Hydrolysis Of Samples Research Articles

Hydrolysis of poly(ester urethane): In-depth mechanistic pathway determination through thermal and chemical characterization

Many structure/property relationships of hydrolyzed poly(ester urethane) (PEU) – a thermoplastic – have been reported. Examples include changes in molecular weight vs. elongation at break and crosslink density vs. mechanical strength. However, the effect of molecular weight (or molar mass) reduction on some physical, thermal, and chemical properties of hydrolyzed PEU have not been reported. Therefore, a large set of hydrolyzed PEU (Estane®5703) samples were obtained from two aging experiments: 1) accelerated aging conducted under various environments (air, nitrogen, moisture) and at 64 °C and below for almost three years, and 2) natural aging conducted under ambient conditions for more than three decades. The hydrolyzed samples were characterized via multi-detection gel permeation chromatography (GPC), thermogravimetric analysis (TGA), modulated differential scanning calorimetry (mDSC), UV–vis spectroscopy, nuclear magnetic resonance (NMR), and Fourier-transform infrared (FTIR) spectroscopy techniques. Hydrolysis of ester linkages in the soft-segments decreases both the molecular weight (Mw) and the melting point (Tm) of Estane (from ∼55 °C to 39 °C). Aging above this Tm, increased mobility of polymer chains and water diffusivity in the PEU matrix alter the PEU degradation pathway from those expected at aging temperatures below this Tm and have significant bearing on the critical molecular weight (MC) at which the physical, chemical, thermal, and mechanical properties of Estane change abruptly. While a MC value of 20 kDa is found for PEU hydrolysis at mild temperatures (e.g., as low as 39 °C), the value of MC increases with increasing aging temperatures. To complement the existing structure/property relationships reported in the literature, more correlations are obtained, which include the effect of Mw on polydispersity, intrinsic viscosity (Mark-Houwink equation), UV extinction coefficient, and dn/dc (GPC analysis) values. Furthermore, we seek to bolster previously reported aging models for PEU by developing a practical model with which the extent of degradation and material performance can be predicted based on aging under different temperature ranges both above and below the melting point of Estane.

Биологически активные гидролизаты белков молока и их комплексы включения с циклодектринами

Enzymatic hydrolysis of dairy proteins increases their nutritional and biological value while reducing their allergenic potential. The subsequent complexation of peptides with cyclodextrins (CDs) reduces the bitterness of the hydrolyzed proteins. The research objective was to obtain hydrolysates of whey proteins and their cyclodextrin inclusion complexes with peptides, as well as to describe the peptide composition of the cleaved dairy proteins, biological activity, and sensory profile of the hydrolysates and inclusion complexes. The research featured enzymatic whey protein hydrolysates with an extensive hydrolysis degree and their inclusion complexes with β- and γ-CDs. Dairy proteins were hydrolyzed with alcalase, and the hydrolysates obtained were subjected to micro- and ultrafiltration (cut-off limit – 10 kDa). The peptide composition of the hydrolyzed proteins was determined by the methods of high-performance liquid chromatography and chromatography-mass spectrometry. The antimutagenic activity was evaluated using the Ames test whereas the antibacterial effect was studied with the impedimetric method. The antioxidant activity was detected with fluorimetry and spectrophotometry. The method of competitive enzyme immunoassay was applied to reveal the antigenic properties. The bitterness of the experimental sample s was determined by a sensory evaluation. The research delivered the optimal modes for whey protein cleavage with alcalase that made it possible to achieve efficient micro- and ultrafiltration. The resulting hypoallergenic peptide fractions and their inclusion complexes with β- and γ-CDs possessed antioxidant, antibacterial, and antimutagenic properties. The whey proteolysis and subsequent filtration with/without tindalization demonstrated a 265/589-fold decrease in the residual antigenicity. The fluorimetric method showed a 1.79/1.90-fold increase in the antioxidant activity of the hydrolysate in complexes with β- and γ-CDs. Binding of β-CDs to peptides enhanced their antimicrobial activity against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538. The hydrolysate samples with β-CDs showed less bitterness. Whey proteolysis with alcalase under optimized conditions and subsequent fractionation resulted in a product with high consumer qualities. Enzymatic hydrolysates of dairy proteins and their CD inclusion complexes were able to substitute native protein components. Their bioactive properties, good taste, and low allergenic potential mean good prospects for the functional food industry.

Soy protein processing impacts the ice recrystallization inhibition activity of protein hydrolysates

AbstractSoy protein hydrolysates have demonstrated moderate ice recrystallization inhibition (IRI) activity, but the properties of the unhydrolyzed protein contributing to this activity are not well known. The objective of this research was to identify the main protein processing factors important to the varying IRI activities observed. Three possible modification reactions were applied to soy protein isolate (SPI): the Maillard reaction that can occur in soy flour before protein isolation, heat denaturation of the fully defatted protein, and heat denaturation of the protein with the presence of residual lipid, mainly polar phospholipids. These modified proteins were hydrolyzed by Alcalase protease (for 2 min) to produce hydrolysates, which were characterized by HPLC and FTIR to investigate how molecular weight (MW) and changes in secondary structure relate to IRI activity. A multilinear regression with the parameters of modification type, surface hydrophobicity, secondary structure profile, and average MW of the hydrolysates was used to investigate their roles in reducing ice crystal size. It was discovered that polar lipids present in the soy hydrolysate samples and MW were the only significant factors (p < 0.05) for IRI activity of SPI hydrolysates which had an ice crystal size reduction from 22.0% to 36.7%. This study demonstrates for the first time a protein hydrolysate‐phospholipid interaction can produce IRI active molecules.

Thermally Stabilised Poly(vinyl alcohol) Nanofibrous Materials Produced by Scalable Electrospinning: Applications in Tissue Engineering.

Electrospinning is a widely employed manufacturing platform for tissue engineering applications because it produces structures that closely mimic the extracellular matrix. Herein, we demonstrate the potential of poly(vinyl alcohol) (PVA) electrospun nanofibers as scaffolds for tissue engineering. Nanofibers were created by needleless direct current electrospinning from PVA with two different degrees of hydrolysis (DH), namely 98% and 99% and subsequently heat treated at 180 °C for up to 16 h to render them insoluble in aqueous environments without the use of toxic cross-linking agents. Despite the small differences in the PVA chemical structure, the changes in the material properties were substantial. The higher degree of hydrolysis resulted in non-woven supports with thinner fibres (285 ± 81 nm c.f. 399 ± 153 nm) that were mechanically stronger by 62% (±11%) and almost twice as more crystalline than those from 98% hydrolysed PVA. Although prolonged heat treatment (16 h) did not influence fibre morphology, it reduced the crystallinity and tensile strength for both sets of materials. All samples demonstrated a lack or very low degree of haemolysis (<5%), and there were no notable changes in their anticoagulant activity (≤3%). Thrombus formation, on the other hand, increased by 82% (±18%) for the 98% hydrolysed samples and by 71% (±10%) for the 99% hydrolysed samples, with heat treatment up to 16 h, as a direct consequence of the preservation of the fibrous morphology. 3T3 mouse fibroblasts showed the best proliferation on scaffolds that were thermally stabilised for 4 and 8 h. Overall these scaffolds show potential as 'greener' alternatives to other electrospun tissue engineering materials, especially in cases where they may be used as delivery vectors for heat tolerant additives.

Angiotensin-Converting Enzyme and Renin-Inhibitory Activities of Protein Hydrolysates Produced by Alcalase Hydrolysis of Peanut Protein.

Hypertension is a major controllable risk factor associated with cardiovascular disease (CVD) and overall mortality worldwide. Most people with hypertension must take medications that are effective in blood pressure management but cause many side effects. Thus, it is important to explore safer antihypertensive alternatives to regulate blood pressure. In this study, peanut protein concentrate (PPC) was hydrolyzed with 3-5% Alcalase for 3-10 h. The in vitro angiotensin-converting enzyme (ACE) and renin-inhibitory activities of the resulting peanut protein hydrolysate (PPH) samples and their fractions of different molecular weight ranges were determined as two measures of their antihypertensive potentials. The results show that the crude PPH produced at 4% Alcalase for 6 h of hydrolysis had the highest ACE-inhibitory activity with IC50 being 5.45 mg/mL. The PPH samples produced with 3-5% Alcalase hydrolysis for 6-8 h also displayed substantial renin-inhibitory activities, which is a great advantage over the animal protein-derived bioactive peptides or hydrolysate. Remarkably higher ACE- and renin-inhibitory activities were observed in fractions smaller than 5 kDa with IC50 being 0.85 and 1.78 mg/mL. Hence, the PPH and its small molecular fraction produced under proper Alcalase hydrolysis conditions have great potential to serve as a cost-effective anti-hypertensive ingredient for blood pressure management.

Interfacial rheological properties of pepsin-hydrolyzed lentil protein isolate at oil-water interfaces

Lentil protein isolate (LPI) was investigated for its potential as a plant protein-based emulsifier. LPI consists mainly of globulins with high molecular weights which are relatively slow to adsorb and stabilize oil-water interfaces. Smaller proteins, such as whey protein, can stabilize the interface much faster, quickly forming a viscoelastic film that slows down the rate of droplet recoalescence, leading to smaller droplets. To increase the rate of adsorption, LPI was enzymatically hydrolyzed by pepsin at 1.5% and 4.5% degree of hydrolysis (DH), to obtain small peptides. The behavior of these peptides was studied at the oil-water interface in comparison to whey protein isolate (WPI) and native lentil protein isolate (LPI). Interfacial properties were investigated using dilatational and interfacial shear rheology in the linear viscoelastic regime (LVE) and nonlinear viscoelastic regime (NLVE) and these were related to protein characteristics and emulsion formation. The 1.5% and 4.5%DH LPI showed high surface hydrophobicity, and consisted mainly of low molecular weight peptides, which contributed to faster adsorption kinetics and consequently covered the interface faster than LPI. In dilatation, the native and hydrolyzed LPI-stabilized interfaces had comparable stiffness and were clearly weaker than WPI-stabilized interfaces, which contrasts with the results in shear deformations, where the former both had higher stiffness than WPI. Both hydrolyzed LPI and WPI formed more brittle and less stretchable interfaces compared to native LPI in both dilatational and shear rheology. In emulsification tests, native LPI produces the largest droplets, and the 4.5% DH LPI and WPI have the smallest droplet size. But, bridging flocculation was observed when emulsions were prepared with both hydrolyzed LPI, which may be attributed to inter-droplet hydrophobic interactions between small polypeptides. This can be linked to an increase in the surface hydrophobicity of hydrolyzed LPI. Thus, the use of native LPI as a stabilizer in emulsion preparation is preferable over the hydrolyzed samples, despite its larger droplet size.

From laboratory to industrial use: Understanding process variation during enzymatic protein hydrolysis with dry film fourier-transform infrared spectroscopy

Industries are continuously looking for dedicated sensor solutions for evaluating the chemical composition of products that can provide valuable insights into process control, process optimization, and product quality. A rapid and robust analytical method, Fourier-Transform Infrared (FTIR) spectroscopy, holds significant potential in this regard as it provides detailed compositional values of food nutrients. The main aim of the present study was to use dry film FTIR spectroscopy as an analytical tool to characterize products from an industrial enzymatic protein hydrolysis process and link this to understand industrial process variations. For this purpose, 463 protein hydrolysate samples were obtained from an industrial enzymatic protein hydrolysis plant. In the same period, systematic variations in process parameters such as raw material composition, enzyme type, and water addition, were performed. All samples were analyzed using dry film FTIR spectroscopy. Two subsets containing 200 and 68 hydrolysate samples were chosen for measuring average molecular weight (AMW) and collagen content respectively, providing reference data for constructing calibration models based on partial least squares regression (PLSR). The percentage of low molecular weight constituents was derived from the molecular weight distribution of size exclusion chromatograms of protein hydrolysates and also used in the modeling. Calibration models for the prediction of AMW, low molecular weight constituents, and collagen content were obtained with a good fit and lower estimation errors. Principal component analysis (PCA) of protein hydrolysates' FTIR spectra effectively differentiated process variations (enzyme types and raw materials) without extensive reference analysis. One-factor analysis of variance (ANOVA) tests was used to observe the impact of process variation on product quality. FTIR proved to be a sensitive method for liquid protein analysis and process control with a significant potential in process optimization approaches. To the authors’ knowledge, this is the first time dry film FTIR spectroscopy has been used to evaluate an industrial bioprocess with designed process variations.

Research of the properties of protein hydrolysates obtained from the broiler chicken gizzards as a potential component of bioactive film coatings

Protein hydrolysates are a promising active component in the production of bioactive film coatings for food products. Some biopolymers can exert the biological activity. More often, however, it is necessary to select biologically active substances to impart these properties to films. On the other hand, not all components allow forming films with the required properties, and therefore there is a need to study the individual technological characteristics of the components used. The purpose of the research is to establish the antioxidant and technological properties of protein hydrolysates obtained by microbial fermenta- tion of poultry by-products in whey with bifidobacteria, propionic acid bacteria and acidophilic bacteria as a potential basis for bioactive film coatings of food products. The hydrolysate obtained by fermentation without the addition of the specified bacterial species was used as a control sample. The functional properties of protein hydrolysates were assessed: antioxidant capacity by coulometric titration on an Expert-006 coulometer using ascorbic acid as a standard, antiradical activity by the DPPH method on a Jenway 6405 UV/Vis spectrophotometer with determination of the IC50 value. The technological proper- ties, solubility, water-holding, fat-holding and fat-emulsifying capacities were also determined by the gravimetric method. In addition, the average hydrodynamic diameter of particles in protein hydrolysates was determined using a Microtrac FLEX particle size analyzer. The results of studies of the antioxidant properties showed that the DPPH antiradical activity was 14.7% higher in the experimental samples of hydrolysates obtained by fermentation with bifidobacteria compared to the control; samples of hydrolysates obtained by fermentation with propionic acid bacteria showed an antioxidant capacity 29.6% higher than that of the control sample. The IC50 value turned out to be the highest in the control hydrolysate sample (2.994 mg/ml), which was 45.5–53.3% higher than that in the experimental hydrolysate samples. The results of determining the technologi- cal properties showed that they differ significantly for protein hydrolysates obtained by fermentation with different types of bacteria. For example, the highest values of fat-holding and fat-emulsifying capacities were found in the hydrolysate obtained by fermentation with bifidobacteria (351.1% and 61%, respectively), which shows its potential for incorporation into the bio- composite in the form of a protein-oil emulsion. The high solubility of the experimental samples of hydrolysates (from 90.1 to 91.4%) suggests their uniform distribution in the aqueous phase when composing the biocomposite of the film. Thus, the research results have shown the prospects of using protein hydrolysates from the gizzards of broiler chickens in whey as an active component of bioactive film coatings. The antioxidant properties of protein hydrolysates allow slowing down oxidative processes in the main food nutrients, which will contribute to an increase in the shelf life of food products packaged in bioac- tive films with this component.

ОПРЕДЕЛЕНИЕ РАЦИОНАЛЬНЫХ РЕЖИМНЫХ ПАРАМЕТРОВ ФЕРМЕНТАЦИИ БИОВОДОРОДА С ИСПОЛЬЗОВАНИЕМ CLOSTRIDIUM BUTYRICUM И ENTEROBACTER CLOACAE НА ГИДРОЛИЗАТАХ СОЛОМЫ, АКТИВИРОВАННОЙ ПАРОВЗРЫВНОЙ ОБРАБОТКОЙ

Studies have been conducted to determine the possibility of using agricultural waste, in particular straw, for the production of biohydrogen. In our work, we used two types of straw hydrolysates (after acidic and enzymatic hydrolysis), pre-activated by steam blasting at steam temperatures of 100, 165, 210 0C. The hydrolysates were diluted with distilled water to a concentration of reducing agents (RR) equal to 1.0, 1.5 and 2.0% of their mass in order to determine the rational concentration. Two cultures of anaerobic microorganisms were tested on 18 samples of hydrolysates: a strain of bacteria of the genus Clostridium Butyricum E.VI .3.2.1 (no. VKPM B-9619), a strain of bacteria of the genus Enterobacter cloacae (no. VKPM B-1980). The fermentation temperature for all samples was set at 37 ± 0.5 0C, pH 5.5 ± 0.1. The released gas accumulated in the gas tank; its samples were taken every 12 hours and analyzed for the volume content of hydrogen. After establishing the rational initial concentration of the substrate, the temperature of steam explosive activation of straw, the type of hydrolysate and the maximum yield of biohydrogen, the cultivation temperature and pH for each culture were further changed in order to determine the rational parameters: for Clostridium Butyricum, the cultivation temperature was set to 32 ±0.5, 37 ±0.5 and 42±0.5 0C for Enterobacter cloacae – 36±0.5, 37±0.5, 38±0.5 0C; the pH for all samples was set in the range of 5...6 in increments of 0.5 ± 0.1). On acidic straw hydrolysates, Clostridium Butyricum demonstrated the best productivity (steam explosion temperature 165 0C) - hydrogen yield was 73 ml/g, on hydrolysates fermented by Enterobacter cloacae (steam explosion temperature 210 0C), hydrogen yield was 50 ml/g. Both cultures are capable of producing the maximum amount of hydrogen at a concentration of reducing substances of 2%. The rational fermentation temperature for Clostridium Butyricum is 37 ± 0.5 0C, for Enterobacter cloacae – 36 ± 0.50 C, for both cultures – pH = 6.

Quantitative at-line monitoring of enzymatic hydrolysis using benchtop diffusion nuclear magnetic resonance spectroscopy.

Benchtop diffusion nuclear magnetic resonance (NMR) spectroscopy was used to perform quantitative monitoring of enzymatic hydrolysis. The study aimed to test the feasibility of the technology to characterize enzymatic hydrolysis processes in real time. Diffusion ordered spectroscopy (DOSY) was used to measure the signal intensity and apparent self-diffusion constant of solubilized protein in hydrolysate. The NMR technique was tested on an enzymatic hydrolysis reaction of red cod, a lean white fish, by the endopeptidase alcalase at 50°C. Hydrolysate samples were manually transferred from the reaction vessel to the NMR equipment. Measurement time was approximately 3 min per time point. The signal intensity from the DOSY experiment was used to measure protein concentration and the apparent self-diffusion constant was converted into an average molecular weight and an estimated degree of hydrolysis. These values were plotted as a function of time and both the rate of solubilization and the rate of protein breakdown could be calculated. In addition to being rapid and noninvasive, DOSY using benchtop NMR spectroscopy has an advantage compared with other enzymatic hydrolysis characterization methods as it gives a direct measure of average protein size; many functional properties of proteins are strongly influenced by protein size. Therefore, a method to give protein concentration and average size in real time will allow operators to more tightly control production from enzymatic hydrolysis. Although only one type of material was tested, it is anticipated that the method should be applicable to a broad variety of enzymatic hydrolysis feedstocks.

A portable dry film FTIR instrument for industrial food and bioprocess applications.

The main objective of this study was to design, build, and test a compact, multi-well, portable dry film FTIR system for industrial food and bioprocess applications. The system features dry film sampling on a circular rotating disc comprising 31 wells, a design that was chosen to simplify potential automation and robotic sample handling at a later stage. Calibration models for average molecular weight (AMW, 200 samples) and collagen content (68 samples) were developed from the measurements of industrially produced protein hydrolysate samples in a controlled laboratory environment. Similarly, calibration models for the prediction of lactate content in samples from cultivation media (59 samples) were also developed. The portable dry film FTIR system showed reliable model characteristics which were benchmarked with a benchtop FTIR system. Subsequently, the portable dry film FTIR system was deployed in a bioprocessing plant, and protein hydrolysate samples were measured at-line in an industrial environment. This industrial testing involved building a calibration model for predicting AMW using 60 protein hydrolysate samples measured at-line using the portable dry film FTIR system and subsequent model validation using a test set of 26 samples. The industrial calibration in terms of coefficient of determination (R2 = 0.94), root mean square of cross-validation (RMSECV = 194 g mol-1), and root mean square of prediction (RMSEP = 162 g mol-1) demonstrated low prediction errors as compared to benchtop FTIR measurements, with no statistical difference between the calibration models of the two FTIR systems. This is to the authors' knowledge the first study for developing and employing a portable dry film FTIR system in the enzymatic protein hydrolysis industry for successful at-line measurements of protein hydrolysate samples. The study therefore suggests that the portable dry film FTIR instrument has huge potential for in/at-line applications in the food and bioprocessing industries.

Recovery of Ionic Liquid from the Model Solution Mixture Mimicking the Catalytically Hydrolyzed Cellulose Product Utilizing Amberlyst Ion-Exchange Resin

The hydrolysis of cellulose using ionic liquid (IL) has been extensively studied but there is limited understanding of the removal of IL from the biomass hydrolysate. Finding a suitable method for the recovery and reuse of IL is one of the biggest challenges before its large-scale application. Selecting an appropriate combined recovery process is very important. This study proposed a facile ion-exchange combined method for the recovery of IL from the modeled cellulose hydrolysate mixture containing sugars as well as γ-valerolactone (GVL) via an adsorption–desorption mechanism using sulfonic acid cation-exchange (Amberlyst 15 (H)) resin. The results showed that the resin could adsorb more than 94% of 1-ethyl-3-methylimidazolium chloride [Emim]Cl IL at ambient conditions within a contact time of 20 min. The other coexisting constituents like glucose and GVL have no significant effect on the adsorption efficiency of IL. The adsorption of IL on Amberlyst 15 (H) resin was observed to be pseudo-second-order adsorption. The regeneration of the adsorbed IL was possible up to 70% using low-cost, easily available sodium chloride (NaCl) solution. Similarly, despite the interference of other unwanted byproducts in the real biomass hydrolysate sample, an IL adsorption efficiency up to 51% was reached under similar operating conditions. This study thus opens the facile possibility of extracting and recycling IL used in the biomass hydrolysis process.

Yessotoxins in Mollusks of the Galician Coast from 2014 to 2022: Variability, Biotransformation, and Resistance to Alkaline Hydrolysis.

The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the samples containing YTX, 45-OH YTX was detected in 37.3% of cases. The maximum recorded levels were 1.4 and 0.16 mg of YTX-equivalentsg-1, for YTX and 45-OH YTX, respectively, which are well below the regulatory limit of the European Union. The YTX and 45-OH YTX toxicities in the raw extracts and extracts subjected to alkaline hydrolysis were strongly and linearly related. Due to the lack of homo-YTX in Galician samples, the effect of alkaline hydrolysis on homo-YTX and 45OH-Homo-YTX was only checked in 23 additional samples, observing no negative effect but a high correlation between raw and hydrolyzed extracts. Hydrolyzed samples can be used instead of raw ones to carry out YTXs determinations in monitoring systems, which may increase the efficiency of those systems where okadaic acid episodes are very frequent and therefore a higher number of hydrolyzed samples are routinely analyzed. The presence of YTX in the studied bivalves varied with the species, with mussels and cockles having the highest percentages of YTX-detected samples. The presence of 45-OH YTX was clearly related to YTX and was detected only in mussels and cockles. Wild populations of mussels contained proportionally more 45-OH YTX than those that were raft-cultured. Spatially, toxin toxicities varied across the sampling area, with higher levels in raft-cultured mussels except those of Ría de Arousa. Ría de Ares (ARE) was the most affected geographical area, although in other northern locations, lower toxin levels were detected. Seasonally, YTX and 45-OH YTX toxicities showed similar patterns, with higher levels in late summer and autumn but lower toxicities of the 45-OH toxin in August. The relationship between the two toxins also varied seasonally, in general with a minimum proportion of 45-OH YTX in July-August but with different maximum levels for raft-cultured and wild mussel populations. Interannually, the average toxicities of YTX decreased from 2014 to 2017 and newly increased from 2018 to 2021, but decreased slightly in 2022. The relationship between 45-OH YTX and YTX also varied over the years, but neither a clear trend nor a similar trend for wild and raft mussels was observed.

Optimisation of low temperature (8ᵒC) enzymatic hydrolysis of acid whey using design of experiments (DOE) for the generation of thermally stable whey protein hydrolysates

The impact of enzymatic hydrolysis at 8ᵒC of liquid whey protein concentrate (WPC35) derived from acid casein manufacture using Prolyve 1000®, a microbial protease preparation, under free-fall pH conditions on the physicochemical and the thermal stability properties of the resultant hydrolysates was investigated. The hydrolysis process was optimised using a design of experiments (DOE) approach. Eleven hydrolysates (H1–H11) were generated using 3 factors x 2 levels, i.e., enzyme:substrate (E:S) 0.25–1.00% (v/w), starting pH 7.5–9.0 and incubation time 10–96 h. Hydrolysate degree of hydrolysis (DH) values ranged from 0.75 (H10) to 4.74% (H6) which increased to 1.91 (H10) and 7.24% (H6) following subsequent thermal processing (mimicking the evaporation process) at 54ᵒC x 15 min and 64ᵒC x 10 min. The apparent viscosity (ηapp) of all hydrolysates was measured to assess their heat stability during heating at 85ᵒC for up to 20 min, this was shown to be E:S and pH-dependent. While the unhydrolysed samples formed a gel on heating at ∼74-80ᵒC. Following DOE analysis, it was found that E:S had a significant impact on the DH, the extent of intact protein degradation (%Deg) and the thermal stability. The %Deg following processing at 64ᵒC showed a strong correlation (R2 = 0.924; p < 0.001) with ηapp, and thus this parameter may be used to predict the thermal stability of the hydrolysate samples. The optimised hydrolysis conditions for the generation of heat stable WPC35 hydrolysates during 8°C incubation were achieved using an E:S = 0.625% at a starting pH = 8.65. Performance of protein hydrolysis reactions at low temperature has potential to enhance process sustainability.

Advancing Quantification of Water-Extractable Arabinoxylan in Beer: A High-Throughput Approach.

Water-extractable arabinoxylan (WEAX) may cause major problems during clarification processes in a brewery owing to its ability to form gel networks. However, high WEAX contents can also enhance the nutritional quality of the final product as they play an important role in the human diet. Therefore, precise quantification of WEAX is required. Current methods are very time- and resource-consuming as well as limited in the number of samples and in some cases provide low accuracy. Thus, a reproducible high-throughput method for the quantification of WEAX optimized for beer was developed, reaching recovery rates (RRs) of almost 100%. The assay is based on Douglas's colorimetric method. Hydrolysis was conducted using glacial acetic acid to induce the formation of red color complexes resulting from the interaction between pentose degradation products and phloroglucinol. The method was successfully transferred to a multi-mode microplate reader to minimize the loss of color intensity over time and to obtain a high throughput. By using 96-well plates, up to 40% of the previous analysis time could be saved, and a larger number of samples could be analyzed in one batch. The collected data determined xylose as an optimal calibration standard due to high accuracy and reproducibility. The respective AX control standards showed RR within the range of 95-105% without exception. To validate and show the ruggedness of the modified method, WEAX concentration in seven commercial German beers (e.g., lager, pilsner, wheat beer, non-alcoholic beer) was quantified. Interfering hexose sugars that lead to measurement errors when analyzing samples with high amounts of fermentable sugars (e.g., non-alcoholic beer produced by limited fermentation) were eliminated by Saccharomyces diastaticus fermentation. Further investigations were carried out by means of LC-MS in order to obtain additional information about the reddish product in the hydrolyzed samples. In this context, C16H12O6 could be identified as one of numerous condensation products, contributing to the coloring. The collected data showed the impact of diverse factors on the measured AX concentration and helped optimize the experimental procedure for a high sample throughput with precise and highly reproducible results. The proposed quantification method should be primarily used in completely fermented finished beer to emphasize the time aspect. Wort samples and non-alcoholic beer produced by limited fermentation can be also analyzed, but only after fermentation with S. diastaticus.

A combined liquid chromatography – trapped ion mobility – tandem high-resolution mass spectrometry and multivariate analysis approach for the determination of enzymatic reactivity descriptors in biomass hydrolysates

Intermediate products such as oxygenated compounds may interfere with bioconversion kinetics of lignocellulosic biomass into bioethanol. This work presents a multidimensional approach, based on liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), tandem high-resolution mass spectrometry (HRMS/MS), and multivariate analysis, for the identification of enzymatic reactivity descriptors in 22 industrial biomass samples, called hydrolysates. The first part of the study is dedicated to the improvement of the chemical diversity assessment of the hydrolysates through an original three-dimensional Van Krevelen diagram displaying the double bond equivalent (DBE) as third dimension. In a second part, the evaluation of data by multivariate data analysis allowed the discrimination of sample according to the biomass type and the level of enzymatic reactivity. In the last part, a potential descriptor of low enzymatic reactivity was selected and used in a case study. An in-depth structural analysis was performed on the feature annotated as carbohydrate derivative. Considering the intricate fragmentation spectrum exhibited by the selected feature, trapped ion mobility was employed to enhance separation prior to the HRMS/MS experiments. This final step improved data interpretation and increased the identification confidence level leading to the characterization of xylotriose, 3,5-dimethoxy-4-hydroxybenzaldehyde and 4-hydroxy-3-methoxy-cinnamaldehyde. This is the first study to present an untargeted multidimensional approach for the identification of enzymatic hydrolysis inhibitors in industrial hydrolysate samples.

Bioethanol Production from Two Varieties (Saccharum Officinarum and Saccharum Barberi) Of Sugarcane Peels

Provision of an alternative clean source of fuel with less cost, will reduce fossil fuel demand. Pollution is the major environmental problem. Air pollution is among the three types of pollution where harmful substances such as CO2 and CO are introduced to the environment and cause harm to humans and animals. Fossil fuel combustion from automobiles and industries causes this type of pollution. There is need to replace another source of fuel with renewable feedstock such as the peels of Saccharum officinarum and Saccharum barberi which are lignocellulose materials to produce bioethanol that has a lower impact on air pollution and the Ozone layer which causes Green-House effect than fossil fuels. Dilute H2SO4 at different concentrations of 1M,2M,3M,4M and 5M were replicated into three different parts respectively as hydrolysing solution. Saccharomyces cerevisiae was used as fermentation enzymes. Different pH values of 5, 6 and 7 was used for fermentation periods of 5, 6 and 7 days, respectively. The Bioethanol was distilled by the use of fractional distillation. Hydrolysed samples of 3M for 6th day of both Saccharum officinarum and Saccharum barberi produced the best yields of bioethanol at the pH of 5 and 5M hydrolysed samples of both Saccharum officinarum and Saccharum barberi produced the least bioethanol for all pH values and fermentation days. The Bioethanol produced was blended with kerosene fuel to create EK10 and EK20 kerosene-bioethanol blends for use as a fuel source in a kerosene lamp. EK10 blend took 126 hours to burn in a rotary kerosene lamp, while EK20 blend took 167 hours to burn-off in the same kerosene lamp. Hydrolysed H2SO4 of 3M, pH 5 and fermentation period of 6 days for both Saccharum officinarum and Saccharum barberi shows the best sample to produce bioethanol. The bioethanol kerosene blend EK10 and E20 was concluded to show long period of burning with clean soot in a rotary kerosene lamp, therefore the bioethanol kerosene blends can be used as substitutes for kerosene fuel.

Nutritional, biological, and structural properties of bioactive peptides from bellflower, Persian-willow, and bitter-orange pollens.

In this study, the composition of amino acids, nutritional characteristics, degree of hydrolysis (DH), antioxidant properties, and antibacterial activity of proteins and hydrolysates of bellflower (Campanula latifolia), Persian willow (Salix aegyptiaca), and bitter orange (Citrus aurantium L.) were investigated under the influence of different proteases (Alcalase: Al, trypsin: Tr, pancreatin: Pa, and pepsin: Pe). Evaluation of the structural features of the proteins showed amide regions (amide A, B, I-III) and secondary structures. Hydrophobic amino acids (∼38%), antioxidants (∼21%), and essential types (∼46%) form a significant part of the structure of flower pollen. The digestibility and nutritional quality (PER) of the hydrolyzed samples (CP: 1.67; CA: 1.89, and PW: 1.93) were more than the original protein. Among proteins and peptides, the highest degree of hydrolysis (34.6%: Al-PWH), inhibition of free radicals DPPH (84.2%: Al-CPH), ABTS (95.2%: Pa-CPH), OH (86.7%: Tr-CAH), NO (57.8%: Al-CPH), reducing power (1.31: Pa-CPH), total antioxidant activity (1.46: Pa-CPH), and chelation of iron ions (80%: Al-CPH and Al-CAH) and copper (50.3%: Pa-CAH) were affected by protein type, enzyme type, and amino acid composition. Also, the highest inhibition of the growth of Escherichia coli (25 mm) and Bacillus cereus (24 mm) were related to CP and PW hydrolysates, respectively. The results of this research showed that hydrolyzed flower pollens can be used as a rich source of essential amino acids as well as natural antioxidants and antibacterial in food and dietary products. PRACTICAL APPLICATION: Enzymatic hydrolysis of Campanula latifolia, Persian willow, and Citrus aurantium pollen proteins was performed. The hydrolyzed ones had high nutritional quality and digestibility (essential amino acids and PER index). Antioxidant properties and chelation of metal ions of peptides were affected by the type of protein and enzyme. The hydrolysates showed inhibitory activity against the growth of Esherichia coli and Bacillus cereus.

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.

Mineral Bioaccessibility and Antioxidant Capacity of Protein Hydrolysates from Salmon (Salmo salar) and Mackerel (Scomber scombrus) Backbones and Heads

Information on the bioaccessibility of minerals is essential to consider a food ingredient as a potential mineral fortifier. In this study, the mineral bioaccessibility of protein hydrolysates from salmon (Salmo salar) and mackerel (Scomber scombrus) backbones and heads was evaluated. For this purpose, the hydrolysates were submitted to simulated gastrointestinal digestion (INFOGEST method), and the mineral content was analyzed before and after the digestive process. Ca, Mg, P, Fe, Zn, and Se were then determined using an inductively coupled plasma spectrometer mass detector (ICP-MS). The highest bioaccessibility of minerals was found in salmon and mackerel head hydrolysates for Fe (≥100%), followed by Se in salmon backbone hydrolysates (95%). The antioxidant capacity of all protein hydrolysate samples, which was measured by Trolox Equivalent Antioxidant Capacity (TEAC), increased (10-46%) after in vitro digestion. The heavy metals As, Hg, Cd, and Pb were determined (ICP-MS) in the raw hydrolysates to confirm the harmlessness of these products. Except for Cd in mackerel hydrolysates, all toxic elements were below the legislation levels for fish commodities. These results suggest the possibility of using protein hydrolysates from salmon and mackerel backbones and heads for food mineral fortification, as well as the need to verify their safety.