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Small peptides such as dipeptides contribute to a great extent to the characteristic taste of dry-cured hams. In this study, hydrophilic interaction liquid chromatography (HILIC) combined to tandem mass spectrometry was used to separate, identify, and quantify seven dipeptides in dry-cured hams sampled at different processing times (6, 12, 18, and 24 months). Results showed an increased concentration of dipeptides DA, DG, EE, ES, and EV with the length of processing, obtaining values up to 23 μg/g of dry-cured ham, which suggests an intense action of muscle enzymes dipeptidyl peptidases during the process. The dipeptide VG significantly decreased from 7 to 4 μg/g of dry-cured ham as the processing increased from 6 to 24 months, whereas the dipeptide PA showed low values between 380 and 550 ng/g of dry-cured ham at all the sampling times. Additionally, in silico analyses reported the sensory characteristics of the studied dipeptides, mostly giving bitter and umami taste, and predicted their allergenicity, toxicity, and physicochemical properties. These results could be useful for further studies related to the pleasant taste of dry-cured hams., Grant AGL2017-89381-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness are acknowledged.

A wide variety of dry-fermented sausages are produced in European countries, where are considered valued traditional products. An intense proteolysis takes place during the processing of dry-fermented sausages due to the combined action of muscle and microbial peptidases, generating large amounts of peptides and free amino acids. These compounds participate in the development of the characteristic flavor of dry-fermented products, but some peptides can also exert certain bioactivities such as antioxidant and ACE inhibitory activities. This study has evaluated the changes in peptide profile and amino acid contents of three European dry-fermented sausages produced in Spain, Italy and Belgium, proving the intense degradation of proteins, mainly myofibrillar, and the generation of high amounts of different size peptides and free amino acids. The changes observed between the profiles of European sausages could be due to differences in product formulation, processing conditions and starter cultures used, which influence the activity of enzymes, both from muscle and bacterial origin. On the other hand, the bioactivity profile of each type of dry-fermented sausage was evaluated through the measurement of the ACE inhibitory and antioxidant activities in water-soluble peptide extracts fractionated by size-exclusion chromatography. Spanish and Belgian dry-fermented sausages showed values of ACE inhibition around 85%, whereas Belgian samples presented the highest DPPH radical-scavenging activity and ferric reducing power capacity. These results evidence the potential of Spanish, Italian and Belgian dry-fermented sausages as natural sources of bioactive peptides, giving an added-value to these traditional products., Authors acknowledge European Union FP7 under Grant Agreement 312090 (BACCHUS). This publication reflects only the authors views and the Community is not liable for any use made of the information contained therein. Grant AGL2014-57367-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness are acknowledged. Juan de la Cierva de Incorporación postdoctoral contract to LM is also acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, (IPT13/0001 - ISCIII-SGEFI/FEDER).

Oxidative modifications of proteins and peptides can negatively affect nutritional, sensory and quality characteristics of dry-cured hams. The aim of this study was to use a peptidomics strategy for the identification and relative quantification of oxidised peptides in two different muscles, the external Semimembranosus (SM) and the internal Biceps femoris (BF), from dry-cured hams of 12 months of curing. The analysis by nanoliquid chromatography-tandem mass spectrometry identified peptides showing oxidation at different amino acid residues such as methionine, cysteine, histidine, tryptophan, and phenylalanine. The highest percentages of oxidised peptides identified in SM and BF muscles were from pyruvate kinase protein (16.7 and 28.6%, respectively). The relative quantitation of oxidised peptides using mass spectrometry in tandem label-free methodology evidenced significant differences between muscles, with myosin and troponin fragments as main responsible peptides for the clustering of data. The existing differences between BF and SM peptides were confirmed using MALDI imaging mass spectrometry technique. The obtained results suggest that differences in physicochemical characteristics such as water and salt contents between SM and BF muscles could affect the quantity of peptide oxidation. This peptidomics approach has allowed to analyse differences in peptide oxidation between dry-cured ham muscles as well as to improve the knowledge about the oxidative processes that occur in dry-cured hams., Grant AGL2014-57367-R from MINECO and FEDER funds are acknowledged. Ramón y Cajal 2016 postdoctoral contract to LM is also acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, (IPT13/0001 - ISCIII-SGEFI/FEDER).

This study aims to examine the effects of non-hydrolyzed octopus (Octopus vulgaris) muscle proteins (NHOPs) and their hydrolysates (OPHs) on alloxan induced diabetes in Wistar rats (AIDR). Animals were allocated into seven groups of six rats each: control group (C), diabetic group (D) and diabetic rats treated with acarbose (D + Acar), non-hydrolyzed octopus proteins (D + NHOPs) and octopus proteins hydrolysates (D + OPHs) groups. The diabetic rats presented a significant increase in glycemic status such as α-amylase activity (in plasma, pancreas and intestine), hepatic glycogen, blood glucose and glycated hemoglobin (HbA1c) levels, as well as a significant decrease in the levels of plasma insulin and total hemoglobin compared to control group. In addition, plasma and liver contents in total cholesterol, triglycerides and LDL-cholesterol significantly increased in AIDR compared to control group. However, the daily administration of OPHs for 30 days improved the glucose tolerance test, the glycemic status of diabetic rats and corrected the lipid profiles. Further, a significant increase in the activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase as well as in the level of plasma bilirubin on diabetic status was observed, indicating considerable hepatocellular injury. OPHs treatment was found to attenuate the increased activities of the plasma enzymes produced by diabetes and caused a subsequent recovery towards normalization compared to the control group. By contrast, the NHOPs treatment was found to increase the glucose metabolic disorders in AIDR. These beneficial effects of OPHs were confirmed by histological findings in the hepatic and pancreatic tissues of diabetic treated rats. Indeed, they avoid lipid accumulation in the hepatocytes and protect the pancreatic β-cells from degeneration. Our results thus suggest that OPHs may be helpful in the preventing from diabetic complications by reversing hepatotoxicity., This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia. The authors wish to express their sincere gratitude to Pr Najiba ZEGHAL, professor in the Sfax Faculty of sciences for her comments that greatly improved the manuscript.

The in vivo wound-healing potential of gels and films based on cuttlefish skin gelatin (CSG) incorporated with aqueous henna extract (AHE) (at 50 and 500 μg/ml) was studied. As wound healing is related to reactive oxygen species (ROS), this study was first conducted to evaluate the antioxidant activity of AHE by DPPH radical-scavenging, β-carotene-linoleate bleaching, and reducing power assays. The major volatile compounds of AHE were lawsone, dimethyl sulfoxide, dimethyl sulfide, eugenol and maltol. The finding of the study showed that the incorporation of AHE into gelatin gels and films enhanced the antioxidant activity in a dose-dependent manner. In the wound-healing study, topical application of gelatin hydrogels or films enriched by AHE, on the wound site in a rat model, enhanced significantly wound healing activity and helped to prevent from inflammation damage, when compared to the control and CICAFLORA®-treated groups. Further, all preparations were found to improve the antioxidant status of treated rats as was evidenced by increased antioxidant enzymatic activities and reduced malondialdehyde (MDA) content. The increase in the activities of catalase (CAT), superoxide dismutase (SOD) and gluthatione peroxydase (GPx) in wound tissues revealed that some bioactive compounds in AHE are able to enhance the healing of wounds by reducing the damage caused by ROS. Wound-healing activity was confirmed by histopathology studies. The obtained results showed that the addition of AHE to the gelatin matrix could improve the wound healing activity., This work was funded by the Ministry of Higher Education and Scientific Research, Tunisia. Emerging Research Group Grant from Generalitat Valenciana in Spain (GV/2015/138) and JAEDOC-CSIC postdoctoral contract of L.M. cofounded by the European Social Found are acknowledged.

Peptides with a similar antioxidant and ACE-inhibitory activity of royal jelly (RJ) generated from Alcalase hydrolysated pollen (AHP) were predicted by Response Surface Methodology (RSM). The model equations were proposed according to the effects of time and enzyme concentration on the antioxidant and ACE-inhibitory activity. The optimum values for Alcalase concentration and hydrolysis time were 1.5% and 4 h, respectively. Later, AHP was prepared and deproteinised to be further analysed using size-exclusion chromatography (SEC). After SEC separation, fractions with the highest activity of ACE-inhibitory, DPPH radical scavenging and ferric-reducing power were purified by RP-HPLC. The highest ACE-inhibitory and DPPH scavenging activity of fractions was found 100% and 66.61%, respectively. The most active fractions were analysed by nano-liquid chromatography and mass spectrometry in tandem (nLC-MS/MS) and a total of 195 peptide sequences were identified. The origins of all peptides were herbal proteins and certain coincidences with previously described bioactive sequences were discussed., This work was funded by the Ministry of Science, Research and Technology of Iran and the Grant AGL2014-57367-R and FEDER funds from the Spanish Ministry of Economy, Industry and Competitiveness. Ramón y Cajal postdoctoral contract to LM is acknowledged. LC-MS/MS analysis was carried out by in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII Proteo Red Proteomics Platform.

This study evaluates the effect of dietary selenium (Se) supplementation source (organic, Se-enriched yeast; SY vs. inorganic, sodium selenite; SS), dose (0.2: L vs. 0.4: H mg/kg) and the combination of Se and vitamin E (VITE + SS) for 26 days on drip loss, TBARS, colour changes, myofibrillar protein pattern and proteolysis in pork. The lowest water losses were observed in the SY-H group when compared to the others. SY-H and VITE + SS groups presented lower myofibrillar protein hydrolysis/oxidation. VITE + SS supplementation also resulted in higher PRO, TRP and PHE content at days 2 and 7, whereas the SY group showed increased GLY and CAR and tended to have higher TAU and ANS at day 2. The myofibrillar fragmentation index was not modified by the dietary treatment; however, at day 8, it tended to be higher in groups supplemented with SeY and VITE + SS. The results of the present study might indicate a possible relation between muscle proteolysis and water loss., This research was supported by projects CDTI IDI-20111017 and S2013/ABI-2913-MEDGAN-CAM.

Thornback ray muscle hydrolysates (TRMHs) prepared by treatment with proteases from Bacillus subtilis A26 (TRMH-A26), Raja clavata crude alkaline protease extract (TRMH-Crude), Alcalase (TRMH-Alcalase) and Neutrase (TRMH-Neutrase) were elaborated and their antioxidant properties and angiotensin I-converting enzyme (ACE) inhibitory activities were tested. TRMHs showed different degrees of hydrolysis (DH from 11 to 22%) and hydrophobic/hydrophilic peptide ratio. Protein content varied from 71 to 74%. Gly, Pro, Asp and Asn were the most prominent amino acids, while hypoxanthine was the major nucleotide related compound present. The antioxidant activity was assayed using various tests. TRMH-Neutrase exhibited the highest antioxidant activity in DPPH scavenging, reducing power and inhibition of β-carotene bleaching tests. However in the total antioxidative efficacy, TRMH-Crude exhibited the highest activity. TRMH-Crude and TRMH-Neutrase were the most potent to prevent DNA oxidation by Fenton reagent. Concerning anti-ACE activity, TRMH-A26 and TRMH-Neutrase exhibited the highest activity with 87% at 5 mg/ml. The results revealed that TRMHs could be employed as a protein source in food additive processing or diets for aquatic organisms and other farmed animals. Biological significance The present study explores for the first time the elaboration of enzymatic hydrolysates from thornback ray R. clavata . The hydrolysates are well characterized and showed an interesting protein content as well as the presence of nucleotide related compounds, essential amino acids and taurine, which make them an interesting source of fish meal in aquaculture feeds. The hydrolysates were found to exhibit ACE inhibitory activity and antioxidant activity. The hydrolysates could serve also as a potential protein source for functional foods.

Thornback ray skin gelatin has been hydrolyzed with two different proteases in order to obtain peptides with ACE inhibitory and antioxidant activity. Hydrolysates with protease from Bacillus subtilis A26 (TRGH-A26) displayed ACE inhibitory activity with an IC 50 value of 0.94 μg/μL whereas Neutrase® hydrolysate from Bacillus amyloliquefaciens (TRGH-Neutrase) showed an IC 50 value of 2.07 μg/μL. Regarding antioxidant activity, IC 50 values of 1.98 and 21.2 μg/μL in TRGH-A26 and TRGH-Neutrase, respectively, were obtained using the DPPH radical-scavenging assay. The most active fractions identified by size-exclusion chromatography were further purified by RP-HPLC and analysed using nanoESI-LC–MS/MS to identify the sequence of the peptides. APGAP was the most active peptide inTRGH-A26 for ACE inhibitory activity with an IC 50 value of 170 μM, whereas GIPGAP showed the best ACE inhibitory activity in TRGH-Neutrase sample with an IC 50 value of 27.9 μM. The highest antioxidant activity was identified in peptide AVGAT, showing a 33% of activity at 3 mg/mL using the DPPH radical-scavenging assay. The obtained results proved the potential of thornback ray skin gelatin hydrolysates as a source of bioactive peptides. Statement of significance This study describes a peptidomic approach for the identification of ACE-inhibitory and antioxidant peptides generated from thornback ray gelatin ( Raja clavata ) hydrolysates from Bacillus subtilis A26 and Bacillus amyloliquefaciens Neutrase® enzymes and expose the potential of thornback ray gelatin hydrolysate as a source of bioactive peptides. In this sense, the decrease of systolic blood pressure is one of the main measurements considered in public health for the treatment of cardiovascular diseases, stroke and even end-stage renal disease. Traditionally, synthetic drugs such as captopril and enalapril have been used as ACE inhibitors despite their secondary effects, but the finding of new sources for the generation of natural bioactive peptides such as thornback ray muscle results is very important in the knowledge of less hostile but highly effective antihypertensive peptides as well as the development of new uses for waste and by-products generated from marine products, helping to solve the already existing environmental problem affecting this industry.

Coenzyme Q10 (CoQ10) or ubiquinone (2,3-dimethoxy-5-methyl-6-multiprenyl-1,4-benzoquinone) is an endogenous hydroxybenzoquinone liposoluble compound which plays important physiological roles that makes it to be considered as a bioactive compound that may be used for clinical practices and as food supplement. The purpose of this work was to analyse CoQ10 in three muscles with different oxidative patterns and determine its variability in different animal species (pork, beef, lamb and rabbit). The content of CoQ10 ranged from 4.3 to 30.9μg/g meat with the highest content in those muscles with oxidative pattern. So, more specific data on type of meat cut and proportion of muscles must be given for this nutrient when reporting its content in food composition databases., Grant PROMETEO/2012/001 from Conselleria d'Educació, Formació i Ocupació of Generalitat Valenciana (Spain) is fully acknowledged.

Pork meat is generally recognised as a food with relevant nutritional properties because of its content in high biological value proteins, group B vitamins, minerals especially heme iron, trace elements and other bioactive compounds. But pork meat also contributes to the intake of fat, saturated fatty acids, cholesterol, and other substances that, in inappropriate amounts, may result in negative physiologically effects. However, there are relevant factors affecting the content of many of these substances and somehow such variability should be taken into consideration. So, genetics, age and even type of muscle have a relevant influence on the amount of fat and the contents in heme iron. Also the composition in fatty acids of triacylglycerols is very sensitive to the contents of cereals in the feed; for instance, polyunsaturated fatty acids may range from 10% to 22% in pork meat. The content of other nutrients, like vitamins E and A, are also depending on the type of feed. Some bioactive substances like coenzyme Q10, taurine, glutamine, creatine, creatinine, carnosine and anserine show a large dependence on the type of muscle. This manuscript describes the main factors affecting the composition of pork meat nutrients and how these changes may affect the general food composition databases. (C) 2012 Elsevier Ltd. All rights reserved., Grant AGL2010-16305 from the Spanish Ministry of Science and Innovation (Madrid, Spain) and FEDER Funds and collaboration of Vaquero Foundation for R+D on Pork Meat (Madrid, Spain) are acknowledged. Grant PROMETEO/2012/001 from Generalitat Valenciana (Spain) is also acknowldeged. Work prepared within the Unidad Asociada IAD (UPV)-IATA (CSIC) framework.

The objective of this study was to purify and identify antioxidant peptides present in a water soluble extract of Spanish dry-cured ham. The initial extract was loaded into a Sephadex G25 column and fractions showing antioxidant activity were collected, pooled together and subjected to reversed-phase chromatography for further purification. Using a nano-LC-MS/MS analysis, 27 peptides were identified in these fractions. Several key peptides were selected for synthesis and the determination of their antioxidant properties using the DPPH radical-scavenging assay and reducing power analysis. The strongest radical-scavenging activity was observed with peptide SAGNPN which showed 50% antioxidant activity at a concentration of 1.5mg/ml. On the other hand, the peptide GLAGA showed the higher reducing power with 0.5 units of absorbance at 700 nm at a concentration of 1mg/ml. Other synthesised sequences showed lower antioxidant activity. The results indicate the potential of Spanish dry-cured ham as a source of antioxidant peptides naturally generated during the dry-curing process.

This work is focused on the determination of compounds of nutritional interest that are naturally present in pork meat and how they are affected during the processing of dry-cured ham. Such compounds are creatine, creatinine, coenzyme Q(10), glutathione, carnosine, anserine, carnitine, taurine, cystine, cysteine and the essential amino acids. Their antioxidant and antyhipertensive functions were evaluated. Of all the assayed substances, only glutathione decreased totally during processing. Carnosine, creatinine, anserine and glutathione showed antioxidant, while cysteine, glutathione and carnosine showed antyhipertensive activity. So, dry-cured ham constitutes an excellent source of essential amino acids (all essential amino acids exhibited a large increase during processing) and other nutritionally interesting compounds such as cystine, cysteine, carnosine, anserine, taurine, carnitine and coenzyme Q(10).

Nitrate and nitrite are commonly added to dry-cured ham to provide protection against pathogen microorganisms, especially Clostridium botulinum. Both nitrate and nitrite were monitored with ion chromatography in dry-cured hams salted with different NaCl formulations (NaCl partially replaced by KCl and/or CaCl2, and MgCl2). Nitrate, that is more stable than nitrite, diffuses into the ham and acts as a reservoir for nitrite generation. A correct nitrate and nitrite penetration was detected from the surface to the inner zones of the hams throughout its processing, independently of the salt formulation. Nitrate and nitrite achieved similar concentrations, around 37 and 2.2 ppm, respectively in the inner zones of the ham for the three assayed salt formulations at the end of the process, which are in compliance with European regulations.

This study examined the antihypertensive and antioxidant activities of water soluble fractions of a Spanish dry-cured ham extract. Antihypertensive activity of a fractionated peptide extract, by size-exclusion chromatography was determined by measuring changes in systolic blood pressure of spontaneously hypertensive rats after oral administration. Every sample exhibited antihypertensive activity, pooled fractions corresponding to 1700 Da or lower were the most antihypertensive with a decrease of 38.38 mm Hg in systolic blood pressure. In vitro experiments revealed marked in vitro angiotensin I-converting enzyme inhibitory activity in fractions corresponding to these elution volumes. Some of the fractions exhibited great 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity, ranging from 39% to 92% as well as superoxide ion extinguishing ability with values ranging from 41.67% to 50.27% of the antioxidant activity, suggesting the presence of peptides with antioxidant activity. These findings suggest that Spanish dry-cured ham contains peptides with antioxidant and antihypertensive activities.

Several muscle compounds (creatine, creatinine, hypoxanthine, inosine, inosine 5' monophosphate, xanthine, adenosine monophosphate, guanosine, and uridine) were studied as possible biological markers of a minimum dry-cured ham processing time. A correlation between the concentration of the compounds and the time of processing was found. The ratios for some of them were calculated to study their behaviour during processing. The Hx/Ino ratio substantially increased up to 5 months of curing and then remained constant (p

The aim of this work was to study how nucleotide degradation during the processing of dry-cured ham is affected when using three types of salting (100% NaCl; 50% NaCl and 50% KCl; 55% NaCl, 25% KCl, 15% CaCl₂ and 5% MgCl₂). Divalent salts in the salting mixture depressed the breakdown rate from the beginning of the process (salting and post-salting) up to the ripening stage (7 months) when the inosine (Ino), hypoxanthine (Hx) and xanthine (X) concentrations matched for the three treatments. The evolution of Hx and Hx+X were analysed by HPLC and an enzyme sensor, respectively, during processing. Time and temperature conditions during the curing time did not affect Hx stability. The usefulness of the enzyme sensor was confirmed and it is a practical tool to determine Hx + X in dry-cured ham, as an index of minimum curing time. A good correlation between enzyme sensor and HPLC data was observed.

A new HPLC method based on hydrophilic interaction chromatography (HILIC) has been developed for the simultaneous analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine (Ino), hypoxanthine (Hx) and nicotinamide adenine dinucleotide (NAD + ) in pork loin muscle. The chromatographic analysis has been developed using a zwitterionic polymeric column and it has been optimised in terms of linearity, repeatability, reproducibility and recovery. Detection limit values of 0.10, 0.15, 0.05, 0.16, 0.16, 1.72 and 0.15 μg/mL for ATP, ADP, AMP, IMP, Ino, Hx and NAD + respectively were achieved. A comparison between the pork meat concentrations of the compounds analysed by hydrophilic interaction chromatography (HILIC), using a ZIC®- p HILIC column, and ion-pair reversed-phase chromatography (IP-RP-HPLC), using a Zorbax Eclipse XDB-C18 column, has been done, concluding that the new chromatographic methodology constitutes a valid and reliable alternative to the existing methods. Finally, this method has also proved to be very effective in the isolation of guanosine, guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), creatinine (Cn) and uric acid (UA) from a standards mixture.

An enzyme sensor employing xanthine oxidase (XO), soluble or immobilised, in combination of an oxygen electrode has been developed and optimised to determinate the hypoxanthine (Hx) content in pork meat at different post-mortem times as measure of meat freshness. The amperometric signal obtained due to the oxygen depletion during the Hx oxidation was related with the consumed oxygen at 190 s in the soluble enzyme sensor or enzymatic rate at 10 s in the immobilised enzyme sensor. In both cases a linear relationship between the signal and the Hx concentration in the range 8.68–26.05 μM (R2 = 0.999) and 15.63–127 μM. (R2 = 0.995), respectively, was found. Both enzyme sensors exhibited very good working conditions and storage stability. A study of Hx oxidation was carried out in order to compare the Hx content measured by both sensors and those measured by high performance liquid chromatography (HPLC) obtaining a good agreement between both techniques. Therefore, the easy preparation and operation of both enzyme sensors suggests a reliable, rapid and an economical alternative for simple or multiple Hx measurements constituting a useful tool as quality control of meat freshness.

The binding ability of pork meat proteins (sarcoplasmic, myofibrillar and isolated actin and actomyosin) was determined by measuring the relative headspace concentration of the volatile compounds in the presence of each protein (expressed as percentages of the free volatiles relative to a standard solution without protein) using solid-phase microextraction (SPME) and gas chromatography analysis. The sarcoplasmic homogenates bound higher quantities of the volatile compounds assayed (3-methylbutanal, 2-methylbutanal, 2-pentanone, hexanal, methional and octanal) than myofibrillar homogenates. The addition of salts also affected the binding ability of sarcoplasmic and myofibrillar proteins. Actomyosin was able to bind all the assayed volatile compounds although the binding depended on protein concentration and conformation, and it was highly affected by frozen storage. On the other hand, G-actin was unable to bind any of the assayed volatile compounds although the polymerized form (F-actin) bound higher quantities of the volatile compounds.

The effect of different salts on the binding ability of porcine soluble protein extracts was studied using solid-phase microextraction (SPME) and GC/MS analysis. Previously, the effect of the different salts (NaCl, KCl, MgCl2 and CaCl2) on the volatile compound headspace concentration in water solution was determined. KCl produced a similar salting out effect as NaCl increasing the volatile compound headspace concentration up to 5–10 times. However, MgCl2 and CaCl2 did not produce a salting out effect on the volatile compounds studied. Finally, NaCl and KCl produced a significant reduction on the binding ability of sarcoplasmic protein extracts to branched aldehydes, hexanal and methional while no effect was produced on octanal and 2-pentanone. On the other hand, the effect of MgCl2 and CaCl2 was much lower even at high ionic strength, except for the branched aldehydes where the presence of MgCl2 at 1.0 ionic strength produced the complete liberation of the volatile compound bound. Consequently, the partial NaCl substitution in meat products by other salts must be considered not only due to their salting out effect but also due to their effect on protein binding ability.

Dipeptidyl peptidases (DPP) constitute a group of proteolytic enzymes able to release a good number of dipeptides from the N-terminus of both synthetic substrates and natural polypeptides. Specific sequences generated by the action of DPP purified from porcine skeletal muscle have been assayed to evaluate their capacity to inhibit the activity of angiotensin-I converting enzyme (ACE; EC 3.4.15.1). A fluorimetric assay based on the hydrolysis of the internally quenched fluorescent substrate o -aminobenzoylglycyl- p -nitrophenylalanylproline by the action of ACE was used for this purpose. The generated fluorescence of the product (the o -aminobenzoylglycine group) was continuously monitored in a microtiter-plate multiscan fluorometer. Among the assayed dipeptides, Arg-Pro showed the strongest ACE inhibitory activity, being able to suppress more than 60% of initial enzyme activity at a concentration of 25 μM. Dipeptides Lys-Ala, Gly-Pro and Ala-Ala also demonstrated to be effective ACE inhibitors, although at a lower degree. Dipeptides Ala-Arg and Gly-Arg caused a more moderate inhibition of ACE activity, and even lower was the inhibition exerted by Arg-Arg. From the data obtained, it is suggested that the proteolytic action of DPP along the ripening period of dry-cured meat products could contribute to the generation of antihypertensive peptides.

Actomyosin and G-actin were isolated from post-rigor porcine muscle and used to study their binding ability with selected volatile aroma compounds (3-methyl-butanal, 2-methyl-butanal, 2-pentanone, hexanal, methional and octanal). The binding ability was determined by measuring the headspace concentration of the volatile compounds in the presence of each protein using solid-phase microextraction (SPME) and gas chromatography analysis. Actomyosin was able to bind to all the assayed volatile compounds, although the binding was dependent on protein concentration and conformation, and highly affected by frozen storage. On the other hand, G-actin was unable to bind any of the assayed volatile compounds and furthermore, it caused the release of several of them (3-methyl-butanal, hexanal, methional and octanal) at the highest protein concentration. However, the polymerized form (F-actin) was the isolated myofibrillar protein that was able to bind higher quantities of the assayed volatile compounds.

The effects of nutrient sources and growth phase of Debaryomyces hansenii on the protease (PrA and PrB) and aminopeptidase (prolyl-[PAP] and arginyl-[AAP] aminopeptidases) activities were investigated. These activities were also monitored during growth on a whole sarcoplasmic muscle protein extract (WSPE) and on an equivalent medium but free of compounds under 10 kDa (SPE>10 kDa). The levels of specific protease and aminopeptidase activities were higher when cells were grown in urea and dipeptides than when grown in either ammonium or free amino acids as nitrogen sources. The level of each aminopeptidase (PAP or AAP) activity was preferentially induced by its own substrate (ProLeu or LysAla), suggesting a role in the utilization of exogenous peptides. Higher specific activities for all proteolytic enzymes were detected when using acetate as carbon source. The time course experiments carried out on urea or sarcoplasmic protein-containing media revealed an increase in all activities during transition and advanced stages of stationary phase of growth. In muscle protein extracts, the absence of low molecular mass nutrients (SPE>10 kDa) initially induced the production of PrA, PrB, and AAP activities, possibly involved in the breakdown of muscle oligopeptides.

A method for the confirmatory analysis of clenbuterol, mabuterol and salbutamol in bovine urine and feeds by gas chromatography–mass spectrometry (GC–MS) is described. β-Agonist residues were extracted and further purified and concentrated by immunoaffinity chromatography. Methyl boronic acid (MBA) was used for the derivatisation of β-agonist residues and time and temperature of derivatisation were optimised. These derivatives allowed the use of shorter columns while improving the chromatographic resolution. Methyl boronic derivatives were stored either at room temperature or at −20 °C and then injected into the gas chromatograph–mass spectrometer at specific time intervals. Methyl boronic derivatives showed a slight decrease for clenbuterol and a more marked loss for mabuterol when stored at −20° C up to 3 days and then remained stable up to 7 days. However, the stability for salbutamol was poorer as it rapidly decreased at 2 days. When derivatives were stored at room temperature, clenbuterol and internal standard (IS) showed good stability up to 6 h while the stability of mabuterol was progressively reduced. Salbutamol dropped to very low values at just the first hour. Under these conditions it is clear that the analysis should be performed as soon as possible after derivatisation. The combination of immunoaffinity chromatography, MBA derivatisation and gas chromatography–mass spectrometry has shown a high sensitivity, good reproducibility and short chromatography time for the control of the studied β-agonists.

The effect of the addition of Debaryomyces spp. on the microbial, chemical properties and degradation of meat proteins in dry fermented sausages was investigated. The manufacture of dry fermented sausages with Debaryomyces spp. produced a slow decline in pH during early drying stage. However, the final product had lower ammonia, and higher acetic and d-lactic acids without producing any effect on the final pH. Sarcoplasmic proteins were not affected by Debaryomyces spp. but the degradation of myofibrillar proteins was accelerated at the beginning of the drying stage even though the final sausage, inoculated with Debaryomyces spp., had lower myofibrillar proteolysis. The content of free amino acids was similar at the beginning of the drying stage for all the studied batches. However, the high differences in the content of free amino acids at the end of the process could be attributed to the Debaryomyces spp. activity. The addition of a higher amount of Debaryomyces spp. did not contribute to a major proteolysis degree.

The stability of a glutaminase from Debaryomyces spp. CECT 11815 was studied. The enzyme showed poor stability at acid pH (half-life below 10 h at pH 5.5). Better stability was observed at pH 7.5–8.5; however, some variability was found, depending on the type of buffer used. The thermal stability of the glutaminase was rather poor at 37 °C (half-life of 7.4 h) but increased at lower temperatures, reaching a half-life of 323.2 h at 4 °C. EDTA and ethylene glycol extended the enzyme half-life while l -Asp, DTT, glycerol and several salts had the opposite effect. In addition, the influence of curing agents on glutaminase activity was investigated. Nitrate, nitrite, glucose and ascorbic acid had no significant effect on enzyme activity at the levels typically used in the processing of dry-cured sausages, while 1 M NaCl inhibited nearly 55% of the glutaminase activity.

The present work is focused on the use of the relative amounts of carnosine and anserine for detecting the presence of proteins of mammalian origin in feeds for ruminants. The methodology is cheap and simple and may serve for the rapid screening of feeds. Animal protein additions to feeds as low as 0.5% can be easily detected based on the cation exchange HPLC determination of the carnosine and/or anserine dipeptides. Furthermore, the molar ratio of these dipeptides was found to be characteristic of the animal species and could be used to discriminate between mammalian and non-mammalian species eventually added to feeds. Feeds having carnosine/anserine molar ratios higher than 0.3 were strongly suspected of containing banned mammalian proteins. Those suspicious feeds might be later confirmed by more specific techniques. The exact source of feeds containing mixtures of animal proteins from different species could not be identified by this method even though the method was able to detect its mammalian origin.

A simple methodology, consisting in cation exchange HPLC separation combined with OPA postcolumn detection, has been succesfully applied to the analysis and detection of histidine dipeptides in feeds. The use of the proposed methodology, which is very simple and relatively fast, allows the detection of 2.63 ppm and 0.58 ppm of carnosine and anserine, respectively, in cattle feed. As balenine (3-methylhistidine) was not commercialy available, it was isolated from pork muscle and used as standard for its quantitation in feeds. These dipeptides have shown good stability against intense heat treatments, even at temperatures as high as 120 °C for 20 min. So, these histidine dipeptides can be feasible used as markers of animal protein content in feeds.

A glutaminase was purified from the cell-free extract of Debaryomyces spp. CECT 11815 by protamine sulphate treatment and several chromatographic procedures including anion exchange chromatography and gel filtration. The purified enzyme consisted of two subunits, with molecular masses of 65 and 50 kDa, respectively. Activity was optimal at 40 degrees C and pH 8.5, and the Km value for L-glutamine was 4.5 mM. The glutaminase exhibited activity against L-gamma-Glu-methyl ester, L-gamma-Glu-hydrazide, and L-albiziin, while L-asparagine, CBZ-L-Gln, CBZ-L-Gln-Gly, glutathione, L-gamma-Glu-pNA and L-gamma-Glu-AMC were not hydrolysed. The enzyme was not affected by PMSF, DTT and EDTA. However, the enzyme was inhibited by sulfhydryl group reagents, DON, L-albizziin, L-asparagine and high concentrations of L-glutamine and ammonium, while L-aspartate did not affect the activity. Phosphate and acetate did not produce any significant effect on the glutaminase activity, but it was slightly stimulated by lactate and borate.

Dipeptidyl peptidase IV (EC 3. 4. 14. 5) from porcine skeletal muscle has been purified to homogeneity by selective protein fractionation with ammonium sulfate and HPLC separations with strong anion-exchange chromatography. Pure DPP IV showed a single band with Mr about 70 kDa by SDS-PAGE and optimum activity at pH 8.0 and 45°C. Substrates best hydrolyzed were those containing a proline residue in the penultimate position at the N-terminal, but it was also possible to hydrolyze (in lower amounts), X-Ala- synthetic and peptide substrates. The presence of diprotin A, puromycin, Co 2+ and Fe 2+ considerably inhibited DPP IV activity. A contribution of DPP IV action to the total proteolytic activity occurring in postmortem muscle during meat storage and ripening of meat products is feasible and could contribute to flavor generation in those products.

Lipolytic and oxidative changes were studied in two typical meat products from pork loin, dry-cured loin (DCL) and pickled-cured loin (PCL). Neither product registered changes in the percentages of the main lipid fractions: non polar lipids, phospholipids and cholesterol. However, in dry cured loin an important decrease was recorded in the main phospholipid classes, phosphatidyl choline and phosphatidyl ethanolamine. Muscle lipolytic enzymes were active in both products, and were accompanied by a significant increase in free fatty acids, from 0.580% (of total lipid) in fresh loin to 5.65 in DCL and 2.95% in PCL. With respect to oxidative changes, the peroxide value decreased in both products, and the TBA number only increased in PCL.

The effects of nitrate and nitrite curing salts on microbial changes and sensory quality of non-fermented sausages of small diameter were investigated. During pre-ripening (day 5), levels of lactic acid bacteria and yeasts were slightly higher in nitrite-made sausages than in those made with nitrate. In contrast, nitrite discouraged the growth of psychrotrophs as occurs in fermented sausages. By the end of ripening (day 26), levels of microorganisms were similar in both batches of sausages except for psychrotrophs being higher in those made with nitrite. Nitrate-made sausages showed higher aroma and taste intensity.

The effect of pre-ripening at 5°C on microbial changes, nitrate reduction and proteolysis in both naturally fermented and inoculated (Lactobacillus sakeandStaphylococcus carnosus) sausages was investigated.Micrococcaceaelevels were significantly higher (P

We compared the inhibitory action of individual amino acids in vitro on the activities of alanyl-, arginyl-, leucyl- and pyroglutamyl aminopeptidases purified from human and porcine skeletal muscle tissues. The range of susceptibility to inhibition by individual amino acids (

Glucoamylase (1,4 α-d glucan glucohydrolase, EC 3.2.1.3) was covalently immobilized on porous glass fibres and beads. The size of the glass beads (125 μm) was chosen to be small enough to avoid diffusional resistance. A comparison of both supports was established and discussed. Enzyme loadings were similar, 1·70–1·85 mg protein/m2 for fibres and 1·65 mg protein/m2 for beads. A 28% w/v maltose and 39% w/v α-amylase-hydrolysed dextrin 12 DE (Dextrose equivalent) syrups were used as model and real feed substrates respectively. A 98·8% maltose conversion was obtained with the glass fibre reactor. However, the hydrolysis of the dextrin syrup (12 DE, 39% dissolved solids, pH 4·5) was incomplete (93·5% conversion with the same reactor and 91·2% with the glass bead reactor). Glucose production from this syrup was poor for both systems: 85·7 and 83·9% respectively. The continuous operational enzyme stability was found to be highly dependent on substrate concentration, up to 57 days being achieved with a feed of 27·6% w/v maltose solution at pH 4·5 and 50°C.

Different procedures for the extraction of myofibrillar and total proteins of both raw and dry-cured ham have been assayed. Three solvents—(1) 0.1 m phosphate buffer + 1.1 m KI, pH = 7.4 , (2) 0.1 m tris/HCl + 1 % ( w/v ) sodium dodecyl sulphate, pH = 7.0 and (3) 8 m urea + 1 % ( v/v ) β-mercaptoethanol-were used for myofibrillar proteins extraction and the homogenates compared quantitatively and by electrophoresis. Solvent (3) gave a higher protein recovery from dry-cured ham than solvent (2) although the electrophoretic patterns showed similar profiles. One relevant fact was the appearance of a 150 KDa fragment and numerous fragments in the 50–100 KDa region. No myosin band was observed.

Different assays of cathepsin D activity in both porcine muscle and dry-cured ham extracts have been tested in order to find the best conditions for a reliable detection of cathepsin D in dry-cured meat products. The enzyme was effectively extracted with 0·2% (v/v) of Triton X-100. Nucleases if present were not observed to interfere with the assays. The best conditions for a reliable detection of cathepsin D in dry-cured ham were found to be the incubation of the enzyme extract for 1 h at 45°C in a reaction mixture containing 0·60% (w/v) of haemoglobin in 0·2 m sodium citrate buffer, pH = 3·7.

Alkali cooking of cellulose at 10% (w/v) consistency, performed at 250°C, gives a liquor containing organic acids as main components (lactic, acetic and succinic acids). A synthetic substrate, representative of this liquor, was used as feed for two anaerobic fluidized-bed reactors operating at 35°C. The effects of hydraulic retention time and influent substrate concentration on substrate consumption and methane generation were studied. Organic removals of up to 95% and methane generation rates of 1·09 litres CH 4 litre R −1 day −1 were achieved at a retention time ( θ R ) of 8·3h and at an influent substrate concentration of 1400 mg COD litre −1 . At θ R = 3·5 h, conversion decreased to 60%. When feeding the cooked liquor, conversions were slightly lower, around 50% at θ R = 4 h and 1300 mg COD litre −1 of influent substrate concentration. However, the methane volumetric production rate was as high as 2·5 litres CH 4 litre R −1 day −1 under the same conditions. The results suggest that alkaline cooking with subsequent methane fermentation offers a viable process for the treament of cellulosic materials, such as municipal solid waste.