J. Richard

Purification, partial characterisation and mode of action of enterococcin EFS2, an antilisterial bacteriocin produced by a strain of Enterococcus faecalis isolated from a cheese

Enterococcus faecalis strain EFS2, isolated from the surface of a traditional cheese, produced a bacteriocin active against Gram-positive bacteria including Listeria spp. and some Staphylococcus aureus strains. The bacteriocin, named enterococcin EFS2, has been purified to homogeneity by ammonium sulphate precipitation and reversed-phase high performance liquid chromatography (RP-HPLC). The molecular weight was determined by mass spectrometry to be 7149.6. The amino acid composition of enterococcin EFS2 revealed that it contained 67 amino acid residues and had a blocked amino-terminal end. Enterococcin EFS2 induced viability loss, efflux of K+ ions and ATP, and cell lysis. Kinetic study of bactericidal activity of enterococcin EFS2 on Listeria innocua strain LIN11 indicated slower cell destruction than by nisin. At pH 7.0, the activity of enterococcin EFS2 was the highest at 35 degrees C and was lost at 15 degrees C. The bacteriocin was more active against L. innocua strain LIN11 in broth adjusted to pH 6.0, 7.0 and 8.0 than to pH 4.5 at 30 degrees C.

Antilisterial activity of three bacteriocins used at sub minimal inhibitory concentrations and cross-resistance of the survivors

The kinetics of inhibition of Listeria innocua strain Lin11 in a nutrient broth containing either nisin, pediocin AcH, or enterococcin EFS2 at concentrations ensuring a 2-3 log reduction of the population were first investigated. The rate of inhibition differed considerably between the bacteriocins. At the time that maximum viability loss occurred in the cultures containing either nisin (12 AU ml(-1)) or pediocin AcH (50 AU ml(-1)) the survivors resumed growth, although the medium retained most of its initial inhibitory activity. The survivors displayed increased resistance not only toward the bacteriocin they were in contact with, but toward the two other bacteriocins under study.

Natural variation in susceptibility of Listeria strains to class IIa bacteriocins

Abstract. Thirty-one Listeria strains were tested for sensitivity to four class IIa bacteriocins, namely, enterocin A, mesentericin Y105, divercin V41, and pediocin AcH, and to nisin A. Class IIa bacteriocins displayed surprisingly similar antimicrobial patterns ranging from highly susceptible to fully resistant strains, whereas nisin A showed a different pattern in which all Listeria strains were inhibited. Particularly, it was observed that the strain Listeria monocytogenes V7 could not be inhibited by any of the class IIa bacteriocins tested. These observations suggest that Listeria strains resistant to the whole range of class IIa bacteriocins may occur in natural environments, which could be of great concern with regard to the use of these peptides as food preservatives.

Isolation and identification of cheese-smear bacteria inhibitory to Listeria spp.

A newly developed hydrophobic grid membrane method was used to rapidly screen 105 traditional French cheeses for surface smear microorganisms inhibitory to Listeria monocytogenes strain V7. Approximately 125,000 colonies comprising a wide variety of bacteria were examined of which less than 0.1% produced visible zones of inhibition. Isolates possessing antilisterial activity consisted of various strains of Enterococcus faecalis, Staphylococcus xylosus, Staphylococcus warneri and coryneform bacteria, including one orange coryneform resembling Brevibacterium linens. All strains of E. faecalis and the orange coryneform that inhibited L. monocytogenes V7 exhibited strong inhibition against a panel of 21 Listeria strains comprised of L. monocytogenes (14 strains), L. innocua (two strains), L. ivanovii (two strains), L. seeligeri (two strains) and L. welshimeri (one strain). The remaining cheese isolates showed moderate to weak inhibition towards the same set of Listeria strains. Inhibitory substances produced by all strains except the orange coryneform were sensitive to one or more of five proteolytic enzymes tested and were therefore classified as bacteriocin-like inhibitory agents.

Catabolism of L-phenylalanine by some microorganisms of cheese origin

The ability of 42 microorganisms isolated from cheese, including strains of yeasts, Geotrichum candidum, Arthrobacter, Moraxella spp., Brevibacterium linens and Staphylococcus saprophyticus spp. to produce phenethyl alcohol (PEA) from L-phenylalanine was studied. All the yeast strains produced labelled PEA from L-[U- 14 C]phenylalanine. Phenylpyruvic acid was detected as an intermediate of PEA production and CO 2 was produced by decarboxylation of this acid. For five strains tested, the level of PEA which had accumulated in the culture at the end of exponential growth phase represented 39–52% of the L-Phe added. None of G. candidum strains nor bacterial isolates produced PEA from L-Phe. Strains of Moraxella spp. and four strains of the S. saprophyticus group produced phenylacetaldehyde. For three strains of Arthrobacter spp., five of B. linens and all the Moraxella , 14 CO 2 produced from uniformly labelled L-Phe represented more than one carbon atom of the L-Phe molecule, suggesting that Phe was catabolized beyond the stage of phenylacetic acid. Production and disappearance of PEA during Camembert cheese ripening is probably related to the metabolic activity of microorganisms present at the cheese surface.

Nutritional requirements of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum for growth in milk

Growth of Leuconostoc mesenteroides in milk was studied with respect to the proteinase and peptidase activities of the strains and their nutritional requirements. L. mesenteroides grew poorly in milk since none of the 14 strains studied exceeded 5 x 10 8 cfu/ml at the end of growth. Few strains displayed proteinase activity, and this did not contribute much to growth. The pattern of peptidase activities varied with the strain. Nitrogen starvation and a high requirement for Mn 2+ were involved in the cause of growth deficiencies. Addition of amino acids, 50 mg Mg 2+ /l and 0.08-0.49 mg Mn 2+ /l stimulated growth of most Leuconostoc strains up to 5 x 10 8 cfu/ml. Addition of 5 g glucose/l to milk containing amino acids, Mg 2+ and Mn 2+ or yeast extract stimulated the growth of seven and eight strains respectively up to 10 9 cfu/ml. No growth advantage was found in a N 2 atmosphere. However, the addition of small amounts of Mn 2+ to milk suppressed the inhibitory effect of aeration on the growth of L. mesenteroides UD23, suggesting a protective role of Mn 2+ against O 2 toxicity.

Nutritional value of the non-protein N that accumulates during growth of proteinase-positive strains of Lactococcus lactis in milk for dairy lactococcal and leuconostoc isolates

To estimate the suitability of cultured milk for the subsequent growth of dairy lactic acid bacteria in cheese manufacturing, control milk was first cultured until the end of the exponential phase with one of four proteolytic (Prt + ) strains of Lactococcus lactis differing in their proteolytic enzymes, and pasteurized after readjusting the pH to its initial value. Nineteen non-proteolytic (Prt − ) strains of Lc. lactis and nine Leuconostoc mesenteroides strains of dairy origin were then grown in the precultured milk until the stationary phase and their growth was compared with that in control milk. Despite the accumulation of non-protein N (NPN) during preculture, the growth of most Prt − strains of Lc. lactis in precultured milk was either reduced or unchanged whereas that of Ln. mesenteroides strains was unchanged or slightly stimulated. This reduction in growth was reversed by adding an NPN source to precultured milk, indicating that it was due to the exhaustion of assimilable NPN in precultured milk. Thus, preculturing milk with Prt + strains of Lc. lactis could not be recommended for promoting the subsequent growth of starter cultures in cheese manufacturing.

Detection of bacteriocin activity in bacteria using hydrophobic grid membrane filters

Seven known bacteriocin‐producing bacterial strains were examined using three newly developed bacteriocin detection methods employing hydrophobic gridmembrane niters (HGMF): (i) filtration, (ii) multiple square inoculation, and (iii) one square inoculation, and the three reference methods: (i) simultaneous direct cross‐streak, (ii) deferred direct cross‐streak, and (iii) agar disc. The HGMF membrane multiple square inoculation method detected bacteriocin activity in all seven strains. This method also yielded similar or slightly better results than those obtained using the deferred direct cross‐streak or agar disc methods. Bacteriocin activity was observed in six of seven and four of seven strains using the one square inoculation and filtration methods, respectively, with corresponding inhibiting zones measuring 2–24 and 6–20 mm in diameter.

Antibacterial activity among Pseudomonas strains of meat origin

Almost 750 Pseudomonas strains from meat, fish, milk or plant were screened for inhibitory potential towards 10 selected Pseudomonas fragi isolated from meat. Only strains of fish origin and a reference Ps. putida strain produced an inhibitory substance which was either a siderophore or a bacteriocin‐like substance. The two systems were efficient in meat extract medium suggesting potential use for controlling meat quality.

Use of bacteriocin producing starters advantageously in dairy industry

Firstly, the technological interest drawn from the determination of MICs (Minimum Inhibitory Concentrations) is discussed along with results demonstrating the efficacy of the bacteriocins in different food products. Then, the results of a reference study on the anti-listerial activity of low concentrations of nisin are presented, using a culture medium as model and a non-pathogenic test organism, Listeria innocua. Addition of nisin to the medium caused a rapid drop in cell viability followed by regrowth of the survivors at the same rate as the control culture. The total decrease in population was used to study the combined effect of pH and temperature on the anti-listerial activity of nisin. It was also observed that survivors to nisin had increased resistance not only to this bacteriocin but towards two other bacteriocins quite different in their molecular structure. Finally, inhibition of Listeria monocytogenes in Camembert cheese made using a nisin-positive starter was compared to that in a control made with a nisin-negative starter. It is shown that unlike the two other bacteriocins under study, inhibition by nisin is limited to a narrow range of milk pH. Inhibition by nisin can be considerably improved by both adding small amounts of this compound in cheese milk when pH is most appropriate for bactericidal activity. Also, heating milk at sublethal temperatures to listeria considerably increase the bactericidal activity of nisin. Thus, use of these improvements may lead to complete eradication of listeria in soft surface-ripened cheeses if raw milk is produced under good hygienic conditions.