Complementary Use of Cultivation and High-Throughput Amplicon Sequencing Reveals High Biodiversity Within Raw Milk Microbiota.

Franziska Breitenwieser,Etienne V Doll,Siegfried Scherer,Mareike Wenning,Thomas Clavel

doi:10.3389/fmicb.2020.01557

Abstract

Raw milk microbiota are complex communities with a significant impact on the hygienic, sensory and technological quality of milk products. However, there is a lack of knowledge on factors determining their composition. In the present study, four bulk tank milk samples of two farms at two different time points were analyzed in detail for their microbiota using cultivation and 16S rRNA amplicon sequencing. Diversity in samples from the first time point was assessed via cultivation of 500 aerobic mesophilic bacterial isolates in each sample. A high biodiversity of 70 and 110 species per sample was determined, of which 25–28% corresponded to yet unknown taxa. The isolates were dominated by Gram-positive members of the genera Staphylococcus, Corynebacterium, Streptococcus, or Janibacter, whilst Chryseobacterium and Acinetobacter were most abundant among the Gram-negative taxa. At the second time point, samples of the same farms were analyzed via both cultivation (1,500 individual colonies each) and high-throughput 16S rRNA gene amplicon sequencing. The latter revealed a threefold higher biodiversity at the genus level, as anaerobic or fastidious species were also detected. However, cultivation identified genera not captured by sequencing, indicating that both approaches are complementary. Using amplicon sequencing, the relative abundance of a few genera was distorted, which seems to be an artifact of sample preparation. Therefore, attention needs to be paid to the library preparation procedure with special emphasis on cell lysis and PCR.

Highlights

The indigenous microbiota of raw milk has re-gained attention during the last years for economic and safety reasons
This increases the risk of zoonotic diseases due to the presence of pathogenic bacteria like Listeria monocytogenes, Salmonella spp., High Biodiversity of Raw Milk Microbiota
Bulk tank milk of two farms in Southern Germany was analyzed in detail for microbial biodiversity

Summary

Introduction

The indigenous microbiota of raw milk has re-gained attention during the last years for economic and safety reasons. Consumers increasingly prefer unpasteurized products such as raw milk cheese or raw milk bought in retail sale or directly at the farm. This increases the risk of zoonotic diseases due to the presence of pathogenic bacteria like Listeria monocytogenes, Salmonella spp., High Biodiversity of Raw Milk Microbiota. Heat-resistant peptidases primarily produced by Gram-negative bacteria such as Pseudomonas or Serratia (Marchand et al, 2009; von Neubeck et al, 2015; Machado et al, 2017) impact the sensory and textural properties of UHT milk during elongated shelf-life and even very low residual activities may lead to bitterness and age gelation (Stoeckel et al, 2016). Besides all negative implications, the raw milk microbiota is valuable and appreciated, for instance for obtaining individual and flavor-rich cheeses (Callon et al, 2005; Montel et al, 2014)

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