Abstract
Bacillus subtilis spores are often used as biological indicators (BI) to monitor decontamination processes with gaseous hydrogen peroxide. Results in practical inactivation validation tests, however, vary considerably with no available explanation so far. This study reports on the effect of cultivation pH on spore surface hydrophobicity. Surface hydrophobicity is suspected to have an impact on the decontamination of technical surfaces such as packaging material when gaseous, condensing hydrogen peroxide is applied. It is the aim of this study to examine the impact of different cultivation pH levels on surface hydrophobicity and resistance of B. subtilis spores. Submersed cultivation of B. subtilis in bioreactors at controlled conditions with different static pH levels led to contact angles ranged between 50° and 80°, which was analyzed with water on a homogeneous layer of spores on a filter sheet. Resistance of spores was also affected by the cultivation pH. The results show that the culturing conditions during BI production should be controlled to obtain BI with specified characteristics in inactivation validation tests.
Highlights
In food and pharma industries sterile surfaces for aseptic packaging and clean rooms are often indispensable
Cultivation at a static pH of 7.00 and 9.00 can be considered successful even with a lower spore yield compared to the other cultivation pH values
Bacillus subtilis spores are surrounded by the outer spore coat consisting of four distinct layers whereat the crust is the outermost one (Imamura et al 2010; McKenney et al 2010)
Summary
In food and pharma industries sterile surfaces for aseptic packaging and clean rooms are often indispensable. The processes to obtain surface sterility are either based on physical or on chemical action. H 2O2, mostly a mixture of 35% H2O2 in water and low amounts of stabilizers, can be applied in its liquid form as immersion bath, as spray or as vapor (Engelhard and Kulozik 2006; Pruß et al 2012). Resistant, nonpathogenic test organisms are applied as biological indicators (BI) as the detection of survivors is the only direct method to assess the inactivation result (Block 2001; Sella et al 2013). Spores of Bacillus subtilis (B. subtilis) and Bacillus atrophaeus (B. atrophaeus) meet the requirements for BI in inactivation processes with high concentrations of H2O2 (Sella et al 2012, VDMA 2006)
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