Daniela M. Guglielmotti

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.

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 TiO2 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.

Nonstarter Lactobacilli Isolated from Soft and Semihard Argentinean Cheeses: Genetic Characterization and Resistance to Biological Barriers

Nonstarter lactic acid bacteria isolated from Argentinean cheeses were identified and characterized by focusing on their resistance to biological barriers, along with other physiological features of potential interest, in the search for future probiotic organisms. Lactobacilli were enumerated and isolated from semihard and soft cheeses made with multistrain Streptococcus thermophilus starters. Lactobacilli counts in 1-week-old cheeses were between 10(5) and 10(7) CFU/g and then reached 10(7) CFU/ g in all 1-month samples, while streptococci were always above 10(9) CFU/g. A total number of 22 lactobacilli isolates were retained, identified, and characterized by in vitro tests. Species identity was determined by carbohydrate metabolism and species-specific PCR assays. Genetic diversity was explored by random amplified polymorphic DNA (RAPD) PCR analysis. The Lactobacillus strains were assigned to the species L. casei, L. plantarum, L. rhamnosus, L. curvatus, L. fermentum, and L. perolens. All the strains studied tolerated 25 ppm of lysozyme, and most of them showed resistance to 0.3% bile. After incubation in gastric solution (pH 2.0), counts decreased by several log units, ranging from 3.2 to 7.0. The strains were able to grow in the presence of bile salts, but only three isolates were capable of deconjugation. The nonstarter lactobacilli that were assayed fermented the prebiotic substrates (especially lactulose and inulin). Some strains showed high cell hydrophobicity and beta-galactosidase activity, as well as inhibitory activity against pathogenic bacteria. It was concluded that most of the lactobacilli isolated in this study demonstrated resistance to biological barriers and physiological characteristics compatible with probiotic properties, which make them suitable for further research in in vivo studies aimed at identifying new probiotic organisms.

Resistance of two temperate Lactobacillus paracasei bacteriophages to high pressure homogenization, thermal treatments and chemical biocides of industrial application

Temperate bacteriophages ф iLp84 and ф iLp1308, previously isolated from mitomycin C-induction of Lactobacillus paracasei strains 84 and CNRZ1308, respectively, were tested for their resistance to several physical and chemical treatments applied in dairy industry. Long-term survival at 4 °C, -20 °C and -80 °C, resistance to either thermal treatments of 63 °C, 72 °C and 90 °C, high pressure homogenization (HPH, 100 MPa) or classic (ethanol, sodium hypochlorite and peracetic acid) and new commercial sanitizers, namely 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), were determined. Phages were almost completely inactivated after eight months of storage at 25 °C, but viability was not affected at 4 °C, -20 °C or -80 °C. Both phages tolerated well HPH treatments. Phage iLp1308 showed higher thermal resistance than ф iLp84, but neither resisted 90 °C for 2 min. Best chemical inactivation was accomplished using peracetic acid or biocides A, C and E, whereas biocides B and D were completely ineffective. These results help to improve selection of chemical agents and physical treatments to effectively fight against phage infections in dairy plants.

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.

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.

Phage-resistance linked to cell heterogeneity in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1

The aim of this work was to study the relationship between the cell morphological heterogeneity and the phage-resistance in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1. Two morphological variants (named C and T) were isolated from this strain. Phage-resistant derivatives were isolated from them and the percentage of occurrence of confirmed phage-resistant cells was 0.001% of the total cellular population. Within these phage-resistant cell derivatives there were T (3 out of 4 total isolates) and C (1 out of 4 total isolates) variants. The study of some technological properties (e.g. proteolytic and acidifying activities) demonstrated that most of phage-resistant derivatives were not as good as the parental strain. However, for one derivative (a T variant), the technological properties were better than those of the parental strain. On the other hand, it was possible to determinate that the system of phage-resistance in the T variants was interference in adsorption step, with adsorption rates <15%. For the C variant derivative it was possible to demonstrate the presence of a restriction/modification system and, moreover, to determinate that this system could be Type I R/M.

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.

High pressure homogenization versus heat treatment: effect on survival, growth, and metabolism of dairy Leuconostoc strains.

The effect of high pressure homogenization (HPH) with respect to a traditional heat treatment on the inactivation, growth at 8°C after treatments, and volatile profile of adventitious Leuconostoc strains isolated from Cremoso Argentino spoiled cheeses and ingredients used for their manufacture was evaluated. Most Leuconostoc strains revealed elevated resistance to HPH (eight passes, 100 MPa), especially when resuspended in skim milk. Heat treatment was more efficient than HPH in inactivating Leuconostoc cells at the three initial levels tested. The levels of alcohols and sulfur compounds increased during incubation at 8°C in HPH-treated samples, while the highest amounts of aldehydes and ketones characterized were in heated samples. Leuconostoc cells resuspended in skim milk and subjected to one single-pass HPH treatment using an industrial-scale machine showed remarkable reductions in viable cell counts only when 300 and 400 MPa were applied. However, the cell counts of treated samples rose rapidly after only 5 days of storage at 8°C. The Leuconostoc strains tested in this work were highly resistant to the inactivation treatments applied. Neither HPH nor heat treatment assured their total destruction, even though they were more sensitive to the thermal treatment. To enhance the inhibitory effect on Leuconostoc cells, HPH should be combined with a mild heat treatment, which in addition to efficient microbial inactivation, could allow maximal retention of the physicochemical properties of the product.