Segregation of Tetragenococcus halophilus and Zygosaccharomyces rouxii using W⁠₁/O/W_⁠2 double emulsion for use in mixed culture fermentation

Antagonism in mixed culture fermentation can result in undesirable metabolic activity and negatively affect the fermentation process. Water-oil-water (W⁠₁/O/W⁠₂) double emulsions (DE) could be utilized in fermentation for segregating multiple species and controlling their release and activity. Zygosaccharomyces rouxii and Tetragenococcus halophilus, two predominant microbial species in soy sauce fermentation, were incorporated in the internal W⁠₁ and external W⁠₂ phase of a W⁠₁/O/W⁠₂, respectively. The suitability of DE for controlling T. halophilus and Z. rouxii in soy sauce fermentation was studied in relation to emulsion stability and microbial release profile. The effects of varying concentrations of Z. rouxii cells (5 and 7 log CFU/mL) and glucose (0%, 6%, 12%, 30% w/v) in the W⁠2 phase were investigated. DE stability was determined by monitoring encapsulation stability (%), oil globule size, and microstructure with fluorescence and optical microscopy. Furthermore, the effect of DE on the interaction between T. halophilus and Z. rouxii was studied in Tryptic Soy Broth containing 10% w/v NaCl and 12% w/v glucose and physicochemical changes (glucose, ethanol, lactic acid, and acetic acid) were monitored. DE destabilization resulted in cell release which was proportional to the glucose concentration in W⁠₂. Encapsulated Z. rouxii presented higher survival during storage (~ 3 log). The application of DE affected microbial cells growth and physiology, which led to the elimination of antagonism. These results demonstrate the potential use of DE as a delivery system of mixed starter cultures in food fermentation, where multiple species are required to act sequentially in a controlled manner.