Silvina Fadda

A review of bacteriocinogenic lactic acid bacteria used as bioprotective cultures in fresh meat produced in Argentina

Several lactic acid bacteria (LAB) associated with meat products are important natural bacteriocin producers. Bacteriocins are proteinaceous antagonistic substances that are important in the control of spoilage and pathogenic microorganisms. The use of LAB as bioprotective cultures to extend the shelf life of fresh meat can improve microbial stability and safety in commercial meat preservation. Lactobacillus curvatus CRL705 used as a protective culture in fresh beef is effective in inhibiting Listeria innocua and Brochothrix thermosphacta as well as the indigenous contaminant LAB, retaining its inhibitory effect at low temperatures and having a negligible effect on meat pH. In addition to the hurdle represented by low temperature and vacuum-packaging, the use of live cells of Lb. curvatus CRL705 seems more feasible from an economic point of view - and without legal restrictions - compared to the addition of purified bacteriocins. A description of meat-borne bacteriocins and their application in meat to extend shelf life is discussed.

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.

Control of Listeria monocytogenes in ground beef by ‘Lactocin 705’, a bacteriocin produced by Lactobacillus casei CRL 705

The effectiveness of Lactobacillus casei CRL 705 as well as that of Lactocin 705, the associated bacteriocin produced, in reducing population levels and growth of Listeria monocytogenes in sterile and non-sterile ground beef was studied. Predetermined numbers of L. monocytogenes and concentrations of Lactocin 705 were added to the meat slurries. Listeria in the bacteriocin-treated and control samples during incubation at 20 degrees C were enumerated as CFU on Bacto blood agar base. Results indicated that reduction in numbers of Listeria was largest with high levels of Lactocin 705 and few initial cell numbers of the pathogen present in the meat slurry. However, when the producer strain was added to the slurry, no significant inhibition was detected. Furthermore, inhibition by listeria was shown to be greater when meat slurries were heat-treated.

Role of lactic acid bacteria during meat conditioning and fermentation: Peptides generated as sensorial and hygienic biomarkers

The microbial ecology of meat fermentation is a complex process in which lactic acid bacteria (LAB) and coagulase-negative cocci play a major role. The present work reviews the most significant developments in which LAB are the main characters acting both as starter cultures improving the sensorial quality and as biopreservative agents. New findings about the identification of low molecular weight peptides arisen from protein hydrolysis in dry fermented sausages and their relation with flavor is presented. Also, a brief description of a proteomic approach is detailed in order to exemplify its application as a tool in the search for improved LAB strains that will contribute to food quality and safety. Finally, the most important features of bacteriocinogenic LAB and its bacteriocins in bioprotection of meat and meat products are analyzed.

Tyramine degradation and tyramine/histamine production by lactic acid bacteria and Kocuria strains

Of 53 strains of lactic acid bacteria and Kocuria, screened for production or degradation of biogenic amines, 29 Kocuria varians and four strains of Enterococcusfaecalisproduced tyramine and, at lower concentrations, histamine. In contrast, Lactobacillus strains that did not possess amino acid decarboxylase activity degraded tyramine. The greatest tyramine oxidase activity was present in the strains L. casei CRL705 (98% degradation) and CRL678 (93%) as well as in L. plantarum CRL681 (69%) and CRL682 (60%).

Proteolytic activity of Lactobacillus strains isolated from dryfermented sausages on muscle sarcoplasmic proteins.

The proteolytic activity of seven strains of Lactobacillus from two species isolated from dry cured sausages was assayed using a soluble muscle extract as a source of proteins, at a temperature of 30 °C. The results indicated that the strains of Lactobacillus plantarum tested had the more active proteolytic system, showing the highest amino acid release in the medium after 72 hr of incubation (L. plantarum CRL 681) when the microorganism was in the stationary phase of growth. The strains of L. casei showed a continued hydrolytic activity with a lower amino acids concentration along the studied period. The SDS-PAGE profiles showed that the major changes in sarcoplasmic proteins were produced in the 13 kDa and 36-40 kDa molecular weights region.

Identification of small peptides arising from hydrolysis of meat proteins in dry fermented sausages.

In this study, proteolysis and low molecular weight (LMW) peptides (<3kDa) from commercial Argentinean fermented sausages were characterized by applying a peptidomic approach. Protein profiles and peptides obtained by Tricine-SDS-PAGE and RP-HPLC-MS, respectively, allowed distinguishing two different types of fermented sausages, although no specific biomarkers relating to commercial brands or quality were recognized. From electrophoresis, α-actin, myoglobin, creatine kinase M-type and L-lactate dehydrogenase were degraded at different intensities. In addition, a partial characterization of fermented sausage peptidome through the identification of 36 peptides, in the range of 1000-2100 Da, arising from sarcoplasmic (28) and myofibrillar (8) proteins was achieved. These peptides had been originated from α-actin, myoglobin, and creatine kinase M-type, but also from the hydrolysis of other proteins not previously reported. Although muscle enzymes exerted a major role on peptidogenesis, microbial contribution cannot be excluded as it was postulated herein. This work represents a first peptidomic approach for fermented sausages, thereby providing a baseline to define key peptides acting as potential biomarkers.

Adaptive response of Lactobacillus sakei 23K during growth in the presence of meat extracts: A proteomic approach

Lactobacillus sakei is a lactic acid bacterium mainly found in meat and meat products. In order to understand the factors favoring its adaptation to meat matrix, growth parameters and survival of the strain L. sakei 23K in the presence of sarcoplasmic or myofibrillar extracts were assessed. Cytosolic proteins putatively involved in the response of this strain to meat proteins were determined using 2D electrophoresis and the significantly regulated proteins were identified by Maldi Tof-MS analyses. From the 31 differentially expressed spots, 16 occurred in the presence of myofibrillar extract while 6 proteins were modulated by the sarcoplasmic extract. Two dipeptidases were overexpressed in the presence of sarcoplasmic proteins, in correlation to the protein degradation patterns obtained by SDS-PAGE. In the presence of the myofibrillar extract, L. sakei 23K overexpressed proteins related to energy and pyrimidine metabolism as well as ala- and tyr-tRNA synthetases, involved in translation, while others corresponding to general stress response, pyrimidine, vitamin and cofactor biosynthesis were down-regulated. The supplementary nutrients furnished by meat extracts modulated the overexpression of proteins related to translation, peptide/amino acid metabolism and energy production while the stress proteins were under regulated. The results obtained here suggest that meat proteins would not represent a stress environment per se for L. sakei 23K in contrast to the harsh conditions during meat processing. This study has extended the understanding of the molecular responses and growth mechanisms of L. sakei 23K in the presence of meat proteins. The transference of genomic information into useful biological insight is an important step for the selection of well-adapted strains for the achievement of high-quality fermented products.

Lactobacillus role during conditioning of refrigerated and vacuum-packaged Argentinean meat.

The role of Lactobacillus strains with bioprotective and technological potential on raw beef during 15days of storage under vacuum at 7°C was investigated. The assayed strains were able to grow on the meat, Lactobacillus curvatus CRL705 and Lactobacillus sakei 23K showing the highest competitiveness. A net increase of amino acids was determined in inoculated samples when compared to the control, this being maximal for Lactobacillus plantarum CRL681. Although an important endogenous activity of meat sarcoplasmic proteins was observed, the disappearance of protein bands and the generation of a new one were detected as a consequence of Lactobacillus growth. A synergistic effect of Lactobacillus in combination with the muscle proteolytic enzyme complex can be suggested. From the studied strains, the bacteriocin producer L. curvatus CRL705 may be considered as a good candidate to contribute to meat ageing by means of small peptides and free amino acids generation while improving shelf life.

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.