Thermophilic spore forming bacilli: attachment and biofilm formation on stainless steel
Studies suggest that the spores of thermophilic bacilli possibly bind to the stainless steel surface in greater numbers than vegetative cells. The aim of this study was to evaluate the attachment and biofilm formation by spore-forming bacteria on stainless steel. The microorganisms (CM12, SL12, SL9, CM3, CH7, and SH6) were isolated from milk powder plants in the USA and belong to the collection of cultures at DPTC/ Cal Poly. To evaluate the spores attachment and biofilm formation, cleaned stainless steel coupons were used. The experiments were carried out using either whole or fat free UHT milk in order to form a fouling film on the coupons surface. The system was kept at 55°C for 20 hours at 200 rpm. After film formation, the coupons were removed from the matrices and immersed into an aqueous spore solution held at the same conditions to verify the ability of attachment and biofilm formation. Spores were enumerated after heat treatment (80ºC/12 minutes) for either 5 minutes or for 20 hours. In addition, a SDS-PAGE gel was performed in order to verify the proteins present in the spores. The effect of the matrices (fat free or whole milk), microorganisms (six different strains), time of exposure (5 minutes or 20 hours), and their interactions on the spores attachment and biofilm formation were evaluated by ANOVA and Tukey's test for comparison between means (p<0.05). Although, the spore strain and the time of exposure significantly affected the attachment and biofilm formation, none of the interactions among the factors were significant. Spores attachment ranged from 3 to 4 log cfu/cm2. The highest average attachment was observed by SL12 and SH6 (4.01 and 4.08 log cfu/cm2, respectively), while CM12 and SL9 showed the lowest average attachment (3.42 and 3.52 log cfu/cm2, respectively). The strains that showed the lowest average attachment (CM12 and SL9) also presented less protein in band density around 25 and 50kDa when compared to the other four strains in the SDS-PAGE. Despite the attachment after 5 minutes was significantly higher than 20 hours, the difference observed was only 0.12 log cfu/cm2. The results suggest that these matrices did not interfere with the spores attachment and biofilm formation. This could be a strain-dependent characteristic since spores protein may play an important role on attachment.
Keywords: dairy, spores, biofilm
Acknowledgments: CNPq, CAPES