Milk

3.4.2.1 Challenge lethality test

Figure 34 shows the combined application of multiple PlyP825 concentrations and pressure levels against a L. monocytogenes cocktail in milk. A synergistic effect at a pressure level of 400 MPa (10 min, 25 °C) was visible at all the PlyP825 concentrations examined. A HHP treatment of 400 MPa alone caused a reduction in cell count of 2.2 log cycles. Prior incubation with 0.34 or 3.4 µg/mL PlyP825, which applied individually did not affect the cell count, allowed for an additional reduction of 1.2 and 1.7 log cycles, respectively. Also after incubation with 34 µg/mL PlyP825, which already individually reduced the cell count by 1.5 log cycles, a synergistic effect of >1 log cycles was observed.

HHP processing at 200 MPa (25 °C, 10 min) only caused a minimal reduction of 0.3 log cycles (Figure 34). However, when cells were first incubated with 3.4 or 34 µg/mL PlyP825, a pressure treatment of 200 MPa further reduced the cell count by 2.0 or 2.1 log cycles (i.e. a synergistic reduction of 1.7 and 1.8 log cycles). Interestingly, although both 3.4 µg/mL PlyP825 and HHP processing at 200 MPa (10 min, 25 °C) individually hardly affect the cell count, their combined application is almost equally effective as a single pressure treatment of 400 MPa (10 min, 25 °C). It was already shown in buffer that the addition of endolysin allows for the inactivation of cells at a lower pressure level (3.2.1.2.4). In this section, it is demonstrated that this also applies to the inactivation of L. monocytogenes in actual foods.

Figure 33: HHP susceptiblity of L. monocytogenes in milk, mozzarella, and smoked salmon. The induced reduction (log10(N0/N)) of stationary-phase L. monocytogenes cells (cocktail with five strains) in milk (black bars), mozzarella (gray bars), or smoked salmon (white bars) by HHP (10 min, 25 °C) with increasing pressure levels (200-500 MPa, increments of

100 MPa). The detection limits (1 log cfu) are shown by the long (milk or mozzarella) and short (smoked salmon) dashed lines.

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3.4.2.2 Challenge storage test

Two milk storage tests were performed. In the first storage test, the efficacy of the combined PlyP825 and HHP treatment was screened at multiple pressure levels and holding times with a somewhat higher inoculation level (3×10⁵ cells/mL). In the second storage test, the efficacy of the combined application was examined at a fixed pressure level and lower inoculation level (7×10³ cells/mL).

3.4.2.2.1 High inoculum

The efficacy of a combined endolysin-HHP treatment in milk was screened with a fixed PlyP825 concentration (3.4 µg/mL), at three pressure levels (200, 300, and 400 MPa), and two pressure holding times (1 and 10 min). In Figure 35, results for a pressure holding time of 10 min are presented. A pressure holding time of 1 min was not sufficient to eliminate Listeria from any of the samples examined and these results are therefore presented in Appendix 8.3 (Figure 48). Treatment with only 3.4 µg/mL PlyP825 reduced the cell count by 1.5 log cycles and growth was inhibited for 1 day, after which cells steadily grew to the upper detection limit of 10⁸ cells/mL at day 13 (). The cell count was reduced by an additional 0.9 log cycles when 3.4 µg/mLPlyP825 was combined with HHP at 200 () or 300 () MPa, but also here rapid growth was visible after day 1. Interestingly, similar to the results in the challenge lethality test, the combination of 3.4 µg/mL PlyP825 with a pressure treatment of 200 MPa () was about equally effective as a pressure treatment of 400 MPa alone (). Incubation with 3.4 µg/mL PlyP825 and subsequent pressure treatment of 400 MPa reduced the cell count until the detection limit and only 2 out of 18 samples became positive for Listeria during storage up to 27 days (one sample at t=1 and one at t=6; ).

Figure 34: Challenge lethality test in milk. The induced reduction (log10(N0/N)) of stationary-phase L. monocytogenes cells (cocktail with five strains at an inoculum of ca. 10⁷ cells/mL) without endolysin (black bars), 0.34 (dark grey bars), 3.4 (light grey bars), or 34 µg/mL PlyP825 (blanc bars) at atmospheric pressure (0.1 MPa) or with an additional HHP treatment at 200

or 400 MPa (10 min, 25 °C). The lower part of the bars represents the calculated additive effect of individual endolysin and HHP treatments, indicated by the letter a. The upper part represents the synergistc inactivation as result of the combined treatment, indicated by the letter s. The detection limit (1.0 log cfu) is shown by the dashed line. Mean values + standard

deviation of three biologically independent experiments are shown (error bars).

72 RESULTS

3.4.2.2.2 Low inoculum

In section 3.3.2.1, it was shown that endolysin can cause a much larger log reduction when the same concentration is applied to a lower number of cells. In the previous section, it was shown that an additional HHP treatment of 200 MPa could further reduce listerial cell count, but was not sufficient to kill all cells. Since the inoculation level in this storage test was higher than typically used to ascertain the microbiological stability of food products (i.e. >10⁵ instead of a recommended 10²-10³ cells/g; FDA, 2001), the storage test was repeated with a pressure level of 200 MPa (10 min, 25 °C) and lowered inoculation level (7×10³ cells/mL; Figure 36).

Although the combined application of 3.4 µg/mL PlyP825 and a HHP treatment of 200 MPa reduced the cell count until the detection limit in two out of three replicates directly after pressure treatment, only four out of the remaining 15 samples remained negative for Listeria during storage (i.e. 12/18 Listeria-positive samples; ). A PlyP825 concentration of 34 µg/mL PlyP825 substantially reduced the initial cell count (until the detection limit in a single sample) and no Listeria-positive samples were detected at later time-points (i.e. only 2/18 Listeria-positive samples; ). Surprisingly, more samples (4/18) became positive for Listeria when 34 µg/mL PlyP825 was combined with 200 MPa (). A possible explanation for the higher number of positive samples after a combined treatment versus endolysin applied individually might be that PlyP825 was (partly) inactivated by the pressure treatment. This is however rather unlikely since previous experiments showed no negative effect on the enzymatic activity of PlyP825 by a pressure treatment of 600 MPa (3.1.2). Since the number of Listeria-positive samples after a combined treatment is only minimal higher and still relatively low (4/18), the small number of replicates and sample variability might be a more likely explanation.

# of Listeria-positive samples

Figure 35: Challenge storage test in milk: high inoculum. The growth (log10(N)) of stationary-phase L. monocytogenes cells (cocktail with five strains at an inoculum of ca. 3×10⁵ cells/mL) without endolysin (blanc symbols) or 3.4 µg/mL PlyP825 (filled red symbols) at atmospheric pressure (upward triangles) or in combination with a HHP treatment (25 °C, 10 min) of 200 (squares), 300 (downward triangles), or 400 MPa (circles). The detection limit (1.0 log cfu) is shown by the dashed line.

Mean values + standard deviation of three biologically independent experiments are shown (error bars). The number of Listeria-positive samples per time-point and in total during storage is shown in the table next to the graph.

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