EFFECT OF CLEANING AND UV TREATMENT ON BACILLUS SPORES.

Extended shelf life (ESL) milk is processed with the aim of obtaining a product encompassing the sensorial characteristics of fresh milk, but with a longer shelf life. To achieve these qualities, a sequence of processes is employed which may include pasteurisation, steam injection, bactofugation and ultra clean filling technologies. Although the technology for ESL milk production is designed to prevent post-pasteurisation contamination, recent studies have shown the occurrence of psychrotrophic spore formers in finished ESL milk products. The spores of Bacillus spp are a problem in the dairy industry as they are exceedingly resistant to heat as well as cleaning and disinfection processes. In ESL milk, B. cereus spores are of particular concern due to their heat resistance as well as the ability of certain strains to germinate and grow at refrigerated temperatures. To counteract these problems the authors cited studied the efficacy of a cleaning in place (CIP) process on B. cereus spores and in another study determined the effect of ultraviolet C (UVC) on the inactivation of spores of Bacillus subtilis and B. velezensis in phosphate buffer saline.  

In the first experiment, the effect of simulated CIP was determined on the structure, attachment and growth of B. cereus spores isolated from raw milk and biofilms in filler nozzles from ESL milk processing lines. Simulated CIP treatment structurally affected more than 98% of B. cereus spores, while 0.1% remained intact. Following simulated CIP treatment, B. cereus spores were able to attach to stainless steel coupons and form biofilms. The spores were also capable of germination and growth under refrigerated conditions for more than 28 days. The authors concluded that contamination with B. cereus spores irrespective of CIP may lead to a reduced shelf life and potentially be a safety risk in ESL milk with a prolonged shelf life.

In the second experiment spores of B. subtilis and B. velezensis were treated with UVC using phosphate buffer saline (PBS) as a suspension medium and their lethality rate was determined for each sample. Purified spore samples of B. velezensis and B. subtilis were treated under one pass in a UVC reactor to inactivate the spores. The resistance pattern of the spores to UVC treatment was determined using dipicolinic acid (Ca-DPA) band of spectral analysis obtained from Raman spectroscopy. The results showed the maximum lethality rate to be 6.5 for B. subtilis strain SRCM103689 (B47) and highest percentage hydrophobicity was 54.9% from the sample B. velezensis strain LPL-K103 (B44). The difference in surface hydrophobicity for all isolates was statistically significant (P < 0.05). Flow cytometry analysis of UVC treated spore suspensions clarifies them further into sub-populations unaccounted for by plate counting on growth media. The Raman spectroscopy identified B4002 as the isolate possessing the highest concentration of Ca-DPA. The study justifies the critical role of Ca-DPA in spore resistance and the possible sub-populations after UVC treatment that may affect product shelf-life and safety. It was concluded that UVC showed a promising application in the inactivation of resistant spores though there is a need to understand the effects at the molecular level to design the best parameters during processing.

References:

C. Pretorius & E. M. Buys, 2020. Extended shelf life milk processing: Effect of simulated cleaning in place on the germination and attachment of Bacillus cereus spores. International Journal of Dairy Technology doi: 10.1111/1471-0307.12744.

J. A. Elegbeleye , R. Gervilla , A. X. Roig-Sagues & E. M. Buys, 2021. Ultraviolet-C inactivation and hydrophobicity of Bacillus subtilis and Bacillus velezensis spores isolated from extended shelf-life milk. International Journal of Food Microbiology 349 (2021) 109231. https://doi.org/10.1016/j.ijfoodmicro.2021.109231