FTIR as a rapid tool for monitoring molecular weight distribution during enzymatic protein hydrolysis of food processing by-products

Wubshet, Sileshi Gizachew; Måge, Ingrid; Böcker, Ulrike; Lindberg, Diana; Knutsen, Svein Halvor; Rieder, Anne; Airado‐Rodríguez, Diego; Afseth, Nils Kristian

doi:10.1039/c7ay00865a

Cited by 60 publications

(43 citation statements)

References 33 publications

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“…The release of peptides with a lag could be seen from the experimental dataset for the tryptic proteolysis of β-LG [23]. These peptides were: f(136-138), f(125-135), f (15)(16)(17)(18)(19)(20), f(79-83), f(21-40).…”

Section: Prediction Of the Kinetics Of Peptide Releasementioning

confidence: 99%

“…An alternative to the degree of hydrolysis could be the degree of changes in a secondary structure and the conformation of protein substrate, as measured by an appropriate spectral technique. In recent years, some progress has been made in comparing spectral data obtained at different times of hydrolysis using fluorescence [12,13] and infrared spectroscopies [14][15][16][17]. It was demonstrated that the degradation of protein substrate can be quantified by a shift in tryptophan fluorescence [12,13].…”

Section: Introductionmentioning

confidence: 99%

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Proteolysis of β-lactoglobulin by Trypsin: Simulation by Two-Step Model and Experimental Verification by Intrinsic Tryptophan Fluorescence

Vorob’ev

2019

Symmetry

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To distinguish differences in enzymatic hydrolysis of various proteins, we propose an algorithm using a dataset of fluorescence spectra obtained at different moments of hydrolysis t. This algorithm was demonstrated in the example of β-lactoglobulin (β-LG) proteolysis by trypsin. The procedure involved processing the spectra to obtain the wavelength of the maximum fluorescence λmax, which was found to be proportional to the fraction of tryptophanes in hydrated proteolysis products (demasked tryptophanes). Then, the dependence λmax(t) was fitted by biexponential function with two exponential terms, one of which was responsible for the fast part of the fluorescence change during proteolysis. The contribution of this term was quite different for various protein substrates—it was positive for β-LG and negative for β-casein. The observed differences in proteolysis of different substrates were explained by different demasking processes. Combining the fluorescence data with the degrees of hydrolysis of peptide bonds allowed us to analyze the hydrolysis of β-LG in the framework of the two-step proteolysis model and estimate the ratio of rate constants of demasking and hydrolysis and the percentages of initially masked and resistant peptide bonds. This model predicted the existence of a bimodal demasking process with a fast part at the beginning of proteolysis and lag-type kinetics of release for some peptides. Compared with monitoring proteolysis in terms of the degree of hydrolysis only, the fluorescence data are helpful for the recognition of proteolysis patterns.

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Section: Prediction Of the Kinetics Of Peptide Releasementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Proteolysis of β-lactoglobulin by Trypsin: Simulation by Two-Step Model and Experimental Verification by Intrinsic Tryptophan Fluorescence

Vorob’ev

2019

Symmetry

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“…Enzymatic hydrolysis and sampling. Hydrolysis samples produced using raw materials from poultry were hydrolysed according to the description by Wubshet et al 11 . The BSA hydrolysates were produced by dissolving 5% (w/v) substrate of BSA in tap water.…”

Section: Raw Materialsmentioning

confidence: 99%

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

Kristoffersen

Amerongen

Böcker

et al. 2020

Sci Rep

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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 dryfilms of protein hydrolysates, the excess TFA will evaporate and the deprotonated acid (CF 3 coo −) 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. Fourier-transform infrared (FTIR) spectroscopy is among the well-established methods for the characterisation of proteins and peptides. The repeated amino acid building blocks constructing the backbone of proteins and peptides give rise to multiple distinctive infrared absorption bands (i.e. the amide bands), containing both chemical and structural information. FTIR is therefore frequently used to study and quantify secondary structures of proteins 1-3. At the same time, the protein information in the FTIR spectra has opened the possibility to study a wide range of protein-derived quality features. This includes parameters such as hydration and solvent effects, pH, peptide size distribution and degree of hydrolysis (DH%) 4-9. Currently, peptide size distribution and DH% are measured using laborious and time-consuming techniques. FTIR, on the other hand, represents a rapid alternative which is applicable to an industrial setting. Recent studies have shown that FTIR spectroscopy can be used to predict parameters such as weight-average molecular weight (M w) derived from the peptide size distribution and DH% with high accuracy 10-12. In recent years, enzymatic protein hydrolysis (EPH) has gained significant attention as a versatile processing technology for protein-rich raw materials. For instance, several value-added peptide products have been developed employing EPH on food-processing by-products. In EPH, proteolytic enzymes are used to break down the proteins into smaller fragments, and the resulting protein hydrolysates are very complex, consisting of thousands

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“…The enzymatic hydrolysis of a protein is a complex process because of the high specificity of protease and the diversity of the substrates involved [ 85 ], accessibility of the peptide bonds [ 86 ] and sensitivity to hydrolysis conditions [ 87 ]. Regardless of the complex factors, the reaction pathway of the enzymatic hydrolysis of a protein may be simply presented as below [ 88 ]:

…”

Section: High-resolution Ultrasonic Spectroscopy (Hr-us)mentioning

confidence: 99%

Advances in Analysis of Milk Proteases Activity at Surfaces and in a Volume by Acoustic Methods

Dizon

Tatarko

Hianik

2020

Sensors

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This review is focused on the application of surface and volume-sensitive acoustic methods for the detection of milk proteases such as trypsin and plasmin. While trypsin is an important protein of human milk, plasmin is a protease that plays an important role in the quality of bovine, sheep and goat milks. The increased activity of plasmin can cause an extensive cleavage of β-casein and, thus, affect the milk gelation and taste. The basic principles of surface-sensitive acoustic methods, as well as high-resolution ultrasonic spectroscopy (HR-US), are presented. The current state-of-the-art examples of the application of acoustic sensors for protease detection in real time are discussed. The application of the HR-US method for studying the kinetics of the enzyme reaction is demonstrated. The sensitivity of the acoustics biosensors and HR-US methods for protease detection are compared.

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FTIR as a rapid tool for monitoring molecular weight distribution during enzymatic protein hydrolysis of food processing by-products

Abstract: An FTIR-based multivariate approach is developed for monitoring molecular weight distribution during enzymatic protein hydrolysis of byproducts.

Cited by 60 publications

References 33 publications

Proteolysis of β-lactoglobulin by Trypsin: Simulation by Two-Step Model and Experimental Verification by Intrinsic Tryptophan Fluorescence

Proteolysis of β-lactoglobulin by Trypsin: Simulation by Two-Step Model and Experimental Verification by Intrinsic Tryptophan Fluorescence

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

Advances in Analysis of Milk Proteases Activity at Surfaces and in a Volume by Acoustic Methods

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