Isolation, Identification and Characterization of Thermoduric Psychrotrophic Bacteria from Market Samples of Pasteurized Milk Collected from Mannuthy, Kerala State, India

Abstract

Aims: To isolate, identify and characterize thermoduric psychrotrophic bacteria from locally available market samples of pasteurized milk. Study Design: Market samples of pasteurized milk were collected, subjected to laboratory pasteurization and thermoduric psychrotrophic bacteria were enumerated after 10 days of storage at 7ºC.The isolates obtained were identified by 16S rRNA sequencing and subsequent blasting. The isolates were characterized in terms of haemolytic, lipolytic and proteolytic activities. Selected isolates were also assessed for their decimal reduction time at 63ºC.          Place and Duration of Study: Verghese Kurien Institute of Dairy and Food Technology,   (Formerly College of Dairy Science and Technology), Mannuthy, Thrissur, Kerala between September, 2019 to March, 2021. Methodology: A total of 42 market samples of pasteurized milk belonging to seven different brands were assessed for their thermoduric psychrotrophic bacterial population. The thermoduric psychrotrophic bacterial isolates were identified by 16S rRNA sequencing and the sequences obtained were searched with the NCBI BLAST program (http://www.ncbi.nlm.nih.gov) for their closest relatives/reference strains with a homology of over or equal to 99 per cent. Haemolytic, proteolytic and lipolytic activities of the isolates  were determined by  streaking  them on blood agar, skim milk agar (SMA) and tributyrin agar respectively and  grading them based on the presence or absence  of zone of clearance around the colonies developed. Decimal reduction time at 63ºC of two selected isolates was also determined. Results: Six thermoduric psychrotrophic bacteria were isolated from the 42 market samples of pasteurized milk i.e., only 14.3 % of tested samples had thermoduric psychrotrophic bacterial population. 16S rRNA sequencing and subsequent blasting identified the isolates as Aeromonas caviae, Moraxella osloensis, Delftia tsuruhatensis, Staphylococcus arlettae and two strains of Carnobacterium maltaromaticum. On assessing the haemolytic activity of the isolates, A.caviae DMV01 exhibited α haemolytic activity whereas Staphylococcus arlettae DMV02, M. osloensis DMV03, C. maltaromaticum DMV05 and C. maltaromaticum DMV06 exhibited ϒ haemolytic activities.  A. caviae DMV01 was found to be lipolytic and the two Carnobacterium strains exhibited proteolytic activities. Staphylococcus arlettae DMV02 was found to be both proteolytic and lipolytic. Aeromonas caviae DMV01 exhibited D63 value of 4 minutes 38 seconds whereas for Moraxella osloensis DMV03 it was 25 minutes 18 seconds. Conclusion:  Exhibition of heat resistance by microorganisms capable of low temperature growth presents quite a challenging situation in terms of quality assurance and shelf life extension of pasteurized milk. So it is high time that dairy industry takes up initiatives to remain updated with the changes happening in pasteurized milk microflora in order to address the challenges such microorganisms may pose in future.