Evaluating the efficacy of non-thermal microbial load reduction treatments of heat labile food components for in vitro fermentation experiments

Authors: PAFF, ANDREW - Pennsylvania State University; COCKBURN, DARRELL - Pennsylvania State University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/3/2023
Publication Date: 3/21/2023
Citation: Paff, A., Cockburn, D.W. 2023. Evaluating the efficacy of non-thermal microbial load reduction treatments of heat labile food components for in vitro fermentation experiments. PLOS ONE. 18(3). Article e0283287. https://doi.org/10.1371/journal.pone.0283287.
DOI: https://doi.org/10.1371/journal.pone.0283287

Interpretive Summary: Simulated colon fermentations are being used as a pre-clinical step to assess the impacts of foods and drugs on the gut microbiota in a cost-effective manner. There is currently a gap in scientific understanding around the effectiveness of liquid sterilants as a non-thermal alternative to autoclaving food materials prior to simulated colonic fermentation. This is particularly important for studying foods that are consumed in a raw form, including many supplements, to allow the study of the impact of these foods on the gut microbiota without interference from native microbes and without thermal destruction of heat sensitive components. In this study, we evaluated the efficacy of three different liquid chemicals (alcohol, hydrogen peroxide, and bleach), commonly accessible at low prices, in addressing the microbial load in green lentil, sprouted green lentil, chickpea, and field pea pulse flours. We measured the microbial load reduction achieved by these sterilants, their effect on starch structure, as well as their downstream effects on simulated in vitro colonic fermentation of these flours. We found that both bleach and hydrogen peroxide are effective treatments and recommend their use by labs that are similarly exploring in vitro fermentations of heat labile food components, such as resistant starch.

Technical Abstract: Increasingly, in vitro simulated colon fermentations are being used as a pre-clinical step to assess the impacts of foods and drugs on the gut microbiota in a cost-effective manner. One challenge in such systems is that they are potentially susceptible to the influences of contaminating microbes in test materials. Simulated gastric and intestinal digestion can relieve some of these concerns, however, live microbes may remain that can confound analysis. Autoclave treatment of test materials is the surest way to eliminate these microbes but presents problems when using heat labile components such as resistant starch. In this study, liquid chemical sterilant alternatives to moist heat sterilization were explored for treating pulse flours for use during in vitro simulated colon fermentation. Key attributes considered in chemical selection were accessibility, impact on treated food components, and effectiveness of the treatments for reducing microbial load. Three chemicals were selected for evaluation, bleach, alcohol, and hydrogen peroxide, at varying concentrations. Flours chosen for testing were from green lentil, field pea, chickpea, or sprouted green lentil. All treatments significantly reduced microbial loads, though there were still detectable levels of microbes after alcohol treatments. Furthermore, in vitro simulated colon fermentations of the treated pulses showed minimal difference from the untreated control both in terms of microbial composition and short chain fatty acid production. Scanning electron microscopy showed minimal impact of sterilization treatments on the gross structure of the pulse flours. Together these results suggest that bleach and hydrogen peroxide treatments can be effective nonthermal treatments to eliminate contaminating microbes in pulse flours without causing significant damage to starch and other fermentable substrates. This is thus also a promising treatment method for other starchy food substrates, though further testing is required.