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Background: The world’s production of whey is estimated to be more than 200 milliontons per year. Although whey is an important source of proteins with high nutritional value andbiotechnological importance, it is still considered as a by-product of the dairy industry with loweconomic value due to low industrial exploitation. There are several challenges in the separation ofwhey proteins: low concentration, the complexity of the material and similar properties (pI, molecularmass) of some proteins.Methods: A narrative review of all the relevant papers on the present methodologies based on ionexchange and adsorption principles for isolation of whey proteins, known to the authors, was conductedResults: Traditional ion exchange techniques are widely used for the separation and purification ofthe bovine whey proteins. These methodologies, based on the anion or cation chromatographicprocedures, as well as the combination of aforementioned techniques are still preferential methodsfor the isolation of the whey proteins on the laboratory scale. However, more recent research on ionexchange membranes for this purpose has been introduced, with promising potential to be appliedon the pilot industrial scale. Newly developed methodologies based either on the ion exchangeseparation (for example: simulated moving bed chromatography, expanded bed adsorption, magneticion exchangers, etc.) or adsorption (for example: adsorption on hydroxyapatite or activatedcarbon, or molecular imprinting) are promising approaches for scaling up of the whey proteins’purification processes.Conclusion: Many procedures based on ion exchange are successfully implemented for the separationand purification of whey proteins, providing protein preparations of moderate-to-high yieldand satisfactory purity. However, the authors anticipate further development of adsorption-basedmethodologies for the separation of whey proteins by targeting the differences in proteins’ structuresrather than targeting the differences in molecular masses and pI. The complex composite multilayeredmatrices, including also inorganic components, are promising materials for simultaneousexploiting of the differences in the masses, pI and structures of whey proteins for the separation. Published manuscript: [https://cer.ihtm.bg.ac.rs/handle/123456789/5555] This is the peer-reviewed version of the following article: (1) Radosavljević, J.; Stanić-Vučinić, D.; Stojadinović, M.; Radomirović, M.; Simović, A.; Radibratović, M.; Veličković, T. Ć. Application of Ion Exchange and Adsorption Techniques for Separation of Whey Proteins from Bovine Milk. Current Analytical Chemistry 18 (3), 341–359. [https://doi.org/10.2174/1573411017666210108092338].

Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58–419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of β-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection. This is the preprint of the article: Đukić, T.; Mladenović, M.; Stanić-Vučinić, D.; Radosavljević, J.; Smiljanić, K.; Sabljić, L.; Dević, M.; Ćujić, D.; Vasović, T.; Simović, A.; Radomirović, M. Ž.; Ćirković-Veličković, T. Expression, Purification and Immunological Characterization of Recombinant Nucleocapsid Protein Fragment from SARS-CoV-2. Virology journal 2021, 557, 15–22. [https://doi.org/10.1016/j.virol.2021.01.004].

In this study, we investigated structural aspects of covalent binding of food derived blue pigment phycocyanobilin (PCB) to bovine beta-lactoglobulin (BLG), major whey protein, by spectroscopic, electrophoretic, mass spectrometry and computational methods. At physiological pH (7.2), we found that covalent pigment binding via free cysteine residue is slow (k(a)=0.065 min(-1)), of moderate affinity (K-a=4x10(4) M-1), and stereo-selective. Binding also occurs at a broad pH range and under simulated gastrointestinal conditions. Adduct formation rises with pH, and in concentrated urea (k(a)=0.101 min(-1)). The BLG-PCB adduct has slightly altered secondary and tertiary protein structure, and bound PCB has higher fluorescence and more stretched conformation than free chromophore. Combination of steered molecular dynamic for disulfide exchange, non-covalent and covalent docking, favours Cys119 residue in protein calyx as target for covalent BLG-PCB adduct formation. Our results suggest that this adduct can serve as delivery system of bioactive PCB. This is peer-reviewed version of the following article: Minic, S.; Radomirovic, M.; Savkovic, N.; Radibratovic, M.; Mihailovic, J.; Vasovic, T.; Nikolic, M.; Milcic, M.; Stanic-Vucinic, D.; Cirkovic Velickovic, T. Covalent Binding of Food-Derived Blue Pigment Phycocyanobilin to Bovine β-Lactoglobulin under Physiological Conditions. Food Chemistry 2018, 269, 43–52.[https://doi.org/10.1016/j.foodchem.2018.06.138] Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3187]

beta-lactoglobulin (BLG) is a major whey protein with numerous techno-functional properties desirable for the food industry. Phycocyanobilin (PCB), a bioactive pigment of Arthrospira platensis with health-promoting effects, covalently binds to BLG at physiological pH. This study investigated the effects of this covalent modification on BLG functional properties. The BLG-PCB adduct possesses enhanced antioxidant properties, and bound PCB protects BLG against free radical-induced oxidation. Despite the similar thermal stabilities of BLG and BLG-PCB, BLG-PCB is less susceptible to covalent and noncovalent aggregation under moderate heat treatment (63 degrees C, 30 min). Blocked thiol group and reduced hydrophobicity due to hindering of hydrophobic residues by bound PCB, as well as the heat-induced transition of beta-sheet to alpha-helix, contributed to the low susceptibility of BLG-PCB to aggregation. BLG-PCB has a higher resistance to pepsin and pancreatin digestion than BLG and unaltered IgEbinding properties. The improved functional properties of BLG-PCB make it a useful ingredient in the food industry.

The worldwide outbreak of COVID-19 was caused by a pathogenic virus called Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Therapies against SARS-CoV-2 target the virus or human cells or the immune system. However, therapies based on specific antibodies, such as vaccines and monoclonal antibodies, may become inefficient enough when the virus changes its antigenicity due to mutations. Polyphenols are the major class of bioactive compounds in nature, exerting diverse health effects based on their direct antioxidant activity and their effects in the modulation of intracellular signaling. There are currently numerous clinical trials investigating the effects of polyphenols in prophylaxis and the treatment of COVID-19, from symptomatic, via moderate and severe COVID-19 treatment, to anti-fibrotic treatment in discharged COVID-19 patients. Antiviral activities of polyphenols and their impact on immune system modulation could serve as a solid basis for developing polyphenol-based natural approaches for preventing and treating COVID-19.

Digestion is the key step for delivering nutrients and bioactive substances to the body. The way different food components interact with each other and with digestive enzymes can modify the digestion process and affect human health. Understanding how food components interact during digestion is essential for the rational design of functional food products. Plant polyphenols have gained much attention for the bioactive roles they play in the human body. However, their strong beneficial effects on human health have also been associated with a negative impact on the digestion process. Due to the generally low absorption of phenolic compounds after food intake, most of the consumed polyphenols remain in the gastrointestinal tract, where they then can exert inhibitory effects on enzymes involved in the degradation of saccharides, lipids, and proteins. While the inhibitory effects of phenolics on the digestion of energy-rich food components (saccharides and lipids) may be regarded as beneficial, primarily in weight-control diets, their inhibitory effects on the digestion of proteins are not desirable for the reason of reduced utilization of amino acids. The effect of polyphenols on protein digestion is reviewed in this article, with an emphasis on food processing methods to improve the antinutritive properties of polyphenols.

The reactions of four cymene-capped ruthenium(II) compounds with pro-apaptotic protein, cytochrome c (Cyt), and anti-proliferating protein lysozyme (Ly) in carbonate buffer were investigated by ESI-MS, UV–Vis absorption and CD spectroscopy. The complexes with two chloride ligands (C2 and C3) were more reactive toward proteins than those with only one (C1 and C4), and the complex with S,N-chelating ligand (C4) was less reactive than one with O,N-chelating ligand (C1). Dehalogenated complexes are most likely species initially coordinating proteins for all tested complexes. During the time, protein adducts vividly exchanged non-arene organic ligand L with CO32- and OH-, while cymene moiety was retained. In water, only dehalogenated adducts were identified suggesting that in vivo, in the presence of various anions, dynamic ligand exchange could generate different intermediate protein species. Although all complexes reduced Cyt, the reduction was not dependent on their reactivity to protein, implying that initially noncovalent binding to Cyt occures, causing its reduction, followed by coordination to protein. Cyt reduction was accompanied with rupture of ferro-Met 80 and occupation of this hem coordination site by a histidine His-33/26. Therefore, in Cyt with C2 and C3 less intensive reduction of hem iron leave more unoccupied target residues for Ru coordination, leading to more efficient formation of covalent adducts, in comparison to C1 and C4. This study contribute to development of new protein-targeted Ru(II) cymene complexes, and to design of new cancer therapies based on targeted delivery of Ru(II) arene complexes bound on pro-apoptotic/anti-proliferating proteins as vehicles. This is the peer-reviewed version of the following article: Stanic-Vucinic, D.; Nikolic, S.; Vlajic, K.; Radomirovic, M.; Mihailovic, J.; Cirkovic Velickovic, T.; Grguric-Sipka, S. The Interactions of the Ruthenium(II)-Cymene Complexes with Lysozyme and Cytochrome c. Journal of Biological Inorganic Chemistry 2020. [https://doi.org/10.1007/s00775-020-01758-3] Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3863]

Resveratrol is a phytoalexin produced by many plants as a defense mechanism against stress-inducing conditions. The richest dietary sources of resveratrol are berries and grapes, their juices and wines. Good bioavailability of resveratrol is not reflected in its high biological activity in vivo because of resveratrol isomerization and its poor solubility in aqueous solutions. Proteins, cyclodextrins and nanomaterials have been explored as innovative delivery vehicles for resveratrol to overcome this limitation. Numerous in vitro and in vivo studies demonstrated beneficial effects of resveratrol in cardiovascular diseases (CVD). Main beneficial effects of resveratrol intake are cardioprotective, anti-hypertensive, vasodilatory, anti-diabetic, and improvement of lipid status. As resveratrol can alleviate the numerous factors associated with CVD, it has potential as a functional supplement to reduce COVID-19 illness severity in patients displaying poor prognosis due to cardio-vascular complications. Resveratrol was shown to mitigate the major pathways involved in the pathogenesis of SARS-CoV-2 including regulation of the renin-angiotensin system and expression of angiotensin-converting enzyme 2, stimulation of immune system and downregulation of pro-inflammatory cytokine release. Therefore, several studies already have anticipated potential implementation of resveratrol in COVID-19 treatment. Regular intake of a resveratrol rich diet, or resveratrol-based complementary medicaments, may contribute to a healthier cardio-vascular system, prevention and control of CVD, including COVID-19 disease related complications of CVD.

Bovine alpha-lactalbumin (ALA) is an important Ca-binding protein of milk. Epigallocatechin-3-gallate (EGCG) is the major and the most biologically active catechin of green tea, which has the highest binding affinity to whey proteins due to galloyl functional group. In this study experimental and computational methods were used to investigate noncovalent interactions of EGCG and ALA. Binding affinity of EGCG for ALA, determined by fluorescence quenching analysis, was in the range described for complexes of EGCG and other dietary proteins, and lower than affinity of some phenolic compounds to ALA. Based on circular dichroism and Fourier transform infrared spectroscopy spectra, binding of EGCG change ALA conformation inducing alpha-helix to beta-structures transition. The isothermal titration calorimetry results suggest that the binding of EGCG to ALA is enthalpically favorable. The docking analysis shows that EGCG binds in the hydrophobic pocket at the entrance of cleft between alpha-helical and beta-sheetrich domains and includes residues of aromatic cluster II. Uptake of ALA by monocytes proceeds at a slower rate in the presence of EGCG suggesting that EGCG binding may impair uptake of ALA by antigen-presenting cells. ALA, being of low cost and widely available protein, can serve as suitable delivery system for EGCG, as well as for food fortification with this bioactive catechin. (C) 2016 Elsevier Ltd. All rights reserved. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3585]

2S albumins of peanuts are seed storage proteins, highly homologous in structure and described as major elicitors of anaphylactic reactions to peanut (allergens Ara h 2 and Ara h 6). Epigallocatechin-3-gallate (EGCG) is the most biologically potent polyphenol of green tea. Non-covalent interactions of EGCG with proteins contribute to its diverse biological activities. Here we used the methods of circular dichroism, fluorescence quenching titration, isothermal titration calorimetry and computational chemistry to elucidate interactions of EGCG and 2S albumins. Similarity in structure and overall fold of 2S albumins yielded similar putative binding sites and similar binding modes with EGCG. Binding affinity determined for Ara h 2 was in the range described for complexes of EGCG and other dietary proteins. Binding of EGCG to 2S albumins affects protein conformation, by causing an alpha-helix to beta-structures transition in both proteins. 2S albumins of peanuts may be good carriers of physiologically active green tea catechin. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3355]

High intensity ultrasound (HIUS) can promote Maillard reaction (MR). Macromolecular crowding conditions accelerate reactions and stabilise protein structure. The aim of this study was to investigate if combined application of ultrasound and macromolecular crowding can improve efficiency of MR. The presence of crowding agent (polyethylene glycol) significantly increased ultrasound-induced whey protein (WP) glycation by arabinose. An increase in glycation efficiency results only in slight change of WP structure. Macromolecular crowding intensifies oxidative modifications of WP, as well as formation of amyloid-like structures by enhancement of MR. Solubility at different pH, thermal stability and antioxidative capacity of glycated WP were increased, especially in the presence of crowding agent, compared to sonicated nonglycated proteins. The application of HIUS under crowding conditions can be a new approach for enhancement of reactions in general, enabling short processing time and mild conditions, while preserving protein structure and minimising protein aggregation. (C) 2015 Elsevier Ltd. All rights reserved. This is the peer-reviewed version of the following article: Peruško, M.; Al-Hanish, A.; Ćirković-Veličković, T.; Stanić-Vučinić, D. Macromolecular Crowding Conditions Enhance Glycation and Oxidation of Whey Proteins in Ultrasound-Induced Maillard Reaction. Food Chemistry 2015, 177, 248–257. [https://doi.org/10.1016/j.foodchem.2015.01.042]. Published version: [https://cherry.chem.bg.ac.rs/handle/123456789/1666]

BackgroundMost food allergens sensitizing via the gastrointestinal tract are stable proteins that are resistant to pepsin digestion, in particular major peanut allergens, Ara h 2 and Ara h 6. Survival of their large fragments is essential for sensitizing capacity. However, the immunoreactive proteins/peptides to which the immune system of the gastrointestinal tract is exposed during digestion of peanut proteins are unknown. Particularly, the IgE reactivity of short digestion-resistant peptides (SDRPs; lt 10kDa) released by gastric digestion under standardized and physiologically relevant invitro conditions has not been investigated. ObjectiveThe aim of this study was to investigate and identify digestion products of major peanut allergens and in particular to examine IgE reactivity of SDRPs released by pepsin digestion of whole peanut grains. MethodsTwo-dimensional gel-based proteomics and shotgun peptidomics, immunoblotting with allergen-specific antibodies from peanut-sensitized patients, enzyme-linked immunosorbent inhibition assay and ImmunoCAP tests, including far ultraviolet-circular dichroism spectroscopy were used to identify and characterize peanut digesta. ResultsAra h 2 and Ara h 6 remained mostly intact, and SDRPs from Ara h 2 were more potent in inhibiting IgE binding than Ara h 1 and Ara 3. Ara h 1 and Ara h 3 exhibited sequential digestion into a series of digestion-resistant peptides with preserved allergenic capacity. A high number of identified SDRPs from Ara h 1, Ara h 2 and Ara h 3 were part of short continuous epitope sequences and possessed substantial allergenic potential. Conclusion and Clinical RelevancePeanut grain digestion by oral and gastric phase enzymes generates mixture of products, where the major peanut allergens remain intact and their digested peptides have preserved allergenic capacity highlighting their important roles in allergic reactions to peanut. This is the peer-reviewed version of the article: Prodić, I.; Stanić-Vučinić, D.; Apostolović, D.; Mihailović-Vesić, J.; Radibratović, M.; Radosavljević, J.; Burazer, L. M.; Milčić, M. K.; Smiljanić, K.; van Hage, M.; Ćirković-Veličković, T. Influence of Peanut Matrix on Stability of Allergens in Gastric-Simulated Digesta: 2S Albumins Are Main Contributors to the IgE Reactivity of Short Digestion-Resistant Peptides. Clinical and Experimental Allergy 2018, 48 (6), 731–740. [https://doi.org/10.1111/cea.13113]. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/3225]

It has been shown before that dietary polyphenols possess cancer chemopreventive effects. As cervical cancer is the second leading genital malignancy in women after breast cancer, the anti-cervical cancer effects of polyphenol extracts of commonly used beverages (green tea, coffee and cocoa) were tested and compared in HeLa cells. All the extracts induced apoptosis of HeLa cells, but green tea was the most potent. However, as opposed to green tea which induced a strong anti-proliferative response in HeLa cells, coffee and cocoa extracts promoted the proliferation of surviving cells. After short-term exposure, green tea and coffee extracts, but not cocoa, induced the formation of intracellular reactive oxygen species. Only the green tea extract increased the production of superoxide anion radicals and decreased reduced glutathione levels. Gene expression of Cu/Zn and Mn-superoxide dismutase or catalase was unaltered in cells treated with extracts, but green tea partially inhibited catalase activity. The cytotoxic activity of green tea and coffee extracts was partially inhibited by vitamin C. The in vitro anti-cervical cancer potency of tested polyphenol extracts is related to their pro-oxidant and anti-proliferative activities and are exhibited in the following order: green tea gt coffee gt cocoa, with only green tea showing both pro-oxidative and anti-proliferative action. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3449]

Human serum albumin (HSA) is an important regulator of the pharmacokinetic properties of bioactive compounds. Phycocyanobilin is a blue tetrapyrrole chromophore of C-phycocyanin with proven health-promoting activities. Despite its structural similarity to bilirubin, the conformation it adopts in aqueous solution is different and the pigment is more soluble than bilirubin. The aim of our study was to examine binding of phycocyanobilin for HSA and to investigate its competition with bilirubin. Based on a computational approach, we demonstrated two putative high-affinity binding pockets on HSA of virtually identical energies for the neutral and anion forms of bilirubin, but with slightly favorable predictions for anion forms of phycocyanobilin. Computational prediction of phycocyanobilin pK(a) values suggested a monoanion form to be the most stable form at physiological conditions. The computationally predicted binding sites for phycocyanobilin were identical to the two previously identified binding sites for bilirubin (subdomains IB and IIA). Results obtained by protein and pigment fluorescence measurements, circular dichroism, and competition experiments confirmed high affinity (binding constant of 2.2 x 10(6) M-1 at 25 degrees C), stereo-selective binding of phycocyanobilin M-conformer to HSA and its competition with bilirubin, warfarin and hemin. Our experimental data confirm that phycocyanobilin binds to IB and IIA binding site of HSA with an affinity similar to bilirubin. In conditions characterized by an increased bilirubin plasma concentration, or intake of drugs binding to IB or IIA binding site, pharmacokinetics of phycocyanobilin may also be changed. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3403]

Phycocyanobilin (PCB) is a blue tetrapyrrole chromophore of C-phycocyanin, the main protein of the microalga Spirulina, with numerous proven health-related benefits. We examined binding of PCB to bovine serum albumin (BSA) and how it affects protein and ligand stability. Protein fluorescence quenching and microscale thermophoresis demonstrated high-affinity binding (Ka = 2 × 106 M−1). Spectroscopic titration with molecular docking analysis revealed two binding sites on BSA, at the inter-domain cleft and at subdomain IB, while CD spectroscopy indicated stereo-selective binding of the P conformer of the pigment to the protein. The PCB protein complex showed increased thermal stability. Although complex formation partly masked the antioxidant properties of PCB and BSA, a mutually protective effect against free radical-induced oxidation was found. BSA could be suitable for delivery of PCB as a food colorant or bioactive component. Our results also highlight subtle differences between PCB binding to bovine vs. human serum albumin. This is peer-reviewed version of the following article: Minic, S.; Stanic-Vucinic, D.; Radomirovic, M.; Radibratovic, M.; Milcic, M.; Nikolic, M.; Cirkovic Velickovic, T. Characterization and Effects of Binding of Food-Derived Bioactive Phycocyanobilin to Bovine Serum Albumin. Food Chemistry 2018, 239, 1090–1099. [https://doi.org/10.1016/j.foodchem.2017.07.066] Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3139]

Beta-lactoglobulin (BLG) is the main whey protein and it is frequently used additive in wide range of food products due to its excellent techno-functional properties, high nutritional value and low cost. It is also considered as acid-resistant drug carrier for delivery of pharmaceutical and nutraceutical agents. However, BLG is the main allergen of milk. A variety of methods have been explored for modification of BLG in attempt to improve its functional properties and to decrease its allergenicity. Due to its compact globular structure BLG is relatively resistant to modifications, especially under mild conditions. BLG can be modified by physical, chemical and enzymatic treatments. Although chemical modifications offer efficient way of alteration of protein structural and functional properties, they are associated with safety concern. In the last decade there is a tendency for application of novel non-thermal physical processing methods, as well as enzymes in order to obtain BLG with desirable properties. The objective of this review is to overview chemical, physical and enzymatic processing techniques utilized to modify BLG and their effects on structure and functional properties of BLG. [Projekat Ministarstva nauke Republike Srbije, br. 172024 and FP7 RegPot project FCUB ERA GA No. 256716]

Warfarin (4-hydroxy coumarin) is Vitamin K (VK) antagonist that inhibits Vitamin K-dependent (VKD) processes, such as blood coagulation. It exerts influence on some non-VKD-related activities as well. In this study, the effect of subacute (30-day) oral warfarin (2 mg L-1 and 1 mg L-1) intake on hematological parameters was examined in two rat strains, Albino Oxford (AO) and Dark Agouti (DA), that differ in their sensitivity to certain chemicals. Greater susceptibility to anticoagulant effect of 2 mg L-1 of warfarin was observed in AO rats and was associated with an increase in relevant hematological parameters in this strain. Although both strains responded to 2 mg L-1 of warfarin with quantitative changes in peripheral blood leukocytes differential bone marrow and lung responses were observed. Strain-related differences in proinflammatory activity of peripheral blood granulocytes and in mononuclear cell IFN-γ production were observed. Recognition of differences in quantitative and qualitative effects of oral warfarin on processes other than hemostasis might be of relevance for those humans who are on warfarin therapy. [Projekat Ministarstva nauke Republike Srbije, br. 173039]

The in vitro gastric digestion of several food allergens (beta-lactoglobulin (BLG), alpha-lactalbumin (LA) and peanut allergens (PE)) in the presence of a catechin-enriched polyphenol extract of green tea (GTC), oxidized polyphenols and phenol oxidase processed food allergens and GTC was investigated. Pepsin-resistant proteins, such as BLG, major peanut allergens, Ara h 1 and Ara h 2, degrade faster in the presence of catechin-enriched green tea polyphenols. Phenol oxidase polymerized GTC affected adversely protein digestion of BLG and LA, but not digestion of PE proteins. Protecting effect of polyphenols correlated well with the ability of proteins to form insoluble complexes with oxidized catechins. Cross-linking of proteins and polyphenols further extended the half-lives of BLG and LA in the in vitro digestion by pepsin. Catechin-enriched green tea polyphenols of food supplements facilitate pepsin digestion of major food allergens, but hamper their digestion if oxidized and polymerized by phenol oxidase. (c) 2012 Elsevier Ltd. All rights reserved.

beta-Lactoglobulin (BLG) is an important nutrient of dairy products and an important allergen in cow's milk allergy. The aim of this study was to investigate the potential of laccase to cross-link BLG in the presence of an apple phenolic extract (APE) and to characterize the obtained products for their digestibility by pepsin and pancreatin. The composition of the apple phenolics used for cross-linking was determined by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The apple phenolic extract contained significant amounts of quercetin glycosides, catechins and chlorogenic acid. The laccase cross-linked BLG in the presence of apple phenolics. The polymerization rendered the protein insoluble in the reaction mixture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the cross-linking reaction mixture revealed a heterogeneous mixture of high molecular masses (cross-linked BLG), with a fraction of the BLG remaining monomeric. Enzymatic processing of BLG by laccase and apple polyphenols as mediators can decrease the biphasal pepsin pancreatin digestibility of the monomeric and cross-linked protein, thus decreasing its nutritional value. In addition, reduced BLG digestibility can decrease its allergenic potential. Apple polyphenols can find usage in the creation of new, more functional food products, designed to prevent obesity and hypersensitivity-related disorders. β-Laktoglobulin (BLG) je važan nutrijent mlečnih proizvoda i važan alergen kod alergija na kravlje mleko. Cilj ove studije je bilo ispitivanje potencijala lakaze da unakrsno poveže BLG u prisustvu fenolnog ekstrakta jabuke (APE), kao i karakterizacija dobijenih proizvoda sa aspekta njihove digestibilnosti pepsinom i pankreatinom. Kompozicija fenola jabuke korišćenih za unakrsno povezivanje određena je pomoću LC-ESI-MS. Fenolni ekstrakt jabuke sadrži znatne količine glikozida kvercetina, katehine i hlorogensku kiselinu. BLG je unakrsno povezan lakazom u prisustvu fenola jabuke, pri čemu je polimerizacija učinila BLG nerastvornim u reakcionoj smeši. SDS-PAGE analiza pokazala je da reakciona smeša sadrži heterogenu smešu velikih molekulskih masa (unakrsno povezan BLG), kao i deo zaostalog monomernog BLG. Enzimsko procesovanje BLG lakazom, u prisustvu polifenola jabuke kao medijatora, može smanjiti bifaznu pepsin-pankreatinsku digestibilnost kako monomernog, tako i unakrsno povezanog BLG, i na taj način smanjiti njegovu nutritivnu vrednost. Takođe, smanjena digestibilnost BLG može smanjiti njegov alergeni potencijal. Polifenoli jabuke mogu se koristiti za kreiranje novih, funkcionalnijih prehrambenih proizvoda, dizajniranih za prevenciju kako gojaznosti, tako i bolesti vezanih za preosetljivost.

Conglutins represent the major peanut allergens and are renowned for their resistance to gastrointestinal digestion. Our aim was to characterize the digestion-resistant peptides (DRPs) of conglutins by biochemical and biophysical methods followed by a molecular dynamics simulation in order to better understand the molecular basis of food protein allergenicity. We have mapped proteolysis sites at the N- and C-termini and at a limited internal segment, while other potential proteolysis sites remained unaffected. Molecular dynamics simulation showed that proteolysis only occurred in the vibrant regions of the proteins. DRPs appeared to be conformationally stable as intact conglutins. Also, the overall secondary structure and IgE-binding potency of DRPs was comparable to that of intact conglutins. The stability of conglutins toward gastro-intestinal digestion, combined with the conformational stability of the resulting DRPs provide conditions for optimal exposure to the intestinal immune system, providing an explanation for the extraordinary allergenicity of peanut conglutins. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3415]

Modified allergens are a safer and more efficient alternative to natural allergens for specific immunotherapy. As the modification of an allergen can diminish its immunogenicity due to the alteration of T-cell epitopes, in this paper we study the effects of a reversible chemical modification of Art v 1, the main allergen of mugwort pollen, on its allergenicity and immunogenicity. Modification of Art v 1 by cis-aconitylation into a polyanionic derivative (CAA) did not result in any significant structural alteration. However, IgE-binding epitopes on CAA were blocked, resulting in a reduced IgE-binding and basophil activation. Both proteins induced proliferation of CD3(+)CD4(+) T-cells in mugwort-allergic patients, but only unmodified allergens increased IL-4, IL-5 and IL-10 production. Rabbit and mouse anti-CAA antibodies exhibited cross-reactivity with native allergens and blocked human IgE-binding to Art v 1. Degradation of CAA by lysosomal fraction enzymes resulted in a similar set of peptides, harboring MHC class II-binding peptides, as unmodified proteins. Thus, cis-aconitylation modified Art v 1 had a significantly reduced allergenicity, whereas its immunogenicity was completely preserved. Acid-environment-responsive modification, which releases a full repertoire of native allergen epitopes within a particular site, can be considered a smart drug delivery system, which is able to deliver a therapeutically-effective dose in a controlled manner, and minimizes adverse side effects. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3528]

Phycocyanobilin (PCB) binds with high affinity (2.2 x 10 6 M-1 at 25 degrees C) to human serum albumin (HSA) at sites located in IB and IIA subdomains. The aim of this study was to examine effects of PCB binding on protein conformation and stability. Using 300 ns molecular dynamics (MD) simulations, UV-VIS spectrophotometry, CD, FT-IR, spectrofluorimetry, thermal denaturation and susceptibility to trypsin digestion, we studied the effects of PCB binding on the stability and rigidity of HSA, as well as the conformational changes in PCB itself upon binding to the protein. MD simulation results demonstrated that HSA with PCB bound at any of the two sites showed greater rigidity and lower overall and individual domain flexibility compared to free HSA. Experimental data demonstrated an increase in the a-helical content of the protein and thermal and proteolytic stability upon ligand binding. PCB bound to HSA undergoes a conformational change to a more elongated conformation in the binding pockets of HSA. PCB binding to HSA stabilizes the structure of this flexible transport protein, making it more thermostable and resistant to proteolysis. The results from this work explain at molecular level, conformational changes and stabilization of HSA structure upon ligand binding. The resultant increased thermal and proteolytic stability of HSA may provide greater longevity to HSA in plasma. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3614]

C-phycocyanin, the major protein of cyanobacteria Spirulina, possesses significant antioxidant, anti-cancer, anti-inflammatory and immunomodulatory effects, ascribed to covalently attached linear tetrapyrrole chromophore phycocyanobilin. There are no literature data about structure and biological activities of released peptides with bound chromophore in C-phycocyanin digest. This study aims to identify chromopeptides obtained after pepsin digestion of C-phycocyanin and to examine their bioactivities. C-phycocyanin is rapidly digested by pepsin in simulated gastric fluid. The structure of released chromopeptides was analyzed by high resolution tandem mass spectrometry and peptides varying in size from 2 to 13 amino acid residues were identified in both subunits of C-phycocyanin. Following separation by HPLC, chromopeptides were analyzed for potential bioactivities. It was shown that all five chromopeptide fractions have significant antioxidant and metal-chelating activities and show cytotoxic effect on human cervical adenocarcinoma and epithelial colonic cancer cell lines. In addition, chromopeptides protect human erythrocytes from free radical-induced hemolysis in antioxidative capacity dependant manner. There was a positive correlation between antioxidative potency and other biological activities of chromopeptides. Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin whose activity is mostly related to the antioxidative potency provided by chromophore. (C) 2016 Elsevier B.V. All rights reserved. This is peer-reviewed version of the following article: Minic, S. L.; Stanic-Vucinic, D.; Mihailovic, J.; Krstic, M.; Nikolic, M. R.; Velickovic, T. C. Digestion by Pepsin Releases Biologically Active Chromopeptides from C-Phycocyanin, a Blue-Colored Biliprotein of Microalga Spirulina. J. Proteomics 2016, 147, 132–139. [https://doi.org/10.1016/j.jprot.2016.03.043] Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3445]

Sonication is a new processing technology in the dairy industry. The aim of this study was to test glycation of beta-lactoglobulin (BLG) in Maillard reaction (MR) induced by high-intensity ultrasound in aqueous solution under neutral conditions at 10-15 degrees C, which is not favourable for the MR. BLG was sonicated in the presence of glucose, galactose, lactose, fructose, ribose and arabinose. Formation of Maillard reaction products (MRPs) was monitored by mass spectrometry, spectrophotometry and fluorimetry. Ultrasound treatment resulted in formation of MRPs with all tested carbohydrates. Ribose induced the highest degree of modification resulting in 76% of BLG modified and an average of three anhydroribose units attached. Circular dichroism spectra analyses indicated only minor alterations in secondary and tertiary structures. MRP obtained by ultrasound exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity and possessed increased iron-chelating activity and reducing power. High-intensity ultrasound efficiently promotes BLG-glycoconjugates formation by MR in aqueous solutions under non-denaturing conditions. Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3525] This is the peer-reviewed version of the following article: Stanic-Vucinic, D.; Prodic, I.; Apostolovic, D.; Nikolic, M.; Cirkovic Velickovic, T. Structure and Antioxidant Activity of β-Lactoglobulin-Glycoconjugates Obtained by High-Intensity-Ultrasound-Induced Maillard Reaction in Aqueous Model Systems under Neutral Conditions. Food Chemistry 2013, 138 (1), 590–599. [https://doi.org/10.1016/j.foodchem.2012.10.087].

Conglutins, the major peanut allergens, Ara h 2 and Ara h 6, are highly structured proteins stabilized by multiple disulfide bridges and are stable towards heat-denaturation and digestion. We sought a way to reduce their potent allergenicity in view of the development of immunotherapy for peanut allergy. Isoforms of conglutin were purified, reduced with dithiothreitol and subsequently alkylated with iodoacetamide. The effect of this modification was assessed on protein folding and IgE-binding. We found that all disulfide bridges were reduced and alkylated. As a result, the secondary structure lost α-helix and gained some β-structure content, and the tertiary structure stability was reduced. On a functional level, the modification led to a strongly decreased IgE-binding. Using conditions for limited reduction and alkylation, partially reduced and alkylated proteins were found with rearranged disulfide bridges and, in some cases, intermolecular cross-links were found. Peptide mass finger printing was applied to control progress of the modification reaction and to map novel disulfide bonds. There was no preference for the order in which disulfides were reduced, and disulfide rearrangement occurred in a non-specific way. Only minor differences in kinetics of reduction and alkylation were found between the different conglutin isoforms. We conclude that the peanut conglutins Ara h 2 and Ara h 6 can be chemically modified by reduction and alkylation, such that they substantially unfold and that their allergenic potency decreases. © 2013 Elsevier B.V.

Common ragweed (Ambrosia atremisiifolia) is one of the most frequent causes of pollen-induced allergic reactions both in humans and dogs. It has not been defined yet, what is the major allergen(s) to which most dogs allergic to ragweed show a positive result on intradermal skin test (IDST). In the present study sensitization to Ambrosia artemisiifolia pollen allergens in dogs with atopic dermatitis was examined with both in vivo and in vitro tests, including IDST and serum allergen specific IgE test. Detection of specific-IgE antibodies against ragweed allergens by immunoblotting in the sera of allergic dogs was optimized, as well. Dogs that were positive, as judged by IDST reactions to ragweed pollen allergens, also had alergen specific IgE antibodies in their sera. Results indicate that major allergens of A. artemisifolia pollen in dogs are Amb a 1 and Amb a 2. Further characterization of ragweed allergens is needed before they could potentially be used in intradermal testing or allergen immunotherapy in affected dogs. Also, we evaluated new Favrots diagnostic criteria for canine atopic dermatitis in dogs allergic to Ambrosia atremisiifolia pollen. It might be concluded that proposed criteria are of great assistance for seting up suspected diagnosis of canine atopic dermatitis, after ruling out other pruritic dermatoses. Kratka ambrozija (Ambrosia artemisiifolia) je jedan od najčešćih uzročnika alergijskih reakcija izazvanih polenom kod ljudi i pasa. Još uvek nije definisano koji je glavni alergen (i), na koji, većina pasa alergičnih na polen ambrozije, ispoljava pozitivnu reakciju na intradermalnom testu. U ovoj studiji je ispitana senzibilizacija na polen ove biljke kod pasa sa simptomima atopijskog dermatitisa in vivo i in vitro testovima, uključujući intradermalni test i dokazivanje prisustva alergen specifičnih IgE antitela u serumu. Optimizovani su uslovi za detekciju IgE specifičnih antitela iz seruma pasa alergičnih na polen ambrozije imunoblot tehnikom. Psi koji su imali pozitivnu reakciju na polen ove biljke na intradermalnom testu, takođe su imali specifična IgE antitela u serumu. Dobijeni rezultati ukazuju da su glavni alergeni Ambrosia artemisiifolia kod pasa Amb a 1 i Amb a 2. Neophodna je dalja karakterizacija alergena ambrozije kako bi se oni mogli primeniti pri rutinskom intradermalnom testiranju ili u alergen specifičnoj imunoterapiji obolelih pasa. Takođe je razmatrana i validnost Favrotovih dijagnostičkih kriterijuma kod pasa alergičnih na polen ambrozije. Može se zaključiti da su predloženi kriterijumi od velike pomoći u postavljanju suspektne dijagnoze atopijskog dermatitisa pasa, nakon isključenja drugih pruritičnih dermatoza.

Food allergy is defined as an abnormal immunological reaction to food proteins, which causes an adverse clinical reaction. Today 4–6% of children and 1–3% of adults are affected with food allergy in Europe (Mills et al., 2007), while in the USA 6– 8% of children and 4% of adults are affected (Gupta et al., 2008). The incidence of food allergy has been increasing over the years, and to date, no effective treatment of food allergies is available. Therapies involving drugs (antihistamines, decongestants, or steroids) only combat the symptoms of an allergic reaction and do not prevent allergenic reactions due to future exposures to the allergen. Specific immunotherapy (SIT) is the only prophylactic desensitizing therapy for allergy (Bousquet et al., 1998; Durham et al., 1999). However, for food allergies immunotherapy safety-profile is extremely poor because allergic reactions are associated with a significant risk of anaphylactic side-effects (Nelson et al., 1997; Skripak et al., 2008), even with fatal outcome. Therefore, allergen modification resulting in almost complete reduction of IgE-binding, while retaining its immunogenicity, could improve formulations for immunotherapy of food allergies. The best way to prevent unintended exposure to a food allergen is the strict avoidance of foodstuffs containing allergens. Such avoidance may not always be possible, and in certain instances impossible. Furthermore, complete elimination of foodstuffs causing allergy also has negative impact on healthy diet as about 90 % of allergens are present in food of high nutrition value (milk, eggs, fish, shellfish, tree nuts, peanuts, wheat, and soybeans) (Thompson et al., 2006). Therefore production of hypoallergenic food become increasingly indispensable part of food industry with growing demand for methods aimed to reduce allergenic potential of food products. There is increasing number of publications describing successful attempts to reduce allergenicity of food allergens. A number of studies of food processing on allergen reactivity are largely focused on thermal treatments. Although thermal treatments can be used on many products to alter food allergen reactivity, thermal processing has also been known to modify organoleptic properties, including flavor, color, and nutrient content (Holdsworth, 1985). Therefore, novel non-thermal food processing techniques are being explored in order to create hypoallergenic products (Shriver & Yang, 2011). Variable success of methods for allergenicity reduction, due to limited knowledge of allergen and epitope structures and the factors governing their stability, imposes need for an empirical approach (Soler-Rivas & Wichers, 2001). Chemical or enzymatic modification of allergen molecule may alter its structure or physically obstruct binding of IgE antibodies to

BACKGROUND: The major whey protein beta-lactoglobulin (BLG) has been widely studied for its functional properties. The aim of this study was to develop an efficient, inexpensive and rapid one-step method for the isolation and purification of BLG while preserving its native structure. RESULTS: BLGwas purified fromdefattedwheyobtainedfromrawcow's milkbyanionexchangechromatography. Protein purity and identitywere determined using reverse phase high-performance liquid chromatography andmass spectrometry. Total BLG yield was 80% with protein purity from 97 to 99%. BLG isoforms A and B were separated into fractions of 91 and 99% purity respectively. The structure and native conformation of the isolated BLGwere compared with those of standard commercial BLG by circular dichroism spectrometry, susceptibility to various crosslinking enzymes and enzyme-linked immunosorbent assay inhibition. CONCLUSION: Theproposedmethodis veryuseful for the rapid preparationofBLGsuitable for studying antigenicandmolecular characteristics of this protein, aswell as the effect of food processing on these properties. The procedure requires only 1 day for the purification of about 300 mgof BLG from a single run using a small column (2.5 cmx20 cm) of diethylaminoethyl Sephadex and has potential for scaling up. (C) 2011 Society of Chemical Industry This is the peer-reviewed version of the article: (1) Stojadinović, M. M.; Burazer, L. M.; Ercili-Cura, D.; Sancho, A.; Buchert, J.; Ćirković-Veličković, T.; Stanić-Vučinić, D. One-Step Method for Isolation and Purification of Native Beta-Lactoglobulin from Bovine Whey. Journal of the Science of Food and Agriculture 2012, 92 (7), 1432–1440. [https://doi.org/10.1002/jsfa.4722]. Published version: [https://cherry.chem.bg.ac.rs/handle/123456789/1275]

α-Lactalbumin (ALA) is a Ca2+-binding protein which constitutes up to 20% of whey protein. At acidic pH, and in the apo-state at elevated temperatures, ALA is the classic 'molten globule' (MG). This study examined epigallo-catechin-3-gallate (EGCG) binding to ALA in its apo form (apoALA) and stabilizing effect on protein structure thereof. EGCG binds to apoALA in both native and MG state. The complex of EGCG and ALA is more stable to thermal denaturation. The docking analysis and molecular dynamic simulation (MDS) showed that Ca2+ removal decreased conformational stability of ALA, because of the local destabilization of Ca2+-binding region. EGCG binding to apoALA increases its stability by reverting of conformation and stability of Ca2+-binding region. Therefore, EGCG-induced thermal stability of apoALA is based on increased apoALA conformational rigidity. This study implies that during gastric digestion of tea with milk EGCG would remain bound to ALA, albeit in the Ca2+-free form. This is the peer-reviewed version of the following article: Radibratović, M.; Al-Hanish, A.; Minić, S. L.; Radomirović, M. Ž.; Milčić, M. K.; Stanić-Vučinić, D.; Ćirković-Veličković, T. Stabilization of Apo Alpha-Lactalbumin by Binding of Epigallocatechin-3-Gallate: Experimental and Molecular Dynamics Study. Food Chemistry 2019, 278, 388–395. [https://doi.org/10.1016/j.foodchem.2018.11.038]. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4849]

Demand for camel milk (CM) is increasing worldwide, due to its high nutritious value and health benefits. In this study, whole CM powders were produced by spray drying (SD) at six inlet temperatures (190 °C–250 °C) and by freeze drying (FD). Physicochemical and functional properties of CM powder proteins were investigated. SD at higher inlet temperatures (230 °C–250 °C) resulted in higher extent of Maillard reaction (MR), in comparison to lower temperatures (190 °C–200 °C) and FD treatment. Both treatments had negative effect on casein solubility, while whey proteins remained soluble and slightly increased its solubility with the extent of MR. The CM powders obtained at higher inlet temperatures demonstrated improved antioxidant activity. Secondary structure of whey proteins did not differ among the samples, while surface hydrophobicity of whey proteins was higher in all SD than in FD samples, suggesting only limited denaturation of camel whey proteins at higher inlet temperatures of drying. Thus, the effects of SD under the conditions applied in our study did not decrease camel whey protein solubility, while drying procedure itself regardless of temperature decreased solubility of camel milk caseins. MR generated during CM processing could be an important means of compensating for the lack of antioxidant protection normally associated with β-lactoglobulin but happens to be absent from this milk. This is peer-reviewed version of the following article: Perusko, M.; Ghnimi, S.; Simovic, A.; Stevanovic, N.; Radomirovic, M.; Gharsallaoui, A.; Smiljanic, K.; Van Haute, S.; Stanic-Vucinic, D.; Cirkovic Velickovic, T. Maillard Reaction Products Formation and Antioxidative Power of Spray Dried Camel Milk Powders Increases with the Inlet Temperature of Drying. LWT 2021, 143, 111091. [https://doi.org/10.1016/j.lwt.2021.111091]. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4334]