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

Resistência de bactérias ácido-láticas a bacteriófagos provenientes de unidades de processamento de queijo Coalho.

Este trabalho teve como objetivos isolar bacteriófagos de amostras de leite, soro e queijo de Coalho e avaliar a resistência de cepas de Lactobacillus paracasei , pertencentes à Coleção de Micro-organismos de Interesse para a Agroindústria Tropical da Embrapa Agroindústria Tropical, aos fagos isolados. Posteriormente, a resistência destas cepas a fagos específicos para L. paracasei, da Coleção do Instituto de Lactología Industrial - INLAIN (Santa Fe, Argentina), também foi avaliada. As amostras para isolamento dos fagos foram obtidas em quatro unidades de processamento de queijo de Coalho, sendo duas artesanais e duas industriais, localizadas no Estado do Ceará. Para o isolamento dos bacteriófagos, foi empregado o teste de lise celular (spot), enquanto que a resistência das culturas aos fagos foi avaliada pelos testes de capacidade de produção de ácido e avaliação da turbidez. As cepas avaliadas foram resistentes aos bacteriófagos provenientes das unidades de processamento de queijo de Coalho e aos bacteriófagos da Coleção do INLAIN. Os resultados obtidos indicaram que as culturas láticas testadas, resistentes aos bacteriófagos, podem ser utilizadas na composição de fermento lático destinado à elaboração de queijo de Coalho, a partir de leite pasteurizado

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.This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; Project PIP 112-201201-00046; Argentina), the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT; Project PICT 2010-0138; Argentina) and the Universidad Nacional del Litoral (UNL, Project CAI+D PI 501 201101 00039 LI; Argentina). S.A.P. was the recipient of an international scholarship awarded by BEC.AR (Becas de formación en el exterior en Ciencia y Tecnología, Presidencia de la Nación, Argentina). M.M.G. thanks Ministerio de Economía y Competitividad (refs. CGL2013-40564-R and SAF2013-49267-EXP), Generalitat Valenciana (ACOMP/2015/133) and Gordon and Betty Moore Foundation (Grant award ref. 5334). F.J.M.M. is funded by the Spanish Ministerio de Economía y Competitividad (BIO2014-53029P) and the European Commission/Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (291815 Era-Net ANIHWA)

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 8uC 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 8uC 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 8uC. 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

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

Temperate bacteriophages v iLp84 and v 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 v 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 plant

Supplementary Material for: Characterization of Two Temperate Lactobacillus paracasei Bacteriophages: Morphology, Kinetics and Adsorption

<p><b><i>Background/Aims:</i></b> Adsorption and kinetic parameters, latent period, burst size and burst time, are characteristics of phage/host systems and can be affected by several environmental factors. As only few studies have focused on temperate dairy phages, we characterized these parameters on temperate <i>Lactobacillus paracasei</i> phages Φ i<i>Lp</i>84 and Φ i<i>Lp</i>1308, infective for probiotic strains. <b><i>Methods:</i></b> Phages were characterized by transmission electron microscopy and genomic DNA restriction. Adsorption under different environmental conditions, phage kinetics and efficiency of plating (EOP) were determined using the double-layer titration method. <b><i>Results:</i></b> Phages Φ i<i>Lp</i>84 and Φ i<i>Lp</i>1308 belong to the <i>Siphoviridae</i> family and have genome sizes of 38 and 34 kbp, respectively. Adsorption was affected by calcium concentration, pH, temperature and host viability, and reached a limit at very high multiplicity of infection. Latency, burst time and burst size were of 85 min, 131 min and 46 for Φ i<i>Lp</i>84, and 51 min, 92 min and 28 for Φ i<i>Lp</i>1308, respectively, at 37°C. A clear influence of temperature on phage kinetics was observed. Regarding EOP, Φ i<i>Lp</i>84 produced plaques on only 1 out of 8 strains tested. <b><i>Conclusion:</i></b> Noticeable differences in adsorption, kinetics and EOP were found for two morphologically identical temperate <i>L. paracasei</i> phages of similar origin.</p

Inactivation of Dairy Bacteriophages by Thermal and Chemical Treatments

This article provides information on the characteristics of diverse phages of lactic acid bacteria and highlights the incidence of their presence in different dairy fermentations. As it is known, thermal treatments on raw milk and use of sanitizers in the disinfection of surfaces and equipment are strategies usually applied in dairy to prevent bacteriophage infections. In this sense, this review mainly focuses on the existing data about the resistance against thermal treatments and sanitizers usually used in the dairy industry worldwide, and the differences found among bacteriophages of diverse genera are remarked upon. Also, we provide information concerning the problems that have arisen as a consequence of the potential presence of bacteriophages in cheese whey powder and derivatives when they are added in fermented dairy product manufacturing. Finally, some important conclusions on each topic are marked and checkpoints to be considered are suggested

Analysis of CRISPR systems of types II-A, I-E and I-C in strains of Lacticaseibacillus

A total of 49 strains of Lacticaseibacillus were screened for the presence of CRISPR-Cas systems. Type II-A CRISPR-Cas loci were sequenced for 8 Lacticaseibacillus paracasei and 3 Lacticaseibacillus rhamnosus strains. They contained 21 to 49 spacers, a few of them with similarity to plasmids. Cas9 identity was well correlated with spacer content of the strains. Only strain L. paracasei 85 contained a type I-E CRISPR locus, uncommon in this species, with highly conserved Cas proteins and 79 spacers not previously reported, a high percentage of them (23%) matching L. paracasei phage genomes. PAM sequences were 5′-NGAA-3´ (type II-A) and 5′-aAA-3´ (type I-E). Protospacer selection in type I-E was highly biased towards genes involved in lysis and morphogenesis. Our results improve the knowledge about distribution and diversity of CRISPR-Cas systems in Lacticaseibacillus strains of industrial interest, not profoundly studied despite the economic impact of phage infections.EEA RafaelaFil: Pujato, Silvina A. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Pujato, Silvina A. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Lactología Industrial; ArgentinaFil: Galliani, Valentina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Galliani, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Lactología Industrial; ArgentinaFil: Irazoqui, Jose Matias. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Irazoqui, Jose Matias. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Amadio, Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Amadio, Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina.Fil: Quiberoni, Andrea. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Quiberoni, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Lactología Industrial; ArgentinaFil: Mercanti, DiegoUniversidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactología Industrial; ArgentinaFil: Mercanti, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Lactología Industrial; Argentin