Identification and confirmation of slit defect-causing bacteria in Cheddar cheese

Abstract

Background: Spoilage microbes remain a significant economic burden for the dairy industry. Validated approaches are needed to identify microbes present in low numbers in those foods and starting ingredients prior to spoilage. Therefore, we applied a combination of propidium monoazide treatment combined with 16S rRNA gene amplicon DNA sequencing for viable cell detection, qPCR for bacterial enumeration, and laboratory culture, isolate identification, and pilot-scale cheese production to identify the causative bacterial agents of slit defects in industrially-produced Cheddar cheese. Because spoilage cannot be predicted in advance, the bacterial composition in milk was measured immediately before and after High Temperature Short Time (HTST) pasteurization over time and on multiple days and in resulting cheese blocks. Results: Milk was sampled over 10 h periods on ten days immediately before and after the final HTST pasteurization step prior to the initiation of cheese fermentations. HTST reduced the alpha-diversity of the viable, but not total, bacterial contents in milk and increased the proportions of thermoduric and endospore-forming bacterial taxa. There was a significant increase in viable bacterial cell numbers over the 10-h run times of the pasteurizer, including 68-fold higher numbers of Thermus. Between 0.22% to 10.9% of the bacteria in cheese were non-starter contaminants comprised mainly of Lactobacillus and Streptococcus, however, only Lactobacillus proportions increased during cheese aging. Lactobacillus, and Lactobacillus fermentum in particular, was also enriched in slit-containing cheeses and in the pre-HTST and post-HTST milk used to make them. Although some endospore-forming bacteria were associated with slits and could be isolated from milk and cheese, none were consistently associated with slit development. Pilot-scale cheeses developed slits when inoculated with L. fermentum, other heterofermentative lactic acid bacteria isolates, or with uncultured bacterial consortia collected from the pre-HTST or post-HTST milk, thus confirming that low abundance taxa in milk can negatively affect cheese quality. Conclusions: We identified and verified that certain low-abundance, bacterial taxa in milk are responsible for causing slit defects in Cheddar cheese. The likelihood for microorganisms in milk to cause defects could be predicted based on comparisons of the bacteria present in the pre- and post-HTST milk used for cheesemaking.

Improved assessments of milk microbiota composition via sample preparation and DNA extraction methods

“…Two raw (unpasteurized) milk from the storage Silos and one HTST pasteurized milk samples were collected by a commercial dairy processor (Hilmar Cheese Company, Hilmar, CA) on 5/23/2018. Bacteria in the milk were collected as previously described [21]. Briefly, each milk sample was split into three 25 mL aliquots, centrifuged at 13,000 g for 5 min at 4 °C, and then the cell pellets were stored at −20 °C until.…”

“…The remaining reads were analyzed using QIIME 1.9.1 [24] and QIIME 2 version 2018.4 [25] with previously described parameters [21]. QIIME 2 generated feature tables, feature sequences, rooted phylogenetic tree, and sample information were imported in R 3.4.2 and analyzed as previously described [21]. Briefly, sequence files were demultiplexed with the demux plugin.…”

“…Reads that were shorter than 200 bases were discarded. The remaining reads were analyzed using QIIME 1.9.1 [24] and QIIME 2 version 2018.4 [25] with previously described parameters [21].…”

“…QIIME 2 generated feature tables, feature sequences, rooted phylogenetic tree, and sample information were imported in R 3.4.2 and analyzed as previously described [21]. Briefly, sequence files were demultiplexed with the demux plugin.…”