A predictive model to set high pressure ... - ISOPOLisopol.esb.ucp.pt/presentations/Bover-Cid ISOPOL.pdf · ISOPOL 2010, Porto. Title: Microsoft PowerPoint - Bover-Cid ISOPOL for

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

*[email protected]

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




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)


HHP

ISOPOL 2010, Porto

(at least in duplicate samples)




Modelling

Methodology

Samplepreparation

HHP


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





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


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




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)

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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


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


Modelling

Validation

Application





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


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


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


Modelling

Validation

Application


L. monocytogenes on dry-cured hamICMFS formula:

H0 + ΣI – ΣR ≤ FSO




Results


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


Modelling

Validation

Application


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

Documents

A predictive model to set high pressure ... - ISOPOLisopol.esb.ucp.pt/presentations/Bover-Cid ISOPOL.pdf · ISOPOL 2010, Porto. Title: Microsoft PowerPoint - Bover-Cid ISOPOL for