Food Protection Trends - January/February 2022 - 44

provided extremely useful data for before and after the sanitation
process because it allowed the biofilm samples to be
examined without destruction. Moreover, it was observed for
the first time that biofilm samples from a dense organic and
inorganic medium such as milk could be analyzed by fluorescent
staining and CLSM.
Hinton et al. (10) demonstrated that caustic solutions
used in cleaning equipment cannot eliminate most bacteria in
product residues. CIP procedures in the food industry often
eliminate problems that may be caused by food pathogens, but
they may not be effective for thermophilic bacilli that negatively
affect food quality (23). Polymeric components (proteins,
carbohydrates, fatty acids, nucleic acids, cations, anions, etc.)
that are released by microbial consortia into the extracellular
environment and that establish the structural or functional
integrity of the biofilm make biofilm cells more resistant to
sanitary agents than their planktonic counterparts (11). The
protein, carbohydrate, and nucleic acid content of thermophilic
biofilms was determined prior to the corresponding study
(data not shown). In this context, the study also focused on
the evaluation of sanitation processes based on extracellular
polymeric components.
The efficacy of polysaccharidases and proteolytic enzymes
Figure 2. Eradication with combined treatment of SDS and TCA
of 18-h biofilms of DSM 465T
, B84a, F81, and DSM 2641T
(biofilms sampled under dynamic conditions).
CLSM images of thermophilic biofilms sampled in milk
are illustrated in Figure 3. They are 3D projections of 18-h-old
biofilms formed under dynamic conditions for strains
G. thermodenitrificans DSM 465T
, A. flavithermus DSM 2641T
,
and A. kamchatkensis subsp. asaccharedens F81.
CLSM image sections showed cells in vegetative form, endospores,
microcolonies, and extracellular DNA (eDNA, yellow
fluorescence) stained with SYTO 9. Dead biofilm cells were
also observed after lysozyme treatment.
From the CLSM analysis, it was clear that the 2-h-old biofilms
of Anoxybacillus and the 5-h-old biofilms of Geobacillus
were formed in a short time on ideal abiotic surfaces. A significant
increase in the thickness of the biofilms was observed
at the end of the 18-h incubation period. Moreover, there were
changes in biofilm thickness and matrix composition depending
on the bacterial strain and abiotic surface. In particular, the
results of the 3D horizontal projection on the z-axis showed
that 2-h-old biofilms consisted of living cells with low matrix
content, whereas 18-h-old biofilms contained eDNA that emitted
intense fluorescence. Increased red fluorescence was observed
when biofilm layers were examined near the substrate. Upper
layers of biofilms contained a large number of living cells
(green fluorescence) (Fig. S1 to S4). This microscopy technique
44
Food Protection Trends January/February
against biofilms formed in product processing lines was
tested by Lequette et al. (17). Serine protease and α-amylase
were found to be the most effective enzymatic agents against
biofilms. Proteolytic enzymes have a broader spectrum compared
to polysaccharidases and are more effective in removing
biofilms; in particular, serine proteases are extremely effective
on Bacillus biofilms. Polysaccharidases are more effective in
removing Pseudomonas biofilms. Enzyme solutions containing
surfactants and chelating agents have also been reported to
give better results in removing Bacillus and Pseudomonas
biofilms (6).
Parkar et al. (24) studied the removal of A. flavithermus
biofilms with cleaning agents in place. They found that it is
important to apply sanitizing agents at ideal concentrations
and temperatures and also that the tested chemical agents were
not sufficient to remove thermophilic biofilms on surfaces.
Parkar et al. (24) also showed that polysaccharides in biofilm
structures can only be degraded by caustic treatment at temperatures
of 60°C and above. It was observed that the biofilm
of A. flavithermus was eradicated only by acid and alkaline
treatment applied at 75°C for 30 min. However, in our study,
it was possible to remove 100% of the A. flavithermus biofilm
in a shorter time using selected sanitation strategies (Table
3). The sanitation agents tested in our study, mainly SDS and
TCA, are the most successful agents for removing all thermophilic
biofilms. The sanitation strategies evaluated in this study
were selected based on the biochemical composition of the
thermophilic biofilms studied. The effectiveness of sanitation
strategies was evaluated considering both biomass (biofilm
matrix) removal from the surface and biofilm cell killing.
Although biofilm cells can be killed using sanitation strategies,
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