HAV detection from milk-based products containing soft fruits: Comparison between four different extraction methods.

Virus detection in food requires appropriate elution and concentration techniques which need to be adapted for different food matrices. ISO/TS-15216-1:2017 and ISO/TS-15216-2:2019 describe standard methods for hepatitis A virus (HAV) research in some food only. Milk-based products containing one or more types of fruit are not covered by ISO procedures, even though they can be contaminated by fruit added to these products or by the food handlers. The aim of this work was to identify an efficient method for the detection of HAV in milk-based products. Four methods were tested to recover HAV from artificially contaminated milk, yoghurt and ice cream containing soft fruits. Results showed that the efficiency of the tested methods depends on the analyzed matrix. In milk we obtained a mean recovery from 13.4% to 1.9%; method based on high speed centrifuge gave the best values. The average recovery in yoghurt was between 3.3% and 114.4%, the latter value achieved by method with beef extract at 3% as eluent. Finally, two methods gave the best results in ice cream with similar recoveries: 29.1% and 27.7% respectively. The first method used glycine as eluent while the other one was based on high speed centrifugation. The ISO method has never proved to be the most efficient in the matrices studied. Therefore, based on the results obtained, a complete rethinking of the ISO method may be necessary to improve its recovery for some products such as milk, while only small changes would be sufficient for other products, such as yoghurt and ice cream.

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species.

Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.

Isolation and characterization of a novel virulent phage (phiLdb) of Lactobacillus delbrueckii.

A new virulent phage (phiLdb) of Lactobacillus delbrueckii subsp. bulgaricus was isolated from a Chinese yogurt sample showing slow acidification. It belonged to the Siphoviridae family with an icosahedral capsid of 47.7+/-0.9 nm in diameter and a long tail of 129.8+/-2 nm. The genome of phage phiLdb was estimated to be approximately 41kbp, and did not contain cohesive ends. One-step growth kinetics of its lytic development revealed latent and burst periods of 45 min and 75 min, respectively, with a burst size of 56+/-2 phage particles per infected cell. Phage phiLdb was highly specific for Lactobacillus delbrueckii subsp. bulgaricus. The presence of calcium or magnesium ions was necessary to accelerate cell lysis and improve plaque formation. Phage phiLdb was able to survive in a pH range between 2 and 10, and resist ethanol and isopropanol. However, a treatment of 90 degrees C for 40 min was observed to inactive phage phiLdb thoroughly. Calcium ions, pH as well as temperature did not show significant influence on phage adsorption, and the adsorption kinetics were similar on viable and nonviable cells. The characterization of this novel phage was helpful to establish a basis for adopting the most effective phage control strategies in industrial plants.

Characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages and the physicochemical analysis of phage adsorption.

AIMS: Three indigenous Lactobacillus delbrueckii subsp. bulgaricus bacteriophages and their adsorption process were characterized. METHODS AND RESULTS: Phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed low burst size and short latent periods. A remarkably high sensitivity to pH was also demonstrated. Indigenous phage genomes were linear and double-stranded DNA molecules of approx. 31-34 kbp, with distinctive restriction patterns. Only one phage genome appeared to contain cohesive ends. Calcium ions did not influence phage adsorption, but it was necessary to accelerate cell lysis and improve plaque formation. The adsorption kinetics were similar on viable and nonviable cells, and the adsorption rates were high between 0 and 50 degrees C. SDS and proteinase K treatments did not influence the phage adsorption but mutanolysin and TCA reduced it appreciably. No significant inhibitory effect on phage adsorption was observed for the saccharides tested. This study also revealed the irreversibility of phage adsorption to their hosts. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on phages of thermophilic lactic acid bacteria.

Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries.

Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp. bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000. Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S. thermophilus strains. The number of viral particles found in samples ranged from 10(2) to 10(9) PFU/ml. The phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed high burst size values and remarkably short latent periods. The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes. The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains. These results suggest that the naturally occurring cultures are inherently more phage resistant.

Thermal inactivation of poliovirus type 1 in water, milk and yoghurt.

Loss of infectivity of poliovirus type 1, strain Sabin, during heating, freezing, and storage in water, milk and yoghurt was determined by plaque-titration in Vero cell cultures. The heating experiments simulated the conditions arising during the processing of milk and yoghurt, for example high-temperature heating (95 degrees C, 15 and 30 s), short-time pasteurization (72 degrees C, 15 and 30 s), long-time pasteurization (62 degrees C, 30 min), and yoghurt-fermentation (42 degrees C, 30 min and 180 min). Only high-temperature heating, long-time pasteurization and short-time pasteurization for 30 s proved to be reliable methods of inactivating polioviruses present in water, milk and yoghurt completely. Short-time pasteurization for 15 s and the conditions of yoghurt-fermentation failed to cause complete inactivation of polioviruses. Additionally, polioviruses mixed in milk or yoghurt withstood these procedures with significantly lower reductions of infectivity than in water. Heating at 55 degrees C for 30 min resulted in complete inactivation of polioviruses, regardless of the suspending medium. The infectivity of polioviruses is scarcely affected by freezing (-20 degrees C, 30 min) and storage (24 days) at low temperatures (4 degrees C) and high humidity (a(w) = 0.99).