The surface characteristics of spores from thermophilic bacilli isolated from a milk powder production line and their influence on adhesion to surfaces

Abstract

Spores of thermophilic bacilli are a common concern during the manufacture of milk powder. Spores are believed to occur in high numbers in milk powder due to their ability to survive pasteurisation, attach to stainless steel surfaces, getminate, grow as biofilms and subsequently enter the product stream and thereby contaminate the final product.

In this study, thirty one thermophilic bacilli isolates were obtained from a New Zealand milk powder production line and identified as either Anoxybacillus flavithermus or Geobacillus spp. using random amplified polymorphic DNA (RAPD) and species-specific PCR. Sporulation media and a polyethylene glycol two-phase separation system were modified to produce high yields of spores free from debris.

The spores of four Geobacillus spp. isolates (CGT-8, D4, E7 and E11) were characterised in terms of structure (electron microscopy), surface charge (zeta potential), hydrophobicity (contact angle and microbial adhesion to hexadecane) and attenuated total reflectance infrared spectroscopy (ATR-IR). Spores from three of the four isolates possessed an exosporium while the fourth did not. However the integrity of the exosporium varied over time. The spores were negatively charged (-10 to -20 mV) at neutral pH and high ionic strength (0.1 M KCl). Both hydrophobicity assays revealed that the spores of the four isolates were relatively hydrophilic while ATR-IR revealed the spores' surfaces consisted of protein and polysaccharides.

The influence of these spore characteristics on adhesion to a variety of substrata under high flow rates was examined using the extended Derjaguin, Landau, Verwey and Overbeek (XDLVO) theory. Spores generally attached in higher numbers to hydrophobic surfaces compared to hydrophilic surfaces, however this observation was more prevalent for isolate D4. This result indicated that a single mechanism could not describe the adhesion of spores from different strains.

A series of glass surfaces with modified characteristics were produced in order to test the antifouling properties on the adhesion of D4 spores. Spores suspended in a high ionic strength medium (0.1 M KCl) attached in greater numbers (1 Log10 CFU cm-2) to positively charged and hydrophobic surfaces compared with negatively charged and hydrophilic surfaces. A clean in place (CIP) procedure, reduced spore numbers on hydrophobic and hydrophilic surfaces by 1.5 and by 2.0 Log10 CFU cm-2 , respectively. When spores were suspended in milk, there was little difference in the number of spores attaching to the different surfaces (ie. 3.5 to 3.8 Log10 CFU cm-2 ), and spore removal from surfaces via a CIP regime was unchanged (1.5 to 2.0 Log10 CFU cm-2 reduction) compared with spores that attached in simple 1: 1 electrolyte media.

The effects of a caustic wash on spore surface characteristics and adhesion was determined. There was a significant reduction in spore viability (2 Log10 CFU mL-1 ) after a 30 min caustic wash at 65 ℃ in the current study, however surviving spores displayed a greater propensity to attach to stainless steel. Surface characterisation results revealed an increase in hydrophobicity and a greater negative charge on the spores' surface after treatment with NaOH. Surviving spores could potentially recontaminate sections of the plant which are cleaned with this recycled caustic wash solution, thereby seeding surfaces with spores at the beginning of the next processing run.

In conclusion, while surfaces that reduce spore adhesion and enhance removal can be produced, exposure to complex solutions such as milk can reduce the anti-fouling effectiveness of such surfaces to spore adhesion.