Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

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

Work performed for WP 2.1Work performed for WP 2.1

Task 2.1.2: Behaviour of the natural microfloraTask 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

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

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)

Experimental design Experimental design Task 2.1.2

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

Experimental procedures Experimental procedures Task 2.1.2 (A)

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Results Results Task 2.1.2 (A)

I) Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis(SDS-PAGE)

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)

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 +.

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Results Results Task 2.1.2 (A)

I) SDS-PAGE

Source+Tempera-ture

(°C)

SDS Group Total isolates

A B C D E F G

Fresh meat 61 6 12

Meatstored

aerobically

0 6 4

70

516

(8,8)2

4(2,2)

1018

(8,10)2

(2,-)

1514

(4,10)2

(2,-)4

(4,-)

Meatstored under

MAP*

09

(5,4)2

(-,2)4

(-,4)

745

12(2,10)

4(4,-)

1010

(10,-)2

(-,2)8

(-,8)

1514

(6,8)2

(2,-)2

(-,2)

Meatstored under

MAP + essential oil**

016

(8,8)2

(-,2)2

(2,-)2

(2,-)

765

18(10,8)

2(-,2)

102

(2,-)18

(8,10)

152

(2,-)12

(2,10)

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 oil1 number of isolates, 2 number of isolates from different time points (middle, final)

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Results 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

Results 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%).

Serratia liquefaciens VK17 Serratia spp. VK5 Serratia liquefaciens VK75 Serratia liquefaciens VK23 Serratia liquefaciens VK40 Serratia grimesii VK108 Serratia proteamaculans VK6 Serratia proteamaculans VK113 Citrobacter freundii VK10

Serratia spp. VK90 Proteus vulgaris VK101 Serratia spp. VK32

Serratia proteamaculans VK25

Hafnia alvei VK20 Hafnia alvei VK60

Proteus vulgaris VK103 Hafnia alvei VK27 Serratia liquefaciens VK74

Hafnia alvei VK52

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Results 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 Serratia liquefaciens.

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 Citrobacter freundii (VK10) and Serratia spp. (VK32) respectively.

Three fingerprints (VK20, VK27 and VK60) were identified as Hafnia alvei (group F), while two fingerprints (VK101 and VK103) assigned to Proteus vulgaris (group G).

Concluding remarks Concluding remarks Task 2.1.2 Task 2.1.2 (A)

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

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 Citrobacter could not be analysed by PFGE, because a continuous smear of DNA rather than well separated fragments was produced.

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)

Concluding remarks Concluding remarks Task 2.1.2 Task 2.1.2 (A)

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

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

Experimental procedures Experimental procedures Task 2.1.2 (B)

Laboratory of Microbiology and Biotechnology of Foods Agricultural University of Athens

Results 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%).