Extraction of Bacillus endospores from water, apple juice concentrate, raw milk and lettuce rinse solutions using tangential flow filtration

As part of investigative sampling there is a need to rapidly concentrate the target from large sample volumes to enable downstream detection using suitable diagnostic platforms. To this end the following describes a method based on tangential flow filtration (0.2 μm pore size) for concentrating Bacillus endospores from sample matrices (water, raw whole milk, apple juice concentrate and lettuce rinse samples). Bacillus endospores were selected as a model system as a representative select agent of significance. From optimization studies it was found that the recovery of endospores was in the order of 2.5% and could not be enhanced through increasing the flow rate or transmembrane pressure (TMP). However, recoveries could be increased to >80% (concentration factor 6.74–8.86) by backwashing the system with 10% w/v Tween 80 solution to detach spores adsorbed onto the internal surface of the filtration unit. The recovery of endospores from raw milk and apple juice concentrate was problematic due to the presence of solids or high viscosity. However, by performing an initial dilution (1:10) prior to passing through the Tangential Flow Filtration (TFF) system it was possible to achieve over 80% endospore recovery with no significant decrease in filter performance (Concentration Factor of 6–10). The recovery of endospores from lettuce surfaces was facilitated by rinsing with 4% w/v Tween 80 solution. When used in conjunction with tangential flow filtration system it was possible to recover endospores derived from lettuce rinse solutions at levels in the order of 300 cfu/ml. The results of the study illustrate that TFF is a useful approach for concentrating microbial targets from a diverse range of food matrices.

► A culture-free method for concentrating endospores from different food matrices.
► We developed a dilution technique to minimize filter membrane fouling by raw milk.
► A method that can be coupled to diagnostic platforms to enhance sensitivity.