Water-insoluble Proteins Research Articles

Membrane protein mediated bilayer communication in networks of droplet interface bilayers

Droplet interface bilayers (DIBs) are model membranes formed between lipid monolayer-encased water droplets in oil. Compared to conventional methods, one of the most unique properties of DIBs is that they can be connected together to generate multi-layered ‘tissue-like’ networks, however introducing communication pathways between these compartments typically relies on water-soluble pores that are unable to gate. Here, we show that network connectivity can instead be achieved using a water-insoluble membrane protein by successfully reconstituting a chemically activatable mutant of the mechanosensitive channel MscL into a network of DIBs. Moreover, we also show how the small molecule activator can diffuse through an open channel and across the neighbouring droplet to activate MscL present in an adjacent bilayer. This demonstration of membrane protein mediated bilayer communication could prove key toward developing the next generation of responsive bilayer networks capable of defining information flow inside a minimal tissue.

Thermogravimetric Analysis and Pyrolysis Kinetics of Tannery Wastes in an Inert Atmosphere

Industrial wastes generated from tanneries contain large quantities of water-insoluble proteins, which may be used for the production of composite materials, renewable chemicals and energy. In this work, the pyrolysis kinetics of powdered sheep fur wastes (SFW) was studied by thermogravimetry (TG) at different heating rates from room temperature to 600°C in nitrogen atmosphere. TG results revealed that there are three stages in this process. The overall apparent activation energy (E) in the main pyrolysis stage was determined to be 275.6 kJ mol-1 by modified Kissinger-Akahira-Sunose (MKAS) method. Because the pyrolysis of SFW could not be described by a single-step reaction, the experimental DTG curve of SFW was deconvoluted into three individual peaks followed by reconstruction of TG curves corresponding to three pseudo components. The average values of E obtained for these pseudo components are 234.7 kJ mol-1, 176.4 kJ mol-1, and 186.2 kJ mol-1, respectively. Generalized master-plots method indicated that the SFW pyrolysis may follow the random nucleation and growth mechanism (Avrami-Erofeev model). Reaction model functions f(?) for these pseudo components could be expressed as: f(?)=3.1(1-?)[-ln(1-?)]0.67; f(?)=3.6(1-?)[-ln(1-?)]0.72, and f(?)=3.9(1-?)[-ln(1-?)]0.74, respectively. These results may provide insight for further studies as well as for future application of pyrolysis technology for tannery wastes.

Characteristics and rheological behavior of Pickering emulsions stabilized by tea water-insoluble protein nanoparticles via high-pressure homogenization

Food-grade Pickering emulsions have attracted lots of researchers' attention. As one type of food-grade particles, tea water-insoluble protein nanoparticles (TWIPN) from tea residues were utilized to prepare Pickering emulsions at an oil-water ratio of 6: 4 (v/v) via high-pressure homogenization (HPH). Characteristics and rheological behavior of TWIPN-stabilized Pickering emulsions (TWIPNPE) were analyzed. The size of TWIPNPE significantly decreased with the increase of homogenization pressure (0–80 MPa) and homogenization times. However, the droplet size of TWIPNPE at 120 MPa significantly increased compared to that at 80 MPa. Furthermore, the droplet size and zeta potential of TWIPNPE prepared at a fixed homogenization pressure of 40 MPa reduced in comparison with those prepared via high-speed homogenization at the same TWIPN concentration (0.5–4.0%). Besides, the emulsions prepared at the TWIPN concentration of 3.0% and 4.0% had no creaming within 50 days. Additionally, HPH improved the gel-like behavior of TWIPNPE. These results suggest that the TWIPNPE have the potential for preparing the high viscoelastic system for the food industry to expand the utility of tea leave byproducts.

Wheat-induced food allergy in childhood: ancient grains seem no way out

Wheat is a frequent elicitor of food allergy in childhood. Especially in popular media, the better digestibility and the lower allergenicity of ancient grains are repeatedly postulated. We addressed the question whether ancient wheat-related grains are less allergenic than modern wheat. Proteins from cultivars of spelt, einkorn, emmer and durum along with durum soft wheat flour, Tritordeum and bread wheat were separated by electrophoresis. Immunoblots were performed with a pool serum of six sera from wheat-sensitized children aged 1-11years (wheat-specific IgE 22 kUA/l). As controls, pool serum from five sera atopic patients aged 3-13years who had no sensitization to wheat (wheat-specific IgE 0.11 kUA/l) and six sera from non-atopics at the age of 3months to 5years (wheat-specific IgE 0.06 kUA/l) was used. Area under the curve (AUC) in Coomassie-stained gels and immunoblots was determined and related. Water/salt-soluble protein patterns were very similar among varieties. In einkorn cultivars, one protein band corresponding to an alpha-amylase/trypsin inhibitor (ATI) was absent. Water-insoluble protein fractions differed markedly among varieties and cultivars. IgE bound to a large number of proteins in all protein fractions both in wheat and in the wheat-related ancient grains. Patients with sensitization to wheat show a significant IgE binding against both modern and ancient grain varieties of the genus Triticum. Therefore, ancient grains do not appear to have a generally reduced risk for wheat allergy sufferers. However, few individuals only sensitized to ATI could benefit from the consumption of einkorn.

Application of Microfluidics in the Production and Analysis of Food Foams.

Emulsifiers play a key role in the stabilization of foam bubbles. In food foams, biopolymers such as proteins are contributing to long-term stability through several effects such as increasing bulk viscosity and the formation of viscoelastic interfaces. Recent studies have identified promising new stabilizers for (food) foams and emulsions, for instance biological particles derived from water-soluble or water-insoluble proteins, (modified) starch as well as chitin. Microfluidic platforms could provide a valuable tool to study foam formation on the single-bubble level, yielding mechanistic insights into the formation and stabilization (as well as destabilization) of foams stabilized by these new stabilizers. Yet, the recent developments in microfluidic technology have mainly focused on emulsions rather than foams. Microfluidic devices have been up-scaled (to some extent) for large-scale emulsion production, and also designed as investigative tools to monitor interfaces at the (sub)millisecond time scale. In this review, we summarize the current state of the art in droplet microfluidics (and, where available, bubble microfluidics), and provide a perspective on the applications for (food) foams. Microfluidic investigations into foam formation and stability are expected to aid in optimization of stabilizer selection and production conditions for food foams, as well as provide a platform for (large-scale) production of monodisperse foams.

Novel food-grade Pickering emulsions stabilized by tea water-insoluble protein nanoparticles from tea residues

Recently, food-grade Pickering particles, particularly plant proteins, have attracted tremendous attention because they are biobased, environmentally-friendly and edible. To explore the potential of tea water-insoluble protein (TWIP) nanoparticles from tea residues for stabilizing Pickering emulsions, the average hydrodynamic diameter (DH), zeta potential and morphologic profiles of TWIP nanoparticles were characterized using dynamic light scattering (DLS), laser Doppler velocimetry (LDV) and atomic force microscopy (AFM), respectively. The results indicated that TWIP nanoparticles were irregular colloidal particles with a DH greater than 300 nm and a negative charge of more than −30 mV at ionic strengths of 0–400 mM and a fixed TWIP nanoparticle concentration (2.0%). Furthermore, the effect of the TWIP nanoparticle concentration (0.5–4.0%, w/v) and oil-water ratio (2:8–6:4) on the characteristics of the Pickering emulsions stabilized via TWIP nanoparticles was investigated. Increasing the TWIP nanoparticle concentration generated a firm and thick TWIP nanoparticle-based interfacial layer, as verified by Cryo-scanning electron microscopy imaging, and decreased the droplet size of Pickering emulsions at an oil-water ratio of 4:6. Additionally, an increase of the oil-water ratio to 6:4 favored the formation of emulsions with extraordinary creaming stability at the fixed TWIP nanoparticle concentration of 2.0%. The present study is the first to suggest TWIP nanoparticles as a type of food-grade Pickering particle.

Functional properties and structural profiles of water-insoluble proteins from three types of tea residues

Green tea water-insoluble protein (GP), oolong tea water-insoluble protein (OP) and black tea water-insoluble protein (BP) were extracted from residues of green tea, oolong tea and black tea by an alkaline method, respectively. The foaming stability and emulsifying capacity of GP were better than that of OP and BP, but the oil absorption capacity (3.00 ± 0.28 mL/g) and foaming capacity (41.00 ± 1.41%) of GP was significantly lower than that of OP and BP. Furthermore, the analysis of protein surface hydrophobicity, amino acid composition, secondary structure, zeta potential and contact angle of different tea water-insoluble proteins (TWIPs) revealed that TWIPs were negatively charged with high hydrophobicity. Moreover, the random coil content (26.70 ± 0.10%) of GP was higher than those of OP and BP in favor of emulsifying capacity. At the oil-water ratio of 5:5 and TWIP concentration of 2.0 g/100 mL, the creaming index of GP, OP and BP emulsions was 33.55 ± 0.93%, 40.79 ± 5.58% and 40.13 ± 0.93% after 6 d. The emulsifying stability of GP better than that of OP and BP was due to the small size (112.67 ± 2.52 μm) and high zeta potential (−36.77 ± 2.47 mV) of GP emulsion droplets. These results suggest that TWIPs, especially GP, are the potentially food-grade emulsifiers for preparing emulsions.

Effects of High Hydrostatic Pressure on the Solubilities and Structures of Alaska Pollock Protein

Alaska pollock is an important protein source which is extensively used in the food industry. Pollock protein isolates (PPI) with significantly enriched protein contents could be prepared using isoelectric solubilization/precipitation (ISP) processing; however, the functional properties of this process is limited by the large amount of water-insoluble proteins. In this study, we investigated the influence of high hydrostatic pressure (HHP) treatment on the solubility and structural changes of PPI. PPI obtained using ISP is treated with hydrostatic pressures of 200, 300, 400, and 500 MPa for up to 15 min, and the HHP-treated samples were observed to exhibit significantly improved solubilities. Further biochemical assays reveal that the continuous HHP treatments reduce the contents of free sulfhydryl groups and promote the formation of macromolecules with better water solubilities, which may induce the solubility improvements of the HHP-treated PPI. Our results indicate that HHP can be utilized to effectively prepare highly water- soluble Alaska pollock protein in food processing.

Fabrication and Characterization of Novel Water-Insoluble Protein Porous Materials Derived from Pickering High Internal-Phase Emulsions Stabilized by Gliadin-Chitosan-Complex Particles.

Pickering high internal-phase emulsions (HIPEs) and porous materials derived from the Pickering HIPEs have received increased attention in various research fields. Nevertheless, nondegradable inorganic and synthetic stabilizers present toxicity risks, thus greatly limiting their wider applications. In this work, we successfully developed nontoxic porous materials through the Pickering HIPE-templating process without chemical reactions. The obtained porous materials exhibited appreciable absorption capacity to corn oil and reached the state of saturated absorption within 3 min. The Pickering HIPE templates were stabilized by gliadin-chitosan complex particles (GCCPs), in which the volume fraction of the dispersed phase (90%) was the highest of all reported food-grade-particle-stabilized Pickering HIPEs so far, further contributing to the interconnected pore structure and high porosity (>90%) of porous materials. The interfacial particle barrier (Pickering mechanism) and three-dimensional network formed by the GCCPs in the continuous phase play crucial roles in stabilization of HIPEs with viscoelastic and self-supporting attributes and also facilitate the development of porous materials with designed pore structure. These materials, with favorable biocompatibility and biodegradability, possess excellent application prospects in foods, pharmaceuticals, materials, environmental applications, and so on.

Solubility of extracted proteins from Chlorella sorokiniana, Phaeodactylum tricornutum, and Nannochloropsis oceanica: Impact of pH-value

Microalgae are a new source of proteins, with yet unknown technofunctionalities. The aim of this work was to study protein solubility – a key technofunctionality - in water in the pH-range of 2–12 of proteins from water-soluble (WSPE) and water-insoluble protein extracts (WISPE) obtained from Chlorella sorokiniana, Phaeodactylum tricornutum, and Nannochloropsis oceanica. Proteins in the WSPEs exhibited an unusual solubility pattern with a solubility ≥39.5 g/100 mL and no complete precipitation in the pH-range 2-12, which was attributed to protein glycosylation and a high amount of hydrophilic amino acids. However, slight aggregation was evident at all pH-values and no transparent solutions were formed. The highest protein solubility was observed for proteins of Nannochloropsis oceanica, followed by Chlorella sorokiniana, and Phaeodactylum tricornutum. In the WISPEs, strong aggregation and a low solubility (≤22.6 g/100 mL) were observed in the pH-range of 2–11, with an increase at pH 12. Overall, the studied proteins exhibited solubility patterns in the pH-range 2-12 that were different to conventional food proteins, and especially the proteins in the WSPE of Nannochloropsis oceanica exhibited a high solubility (≥58.8 g/100 mL) at pH ≤ 5. Thus, this fraction is quite promising for food applications (e.g. protein-rich acidic beverages) and is of interest for further studies.

Wheat Gluten and Its Hydrolysates. Possible Fields of Practical Use

Wheat gluten is a group of water-insoluble proteins of wheat endosperm. As a co-product of the starch and bioethanol production, gluten is available in fairly large quantities. Therefore, the search for optimal ways of its industrial utilization is an important economic task. The food industry is a traditional area for gluten application. However, the study of its structural and functional properties allowed expanding the area of the gluten use due to its various modifications, in particular, enzymatic hydrolysis. As compared to native gluten, the gluten hydrolysates have better foaming and emulsifying abilities, as well as higher water solubility. Biologically active properties of hydrolysates (antioxidant, hepatoprotective and angiotensin-converting enzyme inhibiting) were also shown. The search for and selection of proteolytic enzymes or their combinations, and optimization of the technological process will permit to obtain the hydrolysis products with the required characteristics, expand their use in industry and reduce the cost of their production.

Cod Residual Protein Prevented Blood Pressure Increase in Zucker fa/fa Rats, Possibly by Inhibiting Activities of Angiotensin-Converting Enzyme and Renin.

Hypertension is the leading risk factor for cardiovascular disease, and prevention of high blood pressure through diet and lifestyle should be a preferred approach. High intake of fish is associated with lower blood pressure, possibly mediated through the proteins since peptides with angiotensin-converting enzyme (ACE) inhibiting capacities have been identified in fish skin, backbone, and fillet. The effects of cod meals made from residual materials and fillet on blood pressure were investigated in obese Zucker fa/fa rats which spontaneously develop high blood pressure. Rats were fed diets containing water-soluble (stickwater) or water-insoluble (presscake) fractions of protein-rich meals from cod residual materials (head, gut, backbone with muscle residuals, skin, trimmings) or fillet. Rats were fed diets containing 25% of total protein from cod meal and 75% of protein from casein, or casein as the sole protein source (control group) for four weeks. Results show that a diet containing residual presscake meal with high gut content prevented blood pressure increase, and this cod residual meal also showed the strongest in vitro inhibitions of ACE and renin activities. In conclusion, a diet containing water-insoluble proteins (presscake meal) with high gut content prevented increase in blood pressure in obese Zucker fa/fa rats.

Color-tunable particles through affinity interactions between water-insoluble protein and soluble dyes

This research is focused on the delivery of color using zein particles as a carrier and contributes to understanding the interactions between colorants and zein. Co-precipitation of purified zein protein with anionic dyes such as sulfonate group containing azo-dyes (patent blue sodium salt, azorubine, and fast green FCF) are used to prepare red, green, and blue colored nanoparticles. These dyes had electrostatic interactions with zein and they remained bound in the zein particles even after dialysis. This was not the case for purpurin, for with hydrogen bonding was expected as interaction. Upon increasing dye concentration, the particle sizes increased as a result of a change in the kinetics of precipitation, which was affected by the association between zein and the dye. However, the encapsulation efficiency continued to be very high and no plateau was reached for the used concentrations. The post-addition of dye on undyed zein particles was used to confirm that there is indeed a high affinity between the dye and the protein. The amount of free dye was determined and the affinity was again found to be high (dye adsorption <95%). The amount of dye incorporated into the particles and the resulting color were similar using both techniques. These results show that there is a high affinity between the zein protein and the dye, and may be exploited to produce bio-based and optically-functionalized nanoparticles using zein as a carrier.

Recovery and cryopreservation of insulin amyloid using ionic liquids

Amyloid aggregates containing a high content of β-sheet structure are one of several water-insoluble protein aggregates. In this study, we assessed the use of aqueous ionic liquid (IL) solutions as part of a simple preservation/refolding technique for amyloid aggregates. We investigated the dissolution and refolding of cryopreserved bovine insulin amyloid in concentrated aqueous solutions (X = 20 mol%IL) of six different ionic liquids: three 1-butyl-3-methylimidazolium-based ILs ([bmim][X]; X = SCN, NO3, or Cl), 1-ethyl-3-methylimidazolium nitrate ([emim][NO3]), ethylammonium nitrate (EAN), and propylammonium nitrate (PAN). Dimethyl sulfoxide (DMSO) was used as the reference standard for spectroscopy. The six ILs all exhibited the ability to dissolve insulin amyloid, with imidazolium-based ILs exhibiting increased dissolution capacity compared with ammonium-based ILs or DMSO. Remarkably, >80% of the secondary structure of the folded insulin monomer was recovered from insulin amyloid dissolved in aqueous IL solutions following cryopreservation and IL removal by dialysis. These results indicate that concentrated aqueous IL solutions have potential as a new and simple agent for the dissolution, cryopreservation, and refolding of amyloid aggregates.

Solubility and aggregation behavior of protein fractions from the heterotrophically cultivated microalga Chlorella protothecoides

Protein solubility in water is a key property of food proteins. The aim of this work was to study the solubility and microstructural properties as a function of pH of both protein fractions (water-soluble (WSPE) and water-insoluble protein extracts (WISPE)) obtained from the microalga Chlorella protothecoides, which is promising for food use. Protein solubility was determined as the ratio of protein concentration in the supernatant after centrifugation to total protein concentration. An unusually high solubility and only slight gravitational separation across a very broad pH-range (2−12) were observed for the WSPE with a minimum protein solubility of 84.3 ± 2.2% at pH 2. The origin of this high pH-independent protein solubility was attributed to a high degree of glycosylation and a high amount of hydrophilic amino acids. In contrast, the WISPE was found to contain strongly aggregated proteins, and these large aggregates separated rapidly from solution by gravitation independent of the pH. This corresponded to their protein solubility, which was overall low in the pH-range of 2–11, and only increased at pH 12 to a maximum solubility of 26.9 ± 2.8%. These results suggest that the WSPE of Chlorella protothecoides may exhibit unique properties for food formulations, allowing for example for both acidified, neutral or slightly alkaline foods to be formulated, whereas WISPE may be more suited for foods where phase separation is rather slow.

Stabilization of oil continuous emulsions with colloidal particles from water-insoluble plant proteins

Emulsions stabilized by surface active particles are becoming an attractive alternative to conventional surfactant-stabilized emulsions. Biobased, environmentally friendly, and edible particles are particularly interesting for applications in foods, agriculture, and consumer products. Previously, water insoluble proteins such as prolamins have been only used as a platform for stabilization of water continuous emulsions. In this paper, we investigate the ability of zein particles to stabilize oil continuous emulsions. We synthesized and used zein particles in the form of (i) an aqueous, or (ii) an oil suspension. Aqueous suspensions of zein particles resulted in water-in-oil (w/o) emulsions that were generally stable for more than an hour, with up to 30 vol% water as the dispersed phase. Cryo-SEM images show that only a small fraction of zein particles are adsorbed at the oil-water interface as Pickering stabilizers. Adding an oil soluble surfactant (lecithin) prior to emulsification with zein particles in an aqueous suspension promoted the formation of oil-continuous emulsion with phase inversion point at 40 vol% of water. Zein particles suspended in oil produced less stable emulsions, which showed reversible phase inversion attributed to the electrostatic repulsion of hydrophilic protein loops above their isoelectric point. Our results show that zein particles act as a predominantly hydrophilic stabilizer that requires control prior to emulsification to form w/o emulsions. These emulsions, however, have only a limited stability.

The adsorption of α-amylase on barley proteins affects the in vitro digestion of starch in barley flour

The conversion of barley starch to sugars is a complex enzymic process. Most previous work concerned the biotechnical aspect of in situ barley enzymes. However, the interactions among the macromolecular substrates and their effects on enzymic catalysis has been little examined. Here, we explore the mechanisms whereby interactions of protein and starch in barley flour affect the kinetics of enzymatic hydrolysis of starch in an in vitro system, using digestion rate data and structural analysis by confocal microscopy. The degradation kinetics of both uncooked barley flour and of purified starches are found to be two-step sequential processes. Barley proteins, especially the water-soluble component, are found to retard the digestion of starch degraded by α-amylase: the enzyme binds with water-insoluble protein and with starch granules, leading to reduced starch hydrolysis. These findings are of potential industrial value in both the brewing and food industries.

Zein-based colloidal particles for encapsulation and delivery of epigallocatechin gallate

Zein, a water insoluble plant protein from a renewable natural source, has been identified as a highly promising material for the production of protein-based colloidal particles for the encapsulation of lipophilic compounds. However, the encapsulation of hydrophilic, water-soluble, bioactive molecules within zein colloidal particles is still unexplored. We show that the encapsulation of epigallocatechin gallate (EGCG) is strongly limited by the weak physical interactions occurring with the zein matrix during the precipitation phase. We demonstrate that the use of sodium caseinate, as a colloidal stabilizer to coat the zein particles, enables the modulation of the encapsulation efficiency and functionality of zein colloidal particles for EGCG delivery. In particular, coated zein particles exhibit a larger size, opposite surface charge and significantly different antioxidant activity, owing to the localization of EGCG affected by the different extent of interaction of EGCG with zein and sodium caseinate. Remarkably, particle formulation also tunes the release rate of EGCG during in vitro digestion and modulates the rate of fat digestion, through the combination of the Pickering emulsion stabilization effect and EGCG interaction with lipase enzyme. Zein-based colloidal particles constitute hence remarkable systems for the tunable and multi-functional delivery of EGCG.

Investigation of CO2 precursors in roasted coffee.

Two CO2 formation pathways (chlorogenic acid (CGA) degradation and Maillard reaction) during coffee roasting were investigated. CGA is shown not a major contributor to CO2 formation, as heating of this compound under typical roasting conditions did not release a large quantity of CO2. However, heating of a CGA moiety, caffeic acid, resulted in high yield of CO2 (>98%), suggesting that CGA hydrolysis could be the rate limiting step for CO2 formation from CGA. A large amount of CO2 was detected from glycine-sucrose model system under coffee roasting conditions, implying the importance of Maillard reactions in CO2 formation. Further studies on the heating of various components isolated from green coffee beans showed that CO2 was generated from various green coffee components, including water insoluble proteins and polysaccharides. Around 50% of CO2 was formed from thermal reactions of lower molecular weight compounds that represent ∼25% by weight in green coffee.

One-shot LC-MS/MS analysis of post-translational modifications including oxidation and deamidation of rat lens α- and β-crystallins induced by γ-irradiation.

The eye lens is a transparent organ that functions to focus light and images on the retina. The transparency and high refraction of the lens are maintained by the function of α-, β-, and γ-crystallins. These long-lived proteins are subject to various post-translational modifications, such as oxidation, deamidation, truncation and isomerization, which occur gradually during the aging process. Such modifications, which are generated by UV light and oxidative stress, decrease crystallin solubility and lens transparency, and ultimately lead to the development of age-related cataracts. Here, we irradiated young rat lenses with γ-rays (5-500Gy) and extracted the water-soluble (WS) and water-insoluble (WI) protein fractions. The WS and WI lens proteins were digested with trypsin, and the resulting peptides were analyzed by one-shot LC-MS/MS to determine the specific sites of oxidation of methionine and tryptophan, deamidation sites of asparagine and glutamine, and isomerization of aspartyl in rat α- and β-crystallins in the WS and WI fractions. Oxidation and deamidation occurred in several crystallins after irradiation at more than, respectively, 50 and 5Gy; however, isomerization did not occur in any crystallin even after exposure to 500Gy of irradiation. The number of oxidation and deamidation sites was much higher in the WI than in the WS fraction. Furthermore, the oxidation and deamidation sites in rat crystallins resemble those reported in crystallins from human age-related cataracts. Thus, this study on post-translational modifications of crystallins induced by ionizing irradiation may provide useful information relevant to the formation of human age-related cataracts.