Survival of the biocontrol yeast Pichia anomala after long-term storage in liquid formulations at different temperatures, assessed by flow cytometry

Abstract

Aims: Investigate the survival of liquid formulations of the biocontrol yeast Pichia anomala J121 at different temperatures, and develop a system for comparative studies of different storage conditions and formulations. Methods and Results: The survival of P. anomala in liquid formulations with lactose, starch and trehalose amendments was measured during prolonged storage at temperatures ranging from −20 to +30°C. The relative survival of the stored cells was rapidly estimated by flow cytometry. After 4 weeks incubation at 4 and 10°C, 75–90% of the cells were viable, with no significant differences between the various formulations. Supplementing the storage buffer with lactose or trehalose increased the survival after longer incubations (8 and 12 weeks) at all temperatures (−20 to 30°C). Trehalose was the most effective protectant at 20 and 30°C (>20% viable cells after 12 weeks at 20°C). The biocontrol activity was maintained after formulation and prolonged storage of P. anomala. Conclusions: The storage potential of liquid formulated P. anomala cells can be increased by supplementation with lactose or trehalose. The combination of a custom made incubation chamber and flow cytometry was suitable to evaluate stability of P. anomala formulations. Significance and Impact of the Study: Liquid formulated P. anomala have a long shelf life. The developed test system can be used to study different formulations of other biocontrol agents.

Responses of Yeast Biocontrol Agents to Environmental Stress

“…Using this formulation, a cell count of 10 7 CFU/ml could be maintained for up to 6 months when the formulated product was stored at 4°C. The maintenance of the viability of P. anomala in a liquid medium amended with lactose, starch, or trehalose was evaluated (78). Supplementing the storage medium with either lactose or trehalose allowed a high level of viability to be maintained over an 8-to-12-week period at all the temperatures tested (Ϫ20 to 30°C).…”

“…Antistress compounds. Sugars, polyols, and skim milk are commonly used in formulations of biocontrol agents to help maintain viability during the production process, including liquid culture (33,78,88), freeze-drying (34,35,67,68), spray drying (69,70), vacuum drying (64), and fluidized bed drying (71). Among sugar protectants, trehalose, a nonreducing disaccharide, is generally accepted to function as a protective metabolite.…”

Pichia anomala: cell physiology and biotechnology relative to other yeasts

“…Kalathenos et al 1995;Stratford, 2006), this yeast is generally regarded as a resilient or 'robust' yeast (Fredlund et al 2002;Passoth et al 2006;Lahlali et al 2008), and the stress adaptation mechanisms (both general and specific) it adopts must be very efficient. As testament to the inherent stress tolerance of P. anomala, Melin et al (2005Melin et al ( , 2007 and Mokiou and Magan (2008) have successfully preserved and stabilised this yeast at high viabilities in both liquid and desiccated formulations for environmental biocontrol applications.…”

Biological preservation of plant derived animal feed with antifungal microorganisms: safety and formulation aspects

“…Typically, the quality of the final microbial formulation will be highly dependent on the process parameters and the components used for biomass production. Factors such as the fermentation environment, age of the cultures and composition of the culture media, as well as starvation for different nutritional sources have all been shown to influence the physiological status of cells and their viability following formulation (Melin et al 2006;Schisler et al 2004;Slininger et al 1996;Zhang et al 2005). …”

“…cold storage, and often suffers from a short shelf-life. The storage potential of the biocontrol yeasts, Candida sake and P. anomala, can be increased by supplementing the storage buffer with lactose or preferentially with trehalose Melin et al 2006). Water-in-oil macro-or micro-emulsions provide a form of shielding of the microorganism but these suspensions require storage at low temperatures to maintain a reasonable viability and the stability is often reduced by phase separations Torres et al 2003).…”