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.

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.

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.

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.

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.

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.

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.

Activities of pork muscle proteases in model cured meat systems.

The effect of curing agents (salt, nitrate, ascorbic acid and glucose) and processing parameters (pH, water activity and drying and cooking temperatures) on pork muscle cathepsins B, D, H and L as well as leucyl, arginyl and tyrosyl hydrolysing activities is reported. Salt (60 g/l) showed a powerful inhibitory effect, especially on cathepsin D and aminopeptidase activities where less than 13% of the original activity was recovered. Cathepsin H was also affected (38% of the original activity) while cathepsins B and B+L recovered 72.5 and 63.0%, respectively. Nitrate (0.2-0.25 g/l) and ascorbic acid (0.2-0.4 g/l) did not significantly affect the enzyme activities. On the other hand, 0.5-2 g/l of glucose activated both cathepsins B and D with an increase of 39.5 and 28.5% and also leucyl and arginyl hydrolysing activities which were 75.0 and 24.0%, respectively. No aminopeptidase activity was detected when assayed in 100 mM sodium citrate buffer, pH 5.1. Cathepsin H was also very affected at that pH and only 12.0% of activity was recovered. A decrease in water activity, especially below 0.84, also affected the enzyme activities which were found below 50%. Temperatures in the usual range of the drying process (22 and 30 degrees C) gave substantial enzyme activities (around 40-50 and 80%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

HPLC purification and characterization of porcine muscle aminopeptidase B.

An aminopeptidase B from porcine skeletal muscle was successfully purified by ammonium sulphate fractionation and HPLC anion-exchange. The purified aminopeptidase B eluted at 0.18 M NaCl, had a relative molecular mass of 76,000 Da and was markedly stimulated in the presence of 0.2 M chloride anion. The enzyme exhibited maximum activity for the hydrolysis of the arginine-aminoacyl bond at pH 6.5 and 37 degrees C. Other substrates consisting of phenylalanine, proline and alanine-aminoacyl bonds were cleaved at 5.9, 5.1 and 2.5% of the maximum activity with the arginine-aminoacyl bond. The enzyme did not show endopeptidase activity and was very stable at pH above 6 and temperatures below 35 degrees C. However, the enzyme inactivated very fast when incubated at pH 5 or at 50-65 degrees C. Bestatin (50 microM) completely inhibited the aminopeptidase B activity while EDTA (5 mM) only inhibited 40% of its activity. However, 0.5 mM of E-64 did not cause any inhibition while 0.05 mM amastatin and 1 mM puromycin only inhibited 11% of the enzyme activity.

Myoglobin as an inhibitor of exopeptidases from lactobacillus sake

The effects of myoglobin on exopeptidases of Lactobacillus sake were determined. Inhibition of the aminopeptidases increased as the myoglobin concentration increased; aminopeptidase 3 was the most affected (90% inhibition). Aminopeptidases 1, 2, and 4 showed similar inhibition levels (around 60%). Myoglobin did not affect tripeptidase activity. Thus, myoglobin could limit amino acid generation in meat systems.

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.

Effect of nitrate and nitrite curing salts on microbial changes and sensory quality of non-fermented sausages.

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.

Effect of nitrate and nitrite curing salts on microbial changes and sensory quality of rapid ripened sausages.

The effect of the use of either nitrite or nitrate curing salts on microbial changes and sensory quality in rapid ripened sausages inoculated with a mixed starter culture (Lactobacillus sake and Staphylococcus carnosus) was investigated. Lactic acid bacteria and Micrococcaceae were not greatly affected by the added curing salt. Conversely, the inhibition exerted by nitrite on the undesirable flora (Enterobacteriaceae and psychrotrophs) was evident from the early stages of the processing keeping highly significant differences (P < 0.01) with respect to nitrate made sausages till the end of the ripening stage. The use of nitrite in sausage processing was found to reduce hygienic risks.

Hydrolysis of pork muscle sarcoplasmic proteins by Debaryomyces hansenii.

Strains of Debaryomyces hansenii originally isolated from sausages were screened for proteinase and aminopeptidase activity towards synthetic substrates. On the basis of these results, D. hansenii CT12487 was selected for further assays. The activities of the whole cells (WC), cell-free extracts (CFE) and a combination of both from the selected strain on pork muscle sarcoplasmic protein extracts were determined by protein, peptide and free amino acid analyses. There was a pronounced hydrolysis of protein bands of 110 kDa and 27-64 kDa regardless the incorporation of WC, CFE or a combination of both. The proteolytic activity also resulted in the generation of polar and non-polar peptides showing noticeable differences depending on the addition of WC or CFE. Whole cells generated greater amounts of free amino acids than the cell-free extracts.

Hydrolysis of muscle myofibrillar proteins by Lactobacillus curvatus and Lactobacillus sake.

Proteolytic enzyme activities of whole cells and cell free extracts (CFE) of Lactobacillus curvatus CECT 904 and Lactobacillus sake CECT 4808 were characterised using synthetic chromogenic compounds and myofibrillar proteins as substrates. The hydrolytic action was monitored by SDS-PAGE and reverse phase-HPLC analyses. The CFE of L. sake partially contributed, together with muscle enzymes, to the initial hydrolysis of myofibrillar proteins. Whole-cells of both L. curvatus and L. sake generated peptides considered important for cured-meat taste. The peptide mapping, resulting from the action on the substrates assayed, revealed a profile of extra and intracellular enzymes. Both strains expressed strong amino acid metabolism.

Purification and biochemical properties of dipeptidyl peptidase I from porcine skeletal muscle.

Dipeptidyl peptidase I (DPP I; EC 3.4.14.1) was purified from porcine skeletal muscle after several steps such as heat treatment, ammonium sulfate fractionation, gel filtration chromatography, and HPLC anion exchange chromatography. The purified enzyme showed a native molecular mass of approximately 200 kDa on Sephacryl S-200 column chromatography. Two protein bands of 65 and 42 kDa were obtained by SDS-PAGE, indicating its oligomeric nature. Maximum activity was reached at pH 5.5 and 55 degrees C. DPP I shared some common substrate specificities, both on synthetic derivatives and on real peptides, with porcine muscle DPP III. The enzyme required reducing agents for full activation, although the halide requirement was not proved. DPP I was inhibited by the assayed cysteine peptidase inhibitors except p-CMB. The serine peptidase inhibitor 3, 4-DCI also inhibited the enzyme as did the divalent cations Co(2+), Mn(2+), and Zn(2+). On the basis of its properties, DPP I may contribute to the generation of dipeptides during the processing of meat and/or meat products, including cooked ham.

Hydrolytic action of Lactobacillus casei CRL 705 on pork muscle sarcoplasmic and myofibrillar proteins.

Lactobacillus casei CRL 705 was screened, among other meat isolates, for its proteinase and aminopeptidase activities toward synthetic substrates and, according to that, selected for specific assays on muscle proteins. The hydrolytic effects of whole cells, cell free extracts (CFE), and the combination of both on muscle sarcoplasmic and myofibrillar protein extracts was evaluated by SDS-PAGE and reverse phase HPLC analyses. The proteinase activity of whole cells caused the degradation of a great number of sarcoplasmic protein bands. A partial hydrolysis was also associated with CFE that when combined with whole cells showed an important additional degradation. Peptide profiles from sarcoplasmic protein extracts were greatly modified regardless of the addition of whole cells or CFE, although their combination intensified these changes. The generation of free amino acids was remarkable when whole cells and CFE were incorporated together to sarcoplasmic protein extracts.