Analysis of Nitrite and Nitrate in Foods: Overview of Chemical, Regulatory and Analytical Aspects.

In this chapter, several factors that should be considered for selecting and developing suitable analytical methods for determining nitrite/nitrate are presented. Nitrite and nitrate occurrence and suitability are a controversial issue. Nitrite is an approved additive considered a foremost curing ingredient for the preservation of meat products. Nitrate is a natural constituent of the human diet that, however, raises fears for its suggested potential harmfulness related to carcinogenesis and environmental contamination. Chemical, regulatory, and analytical aspects are discussed in the light of the need to obtain reliable data of nitrite and nitrate for law enforcement purposes, exposure estimates, and investigation of their physiological role in the human body. In addition, current metrological aspects to ensure the "fitness for purpose" of the selected method are suggested and discussed.

Effect of dietary organic selenium on muscle proteolytic activity and water-holding capacity in pork.

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 26days 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.

Lactobacillus sakei CRL1862 improves safety and protein hydrolysis in meat systems.

AIMS: The capacity of Lactobacillus sakei CRL1862 to prevent the growth of pathogens and its ability to degrade sarcoplasmic and myofibrillar proteins in pork meat systems was evaluated. In addition, basic safety aspects of Lact. sakei CRL1862 such as production of biogenic amines and antibiotic susceptibility were addressed. METHODS AND RESULTS: The bacteriocin-producing Lact. sakei CRL1862 showed respectively bactericide and bacteriostatic effect against Listeria monocytogenes and Staphylococcus aureus in beaker sausage assay during 9 days of storage at 22 °C. The hydrolytic effect of Lact. sakei CRL1862 on protein extracts was evaluated by SDS-PAGE and reverse phase HPLC. A more pronounced proteolysis was evidenced in inoculated sarcoplasmic proteins compared with myofibrillar extracts with the generation of predominantly hydrophilic peptides and increase of total free amino acids concentration. Lactobacillus sakei CRL1862 produced neither histamine nor tyrosine and exhibited no resistance to the antibiotics assayed. CONCLUSIONS: Lactobacillus sakei CRL1862 effectively controlled the growth of L. monocytogenes and Staph. aureus; moreover, it was able to hydrolyse pork meat extracts generating peptides and amino acids, which may improve hygienic and sensorial attributes of fermented meat products. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an integrated approach to evaluate the major traits of Lact. sakei CRL1862 showed it can be applied as an autochthonous functional starter in meat fermentation.

Effect of brine thawing/salting on endogenous enzyme activity and sensory quality of Iberian dry-cured ham.

Simultaneous brine thawing/salting process was applied as an alternative to traditional pile salting process using 51 frozen Iberian hams. The effect of this type of salting process on endogenous enzyme activity and sensory quality of Iberian dry-cured hams was analysed. The frozen hams were simultaneously thawed and salted with saturated brine, with and without vacuum pulses, and were compared to hams thawed under refrigeration and traditionally salted. The peptidase and lipase activities were measured at the end of salting and post-salting stages. The activity of cathepsin B+L was reduced in the two brine salted batches while few differences among batches were observed for the other peptidases. Several lipase activities were significantly reduced in the two brine salted batches. The brine thawed processing affected the free fatty acid content at the different stages although the differences were more appreciated at the beginning of the process and no differences were observed at the end. The long ripening time makes these differences negligible and the consumer did not appreciate any differences between the sensory quality of Iberian brine/thawed hams and traditional Iberian thawed pile salted hams.

Small peptides released from muscle glycolytic enzymes during dry-cured ham processing.

Glycolytic enzymes are a group of sarcoplasmic enzymes responsible for the extraction of the energy available from carbohydrates. The glycolytic pathway consists of 10 enzyme-catalyzed steps. Fragments identified in this study, within the range 1100-2600 Da, correspond to glycogen phosphorylase enzyme, which catalyzes the rate limiting step in the degradation of glycogen, enzymes that catalyze steps 6-10 of glycolysis (glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase, respectively), and lactate dehydrogenase, which catalyzes the interconversion of pyruvate and lactate. A total of 45 specific fragments of these enzymes resulting from the processing of dry-cured ham are reported for the first time in this work. This study evidences the intense proteolysis occurring in the sarcoplasmic fraction of dry-cured ham as well as facilitates the choice of the most adequate tools in the identification of naturally generated peptides through comparison between Paragon and Mascot search engines, together with UniProt and NCBInr databases.

Physicochemical changes in dry-cured hams salted with potassium, calcium and magnesium chloride as a partial replacement for sodium chloride.

The reduction of added sodium chloride in dry-cured ham has been proposed to reduce dietary sodium intake in Mediterranean countries. The effect of substituting sodium chloride with potassium chloride, calcium chloride and magnesium chloride on some physicochemical characteristics of dry-cured ham during processing was evaluated. The results showed that hams salted with a mixture of sodium and potassium chloride registered higher salt concentrations and lower water contents and thus, needed less time to reach the required weight loss at the end of the process. The opposite effect was observed when calcium and magnesium chloride were added to the salt mixture. The observed differences in the texture and colour parameters were mainly due to differences in water and salt content.

Nutritional composition of dry-cured ham and its role in a healthy diet.

Dry-cured ham is a traditional product with a strong presence in markets in the Mediterranean area. It is very popular with European consumers and is of enormous economic importance for the meat industry in the Mediterranean area. Although the great palatability of ham largely outweighs other considerations, aspects relating to health and wellbeing are increasingly important factors in consumer decisions. The potential role of ham in a context of healthy nutrition has not been clearly elucidated, especially considering that origins and production methods of dry-cured hams can induce differences in composition. The object of this review was on the one hand to provide an analysis of the components of dry-cured ham and their role in a healthy diet, and on the other hand to suggest possible strategies for improving its nutritional composition.

Physical activity-induced alterations on tissue lipid composition and lipid metabolism in fattening pigs.

In a first experiment one group of pigs was maintained in free-range conditions according to the traditional way in a Mediterranean forest (exercised-1) and another group was housed individually and received acorns (sedentary-1). In a second experiment two groups of pigs were fed a mixed diet for the whole experimental period. One of these groups was housed individually in 8m(2) pens (sedentary-2). The other group was housed in a corridor and forced to walk daily (exercised-2). The subcutaneous fat and neutral lipids of muscle from the exercised pigs fed acorns had higher C18:1n-9, MUFA, C18:1/C18:0, MUFA/SAT and lower C16:0 and SAT when compared with the fat from the pigs fed acorns in confinement. Those exercised animals fed the mixed diet had also lower C16:0 and SAT in subcutaneous fat and lower SAT and higher C18:2, C18:3, PUFA and MUFA/SAT in neutral lipids when compared with the sedentary pigs, which may indicate that delta-9-desaturase activity was higher in exercised than in sedentary pigs. Exercised pigs had higher acid and neutral esterases and lower neutral lipase activity than sedentary pigs. No differences in the α-tocopherol concentration and TBARS values of meat samples among the pigs that received a mixed diet either exercised or sedentary were observed. The moderate exercise reduced the postprandrial concentrations of triglycerides in plasma, but did not reduce other plasma levels.

Influence of sodium replacement on physicochemical properties of dry-cured loin.

The consumption of cured meat products is not recommended to hypertensive consumers due to its high sodium content. This constitutes an important restriction for this industry, which is becoming more and more important due to the current trends in consumption. The partial replacement of sodium chloride by potassium chloride has been proposed as a possible strategy to reduce the sodium content of this type of products. The aim of this study was to evaluate the effect brought about by partial replacement of sodium chloride with potassium chloride (up to 70%) on physicochemical and microbiological parameters of dry-cured pork loin after the curing and drying process. The results showed that it is possible to obtain low sodium dry-cured loin, up to a 50% replacement of sodium by potassium, with similar physicochemical characteristics to the commercial product with usual amounts of sodium.

Effect of prefreezing hams on endogenous enzyme activity during the processing of Iberian dry-cured hams.

The use of frozen/thawed raw material in the processing of Iberian dry-cured ham has been studied to determine its effect on the sensory quality of the final product. The proteolysis and lipolysis processes were measured by the proteolytic and lipolytic enzyme activities and free amino acids and free fatty acids. The thawed Iberian hams had lower salt contents throughout the process. The use of thawing raw material did not affect the proteolytic enzymes, cathepsins, aminopeptidases and dipeptidylpeptidases, only the activity of dipeptidylpeptidase III was reduced due to thawing. Moreover, there were no differences in the content of free amino acids between fresh and thawed hams during the whole process. However, the use of thawing hams affected the lipolytic activity. The activity of phospholipase and neutral lipase were significantly higher in the thawed hams and also the content of free fatty acids, at all the stages analyzed. Consumer sensory analysis showed thawed Iberian hams had the lowest hardness, probably due to an intense proteolysis. The acceptability of the Iberian hams was similar between fresh and thawed hams.

Analysis of protein carbonyls in meat products by using the DNPH-method, fluorescence spectroscopy and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS).

Liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) was applied as an advanced methodology to study the suitability of using α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) as protein oxidation markers in meat products. The results obtained were compared to those obtained by using the DNPH-method and fluorescence spectroscopy for the analysis of protein carbonyls. Lipid oxidation was also investigated in order to elucidate the relationship between lipid and protein oxidation measurements. Both semialdehydes were originally detected in a food system which proves that lysine, arginine and proline are degraded as a result of oxidative reactions to yield AAS and GGS in meat products. A lack of consistency was observed between the MS results for AAS and GGS and the values obtained by the DNPH-method and the fluorescence spectroscopy. Unlike the last two methods, AAS and GGS measurements have proved to be unaffected by the composition or the structure of the food matrix providing precise information about the fate of particular amino acids during processing of muscle foods. These semialdehydes, and particularly GGS, could be used as indicators of protein oxidation in meat products like TBARS numbers are commonly used as lipid oxidation markers. In fact, a significant correlation was found between GGS values and TBARS highlighting the timely interaction between lipid and protein oxidation.

Microbiology and physico-chemical changes of dry-cured ham during the post-salting stage as affected by partial replacement of NaCl by other salts.

Dry-cured ham consumption is restricted by hypertensive consumers due to its high sodium content. This constitutes an important matter for this industry, being relevant due to the current trends in consumption. In order to prevent the problems related to the high sodium intake, one of the possibilities is the total or partial replacement of sodium by other ions, such as potassium, calcium and magnesium. The aim of this study was to characterise the post-salting stage in Spanish cured ham production with the results obtained after salting with low sodium salt content. The results showed that lower sodium hams needed more time of post-salting to reach similar water activity values than those achieved by hams salted with 100% NaCl. Nevertheless, no differences in microbial counts were observed among the studied batches, although a sharp decrease in microbiota was observed when the, post-salting time was prolonged in the lower sodium hams.

Effect of exercise on skeletal muscle proteolytic enzyme activity and meat quality characteristics in Iberian pigs.

The effects of physical activity on performance, carcass traits, Psoas major lysosomal and exoprotease acitivies and meat quality were studied in 24 castrated male Iberian pigs during the last fattening period (from 111.1±SD: 5.2kg). Pigs were randomly distributed in three groups. Two groups receiving the same diet were reared in confinement, one housed in individual pens of 8m(2) (sedentary group) and the other was housed outdoor with daily (up to 2km) forced walking (exercise group). And one group was reared under the traditional production system walking daily several km and fed mostly with acorn from Quercus ilex and Quercus rotundifolia and grass (free-range group). No differences were found in performance and carcass traits. In exercised pigs a lower activity of cathepsin B+L and total cathepsins (P<0.05) was observed. Exercise induced the inhibition of dipeptidyl peptidases II and III and arginyl aminopeptidase and the activation of dipeptidyl peptidases IV and leucyl aminopeptidase (P<0.05). Although no effects on total free amino acids in Psoas major muscle were observed the concentration of branched chain amino acids decreased in the free-range pig group probably related to an increase in physical activity. Exercise had no effects in Psoas major postmortem tenderness and water holding capacity.

Sensory improvement of dry-fermented sausages by the addition of cell-free extracts from Debaryomyces hansenii and Lactobacillus sakei.

The effects of the addition of a combined cell-free extract from Lactobacillus sakei and Debaryomyces hansenii (D+L) or just a D. hansenii cell-free extract (D) to the initial formulation of a dry-fermented sausage were evaluated. The differences found among batches in the main microbial populations, pH, moisture content and global proteolytic and lipolytic indexes (total free amino acids, non protein nitrogen, acidity and tiobarbituric acid index) were not significant. Only, the acidity value of batch D was significantly higher (p<0.05) than that of batch D+L. Thus, cell-free extract from D. hansenii accelerated the lipolysis. Moreover, there were some significant differences (p<0.05) in the amino acid profile and, especially, in the aroma profile. The combination D+L and D promoted the generation of volatile compounds derived from lipid oxidation and carbohydrate fermentation. In batch D, the production of volatile compounds derived from amino acid catabolism and microbial fermentation was also enhanced. The overall quality was improved by both treatments (D+L, D) and also the aroma by addition of the combination of extracts (D+L). It is concluded that the addition of cell-free extracts from D. hansenii and, particularly, D. hansenii plus L. sakei could be useful to improve the final quality of fermented sausages.

Meat flavor: contribution of proteins and peptides to the flavor of beef.

While the majority of meat flavor is lipid in origin, the contribution of peptides and amino acids to overall meat flavor should not be overlooked. Amino acids and peptide levels have been shown to change with postmortem aging in muscle and with dry-curing, a process similar to PMA. Variation in protein, peptide, and amino acid composition have also been shown to occur with heating and with post-heating storage of meat. This makes a large pool of reactive components that may directly affect flavor or indirectly affect flavor by reacting with reducing sugars to form Maillard reaction products and Strecker degradation products that impact meat flavor. Further research in this area should continue with particular emphasis on natural peptide flavor enhancers, modulators, and potentiators.

PCR-based fingerprinting techniques for rapid detection of animal species in meat products.

A reproducible, rapid, and simple method for simultaneous identification of multiple meat species in a single step DNA-based test has been developed based on the generation of species-specific fingerprintings by two different arbitrary DNA amplification approaches (RAPD- and AP-PCR). Samples representative of various species and meat products submitted to different processing conditions were selected to verify the applicability of the techniques. RAPD-PCR fingerprintings allowed the discrimination amongst pork, beef, lamb, chicken and turkey in all cases. Samples corresponding to each species were clustered together at similarity levels ⩾75%. The DNA profiles consisted of a discrete but reproducible number of bands, which made possible the interpretation of the results by simple visual inspection. AP-PCR also allowed identification of the five tested species in every sample although more complex patterns were generated, including some low intensity bands. In both cases, a ramp time between annealing and extension temperatures was introduced to achieve good reproducibility. Overall, the simplicity of RAPD-PCR patterns could make this technique suitable for meat authentication in routine analysis.

Effect of Debaryomyces spp. on the proteolysis of dry-fermented sausages.

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.

Purification and characterisation of a glutaminase from Debaryomyces spp..

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.