PROJECTS

Probiotic microorganisms are known to be very important for intestinal health. Although these microorganisms affect the health positively, they have high sensitivity to various factors. Encapsulation techniques are used to increase the resistance of probiotics towards different conditions. Electrospinning is one of the encapsulation methods used for probiotic microorganisms. In this project, polyvinyl alcohol (PVA) ̶ cellulose acetate (CA) hybrid fibers (PVA/CA fibers) were produced by using angled dual nozzle electrospinning system. Hybrid fibers were produced as a result of overlapping fiber jets. The aim of this study is to produce encapsulation material, resistant to digestive system conditions by using CA. CA was used for the first time for probiotic encapsulation with this project. The toxic effects of CA solvents was prevented by angled double nozzle electrospinning system to avoid the contact of the solvent with the microorganism. Using the developed system, a model probiotic, Escherichia coli Nissle 1917 (EcN), which was inoculated into PVA solution, was encapsulated in hybrid fibers without exposure to the toxic solvent of CA. It was observed that higher viability results were obtained from PVA/CA-EcN fibers, compared to not only free EcN but also kontrol group at in vitro simulation. The results of the study showed that PVA/CA fibers provided remarkeble resistance to digestive system conditions, which is a critical issue for the encapsulation of probiotic microorganisms. This project was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK), project no: 118O860.

The emergence of pathogenic bacterial strains resistant to antibiotics is a major human health threat. In order to overcome this critical problem, it is in urgent need of developing novel antimicrobial agents those either kill or inhibit the growth of bacteria. Most of the antibiotics, commercially available, attack specific target molecules in the cell by similar mechanisms. Targeting the same molecules for the antimicrobial properties limits the development of new antibiotics and facilitates the appearance of resistance strains to the new antibiotic. Therefore, the discovery of new target molecules, that will affect vital functions and/or the pathogenicity of the microorganisms, has been considered as an effective strategy for the development of new antimicrobial agents. Sortase enzymes, found in Gram-positive bacteria, appear as a new target molecule which can be used in the development of antimicrobial agents. Sortase enzymes, a member of transpeptidase family, significantly contribute to the infectivity of bacteria by anchoring the surface proteins of gram-positive bacteria to the cell wall. Although many Gram positive bacteria possess different isoforms of sortase, among them, sortase A (Srt A) is conserved in all pathogenic species. It has been shown that the infectivity of bacteria decreased for those having SrtA gene mutation. As a result, agents, inhibiting the SrtA enzyme activity, have a great potential of showing antimicrobial activity against Gram-positive pathogens. Hence, the development of effective inhibitors against SrtA has become a popular subject for researchers. We aimed to develop novel peptide inhibitors that selectively and effectively inhibit the SrtA enzyme by using high diversity phage displayed peptide libraries. So that, a new antimicrobial peptide will be improved against SrtA which is a different target molecule from those used in the literature. This project was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK) Project No: 114Z947.

Plastics can be found in food packaging, shopping bags, and household items, such as toothbrushes and pens, and facial cleansers. Nanoplastics, having internal or surface structure in the nanosize or any dimension in the nanosize, can be produced by fragmentation or dissociation of microplastic debris. As a result of the widespread use of plastics, human body is exposed to nanoparticles by inhalation, percutaneous or by foods. Some characteristic properties of nanoparticles such as aggregation behavior, morphology and corona structure (observed when biomolecules are adsorbed on nanoparticles) determine the toxicity (cytotoxicity, genotoxicity) based on the reactivity of these nanoparticles. For this reason, it is crucial to determine these characteristic properties to assess the toxicity of the nanoparticles. The characteristic properties of nanoplastics may differ greatly when consumed with foods that may also affect the toxicological properties. Here, we aimed to investigate the interaction between food matrix and nanopolystyrene (one of the most widely used plastics) and determine the effects of these interactions on the particle reactivity. Within the project, nanoplastics having different diameters and concentrations will be incubated with the selected food matrices and the characteristic properties affecting the reactivity of the particles will be determined. Biomimetic 3D-intestinal model will be applied to investigate the translocation and toxic effects of nanoplastics that might potentially cause to the intestinal barrier. Furthermore, the in vivo biodistribution of nanoplastics is another our focus to support the potential risk of nanoplastics in human health and also environments. The safety information of nanoplastics in both 3D-intestinal model and zebrafish model will be important data in food safety aspects. This is a joint project of Turkish and Thai teams (collaborator Dr. Sasitorn Aueviriyavit, NANOTEC, NSTDA) supported by both TÜBİTAK and NSTDA.

Biogenic amines are formed in foods mainly by the microbial decarboxylation of certain amino acids. The presence of biogenic amines in foods poses a threat to public health due to their physiological and toxicological effects after consumption. One of the most frequent biogenic amines in foods, histamine arises by the growth of microorganisms having histidine decarboxylase activity. Thus, the foods having free histidine, such as fish, cheese, wine, can contain high level of histamine. Rapid and sensitive quantification of histamine in food samples has vital importance in terms of human health and food safety. Bioassays and biosensors in which histamine specific enzymes or antibodies are used as biological recognition agents, allow rapid, sensitive and easy detection of histamine. Labor and costly production of biological recognition agents and their instability against environmental conditions enable the development of artificial recognition agents to be used in bioassays. Phage display technology has been used frequently in the selection and development of peptides that show affinity to various target molecules. Phage display peptide libraries not only provide the development of artificial recognition molecules which selectively bind to proteins such as receptors, enzymes, antibodies and toxins, but also used in analysis of protein-protein interactions and mapping specific regions of the protein. Development of peptide ligands by phage display peptide libraries against small molecules such as histamine, enables to overcome the difficulties in antibody production against these molecules having no immune response. In the proposed project, it is aimed to develop artificial recognition molecules against histamine which is the cause of food borne chemical intoxication and use these molecules in biosensor that would be developed for the detection of histamine. In this study, we aimed to select histamine binding phages by using 12 mer phage display peptide library. For this purpose, histidine having a similar structure with histamine, was eliminated in the final cycle of the panning to satisfy the selectivity of the binding phages. Totally, 41 phage clones were isolated, and the binding properties of the selected clones were investigated by phage-ELISA. The variable 4 peptides region of the phage clones having high affinity to histamine, was determined and the peptides were synthesized by solid phase method. The interaction of synthesized peptides with histamine were analyzed by isothermal titration calorimetry, the affinity constant and selectivity of the peptides against histamine was determined. In the last stage of project, surface plasmon resonance-based biosensor will be developed for the detection of histamine by using peptide showing highest affinity and selectivity against histamine. Linearity, limit of detection and limit of quantification of the biosensor will be determined and the selectivity will be examined. Besides, the response of the biosensor against the food samples containing histamine will be investigated in order to state the performance of the developed biosensor in food samples. The propose project will be the first study in literature about the development of artificial recognition agents against histamine. The development of peptide ligands against histamine by phage display peptide libraries will reveal that it would be possible to develop artificial recognition molecules against similar haptens having low molecular weights. The peptides developed at the end of this project can be used in various bioassay and biosensor systems for the detection of histamine. In the scope of this project, artificial recognition molecules binding specifically to histamine will be used in surface plasmon resonance-based biosensor application and the usability of the peptide ligands in bioassay and biosensors will be investigated. In our laboratory, a significant infrastructure has been established about detection of selective peptide sequences to target molecules by the phage display method. With the proposed project, this knowledge will be used on a popular subject, namely the development of artificial recognition molecules having vital importance for human health and food safety. In the scope of this proposed study, significant scientific data will be produced which will lead to new projects. This project was founded by Hacettepe University Scientific Research Projects Coordination Unit for two year. (2018-2020)

Phage display technique has been a useful tool to select and develop inhibitors of enzymes which is of significance for pharmacology and medicine field. In this study, this technique is used to develop a specific and novel peptidic inhibitor against a food enzyme which its activity in milk causes undesirable results. A phage display peptide library was screened against plasmin and the peptides inhibiting target enzyme were selected. The peptides were modified by deleting preferential cleavage site for plasmin and the inhibitory effects of peptides on plasmin activity were investigated. Additionally, the kinetic parameters such as inhibitory constants and types of peptides was found by fluorometric measurements. The determination of affinity constant and driving force of interaction between peptides and plasmin was done with isothermal titration calorimetry (ITC). The selected peptide was highly specific for plasmin as it showed no inhibitory effect on trypsin which belongs to the same protease family with plasmin. Further, the inhibitor activity of the peptide in milk sample was investigated. The results showed that the developed peptide is a promising inhibitor agent against plasmin to be used in food samples. This Project was supported by The Scientific and Technological Research Council of Turkey (TUBİTAK) with Project no: 113O786

Engineered nanoparticles have been used in different fields such as medicine, physics, chemistry and agriculture to solve the stability and efficiency problems. These particles, engineered to have uniform shape, dimension and surface properties, can provide various functions such as specialized catalytic activity, enhanced power, thermal and electrical conductivity and drug targeting. As a result of the widespread use of nanotechnology, human body is exposed to engineered nanoparticles by inhalation, percutaneous or by foods. The hazards of some inorganic nanoparticles (silver, iron, zinc) used as food additives on health have been revealed. However, titanium dioxide (TiO2) and silicon dioxide (SiO2) are widely found in foods which are especially consumed by children of age 0-6 including the baby formulas. There are limited number of studies regarding the changes of TiO2 and SiO2 nanoparticles during digestion when consumed with foods and the effects of these changes on human health. And in these studies, the changes in the nanoparticles due to the interaction with food matrix had not been investigated and extensive toxicity tests had not been performed. In summary, the correlation between the toxicity and the changes in the properties of nanoparticles had not been stated when these particles are consumed with foods and digested. Some characteristic properties of nanoparticles such as aggregation behavior, morphology and corona structure (observed when biomolecules are adsorbed on nanoparticles) determine the toxicity (cytotoxicity, genotoxicity) based on the reactivity of these nanoparticles. For this reason, it is crucial to determine these characteristic properties to assess the toxicity of the nanoparticles. The characteristic properties of SiO2 and TiO2 nanoparticles may differ greatly when consumed with foods that may also affect the toxicological properties. In the proposed study, it is aimed to investigate the interaction between food matrix and TiO2/SiO2 nanoparticles (individually and coupled) and determine the effects of these interactions on the particle reactivity. Milk was chosen as the real food sample and in order to investigate the effects of different food components whole milk, skimmed milk and milk serum will be used in the study. Within the project, SiO2 and TiO2 nanoparticles having different diameters and concentrations will be incubated with the selected food matrices and the characteristic properties affecting the reactivity of the particles will be determined then the cytotoxic effects of digested nanoparticles will be analyzed by cell culture method and in vivo animal testing. The proposed project will be the first study about the investigation of the changes of frequently used food additives, TiO2 and SiO2 nanoparticles when the particles are exposed to a mixture having all of the fundamental food components. In addition, the alteration in the absorption and the toxicity of nanoparticles due to the effect of food matrix will be determined by in-vitro and in-vivo tests. The data obtained from the proposed project will be used to fill a significant gap in the literature by not only investigating the interaction between nanoparticles and food matrix from the point of nanoparticle characteristics and molecular interactions, but also revealing the alterations during digestion and evaluating the changes in in-vivo toxicity of nanoparticles. This project was supported by The Scientific and Technological Research Council of Turkey (TUBİTAK) with Project no: 119O505

Beta-lactoglobulin (β-Lg) is a globular milk protein and a major component of whey with approximately 50% ratio by dry weight. β-Lg strongly binds to various hydrophobic ligands and may act as a transporter for these molecules. Monomeric β-Lg has two disulfide bonds and one free thiol group that play essential roles in the formation of tertiary structures of β-Lg. As a result, β-Lg reacts to pH changes in a very specific manner. In this study, isothermal titration calorimetry (ITC) and circular dichroism (CD) were used to investigate the interaction between β-Lg and allyl isothiocyanate (AITC), a well-known anticarcinogen, at different pH values (pH 3.0, 6.5 and 8.5). The interaction was characterized with attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and molecular docking. The final thermograms from ITC were fitted to three sites of a sequential binding model (Koshland model), revealing the high affinity between β-Lg and AITC with binding constants of 104-105 M−1. ITC and CD results revealed that AITC binding at different pH changes the secondary structure of β-Lg. FTIR results showed the binding of AITC with strong isothiocyanate peaks near 1900-2150 cm−1. Three different AITC-binding sites to β-Lg were confirmed using molecular docking. The results showed that the characteristic properties of the interaction between β-Lg and AITC can be used to predict the nanotransporter capacity of β-Lg for bioactive materials at different pH values.