building homodimers of β‐LG A and β‐LG B and heterodimers of β‐LG AB. Although β‐LG is one of the most intensely characterized lipocalins, the interaction behavior of ligands with hetero‐ and homodimers of β‐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 β‐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 β‐LG in the order β‐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 β‐LG and gives not only important information for β‐LG binding studies, but may also apply for polymorphisms of other self‐aggregating lipocalins. Copyright

Interaction of β-Lactoglobulin with Small Hydrophobic Ligands - Influence of Covalent AITC Modification on β-LG Tryptic Cleavage

Covalent modification of proteins with bioactive organosulfur compounds is suggested to reduce the smell and increase the stability of the compound. Aside from these effects the covalent modification may also alter the physiochemical properties of the protein. In this study, the whey protein β-lactoglobulin (β-LG) was covalently modified with bioactive organosulfur compound allyl isothiocyanate (AITC), originating from cabbage. Native and AITC modified β-LG were subjected to tryptic and chymotryptic digestion to assess the influence of the covalent modification on peptide formation. AITC was shown to modify at least 13 different amino acid residues, containing thiol- or amino groups, in a concentration dependent manner. Therefore, AITC modification can be controlled to some extent. Besides cysteine thiols, the most accessible amino groups for AITC modification were found at the N-terminal end of the protein (residues L1 and K8) along with lysine residues K91, K77 and K83. Higher amount of AITC addition resulted in a significant blocking of several tryptic cleavage sites of β-LG (for example residue K14) resulting in longer peptides, influencing the concentration of certain bioactive peptides following tryptic cleavage.