Diversity of psychrotrophic pseudomonads from raw milk - a method for rapid identification using MALDI-TOF-MS

Introduction: Refrigerated storage of raw milk is a fundamental hygienic measure in the dairy industry. However, under these conditions psychrotrophic bacteria can proliferate in raw milk and secrete enzymes that contribute to product spoilage. Heat-resistant peptidases, which are mainly produced by bacteria of the genus Pseudomonas, can cause spoilage effects of ultrahigh temperature (UHT) treated and sterilized milk which is manifested by symptoms such as bitterness, increased viscosity and gelling. Rapid and reliable identification of pseudomonads in raw milk and tracing their sources on farms and in processing plants is therefore of great importance in order to avoid economic losses and food waste in the milk value chain.

Goals: Our study aimed to rapidly identify spoilage bacteria of the genus Pseudomonas isolated from raw milk by the means of their protein fingerprints using the mass spectrometric technique MALDI-TOF-MS.

Methods: For this purpose, a custom database with more than 260 reference spectra of 104 different food-relevant Pseudomonas species was created, as the Bruker MALDI Biotyper® system could only identify a subset of the raw milk associated pseudomonads. A total of 1,200 isolates from 356 raw milk samples were picked from Pseudomonas CFC selective agar and subsequently identified using the MALDI biotyper.

Results: Overall, 80% of the isolates from CFC agar could be assigned to the genus Pseudomonas (P.), while growth of other genera, such as Stenotrophomonas and Hafnia, was also observed. Twice as many isolates from organic milk could not be identified when compared to isolated from conventional milk. Based on the number of genera and species identified, the diversity in organic milk was higher than in conventionally produced milk. At the species level, P. lundensis was the most frequently detected species present in the raw milk samples investigated.

Summary: The analyses for rapid identification of raw milk isolates using MALDI-TOF indicated a high diversity with a large number of unknown species which may exhibit spoilage characteristics and impact the shelf life of ultrahigh temperature (UHT) treated and sterilized milk.