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

Abstract

The present work focuses on the assessment and comparison of three different formulation technologies and the effect of protectants on cell viability, storage stability, and antagonistic activity of the biocontrol agent Bacillus amyloliquefaciens CPA-8. Cultures were concentrated with different protective substances such as MgSO4, sucrose, and skimmed milk (SM) and subjected to liquid formulation, freeze-drying, and fluid-bed spray-drying. Results showed that CPA-8 freeze-dried cells without protectants or amended with SM suffered the highest losses in cell viability (0.41-0.48 log). Moreover, the cell viability of the tested freeze-dried products decreased after four months of storage at both tested temperatures (4 and 22 ºC). Otherwise, liquid and fluid-bed spray-dried products were stable for four months at 4 ºC and for twelve months at 22, 4 and-20 ºC, respectively, and no effect of the protectants was observed. The most suitable CPA-8 products were then tested against Monilinia laxa and Monilinia fructicola on artificially wounded nectarines and in all cases, the antagonistic activity was maintained similar to fresh cells. The efficacy results revealed that the formulation process did not affect the biocontrol potential of CPA-8. This work led us to conclude that effective formulations with final concentrations ranging from 1.93•10 9 to 2.98•10 9 CFU mL-1 and from 4.76•10 9 to 1.03•10 10 CFU g-1 were obtained for liquid and dried products, respectively. Additionally, the suitability of the fluid-bed spray-drying technology should be taken into account to develop a stable and effective CPA-8 product for practical applications to control brown rot in stone fruit.

Enhanced shelf-life of the formulated biocontrol agent Bacillus amyloliquefaciens CPA-8 combining diverse packaging strategies and storage conditions

“…It is a very remarkable finding since costs of storage and transportation are significantly reduced due to the needless of refrigeration. These results are in agreement with previous reports on CPA-8, in which spray-dried (Yánez-Mendizábal et al, 2012a), freeze-dried and fluid-bed spray-dried cells proved to be shelf-stable after storage at 22 ºC using standard laboratory plastic flasks (Gotor-Vila et al, 2017b;Gotor-Vila et al, 2017d). Taken together all data recorded in this work, higher a w in the samples correlated with poor keeping qualities that mainly appeared in the packaging materials with less protecting barriers.…”

“…Two CPA-8 formulated products were obtained by using a fluid-bed spray-dryer (HüttlinGmbH, Bosch Packaging Technology Company, Schopfheim, Germany) as previously described by Gotor-Vila et al (2017b;2017d). Briefly, CPA-8 cells were mixed with the protective substances 20 % sucrose plus 10 % skimmed milk and with 3.5 g of pregelatinised potato starch as binder and then fluid-bed spray-dried with 300 g of powdered carrier material previously loaded into the drying camera.…”

“…Samples were shaken vigorously for 1 min and then allowed to rehydrate for other 9 min in static. Ten-fold dilutions of each suspension were plated on NYDA to determine cell concentration per gram (CFU g -1 ) (Gotor-Vila, et al 2017b;2017d). To calculate the moisture content of the CPA-8 dried products, samples of 0.5 g each were placed in aluminium-weighing boats and dried in a convection oven at 100 °C for 24 h.…”

Environmental stress responses of the Bacillus amyloliquefaciens CPA-8-formulated products on nectarines and peaches

“…CPA-8 cells were harvested by centrifugation at 9820 g for 12 min at 10 ºC in an Avanti J-20 XP centrifuge (Beckman Coulter, CA, USA) and resuspended approximately at 10 10 CFU mL -1 in the same CPA-8 supernatant medium to include the antifungal lipopeptides synthesised by the bacterium during the production process (Yánez-Mendizábal et al, 2012b). Two CPA-8 formulated products were obtained by using a fluid-bed spray-dryer (HüttlinGmbH, Bosch Packaging Technology Company, Schopfheim, Germany) according to the protocol developed by Gotor-Vila, et al (2017b) and by Gotor-Vila, et al (2017c). Briefly, CPA-8 cells were mixed with the protective substances 20 % sucrose plus 10 % skimmed milk and then fluid-bed spray-dried with 300 g of powdered carrier material previously loaded into the drying camera.…”

Compatibility interactions between the biocontrol agent Penicillium frequentans Pf909 and other existing strategies to brown rot control

“…Bacterium was produced by liquid agitation of 50 ml TEC in 250 ml flasks in an orbital motion (Lab-Line Instruments, Inc., model 3527, Melrose Park Illinois, USA) set to 200 rev min -1 , for 72 h at 30ºC to obtain high endospore concentration (Gotor-Vila et al, 2017b).…”