Abstract
Winemakers add sulphites during winemaking for their antimicrobial (control of indigenous and undesirable microbes) and antioxidant properties. However, the demand for no added sulphite (NAS) or reduced sulphite wines, coupled with a demand for wines with lower alcohol and a distinctive regional character is increasing interest in making wines with indigenous wine yeasts through mixed or spontaneous fermentations.
Pulsed electric field (PEF) processing of grape must has previously been proven to enhance extraction of juice, polyphenols/bioactives and to reduce the microbial levels by either reversible or irreversible electroporation of cell walls. To determine the suitability of using PEF in wine making and to reduce the reliance on the use of sulphites, this study investigated the impact of PEF at levels suitable for extraction and/or sulphites on Saccharomyces and non-Saccharomyces yeasts during the cold soak conditions. The estimation of sub-lethal injury in S. cerevisiae using the traditional approach incorporating a maximum non-inhibitory concentration (MNIC) of sodium chloride (NaCl) was also tested and its accuracy was evaluated in light of the interactions between the initial processing stress and the subsequent selection stress. In addition, studies with PEF, sulphites and the combination of PEF (1.0-1.1 kV/cm and 18-260 kJ/kg) and sulphites (35-350ppm) at 4 and 7 Log CFU/mL at exponential and stationary growth phases in S. cerevisiae, P. kluyveri and H. uvarum were conducted during cold soak conditions for 8 days. Further biochemical analysis using oxidative damage (protein carbonyls and lipid peroxides) and antioxidant enzyme (SOD, CAT, GPx, GR) activity biomarkers was conducted following PEF (1.0 kv/cm,196 kJ/kg), the addition of sulphites (350 ppm) or PEF pre-treatment followed by sulphites in S. cerevisiae, P. kluyveri and H. uvarum over a period of 48h at cold soak conditions.
The results confirm the versatility of PEF for the extraction of grape polyphenols while simultaneously managing the total yeast numbers during maceration, facilitating either mixed/ spontaneous fermentation or the introduction of commercial Saccharomyces spp. The evaluation of MNIC method for quantifying sub-lethal injury showed the shortcomings of the method and the need to delve further into the biochemical and molecular approaches to understand the impact of sub-lethal injury. Screening the impact of a range of PEF and sulphite levels at different initial concentrations and growth phases of the three yeast spp and the biochemical analysis monitoring oxidative stress markers following PEF, sulphites and PEF pre-treatment followed by sulphites showed that there was differential response among the yeast spp. In addition, the pre-treatment of PEF was proven to enhance the impact of sulphites and a differential response in S. cerevisiae, P. kluyveri and H. uvarum. This effect is suggested to be due to PEF (1.0-1.5 kV/cm) inducing reversible electroporation which facilitates the entry of sulphites into the cell. Growth phase, growth temperature and media pH were all found to be key parameters in the selective inactivation process. In conclusion, it is suggested that use of PEF during maceration at cold soak conditions is a feasible technology for the production of reduced sulphite wines.