Modulation of Toll-like receptor-mediated innate immunity in bovine intestinal epithelial cells by lactic acid bacteria isolated from feedlot cattle.

The ability of lactobacilli isolated from feedlot cattle environment to differentially modulate the innate immune response triggered by Toll-like receptors (TLRs) activation in bovine intestinal epithelial (BIE) cells was evaluated. BIE cells were stimulated with Lactobacillus mucosae CRL2069, Lactobacillus acidophilus CRL2074, Lactobacillus fermentum CRL2085 or Lactobacillus rhamnosus CRL2084 and challenged with heat-stable pathogen associated molecular patterns (PAMPs) from enterotoxigenic Escherichia coli (ETEC) to induce the activation of TLR4 or with polyinosinic:polycytidylic acid (poly(I:C)) to activate TLR3. Type I interferons, cytokines, chemokines and negative regulators of TLR signalling were studied by RT-PCR. L. mucosae CRL2069 significantly reduced the expression of interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 in BIE cells in the context of TLR3 activation. L. mucosae CRL2069 also reduced the expression of tumour necrosis factor-α, IL-ß, MCP-1, and IL-8 in heat-stable ETEC PAMPs-challenged BIE cells. In addition, reduced expressions of IL-6, MCP-1, and IL-8 were found in BIE cells stimulated with L. rhamnosus CRL2084, although its effect was significantly lower than that observed for the CRL2069 strain. The reduced levels of pro-inflammatory factors in BIE cells induced by the CRL2069 and CRL2085 strains was related to their ability of increasing the expression of TLR negative regulators. L. mucosae CRL2069 significantly improved the expression of A20-binding inhibitor of NFκ-B activation 3 (ABIN-3), interleukin-1 receptor-associated kinase M (IRAK-M) and mitogen-activated protein kinase 1 (MKP-1) while L. rhamnosus CRL2084 augmented ABIN-3 expression in BIE cells. The results of this work suggest that among the studied strains, L. mucosae CRL2069 was able to regulate TLR3-mediated innate immune response and showed a remarkable capacity to modulate TLR4-mediated inflammation in BIE cells. The CRL2069 strain induce the up-regulation of three TLR negative regulators that would influence nuclear factor kB and mitogen-activated protein kinases signalling pathways while reducing the expression of pro-inflammatory cytokines and chemokines. Therefore, L. mucosae CRL2069 is an interesting immunobiotic candidate for the protection of the bovine host against TLR-mediated intestinal inflammatory damage.

Lactocin AL705 as quorum sensing inhibitor to control Listeria monocytogenes biofilm formation.

AIMS: The control of Listeria monocytogenes biofilm formation using lactocin AL705 bacteriocin at sub-minimum inhibitory concentrations (MICs) through an antiquorum sensing strategy, was preliminarily investigated. METHODS AND RESULTS: The screening for biofilm formation of different Listeria species at 10°C allowed selecting L. monocytogenes FBUNT for its use as biofilm producer. MIC and minimum bactericidal concentration of lactocin AL705 purified extract against the pathogen was determined. Bacteriocin sub-MICs were used to evaluate biofilm reduction. Concentrations between 2·5-20 AU ml-1 of lactocin AL705 produced significant decreases in biofilm formation without affecting the growth of the pathogen after 3 days of incubation. When bacteriocin concentrations (5-20 arbitrary units per millilitre (AU ml-1 )) were investigated as quorum sensing (QS) inhibitors using Vibrio harveyi as reporter strain, a significant reduction in luminescence by lactocin AL705 (20 AU ml-1 ) was observed. Even when L. monocytogenes produced AI-2 like molecules as recognized by the reporter strain, bacteriocins did not interfere with this compound. CONCLUSION: Antilisterial lactocin AL705 used to disrupt QS through a signal molecule inactivation was able to control L. monocytogenes FBUNT biofilm formation. Other molecule(s) different from the AI-2 involved during biofilm formation could be acting as target of the bacteriocin. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of bacteriocins derived from food-grade micro-organisms as a QS inhibition represents an effective strategy to control pathogens as well as an environmentally friendly sanitation method to mitigate postprocessing food contamination.

Increasing the folate content of tuber based foods using potentially probiotic lactic acid bacteria.

It is known that certain lactic acid bacterial (LAB) strains can produce folates, a B-group vitamin that cannot be synthesized by humans and must be exogenously obtained. The aim of this study was to select folate-producing LAB and evaluate their probiotic characteristics in order to obtain a tuber-based food with elevated folate content. Several LAB strains were isolated from a traditional Andean fermented potato product tocosh and cultured in folate-free culture medium. Five folate-producing strains (29-138 ng/mL) were selected to ferment three Andean tubers (potato S. tuberosum spp. andigena, oca Oxalis tuberosa and papalisa Ullucus tuberosus). Sterile purees were inoculated and samples were collected at 0, 6 and 24 h of fermentation and after 28 days of cold storage. Cell growth, pH and total folate were determined. All selected strains were able to grow and produce folates in the substrates and two Lactobacillus sakei strains, CRL 2209 and CRL 2210, produced the highest folate concentrations (730-1484 ng/g after 24 h fermentation). These strains were selected to ferment potato substrates supplemented with amaranth (Amaranthus caudathus) and chia (Salvia hispanica) flour to increase the nutritional value. This addition increased folate synthesis in 89-95%. Furthermore, the ability to survive under simulated gastrointestinal conditions was evaluated and cell counts of the 5 strains remained above the recommended for a probiotic candidate (8.0 log CFU/mL). In conclusion, the selected LAB could be considered potentially probiotic strains and could be used to produce novel tuber based products with elevated folate concentrations. These products could also be used as novel food matrixes for the delivery of probiotic microorganisms.

Biodiversity and technological potential of lactic acid bacteria isolated from spontaneously fermented amaranth sourdough.

UNLABELLED: Spontaneous fermented sourdoughs prepared from amaranth flour were investigated for the presence of autochthonous lactic acid bacteria (LAB) predominating microbiota. The doughs were fermented with daily backslopping on a laboratory scale at 30°C for 10 days. LAB counts ranged from 2·60 to 8·54 log CFU g(-1) with a pH declined from 6·2 to 3·8 throughout fermentation. The combined use of randomly amplified polymorphic DNA (RAPD)-PCR analysis and sequence analysis of 16S rRNA was applied for LAB intraspecies differentiation and taxonomic identification, respectively. Enterococcus, Pediococcus and Lactobacillus species were present in amaranth sourdoughs (AS). After the first refreshment step, Lactobacillus plantarum dominated AS until the end of fermentation. In coincidence, when DGGE analysis was performed, the occurrence of a progressive change in bacterial communities allowed the selection of Lact. plantarum as a dominant species. Moreover, technological, functional and safety characteristics of representative RAPD-biotypes were investigated. Lact. plantarum CRL1898 was selected as a potential candidate for gluten-free amaranth sourdough starter. SIGNIFICANCE AND IMPACT OF THE STUDY: Nowadays, there is an increasing interest in ancient noncereal gluten-free (GF) crops such as amaranth, due to their reported nutritional and health benefits. However, the use of these grains is still limited to traditional foods and bread making processes that are not yet well standardized. Results on the dynamics of autochthonous lactic acid bacteria (LAB) microbiota during laboratory spontaneous amaranth sourdoughs (AS) fermentation will contribute to overcome challenges for GF-fermented products development. In addition, knowledge about LAB diversity involving Enterococcus, Pediococcus and Lactobacillus species, with Lactobacillus plantarum predominating during AS fermentation, and their technological and functional properties provides the basis for the selection of autochthonous strains as starters cultures for novel gluten-free bakery products with enhanced nutritional, sensory and/or safety quality.

Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented quinoa sourdoughs.

AIMS: To analyse lactic acid bacteria (LAB) diversity and technological-functional and safety properties of strains present during spontaneous fermented quinoa sourdoughs. METHODS AND RESULTS: Fermentation was performed by daily backslopping at 30°C for 10 days. Autochthonous LAB microbiota was monitored by a biphasic approach combining random amplified polymorphic DNA (RAPD)-PCR and rRNA gene sequencing with PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Identification and intraspecies differentiation allowed to group isolates within nine LAB species belonging to four genera. A succession of LAB species occurred during 10-days backslopping; Lactobacillus plantarum and Lactobacillus brevis were detected as dominant species in the consortium. The characterization of 15 representative LAB strains was performed based on the acidifying capacity, starch and protein hydrolysis, γ-aminobutyric acid and exopolysaccharides production, antimicrobial activity and antibiotic resistance. CONCLUSION: Strains characterization led to the selection of Lact. plantarum CRL1905 and Leuconostoc mesenteroides CRL1907 as candidates to be assayed as functional starter culture for the gluten-free (GF) quinoa fermented products. SIGNIFICANCE AND IMPACT OF THE STUDY: Results on native LAB microbiota present during quinoa sourdough fermentation will allow the selection of strains with appropriate technological properties to be used as a novel functional starter culture for GF-fermented products.

Brugada Phenocopy as a Dynamic Electrocardiographic Pattern during Acute Anterior Myocardial Infarction.

Brugada phenocopies represent some unusual clinical cases with identical characteristics to Brugada syndrome (BrS) elicited by various clinical circumstances. We report the case of a woman exhibiting "Brugada Phenocopy" during an acute anterior myocardial infarction, highlighting differential diagnosis with true BrS and discussing possible mechanisms underlying its dynamic ECG pattern.

Active polymers containing Lactobacillus curvatus CRL705 bacteriocins: effectiveness assessment in Wieners.

Bacteriocins from lactic acid bacteria have potential as natural food preservatives. In this study two active (synthetic and gluten) films were obtained by the incorporation of lactocin 705 and lactocin AL705, bacteriocins produced by Lactobacillus curvatus CRL705 with antimicrobial activity against spoilage lactic acid bacteria and Listeria. Antimicrobial film effectiveness was determined in Wieners inoculated with Lactobacillus plantarum CRL691 and Listeria innocua 7 (10(4)CFU/g) stored at 5°C during 45days. Active and control (absence of bacteriocins) packages were prepared and bacterial counts in selective media were carried out. Visual inspection and pH measurement of Wieners were also performed. Typical growth of both inoculated microorganisms was observed in control packages which reached 10(6)-10(7)CFU/g at the end of storage period. In the active packages, L. innocua 7 was effectively inhibited (2.5 log cycles reduction at day 45), while L. plantarum CRL691 was only slightly inhibited (0.5 log cycles) up to the second week of storage, then counts around 10(6)-10(7)CFU/g were reached. Changes in pH values from 6.3 to 5.8 were produced and gas formation was observed in active and control packages. The low inhibitory effectiveness against lactic acid bacteria is in correlation with the low activity observed for lactocin 705 in the presence of fat; Wieners fat content (20-30%) may adversely affect antimicrobial activity. This study supports the feasibility of using polymers activated with L. curvatus CRL705 bacteriocins to control Listeria on the surface of Wieners and highlights the importance of evaluating antimicrobial packaging systems for each particular food application.

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.

Protective action of Lactobacillus curvatus CRL705 on vacuum-packaged raw beef. Effect on sensory and structural characteristics.

Lactobacillus curvatus CRL705 was examined for its effectiveness as protective culture in the biopreservation of vacuum-packaged fresh beef stored during 60 days at 2 degrees C. For this purpose, L. curvatus CRL705, producer of lactocin 705 and lactocin AL705, was inoculated on the meat surface (10(6)cfu g(-1)). This microorganism became the dominating population throughout the storage period controlling the growth of Brochothrix thermosphacta and spoilage lactic acid bacteria naturally present on the meat. When the microstructural characteristics of the meat were evaluated using light microscopy, beef samples inoculated with the bioprotective culture showed a 10days delay for the appearance of tissue degradation signs. Sensory analysis demonstrated that beef samples treated with L. curvatus CRL705 only developed an "acid" off-flavor after 60 days of refrigerated storage, and no undesirable off-odors were found. Therefore, inoculation with this bacteriocinogenic strain would provide an additional hurdle to improve storage life of refrigerated vacuum-packaged beef without affecting its sensory and structural characteristics.

Population structure and safety aspects of Enterococcus strains isolated from artisanal dry fermented sausages produced in Argentina.

Enterococci population from Argentinean artisanal dry fermented sausage was identified and their safety aspects were evaluated. Species-specific PCR was used to distinguish between Enterococcus faecium (56%) and Enterococcus faecalis (17%). Other isolates (27%) were identified as Enterococcus durans, Enterococcus casseliflavus and Enterococcus mundtii by using 16S RNA gene sequence. RAPD analyses showed different biotypes for Ent. faecium and Ent. faecalis species. Low incidence of antibiotic resistance and high virulence traits in Ent. casseliflavus and Ent. faecalis were found; the majority of the Ent. faecium strains were shown to be free of virulence factors. The absence of virulence/resistance traits and the anti-Listeria activity of Ent. faecium isolates may be exploited to enhance natural preservation thereby guaranteeing organoleptic/safety characteristics of artisanal fermented sausages.

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.

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.

Development and characterization of an active polyethylene film containing Lactobacillus curvatus CRL705 bacteriocins.

The development and characterization of a bacteriocin-containing polyethylene-based film is described, incorporating lactocin 705 and lactocin AL705, produced by Lactobacillus curvatus CRL705, and nisin. Three different procedures to obtain lactocin 705 and AL705 solution were evaluated, with the partially purified aqueous bacteriocin solution showing the highest inhibitory activity against indicator strains (Lactobacillus plantarum CRL691 and Listeria innocua 7). Pouch contact, soaking and a contact method were compared for incorporating bacteriocins onto PE-based films. Contact between the PE film and bacteriocin solution was the most effective, resulting in a more uniform distribution of bacteriocins on the film surface and using less active solution. The minimal inhibitory concentration of bacteriocin solution was 267 AU cm(-3) (lactocin 705) and 2133 AU cm(-3) (lactocin AL705), while the minimal contact time was 1 h. When relative inhibition area for antilisterial activity of the active films was compared, those treated with L. curvatus CRL705 bacteriocins displayed higher inhibitory activity than nisin-treated films. Functional properties of active PE-films containing lactocin 705 and AL705 showed no differences compared with non-active control films. Bacteriocin-active PE-based films are shown to be highly effective in inhibiting growth of Listeria. The potential use of commercially available packaging films as bacteriocins carriers may benefit active-packaging systems.

Reduction of Escherichia coli population following treatment with bacteriocins from lactic acid bacteria and chelators.

The inhibitory activity of lactocin 705/AL705 (2133 arbitrary units per ml (AU ml(-1))), two bacteriocins produced by Lactobacillus curvatus CRL705 and nisin (1066AU ml(-1)) produced by Lactococcus lactis CRL1109 in combination with chelating agents against Escherichia coli strains in TSB medium at 21 and 6 degrees C was investigated. Treatment with EDTA (500 and 1000 mm) and Na lactate (800 mm) alone produced a variable effect depending on the strain, Na lactate being inhibitory against E. coli NCTC12900 at both assayed temperatures while EDTA (1000 mm) led to its inactivation only at 6 degrees C. Direct and deferred strategies using EDTA and Na lactate showed that the direct addition of bacteriocins and chelators was not as effective as compared to deferred treatments. When the deferred treatment effectiveness was evaluated at 6 degrees C, the use of EDTA (500 and 1000 mm) and Na lactate (800 mm) in combination with lactocin 705/AL705 demonstrated to be the most inhibitory strategy against both E. coli strains. Nevertheless, treatments with chelators and bacteriocins was highly dependent upon strain sensitivity. Permeabilization of the outer membrane of E. coli strains with EDTA and Na lactate combined with lactocin 705/AL705 showed to be valuable in controlling this foodborne bacteria at low temperatures.

Inhibition of Listeria innocua and Brochothrix thermosphacta in vacuum-packaged meat by addition of bacteriocinogenic Lactobacillus curvatus CRL705 and its bacteriocins.

AIMS: To evaluate the inhibition effectiveness of Lactobacillus curvatus CRL705 used as a bioprotective culture and of its bacteriocins, lactocin 705 and lactocin AL705, against Listeria innocua, Brochothrix thermosphacta and indigenous lactic acid bacteria (LAB) in vacuum-packaged meat stored at 2 degrees C. METHODS AND RESULTS: The live culture of Lact. curvatus CRL705 as well as synthetic lactocin 705 and purified lactocin AL705 were shown to be similarly effective in preventing the growth of B. thermosphacta and L. innocua in meat discs in contrast to control samples in which these micro-organisms grew rapidly, their numbers increasing by 3.0- and 2.1-log cycles respectively. In addition, indigenous LAB population showed a lower growth rate in the presence of lactocin 705. Bacteriocin activity was detected in the meat discs during 36 days at 2 degrees C irrespective of the biopreservation strategy applied. Changes in pH were not significantly different in meat discs treated with the protective culture when compared with control samples. CONCLUSIONS: Lactobacillus curvatus CRL705 and the produced bacteriocins, lactocin 705 and lactocin AL 705, were effective in inhibiting L. innocua and B. thermosphacta. The use of the bioprotective culture in refrigerated vacuum-packaged fresh meat would be more feasible from an economic and legal point of view. SIGNIFICANCE AND IMPACT OF THE STUDY: Establishment of biopreservation as a method to ensure the microbiological safety of vacuum-packaged fresh meat at 2 degrees C.

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.

Efficacy of nisin in combination with protective cultures against Listeria monocytogenes Scott A in tofu.

Nisin can be used as a biopreservative to control growth of Listeria monocytogenes in various minimally processed foods. Tofu is an example of a non-fermented soybean product, which may allow growth of Listeria at refrigeration temperatures and in which nisin may be applied to prevent multiplication of Listeria. The efficacy of nisin against Listeria may be compromised by the emergence of spontaneous nisin-resistant mutants. Exposure of L. monocytogenes Scott A to nisin in a culture medium or in a food product results in an initial reduction of Listeria population which is followed by regrowth of survivors to nisin during further incubation. In vitro studies using Standard I Nutrient broth showed that Enterococcus faecium BFE 900-6a and Lactobacillus sakei Lb 706-1a used as protective cultures in combination with nisin were able to suppress proliferation of Listeria cells not killed by nisin at 10 degrees C. Growth and bacteriocin production of these two strains and a third protective culture, Lactococcus lactis BFE 902 was also observed in soymilk and tofu at 10 degrees C. Inoculation studies with tofu prepared with nisin and protective cultures showed that lower amounts of nisin are required for an effective inhibition of L. monocytogenes Scott A when either E. faecium BFE 900-6a or Lc. lactis BFE 902 are used in addition. The combination of nisin with these bacteriocinogenic lactic acid bacteria (LAB) resulted in a complete suppression of listerial growth in homemade tofu stored at 10 degrees C for 1 week. Lb. sakei Lb 706-1a was less effective and did not prevent a slight increase of L. monocytogenes Scott A numbers during storage.

Combined effect of bacteriocins on the survival of various Listeria species in broth and meat system.

The antilisterial efficiency of three bacteriocins from lactic acid bacteria, lactocin 705 (produced by L. casei CRL705, 17000 AU/ml), enterocin CRL35 (produced by E. faecium CRL35, 17000 AU/ml), and nisin (2000 IU/ml), was tested in broth, individually and in combination against Listeria monocytogenes and Listeria innocua. Both Listeria species showed an initial decrease in viable counts followed by the regrowth of the survivors after 1 h in the presence of each bacteriocin. A greater antilisterial effect was observed when the bacteriocins were combined in pairs, maximal inhibition being reached when nisin was involved. When a mix of the three bacteriocins was used, no survivors were observed after 24 h of incubation. Similar results were obtained when the bacteriocin combinations were tested in a meat system, indicating that the use of more than one LAB bacteriocin in combination may be effective in preventing the spontaneous emergence of a bacteriocin-resistant Listeria population.

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.

Purification and amino acid sequence of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705.

Lactobacillus casei CRL 705, isolated from a dry fermented sausage, produces an antibacterial peptide which is active against Listeria monocytogenes. Previous studies have shown that this compound is potentially useful to control food-borne pathogens in ground meat. In view of the potential application of this antimicrobial substance in food fermentation, a detailed biochemical analysis of this peptide is required. In this work, the purification and amino acid sequence of this bacteriocin is presented. The adsorption-desorption pH-dependent property of lactocin 705 was exploited for purification. The active extract was further subjected to RP-HPLC and SDS-PAGE. The active antimicrobial band was electroeluted from an SDS-PAGE gel and its amino acid sequence determined. Lactocin 705 had an estimated molecular weight of 3357.80 and an isoelectric point of 10.03. The peptide contains a high ratio of glycine residues and does not show any modified amino acids, like lanthionine or beta-methyllanthionine. The sequence was unique when compared to several databases.