Διπλωματική εργασία-Καραταΐρη Βαλάντη

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Transcript of Διπλωματική εργασία-Καραταΐρη Βαλάντη

  • :

    , 2015

  • 2

    .

    .

    .

    ,

    .

    .

    .

    ,

    .

    . , HACCP Creta

    Farm

    HACCP .

    ,

    .

    ,

    .

    .

    .

  • 3

    .

    .

    ,

    . real-

    time PCR Enterococcus

    faecalis E. coli .

    (HACCP) .

    HACCP

    ( ..)

    .

  • 4

    ................................................................................................................................................ 2

    ...................................................................................................................................................... 3

    1: ...................................................................................... 6

    1.1 .............................................................................................................. 6

    1.2 ..................................................... 8

    1.2.1 (HURDLES) ......................................................................................... 9

    1.2.2 HACCP .................................................................................................................................................. 10

    1.2.3 (PREDICTIVE MICROBIOLOGY) .................................... 11

    2: .......................................................................................................................... 12

    2.1 ................................................................................................................. 12

    2.2 ........................................................................................ 13

    2.3 ................................................... 13

    2.4

    ...................................................................................................................................................... 14

    2.5 .................................................................................................................. 16

    3: ...................................................................................................... 17

    3.1 .............................................................................................................................................. 18

    3.1.1 (SALMONELLA SP.) ................................................................................................. 20

    3.1.1.1 .................................................................................................................................. 21

    3.1.1.2 ...................................................................................................................................... 23

    3.1.2 ESCHERICHIA COLI ........................................................................................................................... 24

    3.1.2.1 .................................................................................................................................. 25

    3.1.2.2 ...................................................................................................................................... 26

    3.1.3 ENTEROCOCCUS SP. .......................................................................................................................... 27

    3.1.3.1 .................................................................................................................................. 28

    3.1.3.2 ...................................................................................................................................... 28

    3.1.4 LISTERIA SP. ........................................................................................................................................ 28

    3.1.4.1 .................................................................................................................................. 29

    3.1.4.2 ...................................................................................................................................... 30

    3.1.5 STAPHYLOCOCCUS SP. ..................................................................................................................... 31

    3.1.5.1 .................................................................................................................................. 32

  • 5

    3.1.5.2 ...................................................................................................................................... 33

    3.1.6 PSEUDOMONAS SP. ............................................................................................................................ 33

    3.1.6.1 .................................................................................................................................. 34

    3.1.6.2 ...................................................................................................................................... 34

    3.1.7 SHEWANELLA SP. ............................................................................................................................... 35

    3.1.7.1 .................................................................................................................................. 35

    3.1.7.2 ...................................................................................................................................... 36

    4: ............................................................................................................................ 37

    5: ................................................................................................. 38

    5.1 ...................................................................................................................................................... 38

    5.2 ................................................................................................................................ 40

    5.3 ................................................................................................. 42

    5.4 .......................................................................................... 46

    5.5 .............................................................................................. 49

    5.6 .................................. 50

    5.7 PCR .......................................... 51

    6: ........................................................................................................... 53

    6.1 ............................................................................................................... 53

    6.2 ............................................................................................................................... 59

    6.3 .................................................................................... 70

    6.4 .................................................................................. 75

    6.5 ............................................................................................................................. 81

    6.6 REAL-TIME PCR ................................................................................................................................... 87

    7: ISO-HACCP ........................................................................................................................ 91

    7.1 ISO ............................................................................................................................................................. 91

    7.2 HACCP ..................................................................................................................................................... 91

    7.2.1

    HACCP - HACCP ... 100

    7.2.1.2 ........................................................................................................ 101

    7.2.1.3 HACCP ................................................................................. 102

    7.2.2

    .................................................................................................................. 113

    8: ......................................................................................................... 114

    ........................................................... ! .

  • 6

    1:

    ,

    .

    .

    ,

    , ,

    , .

    ,

    , .

    ,

    .

    ,

    .

    ,

    .

    ,

    , .

    , ,

    .

    ,

    .

    1.1

    .

    .

    - -

    .

    .

  • 7

    .

    - -

    .

    ,

    .

    , ,

    ..

    - -

    . (-,

    2009).

    1.1

    . 1.2

    1996 2001

    .

    1.1 . : ,

    - ,2001

    Prions

    Askaris sp. Aeromonas hydrophila Astroviruses Aspergillus sp. PrPres

    Anisakis sp. Bacillus cereus Coxsackie viruses Fusarium sp.

    Blastocystis sp. Brucella melitensis Echo viruses Penicillium sp.

    Cryptosporidium sp. Campylobacter jejuni Hepatitis A. E viruses

    Cyclospora

    cayietanesis

    Citrobacter sp. Norwalk virus group

    Echinococcus

    granulosus

    Clostridium perfringens Poliovirus

    Entamoeba histolytica Clostridium botulinum Rotaviruses

    Fasciola hepatica Edwarsiella sp. Tick borne

    encephalitis virus

    Giardia lamblia Enterobacter sp.

    Isospora sp. Enterococcus faecalis

    Microsporidium sp. Escherichia coli

    Oxyurus sp. Listeria monocytogenes

    Taenia solium Mycobacterium sp.

    Taenia saginata Proteus sp.

    Toxoplasma gondii Providencia sp.

    Trichinella spiralis Pseudomonas sp.

    Sarcocystis sp. Salmonella sp.

    Shigella sp.

    Staphylococcus aureus

    Vibrio sp.

  • 8

    1.2 1996 2001. : Preliminary Foodnet

    Data on the Incidence of Food-borne Illnesses

    1.2

    ,

    .

    .

    ,

    ,

    ( , ..)

    ,

    , ,

    . ,

    107 / g mL

    .

    ,

    . ,

    ,

    (hurdles),

    HACCP (predictive

    microbiology).

  • 9

    1.2.1 (hurdles)

    , ,

    (aw), (pH), , ,

    , , .. 1.2.1

    .

    1.2.1 (hurdles)

    1. (,

    , )

    1. (aw)

    2. 2. (pH) 3. 3.

    (Eh)

    4. 4. NaCl 5. 5. NaNO2 6. Ultrasonication 6. CO2 7. 7. 8.

    ()

    8.

    9. 9. 10. 10.

    11. 12.

    , .

    Leistner 1977 1982 Rodel and Leistner.

    .

    ,

    .

    .

    ,

    .

    1.2.1 .

  • 10

    1.2.1 . : Food Engineering-Vol III-Hurdle Technology

    Leistner, L.

    1.2.2 HACCP

    HACCP Hazard Analysis Critical

    Control Points,

    (CPPs).

    HACCP Pillsburry

    NASA

    .

    FMEA (Failure, Mode and Effect Analysis)

    , ,

    . HACCP

  • 11

    .

    HACCP .

    1.2.3 (predictive microbiology)

    ,

    . ,

    ,

    (pH, aw ..) .

    ,

    /

    / .

    (primary models)

    (secondary models), 1.2.3.

    1.2.3

    :

    A:

    x,y,z:

    0,1n:

    e:

    :

    Gompertz Arrhenius

    EA:

    Richard Belerhadek

    Tmin: max=0

    Barayni

    (Frakruddin et al 2011).

  • 12

    2:

    , ..

    .

    2.1

    .

    .

    1.

    2. .

    3.

    4.

    5. .

    6.

    7.

    8.

    9.

    10.

    11. , ,

    12. , , , ,

    13. ,

    2009/39/

    14. ,

    15.

    16. , 1,3,4

    17. , 2002/46/,

    18. 1 17,

  • 13

    2.2

    .

    , .

    :

    -

    -

    - ,

    -

    -

    -

    -

    -

    - (Belitz et al 2009)

    ,

    . , ,

    .

    2.3

    .

    .

    .

    .

    , ,

    , .

    3.

  • 14

    2.4

    .

    .

    ( ),

    ( ) .

    (pH)

    (aw)

    (Eh-OR)

    ()

    , . 2.4.1, 2.4.2

    2.4.3 , aw pH

    .

    2.4.1 pH

    pH

    minimum optimum maximum

    Clostridium botulinum 4.8-5.0 6.0-8.0 8.5-8.8

    E.coli 4.3-4.4 6.0-8.0 9.0-10.0

    Pseudomonas sp. 5.6 6.6-7.0 8.0

    Pseudomonas aeruginosa 5.6 6.6-7.0 8.0-9.0

    Salmonella sp. 4.5-5.0 6.0-7.5 8.0-9.6

    Staphylococcus aureus 4.0-4.7 6.0-7.0 9.5-9.8

    Listeria monocytogenes 4.4-5.0 7.0-7.5 9

  • 15

    2.4.2

    (oC)

    Minimum optimum maximum

    Aeromonas sp. 10 - 47-50

    Bacillus cereus 5 - 42

    Clostridium sp. 3-10 30-40 42-45

    E. coli 3-10 37-41 48-50

    Pseudomonas sp. -7-4 20-30 31-43

    P. aeruginosa 8 - 42

    P. fluorescens 0-6 20-25 40

    Salmonella sp. 5-10 35-37 46-49

    Staphylococcus sp. 5-10 35-40 46-48

    S. aureus 5-10 35-39 44-48

    L. monocytogenes 4 30-37 45

    2.4.3 aw

    aw

    Minimum

    Aeromonas sp. 0.95-0.98

    Bacillus cereus 0.90-0.99

    Clostridium sp. 0.90-0.98

    E. coli 0.94-0.97

    P. aeruginosa 0.96-0.98

    P. fluoresces 0.94-0.97

    Salmonella sp. 0.93-0.96

    S. aureus 0.83-0.92

    L. monocytogenes 0.92

    .

    ,

    .

    aw (RH)

    , .

    aw

    .

  • 16

    2.5

    .

    .

    , ,

    , .

    .

    .

    ,

    . ,

    .

    ,

    ,

    .

    :

    -

    -

    - (Shafiur Rahman et al 2007)

    .

    :

    (Jay et al 2000)

  • 17

    3:

    . ,

    (0,16m).

    ,

    . Haeckel,

    , :

    1.

    2.

    :

    :

    ,

    . 4 :

    1. ,

    CO2.

    2. ,

    .

    3. , CO2

    .

    4. ,

    .

    .

  • 18

    .

    .

    .

    ,

    .

    ( ),

    .

    . .

    , ,

    , .

    3.1

    .

    :

    -

    p, , ERH

    -

    -

    -

    .

    ,

    .

    ,

    , ,

    ,

    o.

    . 3.1 Gram

    Gram .

    :www.wikipedia.com

  • 19

    .

    .

    Gram (. 3.1).

    Gram

    ,

    (Pepper et al 2009).

    Gram (+):

    Gram.

    15-80 nm

    , . Gram

    -,

    . 1-6

    24-48 .

    S. aureus, Clostridium botulinum

    . Gram

    , ,

    .

    .

    , , ,

    , , .

    , ,

    .

    ,

    .

    .

    Gram (+) : Clostridium botulinum, Clostridium perfringens, Bacillus

    cereus, S. aureus, L. monocytogenes, Bacillus subtilis .

    Gram (-):

    Gram.

    (10nm)

    . Gram

    , (LPS

    ), .

    .

    Gram

    ,

    E.coli O157:H7 (Salton et al 1996). Gram (-)

  • 20

    , , , , - .

    (Good Manufacturing Practices-GMP)

    (Good Hygiene Practices GHP). Gram (-)

    : Salmonella sp., Shigella sp., E.coli, Campylobacter jejuni, Vibrio

    parahaemolyticus.

    3.1.1 (Salmonella sp.)

    , ,

    Gram (. 3.1.1).

    6,5-47 C, pH 4,5,

    aw>0.95

    .

    (36C).

    .

    .

    -

    .

    .

    ,

    . Salmonella typhi

    Salmonella paratyphi A

    (www.wikipedia.com, Overview of Food Microbiology, USDA, 2011, Monaghan

    et al 2010).

    3.1.1 .

    :www.wikipedia.com

    http://www.wikipedia.com/

  • 21

    3.1.1.1

    , ,

    .

    , , .

    , , , , .

    6-48

    1-7

    , ,

    (www.wikipedia.com, Overview of Food

    Microbiology, USDA,2011, Monaghan et al 2010).

    .

    ,

    (,

    2001).

    3.1.1.1 (a) 10 ,

    26 ,2010. : EFSA, ECDC- EU summary report 2010, EFSA Journal 2012; 10(3):2597

  • 22

    3.1.1.1(b)

    2010. : EFSA, ECDC-EU summary report 2010, EFSA Journal 2012;10(3)2597

    3.1.1.1(b)

    0-14 0-4.

    ,

    .

    ,

    .

  • 23

    3.1.1.2(c) , EU/EEA, 2008-2012.

    : European Centre for Disease Prevention and Control. Annual epidemiological report 2014-food and

    waterborne diseases and zoonoses, 2014

    3.1.1.2

    .

    - .

    UV .

    55 oC 90 60

    oC 12 .

    10

    75 C.

    .

  • 24

    3.1.1.2

    2009.

    3.1.1.2 2009. :

    EFSA,ECDC-EU summary report 2009, EFSA Journal 2011;9(3):2090

    3.1.2 Escherichia coli

    Escherichia coli Gram , ,

    , Escherichia

    . E. coli

    .

    . E. coli

    0,1% .

    , .

    3.1.2 E. Coli :

    www.wikipedia.com

  • 25

    (, )

    . E. coli 3-50 C 37-

    41 C, pH 4.3-10.0 aw>0.94 (www.ecdc.europa.eu,

    www.wikipedia.com).

    3.1.2.1

    E. coli

    .

    3-4

    ,

    24 . ,

    8%,

    (HUS).

    3-5%.

    E. coli

    Shiga. Shiga toxin-producing

    E. coli (STEC). 157:7.

    Shiga

    Verocytotoxin-producing E. coli (VTEC).

    , .

    ,

    ,

    .

    STEC VTEC , .

    3.1.2.1 STEC,VTEC 2007-2011

    (Fraser et al 2013, ECDC-annual epidemiologic report,

    Overview of Food Microbiology, USDA, 2011).

  • 26

    3.1.2.1 STEC,VTEC-EU/EEA 2007-2011. : www.ecdc.europa.eu

    3.1.2.2

    . coli ,

    .

    E. coli - .

    .

    , ,

    70C. ,

    .

  • 27

    (www.foodsafety.gov).

    3.1.2.2 : STEC/VTEC ,

    /,2007-2011. :annual epidemiological report 2013 ECDC

    3.1.3 Enterococcus sp.

    Gram .

    (.

    3.1.3).

    .

    , . faecalis (90-95%) .

    faecium (5-10%). Gram ,

    .

    , (10-45C), pH (4.5-10.0)

    (6,5%) (40%).

    3.1.3 :wikipedia

  • 28

    3.1.3.1

    .

    . ,

    . ,

    , (www.wikipedia.com).

    3.1.3.2

    .

    .

    ,

    ,

    . .

    ,

    (www.wikipedia.com).

    3.1.4 Listeria sp.

    Listeria Gram , ,

    (.

    3.1.4).

    30-37 C,

    4 C , pH

    4.4-9.4 aw>0.92. Listeria

    , , ,

    , , ,

    , , ,

    , ,

    . Listeria 3.1.4 L. monocytogenes : www.wikipedia.com

  • 29

    , .

    -, p /

    .

    (www.microbewiki.com).

    Listeria (10) : L. fleischmannii, L.

    grayi, L. innocua, L. ivanovii, L. marthii, L. monocytogenes, L. rocourtiae, L. seeligeri, L.

    weihenstephanensis, L. welshimeri L. dinitrificans.

    (2) .

    3.1.4.1

    . L.monocytogenes ,

    L .ivenovii (Todar, 2012). L.monocytogenes

    (www.microbewiki.com). 1960

    L.monocytogenes

    .

    ,

    .

    L.monocytogenes 5-10%

    . H Listeria

    , ,

    ( AIDS, ..).

    ,

    , .

    .

    , 1.000 .

    Listeria

    .

    , , , ,

    .

    , , .. (Monanghan et al 2010).

    25%

    .

    3.1.4.1 . 2008-2012.

  • 30

    3.1.4.1 , U/, 2008-2012. :

    European Centre of Disease Prevention and Control. Annual epidemiological report 2014-food and waterborne

    diseases and zoonoses 2014

    3.1.4.2

    Listeria,

    .

    . Listeria

    .

    .

    ,

  • 31

    (Todar,2012). Listeria

    ( ).

    hot dog, ..

    Listeria.

    ,

    ,

    ,

    , ,

    ( ,

    ..) hot dog, ..

    (www.foodsafety.gov).

    3.1.5 Staphylococcus sp.

    Gram

    (. 3.1.5).

    ,

    .

    .

    . .

    S.aureus

    S.epidermidis

    (www.microbewiki.com).

    7-45 C 37

    C,

    pH 4.2-9.3,

    25% aw>0,85.

    , ,

    , , , ,

    . .

    S.aureus .

    ,

    .

    3.1.5

    :www.bacteriainphotos.com

  • 32

    50% .

    3.1.5.1

    H

    S.aureus.

    .

    , .

    (CDC).

    S.aureus. S.aureus :

    1.

    2.

    3.

    4.

    5.

    6.

    7.

    8. .

    S.aureus,

    (Todar,2012).

    30 8

    , ,

    ,

    . , , ,

    , .

    . 24-48

    (Overview of Food Microbiology,USDA.2011).

  • 33

    3.1.5.2

    S.aureus

    .

    (.. )

    ,

    .

    .

    ,

    . ,

    (

    )

    .

    (.. )

    (.. ) (verview of Food Microbiology,

    USDA,2011).

    .

    ,

    .

    60C

    4C

    (www.foodsafety.gov).

    3.1.6 Pseudomonas sp.

    Pseudomonas Gram , ,

    (. 3.1.6). Pseudomonas

    ,

    .

    ,

    . Pseudomonas

    aeruginosa

    ,

    ,

    (www.microbewiki.com). 3.1.6 Pseudomonas : www.textofmicrobiology.com

  • 34

    -7-43C 20-30

    C, pH 5.6-8

    aw>0.94.

    .

    3.1.6.1

    Pseudomonas

    .

    , . P. aeruginosa

    .

    , , ..

    P. aeruginosa

    , .

    50%.

    3.1.6.2

    Pseudomonas

    .

    .

    .

  • 35

    3.1.7 Shewanella sp.

    Shewanella Gram , (. 3.1.7), o

    .

    Shewanella H2S

    .

    - (). Shewanella

    .

    Shewanella .

    . Shewanella putrefaciens

    .

    S.putrefaciens

    ()

    ,

    .

    2S. To S.

    prutrefaciens ,

    ,

    . H2S

    , Gram

    .

    H2S (www.ncbi.nlm.nih.gov, Fonnesbech Vogel et al 2005).

    3.1.7.1

    Shewanella ,

    Shewanella. Shewanella

    ,

    .

    , ,

    , ,

    ..

    , ,

    (www.ncbi.nlm.nih.gov, Sharma et al 2010).

    3.1.7 Shewanella sp. :

    www.wikipedia.com

    http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/

  • 36

    3.1.7.2

    ,

    .

    .

    Shewanella

    .

    -

    . ,

    .

    , Shewanella

    .

  • 37

    4:

    HACCP.

    , , , .

    -

    .

    . ,

    Polymerase Chain Reaction (PCR).

    :

    4:

    :

    1)

    2)

    3)

    4) PCR

    5) HACCP

    ()

    E. coli

    Enterococcus sp.

    Staphylococcus sp.

    Salmonella sp.

    Listeria sp.

    Pseudomonas sp.

    Shewanella sp.

  • 38

    5:

    5.1

    - petri 9cm

    - Bactident Oxidase (Merck)

    - // (25:24:1) (Sigma-Aldrich)

    - -Lysozyme molecular biology grade (Applichem)

    -

    -

    - Falcon

    - API tests (Biomerieux)

    - Stomacher bags (Interscience)

    - (Thermo Scientific Heraeus)

    - (TRADE Raypa)

    - (Adventurer OHAUS Balance)

    - Centrifuge 5418R (Eppendorf)

    - (Stuart)

    - Bunsen

    - (Kern)

    - Thermal cycler(Peqlab)

    - Stomacher(interscience)

    - Block heater

    - Vortex

    - StepOne Plus system (Applied Biosystems Inc.)

    - Plate count agar (Lab M)

    - HiCrome (Lab M)

    - Slanetz & Bartley Medium (HiMedia Laboratories)

    - Mannitol Salt Agar (Lab M)

    - Baird-Parker agar (Lab M)

    - XLD agar (HiMedia Laboratories)

    - Listeria Selective Agar Base (Lab M)

    - Listeria Selective Supplement (Lab M)

  • 39

    - Selenite broth (HiMedia Laboratories)

    - Rappaport-Vassiliadis Soya Peptone(RVS) Broth (HiMedia Laboratories)

    - Triple Sugar Iron agar (Lab M)

    - Pseudomonas agar base (Lab M)

    - Nutrient agar (Lab M)

    - Nutrient Broth (Lab M)

    - Egg Yolk (Lab M)

    - Peptone (HiMedia Laboratories)

    - Half-Fraser Broth (Lab M)

    - Fraser Broth Enrichment Supplement (Lab M)

    - Tris-EDTA (TE), 50mM Tris, 50mM EDTA,

    pH 8.0 121C 15 min.

    - Lysozyme lysis buffer 100mM NaCl, 500mM

    Tris pH 8.0, lysozyme 30 mg/mL

    - CTAB-NaCl 4.1g NaCl, 10g CTAB, 100mL dd H2O

    121C 15 min.

    - SDS 10% w/v

    - 20mg/mL

    -

    PCR

    - Primer Efs130F(forward) 0,5

    - Primer Efs490R(reverse) 0,5

    - Primer gadrt-1 0,5

    - Primer gadrt-2 0,5

    - Buffer 1X

    - MgCl2 1,5mM

    - dNTPs 0,25mM

    - Taq 1M

    - Master Mix 2X

    5.1(a): PCR ( 40L)

    PCR mixture

    Buffer 1

    MgCl2 2,5mM

    dNTPs 0,25mM

    Primer 0,5

    Taq 2U

    DNA 2L

  • 40

    5.1(b): PCR

    95oC 1 min.

    95oC 1 min.

    40 cycles 57oC 1 min.

    72oC 45 sec.

    72oC 10 min.

    5.1(c): real-time PCR ( 20L)

    Real-time PCR mixture (L)

    Master Mix(2.0X) 10

    Forward Primer 1

    Reverse primer 1

    DNA 2

    H2O 6

    5.1(d): real-time PCR

    95oC 10 min.

    95oC 30 sec.

    40 cycles 57oC 1 min.

    72oC 30 sec.

    95oC 15 sec.

    59oC 1 min.

    95oC 15 sec.

    5.2

    ,

    .

    ,

    ( ).

    .

    5.2(a), 5.2(b), 5.2(c), 5.2(d), 5.2(e)

    .

  • 41

    5.2(a)

    1

    2

    3

    4

    5

    5.2(b)

    1

    2

    3

    4

    5

    6

    7

    8

    5.2(c)

    1

    2 ,

    3

    4

    5

    5.2(d)

    1

    2

    3

    4

    5

    6

    7

  • 42

    5.2(e)

    1

    2

    3

    4

    5

    .

    5.3

    ,

    .

    Staphylococcus

    sp., Enterococcus sp., E. coli, Salmonella sp., Listeria sp., Pseudomonas sp.

    Shewanella sp. (OMX) .

    .

    5.3(a) .

  • 43

    5.3(a)

    5.3(b)

    Plate Count Agar

    HiCrome E. coli

    Slanetz & Bartley Medium Enterococcus sp.

    Mannitol Salt Agar & Baird-Parker Agar Staphylococcus sp.

    XLD Agar Salmonella sp.

    Listeria Selective Agar Listeria sp.

    Pseudomonas Agar Base Pseudomonas sp.

    Triple Sugar Iron Agar(TSA) Shewanella sp.

    Staphylococcus sp. Enterococcus sp. E. coli Salmonella sp. Listeria sp. Pseudomonas sp. Shewanella sp.

  • 44

    ,

    .

    5.3(b).

    .

    - :

    10g 100mL Peptone water

    Stomacher

    2mL Plate Count Agar

    30oC 48h

    E. coli

    10g 100mL Peptone water

    Stomacher

    300L HiCrome

    37oC 24h

    Enterococcus sp.

    10g 100mL Peptone water

    Stomacher

    300L Slanetz & Barkley Medium

    37oC 24h

    Staphylococcus sp.

    10g 100mL Peptone water

    Stomacher

    300L Mannitol Salt Agar

    Baird-Parker Agar

    37oC 24h

  • 45

    Salmonella sp.

    25g 225mL Buffered Peptone

    water

    Stomacher

    37oC 24h

    1mL 9mL Selenite 0.1mL

    10mL RVS

    Selenite 37oC 48h RVS 37oC

    24h

    300L XLD agar

    37oC 24h

    Listeria sp.

    25g 225mL Half-Fraser Broth

    Stomacher

    30oC 7

    Listeria

    Selective Agar 1, 2 7

    30oC 24h (

    24h )

    Pseudomonas sp.

    25g 225mL Peptone water

    Stomacher

    300L Pseudomonas Agar Base

    37oC 24h

    Shewanella sp.

    25g 225mL Peptone water

    Stomacher

    300L Triple Sugar Agar

    37oC 24h

  • 46

    ,

    Nutrient Agar Base.

    ( ),

    .

    :

    Peptone water ,

    .

    5.4

    .. Staphylococcus,

    . Staphylococcus sp.

    Staphylococcus aureus, S. haemoliticus, S. lentus ..

    . ,

    .

    (Analytical Profile Index)

    Staphylococcus sp.

    1,2,5 TSA 1

    XLD. ,

    .

    tetramethyl-p-

    phenylenediamine,

    .

    cytochrome c oxidase

    20 ( 5.4(a)).

    , 20E ( 5.4(b)).

    Staph ( 5.4(c)).

  • 47

    5.4(b) API 20E

    5.4(a) API 20NE

  • 48

    5.4(c) API Staph

    5.4(b) 5.4(c)

    .

    . (+)

    , (-) (. 5.4(d)).

  • 49

    5.4(d) 20E

    ,

    (-) .

    (https://apiweb.biomerieux.com/servlet/Authenticate?action=prepareLogin)

    .

    5.5

    1. 1mL Eppendorf

    1,5mL

    2. 14.000rpm 5

    3.

    4. 1mL (NaCl 0.9%)

    5. 14.000rpm 5

    6.

    7.

    467L

    8. 30L SDS 10 w/v

    9. 3L - 20mg/mL

    100L Lysozyme lysis buffer.

    10. 65C . Block Heater

    11. 80L CTAB 0.7%

    12. 65C 10

    https://apiweb.biomerieux.com/servlet/Authenticate?action=prepareLogin

  • 50

    13. 750L //

    (25:24:1)

    14. Vortex

    15. 14.000rpm 10

    16. eppendorf

    17. 500L

    18. -20C

    19. 14.000rpm 4C 20

    20. (DNA)

    21. 500L

    70%

    22. 10.000rpm 4C

    23. ,

    70% 10.000rpm 4C 10

    24. eppendorf 37C Block

    heater

    25. 50L

    26. -20C

    5.6

    DNA

    .

    . ,

    DNA

    -.

    , DNA

    260 nm 280 nm. 260 nm

    .

    50 g/mL DNA, 40 g/mL

    DNA RNA 20 g/mL

    .

  • 51

    :

    DNA 1.8.

    .

    DNA

    :

    5.7 PCR

    (PCR)

    DNA,

    . PCR

    ,

    .

    PCR. PCR

    DNA.

    (3) :

    1. DNA

    2. DNA

    3.

    primers

    (bp) 5.7.

  • 52

    5.7 Primers PCR

    Primers primer (53)

    PCR (bp)

    Efs130F -AACCTACCCATCAGAGGG- 360

    Bartosch et al.

    2004 Efs490R -GACGTTCAGTTACTAACG-

    gadrt-1 -

    GCGTTGCGTAAATATGGTTGCCGA- 305 Chen et al. 2006

    gadrt-2 -CGTCACAGGCTTCAATCATGCGTT-

  • 53

    6:

    (5)

    .

    .

    2073/2005 ,

    .

    ,

    .

    .

    (30) , 5

    , 8 , 5 , 7

    (, ) 5 .

    6:

    6.1

    .

    16%

    27%

    17%

    23%

    17%

  • 54

    .

    .

    6.1(a):

    1

    2

    3

    4

    5

    . 6.1(b)

    .

    6.1(b):

    (CFU/g)

    OMX

  • 55

    6.1(a)

    Staphylococcus sp.

    Staphylococcus aureus.

    6.1(c): Staph test

    Staphylococcus sp.

    1 S. xylosus

    2 S. xylosus

    3 S. xylosus

    4 S. aureus

    5 S. xylosus

    S. aureus

    4.

    .

    1,0E+00

    1,0E+01

    1,0E+02

    1,0E+03

    1,0E+04

    1,0E+05

    1,0E+06

    OMX Staphylococcus sp.(M)

    Staphylococcus sp.(B)

    Enterococcus sp.

    E. coli Salmonella sp.

    CFU

    /g

    1

    2

    3

    4

    5

  • 56

    50,00%

    28,13%

    21,88%

    1

    Staphylococcus sp.(M)

    Staphylococcus sp.(B)

    Enterococcus sp.

    E. coli

    Salmonella sp.

    1,20% 0,36%

    93,13%

    5,30%

    2

    Staphylococcus sp.(M)

    Staphylococcus sp.(B)

    Enterococcus sp.

    E. coli

    Salmonella sp.

  • 57

    15,21%

    83,38%

    0,50% 0,91%

    3

    Staphylococcus sp.(M)

    Staphylococcus sp.(B)

    Enterococcus sp.

    E. coli

    Salmonella sp.

    14,54%

    72,70%

    7,09%

    5,67%

    4

    Staphylococcus sp.(M)

    Staphylococcus sp.(B)

    Enterococcus sp.

    E. coli

    Salmonella sp.

  • 58

    CFU/g

    .

    6.1(d):

    Staphylococcus sp.

    Staphylococcus sp.

    E.coli Enterococcus

    sp.

    Salmonella

    sp.

  • 59

    6.1(b)

    6.2

    (8)

    .

    .

    .

    180mL Peptone water

    .

    .

    6.2(a)

    1

    2

    3

    4

    5

    6

    7

    8

    40%

    60%

  • 60

    . 6.2(b)

    ..

    6.2(b):

    (CFU/g)

    OMX

  • 61

    6.2(a)

    6.2(b)

    1,0E+00

    1,0E+01

    1,0E+02

    1,0E+03

    1,0E+04

    1,0E+05

    1,0E+06

    1,0E+07

    OMX Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli Salmonella sp.

    CFU

    /g

    ()

    1

    2

    3

    4

    5

    6

    7

    8

    1,0E+00

    1,0E+01

    1,0E+02

    1,0E+03

    OMX Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp. E. coli Salmonella sp.

    CFU

    /g

    ()

    1

    2

    3

    4

    5

    6

    7

    8

  • 62

    test

    S. aureus.

    6.2(c): Staph test

    Staphylococcus sp.

    1 S. lentus

    1 S. lentus

    2 S. xylosus

    7,26%

    88,85%

    3,89%

    1

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

    100%

    2

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

  • 63

    0,01% Staphylococcus(B)

    93,84%

    6,15%

    3

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

    42,86%

    42,86%

    14,29%

    4

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

  • 64

    4,08%

    95,92%

    5

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

    100%

    6

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

  • 65

    100%

    7

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

    4,48%

    95,52%

    8

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

  • 66

    42,11%

    42,11%

    15,79%

    1

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

    100%

    3

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

  • 67

    100%

    6

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

    100%

    7

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    Salmonella sp.

  • 68

    CFU/g

    .

    6.2(d)

    Staphylococcus sp.

    Staphylococcus sp.

    E.coli Enterococcus

    sp.

    Salmonella

    sp.

  • 69

    6.2(e)

    Staphylococcus sp.

    Staphylococcus sp.

    E.coli Enterococcus

    sp.

    Salmonella

    sp.

  • 70

    6.3

    .

    . .

    6.3(a):

    1

    2 ,

    3

    4

    5

    . 6.3(b)

    .

    6.3(b):

    (CFU/g)

  • 71

    Staphylococcus sp.,

    .

    6.3(a):

    test S. aureus.

    6.3(c): Staph test

    Staphylococcus sp.

    1 (1.1) S. haemolyticus

    1 (1.3) S. sciuri

    3 S. sciuri

    5 S. xylosus

    1,0E+00

    1,0E+01

    1,0E+02

    1,0E+03

    1,0E+04

    1,0E+05

    1,0E+06

    1,0E+07

    OMX Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp. E. coli

    CFU

    /g

    1

    2

    3

    4

    5

  • 72

    17,12%

    6,33%

    76,55%

    1

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    1,25%

    98,75%

    2

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

  • 73

    92,90%

    6,68%

    0,28% 0,14% E. coli

    3

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

    93,33%

    6,15%

    0,51%

    4

    Staphylococcus sp.(B)

    Staphylococcus sp.(M)

    Enterococcus sp.

    E. coli

  • 74

    CFU/g

    .

    6.3(d):

    Staphylococcus sp.

    Staphylococcus sp.

    E.coli Enterococcus

    sp.

  • 75

    6.4

    .

    .

    .

    6.4(a):

    1

    2

    3

    4

    5

    6

    7

    . 6.3(b)

    .

    6.4(b):

    (CFU/g)

    Staphylococcus sp.

  • 76

    6.4(a) .

    .

    . 6.4(a)

    Staphylococcus sp.,

    .

    6.4(a):

    1,0E+00

    1,0E+01

    1,0E+02

    1,0E+03

    1,0E+04

    1,0E+05

    CFU

    /g

    1

    2

    3

    4

    5

    6

    7

  • 77

    42,86%

    51,02%

    0,15% 0,10%

    1

    Staphylococcus(M)

    Staphylococcus(B)

    E.coli

    Listeria spp. I(24h)

    Salmonella spp.

    5,11%

    73,36%

    21,53%

    2

    Staphylococcus(M)

    Staphylococcus(B)

    E.coli

    Listeria spp. I(24h)

    Salmonella spp.

  • 78

    98,46%

    1,54%

    3

    Staphylococcus(M)

    Staphylococcus(B)

    E.coli

    Listeria spp. I(24h)

    Salmonella spp.

    22,22%

    68,52%

    9,26%

    4

    Staphylococcus(M)

    Staphylococcus(B)

    E.coli

    Listeria spp. I(24h)

    Salmonella spp.

  • 79

    55,95%

    1,33%

    5,42%

    37,30%

    5

    Staphylococcus(M)

    Staphylococcus(B)

    E.coli

    Listeria spp. I(24h)

    Salmonella spp.

    100%

    6

    Staphylococcus(M)

    Staphylococcus(B)

    E.coli

    Listeria spp. I(24h)

    Salmonella spp.

  • 80

    CFU/g

    .

    6.4(d):

    Staphylococcus sp.

    Staphylococcus sp.

    E.coli Listeria sp.

    I(24h)

    Listeria sp.

    I(48h)

    Salmonella

    sp.

  • 81

    Listeria

    1,3,4,5,6 , 2 7

    .

    6.4(c)

    6.5

    .

    .

    .

    6.5(a):

    1

    2

    3

    4

    5

    71%

    29%

  • 82

    . 6.5(b)

    .

    6.5(b):

    (CFU/g)

    OMX

  • 83

    6.5(a)

    1,0E+00

    1,0E+01

    1,0E+02

    1,0E+03

    1,0E+04

    1,0E+05

    CFU

    /g

    1

    2

    3

    4

    5

    8,60%

    91,40%

    1

    Staphylococcus(M)

    Staphylococcus(B)

    E. coli

    Pseudomonas spp.

    Shewanella spp.

  • 84

    74,30%

    0,56%

    25,14%

    0,00%

    2

    Staphylococcus(M)

    Staphylococcus(B)

    E. coli

    Pseudomonas spp.

    Shewanella spp.

    97,66%

    2,25% 0,10%

    3

    Staphylococcus(M)

    Staphylococcus(B)

    E. coli

    Pseudomonas spp.

    Shewanella spp.

  • 85

    CFU/g

    .

    95,45%

    4,55%

    4

    Staphylococcus(M)

    Staphylococcus(B)

    E. coli

    Pseudomonas spp.

    Shewanella spp.

    22,22%

    77,78%

    5

    Staphylococcus(M)

    Staphylococcus(B)

    E. coli

    Pseudomonas spp.

    Shewanella spp.

  • 86

    6.5(d):

    Staphylococcus

    sp.

    Staphylococcus

    sp.

    E. coli Pseudomonas

    sp.

    Shewanella

    sp.

  • 87

    6.6 Real-time PCR

    ,

    (. .5.5 ) DNA

    .

    real-time PCR Enterococcus faecalis,

    , E. coli.

    6.6(a).

    6.6(a): real-time PCR

    1 1

    2 2

    4 13

    5 3

    6 4

    8 5

    1 14

    7 6

    1 7

    3 15

    5 8

    1 16

    4 9

    6 17

    7 10

    2 11

    3 18

    5 12

    DNA

    ,

    DNA DNA

    . DNA

    , . 5.6.

    , E. faecalis &

    E. coli, control

    PCR.

    PCR .

    , PCR

    Efs gadrt E. faecalis E. coli, .

  • 88

    6.6(a): PCR gadrt E. coli (

    ) Efs E. faecalis ( )- 1 DNA marker

    100bp, 2-6 PCR product E. coli, 7-11 PCR product E. faecalis

    PCR E. coli 2-6

    gadrt 305bp E. faecalis

    7-11 Efs 360bp.

    E. faecalis E. coli

    (. 6.6(b)) SYBR-GREEN (SYBR

    green method), StepOne Plus System (Applied Biosystems Inc.,

    Foster City, USA).

    1 2 3 4 5 6 7 8 9 10

    00

    11

    1

    E. coli

    E. faecalis

  • 89

    Enterococcus faecalis

    6.6(b): E. faecalis ( 20-2*10-3

    ng)

    PCR ,

    , 0,941

    -3,397 (Frahm and Obst 2003).

    Target: Efs Slope: -3,397 Y-inter: 11,621 R2: 0,941 Eff%: 96,944

  • 90

    Escherichia coli

    Target: gadrt Slope: -3,227 Y-Inter: 10,83 R2: 0,982 Eff%: 104,115

    6.6(c): E. coli ( 20-2*10-3

    ng)

    PCR ,

    , 0,982

    -3,227 (Frahm and Obst 2003).

    real-time PCR

    .

    :

    ,

    PCR.

    PCR

    .

    E. faecalis,

    , Efs

    E. faecalis. .

  • 91

    7: ISO-HACCP

    7.1 ISO

    (ISO- International Organization of

    Standardization) .

    ISO .

    ISO

    ,

    , . ISO

    :

    ISO 9001:2008-

    ISO 18593:2004-

    ISO 22000:2005-

    . ISO 2005 :

    - HACCP

    -

    -

    7.2 HACCP

    ,

    .

    ,

    , ,

    .

    5 852/2004

    ,

    HACCP.

    1 1.2.2, HACCP

  • 92

    .

    HACCP,

    , ,

    .

    HACCP (, , ).

    .

    ,

    .

    HACCP (7) :

    1)

    .

    2) .

    3)

    .

    4) .

    5) .

    6)

    .

    7)

    .

    HACCP,

    HACCP.

  • 93

    1. HACCP

    2.

    3.

    4.

    5.

    6. (1 )

    7. (2 )

    8. (3 )

    9. (4

    )

    10. (5 )

    11. (6 )

    12. (7 )

    HACCP. HACCP HACCP

    . HACCP

    .

    HACCP (CCPs)

    . CCPs

    HACCP

    NACMCF (1992)

    .

  • 94

    ,

    , CCP.

    CCPs .

    CCPs

    HACCP,

    ,

    (, ,1996). 7.1

    CCPs

    CCPs ,

    CCPs

    , CCPs

    . CCPs

    , .

    .

    CCP, ,

    ,

    .

    (5):

    1.

    2.

    3.

    4.

    5.

    ,

    .

    ,

    (

    ).

    .

    ,

    .

  • 95

    *

    7.1 CCPs

    3:

    ;

    CCP

    STOP*

    E4:

    ;

    CCP STOP

    *

    (CCP)

    (CCP)

    NAI

    2:

    ;

    ;

    1: ;

    ,

    CCP STOP

    *

  • 96

    CCP

    (5) :

    1.

    2. ,

    /

    3.

    4.

    5. (..

    )

    (ICMSF,1986) (. 7.1)

    .

    ICMSF :

    - 2 (Two-Class

    Attributes Sampling Plans),

    n

    , c

    m

    - 3 (Three-Class

    Attributes Sampling Plans),

    , n

    , c

    ,

    m

  • 97

    7.1 ( ICMSF)

    *

    1

    3

    n=5, c=3

    2

    3

    n=5, c=2

    3

    3

    n=5, c=1

    ,

    4

    3

    n=5, c=3

    5

    3

    n=5, c=2

    6

    3

    n=5, c=1

    ,

    7

    3

    n=5, c=2

    8

    3

    n=5, c=1

    9

    3

    n=10, c=1

    , ,

    ()

    10

    2

    n=5, c=0

    11

    2

    n=10, c=0

    12

    2

    n=20, c=0

    ,

    13

    2

    n=15, c=0

    14

    2

    n=30, c=0

    15

    2

    n=60, c=0

    *

    . ,

    10%.

    7.2 7.3

    .

  • 98

    7.2: 3

    :

    0,1% 1% 5% 10%

    1 n=5, c=3 0.999 0.999 0.999 0.999

    2 n=5, c=2 0.999 0.999 0.998 0.991

    3 n=5, c=1 0.999 0.999 0.977 0.919

    4 n=5, c=3 0.999 0.999 0.999 0.999

    5 n=5, c=2 0.999 0.999 0.998 0.991

    6 n=5, c=1 0.999 0.998 0.977 0.919

    7 n=5, c=2 0.999 0.999 0.998 0.991

    8 n=5, c=1 0.999 0.998 0.977 0.919

    9 n=10, c=1 0.999 0.995 0.904 0.726

    7.3: 2

    :

    0,1% 1% 5% 10%

    10 n=5, c=0 0,995 0,991 0,774 0,590

    11 n=10, c=0 0,990 0,904 0,599 0,348

    12 n=20, c=0 0,980 0,812 0,358 0,123

    13 n=15, c=0 0,985 0,860 0,463 0,206

    14 n=30, c=0 0,970 0,740 0,215 0,042

    15 n=60, c=0 0,942 0,547 0,046 0,002

    :

    ,

    (DNA, RNA)

    - -

    CCP (

    ) (

  • 99

    )

    . :

    ,

    ,

    ,

    HACCP,

    HACCP CCP ,

    CCPs .

    , FDA. FDA

    (3) : ,

    . 7.4

    .

    7.4: FDA

    ,

    .

    .

    ,

    (..

    ).

    .

    .

    ,

    .

    .

  • 100

    ,

    .

    .

    1. ,

    , .

    2. , ,

    .

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    - ,

    -

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    HACCP - HACCP

    ,

    .

  • 101

    .

    ,

    . HACCP

    .

    ,

    .

    ,

    ,

    ,

    .

    . 6oC

    (..

    S. aureus).

    ,

    . ,

    L. monocytogenes,

    .

    2-7C

    .

    ,

    , ,

    .

    .

    (.. , ),

    .

    7.2.1.2

    (.. ),

  • 102

    .

    HACCP,

    .

    HACCP ,

    .

    .

    pH ,

    , , ,

    .

    (

    ) .

    , S. aureus, ,

    , L.

    monocytogenes, . ,

    S. aureus

    , .

    ,

    pH

    (

    ).

    ( ,

    S. aureus, Mycobacterium

    paratuberculosis).

    ,

    . S.aureus

    ,

    .

    7.2.1.3 HACCP

  • 103

    HACCP

    .

    7.2.1.3 .

    7.2.1.3.

    , ,

    () ,

    () /

    ,

    366 kcal/100g 191 kcal/100g 374 kcal/100g

    20% 11% 10,5%

    30% 19,5% 36%

    () 50% 70% 60%

    NaCl () 1,2% 1,2% 1,4%

    70% 70% 70%

    200g, 1, 1,5, 2kg

    (4C)

    (4C)

    30 90

    . 7.2.1.3.

    ,

    .

    .

    .

    , (, ,

    ),

    .

  • 104

    7.2.1.3:

    CCP4

    4C

    ( )

    ( 24 )

    80-95C/15-30

    ,

    ..

    --

    (4C)

    CCP3

    CCP5

    /

    72C/15

    63C/30

    -

    CCP1

    CCP2

  • 105

    10C,

    2 .

    .

    :

    ( )

    pH

    ,

    .

    6C,

    4 .

    ,

    .

    .

    .

    72C 15 ( )

    63C 30 ( )

    .

    ,

    .

    , .

    . ,

    (

    ,

    ).

  • 106

    .

    32C.

    ,

    ,

    , .

    80-95C 15-20 .

    . 60C

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    .

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    .

  • 107

    (pH 6),

    ,

    ,

    .

    . ,

    ,

    ,

    (, ).

    , , ,

    HACCP.

    HACCP

    .

  • 108

    HACCP

    /

    1 /-

    ,

    CCP1 4C

    2

    CP

    3

    CCP2 72oC/15

    63C/30

    ./

  • 109

    CP

    -

    4

    -

    CP

    -

    >4C (

    )

    (4C)

    ( 24 )

    5 80-95C/15-

    20

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    -

    -

  • 110

    -

    .

    .

    6 :

    -

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    ,

    ,

    ,

    (

    ,

    ,

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    ,

    (

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  • 111

    )

    )

    7

    .

    ,

    CP

    ,

    6

  • 112

    .

    8

    CCP5

    4C

    25g

  • 113

    7.2.2

    HACCP

    ,

    .

    1.

    ,

    ,

    .

    2.

    (..

    )

    3.

    .

    4.

    ,

    5.

    (

    )

    6.

    ,

    7.

    .

  • 114

    8:

    :

    ,

    .

    ,

    ,

    .

    ,

    .

    .

    Salmonella sp.,

    Listeria sp. Staphylococcus aureus. S. aureus

    ,

    S. aureus,

    .

    7,

    .

    HACCP,

    .

    ,

    .

    .

    S. aureus.

    Mannitol salt agar,

    S. aureus.

    .

  • 115

    .

    E. faecalis & E. coli

    real time PCR,

    .

    .

    PCR ()

    .

    HACCP

    ,

    , :

    -

    ,

    .

    -

    .

    -

    .

    -

    3

    , BIO Hellas 2

    .

    .

    .

    .

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