Antilisterial activity of lactic acid bacteria inoculated on cooked ham

This study was conducted to evaluate the ability of Lactobacillus sakei 1, a bacteriocin-producing (bac⁺) lactic acid bacterium (LAB), isolated from Brazilian fresh pork sausage to inhibit two Listeria monocytogenes strains (serotypes 4b and 1/2a) on cooked, sliced vacuum-packaged ham. L. sakei ATCC 15521 was used as a non-bacteriocin producer (bac⁻). L. monocytogenes (ca. 2 log CFU/mL) and LAB (ca. 6 log CFU/ml) were inoculated on the sterilized ham, vacuum-sealed and incubated at 8 °C for 10 days. A treatment with the bacteriocin Chrisin (UI/ml) was included. Both L. monocytogenes strains were significantly inhibited in the presence of either bac⁺ and bac⁻ LAB in comparison to the control (L. monocytogenes alone). Using a bacteriocinogenic strain of LAB did not offer an additional barrier to listerial growth in the studied meat system. The application of Chrisin did not affect at all the growth of L. monocytogenes.     

Some characteristics of lactic acid bacteria present in commercial sucuk samples

A total of 10 sucuk samples, obtained from Denizli, Turkey were analysed for some physical, chemical and microbiological characteristics. In addition, lactic acid bacteria (LAB) strains producing bacteriocin-like metabolites were isolated and identified. The production of some typical metabolites of the cultures isolated was investigated. At the end of the research, the average values of the pH, water and fat content were 5.1, and 37.2% and 30.5%, respectively. Microbiological analyses results were determined: average 8.34 log CFU/g TAMB, 8.91 log CFU/g LAB (at the MRS agar) and average 8.25 log CFU/g LAB (at the Elliker's lactic agar). The average counts of yeast-mould, coliform and Enterobacteriaceae were found to be 5.0 log CFU/g, 3.28 log CFU/g and 3.27 log CFU/g, respectively. In this study, counts of yeast-mould in the two samples, coliform counts in the five samples, and Enterobacteriaceae counts in the three samples were < 1.0 log CFU/g. A total of 6 of 100 LAB isolates obtained from the sucuk samples were found as a strain producing bacteriocin-like metabolites. These 6 strains were identified as follows; 3 strains Lactobacillus plantarum and 3 strains Pediococcus pentosaceus. According to the findings, these strains have the potential to be used as a sucuk starter culture. Additionally, acid and flavour compounds, other undesirable metabolite-producing activities of the strains, were determined in the model system. From these results it was concluded, after the determination of the toxicological properties, that the 4 strains of LAB identified (L. plantarum 13 P. pentosaceus 15 P. pentosaceus 74 and P. pentosaceus 75) would be useful as the starter and protective culture in the processing of the sucuk and similar fermented products.     

In Vitro Assessment of Probiotic Properties of Lactobacillus Strains from Infant Gut Microflora

Lactobacillus isolates from infant feces were identified and investigated in vitro for their probiotic properties. The isolates belonged to the species L. acidophilus, L. rhamnosus, L. paracasei subsp. paracasei, L. fermentum, L. delbrueckii subsp. delbrueckii, L. cellobiosus, L. plantarum, L. brevis and L. crispatus. The strains were examined for acid and bile tolerance, adhesion to Caco-2 cells, antibiotic susceptibility and antimicrobial activity against selected enteric pathogens. According to the obtained results, a L. deblueckii subsp. delbrueckii strain was found to exhibit probiotic properties. Other strains from L. paracasei subsp. paracasei and L. fermentum group also could be further investigated as potential probiotic candidates.     

Selective enumeration and survival of bifidobacteria in fresh cheese

Five species of bifidobacteria (15 strains), two strains of Lactococcus lactis ssp. lactis, two strains of L. lactis ssp. cremoris, and one strain of L. lactis ssp. lactis var diacetylactis were included in a study to develop a selective medium for enumeration of bifidobacteria from fresh cheese. Viable counts of bifidobacteria or lactococci on modified Columbia agar base (CAB with 0.05% cysteine-HCl) plus raffinose (0.5%) containing various selective agents were compared with non-selective media. The mCAB plus raffinose with lithium chloride (2 g L⁻¹) and sodium propionate (3 g L⁻¹) with pH adjusted to 5.1 was used successfully as a selective medium for the enumeration of bifidobacteria from fresh cheese. Using this medium, it was determined that bifidobacteria could survive up to 15 days at a level higher than 10⁶ cfu g⁻¹ in a fresh cheese stored at 4 or 12°C. The decrease in the viable counts of bifidobacteria was faster during storage at 4°C than at 12°C.     

Physiological properties of Lactobacillus paracasei, L. danicus and L. curvatus strains isolated from Estonian semi-hard cheese

Growth and survival of Lactobacillus paracasei (six strains), L. danicus sp. nov. (four isolates, two strains) and L. curvatus (two strains) from semi-hard Estonian cheeses were comparatively studied in different environmental conditions of relevance for their growth in cheese and survival in gastric environment. Maximum specific growth rates for L. paracasei strains varied between 0.40 and 0.57 h⁻¹, and all strains were tolerant to low water activities, heating at 60 °C for 30 min and pH 3. The newly discovered genetically distinct species L. danicus was characterized by low maximum growth rates (0.26–0.38 h⁻¹) and low temperature optimum (<30 °C). It was acid and heat sensitive and inhibited at salt concentrations from 4% and water activities below 0.93. L. curvatus was characterized by the highest growth rates (0.65–0.70 h⁻¹), tolerance to high NaCl concentrations, but sensitivity to heating, bile salts and low pH. The study showed that genetically different LAB species isolated from cheese could be distinguished by simple cultivation experiments.     

Behaviour of Listeria monocytogenes in raw sausages (merguez) in presence of a bacteriocin-producing lactococcal strain as a protective culture

The effectiveness of a bacteriocin produced by Lactococcus lactis subsp. lactis M in reducing population level and growth of Listeria monocytogenes ATCC 7644 in fermented merguez sausage was examined. Two different formulas (with or without added nitrites) were assayed and predetermined numbers of Listeria (ca 10⁶ cfu g⁻¹) were added to sausage mixture. The effect of in situ production of the bacteriocin by Lactococcus lactis M on Listeria monocytogenes ATCC 7644 during fermentation and storage of merguez sausages at room (ca 22 °C) or at refrigeration (ca 7 °C) temperature was tested. Results indicated that counts of Listeria monocytogenes were decreased during fermentation of merguez samples fermented with either the bacteriocin-producing Lactococcus lactis M (Bac⁺) or a nonbacteriocin-producing Lactococcus lactis J (Bac⁻). However, reduction in Listeria cfu's was greater in samples fermented with the Bac⁺ than in those fermented with the Bac⁻ starter. In merguez sausage made without nitrites addition, the Bac⁺ starter induced further decrease in Listeria counts by 1.5 log cycles compared with that induced by the Bac⁻ starter. While in merguez samples with added nitrites (0.4%), the effect of the bacteriocin produced in situ was less important than in those made without nitrites addition.     

Effects of different probiotic strains of Lactobacillus and Bifidobacterium on bacterial translocation and liver injury in an acute liver injury model.

Septic complications represent frequent causes of morbidity in liver diseases and following hepatic operations. Most infections are caused by the individual own intestinal microflora. The intestinal microflora composition is important in physiological and pathophysiological processes in the human gastrointestinal tract, but their influence on liver in different situations is unclear. We therefore studied the effect of different Lactobacillus strains and a Bifidobacterium strain on the extent of liver injury, bacterial translocation and intestinal microflora in an acute liver injury model.

Sprague–Dawley rats were divided into five groups: acute liver injury control, acute liver injury+B. animalis NM2, acute liver injury+L. acidophilus NM1, acute liver injury+L. rhamnosus ATCC 53103, and acute liver injury+L. rhamnosus DSM 6594 and L. plantarum DSM 9843. The bacteria were administered rectally daily for 8 days. Liver injury was induced on the 8th day by intraperitoneal injection of -galactosamine (1.1 g/kg BW). Samples were collected 24 h after the liver injury. Liver enzymes and bilirubin serum levels, bacterial translocation (to arterial and portal blood, liver and mesenteric lymph nodes (MLNs)), and intestinal microflora were evaluated.

L. acidophilus NM1; L. rhamnosus ATCC 53103, and L. rhamnosus DSM 6594+L. plantarum DSM 9843 decreased bacterial translocation compared to the liver injury control group. B. animalis NM2 increased bacterial translocation to the mesenteric lymph nodes. The levels of alanine aminotransferase (ALAT) were significantly lower in the L. acidophilus, L. rhamnosus ATCC 53103, L. rhamnosus DSM 6594+L. plantarum DSM 9843 groups compared to the liver injury group. The L. rhamnosus and L. rhamnosus+L. plantarum groups significantly reduced ALAT levels compared to the B. animalis group. All administered bacteria decreased the Enterobacteriaceae count in the cecum and colon.

Administration of different lactobacilli and a Bifidobacterium strain in an acute liver injury rat model, has shown different effects on bacterial translocation and hepatocellular damage. L. acidophilus, L. rhamnosus, and L. rhamnosus+L. plantarum reduced bacterial translocation and hepatocellular damage. B. animalis NM2 increased bacterial translocation to the mesenteric lymph nodes and did not affect hepatocellular damage.     

Comparison of the microflora isolated from spoiled cold-smoked salmon from three smokehouses

The microflora on spoiled, sliced and vacuum packed, cold-smoked salmon from three smokehouses was quantified and characterized in two independent experiments. Large variations in the microflora were observed both within (i.e. among vacuum packs from the same batch) and among the smokehouses. Lactic acid bacteria dominated the microflora, which reached 10⁷ cfu g⁻¹. Total viable counts of microorganisms alone were not related to quality, though spoilage characteristics were typical for microbiological spoilage. Among the lactic acid bacteria, Lactobacillus curvatus (ca 52–55%) was the most common species in both experiments with Lactobacillus saké, Lactobacillus plantarum, Carnobacterium spp. and Leuconostoc spp. present in smaller numbers. In some cases, large numbers of Enterobacteriaceae were also present and identified species were Serratia liquefaciens, Enterobacter agglomerans and Hafnia alvei. The microflora on cold-smoked salmon appeared to be related to the source of contamination i.e. the raw material and/or the smokehouse rather than being specific for the product, thus rendering the identification of the specific spoilage organisms difficult.     

Studies to determine the critical control points in pork slaughter hazard analysis and critical control point systems

Aerobic mesophilic counts (AMC), coliform (CC) and coliform resuscitation counts (CRCs) were obtained by swabbing 50 cm² areas at three sites (ham, belly and neck) on pig carcasses, after each of seven stages of the slaughter/dressing process (bleeding, scalding, dehairing, singeing, polishing, evisceration and chilling). In most cases, there were no statistical differences (P>0.05) among the counts derived by these three methods. Reductions in counts at individual sites were observed after scalding (3.5 log₁₀ cfu cm⁻²), and singeing (2.5 log₁₀ cfu cm⁻²). Increases in counts at individual sites were observed after dehairing (2.0 log₁₀ cfu cm⁻²) and polishing (1.5 log₁₀ cfu cm⁻²). The incidence of Salmonella on pig carcasses was also obtained by swabbing the outside surfaces of 100 half carcasses. Information on the incidence of Salmonella in scald tank water (108 samples) was also investigated. Carcass swabs and scald tank water were examined for the presence of Salmonella using standard enrichment methods. Salmonella were detected on 31% of carcasses immediately after bleeding, 7% of carcasses immediately after dehairing and evisceration, and 1% of carcasses immediately after scalding. Serovars included Salmonella Typhimurium, Salmonella Hadar, Salmonella Infantis and Salmonella Derby. No Salmonella were recovered from samples of scald tank water. The impact of pig slaughter/dressing processes on carcass microbiology and their potential use as critical control points (CCPs) during pork production are discussed.     

Survival of Lactobacillus acidophilus, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Bifidobacterium animalis and Propionibacterium in cheese-based dips and the suitability of dips as effective carriers of probiotic bacteria

The suitability of cheese-based dips as a delivery vehicle for probiotic bacteria including Lactobacillus acidophilus, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Bifidobacterium animalis, and Propionibacterium freudenreichii subsp. shermanii was studied by evaluating the survival of these organisms in dips. Effects of organic acids, oils and gums, -cysteine and NaHCO₃ on the survival of probiotics in cheese-based dips were also studied. Eight different combinations and five individual bacteria as controls of these probiotic bacteria were added to 21 batches of French onion dip and selective enumeration of these probiotic bacteria was carried out over a period of 10 weeks of storage. The population of L. acidophilus and B. animalis reduced by 1 log and 2 log per g, respectively. However, when the inoculation level of these bacteria were increased to 8 log per g, they maintained a population of more than 6 log over the shelf life. L. paracasei subsp. paracasei and L. rhamnosus remained at the inoculated level or increased slightly during the storage. A rapid increase in the population of P. freudenreichii subsp. shermanii occurred to attain more than the inoculation level following reduction in their number by 3 log. Except bacterial interaction, no other factors showed significant effect on the survival of individual probiotic bacteria. Each of L. acidophilus, B. animalis, L. paracasei subsp. paracasei, and L. rhamnosus showed varied levels of antogonism, while P. freudenreichii subsp. shermanii showed no effect. Any combination of these bacteria can be used as probiotics in cheese-based French onion dip. However, the inoculation level should be 8 log per g for L. acidophilus and B. animalis and 7 log per g for L. paracasei subsp. paracasei, and/or L. rhamnosus to obtain greater than 6 log of individual bacterial population at the end of shelf life.     

Listeria innocua and aerobic mesophiles during chill storage of inoculated mechanically recovered poultry meat treated with high hydrostatic pressure

Mechanically recovered poultry meat (MRPM) was inoculated with Listeria innocua 910 CECT at a level of approximately 10⁸ CFU g⁻¹. Vacuum-packaged samples were treated by combinations of pressure (350, 400, 450 and 500 MPa), time (5, 10, 15 and 30 min) and temperature (2, 10 and 20°C) and later stored at 2°C for 2 months. Counts of L. innocua and aerobic mesophilic bacteria were determined 1, 4, 7, 15, 30 and 60 days after pressurisation. For mesophiles, in most treatments, pressurization at 2°C gave the significantly best results. High pressure caused a marked bactericidal effect on L. innocua: reductions higher than 7.5 log units were achieved in several cases. Some cells were just sublethally injured by pressure. Samples treated at 500 MPa for 30 min at 2°C had counts of only 2.3 log units after 60 days of chill storage. Noninoculated pressurised MRPM did not show Listeria growth throughout storage. These results suggest that high pressure processing can enhance the microbiological quality of MRPM.     

Interactive inhibition of meat spoilage and pathogenic bacteria by lysozyme,nisin and EDTA in the presence of nitrite and sodium chloride at 24 degrees C

To develop a nisin- and lysozyme-based antimicrobial treatment for use with processed ham and bologna, in vitro experiments were conducted to determine whether inhibition enhancing interactions occur between the antimicrobials lysozyme, chrisin (a commercial nisin preparation), EDTA, NaCl and NaNO₂. Inhibitory interactions were observed between a number of agents when used against specific pathogenic and spoilage bacteria. The observed interactions included lysozyme with EDTA (Enterococcus faecalis and Weissella viridescens), chrisin with EDTA (all Gram-positive organisms), EDTA with NaCl (Escherichia coli, Salmonella enterica serovar Typhimurium, Serratia grimesii), EDTA with nitrite (E. coli, Lactobacillus curvatus, Leuconostoc mesenteroides, Listeria monocytogenes, S. Typhimurium), chrisin with nitrite (Lc. mesenteroides, L. monocytogenes), and NaCl with nitrite (S. Typhimurium, Shewanella putrefaciens). Previous reports have described interactions between nisin with EDTA that resulted in enhanced antimicrobial effect against Gram-negative bacteria, or lysozyme with nisin against Gram-positive bacteria. These interactions were not observed in these experiments. We observed that unlike previous studies, these experiments were conducted on growing cells in nutrient broth, rather than under conditions of nutrient limitation. We propose that screening of antimicrobials for use in food systems in nutrient-deficient systems is inappropriate and that new protocols should be developed.     

Observations on the succession dynamics of lactic acid bacteria populations in chill-stored vacuum-packaged beef

Drip samples were collected at 4-week intervals from 10 vacuum-packaged beef striploins stored for 16 weeks at −1.5 °C and assayed for populations of lactic-acid bacteria (LAB), pH and spoilage-causing fermentation products. A total of 15 LAB species were identified using pulsed-field gel electrophoresis and biochemical analysis. A pattern of succession was observed during storage between strains of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. Acetic acid production was associated with increasing LAB populations generally and butyric acid production was associated with the development of a particular strain of Leuconostoc. Changes in pH is postulated as a driver of succession.     

Antioxidant activity of Caryocar brasiliense (pequi) and characterization of components by electrospray ionization mass spectrometry

The Caryocar brasiliense known commonly as pequi is a tropical fruit of Brazilian Cerrado and is considered an important option of income and food for the populations living in this biome. Our previous study indicated that C. brasiliense had high total phenol content (209 g as gallic acid equivalent kg⁻¹) and excellent scavenging activity against 2,2-diphenyl-1-picrylhydrazyl radical (IC₅₀ of 9.44 μg ml⁻¹). In this study, we evaluated the highly efficient antioxidant activity of C. brasiliense using the biological relevant method of chemically induced lipid peroxidation. The half inhibition concentration did not exceed 0.8 μg ml⁻¹. In addition, polar components of pequi ethanolic extract were investigated by direct infusion electrospray ionization mass spectrometry (ESI-MS). The technique revealed the presence of important bioactive components widely reported as potent antioxidants such as gallic acid, quinic acid, quercetin, and quercetin 3-O-arabinose possibly explaining its higher antioxidant activity. This is the first report on the composition by ESI-MS of pequi extract demonstrating excellent antioxidant activity.     

Antagonistic effect on Listeria monocytogenes and L. innocua of a bacteriocin-like metabolite produced by lactic acid bacteria isolated from sucuk

Two Lactobacilli and four Pediococci strains producing bacteriocin-like metabolities isolated from sucuk were tested with agar spot tests and well diffusion assays for their inhibitory activity against 16 Listeria strains, also isolated from sucuk. The production of organic acids and hydrogen peroxide limited, L. sake Lb 706 (used as a bacteriocin producer strain) and the isolated lactic acid bacteria (LAB) showed inhibitory activity against all of the Listeria strains, while L. sake Lb 706-A (used as a bacteriocin non-producer mutant) had the same effects against only two Listeria monocytogenes strains (51, 52) in agar spot tests. In the well diffusion assays, while L sake Lb 706 and four Pediococci isolates (413, 416, 419, 446) exhibited inhibitory activity against all of the Listeria strains tested, L. sake Lb 706-A and two of the Lactobacilli isolates (77, 116) showed no effect on the Listeria strains tested.     

Microbiological quality of freshly shot game in Germany

In the framework of a project on the hygiene status of freshly shot game 289 samples were microbiologically analysed: 127 samples from wild boars, 95 from roe deer and 67 from red deer. The microbiological parameters evaluated were the mesophilic aerobic count (APC), which showed mean log10-counts of 2.6 cfu/cm² for roe deer, 2.9 cfu/cm² for red deer and 3.2 cfu/cm² for wild boars and the numbers of Enterobacteriaceae, which gave mean log10-values of 2.1 cfu/cm² for all three species with differing ranges. The concentrations of coagulase positive staphylococci were >2.0 log10 cfu/cm² between 3.2 and 6.3%, according to species. Listeria was found in 14 samples and three samples gave a positive result for Campylobacter. Salmonella was not found in any of the samples analysed.     

Effect of the lactoperoxidase system on the activity of mesophilic cheese starter cultures in goat milk

The effect of goats’ milk lactoperoxidase (LP) system on the activity of commercially available mesophilic cheese starter cultures was investigated. The growth and acid production of the starter cultures were measured at 2 h intervals for 8 h in goats’ milk kept at 30°C. Most of the starter cultures examined were found to be sensitive to the LP system, but varied in their susceptibility to inhibition. The activity of the mixed starter cultures CHN11, CHN22, CHN19, DCC240 and Flora Danica Normal was strongly inhibited by the LP system. However, the mixed starter culture LL 50C showed resistance to the LP system. The single strain culture Lactococcus lactis subsp. lactis NCDO 605 was inhibited by the LP system. However, the cultures Lactococcus lactis subsp. diacetylactis NCDO 176 and Leuconostoc mesenteroides subsp. cremoris ATCC 33313 were insensitive to the LP system. The results of this study indicate the need for routine screening of starter cultures for resistance to the LP system before using them for cheesemaking from goats’ milk preserved by the LP system.     

Bacteriological quality assurance (BQA) of mechanically deboned meat (MDM)

Adequacy of bacteriological quality assurance during the commercial production of mechanically deboned meat (MDM) was assessed. Lax standards of hygiene during production were observed, resulting in high numbers of Staphylococcus aureus, viz. 10⁴ to 10⁵ cfu g⁻¹, and severe contamination with Enterobacteriaceae: 10⁵ to 10⁶ cfu g⁻¹. These data indicate that measures of hygiene observed during boning of carcasses and during collection, storage and transport of bones or poultry parts should be markedly tightened, while conditions of refrigerated storage of raw materials and MDM should be improved. Use of bones of poor sensory quality (discoloration, abnormal smell) generally resulted in MDM of inferior bacteriological quality.

Phage typing, biotyping and assessment of enterotoxin production was carried out with 136 St. aureus cultures, isolates from mechanically deboned pork produced at one plant. Fifty-five per cent of the isolates was not typable, 28% was typable with human phages, 8% with bovine phages. The majority of the strains could not be explicitly assigned to any Meyer and/or Hájek and Mar álek types. Applying the simplified system of Devriese to eighteen strains isolated in our investigation, ten were found to belong to the poultry ecovar, one to the bovine ecovar, while seven strains were non-host specific. None of the isolates produced enterotoxins A–E.

Microbiological inspection of end products is recommended as part of an integrated quality assurance system. The following reference values for the final product (maximal colony counts to be expected under GMP conditions expressed as 95th percentile) were calculated: Pig MDM: log₁₀ mesophilic colony count 6·8 and log₁₀ cfu mesophilic Enterobacteriaceae g⁻¹ 4·8; Poultry MDM: log₁₀ mesophilic colony count 6·6 and log₁₀ cfu mesophilic Enterobacteriaceae g⁻¹ 4·7.     

Growth energetics of Lactococcus lactis subsp. lactis biovar diacetylactis in cometabolism of citrate and glucose

Certain strains of lactic acid bacteria present in commercial cheese starters, characterized by faint transparent colonies on an agar plate containing 1 mg kg ⁻¹ crystal violet (CVT), were identified as Lactococcus lactis subsp. (ssp) lactis biovar diacetylactis. The effect of citrate on the growth of these strains (CVT strains) in the presence of glucose was studied, in comparison with L. lactis strains. Molar growth yield from glucose (Y_G, g dry weight/mole of glucose consumed) for CVT strains grown on glucose plus citrate was significantly higher than the control (i.e. without citrate), but not for other L. lactis strains tested. Enhanced Y_G was also observed at a pH-controlled experiment, indicating that enhanced Y_G did not result from a buffering effect of citrate. CVT strains, in contrast to other strains of the same species, were shown to obtain enough energy to enhance Y_G on glucose–citrate mixtures.     

Fermentation of salmon fillets with a variety of lactic acid bacteria

The influence of five strains of lactic acid bacteria (four Lactobacillus and one Carnobacterium) on the quality of fermented salmon fillets was studied. Best starter growth (increase of more than 1 log in 3 days) and acidification of muscle (e.g. pH reduction of approximately 0.7 units in 5 days) were achieved with the two commercial strains L. sake LAD and L. alimentarius BJ33. pH reduction was consistently lower (e.g. reduction of 0.2 units in 5 days) with C. piscicola 85. Protein breakdown as observed on SDS-PAGE gels was similar for all strains. In contrast, the starter strain did influence texture and colour changes. Fast acidifying strains L. sake LAD and L. alimentarius BJ33 brought about a firmer overall texture and a lighter colour, while softening of flesh occurred in samples processed with C. piscicola 85. Sensory evaluations indicated that samples processed with fast acidifying strains were preferred. L. sake LAD and L. alimentarius BJ33 are regarded as suitable starters for fermentation of salmon fillets.