Freezing and freeze-drying of the bacterium Rahnella aquatilis BNM 0523: study of protecting agents, rehydration media and freezing temperatures

Abstract

Aims: The aim of the present study was to evaluate and compare freezing and freeze‐drying treatments for conserving Rahnella aquatilis (BNM 0523) with the goal to achieve an adequate commercial formulation of this biocontrol agent. Methods and Results: The effect of several protective agents, rehydration media and freezing temperatures on the viability and functional activity of the R. aquatilis was investigated. The storage stability at 3 months and 4 years was determined by checking the viability of the cells and their biocontrol capability against Botrytis cinerea by measuring the percentage of reduction of disease severity on apple. The best results were obtained by the freeze‐drying of the cells using a mixture of skimmed nonfat milk 10%, yeast extract 0·5% and glucose 1% as the protecting and rehydrating medium, and a quickly freezing (−70°C) before the freeze‐drying. In this case, the viability of the cells after 4 years was 98%, and their antagonistic ability showed a little decrease with respect fresh cells. Conclusions: The studies showed that R. aquatilis was resistant to freezing and freeze‐drying when it was used a mixture of cryoprotectants and that it was possible to obtain inoculums with high viability and good effectiveness for reduction of decay caused by B. cinerea. Significance and Impact of the study: This study is probably the first report about the resistance of R. aquatilis to freezing and freeze‐drying treatments and shows that these operations could be useful for obtaining a commercial formulation of this biocontrol agent.

Formulation of the biocontrol agent Bacillus amyloliquefaciens CPA-8 using different approaches: liquid, freeze-drying and fluid-bed spray-drying

“…In contrast, CPA-8 cells amended with sucrose or sucrose plus SM provided higher degree of cell protection during drying. Other authors have reported the beneficial role of sugars (Costa et al, 2000;Navarta et al, 2011;Zhan et al, 2011), which replace structural water in membrane after rehydration and prevent unfolding and aggregation of proteins (Champagne et al, 1991). The results obtained when MgSO4 was added to CPA-8 cells prior FD -alone or in mixture with SM-are in agreement with the work conducted by Yánez-Mendizábal et al (2012b).…”

“…The SM at concentrations of 1-10 % has often been used for cryopreservation but even more frequently in mixtures with other cryoprotectants (Hubalek, 2003;Li & Tian, 2007). As described Navarta et al (2011), the use of SM in the FD process of the BCA Ranhella aquatilis had lower protective effect than the SM used in combination with other additives such as sugars or polyols.…”

“…The efficacy of the BCA Bacillus amyloliquefaciens CPA-8, formerly known as B. subtilis (Gotor-Vila et al, 2016), has been previously described against postharvest diseases caused by Monilinia spp. based on its ability to produce fengycin-like lipopeptides (Yánez-Mendizábal et al, 2012a;Yánez-Mendizábal et al, 2012d) One of the major bottle necks in the commercialisation of biocontrol products is the development of shelf-stable formulations (Droby et al, 2009;Navarta et al, 2011). A useful microbial formulation should be economical to produce, easy to distribute to the intended environment, contain enough colony forming units (CFU), and provide a long shelf-life (preferentially stored at room temperatures and maintained for at least six months) (Melin et al, 2007;Teixidó et al, 2011).…”

Quantifying the impact of storage procedures for faecal bacteriotherapy in the critically endangered New Zealand Parrot, the Kakapo (Strigops habroptilus)

“…A potential alternative is to freeze pathogen-free faeces with a cryoprotectant in order to suspend microbial activity without damage to the cells. The use of non-toxic cryoprotectant agents is a common practice, with chemicals such as glycerol, skim milk, and sugar compounds frequently cited either as stand-alone reagents [Dan et al, 1989;Bonten et al, 1997;Cody et al, 2008;Savini et al, 2010;Kanmani et al, 2011] or in combination [O'Brien et al, 2001;Kanmani et al, 2011;Navarta et al, 2011]. The relative efficacies of these reagents is highly variable, although as stand-alone reagents glycerol and skim milk additives are consistently more effective than either sugar or other commercial additives [Bonten et al, 1997;Kanmani et al, 2011;Navarta et al, 2011].…”

“…The use of non-toxic cryoprotectant agents is a common practice, with chemicals such as glycerol, skim milk, and sugar compounds frequently cited either as stand-alone reagents [Dan et al, 1989;Bonten et al, 1997;Cody et al, 2008;Savini et al, 2010;Kanmani et al, 2011] or in combination [O'Brien et al, 2001;Kanmani et al, 2011;Navarta et al, 2011]. The relative efficacies of these reagents is highly variable, although as stand-alone reagents glycerol and skim milk additives are consistently more effective than either sugar or other commercial additives [Bonten et al, 1997;Kanmani et al, 2011;Navarta et al, 2011]. Between skim milk and glycerol additives, there are varying reports of comparative efficacy, although these are often confounded by additional factors such as the bacterial strains tested, quantities used and freezing regime [Cody et al, 2008;Savini et al, 2010;Kanmani et al, 2011;Navarta et al, 2011].…”