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

Abstract

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. 𝘭𝘪𝘤𝘩𝘦𝘯𝘪𝘧𝘰𝘳𝘮𝘪𝘴using 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 surface.

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; 72°C, 75°C 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.