Biofilm formation by B. licheniformis isolated from whey protein concentrate 80 powder as a potential source of product contamination : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
This study aimed to examine biofilm formation of Bacillus licheniformis isolated from
whey protein concentrate 80 (WPC80) as a potential source of contamination in the
manufacture of WPC.
Six WPC80 powder samples from one whey processing plant in New Zealand
were used in this study. Six Bacillus species including (percentage of isolates in
brackets) B. licheniformis (66%), Bacillus cereus/Bacillus thuringiensis (18%), Bacillus
subtilis (4%), Bacillus pumilus (4%), Paenibacillus glucanolyticus (2%) and
Lactobacillus plantarum (6%) were identified using colony morphologies, biochemical
tests, species specific PCR and 16S ribosomal DNA gene sequencing and subsequent
analysis using the BLAST and Seqmatch databases.
Preliminary screening for biofilm formation by the predominant contaminant, B.
licheniformis using a microtitre plate assay with the bacteria grown in laboratory
medium tryptic soy broth (TSB) at three different temperatures (30°C, 37°C and 55°C)
showed most biofilm formation at 37°C with 9/33 isolates forming strong biofilm. In
total 13/33 isolates formed strong biofilm at three different temperatures on the
polystyrene microtitre plate surface.
Subsequent tests for biofilm formation on stainless steel (SS) showed an
increased frequency of biofilm formation with 32/33 strains forming strong biofilm in
TSB at 37°C. This demonstrates the limitation of the microtitre plate assay for screening
for biofilm formation and suggests that biofilm growth of B. licheniformis favours a SS
The attachment and biofilm formation was further investigated using SS
coupons and reconstituted whey medium at different concentrations (1%, 5%, and 20%).
The best medium for B. licheniformis isolates to form biofilm on SS at its best growth
temperature (37°C) was 1% reconstituted WPC80. Interestingly, when 1% reconstituted
WPC80 was supplemented with lactose and minerals (mainly calcium and magnesium)
to replicate the composition of Mozzarella cheese whey before ultrafiltration (UF), the B. licheniformis biofilm counts increased at least by one log.
The production of protease enzyme, extracellular polymeric substances (EPS) and nitrate reduction by B. licheniformis showed the potential of B. licheniformis to influence the quality of dairy products. Biosurfactant production by B. licheniformis identified as lichenysin consisting of lipopeptide was detected and this may influence biofilm formation on SS. The inability of the B. licheniformis isolates to ferment lactose as their major carbon source was confirmed by lactose fermentation tests and shows that B. licheniformis is not ideally suited to a dairy environment. The B. licheniformis vegetative cells were found to be heat resistant with a < log10 reduction at the three temperatures tested; 72oC, 75oC and 80°C during 15 s, 30 s and 60 s heating intervals.
In order to thrive in a dairy system, synergistic interactions with other microflora were investigated as a possible mechanism to use lactose that has been broken down by other microflora. Lactobacillus plantarum (L. plantarum), another isolate from the WPC80 samples, has the ability to produce glucose and galactose from lactose. This was grown with each of two B. licheniformis isolates (E30C11 and F30C02) with different abilities to form biofilm. Interestingly this did not enhance the growth of B. licheniformis suggesting that another carbon source, most likely whey protein, must provide the energy source for this bacterium in a whey environment.
A review of the WPC80 processing plant showed the UF membranes had the largest surface area (3500 – 7500 m2), providing most potential for biofilm growth. However, UF was run at 10°C, too low for the growth of B. licheniformis which has a minimum growth temperature of 20°C. The hypothesis that sections of the processing plant before the UF step are the sites for B. licheniformis biofilm growth was supported by analysing several samples from the raw whey balance tank, clarifier, thermaliser and separator where 7 B. licheniformis strains were isolated. This shows that B. licheniformis is present at several early stages of WPC processing, with the most likely areas for growth being the certain sections of the clarifier, thermaliser and the separator where temperatures are close to the best growth temperature for this bacterium (37°C).
Preventing B. licheniformis contamination of WPC needs to focus on adjusting the conditions in these sections of the processing plant to limit biofilm growth.
Keywords: dairy, Bacillus species, L. plantarum, lichenysin, stainless steel, membrane processing plant.