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

Alexandre J Kennang Ouamba,Gisèle Lapointe,Simon Dufour,Denis Roy

doi:10.3390/microorganisms8030368

Alexandre J Kennang Ouamba, Gisèle Lapointe + Show 2 more

Open Access

https://doi.org/10.3390/microorganisms8030368

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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.

Highlights

The interest in culture-independent approaches to study the microbiological quality of dairy milk has continued to rise, especially with the advent of high-throughput omics technologies in the field of dairy science and technology, including metatranscriptomics, metaproteomics, and metagenomics [1]
712 and 977 amplicon sequence variants (ASVs) were inferred for V3-V4 and V6-V8 regions, respectively
We compared the taxonomic profiles resulting from 16S rRNA sequencing of V3-V4 and V6-V8 regions in unpreserved milk samples at day 0

Summary

Introduction

The interest in culture-independent approaches to study the microbiological quality of dairy milk has continued to rise, especially with the advent of high-throughput omics technologies in the field of dairy science and technology, including metatranscriptomics, metaproteomics, and metagenomics [1]. Raw milk samples generally harbor diverse microbial communities of which most can develop due to high nutrient and water contents of the matrix Whether they are spoilage microorganisms [4], disease causing [5], health promoting, or of technological interest [6,7], microbial dynamics is still occurring in raw milk once refrigerated immediately after milking [8,9] and during transportation to dairy processing facilities [10]. This temporal instability of milk microbiota can considerably affect sample microbial integrity, especially in prolonged sampling or when the sample delivery time to the laboratory is delayed, as it frequently happens in large-scale studies. Several preservative chemicals have been developed to prevent milk spoilage or preserve milk constituents, among which bronopol and azidiol are commonly used for milk chemical analysis [15]

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