Non starter lactic acid bacteria biofilms: A means to control the growth of Listeria monocytogenes in soft cheese

The possibility to consider non starter lactic acid bacteria (NSLAB) biofilms as a means to control the growth of Listeria monocytogenes in soft cheeses was evaluated. In particular, the aptitude to form biofilm of four NSLAB strains (Lactobacillus plantarum DSM1055, Lactobacillus casei DSM20011, Lactobacillus curvatus DSM20019 and Lactobacillus paracasei DSM20207) was investigated. All tested strains were able to form biofilm on stainless steel and the highest quantities were produced when NSLAB were present simultaneously (about 6 Log CFU cm^?2). Then, experimental cheeses were made in presence of chips containing 7-days NSLAB biofilms and inoculated with L. monocytogenes (about 2 Log CFU g^?1). Results demonstrated that NSLAB biofilms can be considered a useful means to delay the growth of L. monocytogenes in soft cheeses: the maximum cell load attained at the stationary phase was about 6 Log CFU g^?1 in experimental cheeses against about 7 Log CFU g^?1 observed in control samples.     

Effects of phenyllactic acid as sanitizing agent for inactivation of Listeria monocytogenes biofilms

3-Phenyllactic acid (PLA) has been reported as an antimicrobial compound with broad-spectrum activity, and it can be produced by food-grade microorganisms, including a wide range of lactic acid bacteria species. In this study, the efficacy of PLA to inactivate Listeria monocytogenes planktonic cells and biofilms was determined and compared with the killing effects of lactic acid (LA), and levulinic acid (LVA) with sodium dodecyl sulfate (SDS). L. monocytogenes biofilms of different maturities, i.e., 37 °C for 3 and 7 d and 15 °C for 4 and 7 d, were produced on 24-well flat-bottom polystyrene plates and treated with PLA (0.25%–3%), LA (1% and 3%), and 3% LVA plus 2% SDS for 5, 10, 30, 60 min, respectively. The results of pure culture assays revealed that 1% PLA reduced the population of L. monocytogenes by 7 log CFU/ml within 1 min. The biofilms assays revealed that L. monocytogenes biofilms could be inactivated to different degrees by the sanitizer treatments. The killing effect of PLA treatment was increased as exposure time and PLA concentrations were increased. The sanitizers of 3% PLA and 1% PLA effectively inactivated the early mature biofilm after a 5-min treatment, whereas 3% PLA was better than all the other sanitizers, including 1% PLA, 3% LA, 3% LVA and 2% SDS for inactivation of the late mature biofilm after a 5-min treatment. Confocal laser scanning microscopy analysis revealed that bacterial cell damages in the biofilm were enhanced as the PLA concentrations and exposure times were increased. These results suggested that PLA was effective in inactivating L. monocytogenes and its biofilm, even for the late mature biofilm.     

Antibacterial activity of lactic acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-scale facility: 2—Behaviour of pathogenic and spoilage bacteria in dual species biofilms including a bacteriocin-like-producing lactic acid bacteria

The survival of Listeria innocua ATCC 33090, Staphylococcus aureus E1S-5 and/or Hafnia alvei E1E-25 in dual species biofilms with bacteriocin-like producing lactic acid bacteria (5 Vagococcus carniphilus, 3 Enterococcus faecium, 1 Lactobacillus sakei and 1 Enterococcus sp.) was investigated. The aim was to select strains able to repress the growth of undesirable bacteria in biofilms, i.e., the real mode of bacterial attachment.Two E. faecium and 3 V. carniphilus species were highly antagonistic to L. innocua, S. aureus and H. alvei repressing their growth by reduction levels able to reach 2, 2.7 and 2.4 log₁₀ cfu/ml compared to the positive control made of sole the target microorganism. Furthermore, planktonic cells were more sensitive to the bacteriocin-like substances than sessile ones.First results suggest the possibility of selecting bio-preservatives among the endogenous house flora of the studied small-scale facility, that could be implemented on the processing surfaces to repress the growth of undesirable microorganisms.     

Biomass production and small-scale testing of freeze-dried lactic acid bacteria starter strains for cassava fermentations

Based on their predominance in Gari fermentations, as well as suitable technological properties, Lactobacillus plantarum, Lactobacillus fermentum, Weissella paramesenteroides and Leuconostoc mesenteroides strains were investigated for their suitability for development as starter strains for this African traditional fermented cassava product. The strains were grown in optimized growth media in 2 L fermenters, harvested and freeze dried, and then tested in lab-scale cassava mash fermentation trials for their ability to ferment the cassava. The strains performed well and rapidly increased the titratable acidity from 1.1 to 1.3% at 24 h to 1.3–1.6% at 48 h. The benefit of including starter cultures was that it lowered the pH of the product much faster and to lower levels than in the uninoculated control fermentation. The results furthermore indicated that especially the L. plantarum-group strains could be produced as starter strains at low cost. Overall, the results of this study showed that starter strains could be easily and economically produced, and thus represent a feasible possibility for further development for application in the field.     

Lactic acid bacteria in an alginate film inhibit Listeria monocytogenes growth on smoked salmon

Antimicrobial packaging with lactic acid bacteria incorporated into the film matrix is a novel approach for controlling the growth of food-borne pathogens in ready-to-eat food. The overall objective of this study was to assess the effect of two strains of lactic acid bacteria (LAB) and nisin trapped in an alginate matrix, on Listeria monocytogenes growth on vacuum packed cold-smoked salmon. A film was formulated containing two LAB strains and nisin (100 IU/mL). LAB viability and bacteriocin like substance production (BLS) were assessed using the plate antagonism technique. To check the film antagonistic activity, pieces of salmon (4.0 × 4.0 cm²), inoculated with L. monocytogenes at a final concentration of 10⁴ CFU/cm², were covered with film containing both LAB strains plus nisin and stored at 4 °C. L. monocytogenes colonies on OXA agar were counted after 0, 7, 14, 21 and 28 days to evaluate pathogen inhibition. All treatments led to effective diffusion of the BLS that inhibited L. monocytogenes for 20 days after film preparation, with inhibition zones of 5.7 cm² for film coupons of 8 mm in diameter. After 28 days, salmon pieces covered with the film without inhibitors showed an increase of 2.4 log cycles in L. monocytogenes growth. In contrast, films with either LAB strain or a combination of both strains and nisin had a bacteriostatic effect on the pathogen over a period of 28 days, which exceeds the industrial standard shelf life for smoked salmon. The results demonstrate that these films inhibit L. monocytogenes growth on salmon during refrigerated storage.     

Design of biopolymeric matrices entrapping bioprotective lactic acid bacteria to control Listeria monocytogenes growth: Comparison of alginate and alginate-caseinate matrices entrapping Lactococcus lactis subsp. lactis cells

In order to design biopolymeric matrices entrapping bioprotective lactic acid bacteria (LAB) to control undesirable microorganisms growth in foods, the performances of alginate and alginate-caseinate (an aqueous two-phase system) matrices entrapping Lactococcus lactis subsp. lactis LAB3 cells were compared. Since efficient matrices should preserve the culturability and the antimicrobial activity of entrapped LAB3 cells for prolonged periods, they were both monitored for 12 days storage at 30 °C. Maximal cell density (∼10⁹ CFU mL⁻¹) was reached after 24 h whatever the matrix type. Then, the LAB3 cells population decreased: 10⁷ and 10⁶ CFU mL⁻¹ were enumerated after 12 days in alginate-caseinate matrix and in alginate matrix, respectively. The anti-listerial activity assayed by an agar well method of LAB3 cells entrapped in alginate-caseinate matrices was also higher. LAB3 cells anti-listerial activity has been shown to be due to antimicrobial metabolites: hydrolysis by proteolytic enzymes of LAB3 cell-free culture supernatants (CFS) demonstrated the proteinaceous nature of at least a part of these metabolites. The higher antimicrobial activity of alginate-caseinate matrices might both result from the higher survival rate of bacterial cells and from a higher release of antimicrobial metabolites. To test this latter hypothesis, LAB3 CFS were incorporated in alginate and alginate-caseinate matrices and tested against Listeria innocua and Listeria monocytogenes. The anti-listerial activity of LAB3 CFS was higher in alginate-caseinate matrices indicating a better release of antimicrobial agents from this matrix. Alginate-caseinate matrices are thus better suited for LAB3 cells incorporation both for their survival and to promote the release of their antimicrobial metabolites.     

Effects of lactic acid and hot water treatments on Salmonella Typhimurium and Listeria monocytogenes on beef

The present study is designed to determine alone and combined effects of lactic acid (LA) and hot water (HW) treatments. Hot water and different concentrations of lactic acid were evaluated for their reduction effects on Salmonella Typhimurium and Listeria monocytogenes on contaminated beef during refrigerated storage at 4 °C. The reductions were 0.05–1.19 and 0.09–1.14 log for S. Typhimurium and L. monocytogenes on day 0 respectively, while it was 0.43–1.78 and 1.69–3.84 log respectively on day 5 of storage. Results of this study suggest that LA and HW treatments can be used to reduce S. Typhimurium and L. monocytogenes which provide an additional measure of safety in production line.     

Inhibition of Listeria monocytogenes by resident biofilms present on wooden shelves used for cheese ripening

The purpose of the present study was to characterize the development of Listeria monocytogenes on wooden shelves used for cheese ripening. The fate of two L. monocytogenes strains was analysed over time as a function of the presence of a native biofilm, the farmhouse origin of cheeses, and the wooden shelves properties. In presence of a native microbial flora on the shelves, deposited populations of L. monocytogenes remained stable or even decreased by up to 2 log₁₀(CFU/cm²) after 12 days of incubation at 15 °C in all tested conditions. By contrast, L. monocytogenes populations increased by up to 4 log₁₀(CFU/cm²) when the resident biofilm was thermally inactivated, suggesting a microbial origin of the observed inhibitory effect. All together, our results suggest that the biocontrol of pathogens multiplication on wooden shelves by resident biofilms should be considered for the microbiological safety of traditional ripened cheeses.     

Lactic acid bacteria biodiversity in Italian marinated seafood salad and their interactions on the growth of Listeria monocytogenes

The aim of this research was to study the biodiversity of lactic acid bacteria (LAB) in marinated seafood salad (pH 5.0) and their interaction on the growth of Listeria monocytogenes. LAB were highly present in the samples considered in this study, reaching values of 8.0 log cfu/g at the end of product’s shelf-life. A high biodiversity in terms of LAB species and strains was detected by means of RAPD–PCR within the 171 bacterial isolates collected. Among them Lactobacillus curvatus, Lactobacillus sanfranciscensis and Enterococcus were present in all the salad batches considered. Three challenge tests against L. monocytogenes were carried out in order to assess the growth of this pathogen in the presence of dominant populations of LAB. L. monocytogenes tended to decrease in time, suggesting that a stable concentration of LAB inhibits the development of this pathogenic micro-organism.     

Heterologous production of pediocin for the control of Listeria monocytogenes in dairy foods

Pediocin is an antimicrobial peptide naturally produced by Pediococci with the potential to serve as a food-grade preservative for controlling Listeria contamination. The use of Pediococci in dairy products is limited due to their inability to ferment lactose, thus lactic acid bacteria (LAB) have been considered as potential hosts for the heterologous production of pediocin. In this study the four gene operon (papA-D) required for pediocin production was cloned on the nisin-inducible expression vector pMSP3535H3. The resulting vector, pRSNPed2, was electrotransformed into Streptococcus thermophilus, Lactococcus lactis ssp. lactis and Lactobacillus casei. Transformants containing the properly constructed vector were identified by PCR analysis and shown to inhibit the growth of Listeria monocytogenes Scott A. S. thermophilus transformants were all shown to constitutively express pediocin; however, in L. lactis and L. casei both constitutive and inducer-dependent expression was observed. In all cases nisin-induction resulted in optimal pediocin production. Transformants from each LAB host were also shown to inhibit the growth of L. monocytogenes NR30, a nisin-resistant variant of L. monocytogenes Scott A. These results suggest pediocin has the potential to serve as a hurdle component along with nisin for prevention of Listeria contamination of foods.     

Effects of sanitizing treatments with atmospheric cold plasma,SDS and lactic acid on verotoxin-producing Escherichia coli and Listeria monocytogenes in red chicory (radicchio)

The main objective of this study was to evaluate the synergistic effect of atmospheric cold plasma (ACP), Sodium Dodecyl Sulphate (SDS) and lactic acid (LA) on L. monocytogenes and verotoxin-producing E. coli in red chicory. Experimentally inoculated samples were pre-treated with either SDS, or SDS + LA for 5, 10 or 15 min. Pre-treated samples were then submerged in deionized water and either exposed to ACP generated by dielectric barrier discharge device (DBD: fixed parameters: 19.15 V and 3.15 A) for 15 min or left untreated. All combinations of treatments were evaluated for sensory effects. Viable counts of verotoxin-producing E. coli on red chicory decreased by more than 4 logs (4.78 Log CFU/cm² vs control) after a treatment with LA + SDS for 5 min and ACP for 15 min and often dropped below the limit of quantification. L. monocytogenes showed a higher tolerance to this sanitizing treatment and the level of inactivation was higher than 3 logs (3.77 Log CFU/cm² vs control) only by increasing the duration of the washing step in LA + SDS to 15 min. The different treatments had no detrimental effects on colour, freshness and texture of red chicory, but odour and overall acceptability of the samples treated by ACP slightly decreased during storage. Further optimization of treatment parameters for maintaining fresh characteristics are needed, but the effectiveness of combinations of sanitizers and ACP on other pathogens and fresh produce worth to be investigated.     

Antibacterial mechanism of lactic acid on physiological and morphological properties of Salmonella Enteritidis,Escherichia coli and Listeria monocytogenes

Lactic acid is widely used to inhibit the growth of important microbial pathogens, but its antibacterial mechanism is not yet fully understood. The objective of this study was to investigate the antibacterial mechanism of lactic acid on Salmonella Enteritidis, Escherichia coli and Listeria monocytogenes by size measurement, TEM, and SDS-PAGE analysis. The results indicated that 0.5% lactic acid could completely inhibit the growth of Salmonella Enteritidis, E. coli and L. monocytogenes cells. Meanwhile, lactic acid resulted in leakage of proteins of Salmonella, E. coli and Listeria cells, and the amount of leakage after 6 h exposure were up to 11.36, 11.76 and 16.29 μg/mL, respectively. Measurements of the release of proteins and SDS-PAGE confirmed the disruptive action of lactic acid on cytoplasmic membrane, as well as the content and activity of bacterial proteins. The Z-Average sizes of three pathogens were changed to smaller after lactic acid treatment. The damaged membrane structure and intracellular structure induced by lactic acid could be observed from TEM images. The results suggested that the antimicrobial effect was probably caused by physiological and morphological changes in bacterial cells.     

Novel biocompatible silver nanoparticles for controlling the growth of lactic acid bacteria and acetic acid bacteria in wines

This paper reports the inhibitory potential of two new silver-based biocompatible nanoparticles (Ag NPs), specifically synthesized for this study, against Gram-negative and Gram-positive bacteria, such as Escherichia coli and Staphylococcus aureus, and different wine lactic acid bacteria (LAB) (Oenococcus oeni, Lactobacillus casei, Lactobacillus plantarum and Pediococcus pentosaceus) and acetic acid bacteria (AAB) (Acetobacter aceti and Gluconobacter oxydans). Both solid nanoparticles, dried PEG-Ag NPs 1 (20.01% silver content) and glutathione-stabilized GSH-Ag NPs 2 solution (0.197 mg/mL silver content), showed homogeneous size distribution and physicochemical properties compatible with their use as antimicrobial agents. The nanoparticles showed different antimicrobial spectra, with PEG-Ag NPs 1 being more effective (lower IC₅₀ values) against Gram-negative strains, whereas GSH-Ag NPs 2 showed similar efficiency against Gram-negative and Gram-positive strains, with the exception of O. oeni strains, which turned out to be notably susceptible to them (IC₅₀ ∼1 μg/mL, Ag concentration). For the LAB and AAB tested, these nanoparticles showed IC₅₀ values (particle concentration) of the same order as those for the widely used additive potassium metabisulphite, at least as calculated for PEG-Ag NPs 1. These results confirm the potential of Ag NPs in controlling microbial processes in winemaking, and open new investigations on the design and use of antimicrobial-specific silver-based nanoparticles in oenology.     

Presence of Listeria monocytogenes in Turkish style fermented sausage (sucuk)

Turkish style fermented sausage (sucuk) is a well-known and very popular meat product produced in Turkey. The growth of Listeria monocytogenes in fermented sausage and other meat products may cause serious health problems for consumers. The aim of this study is to assess the presence of L. monocytogenes in sucuk sold in Istanbul. During the period of February 2004–January 2005, a total of 300 sucuk samples were obtained from various markets located in Istanbul and the presence of L. monocytogenes was analyzed according to “Food and Drug Administration” (FDA) method. The results were found to be positive for Listeria spp. and L. monocytogenes as 63 (21%) and 35 (11.6%) respectively.     

Multiple regression model for thermal inactivation of Listeria monocytogenes in liquid food products

A multiple regression model was constructed for thermal inactivation of Listeria monocytogenes in liquid food products, based on 802 sets of data with 51 different strains and 6 cocktails of strains published from 1984 to 2010. Significant variables, other than inactivation temperature, were pH, sodium chloride content, sugar content, the temperature of growth or storage before inactivation, in addition to a heat shock before inactivation. The constructed model for thermal inactivation of L. monocytogenes has a reduced variability as these variables are known to influence the thermal resistance (and these are known or controllable in practice). Mean simulation results of inactivation of L. monocytogenes during pasteurisation (20 s, 76 °C) of raw milk (calculated mean level after growth 14 cfu/l) were comparable with results of a single regression model constructed from inactivation data found in experiments in milk only (175 data sets, 18 strains/cocktails). Both models predicted a probability of survival of less than 1 in a billion litres. The study shows that multiple regression modelling can be used to obtain a model from all data available, with a limited and realistic uncertainty level, while retaining the variability of heat resistance due to the 51 strains and 6 cocktails of strains (unknown and not controllable in practice).     

Predictive model for the growth kinetics of Listeria monocytogenes in raw pork meat as a function of temperature

This study was performed to develop a predictive growth model of Listeria monocytogenes to ensure the safety of raw pork. The pork samples were inoculated with a cocktail of two L. monocytogenes strains ATCC 15313 and L13-2 isolated from pork and were stored at 5, 15, and 25 °C. Results were evaluated using the MicroFit program. To develop primary models, the Baranyi, modified Gompertz, and Logistic model equations were applied to the observed data. The mathematically predicted growth rate parameters were evaluated using the coefficient of determination (R²), bias factor (B_f), accuracy factor (A_f), and mean square error (MSE). The Baranyi model, which showed an R² of 0.998 and MSE of 0.006, was more suitable than the modified Gompertz and Logistic models. In validation study of secondary model, it appeared that MSE's of specific growth rate (SGR) and lag time (LT) were relatively accurate and suitable for modeling the growth of L. monocytogenes. These values indicated that the developed models were acceptable for expressing the growth of microorganisms on raw pork, which can be applied to ensure the safety of meats and to establish standards for avoiding microbial contamination.     

Ripening conditions: A tool for the control of Listeria monocytogenes in uncooked pressed type cheese

The effect of ripening conditions (two temperatures, 9 °C and 13 °C, and two relative humidities 93% and 97%) on the growth of Listeria monocytogenes and other microbial populations was evaluated in the cores and rinds of uncooked pressed type cheeses prepared with pasteurised milk and inoculated either with Streptococcus thermophilus only or with an anti-listerial consortium. Regardless of temperature and relative humidity (RH), inhibition by the anti-listerial consortium was stronger in the cheese cores than in the rinds. Temperature had no significant effect on L. monocytogenes counts in cores or rinds. However, at the beginning of ripening in the consortium cheese, L. monocytogenes growth was more strongly inhibited at 13 °C than at 9 °C. Regardless of inoculation type and ripening temperature, counts of L. monocytogenes were significantly lower in the cores and rinds of cheeses ripened at 93% than at 97% RH. Lactobacilli counts were higher at 13 °C than at 9 °C and at 93% than at 97% RH. Lactobacilli can help to inhibit L. monocytogenes by catabolising galactose and producing lactate. Further investigations will be needed to evaluate the effect of ripening at 13 °C and 93% RH on the sensorial properties of cheese.     

Environmental sampling for Listeria monocytogenes control in food processing facilities reveals three contamination scenarios

Listeria monocytogenes enters the food processing facility via environment, or contaminated raw materials. To increase the understanding of L. monocytogenes environmental contamination in the meat and dairy food sector, six European scientific institutions sampled twelve food processing environments (FPEs) in a harmonized methodological approach. The selection of six previously assumed uncontaminated (UC) FPEs and six contaminated (C) FPEs was based on the L. monocytogenes occurrence information originating from the time prior to the current study. An aim of the study was to highlight, that FPEs regarded for years as uncontaminated, may also become L. monocytogenes contaminated and repeated environmental sampling could help to identify the potential sources of contamination.From a total of 2242 FPE samples, L. monocytogenes was present in 32% and 8.8% of meat and dairy processing environments, respectively. In the actual study, each FPE was contaminated with L. monocytogenes on at least one sampling occasion. Three contamination scenarios could be observed: (i) sporadic contamination in the interface of raw material reception and hygienic areas, (ii) hotspot contamination in the hygienic processing areas (iii), and widely disseminated contamination in the entirely FPE. These data demonstrate that L. monocytogenes are common colonizers of FPEs in the European processing facilities sampled and that a consistent cross-contamination risk exists. To avoid food contamination, a risk assessment approach should assign risk levels to critical control areas (CCAs) and identify those where cross-contamination should be essentially excluded.     

Potential of phenolic compounds for controlling lactic acid bacteria growth in wine

Lactic acid bacteria are important in enology since they undergo the malolactic fermentation, a process which main effect is the reduction of wine acidity and is almost indispensable in red wine-making. However, if this process is not well controlled during the elaboration of wine, alterations in wine quality due to bacteria metabolic activity can happen. Polyphenols are wine natural components in must and wine that can potentially affect the growth of lactic acid bacteria and the malolactic fermentation. In this paper, after describing the main features of the malolactic fermentation in wine, we review the use of different chemical substances to control growth of lactic acid bacteria in enology. Special attention is given to phenolic compounds, being revised the recent studies about the effect of polyphenols on the growth and metabolism of lactic acid bacteria in wine in order to establish the extent to which these compounds are involved in malolactic fermentation during wine-making. Finally, the potential use of phenolic extracts as new antimicrobial agents during wine-making, as a total or partial alternative to traditional treatments mainly using sulphur dioxide (SO₂) is discussed.     

Antibacterial activity of lactic acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-scale facility: 1—Screening and characterization of the antibacterial compounds

A total of 87 lactic acid bacteria (LAB) (36 Lactobacillus sakei, 22 Enterococcus faecium, 16 Lactococcus garvieae, 11 Vagococcus carniphilus and 2 Enterococcus sp.) isolated from a small-scale facility producing traditional dry sausages were screened for antagonistic activity against other LAB and some spoilage and pathogenic microorganisms, also isolated from the same processing facility, except Listeria innocua (in lieu of Listeria monocytogenes) and Escherichia coli. The final goal was to investigate LAB antibacterial activity within the facility microbial ecosystem and to select interesting strains for the role of bio-preservatives.Twenty-one Ec. faecium, 6 Vc. carniphilus, 4 Lc. garvieae, 3 Lb. sakei and 2 Enterococcus sp. were shown to inhibit the growth of some indicator microorganisms in an agar well diffusion assay. Except 2 Lb. sakei and an Enterococcus sp., all these isolates exhibited antibacterial activity against L. innocua, but only 3 Ec. faecium, 5 Vc. carniphilus and 3 Lc. garvieae displayed also antagonistic activity against Staphylococcus aureus. The 5 Vc. carniphilus isolates were also found to be inhibitory for the Gram-negative bacterium Hafnia alvei.Isolates displaying antibacterial activity against L. innocua and/or Sc. aureus were investigated for the nature of antibacterial compounds synthesized against these indicator microorganisms. Bacteriocin-like production could be detected only on agar plated in overlay assays, and was unsuccessfully researched in cell-free culture supernatant fluids under conditions that eliminate acid and hydrogen peroxide inhibition. Results also showed that a Lb. sakei isolate displayed an additional inhibitory effect by H₂O₂ against L. innocua. These isolates will be investigated for their ability to repress the growth of undesirable bacteria in biofilms, i.e., the real mode of bacterial attachment.This is the first report on bacteriocin-like from Vc. carniphilus and on bacteriocin-like from Lc. garvieae active against both L. innocua and Sc. aureus species.