Beatriz Miralles

Functional Characterization of Chitin and Chitosan

Chitin and its deacetylated derivative chitosan are natural polymers composed of randomly distributed � -(1-4)- linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitin is insoluble in aqueous media while chitosan is soluble in acidic conditions due to the free protonable amino groups present in the D-glucosamine units. Due to their natural origin, both chitin and chitosan can not be defined as a unique chemical structure but as a fam- ily of polymers which present a high variability in their chemical and physical properties. This variability is related not only to the origin of the samples but also to their method of preparation. Chitin and chitosan are used in fields as different as food, biomedicine and agriculture, among others. The success of chitin and chitosan in each of these specific applica- tions is directly related to deep research into their physicochemical properties. In recent years, several reviews covering different aspects of the applications of chitin and chitosan have been published. However, these reviews have not taken into account the key role of the physicochemical properties of chitin and chitosan in their possible applications. The aim of this review is to highlight the relationship between the physicochemical properties of the polymers and their behaviour. A functional characterization of chitin and chitosan regarding some biological properties and some specific applications (drug delivery, tissue engineering, functional food, food preservative, biocatalyst immobilization, wastewater treatment, molecular imprinting and metal nanocomposites) is presented. The molecular mechanism of the biological properties such as biocompatibility, mucoadhesion, permeation enhancing effect, anticholesterolemic, and antimicrobial has been up- dated.

Changes in flavour and volatile components during storage of whole and skimmed UHT milk

Abstract Six batches of commercial UHT milk, submitted to direct treatment, three whole and the other three skimmed milk, were stored at 25±2°C for 4 months. Non-casein nitrogen (NCN) and sensorial analysis were carried out on packs opened every month. Volatile composition was analysed every 15 days, using a purge-and-trap concentrator coupled on-line to a GC–MS instrument. NCN increased during storage; the increase was greater in skimmed milk samples. Sensory characteristics were slightly better in the whole samples, although the scores decreased for both groups in the third month. Quantification of about 40 volatile components in whole milks showed no changes until 90 days (the legal shelf-life in Spain); the main change was the increase of methyl ketones. New components appeared in skimmed samples after 65 days storage; they could be related to both proteolysis and Maillard reaction. This is consistent with the poorer sensory quality found in skimmed milk samples.

Improved method for the simultaneous determination of whey proteins, caseins and para-κ-casein in milk and dairy products by capillary electrophoresis☆

A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-kappa-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated capillary, in combination with electrophoresis buffer 0.48 M citric acid-13.6 mM citrate-4.8 M urea at pH 2.3, and a separation voltage of 25 kV, a complete separation of beta-lactoglobulin and para-kappa-casein was achieved, permitting the quantification of both components.

Food-derived peptides stimulate mucin secretion and gene expression in intestinal cells.

In this study, the hypothesis that food-derived opioid peptides besides β-casomorphin 7 might modulate the production of mucin via a direct action on epithelial goblet cells was investigated in HT29-MTX cells used as a model of human colonic epithelium. Seven milk whey or casein peptides, a human milk peptide, and a wheat gluten-derived peptide with proved or probable ability to bind μ- or δ-opioid receptors were tested on the cell culture. Significantly increased secretion of mucins was found after exposure to six of the assayed peptides, besides the previously described β-casomorphin 7, as measured by an enzyme-linked lectin assay (ELLA). Human β-casomorphin 5 and α-lactorphin were selected to study the expression of mucin 5AC gene (MUC5AC), the HT29-MTX major secreted mucin gene. α-Lactorphin showed increased expression of MUC5AC from 4 to 24 h (up to 1.6-fold over basal level expression), although differences were statistically different only after 24 h of exposure. However, this increased expression of MUC5AC did not reach significance after cell treatment with human β-casomorphin 5. In conclusion, six food-derived peptides have been identifed with described or probable opioid activity that induce mucin secretion in HT29-MTX cells. Concretely, α-lactorphin is able to up-regulate the expression of the major secreted mucin gene encoded by these cells.

Bioavailability and Kinetics of the Antihypertensive Casein-Derived Peptide HLPLP in Rats

The aim of this study was to investigate the oral bioavailability and kinetics of the milk casein-derived peptide HLPLP, which had previously demonstrated antihypertensive effect in spontaneously hypertensive rats. HLPLP disposition after single intravenous (4 mg/kg body weight) and oral (40 mg/kg body weight) doses was studied in rats. Plasma concentrations of HLPLP [β-casein fragment f(134-138)], and two derived fragments found after HLPLP administration, LPLP [β-casein fragment f(135-138)] and HLPL [β-casein fragment f(134-137)], were determined by ultrahigh performance liquid chromatography (UPLC) coupled on line to a Q-TOF instrument. For HLPLP, the elimination half-lives (T1/2β) were 7.95 min after intravenous and 11.7 min after oral administration. The volume of distribution at steady state (Vss = 30.8 L/kg) suggests a considerable uptake of HLPLP into tissues. HLPLP was converted to the peptides LPLP and HLPL. After HLPLP intravenous administration, the elimination half-lives (T1/2β) for these biotransformed peptides, LPLP and HLPL, were 8.38 and 10.9 min, respectively. After oral administration, HLPLP was rapidly absorbed with an absorption half-life (T1/2a) of 2.79 min. The oral bioavailability of HLPLP was found to be 5.18%. Our study suggested that HLPLP was rapidly absorbed and eliminated after oral administration, biotransformed into smaller fragments LPLP and HLPL, and distributed throughout the body by the circulation blood. The present pharmacokinetic information from a preclinical kinetic study in rats can also play an important role in designing future kinetic studies in humans for assessing HLPLP dose-response relationship.

Extraction/Fractionation Techniques for Proteins and Peptides and Protein Digestion

In this chapter we outline the state-of-the-art of extraction and fractionation techniques for proteomic analysis of food proteins and peptides. The relevance of food sample preparation in the whole proteome analysis is to ensure an unbiased reliable map that gives accurate representation of all proteins and peptides present in the food, reducing the limitations associated with the broad protein concentration range present in a specific sample. The use of these techniques should not affect the final outcome of protein and peptide separation and their subsequent analysis. Classical and novel extraction and fractionation techniques for food proteins and peptides prior to their analysis by mass spectrometry are presented separately. Appropriate methods for proteome analysis such as cell disruption, protein solubilization, removal of interfering compounds, and protein enrichment methods are discussed. Finally, a brief overview is given on protein digestion, describing the typical enzymes and the most common processes used in this key step of proteomic studies. For peptidomic analysis different techniques are evaluated, among them extraction and preliminary sample cleanup, fractionation by ultrafiltration, low-pressure liquid chromatography, and solid-phase extraction.

Determination of whey protein to total protein ratio in UHT milk using fourth derivative spectroscopy.

Abstract A fourth derivative spectroscopy method was applied for the quantification of whey protein to total protein ratio in UHT milks. Some analytical features such as model compounds, selection of wavelength, linearity, repeatability and interference of milk fat were studied. The effect of refrigerated storage of raw milk, UHT treatment, and storage of UHT milk at room temperature on whey protein to total protein ratio was evaluated. No significant (p

Peptidomic study of Spanish blue cheese (Valdeón) and changes after simulated gastrointestinal digestion.

It is increasingly evident that digestion can affect the biological activity of cheese by the release of new active peptides from their precursors or, on the contrary, giving rise to fragments without activity. The characterization of the peptidome of a Spanish blue cheese, Valdeón, has been conducted before and after gastrointestinal digestion, and the digests have been compared to those obtained from pasteurized skimmed milk powder (SMP) using a bioinformatics platform. Peptidomic profiling of digests revealed several regions that are especially resistant to digestion (among them β‐casein 60–93, 128–140, and 193–209). Some of them correspond to well‐conserved regions between species (human, cow, sheep, and goat) and include peptide sequences with reported bioactivity. The great peptide homology found between both digests, cheese and SMP, suggests that the gastrointestinal digestion could bring closer the profile of products with different proteolytic state. Although most of the biologically active peptides found in cheese after digestion were also present in SMP digest, there were some exceptions that can be attributed to the absence of the relevant precursor peptide before digestion.

Application of capillary electrophoresis to the characterization of processed cheeses.

Capillary electrophoresis using a hydrophilically coated capillary and a low pH buffer containing urea has been used to characterize processed cheeses. Different electrophoretic patterns were obtained depending on the ingredients used in the blend such as acid casein, rennet casein, sodium and calcium caseinates and skim milk powder. Isoelectric casein, and sodium and calcium caseinates were shown to contain intact non-glycosylated kappa-casein (kappa-CN), while rennet casein contained only trace amounts of kappa-CN and mainly para-kappa-CN. Therefore, the addition of casein or caseinate to processed cheeses has been detected by analysing the intact non-glycosylated kappa-CN. Quantitation of intact non-glycosylated kappa-CN in processed cheeses of known and unknown composition was carried out using a regression curve from standard mixtures of 150-550 g isoelectric casein/kg total rennet casein. This capillary electrophoresis method successfully confirmed the addition of isoelectric casein or caseinate to processed cheeses of known composition. The quantitative determination range was 0.605-3.688 mg kappa-CN/ml. This method cannot be used for measuring additions of rennet casein or any caseinates that have been exposed to chymosin.

Immunological behavior of in vitro digested egg-white lysozyme

SCOPE Besides its antimicrobial properties, lysozyme (LYS) is one of the major allergens from hen egg. This paper addresses the identification of the peptides produced upon in vitro gastrointestinal digestion of LYS, together with their IgE-binding and biological activity as a contribution to the understanding of what makes it a relevant allergen. METHODS AND RESULTS Simulated in vitro gastrointestinal digestion together with IgE binding, basophil degranulation, and peripheral blood mononuclear cells stimulation experiments were carried out. Identification of the fragments released was performed by HPLC-MS/MS and the immunoreactive products were analyzed by MALDI-TOF/TOF. Results showed that in vitro gastric and gastroduodenal digests of LYS maintained IgE binding, basophil activation capacity, and preserved T-cell immunogenicity. These biological activities could be attributed to either the persistence of intact LYS, due to incomplete gastric degradation and subsequent duodenal precipitation, the formation of fragment f(24-129) by chymotrypsin action on the soluble intact protein, or the release, upon combined gastric and pancreatic digestion, of immunoreactive peptides linked by disulphide bonds containing the epitopes f(57-83) and f(108-122). CONCLUSION The pH of gastric hydrolysis greatly determined the extent of subsequent duodenal digestion of LYS and the disclosure of relevant epitopes that could increase its allergenic potential.