Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya) — From isolation to application: Characterization of a bacteriocin

Strain ST16PA, isolated from papaya was identified as Lactobacillus plantarum based on biochemical tests, PCR with species-specific primers and 16S rDNA sequencing. L. plantarum ST16PA produces a 6.5 kDa bacteriocin, active against different species from genera Enterobacter, Enterococcus, Lactobacillus, Pseudomonas, Streptococcus and Staphylococcus and different serotypes of Listeria spp. The peptide is inactivated by proteolytic enzymes, but not when treated with ??-amylase, catalase, lipase, Triton X-100, SDS, Tween 20, Tween 80, urea, NaCl and EDTA. However, presence of 1% Triton X-114 deactivates the bacteriocin. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 °C for 120 min or 121 °C for 20 min. The mode of activity against Lactobacillus sakei ATCC 15521, Enterococcus faecalis ATCC 19443 and Listeria innocua 2030C was bactericidal, resulting in cell lysis and enzyme-leakage. No significant differences in cell growth and bacteriocin production were observed when strain ST16Pa was cultured in MRS broth at 26 °C and 30 °C for 24 h (25 600 AU/ml). However, even though strain ST16PA grows well in MRS broth at 15 °C and 37 °C, a reduction of bacteriocin production was observed (400 AU/ml and 1600 AU/ml, respectively). In addition, effect of MRS medium components, different initial pH and additions of glycerol or vitamins to the media on bacteriocin ST16Pa production was studied.Peptide ST16PA adsorbs (400 AU/ml) to producer cells. However, bacteriocin ST16Pa was adsorbed at 50% to cells of L. innocua 2030C and at 75% to L. sakei ATCC 15521 and E. faecalis ATCC 19433 when experiments were conducted at 30 °C and pH 6.5. Adsorption of bacteriocin ST16Pa to target cells at different temperatures, pH and in presence of potassium sorbate, sodium nitrate, sodium chloride, ascorbic acid, Tween 80 and Tween 20 were also studied. To the best of our knowledge, this is the first report on detection of L. plantarum in papaya.     

Characterization of bacteriocins produced by two strains of Lactobacillus plantarum isolated from Beloura and Chouriço, traditional pork products from Portugal

Bacteriocins bacST202Ch and bacST216Ch, produced by Lactobacillus plantarum strains isolated from Beloura and Chouriço, respectively, inhibited the growth of a number of Gram-positive and Gram-negative meat spoilage bacteria. According to trycine–SDS–PAGE, bacST202Ch and bacST216Ch are approximately 3.5 and 10.0 kDa in size, respectively. Maximal activity of bacST202Ch (25,600 AU/ml) was recorded after 27 h of and bacST216Ch (102,400 AU/ml) after 22 h of growth. The mode of activity, as determined against Enterococcus faecium HKLHS, is bactericidal. Both peptides adsorb to the surface of the producer cells, but at very low concentrations. Both peptides remained active after 120 min at 100 ^oC and after 2 h of incubation at pH 2.0–12.0. Treatment for 120 min at 121 ^oC did not affect bacST216Ch activity. Activity of bacST202Ch and bacST216Ch was not affected by 1% Triton X-100, Tween 80, Tween 20, SDS, NaCl, urea and EDTA. Bacteriocin ST216Ch was deactivated in the presence of 1% Triton X-114. The nucleotide sequence of a 1044 bp DNA fragment amplified from L. plantarum ST202Ch is identical to the structural gene encoding pediocin PA-1, suggesting that the two bacteriocins are identical. Based on the broad spectrum of antibacterial activity, strains ST202Ch and ST216Ch may be used as starter cultures in the fermentation of meat products.     

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 °C and 37 °C, reaching a maximum production of 1.0 × 10⁹ AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.     

A bacteriocin-like substance produced from Lactobacillus pentosus 39 is a natural antagonist for the control of Aeromonas hydrophila and Listeria monocytogenes in fresh salmon fillets

We studied the ability of Lactobacillus pentosus 39, a BLS (Bacteriocin-like substance)-producing strain, to control the growth of Aeromonas hydrophila ATCC 14715 and Listeria monocytogenes ATCC 19117 artificially added to fresh salmon fillets at refrigeration temperatures and under simulated cold-chain break conditions.At refrigeration temperatures, Lb. pentosus 39 protective culture and its putative bacteriocin significantly reduced A. hydrophila counts compared with the control (2.1 and 1.4 log CFU/g reductions, respectively). Similar behaviour was observed for L. monocytogenes (3.6 and 1.3 log CFU/g reductions, respectively).Under simulated cold-chain break conditions, an increase in temperature (30°C for 12h) produced an evident increase in the development of A. hydrophila, L. monocytogenes, but also of Lb. pentosus 39, with a consequent increase in BLS production. This condition resulted in a greater reduction of both pathogens compared with samples stored at 4°C throughout the experiment (2.8 log CFU/g reduction for A. hydrophila, 5.8 log CFU/g reduction for L. monocytogenes). In samples treated with the putative bacteriocin alone, a less marked decrease was observed.Our study demonstrates the capability of Lb. pentosus 39 to control the growth of psychrotrophic bacteria in an experimental seafood model system. A similar biopreservation technology could provide more prolonged shelf-life during storage of ready-to-eat seafood, ensuring safety, even under extreme conditions.     

Control of Growth of L. monocytogenes in Fresh Salmon using Microgard and Nisin

Pathogens like Listeria monocytogenes in fresh, or even in cold smoked salmon, have become a major public health concern for the salmon processing industry and government agencies. The effect of bacteriocin solutions (Microgard^™ and Nisin) on reducing total microbial counts, inhibiting Listeria monocytogenes, and prolonging the shelf-life was evaluated. Listeria monocytogenes was inoculated onto chilled and on frozen and thawed salmon samples. The combination of Nisin and Microgard reduced the total aerobic bacteria populations of fresh chilled salmon by 2 log (P<0.05) and increased its shelf-life, at 6 °C, by 3-4 d, as compared with the control. The above bacteriocin combination also reduced the growth of inoculated Listeria monocytogenes in frozen-thawed salmon and increased its shelf-life from 5 to 10 d at 6 °C. The bacteriocin treatment did not affect surface pH values or color of the fish.     

Physicochemical factors differentially affect the biomass and bacteriocin production by bovine Enterococcus mundtii CRL1656

Bovine Enterococcus mundtii CRL1656 (Centro de Referencia para Lactobacilos Culture Collection) produces an anti-Listeria and anti-Streptococcus dysgalactiae bacteriocin identified as mundticin CRL1656. The strain and its bacteriocin are candidates to be included in a beneficial product to prevent bovine mastitis as an alternative to antimicrobial agents. To optimize the production of biomass and mundticin CRL1656 by E. mundtii CRL1656, a complete 3 × 2⁴ factorial design was applied. The effect of culture medium, initial pH, inoculum size, incubation temperature, and agitation conditions on biomass and bacteriocin production was evaluated simultaneously. Growth parameters were determined using the modified Gompertz model. A nonlinear model was used to estimate the effects of the variables on growth parameters. Bacteriocin production was analyzed using a linear mixed model. Optimal biomass and mundticin CRL1656 production by E. mundtii CRL1656 were obtained in different conditions. Maximal growth was recorded in autolyzed yeast, peptone, tryptone, Tween 80, and glucose or M17 broths, pH 6.5, 5.0% inoculum, 30°C, with agitation. However, bacteriocin titers were higher in autolyzed yeast, peptone, tryptone, Tween 80, and glucose or de Man-Rogosa-Sharpe (MRS) broths, pH 6.5, 30°C, both with or without agitation. Knowledge of the optimum conditions for growth and bacteriocin production of E. mundtii CRL1656 will allow the obtainment of high levels of biomass and mundticin CRL1656 as bioingredients of potential products to prevent bovine mastitis.     

Purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides E131

Leuconostoc mesenteroides E131, isolated from Greek traditional fermented sausage, prepared without the addition of starters, produces a bacteriocin which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, and reverse-phase chromatography. Bacteriocin is active at pH values between 4.0 and 9.0 and retains activity after incubation for 1 h at 100 °C. Proteolytic enzymes inactivated the bacteriocin after 1 h of incubation, while renin resulted in full inactivation only after 24 h. Lipase resulted in full inactivation after 4 h. Applying molecular methods, it was determined that the bacteriocin produced, named as mesenterocin E131, was identical to mesenterocin Y105 and was expressed during the exponential growth phase.     

High-salt brines compromise autoinducer-mediated bacteriocinogenic Lactobacillus plantarum survival in Spanish-style green olive fermentations

The effect of NaCl on plantaricin production by five Lactobacillus plantarum strains was investigated. Plantaricin production in these strains was found to be regulated by three-component regulatory systems ruled by two different autoinducer peptides (AIPs), either PLNC8IF or Plantaricin A. Bacteriocin activity exhibited by these strains came to a halt in liquid medium containing NaCl concentrations of or above 2%. In contrast, bacteriocin activity was still observed when the producing strains were growing on solid medium containing up to 4% NaCl. Bacteriocin activity in liquid medium containing up to 2% NaCl could be restored by coculturing the producing strains with a selected plantaricin-production inducing strain of Lactococcus lactis. Growth of these bacteriocinogenic L. plantarum strains was monitored in traditional Spanish-style green olive fermentations. Survival of these strains could not be enhanced when provided with a range of plantaricin-production inducing mechanisms previously described, such as constitutive AIP production or coinoculation with a specific bacteriocin-production inducing strain of L. lactis. Our results suggest that it is advisable the use of constitutive bacteriocin producers, or at least non-AIP-dependant ones, as starters for olive fermentations due to the intrinsic physical characteristics of this food fermentation, especially the high salt concentration of the brines currently used.     

Growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham under refrigerated and temperature abuse conditions

This study examined the growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham at refrigerated and abuse temperatures. A five-strain mixture of L. monocytogenes and a native microflora, consisting of Brochothrix spp., isolated from cooked meat were inoculated alone (monocultured) or co-inoculated (co-cultured) onto cooked ham slices. The growth characteristics, lag phase duration (LPD, h), growth rate (GR, log₁₀ cfu/h), and maximum population density (MPD, log₁₀ cfu/g), of L. monocytogenes and the native microflora in vacuum-packed ham slices stored at 4, 6, 8, 10, and 12 °C for up to 5 weeks were determined. At 4-12 °C, the LPDs of co-cultured L. monocytogenes were not significantly different from those of monocultured L. monocytogenes in ham, indicating the LPDs of L. monocytogenes at 4-12 °C were not influenced by the presence of the native microflora. At 4-8 °C, the GRs of co-cultured L. monocytogenes (0.0114-0.0130 log₁₀ cfu/h) were statistically but marginally lower than those of monocultured L. monocytogenes (0.0132-0.0145 log₁₀ cfu/h), indicating the GRs of L. monocytogenes at 4-8 °C were reduced by the presence of the native microflora. The GRs of L. monocytogenes were reduced by 8-7% with the presence of the native microflora at 4-8 °C, whereas there was less influence of the native microflora on the GRs of L. monocytogenes at 10 and 12 °C. The MPDs of L. monocytogenes at 4-8 °C were also reduced by the presence of the native microflora. Data from this study provide additional information regarding the growth suppression of L. monocytogenes by the native microflora for assessing the survival and growth of L. monocytogenes in ready-to-eat meat products.     

Characterization of a 3944 Da bacteriocin, produced by Enterococcus mundtii ST15, with activity against Gram-positive and Gram-negative bacteria

Strain ST15, isolated from soy beans, and identified as Enterococcus mundtii, produces a 3944 Da bacteriocin that inhibits the growth of Lactobacillus sakei, Enterococcus faecalis, Bacillus cereus, Propionibacterium sp., Clostridium tyrobutyricum, Acinetobacter baumanii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus caprinus. Bacteriocin ST15 is inactivated by proteinase K, pronase, pepsin, protease and Triton X-114, but not when treated with catalase, alpha-amylase, Triton X-100, SDS, Tween 20, Tween 80, urea and EDTA. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 degrees C for 90 min. Activity was, however, lost after treatment at 121 degrees C for 20 min. The mode of activity is bactericidal. The highest level of activity (51200 AU ml(-1)) was recorded when cells were grown in MRS broth, pH 6.5. Bacteriocin ST15 differs from other broad-spectrum bacteriocins described for Enterococcus spp. by being active against Gram-negative bacteria and by being smaller.     

Lack of growth of Listeria monocytogenes and Staphylococcus aureus in temperature abuse of E-beam treated ready-to-eat (RTE) cooked ham

The behaviour of Listeria monocytogenes and Staphylococcus aureus in vacuum-packed cooked ham slices treated with an electron beam and stored at 4, 7 and 10 °C was investigated. Cooked ham slices were inoculated with L. monocytogenes and S. aureus and electron beam treated at 2 and 3 kGy. After treatment, a long temperature-dependent death phase was observed, followed by growth at a slower rate than in untreated samples. Assuming a hypothetical load of 10 cells/g or cm² of L. monocytogenes and S. aureus as an original contamination in an industrial situation, an E-beam treatment of vacuum-packed cooked ham slices at 2 kGy guarantees the microbiological safety of the product along its shelf life, even if a noticeable temperature (10 °C) abuse occur during its storage period. Likewise, the E-beam treatment gave rise to a substantial increase of the RTE cooked ham shelf life off-sensory features associated to the spoilage only were detected in non-treated samples (controls) after 8 and 18 days of storage at 10 °C and 7 °C, respectively.     

Use of Carnobacterium maltaromaticum cultures and hydroalcoholic extract of Lippia sidoides Cham. against Listeria monocytogenes in fish model systems

Minimally processed refrigerated ready-to-eat fishes may offer health risk of severe infection to susceptible individuals due to contamination by the psychrotolerant bacterium L. monocytogenes. In this work, inhibition of L. monocytogenes by a plant extract and lactic acid bacteria (LAB) was studied in model fish systems kept at 5 °C for 35 days. For that, fillets of tropical fish “surubim” (Pseudoplatystoma sp.) and hydroalcoholic extract of the plant Lippia sidoides Cham. (“alecrim pimenta”) were used. Fish peptone broth (FPB), “surubim” broth and “surubim” homogenate were inoculated with combinations of L. monocytogenes and bacteriocin-producing Carnobacterium maltaromaticum (C2 and A9b⁺) and non bacteriocin-producing C. maltaromaticum (A9b^-), in the presence or absence of extract of “alecrim pimenta” (EAP). In all model systems, monocultures of L. monocytogenes and carnobacteria reached final populations ≥ 10⁸ CFU/ml after 35 days, except for L. monocytogenes in “surubim” homogenate (10⁴ CFU/ml). In FPB, EAP alone and combined with cultures of LAB inhibited L. monocytogenes but carnobacteria without EAP were only weakly antilisterial. In “surubim” broth, EAP alone did not prevent L. monocytogenes growth but cultures of carnobacteria combined or not with EAP inhibited L. monocytogenes, with more pronounced effect being observed for C. maltaromaticum C2, which produced bacteriocin. In “surubim” homogenate, EAP alone and combined with cultures of C. maltaromaticum A9b⁻ and A9b⁺ were strongly inhibitory to L. monocytogenes, while C. maltaromaticum C2 with EAP caused transient inhibition of L. monocytogenes. No significant inhibition of L. monocytogenes was observed for carnobacteria in “surubim” homogenate without EAP. In conclusion, it was observed that the use of EAP and cultures of carnobacteria have potential to inhibit L. monocytogenes in fish systems and the applications should be carefully studied, considering the influence of food matrix.     

Coculture with specific bacteria enhances survival of Lactobacillus plantarum NC8, an autoinducer-regulated bacteriocin producer,in olive fermentations

Bacteriocin production in Lactobacillus plantarum NC8 is activated by coculture with specific bacteriocin production-inducing bacterial strains. The system is further regulated by a three-component regulatory system involving a specific autoinducer peptide (PLNC8IF). We have used L. plantarum NC8 as a starter culture in Spanish-style green olive fermentations and examined the influence of coculturing in its survival. We found that L. plantarum NC8 greatly enhanced its growth and survival in the olive fermentations when coinoculated with two specific bacteriocin-production inducing strains, i.e. Enterococcus faecium 6T1a-20 and Pediococcus pentosaceus FBB63, when compared to singly-inoculated fermentations. In addition, a constitutive bacteriocin-producer NC8-derivative strain was used as a control in the olive fermentations and showed also better viability than the parental NC8 strain. Our results suggest the involvement of bacteriocin production in the viability enhancement found in both cases. We postulate that the presence of specific bacteria is recognized by L. plantarum NC8 as an environmental stimulus to switch a specific adaptive response on, most probably involving bacteriocin production. The design of novel bacteriocin-producing starter cultures for food fermentations should consider their constitutive versus regulated character.     

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 °C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 °C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese.     

Fate of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella on fresh-cut celery

Illnesses from Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella have been associated with the consumption of numerous produce items. Little is known about the effect of consumer handling practices on the fate of these pathogens on celery. The objective of this study was to determine pathogen behavior at different temperatures under different storage conditions. Commercial fresh-cut celery was inoculated at ca. 3 log CFU/g onto either freshly cut or outer uncut surfaces and stored in either sealed polyethylene bags or closed containers. Samples were enumerated following storage for 0, 1, 3, 5, and 7 days when held at 4 °C or 12 °C, and after 0, 8, and 17 h, and 1, and 2 days when held at 22 °C. At 4 °C, all populations declined by 0.5–1.0 log CFU/g over 7 days. At 12 °C, E. coli O157:H7 and Salmonella populations did not change, while L. monocytogenes populations increased by ca. 0.5 log CFU/g over 7 days. At 22 °C, E. coli O157:H7, Salmonella, and L. monocytogenes populations increased by ca. 1, 2, or 0.3 log CFU/g, respectively, with the majority of growth occurring during the first 17 h. On occasion, populations on cut surfaces were significantly higher than those on uncut surfaces. Results indicate that populations are reduced under refrigeration, but survive and may grow at elevated temperatures.     

Effect of polythene film activated with enterocin EJ97 in combination with EDTA against Bacillus coagulans

Polythene films coated with the enterococcal bacteriocin enterocin EJ97 alone or in combination with EDTA were tested against Bacillus coagulans CECT 12. Bacteriocin activity was clearly enhanced by EDTA, as shown by viable staining and epifluorescence microscopy observation of treated cells. Activated films were tested in liquids from canned corn and canned peas inoculated with B. coagulans cell suspensions and stored at 4 °C and 20 °C for 24 h. The bacteriocin alone showed highest activity in samples stored at 4 °C, while the maximum performance of EDTA was observed at 20 °C. Films activated with a combination of both antimicrobials showed highest bactericidal activity at 4 °C. In liquid from canned corn and peas stored at 4 °C, the combined treatment reduced the concentrations of viable cells progressively over incubation time. Viable staining revealed an increase in the percentage of dead cells at 20 °C, avoiding proliferation of the bacilli. The bactericidal effect of the combined antimicrobial agents was higher in the liquid of canned peas than that of canned corn. The combined use of viable staining and classical viable cell counts allowed a better estimation of cell damage caused by the antimicrobial treatments.     

Growth,survival, and peptidolytic activity of Lactobacillus plantarum I91 in a hard-cheese model

In this work, we studied the growth, survival, and peptidolytic activity of Lactobacillus plantarum I91 in a hard-cheese model consisting of a sterile extract of Reggianito cheese. To assess the influence of the primary starter and initial proteolysis level on these parameters, we prepared the extracts with cheeses that were produced using 2 different starter strains of Lactobacillus helveticus 138 or 209 (Lh138 or Lh209) at 3 ripening times: 3, 90, and 180 d. The experimental extracts were inoculated with Lb. plantarum I91; the control extracts were not inoculated and the blank extracts were heat-treated to inactivate enzymes and were not inoculated. All extracts were incubated at 34°C for 21 d, and then the pH, microbiological counts, and proteolysis profiles were determined. The basal proteolysis profiles in the extracts of young cheeses made with either strain tested were similar, but many differences between the proteolysis profiles of the extracts of the Lh138 and Lh209 cheeses were found when riper cheeses were used. The pH values in the blank and control extracts did not change, and no microbial growth was detected. In contrast, the pH value in experimental extracts decreased, and this decrease was more pronounced in extracts obtained from either of the young cheeses and from the Lh209 cheese at any stage of ripening. Lactobacillus plantarum I91 grew up to 8 log during the first days of incubation in all of the extracts, but then the number of viable cells decreased, the extent of which depended on the starter strain and the age of the cheese used for the extract. The decrease in the counts of Lb. plantarum I91 was observed mainly in the extracts in which the pH had diminished the most. In addition, the extracts that best supported the viability of Lb. plantarum I91 during incubation had the highest free amino acids content. The effect of Lb. plantarum I91 on the proteolysis profile of the extracts was marginal. Significant changes in the content of free amino acids suggested that the catabolism of free amino acids by Lb. plantarum I91 prevailed in a weakly proteolyzed medium, whereas the release of amino acids due to peptidolysis overcame their catabolism in a medium with high levels of free amino acids. Lactobacillus plantarum I91 was able to use energy sources other than lactose to support its growth because equivalent numbers of cells were observed in extracts containing residual amounts of lactose and in lactose-depleted extracts. The contribution of Lb. plantarum I91 to hard-cooked cheese peptidolysis was negligible compared with that of the starter strain; however, its ability to transform amino acids is a promising feature of this strain.     

Induction of bacteriocin production by coculture is widespread among plantaricin-producing Lactobacillus plantarum strains with different regulatory operons

We describe the bacteriocin-production phenotype in a group of eight singular bacteriocinogenic Lactobacillus plantarum strains with three distinct genotypes regarding the plantaricin locus. Genotyping of these strains revealed the existence of two different plantaricin-production regulatory operons, plNC8-plNC8HK-plnD or plnABCD, involving three-component systems controlled each of them by a specific autoinducer peptide (AIP), i.e. PLNC8IF or PlnA. While all of the strains produced antimicrobial activity when growing on solid medium, most of them halted this production when cultured in broth, thus reflecting the functionality of regulatory mechanisms. Antimicrobial activity in broth cultures was re-established or enhanced when the specific AIP was added to the culture or by coculturing with specific bacterial strains. The latter trait appeared to be widespread in bacteriocinogenic L. plantarum strains independently of the regulatory system used to regulate bacteriocin production or the specific bacteriocins produced. The induction spectrum through coculture, i.e. the pattern of bacterial strains able to induce bacteriocin production, was characteristic of each individual L. plantarum strain. Also, the ability of some bacteria to induce bacteriocin production in L. plantarum by coculture appeared to be strain specific. The fact that induction of bacteriocin production by coculturing appeared to be a common feature in L. plantarum can be exploited accordingly to enhance the viability of this species in food and feed fermentations, as well as to contribute to probiotic functionality when colonising the gastrointestinal tract.     

Heat resistance of Listeria species to liquid whole egg ultrapasteurization treatment

The lethality of ultrapasteurization treatments (70 °C/1.5 min.) applied at constant temperature (isothermal condition) and at a constantly raising temperature of 2 °C/min (non-isothermal condition) in liquid whole egg (LWE) against two strains of Listeria monocytogenes (STCC 5672 and 4032) and one of Listeria innocua has been investigated. Isothermal survival curves up to 71 °C were obtained, which followed first-order inactivation kinetics. The obtained D_t values indicated that L. innocua was significantly (p < 0.05) more heat resistant than L. monocytogenes strains. Non-significant (p > 0.05) differences were observed among z values (12.4 ± 0.4 °C, 13.1 ± 0.4 °C and 12.2 ± 0.7 °C for L. innocua and L. monocytogenes 5672 and 4032, respectively). Based on obtained D_t and z values, isothermal ultrapasteurization treatment (70 °C/1.5 min.) would provide 3.5-, 5.0-, and 6.5-Log₁₀ cycles of L. innocua and L. monocytogenes 5672 and 4032, respectively. Non-isothermal heating lag phase increased the thermotolerance of Listeria species in LWE. The simulated industrial pasteurization treatment for LWE (heating-up phase from 25 to 70 °C followed by 1.5 min. at 70 °C) would attain 5-Log₁₀ reductions of L. monocytogenes 5672 and 4032, and 3.7-Log₁₀ reductions of L. innocua. Therefore, the safety level of industrial ultrapasteurization concerning L. monocytogenes could be lower than that estimated with data obtained under isothermal conditions.     

Isolation and characterization of a nisin-like bacteriocin produced by a Lactococcus lactis strain isolated from charqui,a Brazilian fermented,salted and dried meat product

A Lactococcus lactis subsp. lactis strain (L. lactis 69) capable to produce a heat-stable bacteriocin was isolated from charqui, a Brazilian fermented, salted and sun-dried meat product. The bacteriocin inhibited, in vitro, Listeria monocytogenes, Staphylococcus aureus, several lactic acid bacteria isolated from foods and spoilage halotolerant bacteria isolated from charqui. The activity of the bacteriocin was not affected by pH (2.0–10.0), heating (100 °C), and chemical agents (1% w/v). Treatment of growing cells of L. monocytogenes ScottA with the cell-free supernatant of L. lactis 69 resulted in complete cell inactivation. L. lactis 69 harbored the gene for the production of a nisin-like bacteriocin, and the amino acid sequence of the active peptide was identical to sequences previously described for nisin Z. However, differences were observed regarding the leader peptide. Besides, the isolate was able to survive and produce bacteriocins in culture medium with NaCl content up to 20%, evidencing a potential application as an additional hurdle in the preservation of charqui.