A predictive model to set HP processing criteria for L. monocytogenes inactivation on dry-cured ham
ISOPOL XVII, Porto5-8 May 2010
© Bover-Cid, S (IRTA, Monells, Spain) 1
S. Bover-Cid, N. Belletti, M.Garriga and T. AymerichFood IndustriesFood Safety ProgramFinca Camps i Armet, E-17121 MonellsSpain
A predictive model to set high pressure processing criteria for Listeria monocytogenes inactivation
on dry-cured ham
ü High hydrostatic pressure (HHP) is a non-thermal technology.
ü In-package “cold pasteurization”.
Introduction
120L-equipment (NC Hyperbaric)
ü Listericidal treatment for RTE products(USA, Codex Alimentarius).
ü The efficiency and cost of HHP depends on P, t, Te.Relevant differences among species/strains and food.
ü The modelling approach has been scarcely applied andneeds further development.
ISOPOL 2010, Porto
A predictive model to set HP processing criteria for L. monocytogenes inactivation on dry-cured ham
ISOPOL XVII, Porto5-8 May 2010
© Bover-Cid, S (IRTA, Monells, Spain) 2
To model the HHP induced inactivation of Listeriamonocytogenes on dry-cured ham, as a function ofthe intensity, length and fluid temperature of theprocessing.
To validate the inactivation model to assess itssuitability for predictive purposes.
To apply the inactivation model to set the processcriteria compatible with the Food Safety Objectives.
ISOPOL 2010, Porto
Objectives
Methodology
Samplepreparation
Listeria monocytogenes CTC1034
107
cells/g
(isolated from dry-cured ham)
-80ºCstock
culture
Pre-culture in TSBYE37ºC / 7h
culture in TSBYE
37ºC / 18h
Sliced dry-cured ham(aw= 0.88; pH = 5.84)
Vacuum packaged(plastic bag PET/PE)
HHP
µbiologicalanalysis
Modelling
Validation
LevelsTemperature
(ºC)aHolding time(minutes)b
Pressure(MPa)
-1.68 7.6 2.30 347-1 11 5 4500 16 9 6001 21 13 750
1.68 24.4 15.75 852
High hydrostatic pressure processing (Central Composite Design)
Holdingtime
Temperature
Pressurea: Initial fluid temperature; b: Come up ~220MPa/min; Release almost immediate.
Microbiological determinationsEnumeration
ChromogenicListeria Agar. CLA (Oxoid)@37ºC (48 h)DL= 4 – 10 cfu/g
Presence/absenceTSAYE Enrichment @37ºC for 24-48hStreaked on CLA & PCR confirmation
(Aymerich et al,, 2005)
µbiologicalanalysis
HHP
ISOPOL 2010, Porto
(at least in duplicate samples)
A predictive model to set HP processing criteria for L. monocytogenes inactivation on dry-cured ham
ISOPOL XVII, Porto5-8 May 2010
© Bover-Cid, S (IRTA, Monells, Spain) 3
Modelling
Methodology
Samplepreparation
HHP
µbiologicalanalysis
Modeling
Validation
Mathematical modeling (Statistica v8 for Windows, StatSoft Inc)
Response → Log (N/N0)
Multivariant linear regression – Response surface
Model validationFurther experiments and published data within the assayed experimental domain
...·......·· 52
4210 +++++++= ZXXZXY βββββ
R2adj; P (F-test); P (Lack of fit)
Accuracy factor (Af) Bias factor (Bf)
Bacterial inactivation as Logarithmic reductions:the difference between counts after HHP and the initial inoculum
(Baranyi et al., 1999)
Validation
ISOPOL 2010, Porto
Factors → P, t & T Pressure (MPa) transformed into Log10, time (min) & Temperature (ºC)
-8-6-4-20
Results
Trial Temperature(ºC)
Pressure(MPa)
Time(minutes)
Log (N/N0)mean (SD) *
1 11 450 5 -0.32 (0.10)a
2 11 450 13 -0.60 (0.10)ab
3 11 750 5 -4.86 (0.41)d
4 11 750 13 -6.91 (0.20)e
5 21 450 5 -0.42 (0.01)ab
6 21 450 13 -0.89 (0.08)ab
7 21 750 5 -5.27 (0.33)d
8 21 750 13 -6.20 (1.41)e
9 16 600 9 -2.81 (1.03)c
10 16 600 9 -3.13 (0.26)c
11 16 600 9 -2.94 (0.25)c
15 16 600 15.75 -3.17 (0.13)c
16 16 600 2.30 -1.20 (0.07)b
17 16 852 9 -7.86 (0.00)f
18 16 347 9 -0.05 (0.08)a
19 24 600 9 -2.94 (0.23)c
20 7.6 600 9 -2.61 (0.07)c
HHP inactivation of L. monocytogenes on dry-cured ham
*: mean (standard deviation); values with different superscript letters indicate statistical significant differences according to the LSD test
ISOPOL 2010, Porto
Bacterial inactivation
Modelling
Validation
Application
Bacterial inactivation
A predictive model to set HP processing criteria for L. monocytogenes inactivation on dry-cured ham
ISOPOL XVII, Porto5-8 May 2010
© Bover-Cid, S (IRTA, Monells, Spain) 4
Results
9 min -2.9 Log600 MPa
HHP inactivation of L. monocytogenes on dry-cured ham
aw=0.88
10 min
≥ -5 LogKoseki et al.
(2007)*
≥ -7 LogChen(2007)
Erkmen & Dogan(2004)
≥ -7 LogRitz et al(2000)
Gao et al.(2006)*
≥ -7.5 Log
4.5 min 3-10 min
Phosphatebuffer (pH 7)Citratebuffer (pH 5.7)
1-6 min
* Modelling approach
aw=0.98
ISOPOL 2010, Porto
Bacterial inactivation
Modelling
Validation
Application
Results
Mathematical modelling: polynomial equations
a: R2adj (P): adjusted determination coefficient
(significance level); PLoF: significance of the lack of fit test;
ISOPOL 2010, Porto
Bacterialinactivation
Modelling
Validation
Application
Log(N/N0) = – 380.32 + 292.59[LogP] – 56.13[LogP]2 + 1.41[t] + 0.01[t]2 – 0.64[LogP]·[t]
Observed vs fitted
-9-8-7-6-5-4-3-2-10
-9 -8 -7 -6 -5 -4 -3 -2 -1 0
Fitted
Observed
Internal data
Statistical goodness of fita
R2adj (P) PLoF
0.9884 (<0.0001) 0.268
A predictive model to set HP processing criteria for L. monocytogenes inactivation on dry-cured ham
ISOPOL XVII, Porto5-8 May 2010
© Bover-Cid, S (IRTA, Monells, Spain) 5
Results
Model validation
Validation indexa
Af Bf
1.36 1.06a: Af: Accuracy factor
(+0.1 to +0.15 per factor, Ross et al. 2000); Bf: Bias factor
(0.75 to 1.25, Dalgaard 2000).
Observed vs predicted
-9-8-7-6-5-4-3-2-10
-9 -8 -7 -6 -5 -4 -3 -2 -1 0
Pred
icted
Observed
Internal data
Further experiments Lm CTC1034
Experiments with other Lm
Literature data Tanzi et al. (2004)Morales et al. (2006) Hereu et al. (2009)
ISOPOL 2010, Porto
Bacterialinactivation
Modelling
Validation
Application
-9-8-7-6-5-4-3-2-10
-9 -8 -7 -6 -5 -4 -3 -2 -1 0
Pred
icted
Observed
-9-8-7-6-5-4-3-2-10
-9 -8 -7 -6 -5 -4 -3 -2 -1 0
Pred
icted
Observed
-9-8-7-6-5-4-3-2-10
-9 -8 -7 -6 -5 -4 -3 -2 -1 0
Observed
Log(N/N0) = – 380.32 + 292.59[LogP] – 56.13[LogP]2 + 1.41[t] + 0.01[t]2 – 0.64[LogP]·[t]
Results
Model application : Response surface plot
2.502.55
2.602.65
2.702.75
2.802.85
2.902.95
4
68
1012
14
-10
-8
-6
-4
-2
0
Pressure(Log MPa)
Time(minutes)
347 MPa
450 MPa
852 MPa750 MPa600 MPa
0 Log (N/N -1 Log (N/N -3 Log (N/N -5 Log (N/N -7 Log (N/N -9 Log (N/N
0 Log (N/N0)
Inacti
vatio
n lev
el Lo
g(N/
N 0)
ü HP threshold ≥ 450MPa
ü Tailing phenomenatime ≥ 10 min
ü Temperaturenot significant
Pressure(LogMPa)
time(min)
ISOPOL 2010, Porto
Bacterialinactivation
Modelling
Validation
Application
Log(N/N0) = – 380.32 + 292.59[LogP] – 56.13[LogP]2 + 1.41[t] + 0.01[t]2 – 0.64[LogP]·[t]
A predictive model to set HP processing criteria for L. monocytogenes inactivation on dry-cured ham
ISOPOL XVII, Porto5-8 May 2010
© Bover-Cid, S (IRTA, Monells, Spain) 6
Results
Model application: setting process criteria (P & t)
Pasteurization
300
400
500
600
700
800
900
2 4 6 8 10 12 14 16
Pre
ssur
e (M
Pa)
Time (min)
“Zero tolerance” (FSO)HHS (2008)
NACMCF (2006)Refrigerated RTE products- 6 D800
750
6 10
800 MPa / 5.4 min750 MPa / 9.6 min
ISOPOL 2010, Porto
Bacterial inactivation
Modelling
Validation
Application
Pressure-time isokineticsfor HP-induced inactivation of
L. monocytogenes on dry-cured ham
MC = 100 cfu/g(FSO = 2 Log cfu/g)
H0 = 1 Log cfu/g (ICMSF 2002)ΣI ≤ 5 Log
Results
Model application: setting process criteria (P & t)
300
400
500
600
700
800
900
2 4 6 8 10 12 14 16
Pre
ssur
e (M
Pa)
Time (min)
Refrigerated RTE products- 6 D700
5.2 10
AESAN (2004)
HHP treated RTE meat products
- 4 D650
700 MPa / 5.2 min650 MPa / 9.9 min
ISOPOL 2010, Porto
Bacterial inactivation
Modelling
Validation
Application
Pressure-time isokineticsfor HP-induced inactivation of
L. monocytogenes on dry-cured hamICMFS formula:
H0 + ΣI – ΣR ≤ FSO
A predictive model to set HP processing criteria for L. monocytogenes inactivation on dry-cured ham
ISOPOL XVII, Porto5-8 May 2010
© Bover-Cid, S (IRTA, Monells, Spain) 7
Results
Model application: setting process criteria (P & t)
300
400
500
600
700
800
900
2 4 6 8 10 12 14 16
Pre
ssur
e (M
Pa)
Time (min)
Refrigerated RTE products- 6 D
600
6
HHP treated RTE meat products
- 4 D
Hoz et al. (2008)- 2.39 D RTE
Dry-cured ham
“Zero tolerance”(FSO = -1.39 Log ufc/g)H0 = 1 Log cfu/g (ICMSF 2002)
ΣI ≤ 0 Log
No sanitation treatment would be necessary
600 MPa5.9 min
ISOPOL 2010, Porto
Bacterial inactivation
Modelling
Validation
Application
Pressure-time isokineticsfor HP-induced inactivation of
L. monocytogenes on dry-cured hamICMFS formula:
H0 + ΣI – ΣR ≤ FSO
Conclusions
The modelling approach identified pressure and time asthe most relevant technological factors of the HHPinactivation of L. monocytogenes on dry-cured ham.
An empirical model was developed and both the statisticalperformance and the validation parameters supported itssuitability for predictive purposes.
The outputs of the model enable to define the optimalprocess criteria to achieve the adequate performancecriteria for the HHP inactivation of L. monocytogenes onRTE dry-cured ham, compatible with the FSO.
The model could assist meat industries to evaluate therelation cost/benefit of a potential implementation of HHPtechnology in their production processes.
ISOPOL 2010, Porto