Stabilization of oil-water emulsions with protein concentrates from the microalga Tetradesmus obliquus.

The present work used water-soluble protein concentrates from the microalga Tetradesmus obliquus to stabilize sunflower oil emulsions. Microalgal cells were disrupted by sonication, and proteins were separated from the biomass using two methods, isoelectric and solvent precipitations. The protein extracts were concentrated by lyophilization, and the concentrates were used to produce emulsions with three amounts of Tetradesmus obliquus protein concentrate (TobPC) (0.1, 0.5, and 1.0% w/v). Emulsions were homogenized through sonication and characterized for creaming index, optical microscopy, size distribution, ζ-potential, and rheology. Isoelectric precipitation resulted in TobPC with a high protein content (51.46 ± 2.37%) and a better dispersibility profile. Emulsion stability was higher for both the isoelectric TobPC and control systems than for the TobPC solvent. Solvent TobPC does not efficiently stabilize emulsions at low protein concentrations that showed microscopically larger oil droplets and flocculation spots. A high phase separation velocity was observed for solvent TobPC, probably due to the higher hydrodynamic droplet diameters. The increase in TobPC content in the emulsions resulted in more stable emulsions for all samples. Therefore, Tetradesmus obliquus protein concentrates are a potential emulsifying agent.

Aqueous solutions of glycolic, propionic, or lactic acid in substitution of acetic acid to prepare chitosan dispersions: a study based on rheological and physicochemical properties.

Chitosan (CH) is a biopolymer derived from chitin, which is the second most abundant polysaccharide in nature, after cellulose. Their functional groups -NH2 and -OH can form intermolecular interactions with water and other molecules, enabling a variety of applications for CH. -NH2 groups become protonated in acidic solutions, causing an increase in electrostatic repulsion between CH chains, which facilitates their dispersion in aqueous media. Aqueous solutions of acetic acid and/or acetates buffers have been used to disperse CH, but may not be adequate for technological applications, espeacially because of the strong flavor this acid confers to formulations. In this study, 0.125; 0.250; 0.500; 0.750 and 1.000 g (100 g)-1 CH dispersions were prepared in acidic aqueous media (50 mmol L-1), not only with acetic (AA), but also with glycolic (GA), propionic (PA), or lactic (LA), acid aiming to evaluate the effects of biopolymer concentration and type of organic acid on: electrical conductivity, pH, density and rheological characteristics of dispersions. Moreover, ζ potential values of CH chains dispersed in these acidic aqueous media were assessed. pH, density and consistency index were influenced by the biopolymer concentration, but not by the acid type. At a given biopolymer concentration, ζ potential signs (+) and values suggested that electrostatic interactions between CH chains and counter-anions occurred, regardless of the type of the organic acid. Thus, at least from a physicochemical point of view, GA, PA or LA showed to be suitable to replace AA when preparing dispersions containing from 0.125 to 1.000 g (100 g)-1 CH for technological purposes, such as thickening or stabilizer in formulated food products.

Engineered GH11 xylanases from Orpinomyces sp. PC-2 improve techno-functional properties of bread dough.

BACKGROUND: Endo-1,4-ß-xylanases have marked hydrolytic activity towards arabinoxylans. Xylanases (xynA) produced by the anaerobic fungus Orpinomyces sp. strain PC-2 have been shown to be superior in specific activity, which strongly suggests their applicability in the bakery industry for the processing of whole-wheat flour containing xylans. In the present study, two xylanases from this source, the small wild-type xylanase SWT and the small mutant xylanase SM2 (V108A, A199T), were expressed in Escherichia coli, purified, characterized, tested for their ability to hydrolyze whole-wheat flour and applied in dough processing. RESULTS: Both purified SM2 and SWT showed high specific activity against oat spelt xylan and wheat arabinoxylan, exhibiting maximum activity at pH 3-7 and 60 °C. SM2 was more thermostable than SWT, which suggests that the mutations enhanced its stability. Both SWT and SM2 were able to hydrolyze whole-wheat flour, and evaluation of their applicability in dough processing by the sponge method indicated that use of these enzymes increased dough volume by 60% and reduced texture hardness by more than 50%, while gumminess and chewiness were reduced by 40%. CONCLUSION: The recombinant xylanases showed potential for application in bakery processing and can improve techno-functional properties in sponges. © 2018 Society of Chemical Industry.

Food Protein-polysaccharide Conjugates Obtained via the Maillard Reaction: A Review.

The products formed by glycosylation of food proteins with carbohydrates via the Maillard reaction, also known as conjugates, are agents capable of changing and improving techno-functional characteristics of proteins. The Maillard reaction uses the covalent bond between a group of a reducing carbohydrates and an amino group of a protein. This reaction does not require additional chemicals as it occurs naturally under controlled conditions of temperature, time, pH, and moisture. Moreover, there is growing interest in modifying proteins for industrial food applications. This review analyses the current state of art of the Maillard reaction on food protein functionalities. It also discusses the influence of the Maillard reaction on the conditions and formulation of reagents that improve desirable techno-functional characteristics of food protein.

Chitosan/carboxymethylcellulose polyelectrolyte complexes (PECs) are an effective material for dye and heavy metal adsorption from water.

Granular macroscopic chitosan/carboxymethylcellulose polyelectrolytic complexes (CHS/CMC macro-PECs) were produced and tested as adsorbents for six pollutants often present in wastewaters: sunset yellow (YS), methylene blue (MB), Congo red (CR) and safranin (S), cadmium (Cd2+) and lead (Pb2+). The optimum adsorption pH values at 25 °C were 3.0, 11.0, 2.0, 9.0, 10.0, and 9.0 for YS, MB, CR, S, Cd2+, and Pb2+, respectively. Kinetic studies indicated that the pseudos-econd order model best represented the adsorption kinetics of YS, MB, CR, and Cd2+, whereas the pseudo-first order model was the most suitable for S and Pb2+ adsorption. The Langmuir, Freundlich, and Redlich-Peterson isotherms were fitted to experimental adsorption data, with the Langmuir model providing the best fit. The maximum adsorption capacity (qmáx) of CHS/CMC macro-PECs for the removal of YS, MB, CR, S, Cd2+, and Pb2+ was 37.81, 36.44, 70.86, 72.50, 75.43, and 74.42 mg/g, respectively (corresponding to 98.91 %, 94.71 %, 85.73 %, 94.66 %, 98.46 %, and 97.14 %). Desorption assays showed that CHS/CMC macro-PECs can be regenerated after adsorbing any of the six pollutants studied, with possibility of reuse. These results provide an accurate quantitative characterization of the adsorption of organic and inorganic pollutants on CHS/CMC macro-PECs, indicating a novel technological applicability of these two inexpensive, easy-to-obtain polysaccharides for water decontamination.

Impacts of Ca2+ cation and temperature on bovine α-lactalbumin secondary structures and foamability - Insights from computational molecular dynamics.

We used computational molecular dynamics (MD) to assess molecular conformations of apo- and holo-forms (respectively without and with Ca2+) of bovine α-lactalbumin (α-La) at different temperatures, and to correlate them with the protein's foaming properties. At 4 °C and 25 °C no major protein conformation changes occurred. At 75 °C, lots of changes were evidenced: the Ca2+ depletion triggered the complete loss of h2b, h3c helices and S1, S2 and S3 ß-sheets, and partial losses of H1, H2 and H3 α-helices. The absence of Ca2+ in apo-α-La and its leaving from holo-α-La triggered electrostatic repulsion among Asp82, Asp84 and Asp87, leading to the formation of a hydrophobic cluster involving Phe9, Phe31, Ile1, Va42, Ile55, Phe80 and Leu81. These conformational changes were related to an interfacial tension decrease and to a foaming capacity increase, for both apo-α-La and holo-α-La. This study exemplifies how powerful MD is as a tool to provide a better understanding of the molecular origins of food proteins' techno-functionalities.

Mixed starch/chitosan hydrogels: elastic properties as modelled through simulated annealing algorithm and their ability to strongly reduce yellow sunset (INS 110) release.

Biopolymers mixtures appear as a strategy to improve sensorial/technological characteristics of gel-like products. Thus, self-sustaining starch (S100/C0) hydrogels were prepared with a partial replacement of the gelling agent by 5.0 % (S95/C5), 7.5 % (S92.5/C7.5), or 10.0 % chitosan (S90/C10), and containing yellow sunset (INS 110). Major visual changes or significant differences on L*a*b* parameters were not observed for starch/chitosan hydrogels. Creep-recovery data was modeled using the simulated annealing algorithm, and relative recovery results showed an increase for S95/C5 (82.6 %), when compared to S100/C0 (72.9 %). After 312 h, chitosan strongly reduced the INS 110 release from hydrogels to an ethanolic solution (3.1∙10-4 and 4.1∙10-3 g/100 mL for S95/C5 and S100/C0, respectively) or to a sucrose solution (1.1∙10-3 and 6.5∙10-3 g/100 mL for S95/C5 and S100/C0, respectively). Such results highlighted that chitosan not only presented a techno-functionality on starch hydrogels by improving their elasticity but also by hindering the release of yellow sunset.

Structural and molecular bases of angiotensin-converting enzyme inhibition by bovine casein-derived peptides: an in silico molecular dynamics approach.

The angiotensin-converting enzyme (ACE) plays a key role in blood pressure regulation process, and its inhibition is one of the main drug targets for the treatment of hypertension. Though various peptides from milk proteins are well-known for their ACE-inhibitory capacity, research devoted to understand the molecular bases of such property remain scarce, specifically for such peptides. Therefore, in this work, computational molecular docking and molecular dynamics calculations were performed to enlighten the intermolecular interactions involved in ACE inhibition by six different casein-derived peptides (FFVAPFPEVFGK, FALPQYLK, ALNEINQFYQK, YLGYLEQLLR, HQGLPQEVLNENLLR and NAVPITPTLNR). Two top ranked docking poses for each peptide (one with N- and the other C-terminal peptide extremity oriented towards the ACE active site) were selected for dynamic simulations (50 ns; GROMOS53A6 force field), and the results were correlated to in vitro ACE inhibition capacity. Two molecular features appeared to be essential for peptides to present high ACE inhibition capacity in vitro: i) to interact with the S1 active site residues (Ala354, Glu384, and Tyr523) by hydrogen bonds; ii) to interact with Zn2+ coordinated residues (His383, His387, and Glu411) by short-lenght hydrogen bonds, as observed in the cases of ALNEINQFYQK (IACE = 80.7%), NAVPITPTLNR (IACE = 80.7%), and FALPQYLK (IACE = 79.0%). Regardless of the temporal stability of these strong interactions, they promoted some disruption of Zn2+ tetrahedral coordination during the molecular dynamics trajectories, and were pointed as the main reason for the greatest ACE inhibition by these peptides. On the other hand, peptides with intermediate inhibition capacity (50% < IACE < 45%) interacted mainly by weaker interactions (e.g.: electrostatic and hydrophobic) with the Zn2+ coordinated residues, and were not able to change significantly its tetrahedral coordination structure. These findings may: i) assist the discrimination in silico of "good" and "bad" ACE-inhibitory peptides from other food sources, and/or ii) aid in designing de novo new molecules with ACE-inhibitory capacity. Communicated by Ramaswamy Sarma.

Insights on physicochemical aspects of chitosan dispersion in aqueous solutions of acetic, glycolic, propionic or lactic acid.

Chitosan is a polysaccharide well-known for its applicability as a biocompatible, biodegradable, and non-toxic material to produce drugs excipients and food coatings. Acidic media are required to disperse chitosan, and aqueous solutions of acetic acid have been typically used for this purpose. However, this acid has several sensory drawbacks. In this study, chitosan was dispersed [0.1 g·(100 mL)-1] in aqueous media containing acetic (AA), glycolic (GA), propionic (PA), or lactic (LA) acid, at 10, 20, 30, 40, or 50 mmol·L-1. The increase of acid concentration reduced pH and viscosity of the dispersions, and |ζ potential| of dispersed particles. Conversely, it increased electrical conductivity and density of the dispersions, and hydrodynamic diameter of dispersed particles. At a given concentration, these effects were slightly more pronounced for dispersions formed with GA or LA, compared to AA or PA. FT-IR data suggested more intense attractive interactions of chitosan chains with glycolate and lactate anions, than with acetate and propionate. Chitosan chains interacted more strongly with hydroxylated acids counter-anions than with their non-hydroxylated counterparts, leading to slight quantitative changes of physicochemical properties of these systems. Then, in physicochemical terms, GA, LA or PA are suitable to replace AA when preparing aqueous chitosan dispersions for technological applications.

Use of gelatin and gum arabic for microencapsulation of probiotic cells from Lactobacillus plantarum by a dual process combining double emulsification followed by complex coacervation.

The objectives of this study were i) to microencapsulate probiotic cells of Lactobacillus plantarum through a dual process consisting of emulsification followed by complex coacervation using gelatin and gum arabic, ii) to characterize the lyophilized microcapsules, iii) to evaluate their behavior in simulated in vitro gastrointestinal conditions and iv) to evaluate the survival of microencapsulated probiotic cells during 45 days of storage at 8 °C, 25 °C and -18 °C. The optimized conditions for complex coacervation consisted of a 50:50 biopolymer ratio and pH = 4.0. Emulsification was followed by complex coacervation using gelatin and gum arabic. The microcapsules presented dispersibility of 0.183 ±â€¯0.17 g·mL-1, moisture content of 4.5%, water activity of 0.34 ±â€¯0.03 and hygroscopicity of 9.20 ±â€¯0.43 g of absorbed water per 100 g. Their size ranged from 66.07 ±â€¯3.04 µm to 105.66 ±â€¯3.24 µm. Viability of the encapsulated L. plantarum cells was 8.6 log CFU·g-1 and the encapsulation efficiency was 97.78%. After in vitro simulation of gastrointestinal conditions, viability of the encapsulated cells was 80.4% whereas it was only 25.0% for the free cells at 37 °C. Probiotic cell viability was maintained during storage at 8 °C and - 18 °C for 45 days.

Chitosan dispersed in aqueous solutions of acetic, glycolic, propionic or lactic acid as a thickener/stabilizer agent of O/W emulsions produced by ultrasonic homogenization.

Chitosan is a natural polycationic polysaccharide with several known biotechnological functionalities, but its application in food products as ingredient or additive remains nowadays unusual. Additionally, ultrasonic production of food-grade emulsions is still an open research field, so ultrasound applicability for such purpose must be evaluated case by case. In this study, chitosan was dispersed in acid aqueous media containing acetic, glycolic, propionic or lactic acid (50 mmol·L-1), then added of the emulsifier Tween 20, and finally mixed to sunflower oil, through ultrasonic homogenization (20 kHz, 500 W, 4 min), in order to prepare O/W emulsions (oil fraction = 0.25). In all studied systems, oil droplets with average hydrodynamic diameter < 600 nm were obtained. The increase of chitosan concentration promoted the augment in consistency and the elastic character of the emulsions. Emulsions containing more than 0.500 g·(100 g)-1 of chitosan presented a minor increase of both oil droplets average hydrodynamic diameter and PDI, during storage for 28 days. Furthermore, such systems showed no phase separation when exposed to centrifugation, freeze-thawing, and freeze-thaw-heating cycles. Two main findings may be highlighted from this study: i) ultrasound processing is a promising approach to produce food-grade emulsified systems containing chitosan, and ii) chitosan is a suitable alternative as thickener/stabilizer for acidic emulsions, being its performance influenced by the biopolymer concentration and not by the organic acid present in the medium.

Anti-Hypertensive Peptides Derived from Caseins: Mechanism of Physiological Action, Production Bioprocesses, and Challenges for Food Applications.

This review is focused on the state-of-art of peptides with inhibitory activity towards angiotensin I-converting enzyme (ACE) - thus, with anti-hypertensive potential - derived from enzymatic hydrolysis of caseins. Firstly, molecular characteristics of caseins relevant to a better understanding of this subject were concisely commented. Next, a brief description of the pathophysiology of hypertension was explained, focusing on the ACE role in regulation of blood pressure in human body. Then, casein-derived peptides with ACE inhibitory capacity were specifically addressed. The main in vitro and in vivo bioassays often reported in literature to assess the anti-hypertensive potential of peptides were presented, illustrated with recently published studies, and discussed in terms of advantages and limitations of both approaches. Characteristics related to amino acid composition and sequence of peptides with high ACE-inhibitory potential were also commented. Process parameters of enzymatic hydrolysis (types and origins of casein substrates, types of enzymes, pH, temperature, and times of reactions) were discussed. Patents dealing with casein-derived anti-hypertensive peptides were examined not only in terms of amino acid sequences, but also regarding their novelty claims in hydrolysis process parameters. Finally, some trends, challenges, and opportunities inferred from this literature analysis were commented, emphasizing the importance of this research topic in food products development.

Formation and characterization of supramolecular structures of ß-lactoglobulin and lactoferrin proteins.

Combination of ß-lactoglobulin (ß-Lg) and lactoferrin (Lf), biomacromolecules derived from bovine whey, was used in the formation of supramolecular structures by thermal gelation technique to adjust the pH. Furthermore, the influence of the molar ratio, temperature, pH, and heating time in the formation of supramolecular structures were also studied. The characterization of the protein supramolecular structures was performed using dynamic light scattering, zeta potential measurements, molecular spectrofluorimetry, and circular dichroism spectroscopy. The thermal behavior of the pure proteins was investigated by differential scanning calorimetry. The protein denaturation temperatures were of around 85°C for the ß-Lg and around 52°C and 85°C (a small portion) for the Lf. The protein molar ratio of 2:1 Lf/ß-Lg was used to form the structures, whose characterization showed that the best conditions of supramolecular structure formation occurred at pH6.5 and at temperatures of 62.5°C. In those conditions, more stable systems with reduced hydrophobic surface and average sizes between 30 and 100nm were generated. The correlation between pH and temperature suggests that the method of preparation of the supramolecular structure affects its size during storage.

Acacia gum as modifier of thermal stability, solubility and emulsifying properties of α-lactalbumin.

Protein-polysaccharide conjugates often display improved techno-functional properties when compared to their individual involved biomolecules. α-Lactalbumin:acacia gum (α-la:AG) conjugates were prepared via Maillard reaction by the dry-heating method. Conjugate formation was confirmed using results of absorbance, o-phthalaldehyde test, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography. Techno-functional properties (emulsifying characteristics, solubility, and thermal stability) were evaluated for α-la, α-la/AG mixtures and α-la:AG conjugates. Conjugate thermal stability was improved compared to pure α-la treated at the same conditions of conjugate formation. Response surface methodology was used to establish models to predict solubility and emulsifying activity as functions of the salt concentration, pH and reaction time. α-la:AG conjugate solubility is affected in a complex manner by the three factors analyzed. Emulsifying activity index (EAI) of α-la is significantly affected by pH, while the α-la:AG EAI is affected by the three analyzed factors. Both solubility and EAI are maximized with pH 8.0, NaCl concentration of 0.3 mol L(-1) and two days of Maillard reaction.

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340).

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

Recovery of casein-derived peptides with in vitro inhibitory activity of angiotensin converting enzyme (ACE) using aqueous two-phase systems.

Peptides inhibiting the activity of angiotensin converting enzyme (ACE) were obtained by trypsin-catalyzed hydrolysis of bovine milk casein, performed at 37°C, during 1, 2, 5, 8 and 24h. Results of in vitro inhibitory activity ranged between 13.4% and 78.5%. The highest ACE inhibitory activity was evidenced for hydrolysates obtained after 2h of reaction. Aqueous two-phase systems (ATPS) formed by polyethylene glycol of 1500gmol-1 (PEG 1500)+sodium phosphate or potassium phosphates were produced and evaluated, in terms of partition coefficients (K) and extraction yields (y), to recovery the casein hydrolysates at room temperature. In ATPS containing sodium phosphate, the peptides showed a slightly greater affinity toward the bottom salt-rich phase (0.1≤K≤0.9; 5.7%≤y≤47%). In the case of ATPS containing potassium phosphates, these molecules showed substantially greater affinity toward the top polymer-rich phase (137≤K≤266; y≥99%). These results point out extraction using PEG 1500/potassium phosphate ATPS is an efficient technique to recover casein hydrolysates containing ACE inhibitors peptides. Outlined data will be helpful in integrating such unit operation to larger scale processes.