Combining FTIR-based Screening with Ligand Fishing Technologies for Facilitated Discovery of Antidiabetic Peptides in Protein Hydrolysates

Protein hydrolysates from industrial by-products are valuable sources of peptides of both nutritional and pharmaceutical interest. The current project aims at developing a technology that enables facilitated discovery of health promoting peptides from protein-rich by-products. The technology is based on novel combination of (i) Fourier-transform infrared (FTIR) spectroscopy-based rapid screening and (ii) advanced biochemical detection technologies based on ligand fishing and high-resolution bioassays. Different therapeutic targets related to diabetes have been investigated. These includes alpha-glucosidase, alpha-amylase, protein-tyrosine phosphatase 1B (PTP-1B) and dipeptidyl peptidase IV (DPP-IV). DPP-IV, a well-established therapeutic target for blood sugar regulation, was found to be one of the most promising therapeutic targets for food-derived bioactive peptides. Our extensive screening led to identification of a chicken protein hydrolysate with a potent activity towards DPP-IV. In order to discover the peptides responsible for the observed activity, we have developed and applied a chromatography-coupled bioassay. This led to identification of new peptides with potential antidiabetic activity. The above stated findings are published in the scientific journal Food and Function. Additionally, we have investigated HMG-CoA reductase as a potential therapeutic target for evaluating cholesterol lowering potential of peptides produced from by-products. Our preliminary results show that peptides produced from chicken by-product can inhibit HMG-CoA reductase and, therefore, holds a promising potential as cholesterol lowering agents. Bioactivity screenings against therapeutic targets alpha-glucosidase, alpha-amylase and PTP-1B did not result in positive outcomes. The project was also devoted to developing magnetic ligand fishing as novel technology for facilitated discovery of bioactive peptides. To this end, we have successfully developed angiotensin-converting enzyme (ACE) immobilized on magnetic nanoparticles as a tool for selectively fishing blood pressure lowering peptides from complex protein hydrolysates. Additionally, a novel bio-screening approach based on complementary inhibition and affinity profiles have been developed by combined magnetic ligand fishing and high-resolution inhibition profiling. This technology was demonstrated for the type-2-diabetes therapeutic target alpha-glucosidase and findings are now published on the scientific journal Talanta. In addition, we have successfully immobilized the enzyme DPP-IV onto superparamagnetic beads. The immobilized DPP-IV was demonstrated as a powerful tool for fishing antidiabetic constituents from a model sample (i.e., lingonberry extract). This study represents the first example of ligand fishing using an alkaline protease and a scientific article related to the work is now published in PLoS ONE. Despite successful development of the ligand fishing technologies, our effort to use them for discovery of peptides from hydrolysates was unsuccessful. This was largely due to high degree of none-specific affinity of peptides to the solid support. Therefore, based on our studies so far, we concluded that ligand fishing was better suited for sample matrices such as polyphenols and challenges related none-specific bindings should be borne in mind when considering applications for peptides. One of the sub-aims of the project was to develop FTIR spectroscopy as a rapid screening tool for bioactive peptides. To this end, we have produced a library of 120 different protein hydrolysates from chicken by-products using different process settings. All of these hydrolysates have been screened for three bioactivities: DPP-IV inhibition, ACE inhibition and antioxidant activity. All of the hydrolysates were also fingerprinted using FTIR spectroscopy. Finally, we have successfully developed multivariate models to correlate chemical fingerprints (i.e., FTIR spectra) with the observed bioactivities. These models were demonstrated as valuable tools for guiding selection of potent protein hydrolysates as well as subsequent fractionation and identification. A manuscript summering these results is now in preparation for publication. Similarly, another set of 60 hydrolysates were produced from milk protein and fingerprinted using chromatography and FTIR. Intercorrelation of this with quality parameters such as antioxidant activity and degree of hydrolysis was studied. An article summarizing findings results related to correlation of FTIR and quality parameters was published in Food Chemistry. Overall, we have successfully developed several advanced bioanalytical technologies for discovery and characterization of bioactive constituents in complex mixtures. In addition, the project has demonstrated that bioactive peptides derived from food processing by-products holds promising potential as health promoting ingredients.

PepFishing has inspired a new Collaborative and Knowledge-building Project funded by the research council of Norway (NFR/320086). This project is based on findings in PepFishing related to bioactive peptides. Additionally, PepFishing enabled Nofima (the host institution) to strengthen its strategic initiative related to peptide (Peptec) and analytical (Spectec) technologies by adding an important dimension related to health. Findings in PepFishing have also laid the foundation for future development of high value ingredients (i.e., health promoting peptides) form underutilized by-products from the food industry. The project contributed significantly to the personal career development of the project manager through experience and building a track record. Among the positive consequences of this was the project manager have received 2 new grants (NFR/320086 and MAIBIT/UB0077) as a project managed and 1 new grant as a co-applicant (NFR/312136) related to the field of bioactive peptides.

Protein hydrolysates, produced from food processing byproducts through enzymatic hydrolysis, are invaluable sources of peptides with potential health promoting effect, including antidiabetic activity. However, inherent complexity of food processing byproducts and the chemical changes introduced through enzymatic hydrolysis poses a significant challenge in discovery of antidiabetic peptides. Therefore, a specialized technology for screening and identification of bioactive principles in protein hydrolysates is highly needed in product development. The current project aims at developing a bioanalytical platform combining: (i) Fourier-transform infrared (FTIR) spectroscopy-based rapid screening and (ii) advanced biochemical detection technologies (i.e., ligand fishing and high-resolution bioassay) for facilitated identification of bioactive peptides in protein hydrolysates. This bioanalytical platform will be used to screen protein hydrolysate from poultry and dairy processing byproducts for potential antidiabetic peptides. Chicken carcasses both before and after mechanical de-boning as well as whey will be used as major sources of protein. Both whey and chicken protein hydrolysates have been associated with antidiabetic activities, hence there selection in the current project as relevant protein sources. A successful implementation of this technology will support both Norwegian and international biotech companies to achieve an efficient and high-quality screening program for bioactive constituents in complex food product. While collaboration with two Norwegian food processing industries (Tine and Norilia) provides access to wide range of byproducts, partnership with University of Copenhagen, Denmark and National Institute of Health, USA ensures internationalization.