<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> isolated from cheese: production and partial characterization of bacteriocin B391

Lactobacillus plantarum B391, a strain isolated from an artisanal French cheese, is a producer of a bacteriocin, expressing activity against Enterococcus faecalis NCTC 775, Clostridium perfringens NCTC 13170 and several Listeria monocytogenes strains. High stability was recorded after heat treatment at 121 °C for 20 min and when stored at 4 °C for more than 40 days. A challenge test performed in milk for 11 days showed potential for the control of L. monocytogenes. In the presence of the lytic bacteriocin B391, L. monocytogenes cells present numerous morphology modifications of cell shape and surface structure as well as in the cell division pattern, resulting ultimately in lysis. The high level of Listeria growth inhibition obtained in the presence of Lb. plantarum B391, and the stability of B391 bacteriocin for a long period of time, make this strain potentially interesting to use in milk products to increase food safety.     

Effect of Bacteriocins and Conditions that Mimic Food and Digestive Tract on Biofilm Formation, In Vitro Invasion of Eukaryotic Cells and Internalin Gene Expression by Listeria monocytogenes

In this report, Listeria monocytogenes isolates were evaluated for their ability to form biofilms, for adhesion/invasion of eukaryotic cells and for differential expression of internalin A (inl A) gene, which is related to virulence potential. The presence of bacteriocins of lactic acid bacteria and incubation at 5 °C were the main factors that influenced biofilm formation by L. monocytogenes, in comparison with BHI (control). In general, adhesion and invasion of Caco-2 cells were significantly lower in low pH (4.5), in incubation at 5 °C and in the presence of Oxgall 0.3 %. On the other hand, two L. monocytogenes isolates (INCQS 353 and Reg 26c) showed higher invasion rates when cultivated in the presence of NaCl 5 % (P < 0.05). One L. monocytogenes isolate (H-2) showed the strongest ability to form biofilm and to invade Caco-2 cells, under selected conditions, suggesting there is a relationship between biofilm formation and virulence potential. For all isolates, expression of inl A gene was down-regulated by the presence of bacteriocins, Oxgall 0.3 %, pH 4.5 and incubation at 5 °C. Nonetheless, for one L. monocytogenes isolate (HU 471), expression of inl A gene was eight times higher in the presence of sucrose, indicating that food components can increase the infectiveness of L. monocytogenes.     

Development of Double Loop-Mediated Isothermal Amplification to Detect Listeria monocytogenes in Food

In this study, a double loop-mediated isothermal amplification (dLAMP) based on two target genes hlyA and iap was developed for the rapid detection of Listeria monocytogenes in food. The results revealed that the detection time and temperature of our dLAMP assay for L. monocytogenes were 15 min and 63 °C respectively, with a sensitivity of 10 fg DNA of L. monocytogenes per tube. While normal LAMP (nLAMP) of hlyA or iap was 100 fg DNA of L. monocytogenes per tube for 45 min and 63 °C. Furthermore, mineral oil and GoldViewII nucleic acid stain were chosen as the basic materials to develop a simple visualized identification of the positive samples. A total of 450 food samples were tested for L. monocytogenes using the dLAMP protocol developed in this study. The results showed that the accuracy of the dLAMP and the “gold standard” culture-biotechnical method were 100 % identical, suggesting that the modified dLAMP assay would provide a potential for detection of L. monocytogenes in food products.     

Suppression of Listeria monocytogenes Scott A in Fluid Milk by Free and Liposome-Entrapped Nisin

Nisin is an antimicrobial polypeptide inhibitory toward Gram-positive bacterial pathogens, including Listeria monocytogenes. Encapsulating nisin in lipid nanocapsules (i.e., liposomes) has been shown to protect antimicrobial functionality in complex food matrices. The capacity of liposomes to encapsulate a fluorescent reporter was determined via spectroscopy. Survival and growth of L. monocytogenes incubated in fluid milk containing 50 IU/ml free or liposome-entrapped nisin was assayed via periodic enumeration of survivors. Liposomes were formulated from phosphatidylcholine (PC) and phosphatidyl-DL-glycerol (PG) and prepared as PC, PC/PG 7/3 or PC/PG 6/4 (mol. fraction). Antilisterial activity of nisin-loaded liposomes was determined in ultra-high temperature processed fluid milk containing approximately 4.0 log₁₀ CFU/ml L. monocytogenes Scott A plus liposomal or free nisin at 50 IU/mL. Samples were aerobically held at 5 or 20°C; L. monocytogenes were enumerated via plating after 0, 1, 3, 6, 12, 24, 48, and 72 incubation hours. Liposome entrapment did not enhance pathogen inhibition when compared to free nisin as a function of storage temperature or incubation duration.     

Effectiveness of phages in the decontamination of Listeria monocytogenes adhered to clean stainless steel, stainless steel coated with fish protein, and as a biofilm

Listeria monocytogenes is a food-borne pathogen which causes listeriosis and is difficult to eradicate from seafood processing environments; therefore, more effective control methods need to be developed. This study investigated the effectiveness of three bacteriophages (LiMN4L, LiMN4p and LiMN17), individually or as a three-phage cocktail at ≈9 log₁₀ PFU/ml, in the lysis of three seafood-borne L. monocytogenes strains (19CO9, 19DO3 and 19EO3) adhered to a fish broth layer on stainless steel coupon (FBSSC) and clean stainless steel coupon (SSC), in 7-day biofilm, and dislodged biofilm cells at 15 ± 1 °C. Single phage treatments (LiMN4L, LiMN4p or LiMN17) decreased bacterial cells adhered to FBSSC and SSC by ≈3–4.5 log units. Phage cocktail reduced the cells on both surfaces (≈3.8–4.5 and 4.6–5.4 log₁₀ CFU/cm², respectively), to less than detectable levels after ≈75 min (detection limit = 0.9 log₁₀ CFU/cm²). The phage cocktail at ≈5.8, 6.5 and 7.5 log₁₀ PFU/cm² eliminated Listeria contamination (≈1.5–1.7 log₁₀ CFU/cm²) on SSC in ≈15 min. One-hour phage treatments (LiMN4p, LiMN4L and cocktail) in three consecutive applications resulted in a decrease of 7-day L. monocytogenes biofilms (≈4 log₁₀ CFU/cm²) by ≈2–3 log units. Single phage treatments reduced dislodged biofilm cells of each L. monocytogenes strain by ≈5 log₁₀ CFU/ml in 1 h. The three phages were effective in controlling L. monocytogenes on stainless steel either clean or soiled with fish proteins which is likely to occur in seafood processing environments. Phages were more effective on biofilm cells dislodged from the surface compared with undisturbed biofilm cells. Therefore, for short-term phage treatments of biofilm it should be considered that some disruption of the biofilm cells from the surface prior to phage application will be required.     

Characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery

Lactococcus lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery inhibits the growth of Citrobacter freundii (a bullfrog pathogen) and Listeria monocytogenes by a synergistic effect between lactic acid, hydrogen peroxide and a bacteriocin-like molecule. The chemical characterization of the bacteriocin in cell-free supernatants indicates that it has a proteinaceous nature. Hexadecane and ethyl acetate did not modify the bacteriocin activity, while 10 and 20 % (v/v) chloroform decreased the activity by 29 and 43 %, respectively. The antimicrobial peptide was heat stable since 85 % of residual activity was detected when neutralized supernatants were heated at 80 °C for 30 min. Moreover, no bacteriocin inactivation was observed when supernatants were kept at ?20 °C for 3 months. The synthesis of the bacteriocin was associated with bacterial growth, highest production (2,100 AU/ml) being detected at the end of the exponential growth phase. At pH ranges of 5–6.5 and 5.0–5.5 the inhibitory molecule was stable when stored for 2 days at 4 and 25 °C, respectively. Moreover, it had a bactericidal effect on L. monocytogenes and the ultrastructural studies of pathogenic cells revealed clumping of the cytoplasmic material, increased periplasmic space and cell wall modifications. The deduced amino acid sequence of the bacteriocin was identical to nisin Z and the genetic determinants for its production are harbored in the chromosome. These results, described for the first time in L. lactis from a bullfrog hatchery, will increase knowledge of the bacteriocin under study with a view to its potential inclusion in probiotics for raniculture or biopreservatives.     

Anti-listerial Bactericidal Activity of Lactobacillus plantarum DM5 Isolated from Fermented Beverage Marcha

The strain Lactobacillus plantarum DM5 was isolated from fermented beverage Marcha of Sikkim and explored for its antagonistic activity against food-borne pathogens. The cell-free supernatant of L. plantarum DM5 showed antibacterial activity of 6,400 AU/mL in MRS medium (pH 6.0) against the indicator strain Staphylococcus aureus. MRS medium supplemented with 15 g/L of maltose at 37 °C under static condition yielded highest antimicrobial activity (6,400 AU/mL) with 3 % increase in specific activity when compared to 20 g/L glucose. The antimicrobial compound was heat stable (60 min at 100 °C) and was active over a wide pH range. It showed bactericidal effect on S. aureus and Listeria monocytogenes by causing 96 and 98 % of cell lysis, respectively. The cell morphology of the treated S. aureus and L. monocytogenes was completely deformed as revealed by scanning electron microscopy, suggesting the high potential of L. plantarum DM5 as natural preservatives in food industry. The antimicrobial compound was purified by 80 % ammonium sulphate precipitation and showed antimicrobial activity of 12,800 AU/mL with 19-fold purification and a molecular mass of 15.2 kDa, indicating the proteinaceous nature of the compound.     

Inhibition of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in Hot Dogs by Surface Application of Freeze-Dried Bacteriocin-Containing Powders from Lactic Acid Bacteria

Six lactic acid bacteria (LAB) strains, Lactococcus lactis BFE 920, L. lactis subsp. lactis ATCC 11454, L. lactis subsp. cremoris ATCC 14365, Lactobacillus curvatus L442, Lact. curvatus LTH 1174, and Lact. bavaricus MN, were grown in cheddar cheese whey supplemented with complex nutrient sources. Cell-free culture supernatants were freeze-dried, and the resulting bacteriocin-containing powders were applied on the surface of hot dogs that were inoculated (~4 log cfu/hot dog) with a five-strain Listeria monocytogenes cocktail. Hot dogs were vacuum-sealed and stored at 4 °C for 4 weeks. L. monocytogenes was enumerated, using both tryptic soy agar (TSA) and oxford listeria agar (OXA), on day 0 and at 1, 2, 3, and 4 weeks of the refrigerated storage. In hot dogs containing only the L. monocytogenes inoculum, L. monocytogenes counts increased from 4 up to 7 log cfu/hot dog. All samples containing freeze-dried bacteriocin-containing powders exhibited significantly lowered (P < 0.05) L. monocytogenes populations on the surface of hot dogs throughout the 4-week study except for bavaricin MN powder. Bacterial counts on hot dogs packed without any powder were statistically equal on day 0 when enumerated on OXA. Freeze-dried bacteriocin-containing powders from Lact. curvatus L442 and L. lactis subsp. cremoris ATCC 14365 decreased L. monocytogenes populations on the surface of hot dogs by greater than 2 log cfu/hot dog throughout the 4-week study. For the powdered bacteriocin preparations from L. lactis BFE 920, L. lactis subsp. lactis ATCC 11454, and Lact. curvatus LTH 1174, L. monocytogenes populations were determined to be approximately 3-log cfu/hot dog after 4 weeks of storage.     

Effect of Two Probiotic Strains of Lactobacillus on In Vitro Adherence of Listeria monocytogenes,Streptococcus agalactiae,and Staphylococcus aureus to Vaginal Epithelial Cells

The lactobacilli probiotics maintain a normal vaginal biota and prevent disease recurrence. This microorganisms form a pellicle on the vaginal epithelium that acts as a biologic barrier against colonization by pathogenic bacteria. In this paper were realized assays of exclusion, competition, and displacement. For these test, vaginal epithelial cells, two strains of lactobacilli and pathogenic bacteria (Staphylococcus aureus, Streptococcus agalactiae and Listeria monocytogenes) were used. The lactobacilli strains showed a great capacity of adherence, with a mean of 83.5 ± 26.67 Lactobacillus fermentum cells and 56.2 ± 20.87 Lactobacillus rhamnosus cells per vaginal epithelial cells. L. fermentum and L. rhamnosus were able to reduce the adherence of S. aureus, S. agalactiae and L. monocytogenes in a significant level in this assay (P < 0.01). The lactobacilli used in this study protect the vaginal epithelium through a series of barriers and interference mechanisms. The aim of present study was to assess the ability of vaginal Lactobacillus strains, selected for their probiotic properties, to block the adherence of pathogenic microorganisms in vitro by displacement, competition, and exclusion mechanisms.     

Listeria monocytogenes Can Form Biofilms in Tap Water and Enter Into the Viable but Non-Cultivable State

Listeria monocytogenes is a foodborne pathogen that can be transmitted through contaminated raw food or by ready-to-eat products that have been in contact with contaminated surfaces. Tap water (TW) is used to wash produce, as a processed food constituent and to wash processing surfaces and floors. The main aim of this work was to investigate the formation and survival of L. monocytogenes biofilms on stainless steel (SS) coupons in TW at 4, 22, 30 and 37 °C. For that, coupons with biofilm were visualised in situ while other coupons were scraped to quantify total cells by SYTO 9, cultivable numbers by plating onto brain heart infusion agar and viable numbers by the direct viable count method. Results showed that L. monocytogenes can form biofilms on SS surfaces in TW at any temperature, including at 4 °C. The number of total cells was similar for all the conditions tested while cultivable numbers varied between the level of detection (<8.3 CFU cm^?2) and 3.5?×?10⁵ CFU cm^?2, meaning between 7.0?×?10⁴ and 1.1?×?10⁷ cells cm^?2 have entered the viable but non-cultivable (VBNC) state. This work clearly demonstrates that L. monocytogenes can form biofilms in TW and that sessile cells can remain viable and cultivable in some conditions for at least the 48 h investigated. On the other hand, VBNC adaptation suggests that the pathogen can remain undetectable using traditional culture recovery techniques, which may give a false indication of processing surface hygiene status, leading to potential cross-contamination of food products.     

Extent and Mode of Action of Cationic Legume Proteins against Listeria monocytogenes and Salmonella Enteritidis

The methylated soybean protein and methylated chickpea protein (MSP and MCP) with isoelectric points around pI 8 were prepared by esterifying 83 % of their free carboxyl groups and tested for their interactions with Listeria monocytogenes and Salmonella Enteritidis. The two substances exhibited a concentration-dependent inhibitory action against the two studied bacteria with a minimum inhibitory concentration of about 100 μg/mL. The IC_50 % of the two proteins against L. monocytogenes (17 μg/mL) was comparable to penicillin but comparatively much lower (15 μg/mL) than that of penicillin (85 μg/mL) against S. Enteritidis. The two proteins could inhibit the growth of L. monocytogenes and S. Enteritidis by about 97 and 91 %, respectively, after 6–12 h of incubation at 37 °C. The constituting subunits of MSP (methylated 11S and methylated 7S) were both responsible for its antimicrobial action. Transmission electron microscopy of the protein-treated bacteria showed various signs of cellular deformation. The cationic proteins can electrostatically and hydrophobically interact with cell wall and cell membrane, producing large pores, pore channels and cell wall and cell membrane disintegration, engendering higher cell permeability leading finally to cell emptiness, lysis and death.     

Purification and characterization of plantaricin Y,a novel bacteriocin produced by Lactobacillus plantarum 510

Lactobacillus plantarum 510, previously isolated from a koshu vineyard in Japan, was found to produce a bacteriocin-like inhibitory substance which was purified and characterized. Mass spectrometry analysis showed that the mass of this bacteriocin is 4,296.65 Da. A partial sequence, NH₂- SSSLLNTAWRKFG, was obtained by N-terminal amino acid sequence analysis. A BLAST search revealed that this is a unique sequence; this peptide is thus a novel bacteriocin produced by Lactobacillus plantarum 510 and was termed plantaricin Y. Plantaricin Y shows strong inhibitory activity against Listeria monocytogenes BCRC 14845, but no activity against other pathogens tested. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was completely inactivated by protease K. Furthermore, trypsin-digested bacteriocin product fragments retained activity against L. monocytogenes BCRC 14845 and exhibited a different inhibitory spectrum.     

Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation

We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l^?1 with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS–DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or ?20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1–7.3 × 10⁶ l^?1 after 3 days growth with maximum MS yields (0.7–1.1 × 10⁷ l^?1) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS–DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 10⁹ conidia g^?1). All MS granules showed similar stability after storage at either 26 or ?20 °C for 3.5 months.     

Investigation into the Potential of Bacteriocinogenic Lactobacillus plantarum BFE 5092 for Biopreservation of Raw Turkey Meat

The bacteriocin-producing Lactobacillus plantarum BFE 5092 was assessed for its potential as a protective culture in the biopreservation of aerobically stored turkey meat. This strain produces three bacteriocins, i.e. plantaricins EF, JK and N. The absolute expression of Lactobacillus plantarum BFE 5092 16S rRNA housekeeping gene, as well as l-ldh, plnEF and plnG genes as determined by quantitative, real-time-PCR, revealed that these genes were expressed to similar levels when the strain was grown at 8 and 30 °C in MRS broth. On turkey meat, Lactobacillus plantarum BFE 5092 did not grow but survived, as indicated by similar viable cell numbers during a 9-day storage period at 8 °C. When inoculated at 1 × 10⁷ CFU/g on the turkey meat and subsequently stored at 10 °C, the culture did again not show good growth. Lactobacillus plantarum BFE 5092 could not inhibit the growth of naturally occurring listeriae or Gram-negative bacteria on the turkey meat at 10 °C, or that of Listeria monocytogenes when it was co-inoculated at a level of 1 × 10⁵ CFU/g. Gene expression analyses showed that the bacteriocin genes were expressed on turkey meat stored at 10 °C. Moreover, the investigation into the absolute expression of the three plantaricin genes of Lactobacillus plantarum BFE 5092 in co-culture with Listeria monocytogenes on turkey meat by qRT-PCR showed that the plantaricin genes were indeed expressed during the low-temperature storage condition. The Lactobacillus plantarum BFE 5092 strain overall could not effectively inhibit L. monocytogenes and therefore it would not make a suitable protective culture for biopreservation of turkey meat stored aerobically at low temperature.     

Nisin Z produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> from bullfrog hatchery is active against <Emphasis Type="Italic">Citrobacter freundii</Emphasis>, a red-leg syndrome related pathogen

Lactococcus lactis subsp. lactis CRL 1584 isolated from a bullfrog hatchery produces a bacteriocin that inhibits both indigenous Citrobacter freundii (a Red-Leg Syndrome related pathogen) and Lactobacillus plantarum, and Listeria monocytogenes as well. Considering that probiotics requires high cell densities and/or bacteriocin concentrations, the effect of the temperature on L. lactis growth and bacteriocin production was evaluated to find the optimal conditions. Thus, the growth rate was maximal at 36 °C, whereas the highest biomass and bacteriocin activity was achieved between 20 and 30 °C and 20–25 °C, respectively. The bacteriocin synthesis was closely growth associated reaching the maximal values at the end of the exponential phase. Since bacteriocins co-production has been evidenced in bacterial genera, a purification of the bacteriocin/s from L. lactis culture supernatants was carried out. The active fraction was purified by cationic-exchange chromatography and then, a RP-HPLC was carried out. The purified sample was a peptide with a 3353.05 Da, a molecular mass that matches nisin Z, which turned out to be the only bacteriocin produced by L. lactis CRL 1584. Nisin Z showed bactericidal effect on C. freundii and L. monocytogenes, which increased in the presence l-lactic acid?+?H₂O₂. This is the first report on nisin Z production by L. lactis from a bullfrog hatchery that resulted active on a Gram-negative pathogen. This peptide has potential probiotic for raniculture and as food biopreservative for bullfrog meat.     

Bacteriocin-Producing Lactic Acid Bacteria Isolated from Mangrove Forests in Southern Thailand as Potential Bio-Control Agents: Purification and Characterization of Bacteriocin Produced by Lactococcus lactis subsp. lactis KT2W2L

The aim of this work was to purify and characterize the bacteriocin produced by Lactococcus lactis subsp. lactis KT2W2L previously isolated from mangrove forests in southern Thailand, in order to evaluate its potential as new food protective agent. The active peptide from the cell-free supernatant of this strain was purified in 4 steps: (1) precipitation with 70 % saturated ammonium sulfate, (2) elution on a reversed-phase cartridge using different concentrations of acetonitrile, (3) cation-exchange chromatography and (4) final purification by reversed-phase HPLC on a C₈ column. The molecular mass of 3,329.5254 Da of the purified bacteriocin, determined by mass spectrometry, is nearly identical to that of peptide nisin Z. The activity of the purified bacteriocin was unaffected by pH (2.0–10.0), thermostable but was sensitive to proteolytic enzymes. The bacteriocin activity was stable after 8 weeks of storage at ?20 °C and 7 weeks of storage at 4 °C, but decreased after 3 weeks of storage at 37 °C. It was stable when incubated for 1 month at 4 °C in 0–30 % NaCl. Inhibitory spectrum of this bacteriocin showed a wide range of activity against similar bacterial strains, food-spoilage and food-borne pathogens. L. lactis subsp. lactis KT2W2L was sensitive to kanamycin, penicillin and tetracycline but resistant to ampicillin, gentamicin and vancomycin. The fragment obtained after amplification of genomic DNA from L. lactis subsp. lactis KT2W2L, with specific primers for bacteriocin genes, presented 99 % homology to the nisin Z gene. PCR amplification demonstrated that L. lactis subsp. lactis KT2W2L does not harbor virulence genes cylA, cylB, efaAfs and esp. The bacteriocin and its producing strain may find application as bio-preservatives for reduction in food-spoilage and food-borne pathogens in food products.     

Hybridisation and selective breeding for improvement of low temperature activity of the entomopathogenic nematode Steinernema feltiae

Steinernema feltiae is used to control overwintering larvae of the codling moth Cydia pomonella L. Application is in autumn when efficacy can be limited by low temperature. The objective of this study was to screen for low temperature activity among wild type populations of S. feltiae, hybridise most active strains and further improve low temperature activity by subjection of a hybrid strain to selective breeding. Significant variation was recorded among 22 S. feltiae strains. The temperature at which 50 % (AT₅₀) and 10 % (AT₁₀) of the dauer juveniles (DJs) were active ranged between 2.9 to 5.8 °C and 0.95 to 3.5 °C, respectively. The mean AT₅₀ of 22 S. feltiae strains was 3.83 °C. The five most active strains were crossed. The hybrid strain HYB01 was more active at low temperature than parental and other hybrid strains with an AT₅₀ of 0.52 °C and an AT₁₀ of 0.09 °C. The tolerance was lost after few reproductive cycles in the insect Galleria mellonella, but was recovered after seven selection cycles with exposure to lowering temperatures. The heritability for the low temperature activity was calculated at h ² = 0.45. Negative trade-off effects on virulence against C. pomonella and reproduction on the same insect were not reported. The most virulent strain was a commercial strain with an LD₅₀ of 30.2 at 8 °C and 37.2 DJs per cocooned instar at 15 °C, followed by the selected hybrid with 48.1 and 47.4 DJs, respectively. Offspring production reached 15.000 DJs per instar at 8 °C and was only half at 15 °C. The results well document the potential of a breeding programme for enhancement of the activity of S. feltiae at lower temperature with the objective to improve the control potential of overwintering codling moth C. pomonella.     

Behaviour of Listeria monocytogenes in packaged water buffalo mozzarella cheese

Aims: The aim of the study was to evaluate the behaviour of Listeria monocytogenes in the conditioning liquid of packaged water buffalo mozzarella cheese (WBMC). Methods and Results: The conditioning liquid was contaminated with L. monocytogenes, and the contaminated samples were stored at four different storage temperatures: 5 and 10°C for 22 days; 20°C for 9 days; 20°C for 3 days and then at 5°C for 6 days. The results showed that L. monocytogenes concentration decreased when contaminated samples were stored at 5°C. When WBMC was stored at 20°C and at 10°C, L. monocytogenes started to grow after a lag phase of 3 and 10 days, respectively. When samples were stored at variable temperature conditions, L. monocytogenes numbers showed a lag phase of 5 days. Conclusions: Use of a conditioning liquid characterized by acidity and a correct storage temperature is able to counteract pathogen replication during shelf life. A high concentration of lactic acid bacteria was associated with effective control of L. monocytogenes but the role of lactic acid bacteria in WBMC conditioning liquid requires further investigation. Significance and Impact of the Study: According to European regulations, food producers should be able to justify decision‐making on the shelf life assigned to their products, taking into account reasonable storage conditions and use by consumers. The results of the trial yielded information for producers of WBMC and similar cheeses for decision‐making on product shelf life.     

Screening and Mutation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> UV-20 with a High Yield of Second Generation Bioethanol and High Tolerance of Temperature,Glucose and Ethanol

A wild-type strain was isolated from slightly rotted pears after three rounds of enrichment culture, identified as Saccharomyces cerevisiae 3308, and evaluated for its fermentation capability of second generation bioethanol and tolerance of temperature, glucose and ethanol. S. cerevisiae 3308 was mutated by using the physical and chemical mutagenesis methods, ultraviolet (UV) and diethyl sulfate (DES), respectively. Positive mutated strains were mainly generated by the treatment of UV, but numerous negative mutations emerged under the treatment of DES. A positive mutated strain, UV-20, produced ethanol from 62.33?±?1.34 to 122.22?±?2.80 g/L at 30–45 °C, and had a maximum yield of ethanol at 37 °C. Furthermore, UV-20 produced 121.18?±?2.51 g/L of second generation bioethanol at 37 °C. Simultaneously, UV-20 exhibited superior tolerance to 50% of glucose and 21% of ethanol. In a conclusion, all of these results indicated that UV-20 has a potential industrial application value.     

Prevalence and level of Listeria monocytogenes and other Listeria sp. in ready-to-eat minimally processed and refrigerated vegetables

Minimally processed and refrigerated vegetables can be contaminated with Listeria species bacteria including Listeria monocytogenes due to extensive handling during processing or by cross contamination from the processing environment. The objective of this study was to examine the microbiological quality of ready-to-eat minimally processed and refrigerated vegetables from supermarkets in Osijek, Croatia. 100 samples of ready-to-eat vegetables collected from different supermarkets in Osijek, Croatia, were analyzed for presence of Listeria species and Listeria monocytogenes. The collected samples were cut iceberg lettuces (24 samples), other leafy vegetables (11 samples), delicatessen salads (23 samples), cabbage salads (19 samples), salads from mixed (17 samples) and root vegetables (6 samples). Listeria species was found in 20 samples (20 %) and Listeria monocytogenes was detected in only 1 sample (1 %) of cut red cabbage (less than 100 CFU/g). According to Croatian and EU microbiological criteria these results are satisfactory. However, the presence of Listeria species and Listeria monocytogenes indicates poor hygiene quality. The study showed that these products are often improperly labeled, since 24 % of analyzed samples lacked information about shelf life, and 60 % of samples lacked information about storage conditions. With regard to these facts, cold chain abruption with extended use after expiration date is a probable scenario. Therefore, the microbiological risk for consumers of ready-to-eat minimally processed and refrigerated vegetables is not completely eliminated.