Surviving ratio of milk microflora during storage at freezing temperature : a pilot study

Abstract: Milk contains beneficial and non-beneficial bacteria which contribute with different outcomes to milk and dairy products. Beneficial bacteria are important during manufacturing while non-beneficial bacteria cause spoilage, food related outbreaks but also economical losses for the dairy industry. It is therefore important to control the survival of wanted and unwanted bacteria through microbial analyses. For this purpose, it would be beneficial if milk samples could be stored frozen for some time until analyses will be performed. The aim of this study was to investigate changes in ratio of milk microbiota during storage at two freezing temperatures, -20°C and -80°C, with respect to time. The hypothesis was that no change of milk microbiota would occur. The microbial community in the fresh milk was investigated directly after collection followed by once-a-week cultivation during eight weeks. Analysis was performed through pure culture streaking method both aerobically on Plate Count agar (PCA) and anaerobically on De Man Rogosa and Sharpe (MRS) agar. Bacterial identification was performed using MALDI-TOF MS. Results revealed an effect of storage temperature, where the number of CFU/ml cultivated from milk samples stored at -80°C was higher than after storage at -20°C, irrespectively if bacteria were cultured on PCA or MRS. However, the effect was only significant for bacteria cultured on MRS. There was a significant effect of storage time on both aerobes and anaerobes when cultivating milk samples stored at -80°C and -20°C, respectively. However, for aerobes the difference between these two temperatures was not significant. The colony characterization by MALDI-TOF MS identified 14 different species from PCA and 9 species from MRS media. In conclusion, to maintain the original microbial community in milk during storage at freezing temperatures, storage of milk samples at -80°C is recommended.