Enzymatic protein hydrolysis of food processing by-products is a well-recognized strategy for producing peptide-rich formulations of added value. The biochemical complexity of this process poses a significant challenge in the use of classical analytical methods as process monitoring tools in both laboratory and industrial setups. In the present study, we are reporting an FTIR-based multivariate approach for monitoring the change in the molecular size distribution of proteins during enzymatic hydrolysis of chicken fillets and processing by-products. For 129 protein hydrolysates, weight-average molecular weight derived from size-exclusion chromatographic analysis was established as a pragmatic measure of the extent of hydrolysis. FTIR spectra acquired from dry films of the hydrolysates were used to build multivariate calibration models using partial least squares (PLS) regression. The best predictions were obtained when the data from two different raw materials, i.e., chicken fillets and mechanical chicken deboning residues, were modeled individually. A good prediction model was also achieved for the combined data from the two raw materials using canonical PLS (CPLS) regression or variable selection. The results from the current study underline the potential of FTIR spectroscopy as a rapid analytical tool for monitoring enzymatic protein hydrolysis of complex raw materials.

The potential of dry-film Fourier-transform infrared (FTIR) measurements as a monitoring tool for enzymatic hydrolysis of protein-based substrates is explored in this study. As a proof-of-concept, the enzymatic digestion of bovine serum albumin using Alcalase was monitored. To evaluate the analytical approach on complex substrates with industrial relevance, salmon- and chicken-based substrates were digested for 80 minutes using Alcalase and a total of 12 FTIR spectra were acquired during the course of the hydrolysis. The observed changes in the IR spectral features as a function of hydrolysis time were found to be in agreement with the breakdown of the amide backbone and formation of amino and carboxylate terminals. Some of the most consistent markers for hydrolysis time were the bands at 1516 cm⁻¹ (–NH₃ ⁺) and ∼1400 cm⁻¹ (–COO⁻). Moreover, principal component analysis (PCA) of the FTIR spectra was used to demonstrate the systematic relationship of the hydrolysis time with key variables (wavelengths) in the protein backbone region (800–1800 cm⁻¹). Scores in the first principal component versus the hydrolysis time have been shown to provide an overview of the process dynamics related to protein structural changes. The herein presented results suggest that dry-film FTIR measurements have potential as a rapid tool for monitoring industrial protein hydrolysis processes.

A study of the effects of single and combined protease hydrolysis on myofibrillar versus collagenous proteins of poultry by-products has been conducted. The aim was to contribute with knowledge for increased value creation of all constituents of these complex by-products. A rational approach was implemented for selecting proteases exhibiting the most different activity towards the major protein-rich constituents of mechanically deboned chicken residue (MDCR). An initial activity screening of 18 proteases on chicken meat, turkey tendons and MDCR was conducted. Based on weight yield, size exclusion chromatography (SEC) and SDS-PAGE, stem Bromelain and Endocut-02 were selected. Studies on hydrolysis of four different poultry by-products at 40 °C, evaluated by protein yield, SEC, and SDS-PAGE, indicate that the proteases’ selectivity difference can be utilized in tailor-making hydrolysates, enriched in either meat- and collagen-derived peptides or gelatin. Three modes of stem Bromelain and Endocut-02 combinations during hydrolysis of MDCR were performed and compared with single protease hydrolysis. All modes of the protease combinations resulted in a similar approximately 15% increase in product yield, with products exhibiting similar SEC and SDS-PAGE profiles. This shows that irrespective of the modes of combination, the use of more than one enzyme in hydrolysis of collagen-rich material can provide means to increase the total protein yield and ultimately contribute to increased value creation of poultry by-products.

Inhibition of dipeptidyl peptidase IV (DPP-IV) and stimulation of muscle glucose uptake are two of the key strategies for management of type-2-diabetes (T2D). In the present study, four protein hydrolysates generated by enzymatic hydrolysis of chicken by-product, i.e., mechanical chicken deboning residue, were evaluated for their DPP-IV inhibitory activity as well as their effect on glucose uptake by skeletal muscle cells. The DPP-IV inhibitory assay was performed at two concentrations (1000 μg mL⁻¹ and 10 μg mL⁻¹) for the crude chicken protein hydrolysates. The hydrolysate with the highest DPP-IV inhibition was selected for preparative-scale fractionation using size-exclusion chromatography (SEC). The SEC fractions were tested for DPP-IV inhibitory activity as well as their effect on glucose uptake and metabolic activity of skeletal muscle cells. The muscle cells were treated with the SEC fractions and glucose uptake was measured based on luminescence detection of 2-deoxyglucose-6-phosphate (2DG6P). A fraction with peptides in the lower molecular weight range was shown to promote glucose uptake and to inhibit DPP-IV. Further chromatographic fractionation followed by inhibition assaying of the most potent SEC fraction led to isolation of five refined peptide fractions with more than 80% DPP-IV inhibition, which were subsequently analyzed with LC-HRMS/MS. This led to identification of 14 peptides as potential DPP-IV inhibitors from protein hydrolysates of mechanical chicken deboning residue.