Spores in dairy – new insights in detection, enumeration and risk assessment.

Discipline: contamination; Key words: milk powders, spoilage bacteria, heat treatment, microbial survival, quality assurance, reconstituted milk. 

Bacterial contamination of foods may lead to reduced shelf life due to outgrowth of spoilage organisms and, in the case of pathogens, to food borne illness upon consumption of contaminated products. To inactivate bacteria that may grow in finished products, many food products undergo heat treatment. Pasteurisation results in inactivation of vegetative cells. However, bacterial spores will survive such treatments, after which they may germinate and grow in finished liquid products. To ensure spore inactivation, much higher heat loads such as ultra high-temperature (UHT) treatment or retort sterilisation are required. However, even after such heat treatment, some heat-resistant spores may still survive. Clearly, spores are a main concern for the dairy and general food industry as they are the number one cause of spoilage of a wide range of processed products due to their elevated heat resistance properties. In the study by Dr R.T. Eijlander and colleagues, the germination and outgrowth efficiency was investigated of spores of 38 strains that were isolated from dairy products, ingredients and dairy farm environments. They also collected milk powders from various geographical locations to compare the performance of the ISO method for the ‘Enumeration of the Specially Thermo-resistant Spores of Thermophilic Bacteria in Dried Milk’ [ISO/TS 27265:2009] with a practical method consisting of heating for 30 minutes at 100 °C and culturing isolates on plates containing tryptic soy agar (TSA). Milk powders were furthermore reconstituted in water and subjected to UHT treatment followed by assessment of spoilage of the milk after storage at 37 °C and 55 °C, to link the analytical results to predictability of UHT product spoilage. The results were published in the International Journal of Dairy Technology, Volume 72 of 2019, page 303 to 315, the title being: Spores in dairy – new insights in detection, enumeration and risk assessment.                            

Both methods were tested for predictability of spoilage of UHT treated reconstituted milk. The data showed that heating for 30 minutes at 100 °C has the same predictive value as heating for 30 minutes at 106 °C, provided that specifications are increased 1 log10 [e.g. from 102 coliform units (cfu) per gram to 103 cfu per gram] and the use of TSA as a cultivation medium is recommended over plate count milk agar (PCMA). The results of this study help to develop a practical standard method for the enumeration of highly heat-resistant spores and provide increased insights in the interpretation of analytical results for spoilage risk assessment.  

In summary, there is no plating method for detection of spores in dried milk that could be used in combination with a specification that will result in 100% predictability of spoilage of UHT finished products. Nevertheless, similar predictability of spoilage was found after application of  heat treatment of 30 minutes at 100 °C as for heat treatment of 30 minutes at 106 °C when specifications are increased by 1 log10. In addition, the use of TSA as a plating medium instead of PCMA is highly recommended for increased recovery of spores that can cause spoilage in UHT treated finished products. It should be noted that false positives and (more importantly) false negatives will always occur when using classical plating methods for spore detection, which is most likely due to significant strain-to-strain variation in spore heat resistance and/or germination efficiency. This stresses the importance of understanding spoilage potential of bacterial spores on a deeper level as well as the need for an ability to distinguish this immediately using a practical assay. A better understanding of molecular mechanisms underlying elevated heat resistance, for instance, can help in the development and/or improvement of practical molecular detection methods.