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.

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.

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.

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.

Effect of curing conditions and Lactobacillus casei CRL705 on the hydrolysis of meat proteins.

AIMS: The effect of the common curing conditions used during the manufacture of dry fermented sausage on the proteolytic activity of Lactobacillus casei CRL705 against meat proteins was investigated. METHODS AND RESULTS: Hydrolysis of pork muscle sarcoplasmic and myofibrillar proteins was evaluated by SDS-PAGE and reverse phase-HPLC analysis. Ascorbic acid exerted a stimulatory effect on both sarcoplasmic and myofibrillar protein breakdown by Lact. casei CRL705 with the release of hydrophilic peptides and free amino acids, while NaCl and NaNO2 mainly stimulated myofibrillar degradation. CONCLUSION: Even when processing temperature (25 degrees C) did not positively affect bacterial protein hydrolysis, the presence of curing salts accounted for a remarkable increase in the non-volatile components that constitute taste-active compounds that strongly influence the final flavour of the product. SIGNIFICANCE AND IMPACT OF THE STUDY: To predict the suitability of Lact. casei CRL705 and its proteolytic enzymes as a starter culture for the dry processing of dry fermented sausages.

Evolution of hydrophobic polypeptides during the ageing of exudative and non-exudative pork meat.

Thirty-six carcasses from 6-month-old pigs were classified in different exudative groups based on measurements of pH(2h), pH(24h), the colour parameter L(∗) and drip loss. A fraction containing polypeptides between 66 and 21 kDa was analysed by reverse phase chromatography at 2-h post-mortem and the evolution of 8 polypeptide fractions followed during ageing and related to meat quality. Three polypeptide (fractions P2, P3 and P4) at 2-h post-mortem showed significant lowest area values in the dark firm and dry class. During ageing, the higher content of P4 in exudative meats at 8-h post-mortem could be due to activation of the cathepsin system. On the other hand, P3 and P4 increased in DFD meats during the first 96-h post-mortem probably due to higher calpain activity. Few differences in polypeptides were related to meat qualities although they are important as precursors of small peptides and free amino acids.

Pork meat quality affects peptide and amino acid profiles during the ageing process.

Twenty pork carcasses were classified in different pork meat qualities: red, firm and non-exudative (RFN), pale, soft and exudative (PSE), red, soft and exudative (RSE) and dark, firm and dry (DFD) meat. The content of peptides and free amino acids during the ageing process was analysed and compared within quality classes. Four peptide fractions were isolated through cation-exchange and reverse-phase chromatography. The main significant differences among qualities were obtained for peptide fractions 3 and 4. Peptide fraction 3 at 4 days and peptide fraction 4 at 2 h postmortem were higher in the ideal pork quality (RFN) than in the other quality classes. The ageing of pork meats produced a general increase in all free amino acid concentrations for the studied quality classes except for Gln, ß-Ala, Taurine and Orn and the dipeptides carnosine and anserine. The DFD class showed higher increases in Lys, Ala and Met probably due to the activation of neutral aminopeptidases.

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.

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.

Sensory characteristics of cooked pork loin as affected by nucleotide content and post-mortem meat quality.

Muscle longissimus dorsi excised at 2hr post mortem was utilised to study the effect of post-mortem meat quality, and their content of nucleotide metabolites, on the sensory characteristics of cooked loin. Four different post mortem meat qualities were defined: pale soft exudative (PSE), red soft exudative (RSE), red firm non-exudative (RFN) and dark firm dry (DFD). The DFD meat quality class showed low sour and salty tastes and a high sweet taste, low astringency and high juiciness and tenderness, giving the best overall quality. On the other hand, the RSE quality class produced a meat with a low juiciness and high hardness, and a low overall quality although these differences were not significant (p<0.05) against RFN and PSE meats. The effect of nucleotide metabolite contents on the sensory meat properties were most significant on taste although, the different meat qualities were only significantly different (p<0.05) in the content of hypoxanthine, guanosine, GDP and IDP.

Concentration of free amino acids and dipeptides in porcine skeletal muscles with different oxidative patterns.

Free amino acids and natural dipeptides, have been analyzed in pork muscles having different metabolic type (masseter, trapezius, semimembranosus and longissimus dorsi). Carnosine and anserine showed significantly higher (p < 0.05) concentrations with the glycolytic activity of the muscle while taurine and glutamine were significantly higher in the oxidative muscles. Non-essential free amino acids also showed significant (p < 0.05) increased content in the oxidative muscles.

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.

Cloning and characterization of two genes from Bacillus polymyxa expressing beta-glucosidase activity in Escherichia coli.

DNA fragments from Bacillus polymyxa which encode beta-glucosidase activity were cloned in Escherichia coli by selection of yellow transformants able to hydrolyze the artificial chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside. Restriction endonuclease maps and Southern analysis of the cloned fragments showed the existence of two different genes. Expression of either one of these genes allowed growth of E. coli in minimal medium with cellobiose as the only carbon source. One of the two enzymes was found in the periplasm of E. coli, hydrolyzed arylglucosides more actively than cellobiose, and rendered glucose as the only product upon cellobiose hydrolysis. The other enzyme was located in the cytoplasm, was more active toward cellobiose, and hydrolyzed this disaccharide, yielding glucose and another, unidentified compound, probably a phosphorylated sugar.