Alterantes microbianos atípicos en yogures argentinos: mohos gasógenos y bacterias del género Gluconobacter / Atypical spoilage microorganisms in Argentinean yogurts: Gas-producing molds and bacteria of the genus Gluconobacter

RESUMEN El deterioro microbiológico de alimentos conduce a productos no aptos para consumo, y su descarte, a importantes pérdidas económicas para la industria alimenticia. Durante su almacenamiento, los alimentos frescos representan nichos atractivos para la supervivencia y el crecimiento de microorganismos indeseables. En productos lácteos, la presencia de alterantes o patógenos bacterianos está mejor documentada que la de mohos y levaduras. Estos productos son menos proclives al deterioro por mohos que otros, como frutas y verduras, debido a su almacenamiento refrigerado, su elaboración a partir de leche tratada térmicamente y, para fermentados, a la microbiota dominante, que acidifica el medio. Sin embargo, incluso quesos y yogures pueden sufrir deterioro por mohos. Este trabajo presenta casos atípicos de muestras de yogur con desarrollo de mohos gasógenos y bacterias del géneroGluconobactercomo microorganismos alterantes no reportados previamente como tales en leches fermentadas argentinas. Los organismos alterantes «clásicos¼ de yogur fueron siempre levaduras y, en otros países, mohos del géneroAspergillus.

ABSTRACT Microbial food alterations lead to unfit products for consumption, and their discarding, to significant economic losses for the food industry. During storage, fresh foods offer available niches for the survival and growth of undesirable microorganisms. In dairy products, data regarding spoilage and/or pathogenic bacteria is better documented than those for molds and yeasts. Dairy products are less susceptible to mold's contamination than products such as fruits and vegetables, due to their refrigerated storage; their elaboration from heat-treated milk and, for fermented ones, the dominant microbiota that acidifies the medium. However, even cheeses and yogurts may be susceptible to mold contamination. Atypical cases of yogurt samples containing spoilage microorganisms not previously reported (molds producing gas and bacteria of the genusGluconobacter) in Argentinean fermented milks are presented here. For yogurt, in particular, the "classic" altering organisms were always being yeasts, and in other countries, molds belonging to the genusAspergillus.

[Atypical spoilage microorganisms in Argentinean yogurts: Gas-producing molds and bacteria of the genus Gluconobacter]. / Alterantes microbianos atípicos en yogures argentinos: mohos gasógenos y bacterias del género Gluconobacter.

Microbial food alterations lead to unfit products for consumption, and their discarding, to significant economic losses for the food industry. During storage, fresh foods offer available niches for the survival and growth of undesirable microorganisms. In dairy products, data regarding spoilage and/or pathogenic bacteria is better documented than those for molds and yeasts. Dairy products are less susceptible to mold's contamination than products such as fruits and vegetables, due to their refrigerated storage; their elaboration from heat-treated milk and, for fermented ones, the dominant microbiota that acidifies the medium. However, even cheeses and yogurts may be susceptible to mold contamination. Atypical cases of yogurt samples containing spoilage microorganisms not previously reported (molds producing gas and bacteria of the genus Gluconobacter) in Argentinean fermented milks are presented here. For yogurt, in particular, the "classic" altering organisms were always being yeasts, and in other countries, molds belonging to the genus Aspergillus.

Lactobacillus fermentum: Could EPS production ability be responsible for functional properties?

Exopolysaccharides (EPS) production is a characteristic that has been widely described for many lactic acid bacteria (LAB) of different genera and species, but little is known about the relationship between the functional properties of the producing bacteria and EPS synthesis. Although many studies were addressed towards the application of EPS-producing LAB in the manufacture of several dairy products (fermented milk, cheese) due to their interesting technological properties (increased hardness, water holding capacity, viscosity, etc.), there are not many reports about the functional properties of the EPS extract itself, especially for the genus Lactobacillus. The aim of the present revision is to focus on the species Lactobacillus fermentum with reported functional properties, with particular emphasis on those strains capable of producing EPS, and try to establish if there is any linkage between this property and their functional/probiotic roles, considering the most recent bibliography.

In vivo study of the immunomodulatory capacity and the impact of probiotic strains on physicochemical and sensory characteristics: Case of pasta filata soft cheeses.

Nowadays, there is a great interest of food industries on the development of innovative dairy products containing probiotic bacteria. Beyond yoghurt, cheeses could be a suitable matrix for the incorporation of probiotics. This work aimed to study the impact of the addition of two probiotic lactobacilli (L. rhamnosus GG and L. acidophilus LA5), either individually or combined, on the physicochemical and sensory characteristics of pasta filata soft cheeses and to evaluate the immunomodulating capacity of the product using an in vivo model. Four cheese types were produced: CC (control cheese); L-C (added with L. acidophilus LA5); GG-C (added with L. rhamnosus GG) and GGL-C (added with both probiotics). No differences in gross composition were found. An increase in secondary proteolysis was observed in GGL-C cheeses which correlated with a greater bitter and aftertaste. On the contrary, the L-C cheese received the highest overall quality score. The translocation assay was negative. Moreover, the three experimental cheeses were able to modulate the immune system of mice by reducing the secretion of pro-inflammatory cytokines in the intestine, downregulating the levels of interleukin (IL)-10 and increasing the secretion of Secretory Immunoglobulin A (S-IgA). No synergistic effect was detected when both probiotics were added.

Safety, functional properties and technological performance in whey-based media of probiotic candidates from human breast milk

We aimed at isolating and characterising microorganisms present in human breast milk with probiotic potential. In an 8-week postpartum sampling period, two strains of bifidobacteria (Bifidobacterium longum LM7a and Bifidobacterium dentium LM8a') and four strains of lactobacilli were isolated, all during the first 4-week postpartum. B. longum LM7a and B. dentium LM8a', together with four strains previously isolated from breast milk (Bifidobacterium lactis INL1, INL2, INL4 and INL5), were considered for further studies. Susceptibility of the strains to tetracycline, erythromycin, clindamycin, streptomycin, vancomycin and chloramphenicol was evaluated and the isolates exhibited, in general, the same properties as previously reported for bifidobacteria. All isolates showed low hydrophobicity and B. lactis and B. longum strains had satisfactory resistance to gastric digestion and bile shock, but not to pancreatin. B. lactis INL1, B. longum LM7a and B. dentium LM8a' were selected for some comparative technological studies. In particular, B. lactis INL1 displayed technological potential, with satisfactory growth in cheese whey-based media in biofermentor and resistance to freeze-drying, accelerated storage conditions and simulated gastric digestion

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Effect of storage temperature on the chemical, microbiological, and sensory characteristics of pasta filata soft cheese containing probiotic lactobacilli.

The aim of this study was to manufacture pasta filata cheeses added with two probiotic lactobacilli: Lactobacillus rhamnosus GG and Lactobacillus acidophilus LA5, either individually or combined, and to evaluate the effect of the storage temperature (4 and 12 °C) on their chemical, microbiological, and sensorial characteristics. Three cheese types were made: (i) G: containing L. rhamnosus GG, (ii) L: containing L. acidophilus LA5, and (iii) GL: containing both probiotic strains. Gross composition, pH, microbiological, and sensory characteristics were determined. No differences in gross composition were found among them. pH values remained above 5.2 in cheeses stored at 4 °C. However, a postacidification was observed in cheeses ripened at 12 °C. L. acidophilus LA5 was not able to grow, while L. rhamnosus GG grew 1.5 log10 CFU/g in G and GL cheeses stored at 12 °C, reducing the pH from day 8 onwards. These results emphasize the importance of the storage temperature since the good characteristics of probiotic cheeses are kept if the cold-chain is respected. Thus, the selection of probiotics, together with the food matrix and the starter, should be carefully evaluated.

Intestinal Immunomodulation and Shifts on the Gut Microbiota of BALB/c Mice Promoted by Two Bifidobacterium and Lactobacillus Strains Isolated from Human Samples.

Bifidobacterium animalis subsp. lactis IPLA 20020 and Lactobacillus gasseri IPLA 20212, two strains isolated from human samples, were evaluated for safety and influence over the intestinal microbiota and cytokine production by the intestinal tissue of adult BALB/c mice. Mice were divided into four groups receiving during 8 days PBS or a suspension of each strain, prepared fresh or lyophilized (bifidobacteria), at an amount of 4x108 viable cells/day. This dose could be comparable to the probiotic intake of a human adult who consumed about 100-200 mL of functional fermented milk per day, considering the usual level of probiotics in commercial products. No microbial translocation to liver or alterations in food intake, weight, and behavior were observed in treated mice. Intestinal content of secretory immunoglobulin A (s-IgA) was not affected, discarding any adverse effect on the mucosa-associated immunity. The profile of intestinal proinflammatory/regulatory cytokines after intervention evidenced that the microbial strain administered and its cellular state (fresh or lyophilized) as well as the host tissue analyzed (small or large intestine) influenced the immune response and suggests a moderate shift towards a T helper 1 profile (Th1) in the large intestine after the administration of both strains. Changes on relative levels of some intestinal microbial groups were evidenced after intervention. It is noteworthy that butyrate was positively associated with a balanced pro-Th1 immune response. Therefore, B. animalis subsp. lactis IPLA20020 and L. gasseri IPLA 20212 could be considered potential probiotic candidates to be included in functional foods for balancing the intestinal immune response.

Safety, functional properties and technological performance in whey-based media of probiotic candidates from human breast milk.

We aimed at isolating and characterising microorganisms present in human breast milk with probiotic potential. In an 8-week postpartum sampling period, two strains of bifidobacteria (Bifidobacterium longum LM7a and Bifidobacterium dentium LM8a') and four strains of lactobacilli were isolated, all during the first 4-week postpartum. B. longum LM7a and B. dentium LM8a', together with four strains previously isolated from breast milk (Bifidobacterium lactis INL1, INL2, INL4 and INL5), were considered for further studies. Susceptibility of the strains to tetracycline, erythromycin, clindamycin, streptomycin, vancomycin and chloramphenicol was evaluated and the isolates exhibited, in general, the same properties as previously reported for bifidobacteria. All isolates showed low hydrophobicity and B. lactis and B. longum strains had satisfactory resistance to gastric digestion and bile shock, but not to pancreatin. B. lactis INL1, B. longum LM7a and B. dentium LM8a' were selected for some comparative technological studies. In particular, B. lactis INL1 displayed technological potential, with satisfactory growth in cheese whey-based media in biofermentor and resistance to freeze-drying, accelerated storage conditions and simulated gastric digestion.

Draft Genome Sequence of Lactobacillus fermentum Lf2, an Exopolysaccharide-Producing Strain Isolated from Argentine Cheese.

Lactobacillus fermentum Lf2, an Argentine cheese isolate, can produce high concentrations of exopolysaccharides (EPS). These EPS were shown to improve the texture and rheology of yogurt, as well as to play a protective role in mice exposed to Salmonella enterica serovar Typhimurium. Three gene clusters potentially involved in EPS production were identified in different locations of the L. fermentum Lf2 genome.

Isolation, Characterization and Performance of Autochthonous Spray Dried Lactic Acid Bacteria in Maize Micro and Bucket-Silos.

The aim of this study was to isolate, identify and characterize lactic acid bacteria (LAB) from spontaneously fermented maize silage, and evaluate their performance as spray-dried (SD) cultures to enhance the fermentation and the aerobic stability of maize micro-silos. Eleven strains of LAB were characterized for growth kinetics, the capability to grow in vegetable-based medium (VBM), production of organic acids and the ability to tolerate heat-stress. Three strains (Lactobacillus plantarum Ls71, Pediococcus acidilactici Ls72, and Lactobacillus buchneri Ls141) were selected and further characterized for the ability to grow as single strain or in co-culture in MRS and VMB medium, to survive at freeze and spray-drying process, for their performance as SD bacteria in micro-silos and for the aerobic stability in bucket silos. L. buchneri Ls141 showed the highest growth capability in VBM and produced the highest amount of acetic acid, while L. plantarum Ls71 produced the highest amounts of lactic acid. P. acidilactici Ls72 was the most heat-resistant strain, with a reduction of 0.2 log10 CFU/mL (15 min at 55°C). The three strains satisfactorily tolerated both spray and freeze-drying. After 4 days of fermentation, all the samples reached a pH value of about 3.7-3.8. A significantly lower cell load of filamentous fungi and yeasts (< 3 log10 CFU/g) and a higher concentration of total LAB (> 8.7 log10 CFU/g) was observed after 30 days of fermentation. A greater amount of acetic acid, crude protein, ash and ammonia nitrogen/total nitrogen was detected in inoculated silages. A significant reduction of filamentous fungi and yeasts was also observed in inoculated bucket silos after 50 d of fermentation. The aerobic stability was significantly improved in inoculated silage since the temperature remained stable after 16 days (384 h). On the contrary, an increase of 5°C was observed in control samples after 1 day. The selected strains have the potential to be produced as SD silage inoculant as they were able to accelerate the fermentation process, to control filamentous fungi and yeasts, to improve some nutritional and chemical parameters of silage and to improve aerobic stability.

Variability in gut mucosal secretory IgA in mice along a working day.

OBJECTIVE: To assess the variability of secretory immunoglobulin A (S-IgA) in the lumen and feces of mice along a working day. RESULTS: Mice were maintained under a 12 h light-dark cycle, light period starting at 8 AM. S-IgA was determined in feces and intestinal content (after one or three washes) at three points along the day: at the beginning, in the middle and at the end of the light period (ELP). Significant reduction in the content of S-IgA in the small intestine fluid and in feces was observed at the end of the light cycle, which coincides with the end of a regular working day (8 PM) in any given animal facility. It was also observed that three washes of the small intestine were more effective than one flush to recover a significant higher amount of S-IgA, with the smallest coefficient of variation observed by the ELP. A smaller CV would imply a reduced number of animals needed to achieve the same meaningful results. The results may be useful when designing animal trials for the selection of probiotic candidates based on their capacity of activating S-IgA, since it would imply a more rational use of experimental animals.

Influence of Technological Treatments on the Functionality of Bifidobacterium lactis INL1, a Breast Milk-Derived Probiotic.

The aim of this study is to evaluate the influence of the technological processing on the functionality of the human breast milk probiotic strain Bifidobacterium lactis INL1. In vitro antagonistic activity of B. lactis INL1 was detected for Gram-positive and Gram-negative pathogens. B. lactis INL1 was administered to mice as fresh (F), frozen (Z), spray-dried (S), or lyophilized (L) culture. Immune parameters (IgA, IL-10, and IFN-γ) were determined and histological analysis was performed to assess functionality and protection capacity against Salmonella. In BALB/c mice, F and S cultures induced an increase in the number of IgA-producing cells in the small intestine and IL-10 levels were increased for L culture in the large intestine. In Swiss mice, B. lactis INL1 increased secretory-IgA levels in the small intestine before and after Salmonella infection, both as F or dehydrated culture. Also, an attenuation of damage in the intestinal epithelium and less inflammatory infiltrates were observed in animals that received F and S cultures, whereas in liver only F showed some effect. The anti-inflammatory effect was confirmed in both tissues by myeloperoxidase activity and by IFN-γ levels in the intestinal content. B. lactis INL1 showed inhibitory activity against pathogens and confirmed its probiotic potential in animal models. Technological processing of the probiotic strain affected its functionality. PRACTICAL APPLICATION: This work provides evidence about the influence of technology on the functionality of probiotics, which may help probiotics and functional food manufacturers to take processing into consideration when assessing the functionality of new strains.

Microencapsulation of functional strains by high pressure homogenization for a potential use in fermented milk.

This study was aimed to evaluate the potential of high pressure homogenization for the microencapsulation of two probiotic lactic acid bacteria, Lactobacillus paracasei A13 and Lactobacillus salivarius subsp. salivarius CET 4063 to produce functional fermented milks. Microcapsules of the considered functional microorganisms were obtained by HPH treatments at 50MPa in the presence of sodium alginate and vegetable oil. The microencapsulated microorganisms were then inoculated as adjuncts to produce fermented milks. As controls were used fermented milks in which the two probiotic lactobacilli were inoculated without encapsulation. The viability of the strains was monitored during almost 2months of refrigerated storage. The survival of lactic acid bacteria after the gastric-duodenal simulated test was determined. Fermented milk texture parameters, the presence of exo-polysaccharides and the production of volatile molecules were also evaluated over storage. The microcapsules, for both the considered probiotic strains, were homogeneous and with a size<100µM and therefore did not adversely affect the sensory properties of the fermented milks. The encapsulation decreased the hyperacidity phenomena generally related to the inclusion of probiotic microorganisms in fermented milks. The lower acidity of the products due to the microencapsulation was fundamental for the improvement of the viability of the starter culture and the sensory characteristics of the products. The microencapsulation conditions increased the resistance to the simulated digestion processes, although the strain Lb. paracasei A13 generally showed a higher resistance to the gastric barrier respect to Lb. salivarius CECT 4063. By contrast, the data obtained showed a reduction of EPS production by the microencapsulation. The volatile profiles showed specific profiles in relation to the probiotic strain used and microencapsulation process. In conclusion, the results of this study underlined the applicative potential of HPH microencapsulation of probiotic microorganisms to produce fermented milk with improved functionality and with enhanced sensory properties.

Spray-drying process preserves the protective capacity of a breast milk-derived Bifidobacterium lactis strain on acute and chronic colitis in mice.

Gut microbiota dysbiosis plays a central role in the development and perpetuation of chronic inflammation in inflammatory bowel disease (IBD) and therefore is key target for interventions with high quality and functional probiotics. The local production of stable probiotic formulations at limited cost is considered an advantage as it reduces transportation cost and time, thereby increasing the effective period at the consumer side. In the present study, we compared the anti-inflammatory capacities of the Bifidobacterium animalis subsp. lactis (B. lactis) INL1, a probiotic strain isolated in Argentina from human breast milk, with the commercial strain B. animalis subsp. lactis BB12. The impact of spray-drying, a low-cost alternative of bacterial dehydration, on the functionality of both bifidobacteria was also investigated. We showed for both bacteria that the spray-drying process did not impact on bacterial survival nor on their protective capacities against acute and chronic colitis in mice, opening future perspectives for the use of strain INL1 in populations with IBD.

Exopolysaccharide from Lactobacillus fermentum Lf2 and its functional characterization as a yogurt additive.

Lactobacillus fermentum Lf2 is a strain which is able to produce high levels (approximately 1 g/l) of crude exopolysaccharide (EPS) when it is grown in optimised conditions. The aim of this work was to characterize the functional aspects of this EPS extract, focusing on its application as a dairy food additive. Our findings are consistent with an EPS extract that acts as moderate immunomodulator, modifying s-IgA and IL-6 levels in the small intestine when added to yogurt and milk, respectively. Furthermore, this EPS extract, in a dose feasible to use as a food additive, provides protection against Salmonella infection in a murine model, thus representing a mode of action to elicit positive health benefits. Besides, it contributes to the rheological characteristics of yogurt, and could function as a food additive with both technological and functional roles, making possible the production of a new functional yogurt with improved texture.

Technological, rheological and sensory characterizations of a yogurt containing an exopolysaccharide extract from Lactobacillus fermentum Lf2, a new food additive.

Lactobacillus fermentum Lf2, an autochthonous strain isolated as a non starter culture in Cremoso cheese, produces high EPS levels (~1g/L) in optimized conditions (SDM broth, pH6.0, 30°C, 72h). Technological (texture profile and rheological analysis) and sensory properties of non-fat yogurts with 300 and 600mg EPS/L were studied at 3 and 25days after manufacture. Yogurts with different EPS concentrations showed higher hardness values than the control group at both periods of time, being the only significant difference that remained stable during time. The consistency index was also higher for the treated samples at both times evaluated, being significantly different for samples with 300mg/L of EPS extract, while the flow behavior index was lower for EPS-added yogurts. The thixotropic index was lower (P<0.05) for samples with the highest EPS extract concentration at the end of the storage time. Regarding the sensory analysis, those yogurts with 600mg/L of EPS extract presented the highest values of consistency at 3days of storage. No considerable differences for defects (milk powder, acid, bitter and cooked milk flavors) were perceived between treated and control samples at both times evaluated. Syneresis was also studied and samples with 600mg/L of EPS extract presented the lowest syneresis values at 25days of storage, which considerably decreased with the time of storage. In conclusion, the EPS from L. fermentum Lf2, used as an additive, provided yogurt with creamy consistency and increased hardness, without the presence of unwanted defects and improving the water holding capacity of the product. All the analysis done showed the potential of this extract to be used as a technofunctional natural ingredient, and it should be considered its positive impact on health, according to previous studies.

Phage adsorption and lytic propagation in Lactobacillus plantarum: could host cell starvation affect them?

BACKGROUND: Bacteriophages constitute a great threat to the activity of lactic acid bacteria used in industrial processes. Several factors can influence the infection cycle of bacteriophages. That is the case of the physiological state of host cells, which could produce inhibition or delay of the phage infection process. In the present work, the influence of Lactobacillus plantarum host cell starvation on phage B1 adsorption and propagation was investigated. RESULT: First, cell growth kinetics of L. plantarum ATCC 8014 were determined in MRS, limiting carbon (S-N), limiting nitrogen (S-C) and limiting carbon/nitrogen (S) broth. L. plantarum ATCC 8014 strain showed reduced growth rate under starvation conditions in comparison to the one obtained in MRS broth. Adsorption efficiencies of > 99 % were observed on the starved L. plantarum ATCC 8014 cells. Finally, the influence of cell starvation conditions in phage propagation was investigated through one-step growth curves. In this regard, production of phage progeny was studied when phage infection began before or after cell starvation. When bacterial cells were starved after phage infection, phage B1 was able to propagate in L. plantarum ATCC 8014 strain in a medium devoid of carbon source (S-N) but not when nitrogen (S-C broth) or nitrogen/carbon (S broth) sources were removed. However, addition of nitrogen and carbon/nitrogen compounds to starved infected cells caused the restoration of phage production. When bacterial cells were starved before phage infection, phage B1 propagated in either nitrogen or nitrogen/carbon starved cells only when the favorable conditions of culture (MRS) were used as a propagation medium. Regarding carbon starved cells, phage propagation in either MRS or S-N broth was evidenced. CONCLUSIONS: These results demonstrated that phage B1 could propagate in host cells even in unfavorable culture conditions, becoming a hazardous source of phages that could disseminate to industrial environments.

Effects of sub-lethal high-pressure homogenization treatment on the outermost cellular structures and the volatile-molecule profiles of two strains of probiotic lactobacilli.

Applying sub-lethal levels of high-pressure homogenization (HPH) to lactic acid bacteria has been proposed as a method of enhancing some of their functional properties. Because the principal targets of HPH are the cell-surface structures, the aim of this study was to examine the effect of sub-lethal HPH treatment on the outermost cellular structures and the proteomic profiles of two known probiotic bacterial strains. Moreover, the effect of HPH treatment on the metabolism of probiotic cells within a dairy product during its refrigerated storage was investigated using SPME-GC-MS. Transmission electron microscopy was used to examine the microstructural changes in the outermost cellular structures due to HPH treatment. These alterations may be involved in the changes in some of the technological and functional properties of the strains that were observed after pressure treatment. Moreover, the proteomic profiles of the probiotic strains treated with HPH and incubated at 37°C for various periods showed different peptide patterns compared with those of the untreated cells. In addition, there were differences in the peaks that were observed in the low-mass spectral region (2000-3000 Da) of the spectral profiles of the control and treated samples. Due to pressure treatment, the volatile-molecule profiles of buttermilk inoculated with treated or control cells and stored at 4°C for 30 days exhibited overall changes in the aroma profile and in the production of molecules that improved its sensory profile, although the two different species imparted specific fingerprints to the product. The results of this study will contribute to understanding the changes that occur in the outermost cellular structures and the metabolism of LAB in response to HPH treatment. The findings of this investigation may contribute to elucidating the relationships between these changes and the alterations of the technological and functional properties of LAB induced by pressure treatment.

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.