Bacterial cell recovery after hollow fiber microfiltration sample concentration and washing: most probable bacterial composition in frozen vegetables

Authors: IRWIN, PETER - Collaborator; He, Yiping; Nguyen, Ly Huong; GEHRING, MATTHEW - Pennsylvania State University; Gehring, Andrew; Chen, Chinyi; Capobianco, Joseph

Submitted to: LWT - Food Science and Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2020
Publication Date: 12/4/2020
Citation: Irwin, P., He, Y., Nguyen, L.T., Gehring, M., Gehring, A.G., Chen, C., Capobianco Jr, J.A. 2020. Bacterial cell recovery after hollow fiber microfiltration sample concentration and washing: Most probable bacterial composition in frozen vegetables. LWT - Food Science and Technology. 140:110. https://doi.org/10.1016/j.lwt.2020.110640.
DOI: https://doi.org/10.1016/j.lwt.2020.110640

Interpretive Summary: In order to determine the quantity of pathogens in contaminated food, it is necessary to detect as few as 1 bacterium per sampled volume (or mass) because their numbers are typically very low. Because these organisms are rare, larger volumes are required to be sampled. In this work we have investigated procedures to concentrate food-borne organisms in food samples in such a fashion that a high recovery rate can be obtained without a concomitant alteration in the makeup of the test foods’ microbial flora. Using a hollow fiber filter we measured not only the overall recovery rates but also perturbations to the makeup of the most probable composition using 16S rDNA sequencing due to the concentration process itself. We found that this concentrating process gives relatively high recovery rates (77 ± 4.04%) with no significant alteration in the microbiological makeup. Working with E. coli-spiked buffers we were also able to determine that over 1 Liter of sample (5 CFU per mL) can be efficiently (ca. 86% recovery) reduced in volume to about 2 mL. By collecting an additional 3 mL we were able to increase this recovery rate to 91%. This information is useful to food safety microbiologists in the quantitative assessment of bacterial communities which exist in foods using DNA sequencing-based technologies.

Technical Abstract: In this work the analytics associated with a commercial "concentrating pipette" are scrutinized from the perspective of sample volume reduction in order to augment native bacterial quantitation and identification using 16S rDNA sequence analysis. Results evaluating the relative recovery rate of bacteria in buffers, micron-size particles (latex beads), and the native microbial population in vegetable washes are presented; the perturbation in the native bacterial composition of food due to the filter concentration process were also examined. We found that there was no significant effect of different diluents/eluents on the relative recovery rate of our test bacteria from samples. When concentrating PBS washes from thawed frozen vegetables and enumerating after plates were incubated at either 30 or 37°C, the recovery rates were only slightly lower than above. However, in food wash samples, significantly more mass-normalized bacteria were recovered at the lower culture temperature. Nevertheless, the vegetable wash concentration process did not significantly change the most probable isolate composition relative to that of the controls at either culture temperature. We found no significant difference in recovery of E. coli to those of 0.6-0.8 µm latex beads in water suggesting that there was minimal bacterial injury caused by the InnovaPrep sample processing.