Assessment of milk protein digestion kinetics: effects of denaturation by heat and protein type used.

Knowledge about how molecular properties of proteins affect their digestion kinetics is crucial to understand protein postprandial plasma amino acid (AA) responses. Previously it was found that a native whey protein isolate (NWPI) and heat denatured whey protein isolate (DWPI) elicit comparable postprandial plasma AA peak concentrations in neonatal piglets, while a protein base ingredient for infant formula (PBI, a ß-casein-native whey protein mixture) caused a 39% higher peak AA concentration than NWPI. We hypothesized that both whey protein denaturation by heat as well as changing protein composition by including ß-casein, increases the rate of intact protein loss, and that changing the protein composition (by including ß-casein), but not whey protein denaturation, yields a faster absorbable product release. Therefore NWPI (91% native), DWPI (91% denatured) and PBI hydrolysis was investigated in a semi-dynamic in vitro digestion model (SIM). NWPI and DWPI hydrolysis were also compared in a dynamic digestion model with dialysis (TIM-1) to exclude potential product inhibition effects that may occur in a closed vessel digestion model as SIM. In both models, the degree of hydrolysis (DH), loss of intact protein, and release of absorbable products (SIM: <0.5 kDa peptides and free AA, TIM-1: bioaccessible AA) were monitored. Additionally, in SIM, intermediate product amounts and their characteristics were determined. DWPI showed considerably faster intact protein loss, but similar DH and absorbable product release kinetics compared with NWPI in both models. Furthermore, more, relatively large, intermediate products were released from DWPI than from NWPI. PBI showed increased intact protein loss, similar DH, and absorbable product release kinetics, but more, relatively small, intermediate products than NWPI. In conclusion, both whey protein denaturation and ß-casein inclusion increased the rate of intact protein loss without affecting absorbable product release during in vitro digestion. Our results suggest that intermediate digestion product characteristics are important in relation to postprandial AA responses.

Preparation and of PVA-based compositions with embedded silver, copper and zinc oxide nanoparticles and assessment of their antibacterial properties.

A series of poly(vinyl alcohol) (PVA) based liquid compositions with addition of zinc oxide, silver and copper nanoparticles has been prepared. The compositions also contained other consistency-forming organic components. The physico-chemical properties of the products have been determined. Their pH and density have been assessed. Also, the size of nanoparticles has been defined with using a dynamic light scattering technique. The compositions were subjected to XRD, FT-IR and microscopic analysis as well. Thanks to the incorporation of both metal oxide and metallic nanoparticles, it was possible to enrich the products with antibacterial properties. Their inhibiting properties in the growth of microorganisms have been confirmed against both Gram-negative and Gram-positive strains such as E. coli, S. aureus and P. aeruginosa. Thanks to the ability for solidification, the compositions may be applied on a bacterially contaminated surface, and after destroying the microorganisms and its solidification, it may be peeled off along with the dead bacterial film.

Assessment of physico-chemical changes in UHT milk during storage at different temperatures.

This research communication describes enzymatic and physico-chemical changes during storage of UHT milk. The UHT milk sample was stored at 5 and 30°C for 4 months and analyzed regularly at intervals of one month. During storage of UHT milk, there was a significant (P < 0.001) increase in non-protein nitrogen, non-casein nitrogen, soluble calcium, soluble magnesium and proteolysis, while a significant (P < 0.001) decrease in pH was observed. There was a slight change in the particle size and zeta potential of casein micelles. Changes were more pronounced in milk samples stored at 30°C than in those stored at 5°C. During storage, there occurred changes in pH, viscosity, salt balance and nitrogenous components which adversely affected quality. It was concluded that the proteolysis led to the acidification which had a destabilizing effect on the milk.

Alginate-coated caseinate nanoparticles for doxorubicin delivery: Preparation, characterisation, and in vivo assessment.

Natural protein-based nanoparticles are promising nano-vehicles for the delivery of chemotherapeutic drugs. Caseinate nanoparticles loaded with doxorubicin (CasNPs-DOX) have been surface-modified with the natural polysaccharide alginate to generate the novel nanocarrier Alg-CasNPs-DOX. The fabricated nanoparticles have been characterised by transmission electron microscopy, Fourier-transform infrared spectroscopy, dynamic light scattering, fluorescence spectroscopy, and zeta potential measurement. Drug encapsulation and release profiles were also investigated. In vivo studies were conducted to evaluate the therapeutic efficacy of this novel drug delivery system in tumour-bearing mice. The biodistribution and toxicity of the nano-formulation were also assessed. The results showed that encapsulation of DOX in Alg-CasNPs-DOX not only led to controlled and sustained drug release but also significantly enhanced the effectiveness of DOX against Ehrlich carcinoma. Moreover, no significant changes were observed in liver and kidney enzymes, indicating the selective delivery of DOX to the tumour site, thus minimising DOX toxicity to certain vital organs. Accordingly, Alg-CasNPs-DOX was shown as a promising DOX nanocarrier for improving the therapeutic efficacy of DOX against cancer compared to that of free DOX.

Pre-sleep protein in casein supplement or whole-food form has no impact on resting energy expenditure or hunger in women.

The purpose of this study was to determine the effect of a whole-food protein (cottage cheese, CC) consumed before sleep on next-morning resting energy expenditure (REE), RER and appetite compared with an isoenergetic/isonitrogenous casein protein (CP) supplement and placebo (PL) in active women. In a beverage-blinded, randomised, cross-over design, ten active women (age, 23·1 (sd 1·9) years; body fat, 22·0 (sd 4·6) %) consumed pre-sleep CC (30 g of protein, 10 g of carbohydrate and 0 g of fat) or energy- and protein-matched liquid CP or PL (0 kJ). Participants arrived at 18.00 hours for an overnight stay in the laboratory. At 30-60 min before normal bed time (2 h post standard meal), participants consumed CC, CP or PL before measurement of REE. Upon waking (05.00-08.00 hours), REE was repeated and subjective appetite was recorded. Statistical analyses were conducted using repeated-measures ANOVA (SPSS). Significance was accepted at P≤0·05. There were no significant differences in acute REE (CC, 7217 (sd 1368); CP, 7188 (SD 895); PL, 7075 (sd 1108) kJ/d, P=0·95), acute RER (0·79 (sd 0·05), P=0·56), morning REE (CC, 5840 (sd 1225); CP, 5694 (sd 732); PL, 5991 (sd 903) kJ/d, P=0·79) or morning RER (0·77 (sd 0·03), P=0·52). Subjective measures of appetite were not different between groups. In active women, pre-sleep consumption of CC does not alter REE or RER more than a CP or PL beverage. These data suggest that the metabolic response from whole-food protein do not differ from the metabolic response of liquid protein.

Silver decahedral nanoparticles empowered SPR imaging-SELEX for high throughput screening of aptamers with real-time assessment.

A highly efficient method for aptamer screening with real-time monitoring of the SELEX process was described by silver decahedra nanoparticles (Ag10-NPs) enhanced surface plasmon resonance imaging (SPRI). A microarray chip was developed by immobilization of target protein (Lactoferrin (Lac)) and control proteins (α-lactalbumin (α), ß-lactoglobulin (ß), casein, and bovine serum albumin (BSA)) on the biochip surface. Ag10-NPs were conjugated with an ssDNA library (lib) (Ag10-NPs-library) that consisted of a central 40 nt randomized sequence and a 20 nt fixed primer sequence. Introduction of the Ag10-NPs-library to the SPRI flow channels drastically increased the sensitivity of SPRI signal for real-time monitoring of SELEX. The work allows rapid screening of potential targets, and yields nine aptamers with high affinity (nanomolar range) for Lac after only six-rounds of selection. The aptamer Lac 13-26 was then further tested by SPRI, and the results demonstrated that the aptamer had the capacity to be ultra-sensitive for specific detection of Lac. The novel SPRI-SELEX method demonstrated here showed many advantages of real-time evaluation, high throughput, and high efficiency.

Preparation of konjac glucomannan/casein blending gels optimized by response surface methodology and assessment of the effects of high-pressure processing on their gel properties and structure.

BACKGROUND: In order to improve the compatibility of polysaccharide-protein mixtures and enhance their performance, a response surface methodology was used to optimize the preparation conditions of konjac glucomannan/casein blend gel. Moreover, the effects of high-pressure processing (HPP) on the gel properties and structure were also investigated. RESULTS: The optimal preparation parameters were a temperature of 60 °C, a total concentration 40 g kg-1 , and a dietary alkali concentration 5 g kg-1 . Under these conditions, the experimental value of hardness was 38.7 g, which was close to the predicted value. HPP increased gel hardness by 161-223% and led to a more compact structure at 200-600 MPa/10 min, while a hardness increase of ∼120% and damaged structure were observed at 600 MPa/30 min. Fourier transform infrared spectroscopy showed that noncovalent interactions are likely the most important factor in the modification of gel hardness; indeed, hydrogen bonding interactions in the gels are enhanced when subjected to HPP, but are weakened at 600 MPa/30 min. COUCLUSION: Protein-polysaccharide complexes with excellent properties could be obtained through this method, with broad application prospects in the food industry. © 2018 Society of Chemical Industry.

Physiological relevance of food grade microcapsules: Impact of milk protein based microcapsules on inflammation in mouse models for inflammatory bowel diseases.

In order to increase beneficial effects of bioactive compounds in functional food and dietary supplements, enormous efforts are put in the technological development of microcapsules. Although these products are often tailor-made for disease susceptible consumer, the physiological impact of microcapsule uptake on the respective target consumer has never been addressed. The present study aimed to assess the relevance of this aspect by analyzing the impact of milk protein based microcapsules on experimental inflammatory bowel disease. Long-term feeding of sodium caseinate or rennet gel microcapsules resulted in significant alterations in the intestinal microbiota of healthy mice. In TNFΔARE/wt mice, a model for chronic ileal inflammation, rennet gel microcapsules resulted in further increased splenomegaly, whereas ileal inflammation was unchanged. In IL10(-/-) mice, a model for chronic colitis, both types of microcapsules induced a local increase of the intestinal inflammation. The present study is the first to demonstrate that, independent of their cargo, microcapsules have the potential to affect the intestinal microbiota and to exert unprecedented detrimental effects on disease-susceptible individuals. In conclusion, the impact of microcapsule uptake on the respective target consumer groups should be thoroughly investigated in advance to their commercial use in functional food or dietary supplements.

Identification of bitter peptides in aged cheddar cheese.

The compounds responsible for the bitter taste of aged "sharp" Cheddar cheese were characterized. Sensory-guided fractionation techniques using gel permeation chromatography and multi-dimension semi-preparative reversed-phase high-performance liquid chromatography revealed the presence of multiple bitter compounds. The compounds with the highest perceived bitterness intensity were identified by tandem mass spectrometry de novo peptide sequencing as GPVRGPFPIIV, YQEPVLGPVRGPFPI, MPFPKYPVEP, MAPKHKEMPFPKYPVEPF, and APHGKEMPFPKYPVEPF; all originated from ß-casein. Subsequent quantitative liquid chromatography-tandem mass spectrometry analysis reported that the concentrations of GPVRGPFPIIV, YQEPVLGPVRGPFPI, and MPFPKYPVEP increased during maturation by 28.7-, 3.1-, and 1.8-fold, respectively. When directly compared to young "mild" Cheddar, APHGKEMPFPKYPVEPF was reported only in the sharp Cheddar cheese, whereas the concentration of MAPKHKEMPFPKYPVEPF did not change. Further taste re-engineering sensory experiments confirmed the importance of the identified peptides to the bitterness of sharp Cheddar. The bitter intensity of the aged "sharp" Cheddar model (mild Cheddar with equivalent concentrations of the five bitter peptides in the sharp sample) was rated as not significantly different from the authentic sharp Cheddar cheese. Among the five peptides, GPVRGPFPIIV was reported to be the main contributor to the bitterness intensity of sharp Cheddar. Furthermore, a difference from control sensory test also confirmed the significance of the bitter taste to the overall perception of aged Cheddar flavor. The sharp Cheddar model was reported to be significantly more similar to aged "sharp" Cheddar in comparison to the young "mild" Cheddar cheese sample.

Milk casein and fatty acid fractions in early lactation are affected by nutritional regulation of body condition score at the beginning of the transition period in primiparous and multiparous cows under grazing conditions.

The objective was to evaluate the effect of body condition score (BCS) at 30 days before calving (-30 days) induced by a differential nutritional management, parity and week of lactation (WOL) on milk yield and composition, and milk casein and fatty acid composition. Primiparous and multiparous Holstein cows with high BCS (PH, n = 13; MH, n = 9) and low BCS (PL, n = 9; ML = 8) under grazing conditions were sampled at WOL 2 and 8 (before and after peak of lactation). Milk yield was greater in multiparous than in primiparous cows and tended to decrease from WOL 2 to 8 only in ML cows. Milk protein, fat and casein yields were greater in multiparous than in primiparous cows and decreased from WOL 2 to 8. Milk casein concentration in milk protein was greater in MH cows than in ML, PH and PL cows at WOL 2. Milk κ-casein was greater, and ß-casein was less in multiparous than in primiparous cows. As lactation progressed, proportion of casein fractions were not altered. Only κ-casein fraction was affected by BCS at -30 days as PL showed a higher concentration than PH. The de novo (4:0-15:1) and mixed-origin fatty acids (16:0-16:1) in milk fat increased, whereas preformed fatty acids (≥17:0) decreased from WOL 2 to 8. Saturated (SAT) fatty acids tended to be greater and monounsaturated fatty acids (MUFA) were less in multiparous than in primiparous cows. High-BCS cows had greater concentrations of polyunsaturated (PUFA), conjugated linoleic acid (CLA) as well as n-6 and n-3 fatty acids in milk fat than low-BCS cows. The results indicate that casein and fatty acid fractions in milk were affected by parity and may be modified by a differential nutritional management during the pre-calving period (BCS at -30 days) in cows under grazing conditions.

New routes to food gels and glasses.

We describe the possibility to create solid-like protein samples whose structural and mechanical properties can be varied and tailored over an extremely large range in a very controlled way through an arrested spinodal decomposition process. We use aqueous lysozyme solutions as a model globular protein system. A combination of video microscopy, small-angle neutron and X-ray scattering and reverse Monte Carlo modeling is used to characterize the structure of the bicontinuous network with two coexisting phases of a dilute protein solution and a glassy or arrested dense protein backbone at all relevant length scales. Rheological measurements are then used to determine the complex mechanical response of these protein gels as a function of protein concentration and quench temperature. While in particular the origin of the dependence of the mechanical properties on quench depth and concentration is not well understood currently, it seems ultimately connected to the particular bicontinuous structure of the arrested spinodal network created by the interplay between the early stage of a spinodal decomposition and the position of the glass line. We then generalize this behavior and discuss how this could open up new routes to prepare gel-like food systems with adjustable structural and mechanical properties. We present results from a first feasibility study where we use a depletion interaction caused by the addition of small non-adsorbing polymers to suspensions of casein micelles in order to create food gels with tunable structural and mechanical properties through an arrested spinodal decomposition process.

Adsorption of unstructured protein ß-casein to hydrophobic and charged surfaces.

In this Monte Carlo simulation study we use mesoscopic modeling to show that ß-casein, an unstructured milk protein, adsorbs to surfaces not only due to direct electrostatic and hydrophobic interactions but also due to structural rearrangement and charge regulation due to proton uptake and release. ß-casein acts as an amphiphilic chameleon, changing properties according to the chemical environment, and binding is observed to both positively and negatively charged surfaces. The binding mechanisms, however, are fundamentally different. A detailed, per-residue-level analysis shows that the adsorption process is controlled by a few very specific regions of the protein and that these change dramatically with pH. Caseins, being the most abundant proteins in milk, are crucial for the properties of fermented dairy products, such as nutrition, texture, and viscosity, but may also influence adhesion to packaging materials. The latter leads to product losses of about 10%, leading to economical and environmental problems.

Casein hydrolysate for uterine infection treatment: a patent evaluation (WO2011132191).

Metritis, endometritis and pyometra are common uterus inflammatory diseases, occurring mainly in the early postpartum period of livestock and farm animals. These infections are primarily associated with contamination of the reproductive tract, in particular uterine. Uterine infections bring to uterine and cervical involution as well as sub-fertility; the high economic loss, due to costs for treatment, milk withdrawal, reduced reproductive performance and premature culling, clearly demonstrate that uterine health in the postpartum period requires substantial medical veterinary attention. A wide variety of therapies for endometritis have been reported, including mainly antibiotics administered either by systemic or local somministration. Here, the patent application WO/2011/132191, which describes an alternative treatment for uterine infection, using casein peptides, is evaluated and discussed.

Kappa-casein based electrochemical and surface plasmon resonance biosensors for the assessment of the clotting activity of rennet.

We report for the first time the development of kappa-casein (κ-CN)-based electrochemical and surface plasmon resonance (SPR) biosensors for the assessment of the clotting activity of rennet. Electrochemical biosensors were developed over gold electrodes modified with a self-assembled monolayer of dithiobis-N-succinimidyl propionate, while SPR measurements were performed on regenerated carboxymethylated dextran gold surfaces. In both types of biosensor, κ-CN molecules were immobilized onto modified gold surfaces through covalent bonding. In electrochemical biosensors, interactions between the immobilized κ-CN molecules and chymosin (the active component of rennet) were studied by performing cyclic voltammetry, differential pulsed voltammetry, and electrochemical impedance spectroscopy (EIS) measurements, using hexacyanoferrate(II)/(III) couple as a redox probe. κ-CN is cleaved by rennet at the Phe105-Met106 bond, producing a soluble glycomacropeptide, which is released to the electrolyte, and the positively charged insoluble para-κ-casein molecule, which remains attached to the surface of the electrode. This induced reduction of the net negative charge of the sensing surface, along with the partial degradation of the sensing layer, results in an increase of the flux of the redox probe, which exists in the solution, and consequently, to signal variations, which are associated with the increased electrocatalysis of the hexacyanoferrate(II)/(III) couple on the gold surface. SPR experiments were performed in the absence of the redox probe and the observed SPR angle alterations were solely attributed to the cleavage of the immobilized κ-CN molecules. Various experimental variables were investigated and under the selected conditions the proposed biosensors were successfully tried to real samples. The ratios of the clotting power units in various commercial solid or liquid samples, as they are calculated by the EIS-based data, were almost identical to those obtained with a reference method. In addition, EIS measurements showed an excellent reproducibility, lower than 5%.

Antimicrobial effectiveness of bioactive packaging materials from edible chitosan and casein polymers: assessment on carrot, cheese, and salami.

Antimicrobial packaging is one of the most promising active packaging systems for controlling spoilage and pathogenic microorganisms. In this work, the intrinsic antimicrobial properties of chitosan (CH) were combined with the excellent thermoplastic and film-forming properties of sodium caseinate (SC) to prepare SC/CH film-forming solutions and films. The antimicrobial effectiveness of SC, CH, and SC/CH coatings on the native microfloras of cheese, salami, and carrots was evaluated. In vitro assays through the test tube assay indicated that the most significant antimicrobial effect was achieved by CH and SC/CH solutions on carrot and cheese native microfloras. SC film-forming solutions did not exert antimicrobial activity on any of the native microflora studied. SC, CH, and SC/CH films stored in controlled environments showed that the retention of the antimicrobial action was observed until 5-d storage, at 65% relative humidity in both temperatures (10 °C and 20 °C). In vivo assays were also performed with SC, CH, and SC/CH applied as coatings or wrappers on the 3 food substrates. CH and SC/CH applied at both immersion and wrapper exerted a significant bactericidal action on mesophilic, psychrotrophic, and yeasts and molds counts, showing the 3 microbial populations analyzed a significant reduction (2.0 to 4.5 log CFU/g). An improvement of the bactericidal properties of the CH/SC blend respect to those of the neat CH film is reported. The ionic interaction between both macromolecules enhances its antimicrobial properties. Practical Application: The continuous consumer interest in high quality and food safety, combined with environmental concerns has stimulated the development and study of biodegradable coatings that avoid the use of synthetic materials. Among them, edible coatings, obtained from generally recognized as safe (GRAS) materials, have the potential to reduce weight loss, respiration rate, and improve food appearance and integrity. They can be used in combination with other food preservation techniques in order to extend the effectiveness of the food preservation chain. Moreover, antimicrobial films and coatings have innovated the concept of active packaging and have been developed to reduce, inhibit, or delay the growth of microorganisms on the surface of food in contact with the package. The use of antimicrobials packaging films to control the growth of microorganisms in food can have a significant impact on shelf-life extension and food safety. In addition, antimicrobial films can be prepared by the combination of inherent antimicrobial materials (that is, CH), with good film-forming protein-based ones (that is, SC). Therefore, the objective of this work is to study the performance of 2 biodegradable and edible biopolymers and their combination as natural packages for selected food products.

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry.

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40 °C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60 °C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.

Digestibility and allergenicity assessment of enzymatically crosslinked beta-casein.

Crosslinking enzymes are frequently used in bioprocessing of dairy products. The aim of this study was to examine the effects of enzymatic crosslinking on IgE binding, allergenicity and digestion stability of beta-casein (CN). beta-CN was crosslinked by transglutaminase, tyrosinase, mushroom tyrosinase/caffeic acid and laccase/caffeic acid. The IgE binding to beta-CN was compared in vitro by CAP inhibition assay, ELISA inhibition as well as ex vivo by basophil activation assay. Crosslinked CNs were digested by simulated gastric fluid for 15 and 60 min and obtained digests analyzed for their ability to inhibit IgE binding by CAP inhibition assay and SDS-PAGE. The ability of crosslinked CNs to activate basophils was significantly reduced in seven patients in the case of CN crosslinked by laccase and moderately reduced in the case of tyrosinase/caffeic acid crosslinked CN (in two cow's milk allergy patients tested with different allergen concentrations). The response to various crosslinked CNs differed individually among patients' sera tested by ELISA inhibition assay. The presence of caffeic acid hampered digestion by pepsin, and this effect was most pronounced for the tyrosinase/caffeic acid crosslinked CN. The laccase/caffeic acid and mushroom tyrosinase/caffeic acid had the highest potential in mitigating IgE binding and allergenicity of the beta-CN out of all investigated enzymes. The presence of a small phenolic compound also increased digestion stability of beta-CN.

Bovine chymosin: a computational study of recognition and binding of bovine kappa-casein.

Bovine chymosin is an aspartic protease that selectively cleaves the milk protein kappa-casein. The enzyme is widely used to promote milk clotting in cheese manufacturing. We have developed models of residues 97-112 of bovine kappa-casein complexed with bovine chymosin, using ligand docking, conformational search algorithms, and molecular dynamics simulations. In agreement with limited experimental evidence, the model suggests that the substrate binds in an extended conformation with charged residues on either side of the scissile bond playing an important role in stabilizing the binding pose. Lys111 and Lys112 are observed to bind to the N-terminal domain of chymosin displacing a conserved water molecule. A cluster of histidine and proline residues (His98-Pro99-His100-Pro101-His102) in kappa-casein binds to the C-terminal domain of the protein, where a neighboring conserved arginine residue (Arg97) is found to be important for stabilizing the binding pose. The catalytic site (including the catalytic water molecule) is stable in the starting conformation of the previously proposed general acid/base catalytic mechanism for 18 ns of molecular dynamics simulations.

Gamma irradiated micro system for long-term parenteral contraception: An alternative to synthetic polymers.

An injectable system of levonorgestrel (LNG) was developed using biodegradable polymer of natural origin. The parenteral system was optimized for particle size and higher drug loading. The microparticulate system was characterised by scanning electron microscopy, encapsulation efficiency, moisture content, IR, DSC, XRD, residual solvent content, sterility testing, test of abnormal toxicity and test for pyrogens. The microparticles were sterilised by gamma irradiation (2.5Mrad). The system was injected intramuscularly in rabbits and the blood levels of LNG were determined using radioimmunoassay technique. An optimized drug to polymer ratio of 0.3-1.0 (w/w ratio) gave improved drug loading of about 52%. In vivo studies in rabbits showed that the drug was released in a sustained manner for a period of 1 month. The AUC(0-t) was found to be 9363.6+/-2340pg/mLday(-1) with MRT calculated to be about 16 days and Kel of 0.01day(-1). LNG levels were maintained between 200 and 400pg/mL. In vivo release exhibited an initial burst effect which was not observed in the in vitro dissolution. This promising "Progestin-only" long-term contraceptive with improved user compliance is an alternative to the synthetic expensive polymeric carriers.

Assessment of the immunoglobulin E-mediated immune response to milk-specific proteins in allergic patients using microarrays.

BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available. METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins. RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients. CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.