Impact of pH and protein hydrophobicity on norovirus inactivation by heat-denatured lysozyme.

Norovirus, the leading cause of non-bacterial food poisoning, is responsible for several outbreaks associated with bivalves and ready-to-eat food products worldwide. As norovirus is resistant to alcohol, which is commonly used in food manufacturing processes, sodium hypochlorite is used for its inactivation. However, sodium hypochlorite has two disadvantages: it cannot be added to foods, and its effect is significantly reduced in the presence of organic compounds. Thus, a novel disinfectant against norovirus is urgently required for food hygiene. Thermally denatured egg white lysozyme inactivates norovirus; however, the optimal inactivating conditions and the underlying mechanism are unclear. In the present study, the inactivating mechanism of heat-denatured lysozyme against norovirus was analyzed using murine norovirus strain 1 (MNV-1). We found that the inactivating effect was enhanced by adjusting the pH of the lysozyme solution before thermal denaturation to 6.5 or higher. The reaction of heat-denatured lysozyme and MNV-1 was irreversible, and norovirus was completely inactivated after exposure to heat-denatured lysozyme. Furthermore, it was found that lysozyme residues 5-39 contributed to the norovirus-inactivating effect. Notably, the hydrophobicity and positive charges in this region contributed to the norovirus-inactivating effect, as evidenced by the norovirus inactivation test using mutated residues 5-39. These findings are novel and highlight the possible application of heat-denatured lysozyme as a disinfectant against norovirus in a wide range of food processes.

Evaluation of Inactivation of Murine Norovirus in Inoculated Shell Oysters by High Hydrostatic Pressure Treatment.

One of the major foods causing norovirus gastroenteritis is bivalve shellfish, such as oysters. Depuration and relaying methods have been used to control norovirus. However, these methods may be inadequate to control norovirus gastroenteritis. The present study aimed to investigate the effectiveness of high hydrostatic pressure (HHP) treatment in controlling norovirus in shelled oysters, by evaluating the inactivating effect of HHP on murine norovirus strain 1 (MNV-1) inoculated into a buffer, oyster homogenate, and shelled oysters. First, MNV-1 was inoculated (infectivity of 4.5 log PFU/mL) into the buffer and oyster homogenate, with a pH of 6.3 and salinity (NaCl) of 1.5%, mimicking the habitats of the Pacific oyster (Crassostrea gigas). HHP treatment at 100, 200, 275, and 300 MPa for 2 and 5 min was conducted at an initial temperature of 0 or 5°C. The infectivity of MNV-1 in both the buffer and the oyster homogenate was lower when the initial temperature was 0°C. In the buffer, the infectivity of MNV-1 decreased to 1.8 log PFU/mL after HHP treatment (200 MPa for 5 min at 0°C), and the inactivating effect was higher in the buffer than in the oyster homogenate. MNV-1 was inoculated into shelled oysters (4.8 log PFU per oyster), and HHP treatment was done at 275, 300, and 350 MPa for 5 min at the initial temperature of 0°C. The infectivity of MNV-1 decreased to 2.8 log PFU per oyster after HHP treatment at 275 MPa for 5 min. The results indicate that the inactivating effect of HHP treatment varies, depending on the medium surrounding the viral particles. Inactivation was best in buffer, followed by oyster homogenate and shelled oysters. The data could inform the development of methods to control norovirus in oysters.

Heat-denatured lysozyme could be a novel disinfectant for reducing hepatitis A virus and murine norovirus on berry fruit.

Hepatitis A virus (HAV) is well known worldwide as a causative virus of acute hepatitis. In recent years, numerous cases of HAV infection caused by HAV-contaminated berries have occurred around the world. Because berries are often consumed without prior heating, reliable disinfection of the raw fruit is important in order to prevent HAV outbreaks. Previous studies have found that murine norovirus strain 1 (MNV-1) and human norovirus GII.4 were inactivated in heat-denatured lysozyme solution. In this study, we investigated whether or not heat-denatured lysozyme is effective in inactivating HAV and whether it could be an effective disinfectant for berries contaminated with HAV or MNV-1. We examined the inactivating effect of heat-denatured lysozyme on three strains of HAV and found that it reduced the infectivity of all three strains. We then immersed blueberries and mixed berries into solutions of HAV or MNV-1, and disinfected them by soaking them in 1% heat-denatured lysozyme for 1min. Consequently, the infectious HAV and MNV-1 contaminating the berries were decreased by >3.1 log units in all samples. Our results demonstrate that heat-denatured lysozyme effectively inactivates HAV and suggest that heat-denatured lysozyme may be an effective disinfectant for berry fruit, which is a potential source of HAV food poisoning.