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Optimization of Preservation Methods Allows Deeper Insights into Changes of Raw Milk Microbiota.

ABSTRACT: The temporal instability of raw milk microbiota drastically affects the reliability of microbiome studies. However, little is known about the microbial integrity in preserved samples. Raw cow milk samples were preserved with azidiol or bronopol and stored at 4 °C, or with dimethyl sulfoxide (DMSO) or a mixture of azidiol and DMSO and stored at -20 °C for up to 30 days. Aliquots of 5-, 10-, and 30-day post-storage were treated with propidium monoazide (PMA), then analyzed by sequencing the 16S rRNA gene V3-V4 and V6-V8 regions. The V6-V8 gave a higher richness and lower diversity than the V3-V4 region. After 5-day storage at 4 °C, the microbiota of unpreserved samples was characterized by a drastic decrease in diversity, and a significant shift in community structure. The treatment with azidiol and DMSO conferred the best community stabilization in preserved raw milk. Interestingly, the azidiol treatment performed as well for up to 10 days, thus appearing as a suitable alternative. However, neither azidiol nor bronopol could minimize fungal proliferation as revealed by PMA-qPCR assays. This study demonstrates the preservative ability of a mixture of azidiol and DMSO and provides deeper insights into the microbial changes occurring during the cold storage of preserved raw milk.

SUBMITTER: Kennang Ouamba AJ

PROVIDER: S-EPMC7142718 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Optimization of Preservation Methods Allows Deeper Insights into Changes of Raw Milk Microbiota.

Kennang Ouamba Alexandre J AJ LaPointe Gisèle G Dufour Simon S Roy Denis D

Microorganisms 20200305 3

The temporal instability of raw milk microbiota drastically affects the reliability of microbiome studies. However, little is known about the microbial integrity in preserved samples. Raw cow milk samples were preserved with azidiol or bronopol and stored at 4 °C, or with dimethyl sulfoxide (DMSO) or a mixture of azidiol and DMSO and stored at -20 °C for up to 30 days. Aliquots of 5-, 10-, and 30-day post-storage were treated with propidium monoazide (PMA), then analyzed by sequencing the 16S rR ...[more]

PMID: 32151050

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Project description:As the key contributor to plankton biomass and nutrient cycling in aquatic ecosystems, photosynthetic picoeukaryotes (PPEs) have been recently investigated in freshwater ecosystems. However, the limited access to remote areas creates challenges for PPE sample preservation before sorting and counting by flow cytometry (FCM) in the laboratory. Here, we explored the effects of different preservation methods on the PPE community by combining FCM sorting and high-throughput sequencing. Our results showed that dimethyl sulfoxide (DMSO) cryoprotection could destroy the fluorescence and cell structure of the PPEs, making the subsequent FCM analysis and sorting difficult. Aldehyde fixation maintained the PPE fluorescence, and the fixed samples were of sufficient quality for abundance analysis and sorting by FCM. However, the sequencing results showed that, after preservation by aldehydes, the proportion of PPEs dramatically decreased to approximately 10%, in comparison to 90% in the fresh samples, and the sequences of Ascomycota significantly increased. In contrast, preservation with Pluronic F68 (F68) not only could maintain the PPE abundance close to the initial value but also could keep the PPE community similar to that in the fresh samples over a storage time of 6 months. Thus, F68 cryopreservation is a suitable preservation method for PPE communities from freshwater lakes. IMPORTANCE PPEs contribute significantly to primary productivity in freshwater ecosystems. The combination of FCM sorting and high-throughput sequencing has been shown to be a powerful approach and can largely improve our view of the PPE diversity. However, the water samples could not be counted and sorted immediately after sampling from many lakes due to the inaccessibility of FCM in the field. Thus, the comparison of different preservation methods that allow subsequent analysis of the community structure by high-throughput sequencing is an urgent need. Our results indicated that F68 cryopreservation could maintain the PPE abundance close to the initial value and keep the community similar to that in the fresh samples over a storage time of 6 months.

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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Optimization of Preservation Methods Allows Deeper Insights into Changes of Raw Milk Microbiota.

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Optimization of Preservation Methods Allows Deeper Insights into Changes of Raw Milk Microbiota.

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