Screening of fast biofilm formation on stainless steel by thermophilic sporeformers originated from dairy powder and their resistance against CIP

Introduction:

Thermophilic sporeformers are present in raw milk at very low concentration and resist to pasteurisation applicated to destruct vegetative and pathogenic cells. Those spores can adhere to stainless steel due to their hydrophobicity and can form biofilms. Early stage biofilms are important because it can increase the matrix and the adhesion of other cells. Because of those biofilms, the three main species: Geobacillus stearothermophilus, Anoxybacillus flavithermus and Bacillus licheniformis can resists to Cleaning In Place (CIP) procedure, and contaminate a new process.

 

Material and Methods:

Early stage adhesion was conducted on stainless steel submerged by milk inoculated with a fresh culture of bacteria (G. stearothermophilus (N=15), A. flavithermus (N=32) and B. licheniformis (N=15)) for 6h of growth at 55°C under agitation. The ability of sporeformers to form biofilms under those conditions were measured by image analysis after a fluorescent coloration (acridine orange) and random photography. A coverage percentage was calculated by ImageJ ; and a positive threshold was set up at 5% of covering.

The efficiency of CIP procedures were obtained after a caustic soda and nitric acid treatment during different duration and temperature of treatment. Tested biofilms were formed in milk during 12h at 55°C, in stainless steel microplates (96 wells) on the same species (3 strains for each) under agitation. Surviving spores were enumerated by the microcolony method.

 

Results:

Early stage adhesion shows that 62.5 % (N=20) of A. flavithermus strains can form biofilm within 6h, whereas only 6.7% (N=1) of G. stearothermophilus and 0% (N=0) of B. licheniformis biofilm in 6h at 55°C on submerged stainless steel. However, the maximum covering % on A. flavithermus was 35%; while on the only biofilm forming strain of G. stearothermophilus, this percentage reach 75%. Image analysis also shows biofilm structure from 2D to 3D.

The presence and the resistance of spores to chemical cleaning was highly variable within strains. Nitric acid appears to be more effective than caustic soda against biofilms formed by vegetative cells and spores from these strains.

 

Significance:

Those results shows that strong biofilms are mainly composed of spore and are very resistant to CIP used in dairy industries. That is why a better understanding of control methods can lead to a finer and suitableness use of cleaning products.