The influence of oxygen on the differentiation of temperature-sensitive and temperature-insensitive Lactococcus lactis subsp. cremoris starter strains

The differentiation of temperature-insensitive and temperature-sensitive strains of Lactococcus lactis subsp. cremoris by using a modified sodium-β-glycerophosphate/milk medium is described (temperature-insensitive strains are defined as those that continue to grow at 38°C and temperature-sensitive strains as those that do not grow, or grow poorly, at 38°C). The physiological basis for the differentiation assay was examined by using L. lactis subsp. cremoris strains 2146 (temperature-insensitive) and 2182 (temperature-sensitive) as test strains. After aerobic incubation on the medium, strain 2146 formed uniform colonies, 0·5 mm in diameter, while strain 2182 formed larger colonies, 1·0–1·5 mm in diameter. The differential was dependent on the medium constituents, on an aerobic gas phase, and on the effects of H₂O₂ generated within the colonies. The addition of 0·5% (w/v) pyruvate to the medium facilitated the growth of colonies of strain 2146 to 3-mm diameter, while the colony size of strain 2182 remained at 1-mm diameter, and thus the colony-size differential between strains was reversed. The growth of both strains was inhibited at 0·4–0·8% air saturation during suspension culture in sodium-β-glycerophosphate/milk medium. The inclusion of catalase in the cultures overcame the growth inhibition. There was no observable difference between the two strains in their oxygen sensitivity or NADH oxidase/peroxidase enzymology.     

Carnocin U149, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various Gram-positive bacteria including Listeria sp.

This paper describes a simple purification method for the purification of carnocin U149, a potential biopreservative produced by Carnobacterium piscicola U149. The protocol was also applicable for the isolation of nisin Z, which is a biopreservative produced by Lactococcus lactis SIK-83. The protocol consists of only two purification steps, XAD chromatography and cation exhange chromatography. It is quick, easy, and can be used for large scale purification of these lantibiotics. The bactericidal activity of carnocin U149 against carnobacteria, lactococci and Listeria was compared with that of nisin Z. The carnobacteria showed similar sensitivity towards carnocin U149 and nisin. The nisin producing L. lactis strains were very sensitive towards carnocin U149, while the non-producing L. lactis strains were more sensitive to nisin. The Listeria strains were weakly sensitive to carnocin U149, lower concentrations of nisin were needed to inhibit growth.     

Probiotic white cheese with Lactobacillus acidophilus

The objective of the present study was to determine the effects of Lactobacillus acidophilus on the sensory attributes, ripening time, and composition of Turkish white cheese and to investigate the survival of L. acidophilus during ripening of the cheese stored in vacuum or in brine. Two types of white cheeses, traditional cheese (control, made with Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris) and probiotic cheese (made with Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris and L. acidophilus 593 N), were produced and ripened in vacuum pack or in brine at 4°C for 90 days. Cheese samples were assessed for microbiological and compositional properties, proteolysis, and sensory evaluation at different ripening stages. On ripening in vacuum pack, L. acidophilus survived to numbers >10⁷ cfu g⁻¹, which is necessary for positive effects on health. Protein, dry matter, salt content, and percentage of lactic acid in the vacuum-packed and brine-salted probiotic cheeses were significantly different. Also, the lactic acid content of probiotic cheeses was slightly higher than that of the controls for both vacuum- and brine-packed cheeses. Vacuum-packed probiotic cheese had the highest levels of proteolysis and the highest sensory scores of all cheeses. Consequently, L. acidophilus could be used for the manufacturing of probiotic white cheese to shorten ripening time and vacuum packaging is the preferred storage format.     

The diversity of potential cheese ripening characteristics of lactic acid starter bacteria: 2. The levels and subcellular distributions of peptidase and esterase activities

The levels and subcellular distributions of various peptidase and esterase activities in a range of lactococcal and Streptococcus thermophilus strains were investigated. There was no correlation between the levels of the enzymes in the different strains and the ability of the strains to produce acid when grown in milk. While considerable differences between individual strains were apparent, average levels of X-prolyldipeptidyl aminopeptidase, dipeptidase and tripeptidase were similar in the Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris strains studied, while that of lysylaminopeptidase (i.e. activity assayed using lysine p-nitroanilide as substrate) in the L. lactis subsp. cremoris strains was approximately double that in the L. lactis subsp. lactis strains. The average levels of lysylaminopeptidase and X-prolyldipeptidyl aminopeptidase in the S. thermophilus strains studied were similar to those in the L. lactis subsp. cremoris strains, while the average levels of dipeptidase and tripeptidase were considerably lower. All peptidases studied were recovered predominantly in the cytoplasmic fraction, although in a few strains there was some evidence to suggest that a part of the tripeptidase activity may be associated with cell structures comprising the particulate fraction. The levels of esterase activity in the strains were considerably different between strains. However, the average level of esterase activity detected in the two lactococcal subspecies was similar, while that in the S. thermophilus strains was more than double the lactococcal average. The subcellular distribution of the esterase in all strains studied showed that a significant proportion of the activity is located on the cell surface.     

The diversity of potential cheese ripening characteristics of lactic acid starter bacteria: 1. Resistance to cell lysis and levels and cellular distribution of proteinase activities

By reference to subcellular fraction markers, the resistance to lysis of 23 strains of Lactococcus lactis subsp. cremoris, 30 strains of L. lactis subsp. lactis and five strains of Streptococcus thermophilus and the levels and distribution of proteinase activity in the strains were determined. Strains of L. lactis subsp. cremoris were readily lysed by transfer to hypotonic buffer after treatment with lysozyme alone, whilst strains of L. lactis subsp. lactis and S. thermophilus could be efficiently lysed in this way only after treatment with a combination of lysozyme and mutanolysin. With a few notable exceptions, those strains which gave the fastest rates of acid production also generally presented higher levels of cell surface proteinase, as determined by activity on fluorescein isothiocyanate-labelled β-casein. The highest levels of cell surface proteinase detected were found for strains of L. lactis subsp. cremoris. However, the levels of total proteinase activity in the lactococcal strains did not correlate with the rate of acid production in milk, some slow acid-producers yielding similar or greater total proteinase levels than fast acid-producers. Homology to DNA probes for the lactococcal cell surface proteinase gene and to the conserved region encoding the serine proteinase active site was shown by the fast acid-producing lactococcal strains, but not by most of the slow acid-producing lactococcal strains or by the strains of S. thermophilus. A significant proportion of the total proteinase activity was recovered in the subcellular fractions in which high levels of cytoplasmic marker enzyme activity were found. The total proteinase levels detected in strains ofL. lactis subsp. lactis showed a greater range of variation than in the strains of L. lactis subsp. cremoris. High levels of total proteinase activity were found in the slow acid-producers despite the strains having been grown in the presence of yeast extract. For many of the strains, the levels of proteinase released from the cell surface during cell wall degradation with lytic enzyme treatment were higher than those found using whole cells, suggesting that a significant amount of proteolytic activity was either inaccessible to substrate or present in an inactive form.     

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.     

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.     

A model of the specific growth rate inhibition by weak acids in yeasts based on energy requirements

Zygosaccharomyces bailii, a spoilage yeast, capable of metabolic activity in food environments with low pH, low a_w and in the presence of weak acid preservatives was chosen for a study on the effect of benzoic acid on growth parameters. In batch cultures, under controlled pH, this food preservative inhibited growth, decreasing the specific growth rate (μ) and the yield coefficient (Y_S) on glucose. Data obtained at pH 3.5, 4.0 and 4.5 showed that this inhibition was exclusively promoted by the undissociated form of the acid since the effect was independent of pH when the concentration of the acid was expressed in this form. Moreover, the relationship between the values for μ and Y_S, provided evidence that the specific consumption rate of glucose (q_S) was not affected by benzoic acid, indicating that the inhibition of growth should be completely explained by a decrease of Y_S. The outcome of parallel experiments performed in continuous culture was that the decrease of Y_S was due to an increase of the maintenance coefficient (m), defined as the fraction of q_S diverted from growth to cope with stress, represented in this case by the presence of the preservative. Based on these results a model was built, assuming that m increased hyperbolically with the concentration of benzoic acid, from zero in the absence of the acid up to q_S when growth was completely inhibited. The concentration of the acid, for which m=q_S/2, is a constant (K_W), and represents a measure of the tolerance for a preservative, in this case benzoic acid. The simple equation μ/μ₀=1+W/K_W predicts the value of μ for a concentration (W) of the preservative, requiring the knowledge of two parameters: the specific growth rate in the absence of the preservative (μ₀) and K_W. The equation fitted very well the data of the effect of benzoic acid on the specific growth rate of Z. bailii, having K_W=0.96 mM benzoic acid. The model was also validated with other spoilage yeasts grown in the presence of benzoic acid in microtiter plates in an automated spectrophotometer. The values obtained for K_W under these conditions confirm Z. bailii as the most tolerant (K_W=2.1 mM) followed by Pichia sp. (K_W=0.78 mM), Saccharomyces cerevisiae (K_W=0.53 mM) and Debaryomyces hansenii (K_W=0.11 mM).     

In vitro adherence and other properties of lactobacilli used in probiotic yoghurt-like products

This study compared selected probiotic properties of 9 strains of the Lactobacillus acidophilus group (6 L. acidophilus and 3 L. johnsonii) and 9 strains of the L. casei group, isolated from probiotic dairy products sold on the German market or supplied by a producer of starter cultures. Using an in vitro system simulating gastric transit, strains of the L. acidophilus group were more tolerant to the low pH 2.0 than strains of L. paracasei and L. rhamnosus which rapidly lost their viability after exposure to the simulated gastric juice containing pepsin. Milk showed a protective effect on strains less resistant to the gastric acidity. Bile salt hydrolase activity was detected in all strains of L. acidophilus/L. johnsonii but not in strains of the L. casei group. All lactobacilli were able to adhere to mucus secreting HT29 MTX cells and to human collagen type IV, fibrinogen and fibronectin although differences in the percentage of adhering bacteria were observed between different strains.     

Antihypertensive activity of casein-enriched milk fermented by Lactobacillus helveticus

Milk proteins contain peptidic angiotensin I-converting enzyme (ACE) inhibitors, which can be released by proteolysis during milk fermentation by some strains of Lactobacillus helveticus. Reconstituted milk media containing skim milk powder (12%), skim milk powder (10%) with added sodium caseinate (5%) or whey protein isolate (5%) were fermented by L. helveticus strains R211 and R389, and further tested for bacterial growth, proteolysis (free NH₃ groups) and ACE-inhibitory activity. The antihypertensive activity in spontaneously hypertensive rats (SHR) was also investigated with caseinate-enriched milk unfermented (UFM) and fermented by the two strains of L. helveticus. Caseinate-enriched milk fermented by both strains showed higher proteolysis and ACE-inhibitory activity, indicating that ACE-inhibitory peptides are probably released from caseins during milk fermentation. Significant decreases in mean arterial blood pressure in SHR rats were measured following oral administration of UFM milk at doses of 1.0 and 2.5 g kg⁻¹ of body weight, and milk fermented by R211 or R389 strains at doses of 0.5, 1.0 and 2.5 g kg⁻¹ of body weight. The antihypertensive activity of UFM could be explained by the release of ACE-inhibitory peptides from caseins during the digestion process.     

Phenotypic and genetic diversity of Lactococcus lactis and Enterococcus spp. strains isolated from Northern Spain starter-free farmhouse cheeses

To evaluate a previous phenotypic classification of lactococci, 39 presumed lactococcal strains were classified by molecular techniques. The strains were also subjected to several typing techniques to estimate the phenotypic and genetic diversity present in original populations from starter-free farmhouse cheeses. Partial Amplified rDNA Restriction Analysis (partial ARDRA) with either restriction enzyme MboII or HhaI divided these isolates into four distinctive groups. Sequencing of representative amplicons identified 29 isolates as belonging to Lactococcus lactis subsp. lactis (24) and Lactococcus lactis subsp. cremoris (5). The remaining 10 isolates were shown to be Enterococcus durans (8) and Enterococcus faecalis (2), which were misclassified by the traditional tests. Thus, partial ARDRA was successfully used to classify wild Lactococcus-like strains into Lactococcus and Enterococcus species. The technique also allowed differentiation of L. lactis strains at subspecies level. The 29 strains of L. lactis showed five different fermentation profiles, four distinct Random Amplification of Polymorphic DNA (RAPD) profiles, and 14 unrelated profiles by both Restriction Fragment Length Polymorphism analyzed by Pulsed Field Gel Electrophoresis (RFLP-PFGE) and Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Using the same techniques, the 10 enterococcal strains showed four fermentation profiles, four RADP, and six by RFLP-PFGE and SDS-PAGE, respectively. Several typing techniques, especially RFLP-PFGE and SDS-PAGE, revealed wide phenotypic and genetic variability in both the lactococcal and enterococcal isolates. Two simple, rapid and cheap techniques (partial ARDRA and SDS-PAGE) are proposed as reliable tools for the classification and typing of new lactococcal-like isolates.     

Characterization of Listeria spp. isolated from ready-to-eat products in Argentina using SDS–PAGE and restriction endonuclease

Listeria spp. are considered of interest in public health since their presence indicates the potencial existence of L. monocytogenes. Total cellular proteins and DNA from four strains of L. monocytogenes serotype 4, four strains of L. monocytogenes belonging to serotype 1, twelve strains of L. innocua, four strains of L. seeligeri and two strains of L. welshimeri isolated from ready–to–eat food were studied by SDS–PAGE and restriction endonuclease digestion. SDS–PAGE protein profiles obtained were species specific and could be evaluated by visual comparison. Enzyme for restriction endonuclease analysis was EcoRI, discriminating L. monocytogenes from other Listeria spp. These methodologies might be a helpful tool and a good alternative for epidemiological tracking of listeriosis in laboratories, where other methods are not available.     

rRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime

The rRNA gene restriction patterns (ribotypes) of 69 ropy slime producing Lactobacillus sake strains isolated mainly from vacuum-packaged meat products of ten meat plants were determined. Ribotypes were compared to the corresponding patterns of non-ropy L. sake strains, and also to other species of the genus Lactobacillus, Carnobacterium and Weissella associated with meat products. Ropy slime-producing L. sake strains were divided into four characteristic groups corresponding to the phenotypic carbohydrate grouping. No association between certain ribotypes and individual plants was detected. Ribotyping was unable to distinguish slime producing and non-ropy strains of L. sake group sharing the same carbohydrate pattern. Otherwise, ribotyping distinguished the ropy slime producing strains from the non-ropy L. Sake reference strains and all L. sake strains from other species of the genus Lactobacillus, Carnobacterium and Weissella. These results suggest that ribotyping is a suitable method for detection and surveillance of the contamination of ropy slime-producing L. sake strains but the patterns alone cannot be used as markers of slime production capability.     

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.     

Inhibition of Listeria monocytogenes by a bacteriocinogenic Lactobacillus sake strain in modified atmosphere-packaged Brazilian sausage

Lactobacillus sake 2a is a bacteriocinogenic strain isolated from “lingüiça frescal”, a Brazilian sausage. The combined effect of modified-atmosphere (MA) packaging (100% CO₂ and 50% CO₂/50% N₂) and addition of L. sake 2a on inhibition of growth of Listeria monocytogenes was evaluated in “lingüiça” stored at 6 °C. By the end of the first week, the inhibition of L. monocytogenes due to MA was significant (P0.05) while the presence of L. sake 2a did not influence significantly the growth of the pathogen. After 14 days, a reduction of 1.3–1.4 log in counts of L. monocytogenes was observed in samples containing L. sake 2a only or MA packaged only, while a reduction of 3.5 log was detected in those submitted to both treatments. Results indicate that inhibition of L. monocytogenes in “lingüiça frescal” by the bacteriocinogenic L. sake 2a is enhanced by the packaging of the product in MA.     

PCR confirms multiple Wolbachia strain infection in Australian and international populations of the invasive stored-product psocid Liposcelis bostrychophila Badonnel

Diagnostic Wolbachia polymerase chain reaction (PCR) primers were used to investigate whether Liposcelis bostrychophila individuals from Australian populations were infected by Wolbachia. Wolbachia PCR diagnostic primers were used to amplify Wolbachia from single individuals, individuals subjected to multiple displacement amplification (MDA), and from groups of about 50 pooled individuals each representing an Australian or United Kingdom (UK) L. bostrychophila population. Wolbachia PCR diagnostic primers successfully amplified DNA from pooled L. bostrychophila DNA extractions but not from individual L. bostrychophila DNA extractions or from MDA-subjected samples from both the Australian and UK populations. Use of restriction fragment-length polymorphism (RFLP) techniques produced cleavage sites in Australian and UK L. bostrychophila populations that were diagnostic of Wolbachia strains A and B. This study provides the first evidence of multiple Wolbachia infection with strains A and B in Australian L. bostrychophila populations.     

Evaluation of culture media for enrichment and isolation of Shigella sonnei and S. flexneri

The performance of Gram-negative (GN) broth with (10 μg/ml) and without novobiocin, Shigella broth (SB) with 0.5 and 3.0 μg/ml novobiocin, all incubated at 37 °C (SB with 3.0 μg/ml novobiocin also at 42 °C) and buffered brilliant green bile glucose (EE) broth with 1.0 μg/ml novobiocin incubated at 37 and 42 °C were evaluated for resuscitation and growth of Shigella sonnei and S. flexneri (eight strains; unstressed, chill-stressed and acid-stressed) and non-shigellae (11 strains). GN broth with or without novobiocin supported significantly less growth of Shigella sp. No significant differences in growth of shigellae were obtained between the other culture media. Performance depended more on the Shigella strain used. None of the tested media were significantly superior for suppressing the competitive flora.

Electivity and selectivity of MacConkey agar (MAC), tergitol-7 agar (T7), desoxycholate citrate agar (DCA), xylose lysine desoxycholate agar (XLD), Salmonella Shigella agar and Hektoen enteric agar (HEA) were determined by ecometric testing. HEA confirmed to be a high selective medium for both non-shigellae and stressed Shigella sp. Klebsiella sp., Enterobacter sp., Citrobacter sp., Salmonella sp. and the Escherichia strains can mask the presence of shigellae.

In vitro competition experiments and experiments with artificially contaminated foods showed higher resistance of S. sonnei than S. flexneri towards the stress imposed by the food matrix and its indigenous flora. Reliable detection, however, of shigellae in foods with the current enrichment and isolation media was not achieved.     

Survival of pathogenic microorganisms in an egg-nog-like product containing 7% ethanol

A liquor consisting of whole egg, saccharose (25% w/v) and ethanol (7.0% w/v) was artificially contaminated with Salmonella enteritidis, S. typhimurium, Staphylococcus aureus (three different strains), Bacillus cereus and Listeria monocytogenes. After 3 weeks of incubation at 22°C the numbers of Salmonella, S. aureus and L. monocytogenes decreased more than 3 log₁₀ units. Under such conditions, however, the total number of microorganisms increased 3 log₁₀ units. At 4°C the decrease of pathogenic microorganisms was much slower and a decrease of 3 log₁₀ units was observed only after 7 weeks of incubation. Egg-nog, without ethanol, incubated at 22°C allowed growth of Salmonella and S. aureus, while the numbers of B. cereus spores remained unchanged. Vegetative cells of B. cereus as well as L. monocytogenes decreased in numbers. However, after prolonged incubation the numbers of L. monocytogenes increased significantly.     

Growth of Listeria monocytogenes on vacuum-packaged horsemeat for human consumption

In order to investigate the likelihood of Listeria monocytogenes (serotype 4b, ATCC 19115) growth on vacuum-packaged horsemeat at refrigeration temperature, fourteen horsemeat surface/volume homogeneous 150 g weight pieces were superficially inoculated with serotype 4b L. monocytogenes and vacuum packaged. The samples were stored at 4 ± 1 °C. Two pieces (one for pH determination and one for L. monocytogenes counts) were examined at days 0, 7, 14, 21, 28, 35 and 42. Surface pH did not show significant variations during the experiment. The average L. monocytogenes initial contamination level was 1.77log₁₀ CFU/g. A lag phase of 7 days was recorded. The exponential growth rate between day 7 to day 35 was 0.125log₁₀ CFU/day, corresponding to 3.51log₁₀ CFU/g in 28 days. At the end of the experiment the mean L. monocytogenes log₁₀ CFU/g was 5.78.