Abstract
BackgroundSpoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Identification of these organisms by classic cultivation methods is limited by their ability to form colonies on nutrient agar plates. In this study, we adapted and optimized 16S rRNA amplicon sequencing for quantification of bacterial spores in a canned food matrix and for monitoring the outgrowth of spoilage microbiota in a ready-to-eat food matrix.ResultsThe detection limit of bar-coded 16S rRNA amplicon sequencing was determined for the number of bacterial spores in a canned food matrix. Analysis of samples from a canned food matrix spiked with a mixture of equinumerous spores from the thermophiles, Geobacillus stearothermophilus and Geobacillus thermoglucosidans, and the mesophiles, Bacillus sporothermodurans, Bacillus cereus, and Bacillus subtilis, led to the detection of these spores with an average limit of 2 × 102 spores ml−1. The data were normalized by setting the number of sequences resulting from DNA of an inactivated bacterial species, present in the matrix at the same concentration in all samples, to a fixed value for quantitative sample-to-sample comparisons. The 16S rRNA amplicon sequencing method was also employed to monitor population dynamics in a ready-to-eat rice meal, incubated over a period of 12 days at 7 °C. The most predominant outgrowth was observed by the genera Leuconostoc, Bacillus, and Paenibacillus. Analysis of meals pre-treated with weak acids showed inhibition of outgrowth of these three genera. The specificity of the amplicon synthesis was improved by the design of oligonucleotides that minimize the amplification of 16S rRNA genes from chloroplasts originating from plant-based material present in the food.ConclusionThis study shows that the composition of complex spoilage populations, including bacterial spores, can be monitored in complex food matrices by bar-coded amplicon sequencing in a quantitative manner. In order to allow sample-to-sample comparisons, normalizations based on background DNA are described. This method offers a solution for the identification and quantification of spoilage microbiota, which cannot be cultivated under standard laboratory conditions. The study indicates variable detection limits among species of bacterial spores resulting from differences in DNA extraction efficiencies.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0096-3) contains supplementary material, which is available to authorized users.
Highlights
Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria
Detection of spores in a canned food matrix by colony enumeration The “spike” spore mixture consisting of three mesophilic species, B. subtilis A163, B. cereus thank Jordy Coolen (TNO) 02.0143, and B. sporothermodurans IC4, two thermophilic species, G. thermoglucosidans TNO 09.020 and G. stearothermophilus ATCC 7953, and tenfold dilutions of these five strains were mixed into creamy mushroom soup
Canned food and a RTE meal were chosen as model food matrices as they represent two different relevant food matrices and were suitable for studying different aspects important in food spoilage: the detection limit of amplicon sequencing as detection method for bacterial spore formers in a complex food matrix as well as the possible use of a spikes in order to quantify the detection method or the use of amplicon sequencing in following the dynamics of food spoilage at low temperatures caused by spoilage organisms collected during the production process in the presence and absence of commonly used preservatives (RTE rice meal)
Summary
Spoilage of food products is frequently caused by bacterial spores and lactic acid bacteria. Microbial food spoilage results from metabolic processes that lead to the production of off-smelling flavors or textural changes and renders food unacceptable for human consumption. It is by far the most common cause for food losses. We applied the 16S rRNA amplicon pyrosequencing method for the evaluation of microbial spoilage in complex foods, including a ready-to-eat (RTE) meal as well as processed canned food. These are very different in nature with regard to the spoilage process. Outgrowth of spoilage microorganisms is limited by refrigeration and preservatives, and in the second case, heat processing during manufacturing limits the spoilage problem to outgrowth of bacterial spores that are able to survive the heat regimes applied
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