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BackgroundWheat is one of the most common food allergens in early childhood. In contrast to other food allergies, wheat-specific IgE correlates badly with clinical symptoms and relevant components have been identified mostly for wheat-depended exercise-induced anaphylaxis. Moreover, a high percentage of patients present with immediate type symptoms but wheat-specific IgE cannot be detected with commercial available systems. ObjectiveWe addressed the question whether the IgE recognition pattern between wheat allergic (WA) and clinically tolerant (WT) children differs in order to identify individual proteins useful for component-resolved diagnostics. MethodsSera of 106 children with suspected wheat allergy, of whom 44 children had clinical relevant wheat allergy and 62 were tolerant upon oral food challenge, were analyzed for wheat-specific IgE using the ImmunoCap system as well as immunoblots against water and salt soluble, and water-insoluble protein fractions. 40 randomly selected sera were analyzed for specific IgE to 5-gliadin. ResultsSixty-three percent of the WT and 86% of the WA children were sensitized to wheat with >0.35 kU(A)/l in ImmunoCAP analysis. We could confirm the role of -, ss-, -, and -gliadins, and LMW glutenin subunits as major allergens and found also IgE binding to a broad spectrum of water- and salt-soluble protein bands. It is of great importance that wheat allergic and tolerant patients showed IgE binding to the same protein bands. WT and WA did not significantly differ in levels of 5-gliadin-specific IgE. Conclusions & Clinical RelevanceChildren with challenge proven clinical relevant food allergy and tolerant ones had a similar spectrum of IgE binding to the same protein bands. These findings imply that component-resolved diagnostics might not be helpful in the diagnostic work-up of wheat allergy.
Investigations on the post mortal formation of fluorescent zinc protoporphyrin (ZnPP) IX in pork meat are currently in focus of meat science research. The role of myoglobin degradation in this context appears to be one of the most diversely discussed issues. To address this question meat-extracts of longissimus lumborum (LL) muscle (0.8 mg/mL) were incubated at 30 degrees C for up to 72 h and investigated by HPSEC-UV-fluorescence, SDS-PAGE and MALDI-TOF-MS. Between 0 and 72 h of incubation the fluorescence intensity (lambda(ex)./(em). = 420/590 nm) of the meat-extracts rose significantly (p < 0.001) from 10.9 +/- 0.8 to 34.8 +/- 0.3 (rel. units) while the staining intensity of the SDS-PAGE of myoglobin non-significantly (p > 0.4) changed from 6.2 +/- 0.5 x 105 to 5.0 +/- 0.3 x 105 (rel. units). The results indicate that ZnPP is formed by a Fe(II)-Zn(II)-substitution in myoglobin heme where an accompanying myoglobin degradation is not necessarily obligatory. (C) 2017 Elsevier Ltd. All rights reserved.
The effect of tannins on mediterranean ruminant ingestive behavior the role of the oral cavity
(2011)
Sheep, cattle and goat are domestic ruminants of significant economic interest in the Mediterranean region. Although sharing the same pasture ranges, they ingest different plants and plant parts and, consequently different levels of tannins. This suggests an ability to detect and adapt ingestion according to animal physiological limits of tolerance for plant secondary metabolites. This review will detail the effects of dietary tannins on feeding behavior, and the role of the oral cavity in this process, with focus on such ruminant species. The role of salivary protein profile in tannin perception in the oral cavity, and as a defense mechanism, will be discussed.
The application of technical enzymes is a potential tool in modulating the dough and baking quality of cereal products. No endogenous amylases (alpha- and beta-forms) are present in mature wheat grains; they may be synthesized or activated during germination. Hence, microbial alpha-amylases are added to the dough, being resistant to the endogenous alpha-amylase/trypsin inhibitors. Here, we report on the initial identification of two technical enzymes from a commercial sample based on an in-gel tryptic digestion coupled with MALDI-MS analysis. The primary component of the protein fraction with 51.3 kDa was alpha-amylase from Aspergillus species. A second major protein with 24.8 kDa was identified as endo-1,4-xylanase from Thermomyces lanuginosus. In the following experimental work up, a targeted proteomics approach utilizing the combination of specific proteolytic digestion of the added amylase and xylanase in wheat flour, dough or baked products, solid phase extraction of released peptides and their detection using LC-MS/MS was optimized. The targeted (MRM) MS/MS peptide signals showed that the peptide "ALSSALHER" (MW = 983) originating from amylase and "GWNPGLNAR" (MW = 983) from xylanase can be used to identify the corresponding technical enzymes added. Consequently, locally available baked products were tested and found to contain these enzymes as supplementary ingredients. (C) 2014 Elsevier Ltd. All rights reserved.
The activity of microbial transglutaminase (MTG) and the corresponding secondary structure, measured by circular dichroism (CID), was analyzed before and after treatment at different temperatures (40 and 80 degrees C) and pressures (0.1, 200, 400, 600 MPa). Irreversible enzyme inactivation was achieved after 2 min at 80 degrees C and 0.1 MPa. Enzyme inactivation at 0.1, 200, 400, and 600 MPa and 40 degrees C followed first-order kinetics. The enzyme showed residual activity of 50% after 12 min at 600 MPa and 40 degrees C. Mobility of aromatic side chains of the enzyme molecule was observed in all temperature- and/or pressure-treated samples; however, high-pressure treatment at 600 MPa induced a loss of tertiary structure and a significant decrease in the alpha-helix content. The relative content of beta- strand substructures was significantly increased after 30 min at 600 MPa and 40 degrees C or 2 min at 0.1 MPa and 80 degrees C. We conclude that the active center of MTG, which is located in an expanded 8-strand domain, is resistant to high hydrostatic pressure and pressure-induced inactivation is caused by destruction of cc-helix elements with a corresponding influence on the enzyme stability in solution
Structural changes induced in bovine serum albumin by covalent attachment of chlorogenic acid
(2002)
Bovine serum albumin (BSA) was modifed by covalent attachment of chlorogenic acid using different concentrations at pH 9. The derivatization was accompanied by a reduction of lysine, cysteine and tryptophan residues. The isoelectric points were shifted to lower pH values and formation of high molecular weight fractions was noted. The structural changes were studied using circular dichroism, differential scanning calorimetry (DSC), intrinsic fluorescence, and binding of anilinonaphthalenesulfonic acid. The results showed that the content of alpha-helix decreased with a parallel increase in unordered structures with higher degrees of derivatization. DSC revealed a decrease in both denaturation temperature and enthalpy. Surface hydrophobicity declined, indicating that hydrophilic regions were exposed on the molecular surface. Proteolytic digestion showed that, at a lower degree of derivatization,the tryptic degradation was most adversely effected, whereas the peptic digestion declined with increasing modification. A trypsin inhibitory effect of the breakdown products released from derivatized BSA was also observed.
The carotenoid lutein can improve human health. Since only a fraction is absorbed from food, lutein supplementation might be recommended. Emulsions could be good carrier systems to improve the bioavailability of lutein. Six different emulsifier compositions were used in this study to prepare lutein-loaded emulsions: beta-lactoglobulin, beta-lactoglobulin/lecithin, Biozate 1, Biozate 1/lecithin, Been 20 and Tween 20/lecithin. The droplet size, resistance to creaming, lutein stability, cytotoxicity and lutein uptake by HT29 cells were investigated. The whey protein beta-lactoglobulin, the whey protein hydrolysate Biozate 1 and the combination with lecithin brought the most promising results. The small droplet sizes and resistance to creaming were an indication of physical stable emulsions. Furthermore, these emulsifiers prevented oxidation of lutein. The choice of emulsifier had a strong impact on the uptake by HT29 cells. The highest lutein absorption was observed with the combination of Biozate 1 and lecithin.
Tagetes spp. belongs to the Asteraceae family. It is recognized as a major source of lutein ester (lutein esterified with fatty acids such as lauric, myristic and palmitic acids), a natural colorant belonging to the xanthophylls or oxygenated carotenoids. Four species of Tagetes flower (Tagetes tenuifolia, Tagetes erecta, Tagetes patula, and Tagetes lucida) were used to extract lutein and lutein esters with three different methods. The results showed that T. erecta, type "orangeprinz", is the richest source of lutein esters (14.4 +/- 0.234 mg/g) in comparison to other Tagetes spp. No significant differences between extractions of lutein esters with medium-chain triacylglycerols (MCT) oil, orange oil or solvent (hexane/isopropanol) could be observed. MCT oil also improved stability of lutein esters at 100 degrees C for 40 min. Emulsification of MCT oil improved the stability of lutein ester extract against UV light at 365 nm for 72 h. Finally, an emulsion was prepared under food processing conditions, spray dried and its bioavailability investigated in a preliminary human intervention study. The results show a lower resorption, but further data suggest improvements in implementation of such supplements. (c) 2012 Elsevier Ltd. All rights reserved.
Background: A promising way in diagnostic and therapeutic applications is the development of peptide amphiphiles (PAs). Peptides with a palmitic acid alkylchain were designed and characterized to study the effect of the structure modifications on self-assembling capabilities and the multiple binding capacity to hemagglutinin (HA), the surface protein of influenza virus type A. The peptide amphiphiles consists of a hydrophilic headgroup with a biological functionality of the peptide sequence and a chemically conjugated hydrophobic tail. In solution they self-assemble easily to micelles with a hydrophobic core surrounded by a closely packed peptide-shell.
Results: In this study the effect of a multiple peptide binding partner to the receptor binding site of HA could be determined with surface plasmon resonance measurements. The applied modification of the peptides causes signal amplification in relationship to the unmodified peptide wherein the high constant specificity persists. The molecular assembly of the peptides was characterized by the determination of critical micelle concentration (CMC) with concentration of 10(-5) M and the colloidal size distribution.
Conclusion: The modification of the physico-chemical parameters by producing peptide amphiphiles form monomeric structures which enhances the binding affinity and allows a better examination of the interaction with the virus surface protein hemagglutinin.
Many biochemical processes are involved in regulating the consecutive transition of different phases of dormancy in sweet cherry buds. An evaluation based on a metabolic approach has, as yet, only been partly addressed. The aim of this work, therefore, was to determine which plant metabolites could serve as biomarkers for the different transitions in sweet cherry buds. The focus here was on those metabolites involved in oxidation-reduction processes during bud dormancy, as determined by targeted and untargeted mass spectrometry-based methods. The metabolites addressed included phenolic compounds, ascorbate/dehydroascorbate, reducing sugars, carotenoids and chlorophylls. The results demonstrate that the content of phenolic compounds decrease until the end of endodormancy. After a long period of constancy until the end of ecodormancy, a final phase of further decrease followed up to the phenophase open cluster. The main phenolic compounds were caffeoylquinic acids, coumaroylquinic acids and catechins, as well as quercetin and kaempferol derivatives. The data also support the protective role of ascorbate and glutathione in the para- and endodormancy phases. Consistent trends in the content of reducing sugars can be elucidated for the different phenophases of dormancy, too. The untargeted approach with principle component analysis (PCA) clearly differentiates the different timings of dormancy giving further valuable information.
Many biochemical processes are involved in regulating the consecutive transition of different phases of dormancy in sweet cherry buds. An evaluation based on a metabolic approach has, as yet, only been partly addressed. The aim of this work, therefore, was to determine which plant metabolites could serve as biomarkers for the different transitions in sweet cherry buds. The focus here was on those metabolites involved in oxidation-reduction processes during bud dormancy, as determined by targeted and untargeted mass spectrometry-based methods. The metabolites addressed included phenolic compounds, ascorbate/dehydroascorbate, reducing sugars, carotenoids and chlorophylls. The results demonstrate that the content of phenolic compounds decrease until the end of endodormancy. After a long period of constancy until the end of ecodormancy, a final phase of further decrease followed up to the phenophase open cluster. The main phenolic compounds were caffeoylquinic acids, coumaroylquinic acids and catechins, as well as quercetin and kaempferol derivatives. The data also support the protective role of ascorbate and glutathione in the para- and endodormancy phases. Consistent trends in the content of reducing sugars can be elucidated for the different phenophases of dormancy, too. The untargeted approach with principle component analysis (PCA) clearly differentiates the different timings of dormancy giving further valuable information.
The transition from dormant stage to the beginning of growth was first obvious by markedly changes of the water content. The phase from green tip to tight cluster, with a length of only 4 days, was the period of the most physiological activity in single buds, because of the highest daily accumulation rates of fresh/dry weight, C, N. We assume a concentration dependant regulation of the member of the aspartate family (asparagine, aspartic acid, isoleucine) during dormancy, growth and development in sweet cherry buds. The ABA content showed 2011/12 a clear bimodal pattern which was at lower level similar in 2012/13, but not so strong incisive. In both years, the first peak was probably related to the end of endodormancy. However the ABA-isomer content showed in both seasons a unimodal pattern. The maximum of the ratio of ABA-isomer/ABA indicated the beginning of ontogenetic development which starts 3 and 2 weeks later, respectively. Our results suggest that ABA and the ABA-isomer in the sweet cherry buds regulate differentiated metabolic processes in the dormant stage and during bud growth and development. After replication in the season 2013/14 the estimated dates of release of endodormancy, beginning of ecodormancy and start of ontogenetic development will be used to validate and improve phenological models for the beginning of cherry blossom. (C) 2014 Elsevier B.V. All rights reserved.
Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC–MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.
Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC–MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.
Bromelain was allowed to react with phenolic compounds. The activity and selected physico-chemical properties of the resulting derivatives were characterized. In vitro experiments showed that the proteolytic activity of bromelain was inhibited. Bromelain also serves as a food protein, because food stuffs based on pineapple contain relatively high concentrations of bromelain. In vitro digestion of bromelain derivatives with the main proteolytic enzymes of the gastrointestinal tract was also adversely affected. A covalent attachment of the phenolic compounds was identified at the tryptophan, free amino (lysines and N-terminal) and thiol groups of bromelain. A decrease in solubility of the derivatives was observed. The isoelectric point was shifted to lower pH values and high molecular weight fractions were identified. All effects observed depended on the reactivity of the phenolic substances. Two supplementary food products containing both bromelain and quercetin were also tested in terms of their proteolytic activity and digestibility