Evaluation of a Rapid Approach for the Detection and Direct Identification of Spoilage Microorganisms in Fruit Juices

Introduction: Spoilage microorganisms of the genera Alicyclobacillus, Lactobacillus, Candida, Leuconostoc, Saccharomyces and Zygosaccharomyces are of major concern to the fruit beverage industry. They have different growth characteristics and often require multiple growth media and temperatures for their detection and isolation. A single medium based automated detection system along with Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) based identification can simplify and reduce the total time to quality assessment of fruit beverages.

Purpose:  Evaluation of a single broth based medium in an automated system for the detection of spoilage microorganisms from fruit juices followed by direct identification using MALDI-TOF MS.

Methods: Twenty-five strains of genus Alicyclobacillus, Lactobacillus, Leuconostoc, Candida, Saccharomyces and Zygosaccharomyces were separately inoculated (10-25 CFU) in 15 ml orange and apple juice samples in triplicate. The inoculated samples were mixed in 20 ml BacT/ALERT iLYM medium bottles and incubated at 32±0.5°C in an automated CO₂ sensor based detection system. Samples (2 ml) from positive bottles were centrifuged, washed with sterile water and suspended into 50 µl ethanol, which were directly identified using a MALDI-TOF MS system. Similarly, pure cultures of all the strains were individually detected and identified.

Results: All 25 bacterial and yeast pure cultures were correctly identified using the MALDI-TOF MS system.   All 75 inoculated samples each of apple and orange juices were reported positive within 2-3 days using an automated detector. The yeast and lactic acid bacteria positive samples were correctly identified at genus or species level but identification of the Alicyclobacillus positive samples required additional processing to complete the identification.

Significance: Rapid detection and identification of spoilage microorganisms from fruit beverages could be achieved using a combination of automated detection and MALDI-TOF MS identification, which could significantly reduce the product hold time during quality assessment and allow for earlier intervention and correction if required.