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Microbiota Assessments for the Identification and Confirmation of Slit Defect-Causing Bacteria in Milk and Cheddar Cheese.

ABSTRACT: Validated methods are needed to detect spoilage microbes present in low numbers in foods and ingredients prior to defect onset. We applied propidium monoazide combined with 16S rRNA gene sequencing, qPCR, isolate identification, and pilot-scale cheese making to identify the microorganisms that cause slit defects in industrially produced Cheddar cheese. To investigate milk as the source of spoilage microbes, bacterial composition in milk was measured immediately before and after high-temperature, short-time (HTST) pasteurization over 10-h periods on 10 days and in the resulting cheese blocks. Besides HTST pasteurization-induced changes to milk microbiota composition, a significant increase in numbers of viable bacteria was observed over the 10-h run times of the pasteurizer, including 68-fold-higher numbers of the genus Thermus However, Thermus was not associated with slit development. Milk used to make cheese which developed slits instead contained a lower number of total bacteria, higher alpha diversity, and higher proportions of Lactobacillus, Bacillus, Brevibacillus, and Clostridium Only Lactobacillus proportions were significantly increased during cheese aging, and Limosilactobacillus (Lactobacillus) fermentum, in particular, was enriched in slit-containing cheeses and the pre- and post-HTST-pasteurization milk used to make them. Pilot-scale cheeses developed slits when inoculated with strains of L. fermentum, other heterofermentative lactic acid bacteria, or uncultured bacterial consortia from slit-associated pasteurized milk, thereby confirming that low-abundance taxa in milk can negatively affect cheese quality. The likelihood that certain microorganisms in milk cause slit defects can be predicted based on comparisons of the bacteria present in the milk used for cheese manufacture.IMPORTANCE Food production involves numerous control points for microorganisms to ensure quality and safety. These control points (e.g., pasteurization) are difficult to develop for fermented foods wherein some microbial contaminants are also expected to provide positive contributions to the final product and spoilage microbes may constitute only a small proportion of all microorganisms present. We showed that microbial composition assessments with 16S rRNA marker gene DNA sequencing are sufficiently robust to detect very-low-abundance bacterial taxa responsible for a major but sporadic Cheddar cheese spoilage defect. Bacterial composition in the (pasteurized) milk and cheese was associated with slit defect development. The application of Koch's postulates showed that individual bacterial isolates as well as uncultured bacterial consortia were sufficient to cause slits, even when present in very low numbers. This approach may be useful for detection and control of low-abundance spoilage microorganisms present in other foods.

SUBMITTER: Xue Z

PROVIDER: S-EPMC7883541 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In cheese production, microorganisms are usually added at the beginning of the process as primary starters to drive curd acidification, while secondary microorganisms, with other pro-technological features important for cheese ripening, are added as selected cultures. This research aimed to investigate the possibilities of influencing and selecting the raw milk microbiota using artisanal traditional methods, providing a simple method to produce a natural supplementary culture. We investigated the production of an enriched raw milk whey culture (eRWC), a natural adjunct microbial culture produced from mixing an enriched raw milk (eRM) with a natural whey culture (NWC). The raw milk was enriched by spontaneous fermentation for 21 d at 10°C. Three milk enrichment protocols were tested: heat treatment before incubation, heat treatment plus salt addition, and no treatment. The eRMs were then co-fermented with NWC (ratio of 1:10) at 38°C for 6 h (young eRWC) and 22 h (old eRWC). Microbial diversity during cultures' preparation was evaluated through the determination of colony forming units on selective growth media, and next-generation sequencing (16S rRNA gene amplicon sequencing). The enrichment step increased the streptococci and lactobacilli but reduced microbial richness and diversity of the eRMs. Although the lactic acid bacteria viable count was not significantly different between the eRWCs, they harbored higher microbial richness and diversity than NWC. Natural adjunct cultures were then tested in cheese making trials, following the microbial development, and assessing the chemical quality of the 120 d ripened cheeses. The use of eRWCs slowed the curd acidification in the first hours of cheese making but the pH 24 h after production settled to equal values for all the cheeses. Although the use of diverse eRWCs contributed to having a richer and more diverse microbiota in the early stages of cheese making, their effect decreased over time during ripening, showing an inferior effect to the raw milk microbiota. Even if more research is needed, the optimization of such a tool could be an alternative to the practice of isolating, geno-pheno-typing, and formulating mixed-defined-strain adjunct cultures that require knowledge and facilities not always available for artisanal cheese makers.

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Microbiota Assessments for the Identification and Confirmation of Slit Defect-Causing Bacteria in Milk and Cheddar Cheese.

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