Response of Leuconostoc strains against technological stress factors: Growth performance and volatile profiles.

The ability of twelve strains belonging to three Leuconostoc species (Leuconostoc mesenteroides, Leuconostoc lactis and Leuconostoc pseudomesenteroides) to grow under diverse sub-lethal technological stress conditions (cold, acidic, alkaline and osmotic) was evaluated in MRS broth. Two strains, Leuconostoc lactis Ln N6 and Leuconostoc mesenteroides Ln MB7, were selected based on their growth under sub-lethal conditions, and volatile profiles in RSM (reconstituted skim milk) at optimal and under stress conditions were analyzed. Growth rates under sub-lethal conditions were strain- and not species-dependent. Volatilomes obtained from the two strains studied were rather diverse. Particularly, Ln N6 (Ln. lactis) produced more ethanol and acetic acid than Ln MB7 (Ln. mesenteroides) and higher amounts and diversity of the rest of volatile compounds as well, at all times of incubation. For the two strains studied, most of stress conditions applied diminished the amounts of ethanol and acetic acid produced and the diversity and levels of the rest of volatile compounds. These results were consequence of the different capacity of the strains to grow under each stress condition tested.

Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides bacteriophages: Genomics and cross-species host ranges.

Unveiling virus-host interactions are relevant for understanding the biology and evolution of microbes globally, but in particular, it has also a paramount impact on the manufacture of fermented dairy products. In this study, we aim at characterizing phages infecting the commonly used heterofermentative Leuconostoc spp. on the basis of host range patterns and genome analysis. Host range of six Leuconostoc phages was investigated using three methods (efficiency of plaquing, spot and turbidity tests) against Ln. mesenteroides and Ln. pseudomesenteroides strains. Complete genome sequencing from four out of the six studied Leuconostoc phages were obtained in this work, while the remaining two have been sequenced previously. According to our results, cross-species host specificity was demonstrated, as all phages tested were capable of infecting both Ln. pseudomesenteroides and Ln. mesenteroides strains, although with different efficiency of plaquing (EOP). Phage adsorption rates and ability of low-EOP host strains to propagate phages by crossing the Leuconostoc species' barrier confirm results. At the genome level, phages CHA, CHB, Ln-7, Ln-8 and Ln-9 revealed high similarity with previously characterized phages infecting mostly Ln. mesenteroides strains, while phage LDG was highly similar to phages infecting Ln. pseudomesenteroides. Additionally, correlation between receptor binding protein (RBP) and host range patterns allowed us to unveil a finer clustering of Leuconostoc phages studied into four groups. This is the first report of overlapped phage host ranges between Leuconostoc species.

Phages of dairy Leuconostoc mesenteroides: genomics and factors influencing their adsorption.

Phages infecting Leuconostoc mesenteroides strains can be overlooked during milk fermentation because they do not slowdown the acidification process. However, they can negatively impact the flavor profile of the final product. Yet, the information about these phages is still scarce. In this work, we investigated diverse factors influencing the adsorption of seven virulent Ln. mesenteroides phages, isolated from blue cheese manufacture in Argentina, to their host cells. The addition of calcium ions was generally necessary to observe complete cell lysis and plaque formation for four of the seven phages, but adsorption was very high even in the absence of this cation for all phages. The temperature barely influenced the adsorption process as it was high within the temperature range tested (0 to 50 °C). Moreover, the kinetics of adsorption were similar on viable and non-viable cells, revealing that phage adsorption does not depend on physiological state of the bacterial cells. The adsorption rates were also high at pH values from 4 to 9 for all Ln. mesenteroides phages. We also analyzed the complete genome sequences of two of these phages. Complete nucleotide analysis of phages Ln-8 and Ln-9 showed dsDNA genomes with sizes of 28.5 and 28.9 kb, and the presence of 45 and 48 open reading frames (ORFs), respectively. These genomes were highly similar to those of previously characterized Φ1-A4 (USA, sauerkraut, fermentation) and ΦLN25 (England, whey), both virulent Ln. mesenteroides phages. A detailed understanding of these phages will lead to better control strategies.

Leuconostoc bacteriophages from blue cheese manufacture: long-term survival, resistance to thermal treatments, high pressure homogenization and chemical biocides of industrial application.

Nine Leuconostoc mesenteroides phages were isolated during blue cheese manufacture yielding faulty products with reduced eye formation. Their morphologies, restriction profiles, host ranges and long-term survival rates (25°C, 8°C, -20°C and -80°C) were analysed. Based on restriction analysis, six of them were further examined regarding resistance to physical (heat and high pressure homogenization, HPH) and chemical treatments (ethanol, sodium hypochlorite, peracetic acid, biocides A, C, E and F). According to their morphology, L. mesenteroides phages studied in the present work belonged to the Caudovirales order and Siphoviridae family. Six distinct restriction patterns were obtained with EcoRV, HindIII, ClaI and XhoI enzymes, revealing interesting phage diversity in the dairy environment. No significant reductions in phage counts were observed after ten months of storage at -20°C and -80°C, while slightly and moderate decrease in phage numbers were noticed at 8°C and 25°C, respectively. The phages subjected to heat treatments generally showed high resistance at 63°C and moderate resistance at 72°C. However, 80°C for 30 min and 90°C for 2 min led to complete inactivation of viral particles. In general, the best ethanol concentration tested was 75%, as complete inactivation for most Leuconostoc phages within 30 min of incubation was achieved. Peracetic acid, and biocides A, C, E and F were highly effective when used at the same or at a moderately lower concentration as recommended by the producer. Usually, moderate or high concentrations (600-1,600 ppm) of sodium hypochlorite were necessary to completely inactivate phage particles. Leuconostoc phages were partially inactivated by HPH treatments as remaining viral particles were found even after 8 passes at 100 MPa. This is the first report of L. mesenteroides phages isolated from an Argentinean dairy cheese plant. The results of this work could be useful for establishing the most effective physical and chemical treatments for inactivating phages in industrial plants and laboratory environments.

Leuconostoc citreum MB1 as biocontrol agent of Listeria monocytogenes in milk.

Cell-free supernatant from Leuconostoc citreum MB1 revealed specific antilisterial activity. Preliminary studies demonstrated the proteinaceous, heat-stable, bacteriocin-like trait of the antimicrobial components present in the supernatant. Determination of the genes encoding bacteriocins by PCR and DNA sequencing led to amplification products highly homologous with leucocin A (found in diverse Leuconostoc species) and UviB (found in Leuc. citreum KM20) sequences. Additionally, antimicrobial activity of cell-free supernatant from Leuc. citreum MB1 was revealed by an inhibition halo of the SDS-PAGE gel subjected to a direct detection using Listeria monocytogenes as indicator strain. Different assays were carried out to assess the capacity of Leuc.citreum MB1 to control List. monocytogenes growth: (i) inactivation kinetics of the pathogen by antilisterial compounds present in concentrated cell-free supernatant from Leuc. citreum MB1, (ii) evaluation of optimal Leuc. citreum MB1 initial concentration to obtain maximum List. monocytogenes ATCC 15313 inhibition, and (iii) biocontrol of List. monocytogenes ATCC 15313 with Leuc. citreum MB1 during growth in milk at refrigeration temperature. According to our results, it is unquestionable that at least one bacteriocin is active in Leuc. citreum MB1, since important antilisterial activity was verified either in its cell-free supernatant or in co-culture experiments. Co-culture tests showed that ∼107 CFU/ml Leuc. citreum MB1 was the optimal initial concentration to obtain maximum pathogen inhibition. Moreover, Leuc. citreum MB1 was able to delay List. monocytogenes growth at refrigerated temperature.

Review: efficiency of physical and chemical treatments on the inactivation of dairy bacteriophages.

Bacteriophages can cause great economic losses due to fermentation failure in dairy plants. Hence, physical and chemical treatments of raw material and/or equipment are mandatory to maintain phage levels as low as possible. Regarding thermal treatments used to kill pathogenic bacteria or achieve longer shelf-life of dairy products, neither low temperature long time nor high temperature short time pasteurization were able to inactivate most lactic acid bacteria (LAB) phages. Even though most phages did not survive 90°C for 2 min, there were some that resisted 90°C for more than 15 min (conditions suggested by the International Dairy Federation, for complete phage destruction). Among biocides tested, ethanol showed variable effectiveness in phage inactivation, since only phages infecting dairy cocci and Lactobacillus helveticus were reasonably inactivated by this alcohol, whereas isopropanol was in all cases highly ineffective. In turn, peracetic acid has consistently proved to be very fast and efficient to inactivate dairy phages, whereas efficiency of sodium hypochlorite was variable, even among different phages infecting the same LAB species. Both alkaline chloride foam and ethoxylated non-ylphenol with phosphoric acid were remarkably efficient, trait probably related to their highly alkaline or acidic pH values in solution, respectively. Photocatalysis using UV light and TiO(2) has been recently reported as a feasible option to industrially inactivate phages infecting diverse LAB species. Processes involving high pressure were barely used for phage inactivation, but until now most studied phages revealed high resistance to these treatments. To conclude, and given the great phage diversity found on dairies, it is always advisable to combine different anti-phage treatments (biocides, heat, high pressure, photocatalysis), rather than using them separately at extreme conditions.

Temperate and virulent Lactobacillus delbrueckii bacteriophages: comparison of their thermal and chemical resistance.

The aim of this work was to study the efficiency of diverse chemical and thermal treatments usually used in dairy industries to control the number of virulent and temperate Lactobacillus delbrueckii bacteriophages. Two temperate (Cb1/204 and Cb1/342) and three virulent (BYM, YAB and Ib3) phages were studied. The thermal treatments applied were: 63 degrees C for 30 min (low temperature--long time, LTLT), 72 degrees C for 15 s (high temperature--short time, HTST), 82 degrees C for 5 min (milk destined to yogurt elaboration) and 90 degrees C for 15 min (FIL-IDF). The chemical agents studied were: sodium hypochlorite, ethanol, isopropanol, peracetic acid, biocides A (quaternary ammonium chloride), B (hydrogen peroxide, peracetic acid and peroctanoic acid), C (alkaline chloride foam), D (p-toluensulfonchloroamide, sodium salt) and E (ethoxylated nonylphenol and phosphoric acid). The kinetics of inactivation were drew and T(99) (time necessary to eliminate the 99% of phage particles) calculated. Results obtained showed that temperate phages revealed lower resistance than the virulent ones to the treatment temperatures. Biocides A, C, E and peracetic acid showed a notable efficiency to inactivate high concentrations of temperate and virulent L. delbrueckii phages. Biocide B evidenced, in general, a good capacity to eliminate the phage particles. Particularly for this biocide virulent phage Ib3 showed the highest resistance in comparison to the rest of temperate and virulent ones. On the contrary, biocide D and isopropanol presented a very low capacity to inactivate all phages studied. The efficiency of ethanol and hypochlorite was variable depending to the phages considered. These results allow a better knowledge and give useful information to outline more effective treatments to reduce the phage infections in dairy plants.

Genome analysis of two virulent Streptococcus thermophilus phages isolated in Argentina.

Two Streptococcus thermophilus phages (ALQ13.2 and phiAbc2) were previously isolated from breakdowns of cheese manufacture in Argentina. Complete nucleotide sequence analysis indicated that both phages contained linear double-stranded DNA: 35,525 bp in length for the pac-type phage ALQ13.2 and 34,882 bp for the cos-type phage phiAbc2. Forty-four and 48 open reading frames (ORF) were identified for ALQ13.2 and phiAbc2, respectively. Comparative genomic analysis showed that these isolates shared many similarities with the eight previously studied cos- and pac-phages infecting different S. thermophilus strains. In particular, part of the phiAbc2 genome was highly similar to a region of phage 7201, which was thought to be unique to this latter phage. Protein analysis of the pac-phage ALQ13.2 using SDS polyacrylamide gel electrophoresis (SDS-PAGE) identified three major proteins and seven minor proteins. Parallel structural proteome analysis of phiAbc2 revealed seven protein bands, two of which were related to major structural proteins, as expected for a cos-type phage. Similarities to other S. thermophilus phages suggest that the streptococcal phage diversity is not extensive in worldwide dairy factories possibly because related high-performing bacterial strains are used in starter cultures.

Phage-resistant mutants of Lactobacillus delbrueckii may have functional properties that differ from those of parent strains.

Three commercial phage sensitive Lactobacillus delbrueckii strains (identified as Ab(1), YSD V and Ib(3)), and four spontaneous phage-resistant mutants isolated from them were tested for their capacity to activate the gut mucosal immune response in mice, as indicated by the numbers of IgA-producing cells. Random Amplified Polymorphic DNA (RAPD) analysis revealed a strong genetic homology between the sensitive strains and their respective derivatives. The phage-resistant mutants exhibited high levels of phage resistance, elevated stability of this phenotype and technological properties comparable to those of their respective parent strains. The tolerance to acidic conditions, bile salts and lysozyme was strain dependent and total cell viability losses as a result of exposure to all three stresses ranged from 2.0 to 3.7 log units. All the strains were highly resistant to a simulated gastric solution of pH 3, while significant additional losses in cell viability were observed when acid treated cells were exposed to bile salts and lysozyme. BALB/c mice received pure cultures of Lb. delbrueckii sensitive and phage-resistant strains for 2, 5 or 7 consecutive days. The ability of the parent strains to activate the small intestine immune response was preserved or enhanced in phage-resistant mutants. The maximal proliferation of IgA(+) cells was observed at day 5 or 7, depending on the strain. Mutants isolated in this study using natural selection strategies had improved phage resistance, adequate technological properties and satisfactory gut mucosal immunostimulation ability, and so would be good candidates for industrial applications in functional foods.

Characterization of spontaneous phage-resistant derivatives of Lactobacillus delbrueckii commercial strains.

A total of 44 spontaneous phage-resistant mutants were isolated from three commercial Lactobacillus delbrueckii strains by secondary culture and agar plate methods. Phenotypic characteristics related to their phage-resistance capacities, i.e. plaquing efficiency, phage-resistance stability, lysogeny and adsorption rates were determined. The morphological, biochemical (sugar fermentation patterns) and technological (acidifying and proteolytic activities and acidification kinetics) properties of mutants were also studied. Amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) was applied to confirm strain identity at the subspecies level. Random amplification of polymorphic DNA (RAPD-PCR) was used to determine genetic diversity among the isolates and their respective parent strains. The secondary culture method was the most useful for obtaining phage-resistant mutants. Phage resistance stability was a variable property among the isolates, but a high level of resistance was exhibited as quantified by the efficiency of plaquing. Furthermore, a total absence of spontaneous lysogeny was demonstrated. Adsorption rates were heterogeneously distributed among the three groups of mutants. All mutants isolated from two sensitive strains were similar to them with respect to technological properties. Two groups of mutants with distinctive technological properties were isolated from the other sensitive strain. PCR-ARDRA revealed that two out of three sensitive strains identified commercially as Lb. delbrueckii subsp. bulgaricus were actually Lb. delbrueckii subsp. lactis. Some of the phage-resistant mutants that were obtained might be used in culture rotation programs without regulatory restrictions when commercial strains become sensitive to phages present in industrial environments.

Inactivation of Lactobacillus delbrueckii bacteriophages by heat and biocides.

The effect of several biocides and thermal treatments on the viability of four Lactobacillus delbrueckii phages was investigated. Time to achieve 99% inactivation of phages at 63 and 72 degrees C in three suspension media (Tris Magnesium Gelatin (TMG) buffer, Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)) was calculated. Thermal resistance depended on the phage considered, but a marked heat-resistance was exhibited by one phage (Ib(3)) since its high titre suspensions were completely inactivated only after 45 min at 72 degrees C or 15 min at 90 degrees C. A clear protective effect of the milk was revealed when the three suspension media were compared. As regards to the effects of biocides on phages, only peracetic acid was found to be effective for inactivating high titre suspensions. Ethanol, even at a concentration of 100%, was not suitable to assure no surviving phage particles and isopropanol turned out to be less effective than ethanol. Sodium hypochlorite at 200-400 ppm inactivated the phages completely, except phage Ib(3), which was only destroyed after treatments with 1200 ppm. The diversity observed in the heat and biocide resistance of L. delbrueckii phages is useful to establish a basis for adopting the most effective thermal and chemical treatments for inactivating them in dairy plants and laboratory environments.