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

Discipline: contamination; Keywords: 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, heat treatment is often used. Pasteurisation usually is effective to inactivate vegetative cells, but bacterial spores will survive, 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) treatments or retort sterilisation are required. But even after those heat treatment levels, some (very) heat-resistant spores may still be viable such as sometimes found in bulk tank (raw) milk. These spores are ubiquitously present on the cow and in its environment and can be introduced into the milk at low concentrations (<10 to 7 x 102 cfu per mL) during milking. Furthermore, during the manufacturing of milk powder from raw milk, concentrations of spores may increase in the product due to concentration effects or due to attachment of spores and growth of vegetative cells of these bacterial species in processing equipment, followed by spore formation. The spores that are often encountered in dairy products belong to a wide range of aerobic and anaerobic species with different optimal growth temperatures and growth requirements. Most mesophilic spore formers, including many Bacillus spp. and Brevibacillus spp., grow best at temperatures between 30 °C and 40 °C (and occasionally as high as 46 °C). When spores of such organisms are viable and germinate, this may lead to spoilage when dairy products are stored at ambient temperate in the distribution chain, especially during warm months or at geographical locations with relatively warm climates.                                                                                                                                                                                                                                   

 In the study by Dr R.T. Eijlander and colleagues which was 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, the germination and outgrowth efficiency was investigated of spores of 38 strains that were isolated from dairy products, ingredients and dairy farm environments. Thereafter, milk powders from various geographical locations were collected to compare the performance of the ISO method for the “Enumeration of the Specially Thermoresistant Spores of Thermophilic Bacteria in Dried Milk” (ISO/TS 27265:2009) with a more practical method consisting of heating for 30 min at 100 °C and plating on TSA (tryptic soy agar). 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 data showed that heating for 30 min at 100 °C has the same predictive value as heating for 30 min at 106 °C, provided that specifications are increased 1 log10 and the use of TSA as a cultivation medium is recommended over PCMA (plate count milk agar). However, it should be recognized that the results are limited to the types of milk powders that were used in the study, the bacterial spore species present, and to predictability of spoilage of simple UHT milk products only. In addition, this study does not take into account product spoilage caused by recontamination after UHT treatment. Also, based on the powders used and the spoilage seen for reconstituted milk made thereof, this study focused on thermophilic spore formers, as the milk powders contained very little mesophilic spore formers and no spoilage was observed after incubation at 37 °C.

Summarized discussion: There is no plating method for detection of spores in dried milk that can 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 a heat treatment of 30 min at 100 °C as for a heat treatment of 30 min at 106 °C when specifications are increased by 1 log10 (e.g. from 102 cfu per g to 103 cfu per g). 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 the 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.