The effect of adding antimicrobial peptides to milk inoculated with Staphylococcus aureus and processed by high-intensity pulsed-electric field.

The use of high-intensity pulsed-electric field (HIPEF) and antimicrobial substances of natural origin, such as enterocin AS-48 (AS-48), nisin, and lysozyme, are among the most important nonthermal preservation methods. Thus, the purpose of this study was to evaluate the combined effect on milk inoculated with Staphylococcus aureus of the addition of AS-48 with nisin or lysozyme, or both, together with the use of HIPEF. Synergy was observed in the reduction of Staph. aureus counts with the following combination methods: i) addition of AS-48 and nisin, ii) addition of AS-48 plus use of HIPEF, and iii) addition of AS-48 and nisin plus use of HIPEF. Specifically, when 28 arbitrary units/mL of AS-48 and 20 IU/mL of nisin were added to the milk, and it was treated with HIPEF for 800 mus, over 6 log reductions were observed in the microorganism. In general, Staph. aureus inactivation was dependent on HIPEF treatment time, antimicrobial doses, and medium pH. During storage of the treated milk, survivor population was related to peptide concentration and temperature. Final cell viability was influenced by the sequence in which the treatments were applied: the addition of AS-48 or AS-48 and nisin was more effective before than after HIPEF treatment. The results obtained indicate that the combination of HIPEF and antimicrobials could be of great interest to the dairy industry, although it is necessary to study further the way in which the combined treatments act.

Safety and potential risks of enterococci isolated from traditional fermented capers.

A collection of 17 enterococci isolates obtained from fermentations of capers (the fruits of Capparis sp.) were investigated for incidence of known virulence determinants, antibiotic resistance and production of biogenic amines. Molecular identification revealed the presence of Enterococcus faecium (nine isolates), Enterococcus faecalis (4), E. avium (3) and Enterococcus casseliflavus/flavescens (1). Alpha-haemolytic activity was detected in two E. avium and one E. faecalis isolates, and beta-haemolytic activity was detected in E. casseliflavus/flavescens. The haemolytic component cylB was detected by PCR amplification in three non-haemolytic isolates and in E. casseliflavus/flavescens. The collagen adhesin ace gene and the endocarditis associated antigen gene efaA(fm) were detected in two isolates each. Genes encoding sex pheromone precursors (cpd, cob, ccf) were detected in E. faecalis and E. casseliflavus/flavescens. Other presumed virulence genes (agg, gelE, cylM, cylA and efaA(fs)) were not detected. All isolates were resistant to rifampicin, erythromycin and ciprofloxacin, and some were also resistant to quinupristin/dalfopristin, tetracycline, levofloxacin, gentamicin and streptomycin. Vancomycin resistance was not detected. Tyrosine decarboxylation was detected in all E. faecium isolates. Given the high resistance of enterococci to environmental conditions, and their implication in opportunistic infections, the incidence of potential virulent enterococci in foods (especially those of a higher risk-like home-made foods) should be carefully studied.

Application of the broad-spectrum bacteriocin enterocin AS-48 to inhibit Bacillus coagulans in canned fruit and vegetable foods.

The enterococcal bacteriocin (enterocin) AS-48 is a broad-spectrum cyclic peptide. Enterocin AS-48 was tested against Bacillus coagulans in three vegetable canned foods: tomato paste (pH 4.64), syrup from canned peaches (pH 3.97), and juice from canned pineapple (pH 3.65). When vegetative cells of B. coagulans CECT (Spanish Type Culture Collection) 12 were inoculated in tomato paste supplemented with 6 microg/ml AS-48 and stored at different temperatures, viable cell counts were reduced by approximately 2.37 (4 degrees C), 4.3 (22 degrees C) and 3.0 (37 degrees C) log units within 24 h storage. After 15-days storage, no viable cells were detected in any sample. Strain B. coagulans CECT 561 showed a poor survival in tomato paste, but surviving cells were also killed by AS-48. The bacteriocin was also very active against B. coagulans CECT 12 vegetative cells in juice from canned pineapple stored at 22 degrees C, and slightly less active in syrup from canned peaches. In food samples supplemented with 1.5% lactic acid, enterocin AS-48 (6 microg/ml) rapidly reduced viable counts of vegetative cells below detection limits within 24 h storage. Addition of glucose and sucrose (10% and 20%) significantly increased bacteriocin activity against vegetative cells of B. coagulans CECT 12. Enterocin AS-48 had no significant effect on B. coagulans CECT 12 spores. However, the combined application of AS-48 and heat (80-95 degrees C for 5 min) significantly increased the effect of thermal treatments on spores.

Control of Alicyclobacillus acidoterrestris in fruit juices by enterocin AS-48.

Alicyclobacillus acidoterrestris is a spoilage-causing bacterium in fruit juices. Control of this bacterium by enterocin AS-48 from Enterococcus faecalis A-48-32 is described. Enterocin AS-48 was active against one A. acidocaldarius and three strains of A. acidoterrestris tested. In natural orange and apple juices incubated at 37 degrees C, vegetative cells of A. acidoterrestris DSMZ 2,498 were inactivated by enterocin AS-48 (2.5 microg/ml) and no growth was observed in 14 days. In commercial fruit juices added of AS-48 (2.5 microg/ml) and inoculated with vegetative cells or with endospores of strain DSMZ 2,498, no viable cells were detected during 90 days of incubation at temperatures of 37 degrees C, 15 degrees C or 4 degrees C, except for apple, peach and grapefruit juices inoculated with vegetative cells and incubated at 37 degrees C which were protected efficiently for up to 60 days. Remarkably, in all commercial fruit juices tested, no viable cells were detected as early as 15 min after incubation with the bacteriocin. Endospores incubated for a very short time (1 min) with increasing bacteriocin concentrations were inactivated by 2.5 microg/ml AS-48. Electron microscopy examination of vegetative cells and endospores treated with enterocin AS-48 revealed substantial cell damage and bacterial lysis as well as disorganization of endospore structure.

Effect of virgin and refined olive oil consumption on gut microbiota. Comparison to butter.

There is increasing evidence of the health benefits of olive oil consumption in the diet. Some authors have studied the effect of high fat/high calorie diets and have detected changes on the microbiota. However, these studies are mainly based on saturated fats. Here we present a study on the specific effect on gut bacterial populations of extra virgin olive oil, rich in monounsaturated fatty acids and phenolic compounds, in comparison to refined olive oil, rich in monounsaturated fatty acids but low in phenolic compounds, and to butter, rich in saturated fatty acids and cholesterol. Four groups of animals were studied: one group of mice received a standard chow diet, and the other received three high fat diets, rich in extra virgin olive oil, refined olive oil or butter. Evolution of symbiont population in feces was studied using culture-dependent and culture-independent methods. In the latter, the V3 region of 16S rDNA was amplified and separated by denaturing gradient gel electrophoresis; followed by sequencing of the most representative bands. Culture-dependent studies and comparison of the different DGGE profiles throughout the experiment demonstrated that different dietary fats had different effects on gut microbial composition. Butter-induced changes in the microbial counts resembled those previously described in obese individuals. Interestingly, a different behavior between extra virgin and refined olive oil was also observed, extra virgin olive oil being most different from butter. To our knowledge, no studies have analyzed gut microbiota depending on diets with different fatty acid saturations including different types of olive oil. This may offer new data supporting the benefits for health of extra virgin olive oil, so important in the Mediterranean diet.

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

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

Characterization of lactobacilli isolated from caper berry fermentations.

AIMS: To determine the metabolic and functional properties of lactobacilli isolated from caper fermentation. METHODS AND RESULTS: A collection of 58 lactobacilli from fermentation of caper berries (including species of Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus brevis and Lactobacillus fermentum) was studied. Strains were classified in different clusters according to sugar fermentation patterns. Most strains of L. plantarum (the predominant species in the fermentation) clustered in a single group. Analysis of enzymatic activities revealed a high incidence of leucine aminopeptidase, acid phosphatase, beta-galactosidase and beta-glucosidase among the different strains of lactobacilli. A high number of strains were able to degrade raffinose and stachyose. Phytase activity and bile salt hydrolase activity were only detected in certain strains of L. plantarum. CONCLUSIONS: Lactobacilli from caper fermentation are metabolically diverse, and some strains display functional properties of interest. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of lactobacilli with selected functional properties could be good candidates for future development of commercial starters for industrial caper fermentation.

Bacteriocin production, plasmid content and plasmid location of enterocin P structural gene in enterococci isolated from food sources.

AIMS: To characterize bacteriocin production, antimicrobial spectrum and plasmid content in bacteriocinogenic enterococci from foods. METHODS AND RESULTS: Bacteriocinogenic Enterococcus faecium (14 isolates) and Enterococcus faecalis (three isolates) showed two different patterns of bacteriocin production in liquid broth: exponential-phase and stationary-phase production. Bacteriocin concentrates from all enterococci were inactivated by trypsin, but seldom by heat (100-117 degrees C), extremes of pH (2.0 to 9.0) or reducing agents (such as dithiothreitol). All bacteriocin concentrates were active against Listeria innocua and Listeria monocytogenes, and most were also active against many Ent. faecalis and Ent. faecium isolates. Enterococci clustered in three main groups according to their plasmid content (which included plasmids from 2.0 to 53 kb). Several isolates from different foods showed almost identical plasmid profiles. The enterocin P structural gene (entP) was detected by hybridization on plasmids of c. 19, 26 and/or 35-38 kb. CONCLUSIONS: Enterococci from food show different patterns of bacteriocin production and different plasmid content in spite of carrying similar bacteriocin-encoding genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the diversity of bacteriocinogenic enterococci from food sources carrying apparently similar enterocin genes.

Inhibition of Bacillus licheniformis LMG 19409 from ropy cider by enterocin AS-48.

AIMS: To determine the activity of enterocin AS-48 against ropy-forming Bacillus licheniformis from cider. METHODS AND RESULTS: Enterocin AS-48 was tested on B. licheniformis LMG 19409 from ropy cider in MRS-G broth, fresh-made apple juice and in two commercial apple ciders (A and B). Bacillus licheniformis was rapidly inactivated in MRS-G by 0.5 microg ml(-1)AS-48 and in fresh-made apple juice by 3 microg ml(-1). Concentration-dependent inactivation of this bacterium in two commercial apple ciders (A and B) stored at 4, 15 and 30 degrees C for 15 days was also demonstrated. Counts from heat-activated endospores in cider A plus AS-48 decreased very slowly. Application of combined treatments of heat (95 degrees C) and enterocin AS-48 reduced the time required to achieved complete inactivation of intact spores in cider A to 4 min for 6 microg ml(-1) and to 1 min for 12 microg ml(-1). D and z values also decreased as the bacteriocin concentration increased. CONCLUSION: Enterocin AS-48 can inhibit ropy-forming B. licheniformis in apple cider and increase the heat sensitivity of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study support the potential use of enterocin AS-48 to control B. licheniformis in apple cider.

Molecular characterization, technological properties and safety aspects of enterococci from 'Hussuwa', an African fermented sorghum product.

AIMS: To identify enterococci from Hussuwa, a Sudanese fermented sorghum product, and determine their technological properties and safety for possible inclusion in a starter culture preparation. METHODS AND RESULTS: Twenty-two Enterococcus isolates from Hussuwa were identified as Enterococcus faecium by using phenotypic and genotypic tests such as 16S rDNA gene sequencing, RAPD-PCR and restriction fragment length polymorphism of the 16S/23S intergenic spacer region fingerprinting. Genotyping revealed that strains were not clonally related and exhibited a considerable degree of genomic diversity. Some strains possessed useful technological properties such as production of bacteriocins and H2O2 or utilization of raffinose and stachyose. None produced alpha-amylase or tannase. A safety investigation revealed that all strains were susceptible to the antibiotics ampicillin, gentamicin, chloramphenicol, tetracycline and streptomycin, but some were resistant to ciprofloxacin, erythromycin, penicillin and vancomycin. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some strains. CONCLUSIONS: Enterococcus faecium strains are associated with fermentation of Sudanese Hussuwa. Some strains exhibited useful technological properties such as production of antimicrobial agents and fermentation of indigestible sugars, which may aid in stabilizing and improving the digestibility of the product respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococci were shown to play a role in the fermentation of African foods. While beneficial properties of these bacteria are indicated, their presence in this food may also imply a hygienic risk as a result of antimicrobial resistances or presence of virulence determinants.

Influence of physico-chemical factors on the oligomerization and biological activity of bacteriocin AS-48.

Bacteriocin AS-48 forms a mixture of monomers and oligomers in aqueous solutions. Such oligomers can be clearly differentiated by SDS-PAGE after formaldehyde crosslinking, and we have verified that these associates are stable to acid treatment after fixation. In addition, they show antimicrobial activity and are recognized by anti-AS-48 antibodies. AS-48 oligomers can be dissociated by the detergents SDS and Triton X-100. The degree of oligomerization of AS-48 depends on the pH of the solution and the protein concentration. At pH below 5, AS-48 is in the monomeric state at protein concentrations below 0.55.mg ml(-1), but it also forms dimers above this protein concentration. This bacteriocin forms oligomers at pH values above 5, in agreement with the observation that it is also more hydrophobic at neutral pH. AS-48 is stable to mild heat treatments irrespectively of pH. At 120 degrees C it is more heat resistant under acidic conditions, but it inactivates at neutral pH. Activity of AS-48 against E. faecalis is highest at neutral pH, but it is highest at pH 4 for E. coli. The influence of pH on bacteriocin activity could be owing to changes in the conformation/oligomerization of the bacteriocin peptide as well as to changes in the surface charge of the target bacteria.

Response of Salmonella choleraesuis LT2 spheroplasts and permeabilized cells to the bacteriocin AS-48.

The bacteriocin AS-48 was not active against intact cells of Salmonella choleraesuis LT2 at neutral pH, but it was very effective on spheroplasts, suggesting that the outer membrane (OM) acts as a protective barrier. Cells sublethally injured by heat or treated with OM-permeabilizing agents (i.e., EDTA and Tris) became sensitive to AS-48. The combination of two or more treatments decreased the amount of AS-48 required for cell killing. The activity of AS-48 against heat-injured cells did not change significantly in the pH range of 4.0 to 8.0. AS-48 showed bactericidal activity against intact cells of Salmonella at pH 4.0. The potency of AS-48 increased greatly when the bacteriocin was dissolved at pH 9.0.

Isolation and characterization of enterocin EJ97, a bacteriocin produced by Enterococcus faecalis EJ97.

The bacteriocinogenic strain of Enterococcus faecalis EJ97 has been isolated from municipal waste water. It produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,340 Da) that is very stable under mild heat conditions and is sensitive to proteolytic enzymes. The amino acid sequence of the first 18 N-terminal residues of enterocin EJ97 indicates that it is different from other known protein sequences. Enterocin EJ97 is active on several gram-positive bacteria including enterococci, several species of Bacillus, Listeria, and Staphylococcus aureus. The producer strain is immune to bacteriocin. Enterocin EJ97 has a concentration-dependent bactericidal and bacteriolytic effect on E. faecalis S-47.

Antimicrobial activity of enterocin EJ97 against 'Bacillus macroides/Bacillus maroccanus' isolated from zucchini purée.

AIMS: Activity of the bacteriocin EJ97 produced by Enterococcus faecalis EJ97 against strains of 'Bacillus macroides/B. maroccanus' isolated from spoiled zucchini purée was investigated. METHODS AND RESULTS: The influence of several factors like bacteriocin concentration, incubation temperature, pH, growth medium and chemical perservatives on bacteriocin activity was investigated. Enterocin EJ97 [2 arbitrary units (AU) per millilitre] had a marked bactericidal effect on strain INRA P53-2 after 4 h of incubation at 37 degrees C, 24 h at 15 degrees C or 48 h at 4 degrees C. Activity was markedly reduced at pH values of 5.0 and 9.0, but was potentiated by sodium nitrite, sodium benzoate, sodium lactate and sodium tripolyphosphate. Inhibition of strain INRA P53-2 in a commercial vegetable purée required a 10-fold higher bacteriocin concentration. Strain EJ97 was able to grow and produce bacteriocin on vegetable purée, but no inhibition of strain INRA P53-2 was detected. CONCLUSIONS: The concentration-dependent bactericidal activity of enterocin EJ97 against strain INRA P53-2 was higher at 37 degrees C and neutral pH, and was potentiated by chemical preservatives. Although enterocin EJ97 was less active in vegetable purée, the concentrations providing bactericidal activity in this food matrix are practical for commercial use. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterocin EJ97 may have a potential for use in the prevention of food spoilage caused by 'B. macroides/B. maroccanus'.

Monolayer Characteristics of Bacteriocin AS-48, pH Effect and Interactions with Dipalmitoyl Phosphatidic Acid at the Air-Water Interface.

Bacteriocin AS-48 produced by Enterococcus faecalis S-48 is a ribosomally synthesized cyclic peptide (7.4 kDa) of broad inhibitory spectrum against Gram-positive and Gram-negative bacteria. Simple monolayers of AS-48 and of dipalmitoyl phosphatidic acid (DPPA) at the air-water interface are studied. The AS-48 interfacial behavior in the function of pH explains the biological activity of the peptide. The lipid monolayers show the characteristic behavior of phosphatidic acid at the mentioned interface. The interactions between AS-48 and DPPA, a majority lipid of the bacterial cell membrane, are quantitatively investigated. The results indicate that only when the lipid molecules are charged enough (pH 10.5) is an attractive interaction between AS-48 and DPPA observed, although under these experimental conditions the results seem to indicate that a deformation of the peptide helical structure could take place. Copyright 2001 Academic Press.