2021
40
San iago Condón Aban o
Adding Value o Ready- o-
C us acean P oduc s: P ocess
op imiza ion o "en i e"
c us aceans using no el
echnologies
Depa amen o
Di ec o /es
P oducción Animal y Ciencia de los Alimen os
Raso Pueyo, Ja ie
Lyng, James G.
Ál a ez LAnza o e, Ignacio
© Uni e sidad de Za agoza
Se icio de Publicaciones
ISSN 2254-7606
San iago Condón Aban o
ADDING VALUE TO READY-TO-CRUSTACEAN
PRODUCTS: PROCESS OPTIMIZATION FOR
"ENTIRE" CRUSTACEANS USING NOVEL
TECHNOLOGIES
Di ec o /es
P oducción Animal y Ciencia de los Alimen os
Raso Pueyo, Ja ie
Lyng, James G.
Ál a ez LAnza o e, Ignacio
Tesis Doc o al
Au o
2019
Reposi o io de la Uni e sidad de Za agoza – Zaguan h p://zaguan.uniza .es
UNIVERSIDAD DE ZARAGOZA
Adding Value o Ready- o-ea C us acean P oduc s:
P ocess op imiza ion o “en i e” and “pa icula e”
c us aceans using no el echnologies
by
San iago Condón-Aban o, Dipl. Ing (Ag .), B.Sc., M.Sc.
UNIVERSITY COLLEGE DUBLIN UNIVERSIDAD DE ZARAGOZA
AN
COLÁISTE
OLLSCOILE, BAILE ÁTHA CLIATH
A hesis submi ed o Uni e si y College Dublin and Uni e sidad de
Za agoza o he deg ee o Doc o o Philosophy (PhD) in Food Science
a
UCD Dublin, School o Ag icul u e & Food science, Bel ield, Dublin 4, I eland
and Uni e sidad de Za agoza, Facul ad de Ve e ina ia, Za agoza, España
P incipal Supe iso :
Co-Supe iso :
P o . James Lyng
D . C is ina A oyo
P o . Ignacio Ál a ez
P o . Ja ie Raso

Decla a ion
I he eby decla e ha he wo k he ein submi ed o he deg ee o Doc o o
Philosophy o he Na ional Uni e si y o I eland, Dublin is he esul o my
own in es iga ion, excep whe e he e e ence is made o he publish
li e a u e. I also decla e ha he ma e ial submi ed in his hesis has no
been accep ed o any o he deg ee and cu en ly is only submi ed o he
Uni e si y o Za agoza as ag eeded in he join -PhD con ac signed
be ween bo h ins i u ions (Annex I).
I au ho ise he Na ional Uni e si y o I eland, Dublin and Uni e si y o
Za agoza o lend his hesis o ins i u ions o indi iduals o he pu pose o
schola ship esea ch. I u he he Na ional Uni e si y o I eland, Dublin o
ep oduce his hesis by pho ocopying o byany o he means in o al o in
pa a he eques o o he ins i u ions o he pu pose o schola ly
esea ch, subjec ed o he no mal condi ions o acknowledgemen .
---------------------------------
San iago Condón-Aban o
D. Ignacio Ál a ez Lanza o e y D. Ja ie Raso Pueyo, Po eso i ula y
Ca ed á ico espec i amen e, del Á ea de Tecnología de los Alimen os de
Depa amen o de P oducción Animal y Ciencia de los Alimen os de la
Facul ad de Ve e ina ia de la Uni e sidad de Za agoza,
Ce i ican:
Que la Tesis Doc o al i ulada “Adding Value o Ready- o-ea
C us acean P oduc s: P ocess op imiza ion o “en i e” and “pa icula e”
c us aceans using no el echnologies” de la que es au o San iago Condón
Aban o, ha sido ealizada bajo su di ección y en co u ela con el Uni e si y
College Dublin, donde el p o eso James G. Lyng y la D . C is ina A oyo
han ac uado como supe iso es, y que su con enido co esponde con el
P oyec o de Tesis ap obado en su momen o y cumple con las condiciones
eque idas pa a op a al g ado de Doc o po la Uni e sidad de Za agoza.
Fdo.: D . Ignacio Ál a ez Lanza o e Fdo.: D . Ja ie Raso Pueyo
Table o con en s
TABLE OF CONTENTS
ABBREVIATIONS ...................................................................................................... I
LIST OF FIGURES ...................................................................................................III
LIST OF TABLES ...................................................................................................... X
LIST OF PUBLICATIONS ...................................................................................... XIII
CONFERENCE AND WORKSHOP PRESENTATIONS .............................................. XIV
ACKNOWLEDGEMENT ........................................................................................... XV
ABSTRACT ............................................................................................................ XVI
RESUMEN ........................................................................................................... XVIII
Chap e 1
Gene al in oduc ion and li e a u e e iew
..................................................................... 1
1.1 FOOD PRESERVATION .................................................................................. 4
1.1.1 Food spoilage agen s ............................................................................ 4
1.1.2 Food p ese a ion s a egies ................................................................ 8
1.2. HEAT TREATMENTS FOR FOOD PROCESSING ............................................ 10
1.2.1
Pas eu iza ion
...................................................................................... 11
1.2.2
S e iliza ion
.......................................................................................... 12
1.3 NOVEL TECHNOLOGIES FOR FOOD PROCESSING ...................................... 13
1.3.1
Ionizing adia ion (IR)
......................................................................... 17
1.3.2
Ul asound (US)
................................................................................... 21
1.4 PROCESS OPTIMIZATION ........................................................................... 25
1.4.1
Modelling in he ood indus y
............................................................ 27
1.4.2
Hea p ocess op imiza ion
................................................................... 36
1.5 EDIBLE CRAB-BROWN CRAB (
CANCER PAGURUS
) .................................... 41
1.6 OBJECTIVES ................................................................................................ 54
Chap e 2
Mic obial cha ac e iza ion o edible c ab
(
Cance pagu us
) ............................................ 56
2.1 ABSTRACT/RESUMEN ................................................................................. 57
2.2 INTRODUCTION .......................................................................................... 61
2.3 MATERIAL AND METHODS .......................................................................... 63
2.3.1
Raw ma e ial
........................................................................................ 63
2.3.2
C ab sample p epa a ion
..................................................................... 64
2.3.2.1
Mic obio a in aw c ab
....................................................................... 64
2.3.2.2
Mic obial shel -li e expe imen s
.......................................................... 64
Table o con en s
2.3.2.3
Mic obio a in lab-cooked and comme cial c ab p oduc s
....................... 64
2.3.3
Mic obial analyses and g ow h media
................................................. 65
2.3.4
Isola ion and iden i ica ion
................................................................. 67
2.3.5
Hea esis ance expe imen s
............................................................... 68
2.3.5.1
Cul u e p epa a ion and ea men media
........................................... 68
2.3.5.2
Hea ea men s
............................................................................... 69
2.3.6
Da a and s a is ical analysis
................................................................ 70
2.3.6.1
Shel -li e de e mina ion
..................................................................... 70
2.3.6.2
The mal esis ance de e mina ion
...................................................... 71
2.4 RESULTS AND DISCUSSION ....................................................................... 72
2.4.1
Mic obiological p o ile o aw c ab mea s
........................................... 72
2.4.2
Mic obiological shel -li e o cooked c abs
........................................... 74
2.4.3
Mic obial g ow h modelling (P ima y model)
..................................... 75
2.4.4
E ec o s o age empe a u e (Seconda y model)
............................. 78
2.4.5
Shel -li e de e mina ion (Te ia y model)
........................................... 81
2.4.6
E ec o s o age empe a u e on mic obial lo a p esen in cooked
c ab
……. ............................................................................................................. 84
2.4.7
Bac e ial g oups p esen in lab-cooked and comme cial samples o
eady- o-ea b own c ab
.................................................................................... 86
2.4.8
Bac e ial species isola ed in b own c ab
............................................. 89
2.4.9
The mal esis ance o he main ege a i e cells isola ed om eady-
o-ea b own c ab
............................................................................................... 91
2.4.10
Hea ea men s o eady- o-ea b own c ab
.................................... 96
2.5 CONCLUSIONS ............................................................................................ 99
Chap e 3
The use o ul asound echnology in he i s cooking s ep o eady- o-ea whole b own
c ab
(Cance pagu us) ............................................................................................. 101
3.1 ABSTRACT/RESUMEN ............................................................................... 102
3.2 INTRODUCTION ........................................................................................ 104
3.3 MATERIALS AND METHODS ...................................................................... 105
3.3.1
Raw ma e ial and cooking condi ions
............................................... 105
3.3.2
Hea ans e s udy
............................................................................ 106
3.3.2.1
Hea pene a ion cu es
.................................................................. 106
3.3.2.2
Le hali y and F alue
....................................................................... 108
3.3.3
Mic obiological examina ion o esh c ab mea
.............................. 108
3.3.4
Mass ans e s udy
........................................................................... 109
Lis o Figu es
IV
Figu e 1.14: Example o c abs du ing cooking (le ) and immedia ely a e
cooking wi h no washing ( igh ). ......................................................... 49
Figu e 2.1: Log10 coun s ob ained in di e en selec i e and non-selec i e
aga s (see Table 2.2) o whi e (whi e ba s) and b own (g ey ba s) aw
c ab mea . TVCm, To al iable mesophilic coun s; TVCp, To al iable
psych ophilic coun s; L.H, Long and Hamme aga . ND = no
de ec able. ......................................................................................... 73
Figu e 2.2: E olu ion o TVCm o e s o age ime (hou s) a 2°C (), 5°C (),
7°C (), and 10°C () o whi e (A) and b own (B) mea ob ained
om eady- o-ea whole b own c abs cooked (75°C o 45 minu es)
and packed unde acuum. ................................................................. 76
Figu e 2.3: Measu ed Vs p edic ed coun s o TVCm a all s o age
empe a u es in whi e (A) and b own (B) mea (es ima ed om Eq. 2.6
and 2.7) ............................................................................................. 83
Figu e 2.4: Log10 coun s ob ained o
Bacillus
spp., H2S p oducing bac e ia,
En e obac e iaceae, Lac ic Acid Bac e ia,
Pseudomonas
spp.,
S aphylococcus
spp., To al Viable Coun s, To al Psych ophilic Ae obic
Coun s and o al psych ophilic bac e ia (L.H) o whi e (A) and b own
(B) mea o cooked c ab s o e a 4°C (g ey ba s) and 10°C (whi e
ba s) a he end o he shel -li e (calcula ed om Eq. 2.6). .................... 85
Figu e 2.5: Rela i e pe cen age o in lab-cooked whi e and b own c ab mea
and a ious comme cial samples a he end o hei shel -li e. ............... 88
Figu e 2.6: Log10 educ ions in he popula ions o
S aphylococcus
spp. (A),
Pseudomonas
spp. (B),
Ca nobac e ium
spp. (C),
Shewanella
spp. (D)
and
Co ynebac e ium
spp. (E) ollowing a hea ea men a 60°C o
90 seconds. ........................................................................................ 92
Figu e 2.7: Time (min) necessa y o educe he popula ion o di e en
species by 6 Log10 cycles (6
D
alues) in pH 7 McIl aine ci a e-
phospha e bu e a 60°C. ................................................................... 95
Figu e 2.8: Theo e ical he mal dea h ime cu es (TDT) o achie e 6 Log10
educ ions o
Kocu ia a inae
(black line) and
Lis e ia monocy ogenes
( ed line). The la e based on he ecommended ea men s by FDA
(FDA, 2011). ...................................................................................... 97
Figu e 2.9: Theo e ical TDT lines o achie e 6 Log10 educ ions o he spo e
o ming bac e ia isola ed om c ab based on da a p esen ed in chap e
6 and hea ea men s equi ed o achie e simila educ ions o
C.
bo ulinum
non-p o eoly ic spo es based on he ecommended

Lis o Figu es
V
ea men s by FDA (
F
90ºC7/10ºC = 10 min;
F
90ºC9ºC = 31 min;
F
90ºC8.6ºC =
57 min) (FDA, 2011). .......................................................................... 97
Figu e 3.1: Scheme o he loca ion o he h ee he mocouples du ing he
hea pene a ion expe imen s: abdomen (1), mandibula (2) and claw
(3). Image adap ed om e e ence (BIM, 2017a). .............................. 107
Figu e 3.2: Time- empe a u e p o iles o e a cooking p ocess a 75°C in he
claw (dashed line), mandibula (do ed line) and abdomen (block line)
o a 500g c ab in a con en ional cooking p ocess wi hou ul asound. .. 108
Figu e 3.3: Rela ionship be ween c ab’s weigh ( om 300 o 870g) and he
o al su ace o he c ab’s ca apace (cm2). ......................................... 115
Figu e 3.4:
F
70ºC7.5ºC alue (min) applied du ing he cooking p ocess in he
c ab's cold spo (i.e. abdomen), wi h (dashed line) and wi hou (block
line) ul asound o he (A) small, (B) medium and (C) la ge c abs. The
ho izon al do ed line ep esen s he a ge
F
70ºC7.5ºC o wo minu es. .... 117
Figu e 3.5: Mic obial load o e con en ional (g ey ba s) and ul asound
assis ed cooking p ocesses (black ba s) in (A) whi e mea and (B)
b own mea . Do ed line shows he de ec ion limi o he coun s. ....... 118
Figu e 3.6: (A) Tu bidi y (OD515) and (B) conduc i i y (µS/cm) alues o he
cook wa e du ing he cooking o b own c abs in wa e a 75°C wi h
(black ba s) and wi hou (g ey ba s) ul asound. ................................ 120
Figu e 3.7: Pic u e o he c ab’s exuda e a e a con en ional cooking (A)
and ul asound-assis ed cooking (B). ................................................. 121
Figu e 4.1: Release a e o Cd (% o Cd eleased pe min) in c abs ea ed a
50°C (A), 65°C (B) and 80°C (C) wi h () and wi hou () ul asound. 137
Figu e 4.2: Pe cen age o Cd emo ed in c abs ea ed a di e en
empe a u es wi h o wi hou ul asound. Ba s wi h di e en le e s
indica e s a is ically signi ican di e ences (P≤0.05). .......................... 139
Figu e 5.1: Time/ empe a u e p o iles du ing he mal pas eu iza ion in a
showe e o a 105°C in he c ab’s claw (block line), mandibula
(do ed lines) and abdomen (do ed line) o a 790g c ab. G ey line
shows he he mal p o ile o he au ocla e. ........................................ 147
Figu e 5.2: Diag am o he ubes used o s udy o he colou deg ada ion
kine ics o c ab whi e mea . .............................................................. 151
Lis o Figu es
VI
Figu e 5.3: E ec o c ab weigh on he hea pene a ion pa ame e s
h
(A)
and
j
(B), es ima ed om he i ing o Ball & Olson Equa ion o he
he mal p o iles ob ained in he c ab cold-spo . Figu e 5.3A also shows
he eg ession line (block line) as well as he 95% con idence in e als
(do ed lines). .................................................................................. 154
Figu e 5.4: E ec o he p ocessing ime on he e olu ion o ΔE* o c ab
whi e mea a 95°C (), 105°C () and 115°C (). Figu e also shows
he da a i ing lines o Equa ion 5.10C. E o ba s ep esen he
s anda d de ia ion o h ee eplica es. ............................................... 159
Figu e 5.5: E ec o he ea men empe a u e on he colou change a e
(
k
) (A) and maximum colou change de eloped (
Ymax
) (B) on c ab
whi e mea . E o ba s show he s anda d de ia ion o h ee eplica es.161
Figu e 5.6: Co ela ion be ween B owning index (BI*) and o al colou
change (ΔE*) a e di e en he mal ea men s................................. 162
Figu e 5.7: Quali y sco e associa ed wi h c ab whi e mea colou change
based on he I ish c ab p oduce s pe cep ion. Numbe 4 indica es he
maximum quali y and 1 he lowes . ................................................... 163
Figu e 5.8: Theo e ical op imisa ion g aph o he pas eu iza ion p ocess o
eady- o-ea whole edible c ab based on Equa ions 5.12 and 5.13. G ey
lines ep esen he quali y bounda ies be ween “good” and
“accep able” (dashed line) and be ween “accep able” and “non-
accep able” (block line). Black lines ep esen s he minimum
p ocessing condi ions ( ime/ empe a u e) equi ed o c abs o
di e en weigh s ( om bo om o op 400, 500, 600, 700 and 800
g ams) o achie e an adequa e F alue based on he inac i a ion o
C.
bo ulinum
ype E (
F
90ºC10ºC=10 min). .................................................. 164
Figu e 5.9: Obse ed e sus p edic ed alues o he he mal p o iles (A) and
whi e mea colou deg ada ions (B) o c abs o di e en weigh s ha
we e pas eu ised unde he condi ions de ined in Figu e 5.5. .............. 165
Lis o Figu es
VII
Figu e 6.1: Diag am o he MTS esis ome e . A, MTS esis ome e main uni ;
B, ul asound gene a o ; C, hea ing/cooling wa e ba h; D, agi a ion
mo o ; E, Main essel; F, ea men chambe ; G, empe a u e da a
logge ; 1, wo way al e; 2, mano- educe ; 3, p essu e/ acuum al e;
4, ea men chambe he mocouple; 5, cooling inle (only MS); 6,
main essel illing emp y ube; 7, ul asound p obe housing; 8,
ul asound p obe; 9, ea men chambe illing al e; 10, solenoid
sampling al e; 11, au oma ic injec ion sy inge; 12, agi a ion sha ; 13,
hea ing elemen ; 14, cooling ou le (only MS); 15, main essel
p essu e inle ; 16, main essel he mocouple; 17 and 18 bo om and
op caps. .......................................................................................... 175
Figu e 6.2: Su i al cu es o
Bacillus mycoides
o hea (A) a 80°C (),
85°C (), 90°C () and 95°C () and mano-sonica ion (B) () and
mano- he mo-sonica ion (B) a 50°C (), 65°C (), 80°C () and
90°C () in pH 6.8 ci a e-phospha e bu e . ..................................... 178
Figu e 6.3: Su i al cu es o
Psych obacillus psych odu ans
o hea (A) a
90°C (), 93°C (), 96°C () and 100°C () and mano-sonica ion
(B) () and mano- he mo-sonica ion (B) a 80°C (), 85°C (), 90°C
() and 95°C () in pH 6.8 ci a e-phospha e bu e . ........................ 178
Figu e 6.4: Su i al cu es o
Bacillus weihens ephanensis
o hea (A) a
102.5°C (), 105°C (), 107.5°C () and 110°C () and mano-
sonica ion (B) () and mano- he mo-sonica ion (B) a 80°C (), 85°C
(), 90°C () and 95°C () in pH 6.8 ci a e-phospha e bu e . ........ 178
Figu e 6.5: In luence o he empe a u e on he shoulde leng h calcula ed
wi h he Gee ae d log-linea eg ession plus shoulde model o
B.
weihens ephanensis
(A) and
P. psych odu ans
(B) by hea (open
symbols) and MTS (solid symbols) in pH 6.8 ci a e phospha e bu e .
Do ed line ep esen s he heo e ical DRT cu es o MTS calcula ed
wi h Equa ion 6.6. ............................................................................ 179
Figu e 6.6: In luence o empe a u e on he 4
D
alues calcula ed wi h he
Gee ae d log-linea eg ession plus shoulde model o log-linea model
o
B. mycoides
(),
P. psych odu ans
() and
B. weihens ephanensis
() inac i a ion by hea in pH 6.8 ci a e-phospha e bu e . ............... 180
Figu e 6.7: In luence o empe a u e on he 4
D
alues calcula ed wi h he
Gee ae d log-linea eg ession plus shoulde model o log-linea model
o
B. mycoides
(),
P. psych odu ans
() and
B. weihens ephanensis
() inac i a ion by MS/MTS in pH 6.8 ci a e-phospha e bu e . .......... 182
Lis o Figu es
VIII
Figu e 6.8: Pe cen age o syne gism calcula ed wi h Equa ion 6.6 o he
inac i a ion by MTS o 4 log10 cycles (4
D
alues calcula ed wi h
Gee ae d log-linea eg ession plus shoulde model) o
B. mycoides
(block line), P. psych odu ans (do ed line) and
B. weihens ephanensis
(dashed line). ................................................................................... 184
Figu e 7.1: Su i al cu es o elec on beam ionizing adia ion a oom
empe a u e o
B. mycoides
(A),
B. weihens ephanensis
(B) and
P.
psych odu ans
(C) in ci a e-phospha e bu e o pH 7 and wa e
ac i i y (aw) o >0.99 (), 0.90 () and 0.80 (). E o ba s ep esen
s anda d de ia ion o h ee eplica es. ............................................... 200
Figu e 7.2: Su i al cu es o elec on beam ionizing adia ion a oom
empe a u e o
B. mycoides
(A),
B. weihens ephanensis
(B) and
P.
psych odu ans
(C) in ci a e-phospha e bu e o pH 5.5 and wa e
ac i i y (aw) o >0.99 (), 0.90 () and 0.80 (). E o ba s ep esen
s anda d de ia ion o h ee eplica es. ............................................... 201
Figu e 7.3: Su i al cu es o elec on beam ionizing adia ion a oom
empe a u e o
B. mycoides
(A),
B. weihens ephanensis
(B) and
P.
psych odu ans
(C) in ci a e-phospha e bu e o pH 4 and wa e
ac i i y (aw) o >0.99 (), 0.90 () and 0.80 (). E o ba s ep esen
s anda d de ia ion o h ee eplica es. ............................................... 202
Figu e 7.4: E ec o he wa e ac i i y (aw) on he dose necessa y o educe
6 Log10 cycles o
B. mycoides
(A),
B. weihens ephanensis
(B) and
P.
psych odu ans
(C) a pH 7.0 (), 5.5 () and 4.0 (). E o ba s
ep esen s anda d de ia ion o h ee eplica es. ................................ 205
Figu e 7.5: Su i al cu es o elec on beam ionizing adia ion a oom
empe a u e o
B. mycoides
(),
B. weihens ephanensis
() and
P.
psych odu ans
() in c ab’s whi e mea (A) and b own mea (B). E o
ba s ep esen s anda d de ia ion o h ee eplica es. ......................... 207
Figu e 7.6: Speci ic esis ance o
B. mycoides
(black ba s),
B.
weihens ephanensis
(g ey ba s) and
P. psych odu ans
(whi e ba s) o
di e en inac i a ion echnologies in ci a e-phospha e bu e o pH 7.0
and aw >0.99 (da a o MS, MTS and Hea a e adap ed om Chap e
6). ................................................................................................... 212
Figu e 8.1: Maximum pas eu iza ion empe a u es allow o achie e he a ge
F
90°C10°C= 10 min minmain aining “good quali y” (g een line) and
“accep able quali y” (blue line) depending on he c ab weigh , based
on he equa ions de eloped in Chap e 5. .......................................... 226
Lis o Figu es
IX
Figu e 8.2: Theo e ical op imisa ion g aph o he pas eu iza ion o eady- o-
ea whole edible c ab based on Equa ions 5.12 and 5.13 o c abs o
600g o he inac i a ion o 6 Log10 educ ions o
C. bo ulinum
non-
p o eoly ic ype E based on U.S. FDA ecommenda ions and
B.
weihens ephanensis
based on he he mal esis ance ob ained in his
PhD Thesis (Chap e 6). .................................................................... 227
Figu e 8.3: Log o he ime equi ed o achie e 6 Log10 educ ions o he
popula ion o
B. weihens ephanensis
by hea (black ba s) and MTS
(whi e ba s) a di e en empe a u es in pH 6.8 McIl aine ci a e-
phospha e bu e . ............................................................................. 230
Figu e 8.4: Requi ed adia ion dose o educe 6 Log10 cycles he popula ion
o he main h ee bac e ial spo es isola ed om edible c ab in he wo
ypes o c ab mea ............................................................................ 231
Figu e 8.5: Flow diag am o he di e en al e na i e p ocesses p oposed,
using di e en al e na i e echnologies. ............................................. 234

Lis o Tables
X
LIST OF TABLES
Table 1.1: B ie desc ip ion o he no el he mal and non- he mal
echnologies o ood p ocessing. ........................................................ 15
Table 1.2: Lis o au ho ized ood p oduc s and maximum i adia ion dose in
di e en EU coun ies. ........................................................................ 21
Table 1.3: Volume and alue o landings o edible c ab in he main p oduce s
coun ies in 2013. ............................................................................... 44
Table 2.1: Summa y o comme cial c ab p oduc s e alua ed in his s udy. ...... 63
Table 2.2: Cul u e media and incuba ion condi ions used du ing his
in es iga ion. ...................................................................................... 66
Table 2.3: Lag phase (
λ
) in hou s and maximum g ow h a e (
μmax
) in hou s-1
es ima ed wi h he modi ied Gompe z Equa ion (Zwie e ing
e al
.,
1990) o o al iable coun s in whi e and b own c ab mea om eady-
o-ea whole b own c ab pack unde acuum and s o ed a 2°C, 5°C,
7°C and 10°C. .................................................................................... 78
Table 2.4: Pa ame e s o he squa e oo model (Ra kowsky
e al
., 1982) o
he maximum g ow h a e and Lag phase o o al iable coun s
g ow h on eady- o-ea b own c ab pack unde acuum. ...................... 80
Table 2.5: Equa ions based on he inal Equa ion which de ine he shel -li e
o eady- o-ea whole b own c ab pack unde acuum depending on
he s o age empe a u e. .................................................................... 82
Table 2.6: Vege a i e bac e ial species isola ed om cooked eady- o-ea
b own c ab (
Cance pagu us
) p ocessed unde labo a o y condi ions
and a ious comme cial p oduc s iden i ied by 16S RNA sequencing. .... 90
Table 2.7: The mal esis ance (
D
T and
z
alues) o a selec ion o non-spo e
o ming bac e ial species isola ed om cooked c ab mea . .................... 94
Table 3.1: Hea pene a ion pa ame e s (
h
and
j
, dimensionless) a ising
om he applica ion o he Ball & Olson model o he hea pene a ion
cu es in he cold spo o b own c abs (
Cance pagu us
) o di e en
weigh s and sizes cooked wi h o wi hou ul asound in wa e a 75°C. 112
Table 3.2: Fi s o de Equa ions co ela ing
h
alues (dimensionless) wi h
he weigh o b own c abs (g) du ing con en ional and ul asound-
assis ed cooking. .............................................................................. 114
Lis o Tables
XI
Table 3.3: Sal con en (%) and mois u e con en (%) in whi e and b own
c ab mea cooked wi h o wi hou ul asound in wa e and wa e wi h
5% NaCl. Values ep esen mean alue ± s anda d e o . Fo bo h sal
and mois u e con en , ANOVA ollowed by Tukey’s es s we e
pe o med o whi e and b own mea independen ly. .......................... 122
Table 4.1: DRC-e ICP-MS ope a ing condi ions ............................................ 131
Table 4.2: To al samples weigh s (d y weigh ) and mois u e con en (%) o
whi e and b own mea s o each c ab used in his in es iga ion. The
heo e ical ini ial amoun o Cd was calcula ed based on Equa ion 4.2. 133
Table 4.3: Measu ed cadmium (Cd) concen a ions in whi e and b own
mea s, bo h aw and a e ea men s a di e en empe a u es wi h
and wi hou ul asound (35 kHz, 100%, 200W, 60 minu es) and he
maximum cadmium concen a ion de ec ed in wa e a e each
ea men . Di e en le e s indica e s a is ically signi ican di e ences
wi hin columns (P≤0.05). .................................................................. 134
Table 4.4: Fi s o de Equa ions which co ela e he pe cen age o cadmium
ex ac ed wi h he ea men ime du ing he di e en p ocesses
applied. ............................................................................................ 138
Table 5.1: Hea pene a ion pa ame e s (
h
and
j
, dimensionless) a ising
om he i ing o he Ball & Olson Equa ion o he hea pene a ion
cu es in he cold-spo o a ious edible c abs (
Cance pagu us
), o
di e en weigh s and sizes, du ing he mal pas eu iza ion in a showe
e o a di e en empe a u es. ........................................................ 148
Table 5.2: A e age alues o mois u e, wa e holding capaci y and colou o
whi e and b own mea a e e o pas eu iza ion (
F
90ºC10ºC=10 min) a
di e en empe a u es. ..................................................................... 156
Table 5.3: Colou pa ame e s o c ab’s whi e mea a di e en imes du ing
hea p ocesses a di e en empe a u es. .......................................... 160
Table 6.1: Hea esis ance pa ame e s om he i ing o he Gee ae d log-
linea plus shoulde model o log-linea model o he su i al cu es o
Bacillus mycoides
,
Bacillus weihens ephanensis
and
Psych obacillus
psych odu ans
spo es ea ed in pH 6.8 ci a e-phospha e bu e . ....... 177
Table 6.2: MS/MTS esis ance pa ame e s om he i ing o he Gee ae d
log-linea plus shoulde model o he su i al cu es o
Bacillus
mycoides
,
Bacillus weihens ephanensis
and
Psych obacillus
psych odu ans
spo es ea ed in pH 6.8 ci a e-phospha e bu e . ....... 181
Lis o Tables
XII
Table 7.1: Elec on beam ioniza ion esis ance pa ame e s ob ained om he
i ing o he Gee ae d log-linea plus shoulde model (Equa ion 7.1) o
he su i al cu es o
B. mycoides
,
B. weihens ephanensis
and
P.
psych odu ans
in ci a e-phospha e bu e s o di e en pH and aw. ..... 203
Table 7.2: Elec on beam ioniza ion esis ance pa ame e s ob ained om he
i ing o he Gee ae d log-linea plus shoulde model (Equa ion 7.1) o
he su i al cu es o
B. mycoides
,
B. weihens ephanensis
and
P.
psych odu ans
in whi e and b own c ab mea s. .................................. 206
Lis o Publica ions
XIII
LIST OF PUBLICATIONS
Pee - e iewed a icles
 Condón-Aban o, S., A oyo, C., Ál a ez, I., B un on, N., Why e, P., & Lyng,
J. G. (2018). An assessmen o he applica ion o ul asound in he p ocessing
o eady- o-ea whole b own c ab (
Cance pagu us
). Ul asonics
Sonochemis y, 40, Pa A, 497-504.
 Condón-Aban o, S., A oyo, C., Ál a ez, I., Condón, S., & Lyng, J. G.
(2016). Applica ion o ul asound in combina ion wi h hea and p essu e o
he inac i a ion o spo e o ming bac e ia isola ed om edible c ab (
Cance
pagu us
). In e na ional Jou nal o Food Mic obiology, 223, 9-16.
 Condón-Aban o, S., Ped ós-Ga ido, S., Ceb ián, G., Raso, J., Condón, S.,
Lyng, J. G., & Ál a ez, I. (2018). C ab-mea -isola ed psych ophilic spo e
o ming bac e ia inac i a ion by elec on beam ionizing adia ion. Food
Mic obiology, 76, 374-381.
 Condón-Aban o, S., Raso, J., A oyo, C., Lyng, J.G., Condón, S. and Ál a ez,
I. (2018). E alua ion o he po en ial o ul asound echnology combined wi h
mild empe a u es o educe cadmium con en o edible c ab (
Cance
pagu us
). Ul asonics Sonochemis y, 48, 550-554.
A icles submi ed
 Condón-Aban o, S., Raso, J., A oyo, C., Lyng, J.G. and Ál a ez, I. Quali y-
based he mokine ic op imiza ion o eady- o-ea whole edible c ab (
Cance
pagu us
) pas eu iza ion ea men s. Submi ed o Food and biop ocess
echnology. (Fi s e iew 4/7/2018).
A icles in p epa a ion
 Condón-Aban o, S., Lian F., A oyo, C., Ál a ez, I. and Lyng, J.G. E ec o
s o age empe a u e on mic obial shel -li e and mic obiological p o iles o
eady- o-ea I ish whole b own c ab (
Cance pagu us
).
 Condon-Aban o, S., Ped ós-Ga ido, S., Al a ez, I., Why e, P., Lyng, J.G.
and A oyo, C. Isola ion, iden i ica ion and he mal esis ance o he
mic obio a p esen in eady- o-ea b own c ab (
Cance pagu us
) mea .
Chap e 1
1
Chap e 1
Gene al in oduc ion and li e a u e e iew

Chap e 1
2
Due o hei composi ion, c us aceans such as b own c ab a e conside ed
o be a heal hy choice o consume s due o hei high-quali y p o ein, amino
acid composi ion (Gökoðlu and Ye likaya, 2003; Maul aul ,
e al
., 2012) and
hei low sa u a ed a con en (Ba en o
e al
., 2008a, 2009b). Howe e , i s
consump ion is also associa ed wi h ce ain heal h isks such as high
choles e ol le els in some edible pa s, alle gic eac ions and con amina ion
wi h oxic elemen s (Maul aul
e al
., 2012). Ei he way in case o edible c ab
(
Cance pagu us
) a con inuous inc ease in Eu opean landings has occu ed
o e he las 60 yea s, inc easing om 11,000 onnes o o e 48,000 onnes,
gi ing a o al alue o in excess o 57 million eu os which endo ses he
inc eased in e es in his c us acean. To da e he h ee main Eu opean
p oduce s o edible c ab a e he Uni ed Kingdom, I eland and F ance
(Eu os a , 2018).
Edible c ab p oduc ion in I eland s a ed in 1980s and o e he las h ee
decades has su e ed a signi ican inc ease, wi h I eland being now one o he
op h ee p oduce s o his c us acean in he wo ld wi h p oduc ions anging
be ween 5,000 and 8,000 onnes pe yea , which has a ne alue o o e 8
million Eu os. In e ms o p oduc ion, edible c ab is he second mos exploi ed
species in I eland. This da a shows he impo ance o his c us acean as a
aluable commodi y o he I ish ishing indus y (BIM, 2014, 2017a).
Ou o he o al p oduc ion o edible c ab in I eland, 42% is sold as esh
li e c ab and he emaining 58% is sold in a ious eady- o-ea o ma s. F om
p ocessed c ab only 1% is p esen ed as ans o med p oduc wi h he majo i y
simply p esen ed as cooked whole c ab, cooked claws o cooked mea (bo h
whi e mea om c ab appendages o b own mea om he ca apace) which
is ei he ozen o chilled (Eu os a , 2018). One o he main challenges o he
c ab indus y is o educe losses associa ed wi h anspo a ion. In he case o
expo s o edible c ab as esh li e p oduc , losses o up o a 50% can occu
om c ab dea hs o damage du ing anspo a ion, while he expo in a
eady- o-ea o ma a oids such losses du ing anspo a ion while also adding
Chap e 1
3
alue o he inal p oduc (Ba en o,
e al
., 2008a; 2010b; Uglow,
e al
.,
1986).
The main s eps equi ed in he p oduc ion o eady- o-ea edible c ab
p oduc s a e:
(1) Ini ial cooking (e.g. boiling wa e o 20-30 minu es): Du ing his s ep
a se ies o compounds (e.g. p o eins and c ab di ) a e eleased om he c ab
and a ach o he ca apace which makes a subsequen washing o he c abs
p io o packaging essen ial.
(2) Cooling/washing: This s ep is necessa y o comple e he c ab mea
coagula ion and is applied i s using je s o wa e o di emo al which is
ollowed by cooling in a cold oom. A e cooling c abs a e selec ed depending
on hei inal des ina ion o be sold as ei he whole cooked, ex ac ed mea s,
claws, e c.
(3) Finally a second pas eu iza ion s ep is commonly applied o p oduc s
ollowing hei packagaing o ensu e he mic obiological sa e y o he inal in-
pack p oduc s.
Al hough hese s eps a e gene ally common ac oss he c ab indus y, he
condi ions used in each s ep a e a ying among companies leading o a wide
ange o a iabili y in he quali y o he inal p oduc s.
Addi ionally due o he mic obiological isks associa ed wi h his kind o
p oduc as men ioned, c us acean consump ion is also associa ed wi h o he
heal h isks due o high choles e ol le els in some edible pa s, alle gic
eac ions and con amina ion wi h oxic elemen s (Maul aul
e al
., 2012).
Pa icula ly, in edible c ab high le els o Cadmium (Cd) ha e been epo ed,
especially in he b own mea (Ba en o
e al
., 2009b, 2009c; Bolam
e al
.,
2016; Maul aul
e al
., 2012, 2013). In he EU, he Cd con en o c abs is
only egula ed o he whi e mea , which is loca ed in he c ab appendages
(claws and legs), wi h 0.5 mg/kg being he maximum pe mi ed limi (EU,
Chap e 1
4
2006, 2011). Howe e , se e al s udies ha e wa ned o he isk o exceeding
he o al weekly in ake o Cd se by EFSA (2009) (2.5 μg/kg body weigh ) in
consume s om coun ies whe e b own mea is commonly consumed (Bolam
e al
., 2016; Maul aul
e al
., 2013; Noël
e al
., 2011; Wiech
e al
., 2017). As
opposed o he EU, whe e he e is no egula ion conce ning Cd le els in b own
c ab mea , some o he coun ies such as China ha e ecen ly s a ed o
impose limi s conside ing all c ab edible pa s ins ead o whi e mea only. So,
in a nea u u e his p oblem could ep esen a g ea issue o he edible c ab
p ocessing indus y.
1.1 FOOD PRESERVATION
Mos oods u s a e de i ed om plan s o animals. The inhe en biological
na u e o such p oduc s means ha once ha es ed o slaugh e ed hese
p oduc s unde go a se ies o changes, which modi y he o iginal
cha ac e is ics o he p oduc and ul ima ely hese lead o spoilage. These
changes can be physical o chemical in na u e bu can also be induced by he
me abolic ac i i y o ubiqui ous mic obial con aminan s.
The abili y o p ese e oods and ex end hei shel li e has always been a
key a ea ha has de eloped wi h Human e olu ion. Since ancien imes, and
o many cen u ies, wi h no unde s anding o he unde lying causes o ood
spoilage, a se ies o p ese a ion me hods we e de eloped o ensu e a
cons an a ailabili y o ood. Many o hese me hods a e s ill used oday. The
e olu ion o hese p ese a ion echniques came om expe ience which was in
many ins ances in luenced by he geog aphic loca ions o sub-popula ions.
F om hese ancien imes many ood p ese a ion me hods including d ying,
smoking, sal ing, pickling, eezing (in cold clima es) o e men a ion (in wa m
clima es whe e spoilage occu ed a a as e a e) ha e e ol ed.
1.1.1 Food spoilage agen s
Food spoilage agen s a e in luenced by many ex e nal ac o s such as
empe a u e, humidi y, oxygen concen a ion, ligh and s o age empe a u e
Chap e 1
5
(Casp and Ab il, 2003; Po e and Ho chkiss, 1995). Independen o he e ec
o hese pa ame e s on mic obial g ow h/su i al, signi ican changes can be
also induced on mic obial g ow h due o in insic cha ac e is ics o he ood
ma ix. O e all, he main causes o ood deg ada ion can be classed as
chemical o biological.
Chemical deg ada ion.
The wo main eac ions p oducing ood al e a ions
in his g oup a e non-enzyma ic b owning and lipid oxida ion/ ancidi y.
Non-enzyma ic b owning, also known as he Mailla d eac ion, in ol es a
se ies o complex eac ions be ween educing suga s and p o eins p esen in
oods, which modi y odou and la ou o oods, p oducing da k b own
pigmen s wi h associa ed losses in p o ein (due o dena u a ion) and
nu i ional alue (Pé ez-Locas and Yaylayan, 2010). Addi ionally when hese
eac ions a e e y in ense oxic subs ances can po en ially be p oduced
(Che io
e al.,
2009). The changes p oduced due o hese eac ions migh be
desi able, such as in he case o a oas appea ance o la ou in mea bu
hey can also be undesi able, as in case o a da kened colou o an o la ou
in s e ilized milk. The a e o de elopmen o Mailla d eac ions is in luenced
by a numbe o ex e nal ac o s wi h hea p ocessing condi ions (i.e. ime and
empe a u e) being he mos impo an . Thus he Mailla d eac ion is a key
ac o in ood p ocesses such as cooking, pas eu iza ion, s e iliza ion and
dehyd a ion whe e p oduc s a e exposed o high empe a u es o long imes
(Ma ins
e al.,
2000).
Ano he impo an chemical cause o ood deg ada ion is a ancidi y
(Velasco
e al.,
2010). These eac ions a e esponsible o nu i ional alue
losses and he o ma ion o undesi able ola ile compounds. The wo main
eac ions occu ing wi hin his g oup a e hyd oly ic ancidi y and oxida i e
ancidi y. The i s one is due o he ac ion o lipases which eleases a y acids
om iglyce ides and phospolipids; while he second one e e s o he ac ion
o oxygen and lipoxygenases on unsa u a ed a y acids. These eac ions a e
Chap e 1
6
also in luenced by a numbe o ex e nal ac o s such as empe a u e, p esence
o ca alys s, a ailable oxygen concen a ion and he ype o a y acid among
o he hings (Wa aho
e al
., 2011).
Biological deg ada ion.
Biological de e io a ion is quan i a i ely mo e
impo an han chemical due o he highe equency o i s occu ence and he
se e i y o he consequences p oduced (i.e. ood poisoning and/o spoilage).
Wi hin his g oup enzymes and mic oo ganisms a e he main causa i e agen s
hough pa asi es and i uses a e also o signi ican impo ance (Po e and
Ho chkiss, 1995).
The enzyma ic ac i i y in all li ing cells is p ecisely con olled. In he case
o oods, enzyma ic ac i i y con inues a e ha es ing o slaugh e ing
p oducing di e en changes in oods u s. Some o his enzyma ic ac i i y is
desi able; leading in some cases o posi i e changes such as ui ma u a ion
o mea ende iza ion. Howe e , beyond ce ain limi s con inued enzyma ic
ac ion leads o he decomposi ion o issues which in u n acili a es u he
de e io a ion by mic oo ganisms.
Food spoilage due o he ac ion o mic oo ganisms is a e y a iable
phenomenon, since i is dependen on ype and numbe o mic obial species
p esen which in u n depends on he ype o ood ma ix, p ese a ion
condi ions, including s o age empe a u e and he p esence o absence o
oxygen (Blackbu n, 2006). The ac ion o mic oo ganisms, due o hei di e en
me abolic p ocesses, leads o he deg ada ion o oods making hem un i o
consump ion. A wide ange o mic oo ganisms a e ela ed in di e en ways o
oods, some o which a e na u al con aminan s o oods while o he s a e
consciously o sub-consciously in oduced in he ood chain (Ga g
e al
.,
2016). The h ee main g oups o mic oo ganisms ound in oods u s a e hose
which (a) p oduce, p ese e o de elop cha ac e is ic la ou s (i.e. p ocessing
aids), (b) cause ood spoilage due o o ganolep ic changes o (c) a e
pa hogenic and lead o ood sa e y issues

Chap e 1
7
Al hough, he o al amoun o ood los due o he ac ion o bo h biological
and chemical spoilage is unce ain, is es ima ed ha one hi d o o al global
ood p oduced o human consump ion is los o was ed (FAO, 2011). F om
hese o al losses, i is is es ima ed ha abou 25% o hese losses is di ec ly
ela ed o he ac i i y o mic oo ganisms (Pe uzzi
e al
., 2017). Despi e he
losses p oduced by mic oo ganisms hese biological agen s a e o e en g ea e
conce n o he ood indus y due o hei po en ial as a se ious public heal h
haza d (Ra ishanka and Bai, 2015).
In addi ion o he economic p oblems c ea ed by mic obial spoilage, he
p esence o some o hese mic oo ganisms in addi ion o he p esence o
i uses and pa asi es is a cons an h ea o public heal h and a signi ican
impedimen o socio-economic de elopmen wo ldwide. I has been es ima ed
ha in 2010, he consump ion o con amina ed oods p oduced o e 600
million illnesses and o e 400,000 dea hs wo ldwide (Ha elaa
e al
., 2017).
Figu e 1.1A and 1.1B shows he pe cen ages a ibu ed o he di e en ood
con aminan s ( i uses, bac e ia, pa asi es and chemicals).
As Figu es 1.1A and 1.1B show, abou 60% o he o al illness and dea hs
ela ed o ood a e p oduced by mic oo ganisms, which emphasises he
impo ance o his biological con aminan o he ood indus y. A wide ange
o bac e ia can be hos ed in ood. The main ege a i e h ea s a e
Lis e ia
spp.,
Salmonella
en e ica,
Campylobac e
spp.,
Esche ichia coli
,
Shigella
spp.,
S aphylococcus au eus
,
Vi io chole a
and
B ucella
spp. while he spo e
o ming bac e ia o public heal h signi icance a e
Clos idium bo ulinum
,
Clos idium pe ingens
and
Bacillus ce eus
(Ha elaa
e al
., 2017). F om hese
mic oo ganisms, spo e o ming bac e ia a e o special in e es in ood
echnology due o hei abili y o gene a e o ms which a e esis an o ood
p ese a ion p ocesses.
Chap e 1
8
Figu e 1.1: Pe cen age o (A) o al oodbo ne illnesses and (B) o al oodbo ne dea hs
p oduced by pa asi es (g een), i uses (blue), chemicals (pu ple) and mic oo ganisms ( ed).
Da a ex ac ed om Ha elaa
e al
., (2015).
1.1.2 Food p ese a ion s a egies
In ligh o hese biological and chemical challenges he main objec i es o
ood p ese a ion could be summa ized as: (i) ensu ing he p oduc sa e y
main aining app op ia e o ganolep ic condi ions du ing i s shel -li e; (ii)
minimizing he impac o he p ese a ion p ocess on p oduc quali y
(Blackbu n, 2006). Thanks o cu en knowledge abou he a o e-men ioned
ood spoilage agen s, hei mechanism o ac ion and he in luence o o he
ac o s on hei beha iou , he main s a egies used o ood p ese a ion a e
(Rahaman, 2007a): Inhibi ion o Inac i a ion/elimina ion o he con amina ion.
These p ese a ion s a egies cons i u e he basis o mode n ood echnology
and can be applied using di e en me hods and echnologies.
a. Inhibi ion (i.e. educing mic obial me abolic ac i i y and he a e o
enzyma ic and chemical eac ions). The inhibi ion me hods used o educe he
mic obial ac i i y and enzyma ic/chemical eac ions include:
1)
Reduc ion o wa e ac i i y (aw)
by elimina ion o wa e o addi ion o
solu es. The educ ion o aw in a ood ma ix p olongs he mic obial g ow h lag
phase, educes he exponen ial g ow h a e and also he maximum mic obial
load a ained a he s a iona y phase. The main ad an ages o his p ocess is
i s simplici y and low cos . Howe e , i s p incipal disad an age is i s signi ican
23%
60%
17%
A
14%
60%
22%
4% B
Chap e 1
9
impac on he o ganolep ic cha ac e is ics o he inal p oduc depending o
he sys em used o educe aw (Rhaman and Labuza, 2007).
2)
Reduc ion o s o age empe a u e.
This p ese a ion me hod can be
applied a wo le els, chilling o eezing. Food chilling in ol es empe a u es
anging om -1 o 15°C, and allows a shel -li e ex ension wi h educed e ec s
on ood p ope ies. The main incon enience o chilling is he capabili y o
ce ain pa hogenic mic oo ganisms, such as
Lis e ia
spp. and
Ye sinia
spp., o
g ow a chilling empe a u es (Walke and Be s, 2008). On he o he hand,
eezing leads o a educ ion in p oduc empe a u e, ypically in he ange o
-18°C o -30°C. A hese empe a u es no mic oo ganism is capable o
ep oducing and he e o e chemical eac ions become he dominan
de e io a ion mechanism (Rhaman and Velez-Ruiz, 2007).
3)
Reduc ion o edox po en ial.
This me hod is based on he educ ion o
oxygen concen a ion, which in u n leads o a educ ion o inhibi ion o
ae obic mic obial ac i i y which in u n ex ends he shel -li e o ood. The main
disad an age o his me hod is ha he absence o oxygen p omo es he
de elopmen o anae obic species, such
C. bo ulinum
(bo h p o eoly ic and
non-p o eoly ic species) which is conside ed o be one o he mos haza dous
mic oo ganisms in ood, because o he se e i y o he associa ed poisoning.
This me hod is applied du ing he packaging o ood by: physical emo al o
ai by acuum packaging; chemical abso p ion o oxygen using oxygen
sca enge s; o by changing he gas a mosphe e su ounding he ood using
ine o modi ied a mosphe es (Go is and Peppelenbos, 2007).
4)
Reduc ion o pH (Acidi ica ion).
The educ ion o pH ac s by des oying
some enzymes loca ed in cell en elopes and also by modi ying he cy oplasmic
pH, which in u n signi ican ly educes mic obial de elopmen . F om a
echnical pe spec i e, ood acidi ica ion can be achie ed by adding acids o he
ood o by p omo ing he ac ion o ce ain e men a i e p ocesses. The main
ad an age o his p ocess is i s simplici y and ela i ely low cos . Howe e ,
simila o educ ions in
aw
, his p ocess also p oduces signi ican changes on
he p ope ies and appea ance o ood ma ixes (Rahaman, 2007b).
Chap e 1
10
5)
Addi ion o bac e ios a ic agen s.
T adi ionally his echnique consis ed
o he addi ion o a wide ange o chemical addi i es wi h an imic obial
p ope ies, many o which we e syn he ic as opposed o na u al compounds.
Despi e he e icacy o his me hod, cu en consume pe cep ion o hese
addi i es has changed and nowadays i s use as ood p ese a ion me hod is
declining in popula i y. Nowadays, he ood indus y is looking o mo e
na u al al e na i es wi h an imic obial ac i i y, such as bac e iocins, o ganic
acids, essen ial oils, e c., o eplace he use o syn he ic chemical addi i es
(Smid and Go is, 2007).
These p ese a ion s a egies a e e icien a con olling
mic obial/chemical/enzyma ic ac i i y bu do no elimina e hem. They ha e
ad an ages in e ms o hei simplici y o in e ms o hei low impac on he
quali y o he ood ma e ial. Howe e , he main limi a ion o hese s a egies is
hei inabili y o ensu e ood sa e y by elimina ing he causa i e agen s.
The e o e, he e e ec i eness is highly dependen upon he ini ial quali y o
he aw ma e ial (Rahaman, 2007a).
b. Inac i a ion/elimina ion o con amina ing sou ces (i.e.
mic oo ganisms o spoilage agen s p esen in/on aw ma e ials). Al hough he
inhibi ion s a egies can ex end he shel -li e, a e no able o elimina e he
haza d. Fo his eason ano he g oup o p ese a ion s a egies which ha e
he po en ial o elimina e he pa hogens and spoilage agen s, he eby
ensu ing oods sa e y, exis s. Wi hin hese s a egies, me hods based on
physical p inciples a e he mos impo an . Wi hin ha g oup he mal
p ocessing can be conside ed he mos widely used (Rahaman, 2007a).
1.2. HEAT TREATMENTS FOR FOOD PROCESSING
The use o hea in ood p epa a ion da es back o he disco e y o i e by
man. In mo e ecen imes, i is i ing he pe son who is o en c edi ed as
being he i s ood echnologis is Nicolas Appe who came up wi h he
concep o hea ing oods in sealed ja s o ex end hei shel -li e in 1809
Chap e 1
17
1.3.1
Ionizing adia ion (IR)
The use o ionizing adia ion o ood p ese a ion can be conside ed as
a e-eme gen echnology (Molins, 2001). Food i adia ion was p oposed in
he 19 h cen u y and since hen a wide ange o esea ch has been
pe o med o e alua e he po en ial o his echnology o mic obial
inac i a ion (De La a
e al
., 2002; Jeong and Kang, 2017; Sa ıas
e al
.,
2003) and assess i s in luence on ood p ope ies (Byun
e al
., 2000, 2008;
Lee
e al
., 2001). Despi e he ac ha i shows g ea po en ial o ood
decon amina ion, he use o his echnology a an indus ial scale emains
s alled in he ield o he ood p ocessing in se e al coun ies, due o poo
consume pe cep ion o i adia ed oods. This con used pe cep ion is
gene ally a esul o a lack o in o ma ion, misin o ma ion and misleading
image y and also he con usion among consume s o he di e ence
be ween i adia ed ood and adioac i e con amina ion.
Food ioniza ion consis s o he exposu e o packaged o unpackaged
oods u s o ionizing adia ion o mac o-o ganisms (e.g. insec s, pa asi es)
o mic obial des uc ion in he ood p oduc s (Odueke
e al
., 2016). The
h ee main sou ces o ionizing adia ion o ood p ocessing, a e gamma
ays p oduced om cobal (60Co) (1.17 and 1.33 MeV) o Cesium (137Cs)
(0.662 MeV), gene a o s o elec on beam (max. ene gy 10 MeV) and X
ays (max. ene gy 5MeV) (Codex Alimen a ius Commission, 1984). Apa
om hei di e ing o igins hese i adia ion sou ces ha e di e en ene gy
and pene a ion dep hs. In ood p ocessing he use ulness o elec on beam
i adia ion is limi ed by i s low pene a ion dep h while X ays a e limi ed by
hei low ene ge ic e iciency. By con as , lambda ays (ϒ) ha e an
ele a ed ene gy and pene a ion dep h, which makes hem mo e sui ed o
p ocessing la ge ood masses. Howe e , he use o Y ays is limi ed
because i s main sou ces a e adioiso opes.

Chap e 1
18
Nowadays he main applica ions o his echnology in he ood indus y
a e ocused on he inhibi ion o ege able ge mina ion; disin ec ion and
disinsec ion in ce eals, ui s, mea s and ish; e a da ion o ui and
ege able ipeness; shel -li e ex ension o aw mea and ish; pa hogenic
mic obial inac i a ion; and s e ilisa ion o spices and meals o
immunocomp omissed people and also o space a el (C aw o d and Ru ,
1996; Odueke
e al
., 2016).
In ood ioniza ion he main pa ame e o ake in o accoun is he o al
dose abso ed by he p oduc . The i adia ion dose is he o al amoun o
ene gy abso bed by he i adia ed p oduc . The dose applied is measu ed in
G ay (Gy) o kiloG ay (kGy). In e ms o ene gy 1 G ay equals o 1 Joule
abso bed pe kilog am o i adia ed p oduc . F om a p ac ical poin o iew
h ee di e en anges o doses exis o ood p ocessing: low, medium and
high dose (Fa kas, 2006; Molins, 2001; Odueke
e al
., 2016):
 Radu iza ion o low-dose ea men s in ol e doses below 1kGy.
These ea men s a e used o ge mina ion, inhibi ion, delay o
ipeness, disin es a ion and pa asi es des uc ion.
 Radicida ion o medium dose ea men s in ol e doses be ween 1
and 10kGy. These ea men s a e use ul o he inac i a ion o
spoilage and pa hogenic ege a i e cells bu no o bac e ial spo es.
 Rappe iza ion o high dose ea men s in ol e doses o e 10kGy.
These ea men s a e ocused on he des uc ion o mic oo ganisms
up o le els o s e iliza ion.
The e ec s o ionizing adia ions on biological ma e ial a e he sum o
i s di ec and indi ec e ec s (Figu e 1.2). The di ec e ec is a
consequence o he physical changes p oduced on he molecules as a esul
o hei abso p ion o ene gy. The ene gy abso p ion is p opo ional o he
molecula weigh which makes DNA one o he main a ge molecules due
i s la ge size and complexi y. Fo his eason he i adia ion le hal dose
Chap e 1
19
a ies among di e en o ganisms depending on he complexi y o hei DNA
( i uses (10-200kGy) Bac e ial spo es (>10-50kGy), Vege a i e cells (>0.5-
10kGy), Insec s and pa asi es (>0.01-0.5kGy) and Mammals (>0.005kGy)).
The indi ec e ec is ela ed o he o ma ion o eac i e oxygen species
(ROS) due o he di ec ac ion o adia ion on cell componen s o he media
su ounding, wa e o ins ance (Fa kas, 2006; Lomax
e al
., 2002;
Su he land
e al
., 2000; Yoko a
e al
., 2008).
Figu e 1.2: Scheme o he di ec and indi ec e ec s o i adia ion on DNA
Despi e he g ea po en ial o i adia ion o ood decon amina ion his
p ocess may also p oduce some undesi able changes on oods u s by bo h
di ec and indi ec mechanisms; being wa e , p o eins, o he ni ogen
compounds and a oma ic compounds some o he mos a ec ed by
i adia ion ea men s. The e ec s induced depend among o he hings on
he i adia ion dose, ea men empe a u e, oxygen a ailabili y and
mois u e. Some s a egies o educe he impac o hese ad e se e ec s on
oods u s a e he educ ion o empe a u e ( eezing empe a u es),
educ ion o oxygen a ailabili y, addi ion o ee adicals sca enge s and
dose educ ion (Jay
e al
., 2005).
Chap e 1
20
Cu en ly, a numbe o o ganisa ions wo ldwide ha e accep ed his
echnology as a sa e al e na i e echnology o ood decon amina ion
(WHO, FDA). The Wo ld Heal h O ganiza ion has es ablished 10kGy as he
maximum dose o ood p ocessing wi hou any ad e se e ec on ood
ma ixes (WHO, 1981). Though, a la e s udy concluded ha no limi ing
dose is equi ed (WHO, 1999). Ei he way, nowadays mo e han 60
coun ies wo ldwide ha e egula ions ega ding he use o ionizing adia ion
o ood p oduc s (IAEA, 2017). In ac , he join FAO/IAEA (In e na ional
A omic Ene gy Agency) Di ision o Nuclea Techniques in Food and
Ag icul u e es ima es ha app oxima ely 700,000 onnes o ood we e
i adia ed in 2013 (IAEA, 2015). In he Eu opean Union (EU), only a o al o
5,690.1 onnes o ood was i adia ed in 2015, wi h Belgium being he mos
ac i e coun y wi h a o al o 3,916.9 onnes o ood i adia ed in he same
yea (EU, 2016). Only d ied a oma ic he bs, spices and ege able seasoning
a e au ho ised in he EU o be ea ed wi h ionizing adia ion up o a
maximum dose o 10kGy (EU, 1999). Howe e , some coun ies wi hin he
EU ha e an ex ended lis o ood au ho ized o be ea ed wi h his
echnology. Table 1.2 shows he di e en oods and he maximum doses
au ho ised in hese EU coun ies.
Chap e 1
21
Table 1.2: Lis o au ho ized ood p oduc s and maximum i adia ion dose in di e en EU
coun ies (EU, 2009).
BE, Belgium; CZ, Czech Republic; FR, F ance; IT, I aly; NL, Ne he lands; PL, Poland; UK,
Uni ed Kingdom.
1.3.2
Ul asound (US)
Ul asound echnology consis s o sonic wa es wi h equencies abo e
he h eshold o human hea ing (16-18 kHz). These ul asonic wa es a e
de ined and cha ac e ized by hei equency and hei wa eleng h. Wa es
wi h equencies be ween 20 and 40 kHz a e de ined as high-ene gy o
Chap e 1
22
high-powe ul asound, whe eas hose whose equency anges be ween 40
kHz and 1 MHz a e known as low-powe ul asound (Mason
e al
., 1996).
These wo ypes o ul asonic wa es ha e e y di e en applica ions. High-
powe ul asound is used in he ood indus y o di e en pu poses and is
gene ally applied in p ocesses which in ol e hea ans e , mass ans e o
bo h. On he o he side low-powe ul asound is used o quali y analysis
and p ocess con ol pu poses (e.g. senso s). Ul asonic powe is de ined as
he ene gy ansmi ed by he wa e pe second (W), ul asonic in ensi y as
he powe pe su ace uni (W/cm2) and ul asonic powe densi y as ene gy
pe olume o ea ed p oduc (W/L) (Meullemies e
e al
., 2017).
When an ul asonic wa e is p opaga ed h ough a liquid media, i
c ea es al e na ing comp ession and expansion cycles. When he expansion
cycle has he capaci y o exceed in e molecula o ces and o educe he
absolu e p essu e below apou p essu e, small bubbles a e o med. O e
he cou se o subsequen expansion/comp ession cycles, he bubbles
expand and con ac . A ec i ied di usion p ocess leads o he g ow h o
bubbles un il hey each esonance size. Then, o e he cou se o he nex
acous ic cycle (which co esponds o one oscila ion in Figu e 1.3), he
bubbles g ow o a maximum size and hen collapse. As a consequence o
ha implosion, molecules iolen ly collide wi h each o he , gi ing ise o
shock wa es and c ea ing spo s o e y high empe a u e and p essu e
(Mason
e al
., 1996). These ex eme condi ions usually induce wa e
sonolysis, esul ing in he eme gence o highly eac i e adicals. This
phenomenon is known as ansien ca i a ion, when gene a ed by high
powe ul asound. Ca i a ion is conside ed he main mechanism by which
his o m o ul asound enhances hea and mass ans e phenomena (Kim
e al
., 2004; Zhou
e al
., 2002), hough o he e ec s such us acous ic
s eaming, which is a physical o ce ha p oduces an addi ional mass low
(Solo chuk
e al
., 2011) a e also in ol ed (Legay
e al
., 2011). A scheme o
he ca i a ion p ocess is shown in Figu e 1.3.

Chap e 1
23
Figu e 1.3: Scheme o he e olu ion o ca i a ion bubbles du ing he applica ion o powe
ul asound. Sou ce (Adap ed om Abbas
e al
., 2013).
The e ec s o ul asound on hea ans e has been ex ensi ely s udied
since he 1990s in model sys ems such as wa e , me al ubes, me al balls,
e c. (Huamao
e al
., 1997; Hyun
e al
., 2005; Oh
e al
., 2002; Zhou
e al
.,
2002). Fu he mo e i s abili y o enhance hea ans e in oods, mainly in
p ocessed mea p oduc s, du ing cooking p ocesses (Ala con-Rojo
e al
.,
2015; Reynolds
e al
., 1978; Vimini
e al
., 1983) has been in es iga ed. Also
he heo e ical ad an ages o ul asonic ca i a ion ha e been assessed in
di e en p ocesses ela ed wi h he ood indus y, wi h cooking, ma ina ing
and ex ac ion e alua ed ex ensi ely (Ala con-Rojo
e al
., 2015; Cá cel
e
al
., 2007; Khan
e al
., 2010; Luengo
e al
., 2014; Ma
e al
., 2009; Mason
e
al
., 1996; McDonnell
e al
., 2014; Tu han
e al
., 2013; Vimini
e al
., 1983)
and he use ulness o he applica ion o ul asound o enhance eezing,
d ying, degassing, il a ion, demoulding, de oaming, emulsi ica ion,
oxida ion and cu ing ha e also been explo ed (Ashokkuma , 2015;
Chand apala
e al
., 2012; Chema
e al
., 2011).
Besides he applica ions ela ed o hea and mass ans e p ocesses,
US echnology is one o he new mic obial inac i a ion echnologies ha has
been sugges ed as an al e na i e o con en ional hea ea men s (US FDA,
Chap e 1
24
2000). As men ioned be o e, oday mos au ho s (Condón
e al
., 2011; Lee
e al
., 2009; Wu
e al
., 2015) ag ee ha high powe ul asound inac i a es
cells ia en elope b eakdown as a consequence o shock wa es p oduced
due o ca i a ion phenomena. As a gene al ule he US esis ance o
bac e ial cells dec eases wi h size and is g ea e in coccoid-shaped bac e ia
(Allige , 1975; Condón
e al
., 2005). Fu he mo e, G am-posi i e bac e ia
a e usually mo e esis an han G am-nega i e bac e ia; wi h yeas and
moulds p esen ing in e media e esis ance (Condón
e al
., 2011; Jamb ak
e
al
., 2017; López Malo
e al
., 2005), while bac e ial spo es a e almos no
a ec ed by US ea men s (Jamb ak
e al
., 2017; Sanz
e al
., 1985). Mos
published da a indica e ha he bac e icidal e icacy o ul asound is low
(Jamb ak
e al
., 2017; Lee
e al
., 2013; Meullemies e
e al
., 2017).
The e o e, mos esea che s ha e ied o imp o e he e icacy o he
p ocess by designing combined p ocesses o enhance he o e all le hal
e icacy o ul asound (Lee
e al
., 2013; López-Malo
e al
., 2005; Raso
e
al
., 1998a).
Some o he combined US p ocesses p oposed o da e o inc ease he
le hal e ec o ul asound ha e been classi ied (Chema
e al
., 2011;
Piyasena
e al
., 2003; Sala
e al
., 1995) as he mosonica ion (TS,
combina ion o ul asound and hea ), manosonica ion (MS, combina ion o
ul asound and p essu e), and mano he mosonica ion (MTS, combina ion o
ul asound, p essu e and hea ). The inc emen o he le hal e ec o
ul asound by aising he empe a u e o he ea men media (TS) has
been obse ed on bac e ial ege a i e cells (Lee
e al
., 2009), yeas (Abid
e al
., 2014; Be mudes-Agui e and Ba bosa-Cano as, 2012) and on
bac e ial spo es (Ga cia
e al
., 1989; Milly
e al
., 2007). I has been
sugges ed ha highe empe a u es sensi ize cell en elopes o shock wa es
o igina ed by ca i a ion (Lee
e al
., 2009; Wu
e al
., 2015). In some cases
he le hal e ec o TS inc eases un il eaching a maximum a a ce ain
empe a u e ollowing which he ul asound e ec dec eases. This
Chap e 1
25
dec easing beha iou has been associa ed wi h an inc ease o he apou
p essu e o he medium (Kno
e al
., 2004; Sala
e al
., 1995), which should
acili a e ca i a ion, bu while also educing he in ensi y o ca i a ion
implosion, as he apou wi hin he bubbles ac s as a cushion (Allige ,
1975). When boiling empe a u es a e eached, ca i a ion ceases.
On he o he side, he inac i a ion a e o ege a i e cells due o MS
inc eases when s a ic p essu e is aised (Lee
e al
., 2009, 2013; Mañas
e
al
., 2000). An inc ease in hyd os a ic p essu e makes he o ma ion and
g ow h o bubbles mo e di icul and equi es highe ene gy consump ion
o hei p oduc ion, bu he esul an ca i ies elease mo e ene gy when
hey implode, he eby inc easing he le hal e ec . When p essu e is oo
high in compa ison wi h ul asonic powe , ca i a ion o ma ion is inhibi ed
and ansien ca i a ion ceases. This explains ha he e ec o p essu e
inc eases he bac e icidal e ec i eness o US un il eaching a h eshold,
abo e which he e ec o p essu e p og essi ely dec eases un il i anishes
(Guzel
e al
., 2014; Pagán
e al
., 1999b; Raso
e al
., 1998a).
MTS p ocess was designed in 1992 (Spanish Pa en No. 9200686) wi h
he aim o o e coming he limi a ions o TS and MS, while making use o
hei ad an ages. The au ho s hypo hesized ha p essu e would inc ease
ca i a ion in ensi y and compensa e o changes in he apou p essu e o
he medium as a esul o hea ing, he eby pe mi ing an ad an ageous use
o he mosonica ion e en a high empe a u es (Sala
e al
., 1995). Da a
published o e he las 20 yea s con i ms he alidi y o his hypo hesis o
inac i a e ege a i e mic obial cells, spo es and also enzymes.
1.4 PROCESS OPTIMIZATION
The p e ious sec ion ou lined in mo e de ail he main echnologies ha
we e explo ed in he p esen PhD Thesis. The cu en sec ion p o ides an
o e iew abou he a ea o p ocess op imisa ion and hei usage.
Op imiza ion is de ined as he ac ion o making he bes o mos e ec i e
Chap e 1
26
use o a si ua ion o esou ce (Ox o d dic iona y, 2018). F om he
pe spec i e o ood p ocessing, p ocess op imiza ion can be de ined as he
selec ion o he p ocess pa ame e s which will achie e a sa e/s able
p oduc , while main aining he bes possible nu i ional and o ganolep ic
cha ac e is ics and maximizing he p o i .
The o igins o ood p ocess op imiza ion can be a ibu ed o he
disco e ies o Bigelow, Eas y and Maye in he 1920s, in he labo a o ies o
he Na ional Canne s associa ion in U.S. These esea che s we e
in es iga ing mic obial des uc ion kine ics by hea . The mode n concep o
op imiza ion, which combines mic obial inac i a ion and ood quali y, was
no de eloped un il 1940s when Esselen p o ed he loga i hmic des uc ion
o i amins subjec ed o hea , disco e ing he di e ences on he he mal
sensi i i ies be ween mic oo ganisms and i amins. Nowadays he ole o
p ocess op imiza ion is o minimize p oduc de e io a ion, maximize p o i s
while, a he same ime, ensu e he hygienic and sa e y equi emen s
imposed by na ional o in e na ional egula ions. Nowadays, as a gene al
ule in he ood indus y p ocess op imiza ion is dic a ed by ood sa e y
equi emen s (Vald amidis
e al
., 2012) wi h quali y ha ing a seconda y
ole.
Food p ocess op imiza ion equi es an app op ia e knowledge o he
p ocess and he a iables in ol ed on i . Figu e 1.4 shows he main
in o ma ion and s eps equi ed o pe o m ood p ocess op imiza ion.
Howe e , ob aining all equi ed in o ma ion is a slow and cos ly p ocedu e
which mos o he imes makes i a big challenge. A easonable al e na i e
is o p edic , based on ma hema ical models he impac o he p ocess
a iables on he cha ac e is ics o he p oduc and op imize he p ocess
based on hese p edic ions (Ling
e al
., 2015; S o o os, 1995).
Chap e 1
33
Figu e 1.5: Example o a alida ion g aph. Dashed line ep esen s he bisec ix which
co espond o a pe ec i .
The dashed line in Figu e 1.5 ep esen s he pe ec i . The poin s
u hes om i ep esen he leas accu a e p edic ions. F om Figu e 1.5 i
is also possible o de ine i he model o e es ima es o unde es ima es i s
p edic ions. I he poin s a e loca ed abo e he line, he model would
o e es ima e on i s p edic ions while i he poin s a e loca ed in he a ea
unde he line he model would unde es ima e on i s p edic ions. This
p ocedu e is commonly done in conjunc ion wi h he analysis o esidual
e o s which indica es he di e ences be ween p edic ed and eal alues.
Al hough he g aphical e alua ion is simple and use ul, a ma hema ical
analysis is also necessa y in o de o e alua e he obus ness and goodness
o he model i . The mos common used pa ame e s a e:
a)
Coe icien o de e mina ion (R2).
This pa ame e p o ides a measu e
o how well eal alues a e ep esen ed by he model, based on he
p opo ion o o al a ia ion o ou comes explained by he model. I is
calcula ed as shown in Equa ion 1.1.
0 2 4 6 8 10
0
2
4
6
8
10
Real alues
P edic ed alues

Chap e 1
34
𝑅2=( ∑ (𝑋𝑖−𝑋)(𝑌𝑖−𝑌)
𝑛
𝑖=1
√∑ (𝑋𝑖−𝑋)2
𝑛
𝑖=1 ∑(𝑌𝑖−𝑌)2
𝑛
𝑖=1 )2
(Eq. 1.1)
Whe e Xi ep esen s eal alues alues, 𝑋 he a e age o he eal alues, Yi
ep esen s p edic ed alues, 𝑌 he a e age o p edic ed alues and n is he
o al numbe o obse a ions.
R2
alues close o 1 indica e a be e
co ela ion be ween p edic ed and obse ed alues.
b)
Roo mean squa e e o (RMSE).
This pa ame e quan i ies he
quad a ic e o o he di e ences be ween p edic ed and eal alues. I is
calcula ed as shown Equa ion 1.2.
𝑅𝑀𝑆𝐸=√∑(𝑋𝑖−𝑋0)2
𝑛 (Eq. 1.2)
Whe e Xi ep esen s p edic ed alues, X0 obse ed alues and n is he
o al numbe o obse a ions. Small alues o his pa ame e in ol e highe
ce ain y in he models p edic ion. Howe e , models wi h high
RMSE
alues
s ill can p edic accu a ely as his pa ame e depends on he o de o
magni ude o he alues p edic ed and obse ed.
c)
S anda d e o o p edic ion (SEP).
This pa ame e is simila o
RMSE,
bu quan i ies he e o in ela i e e ms, so i s alue is no
dependen on he magni ude o he da a. I is calcula ed as shown Equa ion
1.3.
𝑆𝐸𝑃=100
𝑋0√∑(𝑋𝑖−𝑋0)2
𝑛 (Eq. 1.3)
Chap e 1
35
Whe e 𝑋0 ep esen s he a e age o he obse ed alues and he o he s
a e de ined in
RMSE
.
d)
Bias ac o (B ) and Accu acy ac o (A ).
These pa ame e s we e
de ined o assess he goodness o i o he p edic i e models in a apid way
and p o ide indica o s o he eliabili y o he models (Ross, 1996). These
pa ame e s a e calcula ed ollowing Equa ions 1.4 and 1.5 espec i ely.
𝐵𝑓=10(∑log(𝑋 𝑝𝑟𝑒𝑑. 𝑋 𝑜𝑏𝑠.
⁄)
𝑛) (Eq. 1.4)
𝐴𝑓=10√(∑(log(𝑋 𝑝𝑟𝑒𝑑. 𝑋 𝑜𝑏𝑠.
⁄))2
𝑛) (Eq. 1.5)
Whe e Xp ed. ep esen s he p edic ed alues, Xobs. ep esen s he
obse ed alues and n is he numbe o obse a ions.
The
B
indica es i he model o e es ima es o unde es ima es in i s
p edic ions. Values o
B
<1 indica es ha obse ed alues a e smalle han
he p edic ed and alues o
B
>1 indica es ha he obse ed alues a e
bigge han he p edic ed. In he case o he pe ec i o he da a,
B
is
equal o 1. Big alues o
A
indica es less accu acy in he p edic ions while,
when he co ela ion be ween obse ed and p edic ed alues is pe ec
A
becomes equal o 1. Ideally he alue o
A
should be 1 bu gene ally
inc eases be ween 0.1 and 0.15 o each a iable, included in he model,
a e accep ed (i.e.
A
alues be ween 1.2 and 1.3 a e accep able o a model
which conside s wo o mo e a iables) (Ross
e al
., 2000).
To da e one o he main examples o he applica ion o modelling o he
p ocessing o he ood indus y is he design and op imiza ion o hea
p ocesses.
Chap e 1
36
1.4.2
Hea p ocess op imiza ion
The Ame ican canning indus y began some ime a e 1810 and is
conside ed o be he o igin o mode n ood echnology. A g ea e
unde s anding o he canning p ocess was de eloped due o he indings o
Louis Pas eu in 1865 and u he ad ances in knowledge o mic obial
inac i a ion and i amin deg ada ion by hea du ing 1920s and 1940s. As
men ioned p e iously, he p ima y objec i e o ood p ocessing is he
p oduc ion o sa e p oduc s which will no pose a po en ial haza d o public
heal h. The e o e, he i s s ep in he op imisa ion o a p ocess is o
e alua e and cha ac e ise he e ec o ha p ocess on he main a ge
mic oo ganisms p esen in he p oduc o be ea ed (S o o s
e al
., 1995).
Fo m he expe iences o Bigelow and Es y in 1920 i was concluded ha
mic obial inac i a ion by hea ollows a i s -o de kine ic a a cons an
empe a u e. Al hough, he echniques ha e e ol ed du ing he yea s and
o he inac i a ion models ha e been epo ed o desc ibe mic obial
inac i a ion by hea (Ceb ián
e al
., 2017), s ill nowadays he indings o
Bigelow and Es y a e being used in he ood indus y o he design o
he mal p ocesses. Due o he exponen ial educ ion o mic obial popula ion
by hea , when he loga i hm o mic obial coun is ep esen ed
e sus
ea men ime a s aigh line is obse ed as shown in Figu e 1.6.
This new knowledge ga e ise o he concep o he
D
T alue, which
can be de ined as he ime necessa y, a a cons an empe a u e, o educe
he mic obial coun by one Log10 cycle (i.e. o educe he o al mic obial
popula ion by 90% o by a ac o o 10). This pa ame e can be calcula ed
as he nega i e in e se o he slope o he line which desc ibes he
mic obial inac i a ion exp essed as Log o mic obial numbe s
s
ea men
ime. This concep could be conside ed as he p ima y model used o de ine
he mal p ocesses.
Chap e 1
37
Figu e 1.6: E ec o ea men ime on he mic obial coun subjec ed o a hea ea men
a a cons an empe a u e. Sou ce (Ceb ián
e al
., 2017).
In addi ion Bigelow (1921) demons a ed he exponen ial e ec o he
ea men empe a u e on he
D
T alue, which ga e ise o he concep o
he
z
alue. This concep is de ined as he equi ed empe a u e ise (°C) o
b ing abou a 1 log change in he
D
T alue (i.e. educe o inc ease he
D
T
by 90% o by a ac o o 10). Simila o he calcula ion o he
D
T,
z
alues
can be calcula ed g aphically as he in e se o he slope o he line
co ela ing he Log10 o
D
T alues and he ea men empe a u e (Figu e
1.7).
Figu e 1.7: E ec o ea men empe a u e on he mic obial inac i a ion a e subjec ed o
a hea ea men . Sou ce (Ceb ián
e al
., 2017).
The concep o he
z
alue can be conside ed as a seconda y model
used o de ine a he mal p ocess. Bu , con a y o he obse a ions wi h
D
T
alues, he concep o he
z
alue has been p o en o be a mo e obus and
use ul model o co ela e he ea men empe a u e wi h he pa ame e s o
o he p ima y models used o desc ibe he hea inac i a ion cu es,
examples including he Weibull dis ibu ion (Fe nández
e al
., 2007; Hassani
e al
., 2005) o he Gee ae d Equa ion (A oyo
e al
., 2011).
Time (min) Time (min)
Mic obial coun
Log mic obial coun
D
Chap e 1
38
D
T and
z
a e co ela ed by Equa ion 1.6 (This Equa ion could be
conside ed as a e ia y model used o calcula e he impac o a he mal
p ocess in e ms o mic obial inac i a ion), which allows he de ini ion o he
D
T
alues a any ea men empe a u e (T) by knowing a e e ence
D
T o
he a ge mic oo ganism and also i s
z
alue. These concep s o m he
basis o classical he mobac e iology and o ms basis o hea p ocess
op imiza ion.
𝐷𝑇=𝐷𝑇𝑟𝑒𝑓10(𝑇𝑟𝑒𝑓−𝑇)𝑧
⁄ (Eq. 1.6)
Simila o wha has been desc ibed o mic obial inac i a ion, he
kine ics o hea induced chemical eac ions in oods can be s udied and
modelled. Howe e , he key eac ion which de ines he quali y o he
p oduc a ies om p oduc o p oduc and om consume o consume .
Fo example wi h cooked bee , ende ness is gene ally he key ac o
a ec ing consume accep ance bu consume s’ a i ude a y in e ms o he
choice o well-done o a e s eak. Fo milk, colou and nu i ional alue
may be key ac o s while in juices he la ou and enzyme inac i a ion may
be key eac ions. Addi ionally, a gene al model o desc ibe hese
deg ada ion kine ics (i.e. p ima y model) does no exis and di e en o de
o eac ions has been obse ed (i.e. ze o-o de , i s -o de , pseudo- i s -
o de , second-o de kine ics) (Ling
e al
., 2015).
Despi e he di e ences in p ima y models be ween mic obial
inac i a ion and quali y deg ada ion, p ocess op imiza ion is possible
because he e ec o ea men empe a u e emains cons an (i.e. p ima y
model pa ame e s a y exponen ially due o empe a u e changes). So an
equi alen o
z
alue, known as Q10 alue, can be de ined. Concep ually,
his pa ame e ela es o quali y changes and has he same meaning as
z
alue o mic obial inac i a ion; hough Q10 alues o chemical eac ions
a e gene ally highe han hose epo ed o
z
, (e.g. 10ºC o mic obial
inac i a ion
s
30ºC o chemical eac ions) which is he basis o he mal

Chap e 1
39
p ocess op imiza ion. This di e ence in
z
alues be ween chemical and
mic obial inac i a ion is exploi ed in UHT p ocessing whe e a 30°C inc ease
in p ocessing empe a u e may p oduce a 1000 old inc ease in he a e o
mic obial inac i a ion bu only a 10 old change in he a e o chemical
eac ions.
The de elopmen o kine ic models o bo h, mic obial inac i a ion and
quali y deg ada ion a e wo o he main componen s in he op imiza ion o
hea p ocesses. Howe e , he applicabili y o hese e ia y models is limi ed
o ea men s pe o med a cons an empe a u es, while in mos hea
p ocesses wi hin he ood indus y he p oduc empe a u e is no cons an
bu a ies h oughou he p ocess (Holdswo h and Simpson, 2007).
The e o e, he las equi ed in o ma ion, in he op imiza ion o a hea
p ocess is he cha ac e iza ion o he ime empe a u e p o ile o he
p oduc in o de o in eg a e he combined e ec o all empe a u es
a ained du ing he ea men ime.
The ime empe a u e p o ile o a oods u du ing hea p ocessing
consis s o , in i s simples o m, a hea ing, holding and cooling cycle and
can be de e mined expe imen ally, by placing he mocouples wi hin a
p oduc , o can be es ima ed ma hema ically by p edic i e models
(S o o os, 1995). The ad an age o he expe imen al p ocedu e is ha he
esul s ob ained will be mo e obus , o subsequen calcula ions, bu in
con as , he applicabili y o he da a is solely ocused on he speci ic
condi ions used o ob ain he da a. Addi ionally, da a collec ion is a cos ly
and ime consuming p ocedu e. On he o he side he use o ma hema ical
models o p edic he ime empe a u e p o ile du ing p ocessing has he
ad an age ha i allows he de ini ion and e alua ion o di e en p ocess
a iables ( he numbe and na u e o hose a iables is dependen on he
ype o model used) bu also he ela i ely educed amoun o expe imen al
da a equi ed o gene a e he model. By con as , he conclusions ex ac ed
ha e highe unce ain y.
Chap e 1
40
Once all he equi ed in o ma ion has been ob ained he nex s ep in
hea p ocess op imiza ion is o calcula e he p ocess condi ions in e ms o
ime and empe a u e o achie e a ce ain limi o mic obial inac i a ion and
quali y cha ac e is ics (Figu e 1.4). To do his, wo equi alen pa ame e s
a e gene ally desc ibed:
F
T
z
o mic obial inac i a ion and
C
T
z
o quali y
changes (Holdswo h and Simpson, 2007). Concep ually,
F
and
C
alues a e
simila han
D
T bu , hese wo alues ep esen he equi alen ea men
ime a a e e ence empe a u e o each a ce ain le el o mic obial
inac i a ion o quali y le el espec i ely. Resul s calcula ed o a p ocess, in
e ms o
C
alue, a e gene ally compa ed o a a ge
F
alue which
ep esen s he ime equi ed o achie e a a ge Log10 educ ion o he mos
pe inen pa hogen in he p oduc . Al hough, he
C
T
z
alue is also c i ical in
e ms o p oduc quali y and nu i ional alue i is less equen ly
conside ed, as he main ocus is p oduc sa e y. These wo alues ep esen
he whole he mal e ec acc ued du ing he hea p ocess.
Figu e 1.8 shows as example he heo e ical g aph o he op imiza ion
o a he mal p ocess ela ed o mic obial inac i a ion (i.e.
F
T
z
) and wo
di e en quali y pa ame e s (i.e.
C
T
z
alues). In he g aph, he black line
ep esen s he minimum equi alen ime- empe a u e combina ions
necessa y in o de o achie e an adequa e le el o mic obial inac i a ion.
The blue line ep esen s he minimum equi alen ime- empe a u e
combina ions equi ed in o de o achie e a minimum quali y a ge and he
ed line ep esen s he maximum ole able equi alen ime- empe a u e
combina ions in o de o keep a speci ic quali y a ibu e unde a de ined
limi . Based on Figu e 1.8, he op imum condi ions in his example a e
hose which accomplish all p oduc equi emen s, ep esen ed by he g een
a ea. In he example, only one mic obiological and wo quali y a iables
ha e been conside ed, bu in his ype o op imiza ion g aph i is possible o
include e en mo e lines ep esen ing o he a iables such as enzyma ic
inac i a ion o e en he economic cos o he p ocess.
Chap e 1
41
Figu e 1.8: Theo e ical lines o he minimum mic obial inac i a ion equi ed (black line),
he maximum quali y deg ada ion accep ed ( ed line) and he minimum quali y equi ed
(blue line) depending on he ea men empe a u e. The g een a ea ep esen s he ime-
empe a u e combina ions which accomplish all p oduc equi emen s (sa e y and quali y in
his example).
1.5 EDIBLE CRAB-BROWN CRAB (
CANCER PAGURUS
)
The de elopmen o he p esen PhD hesis is pa o a la ge p ojec
ocused on I ish b own c ab (
Cance pagu us
). The e o e, he ollowing
sec ion desc ibes he pa icula cha ac e is ics o his oods u and i s
p oduc ion p ocess.
Cance pagu us
, commonly known as edible c ab o b own c ab, is a
species o c ab ound in he No h Sea, No h A lan ic Ocean and in
he Medi e anean Sea. I is a obus c ab o a eddish-b own colou ,
ha ing an o al ca apace wi h a cha ac e is ic "pie c us " edge and black ips
o he claws (Figu e 1.9).
C. pagu us
is a noc u nal p eda o , a ge ing a
ange o molluscs and c us aceans. The maximum age o b own c ab is
a ound 20 yea s and he maximum sizes egis e ed o he ca apace wid h
a e 265mm o males and 225mm o emales and he maximum egis e ed
weigh is 3kg. Fo ishing he minimum size accep ed, o his c ab, is 130-
140mm depending on he egion i is cap u ed and measu ed pe pendicula
o he an e opos e io midline o he ca apace (Council egula ion (EC) No.
T ea men ime
T ea men empe a u e
Mic obial inac i a ion
equi emen s
Limi o quali y e en ion
(i.e. loss o nu i ional alue)
Minimum quali y equi ed
(i.e. cook alue)
Chap e 1
42
850/98, as ammended). This co esponds o a weigh o abou 350 g ams,
and i akes abou 5 o 6 yea s o c ab la ae o each his size.
Figu e 1.9: Female exempla o edible c ab (
Cance pagu us
)
Like all decapods an edible c ab has en appendages a ached o i s
ho ax, he i s being a pai o claws. In case o edible c ab bo h
appendages and ca apace con ain edible issues wi h comme cial in e es .
Muscle mea o whi e mea , is mainly loca ed in claws and legs bu also
some o his mea ype is loca ed in he ho ax. B own mea is ob ained
exclusi ely om he ca apace o he emale exempla s and mainly consis s
o hepa opanc eas and gonads. The composi ion o each mea ype a ies
be ween c abs bu also depends on he ma u i y s age, season and ishing
a ea. In case o whi e mea he o al a con en anges om 0.2 o 0.6%
while he p o ein con en a ies be ween 16.4 and 20.5%. On he o he
side, in b own mea he o al a a ied be ween 3.1-3.6% and p o ein
con en om 19.2-25.5%; while o hepa opanc eas he a con en anges
be ween 10.0 and 16.6% and p o ein con en anges om 9.4 o 15.8%
(Ba en o
e al
., 2009b, 2010a).
Chap e 1
49
a e emo ed. Du ing cooking a se ies o compounds a e eleased om he
c ab, mainly p o eins. These eleased compounds a ach o he ca apace
du ing cooking, as shown in Figu e 1.14, which makes i essen ial o wash
he c abs p io packaging. Fo his eason he i s pa o cooling is ca ied
ou using wa e je s, o cleaning and inalised in a cold oom. The en i e
p ocess has a du a ion o app oxima ely 3-4 hou s. On he o he side,
cooling is e y impo an because allows he comple ion o mea
coagula ion, which acili a es i s emo al om he ca apace and a oids
losses (Edwa ds, 2001).
Figu e 1.14: Example o c abs du ing cooking (le ) and immedia ely a e cooking wi h
no washing ( igh ).
Immedia ely a e cooling c abs, which will be sold as eady- o-ea
cooked whole a e packaged o sale (p emium p oduc s) o ozen. Mos
commonly, a second hea ea men is applied, o ensu e he
mic obiological sa e y o he p oduc . C abs which a e des ined o
manu ac u e o c ab based p oduc s o packaged c ab mea a e ans e ed
o a mea picking a ea. A his s age, whi e mea , om he body claws and
legs and b own mea , om he body o emale c abs, is emo ed. The main
incon enience o his p ocess is ha he main p ocedu e o emo e he
mea is hand picking, al hough comp essed ai je s a e some imes used o
assis emo al o mea om he legs (Edwa ds, 2010). Ei he way his s ep
akes up a signi ican po ion o p ocessing ime. Finally, when he a ious

Chap e 1
50
ypes o mea ha e been emo ed, hey a e packed oge he o sepa a ely,
depending on he in ended inal des ina ion and pas eu ised. In mo e
elabo a ed p oduc s, which equi e a g ea e le el o p ocessing (e.g. pa e,
chowde , bouillion, e c.) a second pas eu iza ion is manda o y due o he
high isk o con amina ion/ econ amina ion (Adams, 2000; Dima
e al
.,
2015; Ghazala and T enholm, 1996).
Due o he na u e o c ab edible issues (high wa e ac i i y, mode a e
salini y and mode a e pH) i p o ides an excellen en i onmen o he
de elopmen o a ious pa hogens, which can be associa ed wi h oodbo ne
diseases due o imp ope ly p ocessed/p ese ed/s o ed low-acid chilled
oods. Acco ding o he U.S. Depa men o Heal h and Human Se ices o
he Food and D ug Adminis a ion (FDA, 2011) he mos impo an
oodbo ne non-spo e- o ming pa hogens, associa ed wi h hese kinds o
p oduc s a e
Lis e ia monocy ogenes, Vib io ulni icus, Vib io
pa ahaemoly icus, Vib io chole ae, Esche ichia coli, Salmonella
spp.
,
Shigella
spp.
, S aphylococcus au eus, Campylobac e jejuni
and
Ye sinia
en e ocoli ica
(FDA, 2011; G am and Huss, 1996).
Fo all o hese ege a i e bac e ia,
Lis e ia
monocy ogenes
is
conside ed as he a ge mic oo ganisms in pas eu isa ion ea men s, since
i is gene ally conside ed o be he mos hea esis an ege a i e cell and is
able o mul iply a chill empe a u es (FDA, 2011).
L. monocy ogenes
may
be ansmi ed o humans ia con amina ed oods (B acke and Beucha ,
1990). I s de elopmen is dependen on in insic ac o s such as he
p oduc pH, wa e ac i i y and sal con en and ex insic ac o s such as
cooking/s o age empe a u es and gas a mosphe e and packaging ype
(FSAI, 2005).
L. monocy ogenes
is able o g ow in a ange o pH om 4.2
o 9.5 and a empe a u es om -1.5C o 45C and a aw o up o 0.9. I is
documen ed ha hey a e able o su i e in en i onmen s down o -18C
and pH alues as low as 4.3 (FSAI, 2005).
Chap e 1
51
Pas eu ized c abmea has been shown o be an adequa e suppo o
he g ow h o
L. monocy ogenes
a 1C and empe a u e ises up o 5C
d ama ically inc ease i s g ow h a e (González
e al
., 2013). Mul iple
po en ial easons o he p esence o
L. monocy ogenes
ha e been
iden i ied, insu icien he mal p ocessing, c oss con amina ion om aw
c ab, ac o y en i onmen o ood-handle s. Acco ding o Regula ion (EC)
No 2073/2005 eady- o-ea oods able o suppo he g ow h o
L.
monocy ogenes
, such as eady- o-ea edible c ab and de i ed p oduc s,
mus comply wi h he ollowing ood sa e y c i e ion: absence o he
pa hogen in 25 g o p oduc be o e he ood has le he immedia e con ol
o he ood business ope a o (FBO) who has p oduced i . Howe e , i he
FBO is able o demons a e, o he sa is ac ion o he compe en au ho i y,
ha he p oduc will no exceed he limi o 100 c u/g h oughou he shel -
li e, hen his la e c i e ion would apply (100 c u/g h oughou he shel -
li e).
Fo pas eu isa ion ea men s, achie ing a six loga i hmic educ ion o
he a ge mic oo ganism is accep ed as an adequa e le el o inac i a ion o
a oid oodbo ne disease (FDA, 2011). In he case o
L. monocy ogenes
, i s
p e alence in aw sea ood is es ima ed as 1 o 103 colony o ming uni s pe
g am (CFU/g) in up o 8% o hese p oduc s. Only 1% o hese p oduc s
a e con amina ed wi h le els g ea e han 103 CFU/g while le els g ea e
han 106 CFU/g a e no obse ed. Howe e , o he majo i y o sea ood
(91%), he con amina ion le el o
L. monocy ogenes
is gene ally lowe han
1 CFU/g (FDA, 2011). I his pa hogen is conside ed as he a ge
mic oo ganisms o pas eu iza ion he ecommended ea men is a
F
70°C7.5°C o wo minu es o an equi alen ea men , in o de o achie e a
p ope inac i a ion le el o
L. monocy ogenes
i.e. in excess o 6 Logs
(CCFRA, 1996; CFA, 2006; ECFF, 1996; FDA, 2011).
Addi ionally o he ege a i e cells enume a ed be o e, eady- o-ea
c ab based p oduc s and sea ood in gene al, ep esen a se ious haza d due
Chap e 1
52
o he p esence o pa hogenic spo e o ming bac e ia, namely:
Clos idium
pe ingens
,
Clos idium bo ulinum
and
Bacillus
spp. which a e he main
h ee spo e o ming bac e ia associa ed wi h sea ood p oduc s (FDA, 2011).
Among hese, g oup II (non-p o eoly ic)
Clos idium bo ulinum
ypes B, F
and specially ype E, a e o pa icula conce n o he sea ood indus y.
C.
bo ulinum
non-p o eoly ic ype E is known o be a na u ally occu ing
ma ine o ganism which can also g ow a e ige a ion empe a u es as low
a 2C (Co ne
e al
., 1989) and i has been also associa ed wi h human
bo ulism cases (Linds öm
e al
., 2006). In addi ion, i has been epo ed
ha he p e alence o g oup II (non-p o eoly ic)
C. bo ulinum
ype E in ish
and o he sea ood may be as high as 40-70% (Bake , 1990; Huss
e al
.,
1974; Johannsen, 1963). Howe e , despi e i s high p e alence in ish and
sea ood, i s coun s in aw oods a e ypically no e y high, a ying om
less han 1 spo e/kg (Bake , 1990; Linds öm
e al
., 2006; Tanasuga n,
2006) o 102 spo es/kg (Hielm
e al
., 1998; Linds öm
e al
., 2006). Fo all
hese easons
C. bo ulinum
non-p o eoly ic ype E is conside ed as a majo
conce n when designing a pas eu iza ion p ocess o eady- o-ea sea ood
based p oduc (FDA, 2011; Sil a and Gibbs, 2010). Gene ally as i is
conside ed o
L. monocy ogenes,
a pas eu iza ion p ocess which ensu es a
6 Log10 educ ion o he popula ion o spo es o
C. bo ulinum
non-
p o eoly ic ype E is conside ed adequa e o any o hese p oduc ypes
(FDA, 2011).
Conside ing he he mal esis ance o
C. bo ulinum
non-p o eoly ic ype
E he mos ecommended hea ea men o be applied is an
F
90°C7o 10°C o
en minu es, o equi alen conside ing
z
o 10°C o ea men
empe a u es ≥90°C o 7°C o ea men empe a u es <90°C (CFA, 2006;
ECFF, 2006; FDA, 2011). These ea men s a e ecommended o all eady-
o-ea e ige a ed p oduc s packed in he absence o oxygen, including ish
and ishe y p oduc s. Howe e , in case o c ab mea -based p oduc s, mo e
se e e hea ea men s ha e been p oposed. I is known ha he p esence
Chap e 1
53
o he enzyme lysozyme imp o es he eco e y o hea - ea ed
Clos idium
spp. spo es and he e o e, a highe hea esis ance is obse ed o his
species (Peck and Fe nandez, 1995; Sco and Be na d, 1982). Mo eo e , i
has also been epo ed ha his enzyme is p esen in ish and sea ood a
le el o up o 200 μg/g (Lie
e al
., 1989; Lund and No e mans, 1992). Fo
his eason, wo mo e se e e al e na i e ea men s we e p oposed by he
FDA o c ab mea
F
90ºC9ºC = 31 min (Blue c ab,
Callinec es sapidus
) and
F
90ºC8.6ºC = 57 min (Dungeness c ab,
Me aca cinus magis e
) o achie e a
sa is ac o y educ ion o
Clos idium bo ulinum
spo es in c ab mea (FDA,
2011; Pe e son
e al
., 1997, 2002).
Addi ionally o he mic obiological isks men ioned, c us acean
consump ion is also associa ed wi h o he heal h isks due o high
choles e ol le els in some edible pa s, alle gic eac ions and con amina ion
o oxic elemen s (Maul aul
e al
., 2012). Pa icula ly, in edible c ab high
le els o Cadmium (Cd) ha e been epo ed, especially in b own mea
(Ba en o
e al
., 2009b, 2009c; Bolam
e al
., 2016; Maul aul
e al
., 2012,
2013). Noël
e al
. (2011) epo ed Cd le els om 0.78 o 27.6 mg/kg o we
weigh (ww) and om 6.38 o 61.8 mg/kg (ww) in he b own mea o
common c abs (
Cance pagu us
), cap u ed in open o coas al a eas o he
A lan ic No h Eas espec i ely, landed in I eland and Uni ed Kingdom.
Also, Bolam
e al.
(2016) analysed Cd le els in he b own mea o
Cance
pagu us
, sampled h oughou he UK, and obse ed Cd concen a ions
be ween 0.11 and 26 mg/kg (ww) and Maul aul
e al.
(2012) epo ed Cd
concen a ions be ween 5.0 o 8.4 mg/kg (ww) in he b own mea o c abs
caugh in he Sco land coas s. Besides, Wiech
e al.
(2017) epo ed Cd
concen a ions om 5.7 o 12 mg/kg (ww) a e di e en p ocesses and
signi ican ly highe Cd le els o up o 44 mg/kg (ww) in c abs om
No he n No way.
In he EU, he Cd con en o c abs is only egula ed o he whi e mea
om c ab appendages (claws and legs); wi h 0.5 mg/kg being he
Chap e 1
54
maximum pe mi ed limi (EU, 2006, 2011). Howe e , se e al s udies ha e
wa ned o he isk o exceeding he o al weekly in ake o Cd se by EFSA
(2009) (2.5 μg/kg body weigh ) in consume s om coun ies whe e b own
mea is commonly consumed, such as in Spain and Po ugal (Bolam
e al
.,
2016; Maul aul
e al
., 2013; Noël
e al
., 2011; Wiech
e al
., 2017). The
main challenge in his ega d is ha he p ocessing echniques cu en ly
used in he c ab indus y ha dly a ec he o iginal concen a ion o Cd
p esen in he c ab (Maul aul
e al
., 2012; Wiech
e al
., 2017), which is
di ec ly dependan on he ishing a ea whe e hey o igina ed om. The e is
no speci ic EU egula ion ega ding he Cd con en in c ab’s b own mea ,
ough o he coun ies such as China ha e s a ed o egula e hose.
The e o e, in a nea u u e Cd could ep esen a g ea issue o he edible
c ab p ocessing indus y.
Conside ing he backg ound p esen ed in his li e a u e e iew
ega ding ood p ese a ion, p ocess op imiza ion and edible c ab he main
objec i es o he p esen PhD Thesis could be summa ize in:
1.6 OBJECTIVES
The main objec i es o he p esen PhD Thesis we e:
 To e alua e he e ec o he s o age empe a u e on he
shel -li e in aw c ab, comme cial c ab p oduc s and eady-
o-ea whole c ab cooked unde mild empe a u es and
packed unde acuum.
 To iden i y he main mic oo ganisms p esen a he end o
he mic obiological shel -li e o aw c ab, comme cial c ab
p oduc s and eady- o-ea whole c ab cooked unde mild
empe a u es and packed unde acuum.
 To de e mine he he mal esis ance o he main
mic oo ganisms p esen in aw c ab, comme cial c ab

Chap e 1
55
p oduc s and eady- o-ea whole c ab cooked unde mild
empe a u es and packed unde acuum.
 To e alua e he e ec i eness o he hea ea men s
ecommended by U.S. Food and D ug Adminis a ion o his
ype o p oduc s.
 To e alua e he po en ial ad an ages o he inco po a ion o
ul asound echnology in he ini ial cooking s ep o edible
c ab o imp o e hea and mass ans e p ocesses (e.g.
educe cooking ime, cleaning and cadmium educ ion).
 To op imise he second pas eu iza ion s ep applied in he
p oduc ion o eady- o-ea edible c ab using a quali y index
deg ada ion kine ic app oach.
 To e alua e he po en ial use o al e na i e echnologies (i.e.
mano-sonica ion, mano- he mo-sonica ion and ionizing
adia ion) o inac i a e he main spo e o ming bac e ia
isola ed om eady- o-ea edible c ab.
Chap e 2
56
Chap e 2
Mic obial cha ac e iza ion o edible c ab
(
Cance pagu us
)
F om his chap e wo a icles a e in p epa a ion o hei submission.
Condón-Aban o, S., Lian F., A oyo, C., Ál a ez, I. and Lyng, J.G. E ec o
s o age empe a u e on mic obial shel -li e and mic obiological p o iles o eady-
o-ea I ish whole b own c ab (
Cance pagu us
). In p epa a ion
Condón-Aban o, S., Ped ós-Ga ido, S., Al a ez, I., Why e, P., Lyng, J.G. and
A oyo, C. Isola ion, iden i ica ion and he mal esis ance o he mic obio a
p esen in eady- o-ea b own c ab (
Cance pagu us
) mea . In p epa a ion
Chap e 2
57
2.1 ABSTRACT/RESUMEN
ABSTRACT
In he pas , he b own c ab (
Cance pagu us
) ma ke mainly consis ed o
esh li e p oduc . Howe e , he opening o new ma ke s (e.g. China and USA)
has now c ea ed a demand o eady- o ea p oduc s. This in u n c ea es an
oppo uni y o add alue bu also a o ds manu ac u e s he chance o imp o e
exis ing comme cial p ocesses (e.g. use o milde hea ea men s o p oduce a
highe quali y p oduc ). Howe e , an impo an conside a ion in all o his is
p oduc shel -li e ollowing hese new p ocesses and he mic obio a p esen in
hese ypes o p oduc . Fo his eason, one o he aims o he p esen s udy
was o assess he e ec o s o age empe a u e on he shel -li e and
mic obiological p o iles o eady- o-ea whole c ab cooked unde mild
empe a u es and packed unde acuum. A e ia y model based on he
modi ied Gompe z Equa ion and he oo squa e model, o mesophilic bac e ial
g ow h a di e en s o age empe a u es was de eloped. The end o shel -li e
was conside ed as he poin whe e he mesophilic bac e ial load eached 6
Log10-cycles. Once he shel -li e was de e mined and he e ec o he s o age
empe a u e was quan i ied, a deepe s udy o he na u al mic obio a p esen in
aw and cooked b own c ab mea and in some comme cial b own c ab p oduc s
was pe o med. Finally, he he mal esis ance o he main mic oo ganisms
iden i ied in c ab mea samples we e de e mined, in e ms o
D
T and
z
alues
and he e ec i eness o he ecommended hea ea men s by U.S. Food and
D ug Adminis a ion o his ype o p oduc s was assessed.
Shel -li es o 362, 257, 204 and 145 hou s o whi e mea and 492, 340,
265 and 183 hou s in case o b own mea , we e calcula ed unde s o age
empe a u es o 2, 5, 7 and 10°C espec i ely. A linea ela ionship was ound
be ween he s o age empe a u e (°C) and he Log10 o shel -li e ega dless o
he mea ype, hough whi e mea was ound as he limi ing componen which
de ined he shel -li e o eady- o-ea b own c ab. In all cases a bigge
he e ogenei y o mic obio a was de ec ed in he case o b own mea . In aw
Chap e 2
58
mea he main mic obial g oups de ec ed we e
Pseudomonas
spp., hen
Bacillus
spp. which was p esen in a simila concen a ion o
S aphylococcus
spp
.
In
cooked c ab he mic obial g oups p esen in he g ea es p opo ion we e
Bacillus
spp. and
S aphylococcus
spp
.,
al hough hei p opo ions a ied
depending on he s o age empe a u e. The 18 bac e ial genus and 31 species
isola ed we e iden i ied by 16S RNA sequencing.
Ca nobac e ium di e gens
and
Shewanella bal ica
we e he mos equen ly isola ed non-spo e o ming
species while
Bacillus mycoides
,
Bacillus weihens ephanensis
and
Psych obacillus psych odu ans
we e he mos equen ly de ec ed spo e o ming
bac e ia. In he he mal esis ance cha ac e iza ion s udy,
Kocu ia a inae
showed he highes le el o esis ance among all ege a i e cells, hough he
ecommended ea men o
Lis e ia monocy ogenes
(F70ºC7.5ºC = 2 min) p o ed
o be e ec i e o inac i a e his mic oo ganism. Fo spo e o ming species, he
mos se e e ea men ecommended o inac i a e
Clos idium bo ulinum
(F90ºC8.6ºC = 57 min in Dungeness c ab (
Me aca cinus magis e
)), p o ed o be
su icien o achie e a 6 Log10 educ ion o
B. mycoides
and
P. psych odu ans
bu no
B. weihens ephanensis.
O e all, his esea ch p o ides aluable
in o ma ion necessa y o design p ocesses o he p oduc ion o sa e cooked
b own c ab p oduc s and highligh s he impo ance o
Bacillus
spp. in his
c us acean.
Chap e 2
65
egula e ige a o (Whi pool mod. WMT552) (7±2°C) up o he end o hei
shel -li e (da e indica ed in he label by he p oduce ).
A e s o age, mea s om lab-cooked c abs we e ex ac ed asep ically and
aliquo s o en g ams o each ype o mea we e p epa ed and used o
mic obiological analysis. Each sample was sampled a leas in duplica e. In case
o comme cial samples, on he las day o hei shel -li e, samples we e opened
and wo aliquo s o 10 g ams each we e p epa ed and used o mic obial
analysis.
2.3.3
Mic obial analyses and g ow h media
Fo he mic obiological analyses, samples we e aken om he
e ige a o on he las day o hei shel -li e. Ten g ams o c ab mea o each
sample we e dilu ed in 90mL o Maximum Reco e y Diluen (MRD) (Oxoid,
Hampshi e, UK) and mixed ho oughly o 2 min wi h a s omache (model 400
ci cula o , Sewa d S omache , UK) a 300 pm. Subsequen ly, en- old dilu ion
se ies we e p epa ed in MRD and aliquo s we e pla ed on a ange o di e en
aga s o e alua e he main mic obial g oups p esen in he samples. The aga s
used in his in es iga ion a e summa ized in Table 2.2.
Fo he enume a ion o colonies g own in/on he pla es a leas wo
eplica es o each sample we e pe o med, conside ing only he pla es wi h
coun s be ween 10 and 300 CFU. Expe imen s we e pe o med in iplica e on
di e en wo king days.

Chap e 2
66
Aga
B and
Bac e ial G oup
Incuba ion
emp. (°C)
Incuba io
n ime (h)
A mosphe e
Obse a ions
TSA
Oxoid
TVCm
25
48
Ae obic
+0.5% NaCl/ pou pla e
TSA
Oxoid
TVCp
10
168
Ae obic
+0.5% NaCl/ pou pla e
L.H.
ecipe
TVCp
10
168-240
Ae obic
Pou pla e
CFC
Oxoid
Pseudomonas
spp.
24
48
Ae obic
Sp ead pla e
VRBGA
Oxoid
En e obac e iaceae
37
48
Ae obic
Double laye /pou pla e
MRS
Oxoid
Lac ic acid bac e ia
25
72
Anae obic
Pou pla e
BP
LAB
S aphylococcus
spp.
35
24
Ae obic
Sp ead pla e
PEMBA
Oxoid
Bacillus
spp.
37
24
Ae obic
Sp ead pla e
I on aga Lyngby
Oxoid
H2S p oducing bac e ia
30
24
Ae obic
Pou pla e/ Black colonies
SPS
Sigma-Ald ich
Clos idium
spp.
37
48
Anae obic
Black colonies
Ox o d
Oxoid
Lis e ia
spp.
30
48
Ae obic
Whi ish colonies/ Pagadala
e
al
., 2012
Table 2. 2: Cul u e media and incuba ion condi ions used du ing his in es iga ion.
TSA, T yp ic Soy Aga ; L.H, Long and Hamme ’s Aga ; CFC, Ce imide Fusidin Cephalo idine aga ; VRBGA, Viole Red Bile Glucose Aga ; MRS, Man
Rogosa Sha pe aga ; BP, Bai d-Pa ke aga ; PEMBA, Bacillus ce eus aga base supplemen ed wi h polymyxin B; SPS Sul i e Polymyxin Sul adizine
aga . TVCm, To al Viable Mesophilic Coun s; TVCp, To al Viable Psych ophilic Coun s.
Chap e 2
67
2.3.4
Isola ion and iden i ica ion
A e enume a ion, a numbe o well de ined colonies we e andomly
selec ed om he di e en aga pla es. Each colony was used o inocula e a
es ube con aining 5mL o T yp ic Soy B o h (TSB) (Oxoid) supplemen ed wi h
0.6% (w/ ) o Yeas Ex ac (YE) (Oxoid) and 0.5% (w/ ) o NaCl (TSB-
supplemen ed) and incuba ed a 25°C unde ei he ae obic o anae obic
condi ions un il u bidi y was de ec ed. Following incuba ion, a loop ul om
each ube was s eaked on a pla e o T yp ic Soy Aga (TSA) (Oxoid)
supplemen ed wi h 0.6% (w/ ) o Yeas Ex ac (YE) (Oxoid) and 0.5% (w/ ) o
NaCl (TSA-supplemen ed), and incuba ed o 24–72h a 25°C unde ae obic o
anae obic condi ions. A single colony was hen used o inocula e es ubes
con aining 5mL o TSB-supplemen ed and e-incuba ed in simila condi ions.
Once u bidi y was de ec ed, 0.1 mL om he ubes we e ans e ed in o a
200mL lask con aining 50mL o TSB-supplemen ed and incuba ed as be o e.
Bac e ial popula ions we e examined pe iodically by mic oscopy using a
Neubaue chambe . When bac e ial cul u es eached he s a iona y phase o
g ow h (i.e. s able coun s o 3 consecu i e days), wo s ock c yo ials we e
p epa ed, coded and s o ed a -80°C.
A o al o 92 isually-dis inc ypes o colonies, 28 om aw c ab mea (bo h
whi e and b own), 40 om cooked mea s and 24 om comme cial p oduc s
we e cul u ed o iden i ica ion. Fo he iden i ica ion wo k using 16S RNA
sequencing, bac e ial popula ions we e eco e ed by inocula ing 5mL TSB-
supplemen ed ubes wi h a loop- ull om he c yo ials and incuba ed a 25°C
unde ei he ae obic o anae obic condi ions un il u bidi y was de ec ed.
G ow h was sub-cul u ed om each ube, by pla ing on o a TSA-supplemen ed
media and incuba ed a 25°C unde ei he ae obic o anae obic condi ions o
24-72h. A single colony was hen ans e ed in o a 5mL TSB-supplemen ed
ube and incuba ed a 25°C unde he condi ions desc ibed p e iously. When
u bidi y was de ec ed, 0.1mL was hen ans e ed in o 50mL TSB-
supplemen ed. Once he s a iona y phase was eached (using a Neubaue
Chap e 2
68
chambe ) wo aliquo s o 1mL we e p epa ed in s e ile Eppendo ubes and a
s ock c yo ial om each cul u e was p epa ed. One o he aliquo s was s o ed
a 4°C and he o he one was used o 16S RNA sequencing.
Samples o 16S RNA sequencing we e p epa ed as ollows. A 1 mL aliquo
o each bac e ial cul u e was cen i uged a 10,000 pm o 5 min a 4°C in an
Eppendo cen i uge (model 5417 R, Eppendo AG 22331, Hambu g,
Ge many). The supe na an was disca ded and he pelle was e-suspended in
s e ile dis illed wa e inside a lamina low cabine . This washing p ocess was
epea ed wice. A e washing, pelle s we e e-suspended in 500μL o lysis
bu e (Fishe Scien i ic, New Hampshi e, Uni ed S a es) and 200μL we e
ans e ed o a 96 deep-well pla e and sealed. The lysed cells we e sen unde
e ige a ion o a comme cial labo a o y (Eu o ins genomics L d.) o he
sequencing. Sequences ( o wa d and e e se) o each bac e ial cul u e we e
ecei ed in AB1 and SEQ ile o ma s and compa ed agains he US Na ional
Cen e o Bio echnology In o ma ion (NCBI) da abase using he Basic Local
Alignmen Sea ch Tool (BLAST) (h ps://blas .ncbi.nlm.nih.go /Blas .cgi).
2.3.5
Hea esis ance expe imen s
A e he iden i ica ion o he mic obial species isola ed, hei hea
esis ance was e alua ed.
2.3.5.1
Cul u e p epa a ion and ea men media
Mic oo ganisms we e i s esusci a ed om he s ock c yo ials as desc ibed
in sec ion 2.3.4. Then, a 0.1mL aliquo o an o e nigh p e-cul u e was
inocula ed in o 200mL Du an bo les con aining 50mL o s e ile TSB-
supplemen ed con aining 6mm diame e s e ile glass beads (app oxima ely
30g) o p e en he o ma ion o cell agg ega es. These we e incuba ed a
25°C in a shaking he mos a ic ba h a 130 pm (mod. LSB18, G an ins umen s
L d., Camb idge, UK) un il cul u es eached he s a iona y phase. All
mic oo ganisms we e incuba ed o 48h excep o
S aphylococcus
spp. which
equi ed 72h o each he s a iona y phase.
Chap e 2
69
2.3.5.2
Hea ea men s
In his s udy, he mal esis ance expe imen s we e pe o med in pH 7.0
McIl aine ci a e-phospha e bu e (Dawson
e al
., 1974) which is simila o he
pH o c ab mea . Fi s ly, he mos hea esis an species wi hin he same genus
was de e mined by means o he end-poin me hod. The p ocedu e used was
as ollows: 1mL o he s a iona y phase cul u es was e-suspended in ubes
con aining 9mL o ci a e-phospha e bu e in o de o achie e an ini ial coun
o app oxima ely 108 CFU/mL. Tubes we e hen subme ged in a he mos a ic
ba h (mod. LSB18, G an ins umen s L d., Camb idge, UK) a 60°C o 90
seconds and immedia ely cooled in iced wa e . Finally, aliquo s o each se ial
dilu ion we e pou pla ed in TSA-supplemen ed aga and incuba ed a 25°C o
72 h. Fu he incuba ion imes did no p o ide highe coun s (da a no shown).
Once he mos hea esis an mic obial species wi hin he same genus was
de e mined ( hose showing lowe Log10 educ ions a e he 60ºC/90s
ea men ), hei decimal educ ion ime alues (
D
T) and
z
alues we e
de e mined using a he mo esis ome e TR-SC (Condón
e al
., 1989, 1993).
This ins umen consis s o a 350mL s ainless s eel essel con aining he
ea men medium and equipped wi h an elec ical hea e and a e ige a ion
coil sys em o main aining a cons an empe a u e, an agi a ion de ice o
ensu e uni o m dis ibu ion and empe a u e homogenei y o he medium and
inoculum and po s o injec ing he mic obial suspension and o sample
ex ac ion. Once he a ge empe a u e a ained s abili y (±0.1°C), 0.2mL o
he co esponding mic obial suspension was injec ed wi h a s e ile sy inge in o
he ea men medium. A e inocula ion, 0.1mL samples we e collec ed a
di e en imes and immedia ely pou pla ed in TSA-supplemen ed aga . Su i al
cu es (log10 numbe o su i o s
e sus
ea men ime) we e ob ained a
di e en empe a u es anging om 54 o 69°C and he co esponding
D
T
alues (min) we e calcula ed. In o de o calcula e
z
alues (°C), ou
empe a u es we e e alua ed o each bac e ial cul u e. All he mal expe imen s
we e ca ied ou a leas in iplica e on di e en wo king days.
Chap e 2
70
2.3.6
Da a and s a is ical analysis
2.3.6.1
Shel -li e de e mina ion
Gompe z Equa ion modi ied by Zwie e ing
e al
., (1990) (Equa ion 2.1) was
i ed o he TVCm g ow h a di e en empe a u es (2, 5, 7 and 10°C):
𝑦=𝐴∗𝑒𝑥𝑝{−𝑒𝑥𝑝[1+𝜇𝑚𝑎𝑥∗𝑒
𝐴∗(𝜆−𝑡)]} (Eq. 2.1)
whe e,
y
indica es he Log10 o he coun eached a he di e en imes,
A
indica es he inc eased Log10 cycles om ini ial coun (y0) o s a iona y phase
(
ymax
) and
is he s o age ime exp essed in hou s;
μmax
ep esen s he
maximum g ow h a e (h-1) obse ed in he linea pa o he g ow h cu e,
λ
ep esen s he du a ion o he lag phase (h) and
e
equals o 2.718.
The es ima ed alues o
λ
and
μmax
we e u he exp essed as a unc ion o
he s o age empe a u e by using he Squa e Roo Model (Ra kowsky
e al
.,
1982) (Equa ions 2.2 and 2.3):
√𝜇𝑚𝑎𝑥=𝑏𝜇∗(𝑇−𝑇minμ) (Eq. 2.2)
√1
𝜆=𝑏𝜆∗(𝑇−𝑇minλ) (Eq. 2.3)
whe e
b
is a cons an ,
T
(°C) is he s o age empe a u e and
Tmin
is he
espec i e heo e ical minimum empe a u e o g ow h (°C), es ima ed by
ex apola ion o he eg ession line when √𝜇𝑚𝑎𝑥 o √1
λ = 0.
All models we e i ed o he da a by using a non-linea eg ession and leas
squa e app oach wi h G aphPad PRISM® 5.0 so wa e (G aphPad so wa e,
Inc., San Diego, CA, USA). The same so wa e was used o pe o m he
s a is ical analyses:
- es and one-way ANOVA ollowed by he
pos hoc
Tukey
es . Di e ences we e conside ed signi ican when P≤0.05.

Chap e 2
71
2.3.6.2
The mal esis ance de e mina ion
Su i al cu es a a cons an empe a u e (T) we e ob ained by plo ing he
Log10 ac ion o su i o s (su i o s a e a ce ain ea men ime
di ided by
he mic obial concen a ion a
=0)
e sus
he ea men ime (
, min). In cases
whe e he p o ile o he su i al cu e appea ed as a downwa d conca e shape,
he Gee ae d log-linea eg ession plus shoulde model was used (Gee ae d
e
al
., 2000). To i he model o he su i al cu es, he Gee ae d and Van Impe
inac i a ion model i ing ool (GInaFiT) was used (Gee ae d
e al
., 2005). The
model (Equa ion 2.4) desc ibes he su i al cu es h ough wo pa ame e s: he
shoulde leng h (
Sl
), de ined as he ime p eceding he exponen ial decay, and
he inac i a ion a e (
kmax
) which co esponds o he slope o he exponen ial
po ion o he su i al cu e.
N0=N0e-kmax (ekmax Sl
1+ (ekmax Sl-1)e-kmax ) (Eq. 2.4)
whe e N0 ep esen s he ini ial coun .
Fo su i al cu es wi h log-linea p o iles, Bigelow and Es y model (Bigelow
& Es y, 1920), which is also included in he GInaFiT so wa e, was used o
calcula e he exponen ial inac i a ion a e (
kmax
) in min-1. The adi ional
decimal educ ion ime alue (
D
T
alue) in minu es was hen calcula ed wi h
Equa ion 2.5.
D
T = 2.303/
kmax
(Eq. 2.5)
The
z
alue (°C), which ep esen s he empe a u e inc ease equi ed o
educe
D
T
alues by 10 old was hen calcula ed om he nega i e in e se o
he eg ession line o he The mal Dea h Time cu es (TDT) ob ained when
Log10
D
T alues a e ep esen ed e sus ea men empe a u e.
Chap e 2
72
Based on he he mal esis ance pa ame e s (
D
T and z alues), an
equi alen ea men o educe 6 Log10 cycles o he mic obial popula ion we e
also calcula ed and compa ed wi h hose p oposed o
L. monocy ogenes
in
case o ege a i e cells and
C. bo ulinum
in case o spo es, based on he
ecommended ea men s by FDA o ishe y p oduc s (FDA, 2011). - es and
one-way ANOVA we e pe o med wi h he G aphPad PRISM 5.0 so wa e
(G aphPad so wa e, Inc., San Diego, USA) and di e ences we e conside ed
signi ican i P≤0.05. The s anda d de ia ions (
SD
) a e gi en in he Figu es as
e o ba s.
2.4 RESULTS AND DISCUSSION
2.4.1
Mic obiological p o ile o aw c ab mea s
Expe imen al coun s o c ab aw esh mea s ob ained on he di e en
speci ic aga s a e shown in Figu e 2.1. O e all, he coun s ob ained in his
esea ch o c ab aw mea s a e in acco dance wi h hose p e iously epo ed
o di e en aw unp ocessed shell ish, which a e be ween 2.5 and 3 Log10
cycles (Go nik
e al
., 2011; Robson
e al
., 2007). Di e ences in coun s be ween
he wo ypes o mea (i.e. whi e
s
b own) we e obse ed o all bac e ial
g oups. In e es ingly, highe coun s we e de ec ed in b own mea . Fagh i
e al
.,
(1984) epo ed simila di e ences in coun s depending on he c ab’s issue
(muscle, gills and hemolymph), ob aining lowe coun s in c ab muscle, which
co esponds o whi e mea han in case o gills and hemolymph. These highe
coun s in b own mea can be explained by he ac ha c abs do no ha e a
closed s uc u ed ci cula o y sys em so he haemolymph, loca ed inside he c ab
body, could be a ese oi o bac e ia (ICMSF, 2005). In bo h ypes o c ab
mea he psych ophilic coun s we e highe han o mesophilic bac e ia. In case
o mesophilic bac e ia he coun s we e 1.2 and 2.5 Log10 cycles o whi e and
b own mea , espec i ely, while o he psych ophilic bac e ia he coun s in PCA
(Pla e Coun Aga , Oxoid) we e 1.8 and 3.0 Log10 cycles. Howe e , when a
speci ic aga was used o ma ine psych ophilic bac e ia (L.H aga ) he coun s
we e 2.1 and 3.7 Log10 cycles in whi e and b own mea , espec i ely, i.e. 0.3
Chap e 2
73
and 0.7 Log10 cycles highe compa ed wi h he non-selec i e PCA. This
misma ch was also obse ed by B oekae
e al
., (2011) who epo ed inc eases
in he coun s be ween 0.3 and 1.1 Log10 cycles when L.H aga was used ins ead
o PCA.
Figu e 2.1: Log10 coun s ob ained in di e en selec i e and non-selec i e aga s (see Table 2.2)
o whi e (whi e ba s) and b own (g ey ba s) aw c ab mea . TVCm, To al iable mesophilic
coun s; TVCp, To al iable psych ophilic coun s; L.H, Long and Hamme aga . ND = no
de ec able.
In case o whi e mea , only
Pseudomonas
spp. and
S aphylococcus
spp.
we e de ec ed and he coun s we e 1.8 and 1.4 Log10 cycles, espec i ely. The
he e ogenei y o he lo a de ec ed in b own mea was la ge han in whi e
mea . Also in b own mea
Pseudomonas
spp. was he genus de ec ed in a
highe p opo ion wi h a o al coun o 3.5 Log10 cycles while
S aphylococcus
spp. and
Bacillus
spp. coun s we e 2.9 and 3 Log10 cycles espec i ely. The
main di e ence obse ed be ween mea s we e he signi ican coun s obse ed
o
Bacillus
spp. (2.9 Log10 cycles) and he p esence o H2S p oducing bac e ia
(0.9 Log10 cycles) and LAB (1.2 Log10 cycles) in b own mea .
O e all, esul s ob ained in his sec ion a e in ag eemen wi h mos
published da a on di e en c ab species and shell ish. Bu in e es ingly, ou
s udy also e ealed a signi ican p esence o
Bacillus
spp. in b own mea hough
Bacillus spp.
S p od. bac e ia
2
HEn e obac e iaceae
Lac ic Acid Bac e ia
Pseudomonas spp.
S aphylococcus spp.
m
TVC
p
TVC
(L.H)
p
TVC
Clos idium spp.
L. monocy ogenes
0
1
2
3
4
ND ND ND ND ND ND
Log10 CFU/g
Chap e 2
74
no in whi e mea . Some au ho s ha e also epo ed he p esence o his spo e
o ming bac e ia in he in es inal con en o
Dungeness c ab
(Lee and P ei e ,
1975). Lin on
e al
., (2003) also desc ibed he p esence o
Bacillus
spp. in
di e en shell ish a e a high hyd os a ic p essu e p ocess, hough some o he
au ho s did no ind his mic oo ganism in aw shell ish (Bozia is
e al
., 2011;
Fagh i
e al
., 1984; Go nik
e al
., 2011). The p esence o
Bacillus
spp. could be
expec ed since di e en au ho s ha e epo ed i s p esence bu mainly in
p ocessed p oduc s (G am and Huss, 1996; Lin on
e al
., 2003). This could be
a ibu able o he low coun s ound in aw p oduc s, which will make hem
unable o compe e wi h o he bac e ia p esen in highe p opo ions.
None heless, he ac ha hey a e spo e- o ming bac e ia wi h a “ a - om-
negligible” esis ance o conse a ion p ocesses gi es hem an impo an ole in
he spoilage o p ocessed sea ood p oduc s. The absence o H2S p oducing
bac e ia and LAB in whi e mea is also in acco dance wi h he s udy o Bozia is
e al
., (2011) wi h lobs e s. Al hough hese wo bac e ial g oups along wi h he
En e obac e iaceae
amily a e conside ed o signi ican ele ance in he
deg ada ion o esh ish (G am and Huss, 1996; G am and Dalgaa d, 2002;
Jay, 2000), hei impo ance in b own c ab seems o be mino due o he lowe
coun s obse ed he e compa ed o epo s on o he species. I is also
in e es ing o no e ha in his esea ch, nei he
Lis e ia
spp
.
no
Clos idium
spp
.
we e de ec ed in any o he samples analysed. This could be a ibu able
o insu icien sample size o due o he small coun s and ela i ely low
p e alence o hese mic oo ganisms in c ab as indigenous bac e ia (Fledhusen,
2000; Rocou
e al
., 2000; Pagadala
e al
., 2012).
2.4.2
Mic obiological shel -li e o cooked c abs
In his sec ion he e ec o he s o age empe a u e on he shel -li e o
c abs cooked a a mild empe a u e (75°C) o he equi ed ime o apply a
leas an equi alen
F
70ºC7.5ºC= 2 min (45 min) as de e mined in Chap e 3, was
e alua ed. To de e mine he e ec o s o age empe a u e on he shel -li e o
his sea ood p oduc , he Gompe z Equa ion modi ied by Zwie e ing
e al
.,
Chap e 2
81
di e en ly depending on he ype o mea .
b
alues, bu no
Tmin
, epo ed he e
a e in he ange o hose epo ed by Kou suomanis (2001) and Kou soumanis
e al
., (2001) o
Pseudomonas
spp. in gil -head seab eam. The di e ences
be ween he calcula ed
b
alues and hose epo ed in he li e a u e would be
a ibu able, as in case o
μmax
, o di e ences in he mic obio a p esen in he
p oduc . Ne e heless, he di e ences ound be ween
bλ
alues o he c ab
mea ypes would need u he explana ion. In his esea ch he shel -li e model
was buil om he esul s ob ained om a mild cooked c ab. As shown in
Chap e 3 he c ab’s cold spo is loca ed in he abdomen. Due o he di e en
hea pene a ion a es obse ed, be ween c ab pa s, signi ican di e ences on
he o al
F
alue applied exis s be ween he wo ypes mea s, so a di e ence on
he mic obio a selec ed a e he ea men could exis . Al hough, he
di e ences be ween mea composi ions could also con ibu e, as has been
discussed p e iously.
2.4.5
Shel -li e de e mina ion (Te ia y model)
Finally, based on he pa ame e s ex ac ed om he p ima y (modi ied
Gompe z Equa ion) and seconda y (Roo Squa e Model) models, wo di e en
inal Equa ions we e de eloped o calcula e he mic obial g ow h in whi e (Eq.
2.6) and b own mea (Eq. 2.7).
𝑦=𝐴×𝑒𝑥𝑝{−𝑒𝑥𝑝[1+[0.0058×(𝑇−(−24.9))]2×𝑒
𝐴∗[(0.014×((𝑇−(−3.7)))2−𝑡]]} (Eq. 2.6)
𝑦=𝐴×𝑒𝑥𝑝{−𝑒𝑥𝑝[1+[0.0061×(𝑇−(−21.1))]2×𝑒
𝐴∗[(0.0096×(𝑇−(−4.4)))2−𝑡]]} (Eq. 2.7)
Whe e
y
indica es he Log10 cycles o he coun s,
A
indica es he di e ence
be ween he coun s (Log10 cycles) in he s a iona y phase (
ymax
) and he ini ial
coun s (y0),
is he s o age ime exp essed in hou s and
T
ep esen he

Chap e 2
82
s o age empe a u e (°C). Figu e 2.3 shows he compa ison be ween obse ed
and p edic ed alues calcula ed om Equa ions 2.6 and 2.7 as well as
R2
and
RMSE
as indica o s o he accu acy o he e ia y Equa ions o whi e (A) and
b own (B) mea . Based on Equa ions 2.6 and 2.7 he shel -li e o whi e and
b own mea we e calcula ed, conside ing 6 Log10 cycles as he end o he shel -
li e. A log-linea ela ionship was ound be ween he shel -li e and he s o age
empe a u e o bo h mea ypes as Dabadé
e al
., (2015) p e iously epo ed
o opical sh imps.
Table 2.5 shows he Equa ions which desc ibe he e ec o he s o age
empe a u e on he shel -li e o each mea ype. In his case, no di e ences
we e ound be ween he slopes ob ained o each mea ype meaning ha he
ela i e e ec o he s o age empe a u e on he shel -li e was independen o
he ype o mea .
F om he Equa ions shown in Table 2.5, a shel -li e o 12 and 16 days we e
calcula ed a 4°C, while 6 and 7 days we e de ined as he shel -li e limi a 10°C
o whi e and b own mea , espec i ely. Acco ding o hese esul s, he eady-
o-ea edible c ab (
Cance pagu us
) shel -li e seems o be limi ed by he whi e
mea , when c abs a e p ocessed in a single s ep cooking a mild empe a u es.
Table 2.5: Equa ions which de ine he shel -li e o eady- o-ea whole b own c ab packed
unde acuum as a ac o o he s o age empe a u e.
Mea ype
Equa ion
Whi e
Log10 Shel -li e
(h) = -0.0497 * T + 2.6588
B own
Log10 Shel -li e
(h) = -0.0537 * T + 2.8001
Chap e 2
83
Figu e 2.3: Measu ed Vs p edic ed coun s o TVCm a all s o age empe a u es in whi e (A) and
b own (B) mea (es ima ed om Eq. 2.6 and 2.7)
A
0 2 4 6 8 10
0
2
4
6
8
10
R2=0.94
RMSE = 0.82
Measu ed alues
P edic ed alues
B
0 2 4 6 8 10
0
2
4
6
8
10
R2=0.91
RMSE = 0.97
Measu ed alues
P edic ed alues
Chap e 2
84
2.4.6
E ec o s o age empe a u e on mic obial lo a p esen in
cooked c ab
Based on he de eloped model (Sec ion 2.4.5), a s udy o he main
mic obio a p esen a he end o he shel -li e i.e. a e 12 days a 4°C and a e
6 days a 10°C was pe o med. Simila o aw mea s (Figu e 2.1), Figu e 2.4
shows he mic obiological p o iles o cooked c ab a he end o shel li e, using
di e en selec i e and non-selec i e aga s (Table 2.2) o whi e (A) and b own
(B) c ab mea . In gene al, no di e ences be ween coun s we e obse ed a 4
and 10°C in he case whe e he same bac e ial g oup was de ec ed a bo h
empe a u es; excep o
Bacillus
spp. in whi e mea and
S aphylococcus
spp. in
b own mea . This esul suppo s he e ec i eness o he model de eloped in
he p e ious sec ion.
The maximum coun s egis e ed o TVCm (Figu e 2.4) we e 5.3 and 4.8
Log10 CFU/g in whi e mea and 5.9 and 6.0 Log10 CFU/g in b own mea a 4 and
10°C, espec i ely. As i was obse ed wi h he aw mea s (Figu e 2.1), in he
case o cooked c ab mea s, he psych ophilic bac e ia coun s we e highe and
he highes coun s we e de ec ed in L.H aga (6.1 and 4.9 Log10 CFU/g in he
case o whi e mea and 6.4 and 6.1 Log10 cycles in he case o b own mea ).
Fo whi e mea (Figu e 2.4A), H2S p oducing bac e ia we e only ound in he
mea s o ed a 10°C in a le el o 2.7 Log10 CFU/g. In he case o
Pseudomonas
spp
.
no di e ences we e de ec ed be ween he absolu e coun s a 4 and 10°C
(2.5 Log10 CFU/g). Simila ly, no di e ences be ween coun s we e obse ed
be ween
S aphylococcus
spp
.
coun s a 4 and 10°C (3.4 Log10 CFU/g). Only in
he case o
Bacillus
spp
.
signi ican di e ences we e obse ed in coun s 3.9
Log10 a 10°C and 5.3 Log10 a 4°C. Fo b own mea (Figu e 2.4B), H2S
p oducing bac e ia,
En e obac e iaceae
and LAB (Lac ic Acid Bac e ia) we e only
de ec ed when he mea was s o ed a 10°C. The p esence o
S aphylococcus
spp. was dependen on he s o age empe a u e: a 4°C his bac e ia coun was
3.2 Log10 cycles and 5.7 Log10 cycles o samples s o ed a 10°C. On he o he
Chap e 2
85
side, in case o
Bacillus
spp
.
, no signi ican di e ences we e de ec ed be ween
he coun s a 4 and 10°C (be ween 5.5 and 6 Log10 CFU/g).
Figu e 2.4: Log10 coun s ob ained o
Bacillus
spp., H2S p oducing bac e ia,
En e obac e iaceae
, Lac ic Acid Bac e ia,
Pseudomonas
spp.,
S aphylococcus
spp., To al Viable
Coun s, To al Psych ophilic Ae obic Coun s and o al psych ophilic bac e ia (L.H) o whi e (A)
and b own (B) mea o cooked c ab s o e a 4°C (g ey ba s) and 10°C (whi e ba s) a he end
o he shel -li e (calcula ed om Eq. 2.6).
A
Bacillus spp.
S p od. bac e ia
2
HEn e obac e iaceae
Lac ic Acid Bac e ia
Pseudomonas spp.
S aphylococcus spp.
m
TVC
p
TVC
(L.H)
p
TVC
Clos idium spp.
L. monocy ogenes
0
2
4
6
84C
10C
ND ND
NDNDND
Log10 CFU/g
B
Bacillus spp.
S p od. bac e ia
2
HEn e obac e iaceae
Lac ic Acid Bac e ia
Pseudomonas spp.
S aphylococcus spp.
TVCm
TVCp
TVCp (L.H)
Clos idium spp.
L. monocy ogenes
0
2
4
6
84C
10C
ND ND ND ND ND
Log10 CFU/g
Chap e 2
86
Based on he esul s o his sec ion,
Bacillus
spp
.
p o ed o be he species
o majo signi icance in eady- o-ea whole b own c ab ollowed by
S aphylococcus
spp. especially a highe s o age empe a u es. The p esence o
o he SSO bac e ia, which is conside ed o impo ance, was p ac ically non-
exis en . Wa d
e al
., (1977) also epo ed he p esence o
Bacillus
spp. in
pas eu ized c ab mea s o ed a 3.3 °C. Howe e he same au ho s also
epo ed an impo an ele ance o
Pseudomonas
spp., which has no been
de ec ed in his esea ch. Lo en zen
e al
., (2014) also epo ed he impo ance
o H2S p oducing bac e ia and
Pseudomonas
spp. in cooked King c ab clus e s
s o ed a 4°C. The p esence o
Pseudomonas
spp. as impo an SSO in cooked
b own c ab was also epo ed by Anacle o
e al
., (2011). Bu o he au ho s
ha e also epo ed he impo ance o
Bacillus
spp
.
in p ocessed ish and ishe y
p oduc s (G am and Huss, 1996; Lin on
e al
., 2003). Howe e , his is he i s
wo k which shows he impo ance o
S aphylococcus
spp. as species o
signi icance in a p ocessed sea ood s o ed a 10ºC. The disc epancies obse ed
wi h p e ious s udies, whe e he p esence o
S aphylococcus
spp. has no been
highligh ed, could be a ibu able o di e en aw ma e ials, he empe a u es
used in he expe imen al plan o he di e en ea men s applied. In he
p esen s udy a he mal ea men speci ically designed o inac i a e
L.
monocy ogenes
in sea ood, based on he US FDA ecommenda ions, was
applied. This would explain he low coun s o ege a i e bac e ia cells and he
s ong p esence o
Bacillus
spp. as hose a e able o p oduce hea esis an
spo e o ms. I is also no ewo hy o men ion ha in he majo i y o he
published s udies he he mal ea men condi ions applied a e no epo ed
hence he di e ences in he mic obio a could be also associa ed o he di e en
he mal ea men s applied.
2.4.7
Bac e ial g oups p esen in lab-cooked and comme cial samples
o eady- o-ea b own c ab
Once a sui able model o desc ibe he shel -li e o eady- o-ea b own c ab
mea samples depending on he s o age empe a u e has been de ined, he

Chap e 2
87
mic obio a o a numbe o c abs cooked, as desc ibed in sec ion 3.2, packed
unde acuum and s o ed a 7±1°C we e analysed on he las day o hei shel -
li e (de ined based on p e ious esul s). Addi ionally, he mic obial g oups o a
numbe o comme cial samples (see Table 2.1) we e also analysed a he end
o hei shel -li e, as indica ed in he label. The pe cen ages calcula ed in his
sec ion a e calcula ed based on he p esump i e coun s in speci ic aga s using
Equa ion 2.8.
Rep esen a i i y %= Coun in speci ic aga
Coun in non−speci ic aga (𝑇𝑉𝐶𝑚)×100 (Eq. 2.8)
Fo labo a o y p ocessed c ab mea s o ed o 7 days (i.e. he end o hei
shel -li e as de ined by he equa ions de eloped in sec ion 2.4.5), he maximum
TVCm coun s obse ed we e 5.1 and 7.1 log10 CFU/g o whi e and b own mea ,
espec i ely. In con as o aw mea , he highes coun s we e de ec ed o
TVCm ins ead o TVCp. The p opo ions o he di e en bac e ial g oups
de ec ed a e p esen ed in Figu e 2.5. Fo whi e mea , he main gene a de ec ed
we e:
Bacillus
spp
.
(14%),
S aphylococcus
spp. (13%),
Pseudomonas
spp
.
(0.7%) and H2S p oducing bac e ia (0.1%), while unde ined bac e ial g oups
ep esen ed 71% o he o al mic obio a, showing he limi a ions o
p esump i e coun s in selec i e aga s. Fo b own mea , uniden i ied bac e ial
g oups made up 9%, wi h
Bacillus
spp. mos equen ly de ec ed (59.4%)
ollowed by
S aphylococcus
spp. (29.7%). Fu he mo e,
En e obac e iaceae
,
LAB and
Pseudomonas
spp. we e de ec ed in low p opo ions co esponding o
0.3%, 0.5% and 0.2%, espec i ely. As o aw c ab mea ,
H2S p oducing
bac e ia,
Lis e ia
spp. and
Clos idium
spp. we e no de ec ed in ei he whi e o
b own cooked mea s samples.
The TVCm coun s enume a ed in comme cial samples a he end o hei
shel -li e we e 8.3, 2.1, 6.8, 5.4, 6.7 and 3.6 Log10 CFU/g o samples A, B, C,
D, E and F, espec i ely (see Table 2.1). These esul s highligh subs an ial
a iabili y in TVCm coun s among comme cial c ab p oduc s. The highes coun s
we e de ec ed in pa icula e whi e mea packed unde modi ied a mosphe e
Chap e 2
88
condi ions, whe e he main bac e ia de ec ed we e H2S p oducing bac e ia
(77.6%), ollowed by
Bacillus
spp. (4.9%) and
Pseudomonas
spp
.
(3.8%), while
LAB and
S aphylococcus
spp. ep esen ed only 1% o he o al mic obial
concen a ion, as shown in Figu e 2.5. Ready- o-ea whole c ab claws (sample
B) we e ound o ha e he lowes coun s a he end o shel -li e. In samples C
and F no speci ic bac e ial g oups we e de ec ed wi h he selec i e aga s used.
S aphylococcus
spp. ep esen ed 20% o he lo a in p oduc E, while
Pseudomonas
spp. and H2S p oducing bac e ia we e de ec ed in p opo ions o
16.5% and 5% espec i ely in sample D (Figu e 2.5).
Figu e 2.5: Rela i e pe cen age o in lab-cooked whi e and b own c ab mea and a ious
comme cial samples a he end o hei shel -li e.
The he e ogenei y obse ed among he comme cial samples may be ela ed
o he di e en p ocesses used by indi idual companies wi hin he c ab
p ocessing sec o . Di e ences in he mic obio a p esen in mea cooked in a
single s ep unde con olled condi ions e sus comme cial could be ela ed o
he ac ha comme cial c ab p oduc s a e pas eu ised a e packaging and
hey a e gene ally designed o a ge non-p o eoly ic
Clos idium bo ulinum
ype E (Edwa ds and Ea ly, 2001). The e o e, he low p esence o spo e-
o ming bac e ia??? is no su p ising and his is likely he eason why nei he
Lis e ia
no
Clos idium
we e de ec ed.
whi e mea
b own mea
Comme cial A
Comme cial B
Comme cial C
Comme cial D
Comme cial E
Comme cial F
0
10
20
30
40
50
60
70
80
90
100 Bacillus spp.
H2S p od. Bac e ia
En e obac e iaceae
Lac ic acid bac e ia
Pseudomonas spp.
S aphylococcus spp.
o he s
species %
Chap e 2
89
2.4.8
Bac e ial species isola ed in b own c ab
Table 2.6 shows he main bac e ial species iden i ied using 16S RNA
sequencing in c ab and c ab p oduc s. In e es ingly, in aw c ab mea 14% o
he isola es iden i ied we e con i med as
Shewanella bal ica
in bo h b own and
whi e mea e en hough no H2S p oducing bac e ia we e de ec ed in i on
Lyngby aga , which is expec ed o isola e his species as i is a sulphi e
p oducing bac e ia. Howe e , he de ec ion o his bac e ium is no su p ising
gi en ha hey a e conside ed a common speci ic spoilage o ganism (SSO) in
sea ood.
Ca noba e ium di e gens
was iden i ied in 28.5% o he bac e ial
isola es and was he mos equen ly isola ed bac e ium i espec i e o c ab
mea ype e en hough he LAB coun s ( he g oup o which i belongs) we e
e y low in MRS aga . The emaining bac e ial species isola ed om c ab mea
ep esen ed om 3.5 o 7% o he o al bac e ia isola ed in aw c ab mea s and
included se e al species o
S aphylococcus
,
Pseudomonas
,
Psych obac e
,
Kocu ia
and di e en gene a such us
En e ococcus
,
Die zia
,
Vib io
,
Planococcus
,
B ocho ix
,
Oe sko ia
,
Ch yseobac e ium
,
Co ynebac e ium
,
P o idencia
and
Pseudoal e omonas
.
Fo comme cial samples, a numbe o ege a i e cells we e iden i ied,
hough
S aphylococcus
spp.,
Ca nobac e ium
spp. and
Co ynebac e ium
spp.
we e he dominan species iden i ied. In case o spo e- o ming bac e ia, only
Psych obacillus psych odu ans
and
Bacillus weihnes ephanensis
we e iden i ied,
wi h
P. psych odu ans
being he mos p e alen in comme cial samples. Fo
c ab mea s cooked in a single s ep unde labo a o y condi ions, a numbe o
ege a i e gene a we e also de ec ed including
Pseudomonas
and
S aphylococci
. Fo spo e- o ming bac e ia,
Bacillus mycoides
,
B.
weihens ephanensis
and
P. psych odu ans
we e he mos p e alen species
eco e ed wi h p opo ions o 35%, 30% and 7.5% iden i ied, espec i ely.
Chap e 2
90
Table 2.6: Vege a i e bac e ial species isola ed om cooked eady- o-ea b own c ab (
Cance
pagu us
) p ocessed unde labo a o y condi ions and a ious comme cial p oduc s and iden i ied
by 16S RNA sequencing.
Vege a i e Cells
Sample Type
Bac e ial genus
species
Whi e mea
B own mea
Comme cial
mea
Raw
P ocessed
Raw
P ocessed
P ocessed
S aphylococccus
equo um
1
1
1
-
-
capi is
1
1
-
-
-
xylosus
1
-
-
-
3
luo e ii
-
-
-
-
1
Shewanella
bal ica
2
1
1
-
1
Ca nobac e ium
di e gens
4
1
4
1
2
undi um
-
-
-
-
1
En e ococcus
aquima inus
-
-
-
-
1
Die zia
ma is
-
-
1
-
-
Pseudomonas
decep ionensis
-
1
-
-
-
lundensis
-
1
-
1
-
luo escens
-
-
-
1
-
synxan ha
-
-
1
-
-
psych ophila
-
1
-
-
-
Psych obac e
ozii
1
-
1
-
-
ma i imus
-
-
1
-
-
a icus
-
-
1
-
-
aecalis
-
-
1
-
-
Vib io
anguilla um
-
-
1
-
-
Planococcus
haloc yophilus
-
-
1
-
-
B ocho ix
he mosphac a
-
-
-
1
-
Oe sko ia
u ba a
-
-
1
-
-
Kocu ia
salsiccia
-
-
1
-
-
a inae
-
-
-
-
1
Ch yseobac e ium
humi
-
-
1
-
-
Co ynebac e ium
es udino is
-
-
-
-
3
P o idencia
heimbachae
-
-
-
-
1
Pseudoal e omonas
elyako ii
-
-
1
-
-
Spo e o ming
Bacillus
mycoides
-
9
-
5
-
weihens ephanensis
-
8
1
4
1
Psych obacillus
psych odu ans
-
-
-
3
9
Chap e 2
97
Figu e 2.8: Theo e ical he mal dea h ime cu es (TDT) o achie e 6 Log10 educ ions o
Kocu ia a inae
(black line) and
Lis e ia monocy ogenes
( ed line). The la e based on he
ecommended ea men s by FDA (FDA, 2011).
Figu e 2.9: Theo e ical TDT lines o achie e 6 Log10 educ ions o he spo e o ming bac e ia
isola ed om c ab based on da a p esen ed in chap e 6 and hea ea men s equi ed o
achie e simila educ ions o
C. bo ulinum
non-p o eoly ic spo es based on he ecommended
ea men s by FDA (
F
90ºC7o 10ºC = 10 min;
F
90ºC9ºC = 31 min;
F
90ºC8.6ºC = 57 min) (FDA, 2011).
A
50 60 70 80 90
-3
-2
-1
0
1
2
3Koku ia
L. monocy ogenes
T ea men empe a u e (C)
Log10 6DT(min)
70 80 90 100 110 120
-3
-2
-1
0
1
2
3
4
F907o 10= 10min
F908.6= 57min
F909= 31min
B. mycoides
B. weihens ephanensisP. psych odu ans
T ea men empe a u e (C)
Log10 6DT(min)
70 80 90 100 110 120
-3
-2
-1
0
1
2
3
4
F90C7o 10C= 10min
F90C8.6C= 57min
F90C9C= 31min
B. mycoides
B. weihens ephanensisP. psych odu ans
T ea men empe a u e (C)
Log10 6DT(min)

Chap e 2
98
On he o he side, Figu e 2.9 shows he he mal ea men s p oposed o
c ab and c ab based p oduc s conside ing
C. bo ulinum
non-p o eoly ic ype E
as he a ge mic oo ganism.
F
90°C= 10 min o equi alen , conside ing
z
alues
o 7°C o empe a u es below 90°C, o 10°C o empe a u es o e 90°C. As i
can be seen, his ea men would no eliably p o ide a educ ion o 6 log10
cycles o
B. mycoides,
he mos hea sensi i e spo e isola ed om c ab
samples as i is shown in sec ion 6.4.1. Howe e , some esea che s conside
c ab mea as an excep ion wi h ega d o he inac i a ion o
Clos idium
spo es
due o i s high concen a ion o lysozyme (Pe e son
e al
., 1997). I is known
ha he p esence o lysozyme imp o es he eco e y o hea - ea ed
Clos idium
spo es and he e o e i s hea esis ance (Peck and Fe nadez, 1995;
Sco and Be na d, 1982). I has also been epo ed ha his enzyme is p esen
in ish and sea ood up o le els o 200 μg/g (Lie
e al
., 1989; Lund and
No e mans, 1992). Fo his eason, wo mo e se e e al e na i e ea men s
we e p oposed by he FDA o c ab mea :
F
90ºC9ºC= 31 min (Blue c ab,
Callinec es sapidus
) and
F
90ºC8.6ºC= 57 min (Dungeness c ab,
Me aca cinus
magis e
) in o de o achie e a sa is ac o y educ ion o
Clos idium
spo es
(FDA, 2011; Pe e son
e al
., 1997, 2002). These wo al e na i e ea men s a e
also ep esen ed in Figu e 2.9. As he Figu e shows, i he mos se e e
ecommended ea men , i.e. o Dungeness c ab mea (
F
90ºC8.6ºC= 57 min), is
o be applied o b own c ab, an adequa e educ ion o wo o he h ee spo e
o me s isola ed in his hesis (
B. mycoides
and
P. psych odu ans
) would be
achie ed. Howe e , his ea men would no be su icien o achie e a simila
educ ion in he popula ion o
B. weihens ephanensis
spo es, which would need
ea men imes 10- old longe . Van Assel and Zwie e ing (2006) epo ed
simila di e ences when compa ing he he mal esis ance o a ious
Clos idium bo ulinum
and
Bacillus ce eus
s ains
.
In 1998
Bacillus weihens ephanensis
was p oposed as a new species o
accommoda e some o he psych o ophic s ains o
Bacillus ce eus
(Lechne
e
al
., 1998). This spo e- o ming species is able o g ow a empe a u es o 5°C o
less and he abili y o p oduce eme ic and dia heal oxins, no mally associa ed
Chap e 2
99
wi h
B. ce eus
, has also been epo ed (S en o s and G anum, 2001; S en o s
e
al
., 2002; Tho sen
e al
., 2006, 2009). As a esul , he isola ion o his species
in b own c ab combined wi h i s high hea esis ance (highe han non-
p o eoly ic species o
C. bo ulinum
) highligh s hei po en ial signi icance in
e ms o ood sa e y. Howe e , no disease associa ed wi h
Bacillus
spp. in
b own c ab p oduc s ha e been epo ed, p obably due o p oduc s being
packaged unde acuum, which can hinde he g ow h o
B. wiehen ephanensis
(Samapundo
e al
., 2011). O e all, he esul s o his in es iga ion showed ha
spo es na u ally p esen in b own c ab mea could be mo e he mo- ole an
han he non-p o eoly ic s ains o
C. bo ulinum
. The e o e, hea ea men s
designed o hese spo e- o ming bac e ia may no be su icien o educe
popula ions o sa e le els.
2.5 CONCLUSIONS
F om he ob ained esul s in his chap e i can be concluded ha
Bacillus
spp
.
,
Pseudomonas
spp. and
S aphylococcus
spp. a e he main mic obial g oups
p esen in aw whi e and b own c ab mea . F om he shel -li e s udy i can be
concluded ha he s o age empe a u e has a g ea e e ec on he lag phase
han on he g ow h a e o he TVCm du ing s o age. A log-linea ela ionship
was ound be ween he s o age empe a u e and he loga i hm o shel -li e
(hou s) o bo h mea ypes (i.e. whi e and b own).
Bacillus
spp. and
S aphylococcus
spp
.
p o ed o be he bac e ial g oups p esen in a majo
p opo ion a he end o he shel -li e o he p oduc . Howe e , hei p opo ions
changed conside ably depending on he s o age empe a u e. Raw b own c ab
mea shows a g ea e he e ogenei y in mic obio a han whi e mea . In case o
aw mea ,
Ca nobac e ium di e gens
and
Sewanella bal ica
we e he mos
equen ly isola ed mic oo ganisms ega dless o he mea ype, while o
cooked mea s o ed a 7-8°C,
B. mycoides
,
B. weihens ephanensis
and
P.
psych odu ans
we e he mos equen ly de ec ed in all comme cial mea s and
c abs cooked unde con olled condi ions a mild empe a u es in he
Chap e 2
100
labo a o y. Fo ege a i e cells,
Kocu ia a inae
showed he highes le el o
hea esis ance among non-spo e o ming bac e ial species isola ed om b own
c ab mea , hough impo an ly, he hea ea men ecommended by he FDA o
inac i a e
L. monocy ogenes
(
F
70ºC7.5ºC= 2 min) p o ed o be su icien o
inac i a e i . In con as , he mos se e e hea ea men ecommended o
inac i a e non-p o eoly ic species o
C. bo ulinum
was enough o educe 6 Log10
cycles o
B. mycoides
and
P. psych odu ans
bu no
B. weihens ephanensis,
which would equi e a ea men ime 10- old longe . O e all, his sec ion
p o ides signi ican knowledge abou he mic obio a and he shel -li e o eady-
o-ea b own c ab (
Cance pagu us
), a p oduc o ample ma ke bu
unde es ima ed om a scien i ic poin o iew and highligh s he impo ance o
Bacillus
spp. al hough addi ional s udies would be equi ed o de e mine he
po en ial ood sa e y isks associa ed wi h
B. weihens ephanensis
in b own c ab
based p oduc s.
Chap e 3
101
Chap e 3
The use o ul asound echnology in he i s cooking s ep o
eady- o-ea whole b own c ab
(Cance pagu us)
This chap e is as publish in Ul asonics Sonochemis y, wi h some mino
modi ica ions o a oid duplica ion be ween chap e s.
Condón-Aban o, S., A oyo, C., Ál a ez, I., B un on, N., Why e, P., & Lyng, J.
G. (2018). An assessmen o he applica ion o ul asound in he p ocessing o
eady- o-ea whole b own c ab (
Cance pagu us
). Ul asonics Sonochemis y,
40, Pa A, 497-504.
Chap e 3
102
3.1 ABSTRACT/RESUMEN
ABSTRACT
This s udy assesses he po en ial o inco po a ing ul asound as a
p ocessing aid in he p oduc ion o whole cooked b own c ab (
Cance pagu us
).
The FDA ecommended hea ea men o educe
Lis e ia monocy ogenes
by 6
Log10 cycles in his p oduc is a
F
70ºC7.5ºC o 2 min. An equi alen
F
alue was
applied a 75°C in p esence and absence o ul asound in wa e alone o in
wa e wi h 5% w/ NaCl added. Hea pene a ion, u bidi y and conduc i i y o
he cook wa e and also sal and mois u e con en o he c ab mea (bo h whi e
and b own mea ) we e de e mined. Ul asound assis ed cooking allowed a
educ ion o he cooking ime by up o 15% while s ill main aining an
F
70ºC7.5ºC
o 2 min. Ul asound also enhanced he a e and o al amoun o compounds
eleased om he c ab, which sugges s ha c abs cooked in he p esence o
ul asound would be expec ed o be cleane . Ul asound also p o ed o be
e ec i e in educing he sal con en bu ha dly a ec ed he inal mois u e
con en o he c ab mea .

Chap e 3
103
RESUMEN
Es e es udio se ealizó pa a e alua el po encial de la inco po ación de la
ecnología de los ul asonidos como coadyu an e en la p oducción de buey de
ma (
Cance pagu us
) cocinado en e o lis o pa a el consumo. El a amien o
ecomendado po la FDA pa a inac i a 6 ciclos loga í micos de
Lis e ia
monocy ogenes
en es e ipo de p oduc os es un
F
70ºC7.5ºC de 2 minu os. Se
aplicó un
F
equi alen e a 75°C en agua y agua con un 5% p/ de NaCl
u ilizando o no ul asonidos du an e el a amien o. Se e aluó la pene ación de
calo y se midió la u bidez y conduc i idad eléc ica del agua de cocinado y el
con enido de sal y humedad de ambos ipos de ca ne (blanca y ma ón) de
cang ejo as los dis in os a amien os. El a amien o con ul asonidos pe mi ió
educi has a un 15% el iempo o al de a amien o man eniendo un
F
70ºC7.5ºC
equi alen e de 2 minu os. La aplicación de ul asonidos du an e el cocinado
ambién mejo ó la elocidad de salida de compues os del cang ejo así como la
can idad o al de es os compues os, lo cual sugie e que los cang ejos cocinados
en p esencia de ul asonidos debe ían de es a más limpios. Los ul asonidos
ue on ambién e ec i os pa a educi el con enido de sal de la ca ne de
cang ejo, pe o p ác icamen e no a ec a on a su con enido de humedad. En
conclusión, los esul ados ob enidos en es e capí ulo pe mi i ían educi el
iempo de cocción así como elimina la e apa de la ado del p oceso adicional
del cang ejo con las consiguien es consecuencias económicas y de calidad del
p oduc o.
Chap e 3
104
3.2 INTRODUCTION
As men ioned be o e he p oduc ion o eady- o-ea edible c ab in ol e a
se ies o gene al s eps which ha e no e ol ed in line wi h cu en echnological
de elopmen s. Many p oduce s s ill use adi ional echniques and de ine hei
own cooking condi ions in e ms o ime and empe a u e which leads o
he e ogenei y in he quali y o ma ke able p oduc s (e.g. o e o
unde cooking). The size o hese companies is usually small and hei
in es men in echnology and p ocess op imisa ion is gene ally low. Howe e ,
no el p ocessing echnologies such as ul asound ha e many bene i s o o e
hem. Fo example, ul asound echnology is widely used in he ood indus y o
enhance hea and mass ans e p ocesses (Chand apala,
e al
., 2012; Chema ,
e al
., 2011) which could ha e g ea ele ance and be easily adop ed o
imp o e adi ional imme sion cooking p ocesses used in he p oduc ion o
eady- o-ea c ab.
High-in ensi y ul asound in ol es in ensi ies g ea e han 1 W/cm2 and is
pe o med a equencies anging om 18 o 100 kHz. Ca i a ion is conside ed
he main mechanism by which his o m o ul asound enhances hea and mass
ans e phenomena (Kim,
e al
., 2004) hough o he e ec s such as acous ic
s eaming a e also in ol ed (Legay,
e al
., 2011). The e ec s o ul asound on
hea ans e ha e been ex ensi ely s udied since he 1990s in model sys ems
such as wa e , me al ubes, me al balls, e c. (Huamao,
e al
., 1997; Hyun,
e
al
., 2005; Oh,
e al
., 2002) and i s abili y o enhance hea ans e in oods,
mainly in p ocessed mea p oduc s, du ing cooking p ocesses has also been
p o en (Ala con-Rojo,
e al
., 2015; Vimini,
e al
., 1983). The po en ial o
ul asound o assis di e en p ocesses such as ex ac ion (Khan,
e al
., 2012;
Luengo,
e al
., 2014; Ma,
e al
., 2009), cooking (Mason,
e al
., 1996) and
ma ina ing (Cá cel,
e al
., 2007; McDonnell,
e al
., 2014; Tu shan,
e al
., 2013;
Vinimi,
e al
., 1983) ha e been assessed in ege ables issues, mea s and ish.
Howe e , i s e ec s on he indus ial hea p ocessing o eady- o-ea
c us aceans p oduc s ha e ne e been explo ed. The e o e, he aim o his
Chap e 3
105
s udy was o assess he po en ial o ul asound o he cooking o b own c ab
by accele a ing hea and mass ans e p ocesses. The inc ease o hea
pene a ion should lead o a educ ion in cooking imes which in u n should
enhance p oduc quali y whils ensu ing adequa e le els o sa e y. In addi ion,
he p oduc ion o eady- o-ea b own c ab in ol es a cleaning/cooling s ep
which is needed o emo e c ab di and cook exuda e deposi s be o e packing.
This s ep akes 3-4 h and cons i u es a mic obiological isk due o a possible e-
con amina ion o he p oduc , hence equi ing a subsequen pas eu iza ion s ep
wi h he sole pu pose o elimina ing mic obial con amina ion (Ghazala and
T enholm, 1996; Pagadala,
e al
., 2012). The abili y o ul asound o enhance
mass ans e could also be used o emo e di and exuda e om c ab shells
du ing cooking hus elimina ing o educing he se e i y o he subsequen
pas eu iza ion ul ima ely esul ing in g ea e yields, less ene gy inpu and a
milde hea - ea ed highe quali y p oduc .
The e o e he objec i e o his esea ch was o e alua e he po en ial
imp o emen s induced by he applica ion o ul asound in he cooking p ocess
o eady- o-ea whole b own c ab, wi h pa icula e e ence o he bene i s o
associa ed hea and mass ans e phenomena.
3.3 MATERIALS AND METHODS
3.3.1
Raw ma e ial and cooking condi ions
All pe o med expe imen s we e ca ied ou wi h emale c abs wi h weigh s
anging om 375 o 732g, landed in I eland in he win e o 2014. Those we e
ob ained om a local ishmonge and main ained ali e a 4ºC in d y condi ions
o a maximum o 48 h. A e s o age, c abs we e adequa ely eu hanized (Ro h
and Øines, 2010) while main aining he in eg i y o he ca apace. Be o e
cooking, each c ab was cha ac e ised by measu ing weigh (g ams) and
dimensions (cm2) (assuming ha he c ab shape was o al, he a ea o which
was mul iplied by wo in o de o accoun o bo h sides o he c ab). A e
cooking he wo ypes o c ab mea , whi e and b own, we e e alua ed
Chap e 3
106
sepa a ely. The whi e mea is he mea loca ed in claws and legs and he b own
mea inside he ca apace. The dis inc ion be ween he wo ypes o mea was
conside ed due o hei di e en composi ion and ma ke alue.
Cooking expe imen s wi h and wi hou ul asound we e pe o med
immedia ely a e eu hanasia in an ul asonic ba h (Guyson mod. KS MK3 525,
No h Yo kshi e, UK) wi h a ank capaci y o 55L, a maximum ul asound powe
o 900 W and a hea ing powe o 2000 W. All ials we e ca ied ou by using
he maximum olume o wa e (55L). Once he empe a u e o he wa e
eached 75ºC, eigh c abs we e subme ged and cooked o 45 min. P elimina y
expe imen s (da a no shown) showed ha 45 min was a su icien cooking
ime o apply an equi alen
F
70ºC7.5ºC o 2 min, which co esponds o he FDA
ecommended hea ea men o eady- o-ea sea ood p oduc s. This hea
ea men ensu es he inac i a ion o a leas 6 Log10 cycles o
Lis e ia
monocy ogenes
, he a ge mic oo ganism in pas eu ised sea ood p oduc s
(FDA, 2011). Fo hose expe imen s applying ul asound, he maximum
ul asonic powe o he ank was used, 900W (ul asonic ene gy consump ion).
In o de o s anda dize he cooking condi ions as much as possible, he o al
weigh o all ba ches anged om 4.0 o 4.3kg. A leas h ee independen
eplica es o each cooking condi ions we e pe o med on di e en wo king
days.
3.3.2
Hea ans e s udy
To assess he e ec o ul asound on he hea ans e phenomena in c abs
wo di e en compa isons, based on ma hema ical models, we e ca ied ou .
3.3.2.1
Hea pene a ion cu es
Hea pene a ion cu es we e ob ained by placing a K ype he mocouple
(Alhbo n, Holziki chen, Ge many) in he abdomen o he c ab which
co esponds o i s cold spo (Figu e 3.1) which had been p e iously iden i ied in
p elimina y expe imen s (Figu e 3.2).
Chap e 3
113
Table 3.1 Con inued
Weigh (g)
Cooking ype
Leng h
(cm)
Wid h
(cm)
h
j
R2
RMSE
300.0
Con en ional
13.0
8.0
18.45
1
0.95
4.19
348.0
Con en ional
14.0
9.0
21.44
1.05
0.97
1.54
375.5
Con en ional
14.0
9.0
31.48
1.21
0.99
1.15
388.0
Con en ional
14.0
8.5
20.68
1
0.95
3.47
398.0
Con en ional
14.5
9.0
29.09
1.11
0.99
0.31
404.0
Con en ional
14.5
8.5
26.45
1.02
0.99
0.55
411.4
Con en ional
15.0
9.5
22.79
1.07
0.99
0.59
428.0
Con en ional
14.0
9.5
17.12
1
0.98
1.09
432.0
Con en ional
14.5
9.0
24.15
1.47
0.99
1.01
455.4
Con en ional
14.5
9.0
33.99
1.4
0.99
0.91
462.8
Con en ional
15.0
10.5
27.68
1.48
0.99
0.69
472.0
Con en ional
16.0
9.0
27.55
1.27
0.99
0.03
478.0
Con en ional
14.5
9.0
30.12
1.36
0.99
0.21
484.0
Con en ional
15.0
9.0
34.14
1.23
0.99
0.39
500.0
Con en ional
16.5
10.5
35.45
1.58
0.99
0.47
538.0
Con en ional
17.0
10.5
37.88
1.13
0.99
0.19
553.6
Con en ional
16.0
10.0
40.62
1.11
0.99
0.31
586.9
Con en ional
16.5
10.0
35
1.06
0.99
1.62
614.6
Con en ional
16.0
10.0
39.91
1.47
0.99
0.48
630.5
Con en ional
16.5
11.0
43.4
1.09
0.99
0.24
640.7
Con en ional
18.8
11.0
43.67
1.5
0.99
0.47
714.0
Con en ional
18.0
11.5
41.35
1.4
0.99
0.69
732.7
Con en ional
17.5
11.0
42.99
1.05
0.99
0.30
799.4
Con en ional
17.5
11.0
40.91
1.06
0.99
1.91
816.8
Con en ional
19.0
12.5
42.65
1.28
0.98
1.60
829.7
Con en ional
18.0
11.0
40.97
1.29
0.99
0.64
869.7
Con en ional
19.0
13.5
64.32
1
0.90
0.06

Chap e 3
114
In addi ion, a linea ela ionship be ween
h
alues and c ab weigh was
no ed in each cooking p ocess indica ing ha hea pene a ion in he c ab cold
spo is weigh -dependen . Table 3.2 includes he i s o de Equa ions which
co ela e he inc ease in
h
alues wi h he inc ease o he weigh o he c ab in
bo h cooking p ocesses. Signi ican ly di e en slopes we e obse ed be ween
Equa ions (P≤0.05), indica ing ha he c ab weigh a ec ed hea pene a ion o
a di e ing ex en depending on he cooking p ocess. When ul asound was
used o assis he cooking he slope was 2.6- old smalle , meaning ha he
weigh o he c ab had a much smalle e ec on inc eases in
h
alue. Hence,
he la ge he c ab he g ea e he impac o ul asound in enhancing he
hea ing a e as indica ed in Table 3.2.
Table 3.2: Fi s o de Equa ions co ela ing
h
alues (dimensionless) wi h he weigh o b own
c abs (g) du ing con en ional and ul asound-assis ed cooking.
Cooking ype
Equa ion
RMSE
B
A
Con en ional
𝑓ℎ=0.053× 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) +4.9
5.57
1.01
1.14
Ul asound-assis ed
𝑓ℎ=0.020×𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) + 20.4
4.23
1.01
1.12
Much o he wo k published o da e a ibu es he ul asonic enhancemen o
hea ans e o he o ma ion o ca i a ion bubbles (Wong and Chon, 1969),
al hough o he au ho s ha e also sugges ed ha imp o emen s in con ec ion
hea ans e may also be due o acous ic s eaming (Gould, 1966; Hyun,
e al
.,
2005). Ei he way, i is gene ally accep ed ha ul asonically induced hea ing is
a esul o ene gy dissipa ion om he accumula ion o ca i a ion bubbles a he
in e ace o he subme ged body (Ba igi and Ba oli, 2012; Kiani,
e al
., 2012).
Addi ionally i is accep ed ha he numbe and densi y o ca i a ion bubbles can
play an impo an ole in he hea ans e caused by ul asound and also ha
he ca i a ion o bubbles inc eases he mic o-con ec ion e ec a he p oduc
su ace (Kim,
e al
., 2004).
In ela ion o he su ace a ea, some esea che s ha e epo ed a
ela ionship be ween he weigh and dimensions (wid h and leng h) o he
Chap e 3
115
ca apace o a c ab (Klaouda os,
e al
., 2013). In he p esen s udy a linea
ela ionship ( ep esen ed by Equa ion 3.5) was ound be ween he su ace a ea
(cm2) o he ca apace and he c ab weigh (g) (Figu e 3.3).
Figu e 3.3: Rela ionship be ween c ab’s weigh ( om 300 o 870 g) and he o al su ace o
he c ab’s ca apace (cm2).
𝑆𝑢𝑟𝑓𝑎𝑐𝑒 (𝑐𝑚2)=0.362× 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑔) + 54.83 (
R
2 = 0.85) (Eq. 3.5)
This ela ionship indica es ha hea ie c abs had highe ca apace su ace
a eas (Figu e 3.3). The e o e he g ea e impac o he ul asonic ield on he
educ ion o
h
alues in hea ie c abs could be a ibu ed o hei la ge su ace
a eas and as a consequence a g ea e amoun o ca i a ion bubbles a ound
hei su ace.
The e ec i eness o a hea ea men in e ms o mic obial inac i a ion is
gi en by he applied
F
alue. As indica ed be o e, he cooking p ocesses applied
in he cu en s udy we e designed ollowing FDA ecommenda ions (FDA,
2011) o ensu e a 6 Log10 educ ion o
L. monocy ogenes
in sea ood p oduc s
(
F
70ºC7.5ºC = 2 min). A cooking p ocess o 45 min in wa e a 75°C was
p e iously demons a ed o achie e his minimum ecommended
F
alue in all
0200 400 600 800 1000
0
100
200
300
400
500
weigh (g)
su ace (cm2)
Chap e 3
116
c abs i espec i e o hei weigh (da a no shown). Fo each cooking p ocess,
ei he wi h o wi hou ul asound, he ac ual equi alen
F
70ºC7.5ºC alue applied
was calcula ed based on he co esponding hea pene a ion cu es. Figu es
3.4A, 3.4B and 3.4C show he
F
70ºC7.5ºC alues a ained du ing he cooking o
c ab a 75°C wi hou ul asound (block line) and wi h ul asound (dashed line)
in small, medium and la ge c abs, espec i ely. Figu e 3.4 also shows he
h eshold o he a ge
F
70ºC7.5ºC o 2 min (ho izon al do ed line).
The
F
alue applied in he ul asound-assis ed cooking was 2.5-, 3.2- and
2.2- old highe han he con en ional hea -only cooking o he small, medium
and la ge c abs, espec i ely. In o he wo ds, he cooking ime was educed by
15.5% ( om 45 o 38 min), 16.9% ( om 45 o 37.4 min) and 12.7% ( om 45
o 39.3 min), espec i ely, while applying he same
F
alue when ul asound
was used du ing cooking.
Chap e 3
117
Figu e 3.4:
F
70ºC7.5ºC alue (min) applied du ing he cooking p ocess in he c ab's cold spo (i.e.
abdomen), wi h (dashed line) and wi hou (block line) ul asound o he (A) small, (B) medium
and (C) la ge c abs. The ho izon al do ed line ep esen s he a ge
F
70ºC7.5ºC o wo minu es.
A
0 5 10 15 20 25 30 35 40 45
0
5
10
15
20
25
30
C ooking im e (m in)
F707 .5 alue (m in)
Ta ge F707.5
B
0 5 10 15 20 25 30 35 40 45
0
5
10
15
20
25
30
C ooking im e (m in)
F707 .5 alue (m in)
Ta ge F707.5
C
0 5 10 15 20 25 30 35 40 45
0
5
10
15
20
25
30
C ooking im e (m in)
F707 .5 alue (m in)
Ta ge F707.5
Chap e 3
118
The e icacy o he wo cooking p ocesses was also e alua ed by
enume a ing o al bac e ial le els in whi e (Figu e 3.5A) and b own c ab mea
(Figu e 3.5B). A e 5 min o ul asound-assis ed cooking he mic obial load in
whi e mea was 0.6 Log10 cycles lowe han in samples which had unde gone
egula cooking (2.4
s
1.8 Log10 cycles), while b own mea needed a 10 min
longe ea men o achie e simila educ ions (2.3
s
1.8
log10 cycles). These
esul s indica e ha he mic obial educ ions in c ab a e signi ican ly highe in
p oduc s exposed o an ul asound assis ed cooking. In addi ion, he e ec o
ul asound alone was e alua ed by measu ing mic obial loads in whi e and
b own mea ea ed a empe a u es below 30ºC o 45 min. In his case no
di e ences we e de ec ed in he mic obial loads ega dless o he mea ype
(
p
>0.05).
Figu e 3.5: Mic obial load o e con en ional (g ey ba s) and ul asound assis ed cooking
p ocesses (black ba s) in (A) whi e mea and (B) b own mea . Do ed line shows he de ec ion
limi o he coun s.
A
0
5
10
15
30
45
0
1
2
3
4
5Regula cooking
Ul asonic cooking
Cooking ime (min)
Log c u/g
B
0
5
10
15
30
45
0
1
2
3
4
5Regula cooking
Ul asonic cooking
Cooking ime (min)
Log c u/g

Chap e 3
119
Some au ho s ha e sugges ed ha he highe mic obial inac i a ion le els
o en obse ed when ul asound and hea a e combined (i.e. he mo-
sonica ion) is due o addi i e o syne gis ic e ec s be ween he wo
echnologies (A oyo,
e al
., 2012; Raso,
e al
., 1998). Howe e , in he case o
c ab cooking, when ul asound was applied a low empe a u es he mic obial
loads did no dec ease. The e o e, he g ea e mic obial educ ion obse ed
could be a ibu ed o he ac ha ul asound imp o ed hea pene a ion in he
c ab a he han as a esul o he e ec o ul asound i sel . Fo example, as a
consequence o mo e apid hea pene a ion he cumula i e
F
alue inc eased
a a highe a e which would esul in g ea e educ ion in mic obial loads
du ing ul asonic cooking. Fu he mo e, ul asound may ha e sub-le hally
damaged bac e ial cell en elopes hus educing hea ole ance and he e o e
esul ing in g ea e mic obial educ ion alues compa ed o hose samples ha
ecei ed he hea only ea men (Sala,
e al
., 1995).
3.4.2
Mass ans e
Ul asound echnology is widely used in he ood indus y o imp o e
p ocesses in ol ing mass ans e phenomena such as cleaning, ex ac ion,
b ining, pickling, ma ina ing and cu ing (Chema ,
e al
., 2011; McDonnell,
e al
.,
2014). In his sec ion he e ec o ul asound on he mass ans e phenomena
occu ing du ing he cooking o c abs is quan i ied using wo di e en me hods.
3.4.2.1
Me hod 1: Tu bidi y and conduc i i y o cook wa e
Figu e 3.6A illus a es he mean cook wa e u bidi y alue a e cooking
wi h and wi hou ul asound. I is e y clea ha he u bidi y o he cook wa e
inc eased mo e apidly a e 15 min when ul asound was used o assis he
p ocess. A e 40 min o cooking wi h ul asound he u bidi y o he cook wa e
eached a maximum o 1.04 abso bance uni s which cons i u e a 113.7%
inc ease compa ed o u bidi y in con en ional cook wa e . The conduc i i y o
he cook wa e (Figu e 6B) also inc eased in he p esence o ul asound a e 10
min indica ing a as e a e o ionic compound elease om he c ab. The
Chap e 3
120
maximum inc ease in cook wa e conduc i i y was eached a e 35 min o
cooking, and was 55.7% highe han alues obse ed in wa e used o he
cooking wi hou ul asound. Regula comme cial p ac ice in he I ish c ab
indus y in ol es a cleaning/cooling s ep wi h esh wa e immedia ely a e
cooking. This s ep is c i ical in e ms o mic obial sa e y as econ amina ion can
po en ially occu . As a esul , immedia ely a e he cleaning/cooling s ep c abs
a e packed and pas eu ized (Edwa ds and Ea ly, 2001; Lali ha and Thampu an,
2012). I he u bidi y o he cook wa e is conside ed as an indica o o he
emo al o di om he c ab’s su ace, as Figu e 3.7 sugges s, he esul s
ob ained wi h ul asound indica e ha he use o his echnology may ha e he
po en ial o elimina ing he cleaning s ep as his ul asonically induced cleaning
would be done concu enly wi h he cooking.
Figu e 3.6: (A) Tu bidi y (OD515) and (B) conduc i i y (µS/cm) alues o he cook wa e
du ing he cooking o b own c abs in wa e a 75°C wi h (black ba s) and wi hou (g ey ba s)
ul asound.
A
0510 15 20 25 30 35 40 45
0.0
0.5
1.0
1.5
Cooking ime (min)
Abs (515 nm)
B
0510 15 20 25 30 35 40 45
0
200
400
600
Cooking ime (min)
conduc i i y (S/cm)
Chap e 3
121
Figu e 3.7: Pic u e o he c ab’s exuda e a e a con en ional cooking (A) and ul asound-
assis ed cooking (B).
3.4.2.2
Me hod 2: Sal con en o he c ab mea
The sal con en in bo h whi e and b own c ab mea was measu ed o
assess he po en ial o ul asound o ans e subs ances om he cook wa e
in o he c ab mea . The ini ial sal con en o aw c ab was 1.43% in he whi e
mea , which is loca ed mainly in claws and legs, and 0.89% in he b own mea ,
which is loca ed inside he ca apace.
Sal con en be o e and a e cooking wi h and wi hou ul asound and in he
p esence o absence o 5% NaCl in he cook wa e is shown in Table 3.3. When
he c abs we e cooked in wa e wi hou NaCl added he inal sal con en in
whi e mea was educed by 33.6% using egula cooking and by 46.1% o he
ul asound-assis ed cooking. When 5% NaCl was added o he cook wa e , he
sal con en in whi e mea emained s able a e egula cooking and was
educed by 21.1% ollowing ul asound assis ed cooking. Fo b own mea , he
sal con en emained he same when c abs we e cooked ei he wi h o wi hou
ul asound in wa e wi hou 5% NaCl added as opposed o whi e mea .
Howe e , when c abs we e cooked in wa e wi h 5% NaCl added, he sal
con en in b own mea sligh ly inc eased om 0.89% ( aw) up o 1.34% in he
absence o ul asound and emained unchanged wi h he applica ion o
ul asound. Ou esul s sugges ha ul asound did no acili a e he up ake o
Chap e 3
122
sal du ing cooking in wa e wi h 5% NaCl added. This e ec may be due o he
physical ba ie o he c ab ca apace which could ac as a esonan box on
which ca i a ion bubbles a e p oduced on he in e nal su aces. These bubbles
could c ea e mic o-cu en s which could aid in he elease o he sal om he
mea o he cook wa e e en agains an osmo ic g adien .
The e ec on whi e mea when c abs we e cooked in wa e wi hou NaCl
added sugges s ha ul asound enhances he elease o sal om he mea .
Some au ho s obse ed a simila e ec wi h enhanced ex ac ion when
ege able issues such as oma o peel we e ea ed wi h ul asound (Luengo,
e
al
., 2014; Rie a,
e al
., 2004). O he s udies assessing he e ec i eness o
ul asound o accele a ing he ma ina ing o mea s epo ed con as ing
indings o hose in he cu en s udy (Cá cel,
e al
., 2007; Mason,
e al
., 1996;
McDonnell,
e al
., 2014) showed ha he con en o sal in slices o po k
ende loin inc eased when hey we e soaked in a sa u a ed sal solu ion o 45
min wi h inc easing ul asound in ensi y. Also Si ó
e al
., (2009) showed a
signi ican imp o emen in sal di ussion in po k loins when ul asound was
applied. A simila e ec was obse ed by Tu han
e al
., (2013) who epo ed
enhanced a es o ma ina ing in ancho ies when ul asound was used.
Table 3.3: Sal con en (%) and mois u e con en (%) in whi e and b own c ab mea cooked
wi h o wi hou ul asound in wa e and wa e wi h 5% NaCl. Values ep esen mean alue ±
s anda d e o . Fo bo h sal and mois u e con en , ANOVA ollowed by Tukey’s es s we e
pe o med o whi e and b own mea independen ly.
Raw: Raw mea , CC: Con en ional cooking, USC: Ul asound-assis ed cooking. Di e en
supe sc ip le e s indica e signi ican di e ences (P≤0.05).
Whi e mea
B own mea
Wa e
Wa e wi h
5% NaCl
Wa e
Wa e wi h
5% NaCl
Sal con en
(%)
Raw
1.43 (0.03)c
1.43 (0.03)c
0.89 (0.03)a
0.89 (0.03)a
CC
1.06 (0.03)b
1.33 (0.03)c
0.96 (0.12)a
1.34 (0.02)b
USC
0.84 (0.02)a
1.17 (0.05)b
0.98 (0.02)a
0.81 (0.04)a
Mois u e
con en (%)
Raw
75.83 (3.85)b
75.83 (3.85)b
49.08 (0.54)a
49.08 (0.54)a
CC
75.02 (1.65)b
60.75 (0.92)a
56.61 (1.44)b
45.20 (0.20)a
USC
70.08 (0.89)ab
69.98 (2.85)ab
56.32 (1.70)b
63.60 (1.51)c
Chap e 8
225
Mo eo e and in pa allel o he colou change kine ics s udy, he quali y
pe cep ion o he whi e mea was co ela ed wi h a ΔE* pa ame e du ing a
se ies o ocus g oups wi h he main edible c ab p oduce s o I eland. Based on
he ob ained esul s whi e mea was classi ied, as “good quali y” (ΔE* ≤ 7),
“accep able quali y” (7< ΔE* <9) and “non-accep able quali y” (ΔE* ≥ 9) based
on hei colou (Figu e 5.7 in Chap e 5). One o he main ad an ages o his
s udy is ha , he Equa ions de eloped o he colou change, could be easily
ans e ed o o he c ab p oduc s such as picked whi e mea o c ab claws.
Using he limi s es ablished abo e in e ms o he ΔE* pa ame e , he
he mal p o ile o c ab claws and c ab’s “cold-spo ”, an op imiza ion g aph was
cons uc ed (Figu e 5.8 in Chap e 5). As obse ed, depending on i s weigh ,
di e en hea ea men s we e equi ed o achie e he goal o
F
90°C7o 10°C=
10min, in he c ab’s “cold-spo ”. To e alua e he e ec o he c ab weigh on
he pas eu iza ion pa ame e s, Figu e 8.1 shows he maximum empe a u es a
which i would be possible o pas eu ize whole cooked c abs o main ain a
“good quali y” o “accep able quali y” in hei whi e mea , based on he
equi ed ime a each empe a u e o achie e an app op ia e le el o
C.
bo ulinum
non-p o eoly ic ype E inac i a ion. In his Figu e all empe a u es
ep esen ed by he g een line o below would p oduce c abs wi h a “good
quali y” in e ms o whi e mea colou , while he empe a u es in he a ea
be ween g een and blue line would p oduce an “accep able quali y”. Also in he
Figu e i can be obse ed ha inc eases in he weigh o he c ab suppose a
educ ion o he maximum pas eu iza ion empe a u es, o main ain a ce ain
quali y, in a p opo ion o 1°C o an inc ease o 100g. All o hese calculi a e
e e ed o he quali y associa ed wi h he colou o whi e mea .

Chap e 8
226
Figu e 8.1: Maximum pas eu iza ion empe a u es allow o achie e he a ge
F
90°C10°C = 10
minmain aining “good quali y” (g een line) and “accep able quali y” (blue line) depending on
he c ab weigh , based on he equa ions de eloped in Chap e 5.
The e ec o c ab weigh on pas eu iza ion pa ame e s is a consequence o
he di e en he mal p o iles o c ab claws (whe e he whi e mea is loca ed)
and c ab abdomen (whe e he “cold-spo ” was de ined) as is ep esen ed
Figu e 5.1 (Chap e 5). While he c ab weigh did no a ec he he mal p o ile
o he claws, because small di e ences be ween claws exis ega dless he size
o he c abs. The impac o c ab weigh has a big impac on he he mal p o ile
o he “cold-spo ” (i.e. c ab’s abdomen). The e o e o he pas eu iza ion o
bigge c abs a educ ion o he pas eu iza ion empe a u e is equi ed, based
on he colou change kine ics desc ibed, due o he longe pas eu iza ion ime
necessa y o achie e he pas eu iza ion objec i e in he “cold-spo ”. These
esul s endo se, as in case o cooking, he impo ance o he classi ica ion s ep
p io o p ocessing in o de o a oid he o e -hea ing o unde -hea ing wi hin
c abs in he same ba ch.
In based o he esul s ob ained in his PhD Thesis conside ing mic obial
and quali y aspec s al eady discussed wi h espec o he FDA ecommended
pas eu iza ion ea men , a heo e ical op imisa ion g aph o he pas eu iza ion
o eady- o-ea whole edible c ab has been gene a ed. This g aph has been
gene a ed hanks o he equa ions de eloped in Chap e 5 (i.e. Eq. 5.9 and Eq.
300 400 500 600 700 800 900
85
90
95
100
105
110
115 T max. Good
T max. Accep able
C ab weigh (g)
Max. pas . Tmp. (C)
Chap e 8
227
5.12) and Chap e 3 (Eq. 3.2 and Eq. 3.3). I has o be poin ed ou ha he
ma hema ical Equa ions de eloped in Chap e 5 allow he de ini ion o
ea men condi ions o whiche e he a ge mic oo ganism may be and no
only cen ed on
C. bo ulinum
non-p o eoly ic ype E. Figu e 8.2 compa es he
ea men s equi ed o
C. bo ulinum
(
F
90°C10°C= 10 min)
s.
hose p oposed o
B. weihens ephanensis
(
F
105°C7.6°C= 6 min) wi h he impac on he quali y o
whi e mea .
Figu e 8.2: Theo e ical op imisa ion g aph o he pas eu iza ion o eady- o-ea whole edible
c ab based on Equa ions 5.12 and 5.13 o c abs o 600g o he inac i a ion o 6 Log10
educ ions o
C. bo ulinum
non-p o eoly ic ype E based on U.S. FDA ecommenda ions and
B.
weihens ephanensis
based on he he mal esis ance ob ained in his PhD Thesis (Chap e 6).
As Figu e 8.2 shows, wi h
B. weihens ephanensis
conside ed as he a ge
mic oo ganism he pas eu iza ion pa ame e s (i.e. ime/ empe a u e
combina ions) equi ed o achie e a simila inac i a ion le el o
C. bo ulinum
non-p o eoly ic ype E (i.e. 6 Log10 cycles) would p oduce conside able changes
in he colou o c ab whi e mea . In ac , based on Figu e 8.2 in he ange o
empe a u es om 90 o 120°C, he equi ed imes o educe he popula ion o
B. weihens ephanensis
by 6 Log10 cycles will p oduce p oduc s wi h only
“accep able” quali y. I a “good” quali y is o be main ained, he pas eu iza ion
80 90 100 110 120 130
1.0
1.5
2.0
2.5
B. weihens ephanensis
Accep able Good excellen
C bo ulinu,
T ea men empe a u e (C)
Log10 pas e uiza ion ime (min)
No pas eu ize
“Good”
Pas eu ize
C. bo ulinum
“Good”
Pas eu ize
C. bo ulinum
“Accep able”
Pas eu ize
B. weihens ephanensis
“Accep able”
Pas eu ize
B. weihens ephanensis
“No Accep able”
Chap e 8
228
empe a u es mus be ≤90°C. Howe e , due o he high he mal esis ance o
B. weihens ephanensis
, hese ea men s would equi e an excessi ely long
ea men ime (i.e. o e 3 hou s).
These esul s sugges he need o looking o s a egies which enable o
imp o e he le hal e ec i eness o he cu en hea ea men s o o ind
echnologies which gua an y he sa e y and s abili y o he de ined hea
pas eu iza ion ea men s while minimally impac ing c ab quali y. As men ioned
in Chap e 1 se e al al e na i e echnologies could be employed o educe o
a oid he nega i e hea induced impac when imposing hea ea men s o he
inac i a ion o spo es. Du ing he de elopmen o his PhD Thesis wo o hese
al e na i e echnologies, ul asound in combina ion wi h p essu e and hea and
elec on beam ionizing adia ion, we e explo ed in e ms o hei po en ial o
inac i a e he main bac e ial spo es isola ed o m edible c ab.
As p e iously discussed, ul asound has he po en ial o imp o e he
cooking and cleaning o edible c ab. Also in Chap e 3 he po en ial le hali y o
ul asound du ing cooking was discussed (Figu e 3.5 A and B). The esul s
ob ained sugges ha ul asound can inc ease mic obial inac i a ion du ing
cooking. Howe e , unde a mosphe ic p essu es and he low ul asonic powe
densi y employed in he p esen s udy, his e ec is mo e likely due o
ul asonically induced imp o emen s in hea ans e . Using he same ul asonic
condi ions, bu a oom empe a u e (i.e. 20°C) no mic obial inac i a ion was
obse ed. Unde ce ain condi ions ul asound is conside ed as one o he new
al e na i e mic obial inac i a ion echnologies o con en ional hea ea men s
(U.S. FDA, 2000) when applied a empe a u es simila o hose used in he
p esen s udy. Howe e , o e ec i e mic obial inac i a ion he ul asonic powe
densi y equi ed is conside ably highe han ha employed in he p esen s udy
and also ul asound is gene ally applied unde p essu e. Due o he low
bac e icidal e icacy o ul asound a oom empe a u e (Lee
e al
., 2013;
Meullemies e
e al
.,
2017; Jamb ak,
e al
., 2017), mos esea che s ha e ied
o design combined p ocesses o enhance he o e all le hal e icacy o
Chap e 8
229
ul asound (López-Malo
e al
.,
2005; Lee
e al
., 2013; Raso
e al
.,
1998a). Some
o he combined ul asound p ocesses p oposed o da e which inc ease he
le hal e ec o ul asound a e he mosonica ion (TS), manosonica ion (MS) and
mano he mosonica ion (MTS) (Chema
e al
., 2011; Piyasena
e al
., 2003; Sala
e al
.,
1995).
Resul s ob ained in Chap e 3 showed ha he applied TS ea men (i.e.
cooking a 75°C in p esence o ul asound in an ul asonic ba h) did no
p oduce a subs an ial inc ease o mic obial inac i a ion, hough MS and MTS
ea men s p o ed o be e ec i e o he inac i a ion o spo es isola ed om
c ab, in liquid media (Chap e 6). I has been epo ed ha he bac e icidal
e icacy o ul asound is di ec ly co ela ed wi h he ul asonic powe ans e ed
o he media (Mañas
e al
., 2000). In Chap e 6 all ials we e ca ied ou in a
speci ic de ice specially designed o e alua e he bac e icidal po en ial o TS,
MS and MTS ea men s, which allows he applica ion o ul asonic powe
densi ies anging be ween 1600 and 8000W/L a 20kHz. In Chap e 6 an
ul asonic powe o 4800W/L and 20kHz was used o e alua e he po en ial o
MS and MTS ea men s while o he TS ea men , which was applied in an
ul asonic ba h o c ab cooking a a mosphe ic p essu e, a maximum ul asonic
powe densi y o 10W/L and 35kHz was used. Al hough, ul asonic equency
could ha e an in luence on he mic obial inac i a ion, he di e ence on he
applied ul asonic powe would mo e han likely be he main eason why in case
o TS no bac e icidal e ec was obse ed while MS and MTS ea men s p o ed
o be e ec i e o bac e ial spo e inac i a ion.
Al hough he esul s ob ained in Chap e 6 show g ea po en ial o MTS
ea men (using empe a u es o e 80°C and an ul asonic inpu powe o
4800 W/L) o inac i a e he main bac e ial spo es isola ed om c ab, o his
p ocess is gene ally only conside ed o pumpable liquids and is no eadily
adap able o whole c ab, hough i s ad an ages could be exploi ed only o
liquid p oduc s such as c ab soups. Howe e , he high ul asonic ene gy
equi ed o bac e ial spo e inac i a ion using MTS ea men s, (4800W/L)
Chap e 8
230
compa ed o o he ul asound applica ions such as assis ed eezing ( om 7.3
o 25.89W/L) (Li and Sun, 2002) o ma ina ing ( om 800 o 1200W/L) (Tu han
e al
., 2013) makes i s indus ial applica ion di icul due o he high ene gies
in ol ed. Ei he way, conside ing he esul s ob ained o bac e ial spo es
inac i a ion using ul asound in combina ion wi h hea plus p essu e (MTS)
(Chap e 6), i enough ul asonic powe is applied du ing he pas eu iza ion
p ocess , wi h
B. weihens ephanensis
conside ed as a a ge mic oo ganism, a
subs an ial educ ion, o o e he 80%, o he pas eu iza ion ime could be
eached as shows Figu e 8.3.
Figu e 8.3: Log o he ime equi ed o achie e 6 Log10 educ ions o he popula ion o
B.
weihens ephanensis
by hea (black ba s) and MTS (whi e ba s) a di e en empe a u es in pH
6.8 McIl aine ci a e-phospha e bu e .
Besides MTS echnology, in his PhD Thesis Elec on Beam Ionizing
adia ion (EBI) was also e alua ed. Ini ially, his echnology would ha e mo e
iabili y, i legally pe mi ed, since i could be applied o cooked whole c ab and
c ab p oduc s in pack. As i is discussed in Chap e 7 one o he majo
ad an ages o ionizing adia ion is i s capabili y o inac i a e he main bac e ial
spo es p esen in c ab a low empe a u es (e en ozen) which in u n would
a oid he ad e se e ec s p oduced du ing hea pas eu iza ion. F om he esul s
shown in Chap e 7, Figu e 8.4 shows he equi ed dose o educe he
popula ion o he main h ee bac e ial spo es isola ed om edible c ab by 6
Log10 cycles. Based on Figu e 8.4, ioniza ion ea men s below 8kGy appea o
80
85
90
95
0
1
2
3
4
5Hea ea men
MTS ea men
T ea men empe a u e (C)
Log 6D (min)

Chap e 8
231
be su icien o achie e a p ope inac i a ion le el o he h ee main spo es
isola ed om edible c ab, iden i ied in his PhD Thesis. The esul s ob ained also
showed ha despi e he di e en composi ion and chemical cha ac e is ics o
he wo ypes o c ab mea (Anacle o
e al
.,
2011; Ba en o
e al
.,
2010a), he
esis ance o he bac e ial spo es was ba ely a ec ed by he mea ype.
The e o e, i he pene a ion dep h, which depends on he adia ion sou ce, is
su icien hese esul s indica e ha a homogeneous inac i a ion will be
p oduced du ing he ioniza ion ea men wi hin he c ab. Addi ionally, he
speci ic esis ances o he bac e ial spo e species in c ab mea we e simila o
hose de ec ed in lab media a simila pH (7) and aw (>0.99). The e o e,
conside ing he esul s ob ained in he p esen PhD hesis, he adia ion dose
calcula ed in lab media o a speci ic mic oo ganism could be used as e e ence
o calcula e he necessa y ea men s o achie e a ce ain le el o sa e y o
s abili y o a speci ic oods u .
Figu e 8.4: Requi ed adia ion dose o educe 6 Log10 cycles he popula ion o he main h ee
bac e ial spo es isola ed om edible c ab in he wo ypes o c ab mea .
Conside ing ha he maximum pe mi ed adia ion dose o oods is10 kGy
(WHO 1981), he esul s ob ained endo sed he use ulness o his echnology
o he pas eu iza ion o edible c ab, since an adequa e le el o inac i a ion o
Da a 1
B. mycoides
B. weihens ephanensis
P. psych odu ans
0
2
4
6
8
10 Whi e mea
B own mea
6D i adia ion dose (kGy)
Chap e 8
232
he bac e ial spo es isola ed om c ab could be eached by applying adia ion
doses below 10kGy. Howe e , he downside is ha al hough mo e han 60
coun ies wo ldwide now ha e egula ions ega ding he use o ionizing
adia ion o ood p oduc s (IAEA, 2017) i s use in Eu ope is s ill e y limi ed o
ood pas eu iza ion especially o sea ood p oduc s. Only Belgium, Czech
Republic and Uni ed Kingdom ha e au ho ized he i adia ion o ish and
sea ood and only up o a maximum dose o 3kGy. I has been also epo ed
ha a adia ion dose ≤ 2kGy p oduces a signi ican ex ension o he shel -li e o
di e en c ab p oduc s hough; ea men s o e his dose migh p oduce
signi ican impac on he senso y pe cep ion o hese p oduc s (Chen
e al
.,
1996; ICGFI, 1998). The e o e in o de o inalise his s udy, u u e wo k would
in ol e a senso y e alua ion o de e mine he impac o i adia ion on he c ab
mea quali y and he maximum applicable doses o a oid possible undesi able
changes on i s senso y cha ac e is ics, hough his was beyond he scope o he
p esen s udy.
Conside ing he esul s ob ained du ing he de elopmen o his PhD
Thesis, a numbe o di e en al e na i e p ocesses ha e eme ged which can
imp o e he p oduc ion o eady- o-ea edible c ab and c ab based p oduc s.
Figu e 8.5 shows hese p ocesses o c ab p oduc s based on di e en
al e na i e echnologies a di e en s ages in c ab p ocessing. As Figu e 8.5
shows, di e en al e na i e p ocesses can be applied o he p oduc ion o
eady- o-ea c ab p oduc s using no el echnologies. Blue shadow boxes
ep esen he adi ional s eps cu en ly applied in he c ab indus y while,
whi e boxes ep esen he possible al e na i e applica ions, a di e en s ages
o he p oduc ion line. On he o he side, black lines connec he common s eps
applied o all c ab p oduc s o ea ly p ocessing s age, independen o i s
o ma . While g een lines connec he speci ic p ocesses depending on he
di e en o ma s i.e. whole cooked c ab (G een block lines), picked whi e and
b own mea s o claws (G een dashed lines) o ans o med c ab p oduc s
(G een do ed line).
Chap e 8
233
On one side, he esul s ob ained in his PhD Thesis p o ed he possibili y
o imp o ing he adi ional p ocesses conside ing he ob ained da a ega ding
he mic obio a p esen in edible c ab (Chap e 2), cooking p ocess (Chap e 3)
and he e ec o second pas eu iza ion on quali y cha ac e is ics o edible c ab.
Rega ding he use o he di e en al e na i e echnologies he esul s ob ained
in his PhD Thesis showed ha ul asound echnology could be used a di e en
s ages wi h di e en goals. This echnology could be applied in he ea ly
p ocessing s ages as a p e- ea men p io o cooking o educe he Cd con en
o he c ab (Chap e 4) o could be also implemen ed in he cooking p ocess
(Chap e 3) whe e, on one side would enhance hea ing a es in he c ab cold-
spo and on he o he side would allow o a oid he washing s ep, a e
cooking, which in u n would limi he econ amina ion o he cooked c ab
(Figu e 8.5). Addi ionally ul asound echnology could be also used in
combina ion wi h hea and p essu e (MTS) (Chap e 6), in ce ain ype o c ab
based p oduc s such as c ab soups. Howe e , he down side o his echnology
o pas eu iza ion pu poses is he ele a ed ene gy equi ed, which makes e y
di icul i s indus ial applica ion. ,
Ano he echnical op ion o edible c ab pas eu iza ion is elec on beam
ionizing adia ion, which has been shown in his PhD Thesis (Chap e 7) o be
an e ec i e echnology o inac i a ing he main bac e ial spo es isola ed om
edible c ab. Un o una ely, while i is e y e ec i e, i s use o sea ood
pas eu iza ion is cu en ly no pe mi ed in mos EU ju isdic ions. In coun ies
whe e i s use is app o ed a maximum ea men dose o 3kGy is all ha is
pe mi ed hough he esul s ob ained in Chap e 7 o he p esen s udy sugges
ha ea men s o be ween 6 and 8kGy a e necessa y in o de o a ain
su icien inac i a ion o bac e ial spo es isola ed om c ab.
Thus, new s a egies based on ei he he op imiza ion o adi ional
p ocesses o on he e o i ing o al e na i e echnologies in o adi ional
p ocesses, could be used o o p ocess in ensi ica ion and o enhancing
compe i i eness in he c ab p ocessing sec o .
Chap e 8
234
Shells disca ded
C ab anspo o p ocessing plan
C ab classi ica ion
C ab slaugh e
C ab Ul asonic Cooking/Cleaning
Ul asonic p e- ea men (Cd emo al)
C ab Cooking
C ab Cooling/Cleaning
C ab Cooling
Shelling
P ocessing/T ans o ma ion
Re o pas eu iza ion
Radicida ion
MTS pas eu iza ion (Liquid p oduc s)
Asep ic packaging
End p oduc s s o age and ma ke
C ab/Mea s/T ans o med p oduc s Packaging
En i e c ab p ocessing
Claws and c ab mea s p ocessing
T ans o med c ab p oduc s p ocessing
Figu e 8. 5: Flow diag am o he di e en al e na i e p ocesses p oposed, using di e en al e na i e echnologies.
Chap e 9
241
mycoides
and
P. psych odu ans
espec i ely. Also om he esul s ob ained i
can be deduc ed ha an ag eemen be ween spo e esis ance in c ab mea s
and lab media, wi h simila cha ac e is ics in pH and aw, exis which opens he
possibili y o ex apola e he esul s ob ained in- i o o di e en eady- o-ea
c ab based p oduc s
O e all, om he inac i a ion esul s ob ained i can be concluded ha he
esis ance a iabili y among he main h ee spo e o me s species agains hea
ea men s is much highe han he a iabili y obse ed o MTS o EBI
ea men s. EBI ea men showed he lowes a iabili y among bac e ial spo es
esis ance (20%) ollowed by MS (70%), MTS (440%) and inally hea
(4400%). The e o e, he use o hese al e na i e echnologies would educe he
po en ial isk associa e wi h he pas eu iza ion o p oduc s due o he w ong
choice o he a ge mic oo ganism.
The esul s p esen ed in his PhD hesis poin ou he possibili y o
imp o ing he p oduc ion o eady- o-ea b own c ab and b own c ab based
p oduc s by in oducing di e en echnologies a di e en s ages o he
p oduc ion p ocess o by op imizing cu en p ac ices, o sol e he challenges
associa ed wi h he p ocessing o his ype o p oduc s.

Chap e 10
242
Chap e 10
Resumen y Conclusiones
Chap e 10
243
La indus ia alimen a ia es á en con inua compe encia y e olución, lo que
equie e una g an ac i idad inno ado a apoyada de una g an labo de
in es igación y desa ollo. En el caso del sec o pesque o y más conc e amen e
en la p oducción de buey de ma y sus p oduc os lis os pa a el consumo, la
I+D+i se ha con e ido en una pa e undamen al. El desa ollo de la p esen e
Tesis Doc o al da espues a a muchas necesidades del sec o pe mi iendo
ob ene a ances conside ables pa a el sec o al in oduci nue as ecnologías
de p ocesado en su cadena p oduc i a.
En la p oducción de buey de ma y sus p oduc os lis os pa a el consumo se
lle an a cabo di e sas e apas que a con inuación se de allan y en las que se
pueden abo da nue os desa ollos. Así, el p ocesado equie e de los siguien es
pasos: una ez sac i icado el cang ejo, es os son cocinados, gene almen e en
agua hi iendo du an e 20-30 minu os (p ime a amien o é mico). T as el
cocinado, se ealiza un p oceso de en iamien o y la ado necesa io pa a e i a
la suciedad y los exudados del capa azón del cang ejo, p oducidos du an e el
cocinado. Una ez en iados los cang ejos que an a se des inados a la en a
como en e os, es os son en asados, gene almen e a acío, y se les aplica un
segundo a amien o é mico, en es e caso de pas eu ización. Po o o lado, los
cang ejos des inados a la p oducción de p oduc os elabo ados o simplemen e
pa a la en a de las ca nes de cang ejo po sepa ado, se asladan a la zona de
descasca illado as el en iamien o con el in de e i a ambos ipos de ca ne
(la ca ne blanca de las pinzas y pa as y la ca ne ma ón del in e io del
capa azón). Es e p oceso se ealiza p incipalmen e de mane a manual.
Finalmen e, las ca nes de cang ejo son en asadas, de di e en es o mas y
u ilizando di e en es o ma os según con enga, an es de aplica les un
a amien o de pas eu ización. Aunque es os pasos son comunes pa a odos los
p oduc o es de buey de ma lis o pa a el consumo, exis e una g an a iabilidad
en la calidad en e p oduc os de simila es ca ac e ís icas y o ma os. Es a
a iabilidad se pod ía a ibui a una al a de es anda ización de los p ocesos
debido al pequeño amaño de las compañías (que gene almen e son emp esas
Chap e 10
244
amilia es) y a su educida in e sión en ecnología y op imización de los
p ocesos. Pa a ealiza la op imización de un p oceso, se equie e un
conocimien o adecuado an o de la ma e ia p ima como de las a iables
in oluc adas en el p ocesado. Además, es ambién necesa io iden i ica los
iesgos y eque imien os de la ma e ia que deben se cubie os po el p oceso
(S o o os, 1995).
En el caso del buey de ma lis o pa a el consumo, muy pocos es udios se
han cen ado en los mic oo ganismos esponsables de su de e io o y ninguno
de ellos los ha iden i icado. La p ime a pa e de es a Tesis Doc o al (Capí ulo 2)
se cen ó en la ca ac e ización e iden i icación de los p incipales
mic oo ganismos p esen es en el buey de ma c udo y cocinado lis o pa a el
consumo así como la iden i icación de los mic oo ganismos esponsables de su
al e ación y el es udio de su e mo esis encia con el in de alo a la e icacia
de los ac uales a amien os é micos aplicados a es os p oduc os según la FDA
de los EE.UU. Los esul ados ob enidos mos a on que los g upos bac e ianos
p esen es en mayo p opo ción en el buey de ma c udo ue on
Pseudomonas
spp.,
Bacillus
spp. y
S aphylococcus
spp. Po o o lado, los g upos bac e ianos
de mayo impo ancia en el buey de ma cocinado ue on
Bacillus
spp. y
S aphylococcus
spp. aunque sus p opo ciones a ia on dependiendo de la
empe a u a de almacenamien o. De las bac e ias aisladas du an e el mismo
es udio, se iden i ica on median e la secuenciación del ARN 16S un o al de 18
géne os y 31 especies bac e ianos di e en es. De las bac e ias no o mado as
de espo as,
Ca nobac e ium di e gens
y
Shewanella bal ica
ue on las aisladas
con más ecuencia y
Bacillus mycoides
,
Bacillus weihens ephanensis
y
Psych obacillus psych odu ans
las bac e ias o mado as de espo as más
ecuen es.
Los esul ados del es udio de e mo esis encia de los mic o ganismos
aislados e ela on a
Kocu ia a inae
como la bac e ia ege a i a más
e mo esis en e de odas las aisladas del buey de ma ; sin emba go, el
a amien o é mico ecomendado pa a inac i a
Lis e ia monocy ogenes
, en
Chap e 10
245
p oduc os del ma p ocesados (
F
70°C7.5°C = 2 min) ue su icien e pa a consegui
un ni el de inac i ación adecuado de es e mic oo ganismo. De las bac e ias
o mado as de espo as,
B. weihens ephanensis
mos ó la mayo ole ancia al
calo seguido po
P. psych odu ans
y inalmen e
B. mycoides
(Capí ulo 6). Al
compa a los a amien os é micos eque idos pa a alcanza unos ni eles de
inac i ación adecuados de es as espo as bac e ianas con el a amien o é mico
más se e o ecomendado pa a inac i a
Clos idium bo ulinum
no p o eolí ico
ipo E (
F
90°C8.6°C = 57 min) en es os p oduc os, se de ec ó que el a amien o
e a adecuado pa a consegui la inac i ación de
P. psych odu ans
y
B. mycoides
,
pe o no pa a inac i a
B. weihens ephanensis
. En gene al, es os esul ados
demues an la impo ancia de
Bacillus
spp. en el buey de ma an o po sus
ni eles de con aminación y p e alencia como po su ele ada esis encia a los
a amien os é micos.
Como se ha indicado, en el p ocesado con encional de buey de ma lis o
pa a su consumo, es necesa io un p oceso de la ado del cang ejo, an es del
en asado, pa a e i a la suciedad del capa azón del cang ejo p oceden e del
ambien e ma ino así como los exudados p o eicos p oducidos du an e el
cocinado. Es e p oceso de la ado puede supone una econ aminación del
cang ejo cocinado lo cual hace necesa ia una pas eu ización del mismo as el
en asado con el in de ga an iza su segu idad sani a ia así como pa a ala ga
la ida ú il del p oduc o. Además, es e p oceso de la ado supone un iempo
conside able en la p oducción y un ele ado consumo de agua.
Además, de los iesgos mic obiológicos asociados a los pescados y
ma iscos, en el caso conc e o del buey de ma a ios es udios han ale ado del
iesgo de sob epasa la inges a semanal ole able de cadmio, es ablecida po la
EFSA (2009), al consumi es e ma isco debido a la ele ada concen ación de
es e me al pesado, especialmen e en la ca ne ma ón del buey de ma (Bolam
e al
., 2016; Maul aul ,
e al
., 2013; Noël
e al
., 2011; Wiech
e al
., 2017).
Chap e 10
246
Una de las soluciones p opues as pa a a on a algunos de es os p oblemas
de p ocesado del buey de ma ue la inco po ación de la ecnología de
ul asonidos du an e el cocinado. La e icacia de los ul asonidos pa a mejo a
los p ocesos de ans e encia de calo y de masa, así como pa a la inac i ación
mic obiana han sido p obados ex ensamen e en di e en es p ocesos u ilizando
dis in as ma ices alimen a ias (Chand apala,
e al
., 2012; Chema ,
e al
.,
2011). Es po ello que se e alua on las posibles en ajas del uso de es a
ecnología en el p ocesado del buey de ma . Los esul ados ob enidos en es a
Tesis Doc o al p ueban que la aplicación de ul asonidos du an e el cocinado del
buey de ma mejo a la ans e encia de calo en el pun o io del animal
(abdomen) además de se ú il pa a educi el e ec o del amaño y peso de los
cang ejos sob e las elocidades de calen amien o (Capí ulo 3). Es e e ec o
pe mi ió po un lado educi el iempo o al de cocinado (has a un 15%)
man eniendo el un alo
F
ac ualmen e aplicado o po o o lado aumen a en e
2.2 y 3.2 eces el
F
equi alen e o al aplicado man eniendo el mismo iempo de
cocinado. Es a ecnología ambién p obó su e ec i idad pa a inc emen a la
salida de compues os ( ales como la suciedad na u al, los exudados p oducidos
du an e el cocinado y compues os iónicos) desde el cang ejo al agua de cocción
lo cual en un p incipio pod ía pe mi i omi i el la ado de los cang ejos as el
cocinado. Además, la aplicación de ul asonidos e i o la inco po ación de sal a
la ca ne de cang ejo du an e el cocinado en agua con un 5% p/ de NaCl, al
con a io de lo que sucede en el cocinado con encional.
Dado el po encial que mos a on los ul asonidos pa a elimina sus ancias
del buey de ma , ambién se e alúo su po encial pa a educi la concen ación
de Cd de es e c us áceo (Capi ulo 4). Pa a ealiza es e es udio, se a a on
cang ejos hemb a a di e en es empe a u as (50, 65 y 80°C) en p esencia y
ausencia de ul asonidos, moni o izando la concen ación de Cd en el agua de
a amien o. Además, as el co espondien e a amien o, se cuan i icó la
concen ación de es e me al pesado en las di e en es ca nes del buey de ma .
La empe a u a de a amien o no mos ó una in luencia signi ica i a en la

Chap e 10
247
elocidad de salida de Cd desde el cang ejo en aquellos a amien os sin
ul asonidos, pe o mos ó una g an impo ancia en los a amien os en
p esencia de ul asonidos. En los a amien os con encionales (en ausencia de
ul asonidos), la elocidad de salida de Cd ambién ue independien e de la
empe a u a de e minándose como máximo una educción del 3% de Cd en el
cang ejo. Po o o lado, en los a amien os combinados, la p esencia de
ul asonidos inc emen ó la salida de Cd de los cang ejos 8,2, 2,1 y 2,7 eces a
las empe a u as de a amien o de 50, 65 y 80°C, espec i amen e. La mayo
educción de Cd de ec ada en los cang ejos ue de un 22,8% as el
a amien o combinado a 50°C u ilizando una ene gía ul asónica de 200W.
Es os esul ados mues an el po encial del uso de la ecnología de ul asonidos
pa a educi el con enido de Cd del buey de ma , aunque se eque i ía una
in es igación más en p o undidad pa a op imiza las condiciones ul asónicas de
a amien o pa a maximiza la educción de es e me al pesado. En base a los
esul ados ob enidos en es a Tesis Doc o al, la ecnología de ul asonidos se
pod ía implemen a en la indus ia del buey de ma du an e los p ocesos de
cocción o la ado o incluso se pod ía p opone un a amien o p e io a la
cocción con el obje i o de educi el con enido en Cd, educiendo así los iesgos
de inges a asociados con el consumo de es e p oduc o. De cualquie mane a,
en es a Tesis Doc o al se ha demos ado po p ime a ez que la ecnología de
ul asonidos es un p ocedimien o ísico e ec i o pa a educi la concen ación de
Cd du an e la cocción del buey de ma .
Como ya se ha expues o, los a amien os é micos inducen una se ie de
modi icaciones ísico-químicas en los alimen os que al e an sus ca ac e ís icas.
Es a ci cuns ancia ambién se p oduce en el buey de ma y sus p oduc os. Po
esa azón, en es a Tesis Doc o al, se es udió el e ec o de los a amien os
é micos de pas eu ización sob e di e en es pa áme os que de e minan la
calidad del buey de ma y, según los esul ados ob enidos, se p opusie on
di e en es condiciones de pas eu ización basadas en ecuaciones ma emá icas
desa olladas (Capí ulo 5).
Chap e 10
248
La empe a u a de pas eu ización no a ec ó en el con enido en agua ni en la
capacidad de e ención de agua de ninguno de los ipos de ca ne del cang ejo
ni ampoco a ec ó al colo de la ca ne ma ón siendo el colo de la ca ne blanca
el pa áme o más a ec ado po el a amien o é mico. Po ello, el colo de la
ca ne blanca ue seleccionado como indicado pa a ealiza la op imización del
a amien o de pas eu ización del buey de ma .
El es udio de la ciné ica de cambio de colo de la ca ne blanca e eló que la
deg adación del colo de es e ipo de ca ne del buey de ma sigue una ciné ica
de pseudo-p ime o den du an e el a amien o é mico. Además, an o el
máximo cambio de colo p oducido po los a amien os como la elocidad del
cambio de colo aumen a on exponencialmen e con la empe a u a de
a amien o. Con el in de es ablece los lími es de calidad en los que basa la
op imización y an es de lle a a cabo dichos cálculos de op imización, se ealizó
un “ ocus g oup” con indus iales del sec o pa a de ini la calidad de los
p oduc os en base al colo de la ca ne blanca. G acias a es e abajo, se ha
podido es ablece po p ime a ez un anking de ca ego ías habiéndose
de inido los siguien es angos de calidad en base al cambio de colo : “Buena
calidad” (ΔE* ≤7), “calidad acep able” (7< ΔE* <9) y “calidad inacep able”
(ΔE* ≥9).
En base a las ecuaciones desa olladas las empe a u as de pas eu ización
máximas acep ables pa a el buey de ma en e o se ían en e 96 y 100°C y
en e 104 y 108°C (dependiendo del peso de los cang ejos) pa a ob ene
calidades buenas o acep ables, espe i amen e. Como se de alla en el Capí ulo
5, es as empe a u as de pas eu ización se p opusie on en base al a amien o
ecomendado po la FDA de los EE.UU. pa a inac i a
C. bo ulinum
no
p o eolí ico ipo E (
F
90ºC10ºC= 10 min). Sin emba go y como se ha demos ado
en es a Tesis Doc o al, o as especies de bac e ias o mado as de espo as, más
e mo ole an es que
C. bo ulinum
ipo E, como
B. weihens ephanensis
pueden
es a p esen es en el buey de ma lis o pa a su consumo y sus p oduc os
de i ados.
Chap e 10
249
Po es a azón en es a Tesis Doc o al se e aluó el po encial de ecnologías
al e na i as al calo en conc e o, los ul asonidos y adiaciones ionizan es pa a
la inac i ación mic obiana con el obje i o de la pas eu ización del buey de ma
lis o pa a su consumo y sus p oduc os.
En el caso de los ul asonidos, además de las aplicaciones elacionadas con
los p ocesos de ans e encia de masa y de ene gía, es a ecnología es ambién
conside ada como una de las nue as ecnologías ú iles pa a la inac i ación
mic obiana y ha sido suge ida como al e na i a a los a amien os é micos
con encionales pa a la pas eu ización de alimen os (U.S. FDA. 2000). Como
egla gene al, la esis encia bac e iana a los a amien os de ul asonidos
disminuye con el amaño celula siendo mayo en las de o ma cocoide (Allige ,
1975; Condón
e al
., 2005). Sin emba go, la mayo ía de los da os publicados
indican que la e icacia bac e icida de la ecnología de ul asonidos es educida
(Lee,
e al
., 2013; Meullemies e
e al
., 2017; Jamba k,
e al
., 2017). Po ello,
se ha a ado de mejo a la e icacia bac e icida de es a ecnología diseñando
p ocesos combinados (López-Malo
e al
., 2005; Lee,
e al
., 2013; Raso
e al
.,
1998a). Algunos de los p ocesos de ul asonidos combinados que mejo an la
e icacia le al de la ecnología p opues os has a la echa son la e mosonicacion
(TS), manosonicacion (MS) y mano e mosonicacion (MTS) (Chema ,
e al
.,
2011; Piyasena,
e al
., 2003; Sala,
e al
., 1995).
En es a Tesis Doc o al, se e aluó el po encial de los a amien os
combinados de ul asonidos con p esión (manosonicacion) y la combinación de
ul asonidos con empe a u a y p esión (mano e mosonicacion) pa a inac i a
las p incipales bac e ias espo uladas aisladas del buey de ma (
B. mycoides
,
B.
weihens ephanensis
y
P. psych odu ans
) (Capí ulo 6). De los esul ados
ob enidos, puede deduci se que los pe iles de las cu as de supe i encia,
an o pa a los a amien os é micos como pa a los a amien os de ul asonidos
son dependien es de las especies bac e ianas. En aquellas especies en las que
se obse a on homb os en las cu as de inac i ación en e a los a amien os
Chap e 10
250
é micos ambién se obse a on homb os en los a amien os MS/MTS, aunque
la aplicación de ul asonidos edujo su du ación.
La e icacia le al de los a amien os de MTS es u o di ec amen e
elacionada con la e mo esis encia de las especies espo uladas.
B. mycoides
mos ó la mayo esis encia en e a los a amien os de MS y la meno a los
a amien os é micos y como esul ado ue la especie espo ulada más sensible
a los a amien os de MTS. El a amien o combinado de ul asonidos bajo
p esión con empe a u a mani es ó un e ec o siné gico pa a la inac i ación de
odas bac e ias espo uladas es udiadas. El mayo po cen aje de sine gia se
de ec ó en la especie bac e iana con el mayo alo de
z
T (
B. mycoides
)
mien as que la mayo empe a u a a la que se de ec ó el e ec o siné gico se
cuan i icó en la especie espo ulada más e mo esis en e (
B.
weihens ephanensis
). Los esul ados ob enidos demues an po an o el
po encial de la ecnología de los ul asonidos pa a pas eu iza p oduc os
líquidos a base de buey de ma (como sopas o caldos) pe mi iendo educi los
iempos de a amien o, en e un 13% y un 85%, dependiendo del
mic oo ganismo diana, o las empe a u as del p ocesado. Es o pe mi i ía
man ene los ni eles de inac i ación a ec ando en meno medida a las
p opiedades del p oduc o inal.
En el caso de las adiaciones ionizan es, se e aluó el po encial de las
mismas aplicadas con elec ones acele ados pa a la inac i ación de es os
espo os bac e ianos en ca ne de buey de ma y en medios de di e en es pH y
aw. De los esul ados, se dedujo que el e ec o del pH sob e la esis encia en e
a las adiaciones ionizan es depende de la especie espo ulada más que de las
condiciones de a amien o. Po o o lado, se de ec ó un impo an e e ec o
p o ec o de las bajas aw en e a la adiación ionizan e aunque la magni ud de
su impac o ambién dependió de la especie espo ulada. Además, el mayo
e ec o p o ec o de la educción de la ac i idad de agua se de ec ó en el ango
de >0,99 a 0,90, siendo es e e ec o en es e ango ambién independien e de la
especie espo ulada a ada.
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Annex I
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Annex I
Join -PhD ag eemen