Microbiota dynamics and bacterial pathogen survival during milk kefir fermentation and storage

This study identified and investigated the relative abundance of bacterial and fungal (predominantly yeast) species in the milk kefir grains, during a typical fermentation (25°C for 48 h) and subsequent chilled storage (4°C for up to 30d) using Nanopore sequencing (16S and ITS amplicon). Challenge studies were also undertaken to examine the behaviour of Listeria monocytogenes , Salmonella Senftenberg, Escherichia coli O157:H7, Staphylococcus aureus as well as non-toxigenic mesophilic and psychrophilic Clostridium spp. ( Clostridium sporogenes and Clostridium gasigenes , respectively). Lactobacillus and Lactococcus were the predominant bacterial genera with relative abundance of 91% and 2% in milk kefir grains, 32% and 63.7% after 24 h of the fermentation, 87% and 2% after 48 h, respectively. Other bacteria detected included Lentilactobacillus , Leuconostoc , Acetobacter, Asaia and Entomobacter . The most abundant fungal genus in the milk kefir grains was the yeast, Brettanomyces (90%), consisting solely of the species Brettanomyces anomalus , which remained dominant at 24 h (80.7%), 14d (71.7%) and 21d (75.6%). Zygotorulaspora florentina was most abundant immediately after adding the grains to the milk (42.8%) and after 7d (33.9%). Other significant yeast genera detected included Kazachstania (mainly K. unispora ) and Grigorovia (mainly G. humatica ). In the challenge studies, the L. monocytogenes population increased by 1.3 log 10 cfu ml -1 after 8 h, remained static until 32 h, decreased by 2.9 log 10 cfu ml -1 (48 h) with the residual population of approximately 0.6 log 10 cfu ml -1 being maintained during chilled storage. S. Senftenberg and E. coli O157:H7 also increased by approximately 1.3 and 2.8 log 10 cfu ml -1 after 8 h and 24 h, respectively. The former were not detectable after 48 h while the E. coli O157:H7 population had decreased by approximately 3 log 10 cfu ml -1 leaving a sub-population of approximately 0.5 log 10 cfu ml -1 that was still detectable up to 14 days. In contrast, S. aureus growth was limited and these bacteria were not detected at 48 h, while C. sporogenes and C. gasigenes populations were stable at 1.8-2.1 log 10 cfu ml -1 and 1.6-2.4 log 10 cfu ml -1 , respectively, throughout the kefir fermentation and during chilled storage. It was concluded that the dominant bacterial and fungal species in the milk kefir grains and product were Lactobacillus kefiranofaciens and Brettanomyces anomalus , respectively, although other bacteria and yeasts were also present. Despite the production of lactic acid with rapid acidification, bacterial pathogens survived suggesting the safety of these products is reliant on pasteurisation of the milk, good hygiene practices throughout the process and efficient chilled storage of the final product.