Exploring Effects of Protease Choice and Protease Combinations in Enzymatic Protein Hydrolysis of Poultry By-Products.

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.

(Acetonitrile){2-[bis-(pyridin-2-ylmethyl-κ(2)N)amino-κN]-N-(2,6-dimethyl-phen-yl)acetamide-κO}(perchlorato-κO)zinc (acetonitrile){2-[bis-(pyridin-2-ylmethyl-κ(2)N)amino-κN]-N-(2,6-dimethyl-phen-yl)acetamide-κO}zinc tris-(perchlorate).

In the title salt, [Zn(C(22)H(24)N(4)O)(CH(3)CN)][Zn(ClO(4))(C(22)H(24)N(4)O)(CH(3)CN)](ClO(4))(3), two differently coordinated zinc cations occur. In the first complex, the metal ion is coordinated by the N,N',N'',O-tetra-dentate acetamide ligand and an acetonitrile N atom, generating an approximate trigonal-bipyramidal coordination geometry, with the O atom in an equatorial site and the acetonitrile N atom in an axial site. In the second complex ion, a perchlorate ion is also bonded to the zinc ion, generating a distorted trans-ZnO(2)N(4) octa-hedron. Of the uncoordinating perchlorate ions, one lies on a crystallographic twofold axis and one lies close to a twofold axis and has a site occupancy of 0.5. N-H⋯O and N-H⋯(O,O) hydrogen bonds are observed in the crystal. Disordered solvent mol-ecules occupy about 11% of the unit-cell volume; their contribution to the scattering was removed with the SQUEEZE routine of the PLATON program [Spek (2009 ▶). Acta Cryst. D65, 148-155.].

FTIR-based prediction of collagen content in hydrolyzed protein samples.

Fourier transform infrared spectroscopy (FTIR) is a powerful analytical tool that has been used for protein and peptide characterization for decades. In the present study, the objective was to investigate if FTIR can be used to predict collagen content in hydrolyzed protein samples. All samples were obtained from enzymatic protein hydrolysis (EPH) of poultry by-products providing a span in collagen content from 0.3% to 37.9% (dry weight), and the FTIR analysis was performed using dry film FTIR. Since nonlinear effects were revealed by calibration using standard partial least squares (PLS) regression, Hierarchical Cluster-based PLS (HC-PLS) calibration models were constructed. The HC-PLS model provided a low prediction error when validated using an independent test set (RMSE = 3.3% collagen), while validation using real industrial samples also showed satisfying results (RMSE = 3.2%). The results corresponded well with previously published FTIR-based studies of collagen, and characteristic spectral features for collagen were well identified in the regression models. Covariance between collagen content and other EPH related processing parameters could also be ruled out in the regression models. To the authors' knowledge, this is the first time that collagen content has been systematically studied in solutions of hydrolysed proteins using FTIR. This is also one of few examples where FTIR is successfully used to quantify protein composition. The dry-film FTIR approach presented in the study is expected to be an important tool in the growing industrial segment that is based on sustainable utilization of collagen-rich biomass.

Production of food-grade microcarriers based on by-products from the food industry to facilitate the expansion of bovine skeletal muscle satellite cells for cultured meat production.

A major challenge for successful cultured meat production is the requirement for large quantities of skeletal muscle satellite cells (MuSCs). Commercial microcarriers (MCs), such as Cytodex®1, enable extensive cell expansion by offering a large surface-to-volume ratio. However, the cell-dissociation step post cell expansion makes the cell expansion less efficient. A solution is using food-grade MCs made of sustainable raw materials that do not require a dissociation step and can be included in the final meat product. This study aimed to produce food-grade MCs from food industry by-products (i.e., turkey collagen and eggshell membrane) and testing their ability to expand bovine MuSCs in spinner flask systems for eight days. The MCs' physical properties were characterized, followed by analyzing the cell adhesion, growth, and metabolic activity. All MCs had an interconnected porous structure. Hybrid MCs composed of eggshell membrane and collagen increased the mechanical hardness and stabilized the buoyancy compared to pure collagen MCs. The MuSCs successively attached and covered the entire surface of all MCs while expressing high cell proliferation, metabolic activity, and low cell cytotoxicity. Cytodex®1 MCs were included in the study. Relative gene expression of skeletal muscle markers showed reduced PAX7 and increased MYF5, which together with augmented proliferation marker MKI67 indicated activated and proliferating MuSCs on all MCs. Furthermore, the expression pattern of cell adhesion receptors (ITGb5 and SDC4) and focal adhesion marker VCL varied between the distinct MCs, indicating different specific cell receptor interactions with the various biomaterials. Altogether, our results demonstrate that these biomaterials are promising prospects to produce custom-fabricated food-grade MCs intended to expand MuSCs.

Post-enzymatic hydrolysis heat treatment as an essential unit operation for collagen solubilization from poultry by-products.

Enzymatic protein hydrolysis (EPH) is an invaluable process to increase the value of food processing by-products. In the current work the aim was to study the role of standard thermal inactivation in collagen solubilization during EPH of poultry by-products. Hundred and eighty hydrolysates were produced using two proteases (stem Bromelain and Endocut-02) and two collagen-rich poultry by-products (turkey tendons and carcasses). Thermal inactivation was performed with and without the sediment to study the effect of heat on collagen solubilization. A large difference in molecular weight distribution profiles was observed when comparing hydrolysate time series of the two proteases. In addition, it was shown that 15 min heat treatment, conventionally used for inactivating proteases, is essential in solubilizing collagen fragments, which significantly contributes to increasing the protein yield of the entire process. The study thus demonstrated the possibility of producing tailored products of different quality by exploiting standard heat inactivation in EPH.

Fourier-transform infrared spectroscopy for monitoring proteolytic reactions using dry-films treated with trifluoroacetic acid.

In this study we explore the potential of using Fourier-transform infrared (FTIR) spectra of trifluoroacetate-protein and peptide complexes for monitoring proteolytic reactions. The idea of treating dry-films of protein hydrolysates with trifluoroacetic acid (TFA) prior to FTIR analysis is based on the unique properties of TFA. By adding a large excess of TFA to protein hydrolysate samples, the possible protonation sites of the proteins and peptides will be saturated. In addition, TFA has a low boiling point when protonated as well as complex-forming abilities. When forming TFA-treated dry-films of protein hydrolysates, the excess TFA will evaporate and the deprotonated acid (CF3COO-) will interact as a counter ion with the positive charges on the sample materials. In the study, spectral changes in TFA-treated dry-films of protein hydrolysates from a pure protein and poultry by-products, were compared to the FTIR fingerprints of untreated dry-films. The results show that time-dependent information related to proteolytic reactions and, consequently, on the characteristics of the protein hydrolysates can be obtained. With additional developments, FTIR on dry-films treated with TFA may be regarded as a potential future tool for the analysis of all types of proteolytic reactions in the laboratory as well as in industry.

Average molecular weight, degree of hydrolysis and dry-film FTIR fingerprint of milk protein hydrolysates: Intercorrelation and application in process monitoring.

Fourier-transform infrared (FTIR) spectroscopy was applied to predict the degree of hydrolysis (DH%) and weight-average molecular weight (Mw) in milk protein hydrolysates. Both DH% and Mw are important quality parameters of protein hydrolysates. Measuring these parameters and following their development during proteolytic reactions is therefore essential for process control and optimization in industry. In the present study the intercorrelation and the complimentary nature of these parameters were investigated and a partial least squares regression (PLSR) model was developed for the prediction of DH% from molecular weight distributions. Finally, we developed PLSR models based on dry-film FTIR spectroscopy for the prediction of both DH% and Mw. Here spectral changes in the amide region were found to be important for the two calibration models, underlining the advantage of dry-film FTIR measurement. This shows that dry-film infrared spectroscopy is a promising tool for dual prediction of DH% and Mw.

FTIR-based hierarchical modeling for prediction of average molecular weights of protein hydrolysates.

In the presented study, Fourier-transform infrared (FTIR) spectroscopy is used to predict the average molecular weight of protein hydrolysates produced from protein-rich by-products from food industry using commercial enzymes. Enzymatic protein hydrolysis is a well-established method for production of protein-rich formulations, recognized for its potential to valorize food-processing by-products. The monitoring of such processes is still a significant challenge as the existing classical analytical methods are not easily applicable to industrial setups. In this study, we are reporting a generic FTIR-based approach for monitoring the average molecular weights of proteins during enzymatic hydrolysis of by-products from the food industry. A total of 885 hydrolysate samples from enzymatic protein hydrolysis reactions of poultry and fish by-products using different enzymes were studied. FTIR spectra acquired from dry-films of the hydrolysates were used to build partial least squares regression (PLSR) models. The most accurate predictions were obtained using a hierarchical PLSR approach involving supervised classification of the FTIR spectra according to raw material quality and enzyme used in the hydrolysis process, and subsequent local regression models tuned to specific enzyme-raw material combinations. The results clearly underline the potential of using FTIR for monitoring protein sizes during enzymatic protein hydrolysis in industrial settings, while also paving the way for measurements of protein sizes in other applications.

Crystal structures of di-bromido-{N-[(pyridin-2-yl-κN)methyl-idene]picolinohydrazide-κ2N',O}cadmium methanol monosolvate and di-iodido{N-[(pyridin-2-yl-κN)methyl-idene]picolinohydrazide-κ2N',O}cadmium.

The title compounds, [CdBr2(C12H10N4O)]·CH3OH, (I), and [CdI2(C12H10N4O)], (II), are cadmium bromide and cadmium iodide complexes of the ligand (E)-N'-(pyridin-2-yl-methyl-ene)picolinohydrazide. Complex (I) crystallizes as the methanol monosolvate. In both compounds, the Cd2+ cation is ligated by one O atom and two N atoms of the tridentate ligand, and by two bromide anions forming a Br2N2O penta-coordination sphere for (I), and by two iodide anions forming an I2N2O penta-coordination sphere for (II), both with a distorted square-pyramidal geometry. In the crystal of complex (I), mol-ecules are linked by pairs of N-H⋯O and O-H⋯Br hydrogen bonds, involving the solvent mol-ecule, forming dimeric units, which are linked by C-H⋯Br hydrogen bonds forming layers parallel to (101). In the crystal of complex (II), mol-ecules are linked by N-H⋯I hydrogen bonds, forming chains propagating along [010]. In complex (II), measured at room temperature, the two iodide anions are each disordered over two sites; the refined occupancy ratio is 0.75 (2):0.25 (2).