Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

1. Pillar 2: Control and intervention strategies which can be implemented along the fork to farm chain to ensure safe beef Workpackage 2.1: Distribution and consumers strategies Progress during 30 - 36 months Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

2. Work performed for WP 2.1 Task 2.1.2: Behaviour of the natural microflora • Apply molecular tools to monitor the changes of the spoilage related microbial flora during storage of beef Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

3. Experimental design Task 2.1.2 Product: minced beef Packaging: different packaging systems (e.g. air, MAP, MAP+EO*) Storage: various temperatures * Smart packaging  MAP with oregano essential oil (2% v/w) Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

4. Experimental procedures Task 2.1.2 (A) Identification of Enterobacteriaceae • Molecular tools : Pulsed Field Gel Electrophoresis (PFGE) Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis(SDS-PAGE) Sequencing analysis Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

5. Results Task 2.1.2 (A) I) Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis(SDS-PAGE) • The SDS – PAGE fingerprints obtained from whole cell protein analysis of the Enterobacteriaceae isolates revealed seven groups • The group A was common for all packaging and temperature conditions, but 10°C and 15°C under MAP +. • The isolates belonging to group E were recovered only when beef stored aerobically, • The isolates belonging to group F and G were recovered under MAP - and MAP +. Figure 1. SDS – PAGE profiles from whole cell proteins of SDS – group: A (VK2, VK3, VK33, VK34, VK35), B (VK4, VK5, VK6), E (VK32) and F (VK31) Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

6. Results Task 2.1.2 (A) I) SDS-PAGE Table 1. Number of isolates that shorted through the seven groups in all conditions adopted * modified atmosphere packaging (40% CO2/30% O2/30% N2), ** volatile compounds of 2% v/w oregano essential oil 1 number of isolates, 2 number of isolates from different time points (middle, final) Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

7. Results Task 2.1.2 (A) II) Pulsed Field Gel Electrophoresis (PFGE) • A modified protocol was applied in order to prevent DNA degradation of Enterobacteriaceae (group A, B, D, E, G based on SDS – PAGE analysis) PFGE pattern of Enterobacteriaceae isolates after XbaI digestion of their genomic DNA performed without (Lanes 1-10), and with the modification (Lanes 11-15). (Lanes M: Low range PFG marker, Lanes 1, 6 and 11 positive control). Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

8. Results Task 2.1.2 (A) II) Pulsed Field Gel Electrophoresis (PFGE) Cluster analysis of PFGE XbaI digestion fragments of the Enterobacteriaceae isolates calculated by the unweighted average pair grouping method. The distance between the pattern of each strain is indicated by the mean correlation coefficient (r%). Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

9. Results Task 2.1.2 (A) • Five fingerprints (VK17, VK23, VK40, VK74 and VK75), which belonging to SDS – PAGE group A cound be assigned to Serratialiquefaciens. • From the four different fingerprints grouped to B based on SDS – PAGE profiles, VK6, VK25, VK53 and VK113 could be assigned to Serratia proteamaculans, whereas VK5 to Serratia spp. • Similar results were revealed with the group D, where VK90 and VK108 were assigned to Serratia spp. and Serratia grimesii respectively. • Fingerprints that were belonging to group C and E were attributed to Citrobacterfreundii (VK10) and Serratia spp. (VK32) respectively. • Three fingerprints (VK20, VK27 and VK60) were identified as Hafniaalvei (group F), while two fingerprints (VK101 and VK103) assigned to Proteusvulgaris (group G). Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

10. Concluding remarks Task 2.1.2 (A) • Enterobacteriaceae isolates appeared sensitive to rapid DNA degradation during the course of PFGE pattern analysis using the standard preparation of agarose embedded bacterial DNA. To obtain better results, it was necessary to include some additional steps in the preparation of PFGE samples. • Indeed, fingerprints assigned to genus Serratia and Proteus and Citrobactercould not be analysed by PFGE, because a continuous smear of DNA rather than well separated fragments was produced. Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

11. Concluding remarks Task 2.1.2 (A) • PFGE and SDS – PAGE analysis demonstrated that the storage temperature and the modification of package influenced the microbiota of minced beef whereas different species of Enterobacteriaceae were isolated. • S.liquefaciens was the most common isolate in most conditions adopted while S. proteamaculans was dominated the Enterobacteriaceae community of fresh meat • Hafnia alvei dominated the Enterobacteriaceae community at the final stage of storage at 10°C under MAP and at 10 and 15°C (middle and final stage of storage) Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

12. Experimental procedures Task 2.1.2 (B) Identification of pseudomonads (in progress) • Molecular tools : Pulsed Field Gel Electrophoresis (PFGE) Sequencing analysis (in progress) Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

13. Results Task 2.1.2 (B) Pulsed Field Gel Electrophoresis (PFGE) Cluster analysis of PFGE SpeI digestion fragments of the pseudomonads isolates calculated by the unweighted average pair grouping method. The distance between the pattern of each strain is indicated by the mean correlation coefficient (r%). Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens