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Soybean MON 87751 was developed through Agrobacterium tumefaciens‐mediated transformation to provide protection certain specific lepidopteran pests by the expression of the Cry1A.105 and Cry2Ab2 proteins derived from Bacillus thuringiensis. The molecular characterisation data and bioinformatic analyses did not identify issues requiring assessment for food and feed safety. None of the compositional, agronomic and phenotypic differences identified between soybean MON 87751 and the conventional counterpart required further assessment. The GMO Panel did not identify safety concerns regarding the toxicity and allergenicity of the Cry1A.105 and Cry2Ab2 proteins as expressed in soybean MON 87751, and found no evidence that the genetic modification might significantly change the overall allergenicity of soybean MON 87751. The nutritional impact of soybean MON 87751‐derived food and feed is expected to be the same as those derived from the conventional counterpart and non‐GM commercial reference varieties. The GMO Panel concludes that soybean MON 87751, as described in this application, is nutritionally equivalent to and as safe as the conventional counterpart and the non‐GM soybean reference varieties tested, and no post‐market monitoring of food and feed is considered necessary. In the case of accidental release of viable soybean MON 87751 seeds into the environment, soybean MON 87751 would not raise environmental safety concerns. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of soybean MON 87751. In conclusion, soybean MON 87751, as described in this application, is as safe as its conventional counterpart and the tested non‐GM soybean reference varieties with respect to potential effects on human and animal health and the environment.

Proteins from Juglans regia L. were isolated. Then, proteins were hydrolyzed with different enzymes. Antiproliferative activity of proteins and of the protein hydrolysates of J. regia L. were evaluated using the sulforhodamine B method. Glutelin and prolamin proteins presented a high antiproliferative activity against cancer cells PC-3 (prostate) and K-562 (leukemia) with values of 43.9 and 84.4 μg/mL, respectively. The highest inhibitory effect observed was 50% at 0.25 μg/mL concentration in gastrointestinal digestion with pepsin and corolase pp in a dose-dependent manner against cancer cell UACC62 (melanoma). Pepsin hydrolysate showed inhibitory effects against cancer cell UACC62 (melanoma) with a concentration of 71.0 μg/mL. The effects were studied in a dose-dependent manner. The hydrolysate obtained with neutrase enzyme presented inhibitory effects against cancer cell UACC62 (melanoma) at a concentration of 25 μg/mL. Neither proteins nor protein hydrolysates presented cytotoxicity against normal cell assay VERO (epithelial)., This study was supported with project number AGL2015-66886-R of Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), Madrid, Spain, Consolider Ingenio 2010 FUN-C-Food CSD2007-063, CYTED110AC0386 IBEROFUN, and CNPq (Brazil), and project 1373-2014-UTA, Universidad Técnica de Ambato. This work has been reviewed in the English edition by Emilio Labrador. W.C. thanks Comunidad Autónoma de Madrid for the research contract to complete his PhD and CYTED IBERFORUN for the grant of study interchange with Brazil.

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, Valdeon, 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.

Hen egg lysozyme was hydrolyzed with pepsin in situ on a cation-exchange column to isolate antioxidant peptides. The most cationic fraction was eluted with 1 M NaCl. Five positively charged peptides f(109–119) VAWRNRCKGTD, f(111–119) WRNRCKGTD, f(122–129) AWIRGCRL, f(123–129) WIRGCRL and f(124–129) IRGCRL were identified using tandem mass spectrometry. Using ORAC-FL , all five peptides presented antioxidant activity with values of (1970, 3123, 2743, 2393 and 0.313 µmol Trolox/µmol peptide), respectively. Using method TBARS in Zebrafish larvae, all five synthetic peptides were found to efficiently inhibit lipid peroxidation (36.8, 51.6, 55.56, 63.2, 61.0 % inhibition of lipid peroxidation), respectively. None of the five peptides were toxic in Zebrafish eggs and larvae at concentrations lower than 50 µg/ml. Concentrations higher than 50 µg/ml were toxic for both Zebrafish eggs and larvae., This study was supported with Project Number AGL2015-66886-R of Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), Madrid, Spain, and project number CPU-1373-2014-UTA of Universidad Técnica de Ambato, Ecuador/Universidad Nacional Rio Negro, Argentina. Carrillo W. thanks Comunidad Autónoma de Madrid for the research contract to complete his PhD. Daniel Alejandro Barrio is member of the Carrera del Investigador, CONICET, Argentina.

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon-farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk-derived proteins as bovine caseins or casein-derived peptides at different stages during the course of IHNV infection. The results indicate that the 3-h fraction of casein and αS2-casein hydrolysates reduced the yield of infectious IHNV in a dose-dependent manner and impaired the production of IHNV-specific antigens. Hydrolysates of total casein and αS2-casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein-treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV-inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections. © 2012 Blackwell Publishing Ltd., Funded by: Ministerio de Ciencia e Innovación (MICIIN). Grant Numbers: CSD 2007-0063, AGL2008-01713, AGL2007-60256/ACU, AGL2010-1854 and Consejo Superior de Investigaciones Científicas. Grant Number: CSIC200520F113.

El pdf del artículo es la versión pre-print., Cheese whey, also known as sweet whey, is a by-product of cheese-making that contains many valuable constituents. Among them, whey proteins stand out for their high nutritional value in terms of biological value and composition in essential amino acids. In recent years, the increasing demand for caprine and ovine cheeses has produced important amounts of whey from these species, boosting research on the biological activities of its constituent proteins. Different bioactivities have been associated to these proteins, among them antihypertensive, antimicrobial, opioid, antioxidant and immunomodulant activity being the most studied. Although biological activities are present in the intact proteins, in many cases whey proteins act as precursors of bioactive peptides that are released from the hydrolysis of these proteins with different enzymes. This review presents an overview of the different biological activities described for caprine and ovine cheese whey proteins as well as for other whey components such as lactose, oligosaccharides or minerals. © 2011 Elsevier B.V., This work has received financial support from projects AGL2007-65035, AGL2008-01713, Consolider Ingenio 2010 FUN-C-Food CSD2007-00063 and P2009/AGR-1469. B.H.-L. thanks CSIC for her post-doctoral research contract.

Potent angiotensin I-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of β-lactoglobulin (βLg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to βLg f(36–42) and had an IC 50 value of 8 μ m , which is considerably lower than the most potent ACE inhibitory peptides derived from bovine βLg reported so far.

This paper shows the potential role of different ovine casein fractions and their hydrolysates to exert antioxidant activity. ABTS·+ decolorization assay was used to evaluate the antioxidant activity of the casein fractions (β-, κ- and αs-caseins) before and after their hydrolysis by pepsin, trypsin and chymotrypsin. Although the antioxidant activity increased in all the fractions after hydrolysis, the effect was particularly remarkable in the κ-casein fraction, which increased its antioxidant activity almost threefold. Further assays in a linoleic acid oxidation system showed that κ-casein hydrolysate inhibited lipid peroxidation. Analysis of the ovine κ-casein hydrolysate by RP-HPLC–MS/MS allowed the identification of 12 peptide sequences with potential antioxidant properties. One of the most abundant peptides, the fragment HPHPHLSF [f(98–105)] was chemically synthesized. Results showed that this κ-casein-derived peptide was a potent inhibitor of linoleic acid oxidation with an activity similar to that obtained with the synthetic antioxidant BHT. Although other peptides might also contribute, HPHPHLSF was the one most likely to be responsible for the activity found in the κ-casein hydrolysate., This study has received financial support from the projects AGL2005-03381 and CM-S0505-AGR-0153 financed by Ministerio Educación y Ciencia and Comunidad de Madrid, respectively.

The in vitro antioxidant activity and the protective effect against human low density lipoprotein oxidation of coffees prepared using different degrees of roasting was evaluated. Coffees with the highest amount of brown pigments (dark coffee) showed the highest peroxyl radical scavenging activity. These coffees also protected human low-density lipoprotein (LDL) against oxidation, although green coffee extracts showed more protection. In a different experiment, coffee extracts were incubated with human plasma prior to isolation of LDL particles. This showed, for the first time, that incubation of plasma with dark, but not green coffee extracts protected the LDL against oxidation by copper or by the thermolabile azo compound AAPH. Antioxidants in the dark coffee extracts must therefore have become associated with the LDL particles. Brown compounds, especially those derived from the Maillard reaction, are the compounds most likely to be responsible for this activity.

The aim of this work was to isolate and identify antibacterial peptides present in a pepsin digest of ovine α s2 -casein. A protein digest with antibacterial properties was first separated by ion exchange chromatography. The fractions most active against Escherichia coli ATCC 25922 were fractionated by semi-preparative RP-HPLC, and the identification of the active peptides was carried out by on-line and off-line RP-HPLC-ESI-MS/MS. Following this strategy, 10 different peptides were identified, all corresponding to the C-terminal region of the ovine α s2 -casein. Four of them were chemically synthesized and showed antibacterial activity against several Gram-positive and Gram-negative bacteria. Among the synthesized peptides, ovine α s2 -casein f(165–181) exhibited the highest antibacterial potency against all bacteria tested. The antimicrobial activity was compared with that of other previously described peptides like lactoferricin and fragment f(183–207) of bovine α s2 -casein.

Different Spanish cheeses (Cabrales, Idiazábal, Roncal, Manchego, Mahón and a goat's milk cheese), made with diverse technologies and milk from different species, were studied for their angiotensin converting enzyme (ACE)-inhibitory activity. Among the water-soluble extract WSE, This work has received financial support from project AGL-2004-06903-C02-01. J.A. Gómez-Ruiz was the recipient of a fellowship from the Comunidad Autónoma de Madrid, Spain. G. Taborda expresses his appreciation to Colciencias for a fellowship and to the Universidad de Caldas (Manizales, Colombia).

In this study, several peptides, which had previously been identified in active HPLC fractions from Manchego cheese, were synthesised and their angiotensin converting enzyme (ACE)-inhibitory activities were measured. From 11 peptides, which were selected based on their structures, only two, VRYL and KKYNVPQL, showed considerable ACE-inhibitory activity with IC50 values of 24.1 and 77.1μM, respectively. Subsequently, the impact of the gastrointestinal digestion on ACE-inhibitory activity was evaluated. Some of the peptides selected were resistant to the incubation with pepsin followed by hydrolysis with a pancreatic extract. The ACE-inhibitory activity after simulated digestion did not change drastically except for peptide αs2-CN f(195-204) (TQPKTNAIPY) that exhibited an activity 6 times greater after simulated digestion. In contrast, after simulated digestion, the activities of peptides VRYL and KKYNVPQL decreased. The peptides not hydrolysed by gastrointestinal enzymes and peptide VRYL, which was only partly hydrolysed, were incubated with ACE and were found to be true inhibitors of the enzyme and to have a competitive inhibition pattern. © 2004 Elsevier Ltd. All rights reserved., This work has received financial support from Ministerio de Ciencia y Tecnologı́a, projects AGL 2001-1261 and CAL01-046-C2.

A capillary electrophoresis (CE) method has been applied for the quantitative determination of alpha(s1)- and alpha(s2)-CN in goat's milk. Several analytical parameters were evaluated showing the reliability of this CE method. Coefficients of determination (R2) greater than 99% were obtained and determination limits of 1.23 and 0.98 mg/ml were achieved for alpha(s1)- and alpha(s2)-CN, respectively. The analytical parameters studied in terms of accuracy, precision and recovery were within acceptable limits. Among 18 samples of 4 different genotypes (BB, EE, BF and FF) for alpha(s1)-CN were analysed, different amounts were obtained from the genotypes.

Manchego cheese, produced from local ewe's milk in the La Mancha region of Spain un- der a Protected Designation of Origin (PDO), is Spain's most popular cheese variety and may be made from raw milk (artisanal cheese) or pasteurised milk (industrial cheese). As a consequence, the cheeses so manufactured may show differences in their organoleptic properties, associated with dif- ferences in volatile composition, important for cheese aroma. Samples of Manchego cheese made from raw milk and from pasteurised milk and aged for 2, 4, 6 and 12 months were analysed in this study of the volatile composition of the different cheese types, performed using two different analyti- cal methods, Simultaneous Distillation Extraction (SDE) and Automatic Dynamic Headspace (P&T). Approximately 79 compounds were detected by SDE and 44 by headspace, falling in the chemical groups free fatty acids (FFAs), ketones, esters, alcohols and others. Also, olfactory profiles of the cheese samples were assessed by sensory analysis. Nine odour attributes (odour intensity, milk odour, lactic/yoghurt odour, briny odour, rancid odour, butter odour, dry hay odour, fruity odour and pungent sensation) were assessed sensorially. Both the concentrations of the volatile compounds de- tected and the intensity of the sensory odour attributes evaluated were found to vary during ripening. The results were distinctly different for the artisanal and industrial Manchego cheese samples. Manchego cheese / ripening / volatile compound / sensory analysis

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.

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., This work was supported by projects Consolider-Ingenio FUNC-Food CSD 2007-063, FP7-SME-2012-315349 (FOFIND), and AGL2011-24643 from Ministerio de Economiá y Competitividad, projects P2009/AGR-1469 and (ALIBIRDCM) S2013/ABI-2728 from Comunidad de Madrid, and project 920204 UCM-BSCH from Universidad Complutense de Madrid.

Peptides released during the shelf life of cheeses packaged using 2 different technologies, vacuum packaging (VP) and modified-atmosphere packaging (MAP), were identified by on-line reverse phase-HPLC-tandem mass spectrometry. A total of 22 peptides from the N-terminal domain of αS1-casein (CN) and 26 from β-CN were identified, the latter more evenly distributed over the whole sequence. Peptides were monitored during the shelf life of these cheeses when stored at 4°C, revealing that the peptide profile changed significantly with the storage time. Qualitative differences between VP and MAP cheeses were only found for 3 αS1-CN peptides, which were absent in MAP cheeses. Semiquantitative analysis of peptides revealed some differences between cheeses packaged using different technologies. However, evolution of peptides during storage followed a common trend in both types of cheeses. In addition, the presence of certain peptides, which had been previously described because of their potential bioactivity, is illustrated. For instance, some of the identified peptides had been previously reported as antihypertensive peptides, such as peptide αS1-CN (1-9) or β-CN f(201-209). © 2013 American Dairy Science Association., This work was supported by projects CENIT Pronaos AGL2011-24643 and Consolider-Ingenio FUN-C-Food CSD 2007-063 from Ministerio de Economía y Competitividad (Madrid, Spain). Laura Sánchez-Rivera acknowledges Consejo Superior de Investigaciones Científicas (CSIC) for a Junta para la Ampliación de Estudios (JAE) Program fellowship.

Sea cucumber is a benthic marine organism distributed worldwide and used as food in several Asian countries. The species Isostichopus badionotus is captured intensively off the Yucatan Peninsula, Mexico. Boiled I. badionotus was subjected to in vitro simulated gastrointestinal digestion using pepsin and a pepsin-Corolase PP® mixture. ACE-inhibitory and radical scavenging activities, iron reducing capacity and cytotoxic effects against colorectal cancer cells were evaluated in the hydrolysates and their ultrafiltered fractions. ACE-inhibitory activity was potent in fractions containing peptides, The research reported here forms part of a project financed by the FOMIX-Yucatan (Code M0023. No. 108373). B.H.L. thanks the Spanish National Research Council (CSIC) for her Ramón y Cajal post-doctoral research contract.

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.

Donkeys' milk was subjected to in vitro simulated gastrointestinal digestion using pepsin and a mixture of pancreatic enzymes. Analysis of the hydrolysate by high pressure liquid chromatography coupled to tandem mass spectrometry allowed the identification of 46 peptides, of which 30 peptides belonged to β-casein (β-CN). The gastrointestinal digest possessed an important angiotensin converting enzyme (ACE)-inhibitory activity with an IC 50 of 273.0±27.9μgmL -1. The β-CN fragment f(176-185) [VAPFPQPVVP], one of the most abundant peptides in the hydrolysate, was synthesized and its ACE-inhibitory activity measured. This peptide showed very potent activity with an IC 50 of 48.8±2.3μm. To our knowledge, this is the first time that a bioactive peptide from donkeys' milk has been reported. © 2011 Elsevier Ltd., This work has received financial support from projects AGL2008-01713, Consolider Ingenio 2010 FUN-C Food CSD2007-00063 and P2009/AGR-1469.

6 páginas, 3 figuras, 1 tabla., In this study we evaluated the short-term oral antihypertensive effect of several peptide sequences isolated from casein fractions, previously characterized as in vitro angiotensin-converting enzyme-inhibitors, in spontaneously hypertensive rats (SHR). Systolic blood pressure (SBP) and the diastolic blood pressure (DBP) of the rats were measured by the tail cuff method before administration and also 2, 4, 6, 8 and 24 h post-administration. The sequences LVYPFTGPIPN, HLPLP, IAK, YAKPVA and WQVLPNAVPAK showed a clear decrease in SBP and DBP in SHR. HPHPHLSF caused a significant decrease of the DBP in the SHR, but this sequence did not modify the SBP of these animals in a significant manner. KKYNVPQL did not modify SBP in the SHR, and caused a slight, but significant and maintained, decrease in DBP in these animals. SBP and DBP returned to baseline values 24 h post-administration of all peptides. In conclusion, these peptides are bioactive ingredients with potential benefit in the prevention and treatment of hypertension or other associated disorders., This study has received financial support from the projects AGL2007-65035, AGL2008-01713 and CSD-2007-00063. Marta Miguel is the recipient of a Ramón y Cajal contract from MICINN.

A collaborative study on the determination of the 15. +. 1 EU priority PAHs in Primary Smoke Condensate (PSC) investigating the performance profile of EU Member States' laboratories and supporting the work carried out by the European Food Safety Authority on smoke flavourings was organised. Two spiked liquid smoke condensate materials were employed in this study. The results of 25 laboratories from across the European Union, using either high performance liquid chromatography with fluorescence detection or gas chromatography with mass selective detection, were evaluated by application of robust statistics. The assessment of the data indicated broadened Gaussian distributions for all analytes. For benzo[. a]pyrene (BaP), benzo[. a]anthracene (BaA), chrysene and 5-methylchrysene more than 80% of the respective reported values gave rise to a satisfactory score of |z|≤ 2. For benzo[. b]fluoranthene, benzo[. k]fluoranthene, and dibenzo[a, l]pyrene 70-80%, for the remaining analytes less than 70%, and for dibenzo[ a, i]pyrene 52% of the scores were satisfactory. No systematic differences could be detected between values reported by laboratories using methods based on HPLC and the values related to methods based on GC for most of the analytes, except for benzo[. k]fluoranthene and benzo[. c]fluorene. In both cases laboratories using GC based methods reported about 50% higher values than laboratories using HPLC based methods. The overview on all z-scores sorted by laboratory revealed broad distributions and/or laboratory biases for several laboratories. An assessment of the reported method performance parameters revealed that for the two regulated compounds, BaP and BaA, only two thirds of the reported data were in compliance with Regulation (EC) 627/2006. Overall the methods used in the participating laboratories were - with ample room for improvement - well on the way to comply with European legislation. © 2010 Elsevier Ltd.

This paper presents the optimisation of a programmed temperature vaporization solvent vent (PTV-SV) injection gas chromatographic mass spectrometric (GC-MS) method for the analysis of the 15+1 EU-priority PAHs in food extracts. Three operation parameters (vent time, vent flow and vent pressure) were optimised by applying a D-optimal experimental design. Among these variables, vent time showed the highest effect on the analytical response (signal intensity) of the target PAHs. The 15+1 EU-priority PAHs were analysed in solvent solutions and in extracts of fortified sausage. In addition, blank lamb meat extracts were prepared and spiked with the target PAHs prior to GC-MS analysis. The performance of the optimised PTV-SV injection GC-MS method was scrutinised for linearity, precision, matrix effects and robustness. All parameters were found satisfactorily. Compared to PTV injection in splitless mode, the PTV-SV injection method provided an enhancement of sensitivity for all target PAHs. Especially significant was the improvement of the S/N ratios of the compounds with the highest molecular mass.

Three different stationary phases were investigated for the analysis of the 15 + 1 EU-priority polycyclic aromatic hydrocarbons (PAHs) by gas chromatography-mass spectrometry. In addition to the most commonly used 5% phenyl methylpolysiloxane, a mid-polar phase (50% phenyl methylpolysiloxane) and a recently commercialised mid-polar to polar phase (Optima delta-6), were evaluated. Challenging groups of PAHs in terms of separation, such as the pair dibenz[a,h]anthracene-indeno[1,2,3,-cd]pyrene and the two groups benzo[b]fluoranthene-benzo[k]fluoranthene-benzo[j]fluoranthene and cyclopenta[cd]pyrene-benz[a]anthracene-chrysene, were satisfactorily separated by using the mid-polar phase. Moreover, discrimination in terms of peak height for the heaviest PAHs (caused from the strong interaction of these compounds with the stationary phase) was reduced without compromising the resolution of the other target analytes when applying the mid-polar phase in a tailor-made column geometry (20 m x 0.18 mm internal diameter and 0.14 microm film thickness) in combination with optimised chromatographic conditions. A significant enhancement of the analytical sensitivity for dibenzopyrenes is demonstrated with an almost threefold increase of the signal-to-noise (S/N) ratio for dibenzo[a,h]pyrene, the last eluting PAH. The ability of the selected column to separate potentially interfering PAHs from the target analytes in both solvent solutions and food extracts is demonstrated.

10 pages, 1 figure, 6 tables.-- Available online Jul 18, 2007., Three batches of Manchego cheese were manufactured using one of the following starter culture systems: (1) a defined strain starter culture comprising Lactococcus lactis subsp. lactis and Leuconostoc mesenteroides subsp. dextranicum; (2) the above-defined strain starter culture and an adjunct culture (Lactobacillus plantarum), all these strains being isolated from high-quality Manchego cheeses and (3) a commercial starter consisting of two strains of Lactococcus lactis. Differences in volatile profile and the sensory characteristics of these cheeses were studied. After 4 months of ripening, the two batches of cheese made with the defined strain starter cultures obtained the highest scores for sensory attributes and for the overall impression. Additionally, Purge & Trap and SDE analysis showed a more complex volatile profile in these cheeses than in those made with the commercial starter. Extending the maturation time to 8 months for cheeses made with the defined starter cultures led to significant higher levels of free fatty acids and ethyl esters in those cheeses made without adjunct culture. However, panelists did not find significant differences among the sensory characteristics of the two cheeses., This work was supported by the Spanish Ministry of Education and Culture and EU (FEDER Program 1FD97-0166). J.A. Gómez-Ruiz acknowledges Comunidad de Madrid for a scholarship.

In this report, we present the use of CE-MS as complement to RP separation for the identification of novel angiotensin-converting enzyme-inhibitory (ACEI) peptides from a complex milk protein hydrolysate. As preliminary step, fast protein LC (FPLC) was used to isolate the different casein fractions from raw ovine milk. Enzymatic hydrolysis of these fractions was performed by using proteolytic enzymes of gastrointestinal origin. The most active hydrolysate, corresponding to kappa-casein hydrolyzed with pepsin, chymotrypsin, and trypsin, was fractionated by RP-HPLC and the peptides contained in the active fractions were sequenced by CE coupled to IT-MS (CE-MS). The use of CE-MS allowed the identification of short peptides with ACEI activity included in the scarcely retained fraction obtained by semipreparative RP-HPLC. Among the identified peptides, those with hydrophobic or positively charged residues at the C-terminal tripeptide were chemically synthesized to determine their ACEI activity. This procedure allowed us to identify four novel potent ACEI peptides from kappa-casein with sequences IAK, YQQRPVA, WQVLPNAVPAK, and HPHPHLSF. These active sequences could be obtained by enzymatic hydrolysis either of the individual kappa-casein fraction or the total casein fraction from ovine milk.

8 pages, 3 figures, 4 tables.-- Printed version published Jun 2008.-- Supplementary information (2 tables, 3 pages) available at: http://www.springerlink.com/content/c4536l80871rjj13/217_2007_Article_769_ESM.html, The correlation between chemical and sensory analysis of the water-soluble extract compounds with molecular weight less than 1,000 Da (WSE < 1,000 Da) from Manchego cheese at different stages of ripening, as well as the influence of the pasteurization processes, has been studied. Free amino acids, peptides and volatile compounds were more abundant in raw milk cheeses WSE < 1,000 Da. Further fractionation of the WSE < 1,000 Da was carried on by gel permeation chromatography and amino acids, peptides, ions and volatiles were determined in the fractions. The same fractions were also subjected to organoleptic evaluation. Volatile compounds were related to the aroma and small peptides and amino acids to the taste. Volatile compounds were mainly alcohols and ketones. Fresh and floral notes were detected in the 4-month-old cheeses, which evolved into more intense and complex flavors in the 8-month-old cheeses. Peptides detected in the fractions were mainly hydrophilic. Umami taste was predominant in most of the fractions and it could be related to the presence of glutamic and aspartic acids in these fractions. Salty taste was perceived with high intensity in the fractions with a high content of salts. Bitter taste was mainly detected in the last eluting fractions and it was attributed to the Tyr, Phe and Trp residues found in these fractions., This work was financially supported by the projects AGL2005-03381 and S-505/AGR/0153-01. Dr. G. Taborda wishes to express his appreciation to the University of Caldas and Colciencias (Colombia) for financial fellowship.

Lysinoalanine (LAL) is an unwanted byproduct, which is formed during the processing of protein and protein-containing foods and feeds. A GC method for the quantitative analysis of LAL under conventional chromatographic conditions has been developed. The method was applied to the analysis of pure standard substances, boiled eggs, commercial caseinates, fresh cheese, fresh cheese made from milk supplemented with caseinate, and fresh cheeses adulterated with caseinate after cheese making process. Results demonstrated the reliability of the GC capillary chromatography for the analysis of LAL in protein containing foods. LOD and LOQ of 50 and 152 ppm of LAL in protein, respectively, were achieved. Range of linearity, precision, and accuracy of the method, measured using diaminopimelic acid as internal standard, were satisfactory for quantification purpose. The method might also be suitable for the quantitative analysis of other amino acids such as lysine and arginine. Results also indicated the utility of this methodology for detecting protein quality of egg products and caseinates as well as fresh cheese adulterations., This study was funded by the Spanish Ministry of Education and Science (project AGL2004-05031).

Water-soluble fractions from Protected Denomination of Origin Manchego cheese, with molecular weight

Some of the most potent ACE-inhibitory peptides described in food have a proline at the end of their sequence, a characteristic that can cause problems in the synthesis procedures. In this work, we studied two different preparations of peptide Asp-Lys-Ile-His-Pro (DKIHP), which were obtained by two different synthetic procedures (Boc and Fmoc). The peptide synthesized by the Boc method yielded a unique conformer, containing trans-Pro, and significant ACE-inhibitory activity (IC(50) = 113.18 microM). The chromatographic and NMR data of this active conformer are reported. The peptide synthesized by Fmoc chemistry yielded three conformers, two of them containing trans-Pro and a third one containing cis-Pro, and showed a lower activity (IC(50) = 577.92 microM). This was attributed to the presence of conformers with less (or none) activity. We have pointed out the importance of performing structural studies on these type of peptides before testing their ACE-inhibitory activity.

The hydrolysis of crude egg white with pepsin, trypsin, and chymotrypsin produced peptides with angiotensin-converting enzyme (ACE) inhibitory properties. These peptides were mainly derived from the proteolysis of ovalbumin. The most active hydrolysates were obtained after treatment with pepsin (50% inhibitory concentration [IC50], 55.3 μg/ml), with the fraction having a molecular mass lower than 3,000 Da giving the highest ACE inhibitory activity (IC50, 34.5 μg/ml). Nine subfractions were collected from the fraction with a molecular mass lower than 3,000 Da using semipreparative reversed-phase high-performance liquid chromatography. Considerable ACE inhibitory activity (IC50 < 40 μg/ml) was found in three of them. These subfractions were analyzed by reversed-phase high-performance liquid chromatography–tandem mass spectrometry, and 14 peptides were identified. These sequences were synthesized, and their ACE inhibitory activities were measured. Among the identified peptides, two novel sequences with potent ACE inhibitory activity were found. The amino acid sequences of these inhibitors were identified as Arg-Ala-Asp-His-Pro-Phe-Leu and Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu and showed IC50 values of 6.2 and 4.7 μM, respectively., This work was supported by the Comisión Interministerial de Ciencia y Tecnología (CICYT) project AGL2001-1261 and by the Comunidad de Madrid, Project 07G/0036/2003 1.

A total of 75 peptides included in the fraction with molecular mass below 3000 from an 8-month-old Manchego cheese could be identified using HPLC coupled on line to an ion trap mass spectrometer. Some previously described peptides with antihypertensive and/or angiotensin-converting enzyme (ACE)-inhibitory activity were detected. The formation of five active sequences was followed during cheese ripening in four different batches of Manchego cheese. Two experimental batches of Manchego cheese elaborated with selected bacterial strains with the aim of improve the organoleptic characteristics demonstrated also a good performance in the formation of peptides with ACE-inhibitory activity during cheese ripening., This work has received financial support from the project AGL2001-1261. J.Á.G.-R. was the recipient of a fellowship from Comunidad Autónoma de Madrid, Spain.

In this paper we report the antioxidant activity of different compounds which are present in coffee or are produced as a result of the metabolism of this beverage. In vitro methods such as the ABTS*+ [ABTS = 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] decolorization assay and the oxygen radical absorbance capacity assay (ORAC) were used to assess the capacity of coffee compounds to scavenge free radicals. The importance of caffeine metabolites and colonic metabolites in the overall antioxidant activity associated with coffee consumption is shown. Colonic metabolites such as m-coumaric acid and dihydroferulic acid showed high antioxidant activity. The ability of these compounds to protect human low-density lipoprotein (LDL) oxidation by copper and 2,2'-azobis(2-amidinopropane) dihydrochloride was also explored. 1-Methyluric acid was particularly effective at inhibiting LDL oxidative modification. Different experiments showed that this caffeine metabolite is not incorporated into LDL particles. However, at physiologically relevant concentrations, it was able to delay for more than 13 h LDL oxidation by copper.