Characterization of antiviral activity of green tea extract and applications for improving food safety

Resumen

Norovirus, hepatitis A virus (HAV) and more recently hepatitis E virus (HEV) are some of the main health risks associated with food consumption having a high impact on food safety and being responsible for diverse pathologies in consumers (from moderate gastroenteritis to more serious pathologies such as hepatitis or even meningitis or encephalitis). Because enteric viruses are mainly transmitted by the faecal-oral route, their presence can occur in food which has been directly contaminated with faecal material or contaminated water. The main foodstuffs involved in foodborne infections are mollusc bivalves, vegetables and salads, berries, and ready-to-eat food which have been contaminated by improper handling after their preparation or cooking. As a consequence of the increasing number of foodborne outbreaks, international organisms, such as the World Health Organization (WHO) or the European Food Safety Authority (EFSA), propose studies on the efficacy of food processes for virus inactivation. Moreover, the WHO is promoting the development of alternative methods for the decontamination of food. To this end, the present doctoral thesis has focused on the effect of green tea extract (GTE) against human enteric viruses and its potential application in food products or food contact surfaces to enhance food safety. Initially, the antiviral activity and characterization of GTE was evaluated on murine norovirus (MNV), a human norovirus surrogate, and HAV with different temperatures, exposure times and pH conditions showing effectiveness against both viruses depending on pH with higher reductions observed in alkaline and neutral conditions. At the same time, different concentrations of GTE (0.5 and 5 mg/mL) were mixed with viral suspensions and incubated for 2 or 16h (overnight) at 4, 25 and 37°C. Complete viral inactivation was achieved after overnight exposure at 37°C for both viruses and also at 25°C for HAV. In following assays, it was observed that GTE enhanced its antiviral activity when the GTE solution was prepared 24h before its application (aged-GTE). Additional HPLC/MS analyses demonstrated that the enhanced antiviral activity was related to changes in the chemical composition resulting from the formation of catechin derivatives during storage, mainly epigallocatechin gallate (EGCG) that accounted for around 40-50% of GTE composition. Furthermore, norovirus inactivation by GTE was assessed indirectly by testing the effect of aged-GTE on virus-like particles (VLPs) of human norovirus by a porcine gastric mucine (PGM) binding ELISA and transmission electron microscopy (TEM), and on human norovirus suspensions by an in situ capture-RT-qPCR method. Viability RT-qPCR was also evaluated by treating norovirus with EGCG in combination with PMAxx as intercalating dye. Results displayed a significant reduction of close to 50% in the binding to PGM. However, results from viability RT-qPCR indicated that EGCG did not dramatically affect the viral capsid. With the aim to apply GTE to enhance food safety, the potential application of aged-GTE was evaluated in different scenarios. Firstly, the stability of GTE was evaluated in simulated gastric conditions (salivary, gastric and intestinal) to shed light on its capacity as a therapeutical antiviral compound to fight enteric viruses inside the organism. MNV titers decreased to undetectable levels while HAV infectivity reduced by 2 log. Moreover, aged-GTE was incorporated into different contaminated beverages (orange and apple juice, horchata and milk). Results showed a reduction of infectious titers to undetectable limits for MNV in apple juice, while the best HAV reductions were reported in apple juice with a more than 2 log decrease when treated at 5 mg/mL of aged-GTE at 37⁰C for 16 hours. When food models were exposed to moderate heat treatments combined with aged-GTE, increased MNV inactivation was reported. Nevertheless, no such synergistic effect was observed for HAV. Subsequent assays were on track to elucidate the antiviral behaviour of GTE as natural sanitizers for food contact surfaces and washing solution for vegetables. Based on the ISO 13697:2001 standard, suspensions of MNV and HAV were spotted and dried on stainless steel and glass food contact surfaces. Then, aged-GTE was pipetted above the viral inoculums for 15 and 30 min and finally recovered. A similar methodology was performed in the sanitation tests for fresh lettuce and spinach leaves. Results reported a 1.5 log reduction and complete inactivation was observed for MNV and HAV on stainless steel and glass surfaces that had been treated with 10 mg/mL of GTE for 30 min. In lettuce and spinach, MNV and HAV titers were reduced by more than 1.5 log after a 30 min treatment. Finally, GTE was incorporated into edible coatings with the goal of controlling virus contamination in berries. The addition of aged-GTE to carrageenan and alginate/oleic matrices applied on blueberries and raspberries improved the antiviral activity at both refrigerated and ambient temperatures, being slightly more effective in the case of MNV. Lastly, supported by the results of the present thesis, it can be concluding that GTE is a natural and inexpensive option to improve viral food safety.