Enzymatic cross-linking of β-casein and its impact on digestibility and allergenicity

Protein modification via enzymatic cross-linking is an attractive way for altering food structure so as to create products with increased quality and nutritional value. These modifications are expected to affect not only the structure and physico-chemical properties of proteins but also their physiological characteristics, such as digestibility in the GI-tract and allergenicity. Protein cross-linking enzymes such as transglutaminases are currently commercially available, but also other types of cross-linking enzymes are being explored intensively. In this study, enzymatic cross-linking of β-casein, the most abundant bovine milk protein, was studied. Enzymatic cross-linking reactions were performed by fungal Trichoderma reesei tyrosinase (TrTyr) and the performance of the enzyme was compared to that of transglutaminase from Streptoverticillium mobaraense (Tgase). Enzymatic cross-linking reactions were followed by different analytical techniques, such as size exclusion chromatography -Ultra violet/Visible multi angle light scattering (SEC-UV/Vis-MALLS), phosphorus nuclear magnetic resonance spectroscopy (31P-NMR), atomic force (AFM) and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS). The research results showed that in both cases cross-linking of β-casein resulted in the formation of high molecular mass (MM ca. 1 350 kg mol-1), disk-shaped nanoparticles when the highest enzyme dosage and longest incubation times were used. According to SEC-UV/Vis-MALLS data, commercial β-casein was cross-linked almost completely when TrTyr and Tgase were used as cross-linking enzymes. In the case of TrTyr, high degree of cross-linking was confirmed by 31P-NMR where it was shown that 91 % of the tyrosine side-chains were involved in the cross-linking. The impact of enzymatic cross-linking of β-casein on in vitro digestibility by pepsin was followed by various analytical techniques. The research results demonstrated that enzymatically cross-linked β-casein was stable under the acidic conditions present in the stomach. Furthermore, it was found that cross-linked β-casein was more resistant to pepsin digestion when compared to that of non modified β-casein. The effects of enzymatic cross-linking of β-casein on allergenicity were also studied by different biochemical test methods. On the basis of the research results, enzymatic cross-linking decreased allergenicity of native β-casein by 14 % when cross-linked by TrTyr and by 6 % after treatment by Tgase. It can be concluded that in addition to the basic understanding of the reaction mechanism of TrTyr on protein matrix, the research results obtained in this study can have high impact on various applications like food, cosmetic, medical, textile and packing sectors.Proteiinien modifiointi entsyymaattisen ristisidonnan avulla on kiinnostava tapa muokata elintarvikkeen rakennetta tuotteen laadun parantamiseksi. Proteiinien kolmiulotteisen rakenteen ja fysikaalis-kemiallisten ominaisuuksien lisäksi myös proteiinien fysiologiset ominaisuudet, kuten sulavuus ruoansulatuskanavassa sekä allergeenisuus voivat muuttua parempaan suuntaan. Proteiinien ristisidontaan soveltuvia entsyymejä, kuten transglutaminaaseja, on tällä hetkellä kaupallisesti saatavilla, mutta parhaillaan myös muuntyyppisiä ristisidontaan kykeneviä entsyymejä tutkitaan ja kehitetään runsaasti. Tässä väitöskirjassa tutkittiin naudan yleisimmän maitoproteiinin β-kaseiinin entsyymaattista ristisidontaa molekyyli- ja kemiallisen sidoksen tasolla eri analyysitekniikoita hyodyntäen. Entsyymaattiset ristisidontareaktiot toteutettiin Trichoderma reesei -homeen tyrosinaasilla (TrTyr) ja niitä verrattiin Streptoverticillium mobaraense -transglutaminaasin (Tgase) aiheuttamiin vastaavissa olosuhteissa tehtyihin reaktioihin. Tutkimustulokset osoittivat, että kummassakin tapauksessa β-kaseiini pystyttiin lähes täydellisesti polymeroimaan ja että reaktiotuotteet olivat rakenteeltaan levyn muotoisia nanopartikkeleita. Tyrosinaasia käytettäessä partikkelit olivat kuitenkin noin 4-5 kertaa suurempia ja väriltään rusehtavia. β-Kaseiinin entsyymaattisen ristisidonnan vaikutusta proteiinin sulavuuteen tutkittiin in vitro seuraamalla β-kaseiinin pilkkoutumista pepsiinin vaikutuksesta useilla eri menetelmillä. Tutkimustulokset osoittivat, että entsyymaattisesti ristisidottu β-kaseiini on stabiili vatsalaukun happamissa olosuhteissa. Sen lisäksi havaittiin, että ristisidottua β-kaseiinia ei pystytä pilkkomaan pepsiinillä yhtä tehokkaasti kuin käsittelemätön β-kaseiini. Entsyymien välillä ei havaittu suuria eroja. Entsyymaattisen ristisidonnan vaikutusta β-kaseiinin allergeenisuuteen arvioitiin myös erilaisin biokemiallisin tesimenetelmin. Tutkimustulosten perusteella entsyymaattinen ristisidonta vähensi β-kaseiinin allergeenisuutta 14 %, kun se ristisidottiin TrTyr -entsyymillä ja 6 %, kun se modifioitiin Tgase -entsyymillä. Yhteenvetona voidaan todeta, että tutkimustuloksilla on merkitystä paitsi tyrosinaasientsyymin perustutkimukselle, mutta arvoa myös elintarvike, kosmetiikka, lääke, tekstiili ja pakkaus sovelluksille

Development of a Certified Reference Material for myeloperoxidase-anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA).

A serum Certified Reference Material (CRM) for supporting reliable autoimmune diagnostics was recently released by the Institute for Reference Materials and Measurements (IRMM) of the Joint Research Centre of the European Commission. It was produced in collaboration with a Working Group on the Harmonisation of Autoimmune Tests of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC WG-HAT). This material is aimed at facilitating the standardisation of measurements of anti-myeloperoxidase immunoglobulin G antibodies. The CRM could be used as a common calibrant by clinicians and manufacturers thereby significantly improving the comparability of results from commercial immunoassays used for IgG anti-MPO measurements. This paper provides information on the new CRM and its intended use

Standardization of autoimmune testing - is it feasible?

Correct measurement of autoantibodies is essential for the diagnosis of autoimmune diseases. However, due to the variability of autoantibody results and the heterogeneity of testing, wrong diagnosis is a reality. For this and more reasons, harmonization of testing is of the outmost importance. In this review we have summarized the factors contributing to this variability. The ways with which the working group on harmonization of autoantibody testing of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has been trying to tackle the issue with the production and correct use of certified reference materials (CRMs), is discussed. Finally the advantages and the limitations of the use of CRMs are presented

Certified reference material against PR3 ANCA IgG autoantibodies. From development to certification.

Background The importance of the standardisation of immunoassays for autoantibodies has been widely discussed. The appropriate use of certified reference materials (CRM) could contribute to a more accurate diagnosis and follow-up of a series of diseases such as small vessel-associated vasculitis. This is a systemic autoimmune disorder during which two autoantibodies can be present, MPO ANCA IgG and PR3 ANCA IgG. Results from different commercially available immunoassays used for PR3 ANCA IgG measurement can vary significantly. Therefore the potential for improvement using a suitable certified reference material was assessed and led to the development of a CRM. Methods Thirty clinical samples were evaluated using 10 immunoassays. The correlation between results from these assays was assessed in a pairwise manner. Feasibility studies were conducted in order to find a reference material format most suitable for the preparation of a CRM. Results The evaluation of two sets of 30 clinical samples with 10 assays showed that differences between assays can result in different interpretations for individual clinical samples. Most of the samples had the same result classification in all assays. However, six of the samples tested led to inconsistent results. Conclusions The correlation between results from clinical samples was systematically good for combinations of eight of those assays. Therefore, it should be possible to improve the comparability of results using a commutable CRM for calibration. Based on these studies, a final format for the CRM was selected and eventually produced and certified for its PR3 ANCA IgG content

The certification of anti-myeloperoxidase immunoglobulin G in human serum ERM® - DA476/IFCC

This report describes the production and certification of ERM-DA476/IFCC, a new serum protein reference material intended for the standardisation of measurements of anti-myeloperoxidase immunoglobulin G (anti-MPO IgG) antibodies. The material was produced according to ISO Guide 34:2009. The raw material used to prepare ERM-DA476/IFCC was a plasmapheresis material containing a high concentration of anti-MPO IgG. After a thorough commutability study lyophilised serum was selected as the format for the candidate reference material. Serum processing was performed based on the procedure used for the reference material ERM-DA470k/IFCC. The plasma was converted into serum which was then delipidated. After the addition of preservatives the processed serum was diluted with plasmapheresis solution containing albumin, prior to the transfer of 1 mL aliquots to glass vials. The serum was then lyophilised and the vials closed with rubber stoppers and screw caps under nitrogen atmosphere prior to storage at -70 °C. The between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The material was characterised by an inter-laboratory comparison exercise performed by laboratories of demonstrated competence and with adherence to ISO/IEC 17025 , using a purified anti-MPO IgG preparation as calibrant. This was achieved using a value transfer protocol previously used in the characterisation of ERM-DA470k/IFCC. Technically invalid results were removed. However no other outliers were eliminated on statistical grounds only. Uncertainties of the certified values were calculated in accordance to the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties relating to possible lack of homogeneity, instability and characterisation. The material is intended for the calibration of methods and quality control. As any reference material, it can also be used for control charts or validation studies. The CRM is available in glass vials containing the lyophilised residue of 1 g serum. The minimum amount of reconstituted sample to be used is 10 μL. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium

The certification of the mass concentration of immunoglobulin G proteinase 3 anti-neutrophil cytoplasmic autoantibodies (IgG PR3 ANCA) in human serum: ERM® - DA483/IFCC

This report describes the production and certification of ERM-DA483/IFCC, a serum protein reference material intended for the standardisation of measurements of immunoglobulin G proteinase 3 anti-neutrophil cytoplasmic autoantibodies (IgG PR3 ANCA). The material was produced according to ISO Guide 34:2009 and is certified in accordance with ISO Guide 35:2006. The raw material used to prepare ERM-DA483/IFCC was a plasmapheresis material containing a high concentration of IgG PR3 ANCA. After a prior commutability study lyophilised serum was selected as the best format for the reference material. The processing of the serum was based on the procedure used for the reference material ERM-DA470k/IFCC . The plasma was converted into serum which was then delipidated. After the addition of preservatives the processed serum was diluted with plasmapheresis buffer containing albumin, prior to the transfer of 1 mL aliquots to glass vials. The serum was then lyophilised and the vials were closed with rubber stoppers and screw caps under argon atmosphere prior to storage at -70 °C. The between unit-homogeneity was quantified and stability during dispatch and storage was assessed in accordance with ISO Guide 35:2006. The material was characterised by an inter-laboratory comparison exercise performed by laboratories of demonstrated competence, using a purified IgG PR3 ANCA preparation as calibrant. This was achieved by applying a value transfer protocol previously used in the characterisation of ERM-DA470k/IFCC. Technically invalid results were removed, but no outliers were eliminated on statistical grounds alone. The uncertainty of the certified value was estimated in accordance to the Guide to the Expression of Uncertainty in Measurement (GUM) and included components relating to possible lack of homogeneity, stability and the property value measured during characterisation. The main purpose of this material is to be used for the calibration of immunoassay-based in vitro diagnostic devices or control products for IgG PR3 ANCA measurements. As any reference material, it can also be used for control charts or validation studies. The CRM is available in glass vials containing approximately 0.1 g of dried powder. The minimum sample intake to be used after reconstitution of the material is 5 μL. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium

Development of a certified reference material for anti-β2-glycoprotein I IgG - commutability studies.

Objectives: In this paper, we describe the steps followed for the development of a certified reference material for immunoglobulin G antibodies against β2-glycoprotein I (also known as apolipoprotein H). These steps include processing of the material, commutability, the impact of dilution, the appropriate reconstitution conditions, homogeneity and stability during transport and storage. Methods: We analysed 69 clinical samples from patients suffering from antiphospholipid syndrome with several commercial enzyme-linked immunosorbent assays (ELISA) purchased from in vitro diagnostic manufacturers. Results: Analysis of the results indicated that the candidate reference material can be safely freeze-dried, and that the user should carefully follow the reconstitution instructions as small changes in e.g. temperature may have unwanted effects. The statistical analysis of the commutability studies indicated that the analytical response of the reference material upon dilution is similar to that of clinical samples, and that correlation between results may differ from assay to assay. Finally yet importantly, the presented and developed candidate reference material is commutable for most assays tested, homogeneous and stable. Conclusions: Immunoglobulin G antibodies against β2-glycoprotein I are associated with a higher risk of thrombosis and pregnancy complications. Their measurement is essential for the diagnosis and monitoring of antiphospholipid syndrome. These antibodies are detected by specific immunoassays, routinely used in clinical diagnostics, but various of these methods show enormous variability, in part due to the lack of a reference material

Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules

Peer reviewe

A synthetically modified hydrophobin showing enhanced fluorous affinity

Hydrophobins are natural surfactant proteins endowed with exceptional surface activity and film-forming capabilities and their use as effective “fluorine-free fluorosurfactants” has been recently reported. In order to increase their fluorophilicity further, here we report the preparation of a unique fluorous-modified hydrophobin, named F-HFBI. F-HFBI was found to be more effective than its wild-type parent protein HFBI at reducing interface tension of water at both air/water and oil/water interfaces, being particularly effective at the fluorous/water interface. F-HFBI was also found to largely retain the exceptionally good capability of forming strong and elastic films, typical of the hydrophobin family. Further studies by interface shear rheology and isothermal compression, alongside Quartz Crystal Microbalance and Atomic Force Microscopy, demonstrated the tendency of F-HFBI to form thicker films compared to the wild-type protein. These results suggest that F-HFBI may function as an effective compatibilizer for biphasic systems comprising a fluorous phase

Hydrophobin: fluorosurfactant-like properties without fluorine

The stabilization of fluorous oil droplets in aqueous environment is a critical issue in the preparation of emulsified systems for biomedical applications and in emulsion polymerization technology, due to the extreme immiscibility of aqueous and fluorous phases. We present here a detailed study on the behavior of the hydrophobin HFBI, i.e. a small natural protein endowed with exceptional surface activity, at the interface between aqueous and fluorous phases. HFBI behaves as an efficient and sustainable surfactant at remarkably low concentrations and forms a strong and elastic film at the interface between the two phases. We also show proof-of-concept experiments on the use of HFBI as a surfactant in fluorous oil/water emulsified systems and in microfluidic circuits. This journal is © The Royal Society of Chemistry 2013