Acid-induced gelation of heat-treated milk studied by diffusing wave spectroscopy

Fresh skim milk is a stable colloidal system containing casein micelles and whey proteins. By decreasing the pH, the casein micelles become unstable and a gel is formed. During heat treatment at temperatures higher than 70 degrees C, the major whey proteins, e.g. alpha-lactalbumin and beta-lactoglobulin denature and start to interact with each other and with casein micelles. This changes the colloidal properties of the casein micelles. In this article, the pH-induced gel formation of heat-treated milk and the role of whey proteins was studied. Heat treatment in the range 70-90 degrees C induced a shift in gelation pH of skim milk to more alkaline pH values. This shift was directly related to whey protein denaturation. By using WPF milk it was shown that beta-lactoglobulin is principally responsible for the shift in gelation pH. alpha-lactalbumin caused neither alone nor in combination with beta-lg, an effect on the gelation pH. Heat treatment of milk for 10 min at 90 degrees C resulted in complete denaturation of the beta-lg present in skim milk but it is estimated that the casein micelles are coated only up to 40% by whey proteins when compared with pure whey protein aggregates.     

Drug Delivery Vehicles Based on Albumin–Polymer Conjugates

Albumin has been a popular building block to create nanoparticles for drug delivery purposes. The performance of albumin as a drug carrier can be enhanced by combining protein with polymers, which allows the design of carriers to encompass a broader spectrum of drugs while features unique to synthetic polymers such as stimuli‐responsiveness are introduced. Nanoparticles based on polymer–albumin hybrids can be divided into two classes: one that carries album as a bioactive surface coating and the other that uses albumin as biocompatible, although nonbioactive, building block. Nanoparticles with bioactive albumin surface coating can either be prepared by self‐assembly of albumin–polymer conjugates or by postcoating of existing nanoparticles with albumin. Albumin has also been used as building block, either in its native or denatured form. Existing albumin nanoparticles are coated with polymers, which can influence the degradation of albumin or impact on the drug release. Finally, an alternative way of using albumin by denaturing the protein to generate a highly functional chain, which can be modified with polymer, has been presented. These albumin nanoparticles are designed to be extremely versatile so that they can deliver a wide variety of drugs, including traditional hydrophobic drugs, metal‐based drugs and even therapeutic proteins and siRNA.

     

Formation and characterization of polyglutamate core-shell microspheres

The need for organ-targeted delivery of drugs and imaging agents creates an interest in biocompatible, biodegradable vesicles. We make protein microspheres using high-intensity ultrasound; these microspheres have a protein shell and a hydrophobic interior, making them ideal for delivering hydrophobic materials. We have previously shown that various proteins, e.g., bovine serum albumin (BSA), form a microsphere shell stabilized by interprotein cross-linking of cysteine residues. In this study, polyglutamate was used to form core-shell microspheres at slightly basic pH using sonication. These particles are smaller than our previous protein microspheres and are stable under conditions encountered in vivo. The stability of polyglutamate microspheres appears to be due to hydrogen bonding networks and not covalent cross-linking.     

Surfactant-Free Multiple Pickering Emulsions Stabilized by Combining Hydrophobic and Hydrophilic Nanoparticles

A series of W/O/W or O/W/O emulsion stabilized solely by two different types of solid nanoparticles were prepared by a two-step method. We explored the option of particular emulsifiers for the multiple Pickering emulsions, and a variety of nanoparticles (silica, iron oxide, and clay) only differing in their wettability was used. The primary W/O emulsion was obtained by the hydrophobic nanoparticles, and then the hydrophilic nanoparticles were used as emulsifier in the secondary emulsification to prepare the W/O/W emulsion. In a similar way, the primary O/W emulsion of the O/W/O emulsion was stabilized by the hydrophilic nanoparticles, while the secondary emulsification to prepare the O/W/O emulsion was effected with the hydrophobic nanoparticles. The resultant multiple Pickering emulsion was stable to coalescence for more than 3 months, except the W/O/W emulsions of which the secondary emulsion stabilized by clay nanoparticles became a simple O/W emulsion in a day after preparation. Moreover, the temperature and pH sensitive poly(N-isopropylacrylamide-co-methacrylic acid) (P(NIPAm-co-MAA)) microgels were introduced as an emulsifier for the secondary emulsification to obtain the stimulus-responsive multiple W/O/W emulsion. Such microgel-stabilized multiple emulsions could realize the efficient controlled release of water-soluble dye, Rhodamine B (RB) on demand in a multiple-emulsion delivery system.      

Hydrocolloid gels of polysaccharides and proteins

Recent developments from 1997 to 2000 in hydrocolloid gels which consist of dispersed phase (polysaccharide or protein) and dispersing medium (water) are reviewed in the present paper. Gels and gelling processes of polysaccharides such as gellan gum, methylcellulose, xyloglucan, curdlan, konjac glucomannan and starch are described. Fluid gels and galactomannan gels prepared by freeze-thaw cycling are also described. Effects of pH and ionic strength on the gelation of proteins such as casein and β-lactoglobulin are described. Fractal treatment is introduced to study the structure–property relationship for globular protein gels. Gels formed by different hydrocolloids are also described briefly.     

Preparation of Polyclonal Antibodies with Application for an Organophosphorus Pesticide Immunoassay

Haptens of dichlorvos and paraoxon were conjugated to the carrier proteins of bovine serum albumin. The obtained conjugates were characterized by infrared and ultraviolet–visible spectroscopy. The binding ratios of dichlorvos and paraoxon-to-carrier proteins were also evaluated. The number of hapten molecules per protein molecule of dichlorvos–cationized bovine serum albumin conjugate was higher than for paraoxon–bovine serum albumin conjugate. The sheep polyclonal antibodies were produced against the dichlorvos and paraoxon. New multipolyclonal antibodies were obtained and characterized following the immunization of a 1:1 mixture of the immunogens for the simultaneous determination of dichlorvos and paraoxon by the immunoassay. An indirect enzyme-linked immunosorbent assay was used to characterize the reactivity of the antibodies to hapten conjugates. The multiantibodies showed lower affinities than the separate antibodies, but their affinities were sufficient for an immunoassay for the simultaneous determination of the analytes. The detection limit and linear range for the determination of dichlorvos and paraoxon alone and together were determined. The recovery was characterized to determine dichlorvos and paraoxon fortified in model solutions and milk. These results demonstrate the potential of this immunoassay for the quantitative screening of dichlorvos and paraoxon.     

Interactions in the aqueous phase and adsorption at the air–water interface of caseinoglycomacropeptide (GMP) and β-lactoglobulin mixed systems

The aim of this work was to study the interactions and adsorption of caseinoglycomacropeptide (GMP) and GMP:β-lactoglobulin (β-lg) mixed system in the aqueous phase and at the air–water interface. The existence of associative interactions between GMP and β-lg in the aqueous phase was investigated by dynamic light scattering, differential scanning calorimetry (DSC), fluorometry and native PAGE-electrophoresis. The surface pressure isotherm and the static and dynamic surface pressure were determined by tensiometry and surface dilatational properties. The results showed that GMP presented higher surface activity than β-lg at a concentration of 4% wt but β-lg showed higher film forming ability. In the mixed systems β-lg dominated the static and dynamic surface pressure and the rheological properties of interfacial films suggesting that β-lg hinders GMP adsorption because, in simple competition, GMP should dominate because of its higher surface activity. The surface predominance of β-lg can be attributed to binding of GMP to β-lg in the aqueous phase that prevents GMP adsorption on its own.     

Effects of degree of substitution on stearic acid-modified Bletilla striata polysaccharides nanoparticles and interactions between nanoparticles and bovine serum albumin

A stearic acid-modified Bletilla striata polysaccharides nanoparticle was fabricated. The effects of degree of substitution on its characteristic properties, the interactions of nanoparticles with bovine serum albumin and cell toxicity were assessed.     

Covalently cross-linked proteins & polysaccharides: Formation,characterisation and potential applications

This review presents recent research conducted on the development of various protein-polysaccharide conjugates, their functional properties and industrial applications. These conjugates are formed by the glycosylation of food proteins with carbohydrates via the Maillard reaction and are capable of improving the functional properties of proteins. The Maillard reaction facilitates covalent bonding between a reducing group of a carbohydrate and an amino group of a protein under controlled conditions of temperature, time, pH, and relative humidity. There is a great deal of interest in modifying the functional properties of proteins and in the use of novel conjugates for various industrial applications. This review discusses various methods and their implications for preparing and characterising these conjugates. Furthermore, the physicochemical properties of conjugates such as solubility, thermal stability, emulsifying activity, emulsion stabilising properties, gelling and foaming properties are also analysed. A novel processing technology, a spinning disc reactor, could be an alternative process for the production of protein–polysaccharide conjugates, with desirable functionality in different food systems.     

Influence of whey protein denaturation on κ-carrageenan gelation

Response surface methodology was applied to study the effect of different heating temperature/time treatments on whey protein denaturation and its effect on κ-carrageenan gelation in milk. The path of gel formation was followed using small deformation rheology and the extent of whey protein denaturation was determined by gel permeation chromatography. κ-Carrageenan did not influence the rate of whey protein denaturation and it was unlikely that whey protein denaturation played a significant role on κ-carrageenan gelation in milk. In skim milk serum or skim milk ultrafiltrate the path of gel formation and gel strength were not influenced by the severity of heat treatment but increasing the concentration of whey proteins enhanced the gel strength. Heat treatment became important for carrageenan gelation in skim or recombined milks (i.e. in the presence of casein micelles) by influencing the gelation temperature and gel strength. Increasing the concentration of whey proteins in the recombined milks had a beneficial effect on gel strength.     

Encapsulation,protection, and release of hydrophilic active components: Potential and limitations of colloidal delivery systems

There have been major advances in the development of edible colloidal delivery systems for hydrophobic bioactives in recent years. However, there are still many challenges associated with the development of effective delivery systems for hydrophilic bioactives. This review highlights the major challenges associated with developing colloidal delivery systems for hydrophilic bioactive components that can be utilized in foods, pharmaceuticals, and other products intended for oral ingestion. Special emphasis is given to the fundamental physicochemical phenomena associated with encapsulation, stabilization, and release of these bioactive components, such as solubility, partitioning, barriers, and mass transport processes. Delivery systems suitable for encapsulating hydrophilic bioactive components are then reviewed, including liposomes, multiple emulsions, solid fat particles, multiple emulsions, biopolymer particles, cubosomes, and biologically-derived systems. The advantages and limitations of each of these delivery systems are highlighted. This information should facilitate the rational selection of the most appropriate colloidal delivery systems for particular applications in the food and other industries.     

Buffalo Colostrum β-lactoglobulin Inhibits VEGF-Induced Angiogenesis by Interacting with G protein-Coupled Receptor Kinase

β-lactoglobulin (β-lg), a major whey protein was purified and characterised from buffalo colostrum. The in silico analysis of the tryptic peptides based on LC-CID-MS/MS facilitated the identification of protein as β-lg. The sequences IIVTQ f[1–5] and LSFNPTQLEEQCHV f(149–162) of m/z 933⁺ and 851²⁺ were found to match N- and C-extreme of β-lg while IDALNENK f(84–91) and TPEVDDEALEKFDK f(125–138) sequences deduced for m/z 916⁺ and 818²⁺ were in compliance to buffalo milk β-lg. Considering the sequence similarity of β-lg to glycodelin, a proven angiogenic protein, similar role for β-lg from buffalo colostrum (BLG-col) was examined. Interestingly, BLG-col exhibited anti-angiogenic activity by potently inhibiting cell proliferation, micro-vessel sprouting, cell migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but having varied effect on Ehrlich ascites tumor cells, MCF-7, MDA-MB 435 and MDA-MB 231 cell lines. The anti-angiogenic potential of BLG-col was found to be vascular endothelial growth factor mediated. The immunolocalisation of BLG-col on the cell surface of HUVECs evidenced using FITC-labelled β-lg antibody indicated its extra-cellular binding. Furthermore, BLG-col interacting HUVEC membrane protein (64 kDa) was detected by immunoblot and its identity was established by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis, which showed peptide sequence homology to G protein-coupled receptor kinase 4.     

Ultrasound-Assisted Microencapsulation of Soybean Oil and Vitamin D Using Bare Glycogen Nanoparticles

Ultrasonically synthesized core-shell microcapsules can be made of synthetic polymers or natural biopolymers, such as proteins and polysaccharides, and have found applications in food, drug delivery and cosmetics. This study reports on the ultrasonic synthesis of microcapsules using unmodified (natural) and biodegradable glycogen nanoparticles derived from various sources, such as rabbit and bovine liver, oyster and sweet corn, for the encapsulation of soybean oil and vitamin D. Depending on their source, glycogen nanoparticles exhibited differences in size and ‘bound’ proteins. We optimized various synthetic parameters, such as ultrasonic power, time and concentration of glycogens and the oil phase to obtain stable core-shell microcapsules. Particularly, under ultrasound-induced emulsification conditions (sonication time 45 s and sonication power 160 W), native glycogens formed microcapsules with diameter between 0.3 μm and 8 μm. It was found that the size of glycogen as well as the protein component play an important role in stabilizing the Pickering emulsion and the microcapsules shell. This study highlights that native glycogen nanoparticles without any further tedious chemical modification steps can be successfully used for the encapsulation of nutrients.     

Spatial structure formation in protein gels.

The present article is concerned with the practical and theoretical aspects of gelation in protein systems. The processes involved are, inter alia, so complicated that several types of protein molecule assemblies and conformational changes during the association have to be considered. The gel network can be stabilized by five types of interaction: covalent crosslinking, polar interactions, hydrogen bonding, salt bridges and hydrophobic interactions. The microstructures of gels formed from fibrillated proteins (collagen) and from globular proteins (e.g. α- and β- casein) differ considerably. Of practical interest are methods of controlling the solubility of gels, including e.g. the use of cross-linking agents and addition of salts.     

Proteins conjugation with ZnO sol–gel nanopowders

The conjugation between probe biomolecules and inorganic nanoparticles has been studied. Three different and biologically relevant proteins, bovine serum albumin (BSA), lysozyme (LSZ) and Ribonuclease A (RNAseA), have been selected as model systems because of their difference in size and isoelectric point. Zinc oxide nanoparticles, synthesized via sol–gel, have been thoroughly characterized by X-ray Photoelectron Spectroscopy, Scanning Electron Microscopy and X-ray Diffraction, and subsequently used as platforms for immobilization of the biomolecules. The interaction of the three proteins with the ZnO surface was performed in phosphate buffer solutions at pH 7.2 in order to mimic physiological fluids and was investigated through fluorescence experiments. The obtained results indicate that conjugation of BSA, LZS and RNAseA on the oxide nanoparticles was mostly dictated by the overall charge of the different proteins. Electrostatic bonds dominate the formation of the protein/ZnO conjugates, whereas the size of the proteins seems to play a negligible role under the adopted experimental conditions.     

球状苯基纤维素疏水性吸附树脂的研制

本文详细报道了在再生纤维素白球上,采用 Ｃｅ_（４＋）氧化还原体系引发纤维素与苯乙烯接枝,研制球状苯基纤维素疏水性吸附树脂的全过程。并详细考察了球状苯基纤维素疏水性吸附树脂对酪蛋白的吸附,其动态吸附量可达 １０ｍｇ／ｍｌ树脂以上。对几种生物活性蛋白质（精制木瓜酶、中性蛋白酶,天然菠萝蛋白酶）的吸附情况也给予了简介。     

Gelation of β-lactoglobulin in the presence of propylene glycol alginate: kinetics and gel properties

The role of the non-gelling polysaccharide, propyleneglycol alginate (PGA), on the dynamics of gelation and gel properties of β-lactoglobulin (β-lg) under conditions where the protein alone does not gel (6%) was analyzed. To this end, the kinetics of gelation, aggregation and denaturation of β-lg in the mixed systems (pH 7) were studied at different temperatures (64–88 °C). The presence of PGA increased thermal stability of β-lg. The rate of β-lg denaturation was decreased and the onset and peak denaturation temperatures increased by 2.2–2.4 °C. PGA promoted the formation of larger aggregates that continued to grow in time. An average aggregate diameter of approximately 300 nm is reached at the gel point in the mixed β-lg+PGA systems, irrespective of the heating temperature. Comparing the activation energies for the aggregation (193 kJ/mol), denaturation (422 kJ/mol) and formation of the primary gel structure (1/t_gel) (256 kJ/mol) processes in the mixed protein–polysaccharide system, it can be concluded that the rate determining step in the formation of the primary gel structure would be the aggregation of protein. E_a values for the processes after the gel point (solid phase gelation) suggest a diffusion limited process because of the high viscosity of the solid gelling matrix. The characteristics of the mixed β-lg+PGA gels in terms of rheological and textural parameters, water loss and microstructure were studied as a function of heating temperature and time. The extent of aggregation and the type of interactions involved, prior to denaturation seem to be very important in determining the gel structure and its properties.     

Rational Design of Self-Assembled Mitochondria-Targeting Lytic Peptide Conjugates with Enhanced Tumor Selectivity

Membrane lytic peptides (MLP) are widely explored as cellular delivery vehicles or antitumor/antibacterial agents. However, the poor selectivity between cancer and normal cells slims their prospects as potential anti-tumor drugs. Herein, we have developed a rationally designed self-assembly strategy to enhance tumor selectivity of MLP-based conjugates, incorporating a hydrophobic triphenylphosphonium (TPP) group for mitochondria targeting, and a hydrophilic arginine-glycine-aspartic acid (RGD) sequence targeting integrins. The self-assembly nanoparticles can enhance the stability of the peptides in vitro plasma and be endocytosed selectively into the cancer cells. The histidine-rich lytic peptide component assists the disruption of endosomal/lysosomal membranes and subsequent the mitochondria membrane, which leads to apoptosis. This rational design of MLP-based conjugates provides a practical strategy to increase the application prospects of lytic peptides in cancer treatment.     

Application of colloidal palladium nanoparticles for labeling in electron microscopy

The application of palladium nanoparticles as electron-dense markers for labeling in both transmission and scanning electron microscopy requires their conjugation to a specific protein. The conjugation protocol described here includes the dihydrolipoic acid (DHLA) capping of Pd nanoparticles (8 nm equivalent diameter) and their subsequent covalent attachment to functional protein molecules such as streptavidin, protein A, or avidin. The single-step reaction was mediated using the cross-linking agent ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The final Pd conjugates were fully functional, as demonstrated by labeling of ultrathin resin sections of either bovine serum albumin or secretory granules of the salivary gland isolated from the partially fed female Ixodes ricinus tick. The results of bovine serum labeling were quantified, statistically evaluated, and compared with results obtained using commercially available gold particle conjugates (10 nm diameter). The highest values of labeling density were achieved using both streptavidin-Pd (106 ± 7 particles/μm2) and protein A-Au conjugates (130 ± 18 particles/μm2) compared to a commercial streptavidin-Au (66 ± 16 particles/μm2) and protein A-Pd conjugates (70 ± 11 particles/μm2). The concentrations of both DHLA and EDC, pH during conjugation, and finally thorough washing away of unbound proteins crucially influenced conjugation.