Natural antimicrobials in sanitationin sanitation… · 4. Canadian Meat Council’s Technical Symposium October 1-2, 2009 Natural antimicrobials Introduction Antimicrobials from

Natural antimicrobials in sanitationin sanitation…

… an alternative to enhance microbial food safety ?microbial food safety ?

Florence Dubois-BrissonnetAssociate Professor

AgroParisTech – INRAUnité Mixte de Recherche “Bioadhésion et Hygiènedes matériaux”, Massy, FranceResearch unit « Bioadhesion and hygiene of materials »

1Canadian Meat Council’s 2009 Technical Symposium

“Advanced Listeria monocytogenes Control Measures in RTE Meats and Poultry Products”October 1-2, 2009

Canadian Meat Council’s Technical Symposium October 1-2, 2009

General outlinesIntroduction

Applications of natural antimicrobials:

General outlines

Applications of natural antimicrobials:

as preservatives in food products

for surface decontamination

in active-packaging

Hurdle-technology and applications

Conclusion

2


IntroductionIntroduction

Foodborne illness still has a huge heath and economic impact76 illi f db i t i ti i USA (MMWR 2002 1 32 329)76 millions foodborne intoxications per year in USA (MMWR. 2002. 51:325-329)11-13 millions per year in Canada (http://www.inspection.gc.ca)67000 per year in France (estimation by the National French Food Safety Agency in 2004)

Consumers demand:High quality, safe food productsg q y,

natural flavor and tasted d h lf lif

pconvenient and innovative

less salt, less acid and less chemicalsextended shelf-life

3

Innovative alternative technologies


Challenges

Introduction

Available solutions to improve microbial safety and quality of food products?

Challenges

and quality of food products?

CHEMICALS NATURAL PACKAGINGPROCESSESCHEMICALS ANTIMICROBIALSPACKAGINGPROCESSES

Th l P l filBiocides:

chlorine, PAA, QACs

Thermal processes Bacteriocins,

organic acids, enzymes, Non-thermal

Polymer filmsMAP, etc…

ActiveQACs, aldehydes…),

etc…

y ,animal or plant extracts, etc…

processes: Irradiation,

High Hydrostatic Pressure, etc…

Active-packaging

4


Natural antimicrobials

Introduction

Antimicrobials from micro-organismsBacteriocins: nisin pediocin

Natural antimicrobials

Bacteriocins: nisin, pediocin…Organic acids: acetic, lactic acids…

Plant-derived antimicrobials

.DB

-A

groP

aris

Tech

Organic acids: citric, sorbic acids… Phyto-phenols, adhehydes, flavonoids, catechins, saponins …

Animal-derived antimicrobials

F

Animal-derived antimicrobialsLacto-antimicrobials : lactoferrin, lactoperoxydase, lactoglobulins, lactolipidsOvo-antimicrobials : lysosyme, ovotransferrin, ovoglobulin

sTec

h

OthersSodium chlorideWood smoke

F.D

B -

Agr

oPar

is

.DB

-A

groP

aris

Tech

5Natural doesn’t mean no toxic

F.


I Natural antimicrobials as foodI- Natural antimicrobials as food preservatives

I hibiti f i bi l th

Log N ControlC1C2 > C1

Inhibition of microbial growth

C3 > C2C4 > C3

C5 > C4

Food matrixTime

C6 > C5

Agr

oPar

isTe

ch

Tech

-Agr

oPar

isTe

ch

6

F.D

B -

F.D

B -

Agr

oPar

isT

F.D

B


Inhibitory effect of preservatives

Food preservatives

Dilution method: bacterial growth is followed in presence of differentconcentrations of an antimicrobial.

y p

2 x 100 wells200 µl

ch

Bioscreen (Labsystems, France)

01 8111 91

2131

4151

6171

0101 81811111 9191

21213131

41415151

61617171

F.D

B -

Agr

oPar

isTe

c

1,4

Salmonella Typhimurium grown in presence of α-terpineol

( y , )

0,8

1

1,2

00 n

m0

nm

Minimal Inhibitory Concentration

0,2

0,4

0,6

DO

60

0 mM1 mM2 mM3 mM4 mM

OD

600

24h-MIC is between 4 and 4 5 mM

7

0

,

0 20 40 60 80Temps (h)

4,5 mM

Time (h)

4 and 4.5 mM



Food preservatives

Dose-response curve modelingy p

OD

600

nm

µmax

Maximum growth rateFor each antimicrobial concentration,

modeling of growth response

ln OD(t)( )= ln OD0( )+ A ⋅ exp −exp μmax ⋅ eA

⋅ lag − t( )+1⎛ ⎝ ⎜

⎞ ⎠ ⎟

⎛

⎝ ⎜

⎞

⎠ ⎟

Time (h)

modeling of growth response

OD= f(time)

µmax =f(C) Lambert-Pearson model (2000)

Growth inhibition of Salmonella Typhimurium by α-terpineol

( )⎤⎡

⎞⎛ ⎟⎟⎞

⎜⎜⎛ −e

( ) ( )cgcc ⋅== 0)( maxmax μμ

with

( ) ( )

( )

⎥⎥⎥⎥⎥⎥

⎦

⎤

⎢⎢⎢⎢⎢⎢

⎣

⎡

⎟⎟⎟⎟

⎠

⎞

⎜⎜⎜⎜

⎝

⎛

⎟⎠⎞

⎜⎝⎛

−

−=

⎟⎟⎠

⎜⎜⎝ CMICNI

eCMICNICMI

ccg

ln

lnexpexp

At 24hNIC = 2.3 mM ±0.75MIC = 4.2 mM ±0.42

8MIC = Minimal Inhibitory Concentration

NIC = Non-Inhibitory Concentration Guillier et al (2007) JFP,70, 2243-2250


Inhibitory activity of phyto-phenols

Food preservatives

1 41,6

1 41,6

y y p y p

CarvacrolThymol 1,6 α-Terpineol

on Salmonella Typhimurium growth

B -

Agr

oPar

isTe

ch

-Agr

oPar

isTe

ch

0,40,60,81,01,21,4

µmax

0,5

0,40,60,81,01,21,4

µmax

0,5

y

0 40,60,81,01,21,4

µmax

0,5

α Terpineol

At 24hNIC = 2 3 mM

At 24hNIC = 0 7 mM

At 24hNIC = 0 7 mM

Thyme RosemaryOregano

F.D

F.D

B

0,00,20,4

0,0 0,2 0,4 0,6 0,8 1,0 1,2Concentration (mM)

0,00,20,4

0,0 0,2 0,4 0,6 0,8 1,0 1,2Concentration

0,00,20,4

0 1 2 3 4Concentration (mM)

NIC = 2.3 mMMIC = 4.2 mM



1 01,21,41,6

,5

Eugenol

1,21,41,6

5

Menthol

1 01,21,41,6

5

Geraniol

MintClove F.D

B -

Agr

oPar

isTe

ch

-A

groP

aris

Tech

0,00,20,40,60,81,0

µmax

0

0,00,20,40,60,81,0

µmax

0,5

0,00,20,40,60,81,0

µmax

0,

At 24hNIC = 1.1 mMMIC = 3.0 mM



BasilClove

F.D

B

9Guillier et al (2007) JFP,70, 2243-2250

0 1 2 3 4Concentration (mM) 0 1 2 3 4

Concentration (mM)

,0 1 2 3 4

Concentration (mM)



We have to be very careful with the MIC absolute values. - the growth medium and conditionsThey depend on:

y p

the growth medium and conditions- the inoculum size- the incubation time- the strain

They depend on:

910

/mL) Control TSB TSB + green tea (1% p/v)) TSB + Rosemary (1% p/v)

They are only comparative.

456789

ount

(log

CFU

ter 2

4h

01234

B ill S l ll E t b t S E li O157H7 Li t i

Bac

teria

l co aft

10

Bacillus cereus SalmonellaTyphimurium

Enterobactersakazakii

S.aureus E.coli O157H7 Listeriamonocytogenes

Lee et al, 2009, JFP, 72,1107-1111


Natural antimicrobials activity in food systems

Food preservatives

6

8 ControlGreen tea extract (1% p/v))Rosemary extract (1% p/v) 8

10 ControlGreen tea extract (1% p/v))R t t (1% / )

y y

4h4hStaphylococcus

aureusBacillus cereus

2

4

6 Rosemary extract (1% p/v)

2

4

6

8 Rosemary extract (1% p/v)

logN

afte

r 24

logN

afte

r 24

Lee et al, 2009, JFP, 72,1107-1111

0TSB rice cake

0

2

TSB rice cake

l

Levels of natural preservatives in-vitro efficientLevels of natural preservatives required in food systems >> in-vitro efficient

concentration

Ratio of t ti Antimicrobial Micro-organism Matrix Referenceconcentration g

Twofold Carvacrol L.monocytogenes Skimmed-milk Karatzas et al, 2001, JAM, 90,463-469

Ten-fold Rosemary extract L.monocytogenes Pork liver sausage Pandit et Shelef, 1994, Food Microbiology, 11, 57-63

11

50-fold Carvacrol B.cereus Soup Ultee and Smid, 2001, IFFM, 64,373-378

25- to 100-fold Plant essential oil from Spain L.monocytogenes Soft-cheese Mendoza-Yepes et al, 1997, JFS, 17,47-55


II- Natural antimicrobials in surfaceII Natural antimicrobials in surface decontamination

Log NLog N

Bacterial inhibition

Time

Bacterial inactivation

Bacterial inhibition

Food surfaces Food contact surfacesMeat Fr its andMeat

carcassesFruits and vegetables

chech

12

F.D

B -

Agr

oPar

isTe

F.D

B -

Agr

oPar

isTe


Decontamination of meat products by lactic acid

Surface decontamination

Decontamination of meat products by lactic acidInoculation of chicken carcasses with 7-8 log/cm2 Listeria monocytogenes orSalmonella Enteritidis.Decontamination by immersion in lactic acid at different concentrations during 20 minDecontamination by immersion in lactic acid at different concentrations during 20 min

1,61,8

min

0,5% 1% 1,5% 2%

min

Anang et al (2007) Food Control,18, 961-969

0,81

1,21,41,6

ion

en 2

0 m

ctio

n at

20m

00,20,40,60,8

Log

rédu

ctLo

g re

duc

In USA, lactic acid is recognized as antilisterial agent and is allowed in food

Listeria Salmonella

13

products (Food Safety and Inspection Service, 2000)

In Europe it is approved by the European commission (directive 95/2/CE)


Decontamination of meat products by nisin


Inoculation of meat pieces with 4.5 log UFC/cm² of Listeria innocua.Decontamination by spraying nisin solution at 5000 UI/mL on meat pieces.

p y

910

m2)

78

m2)

Cutter et Siragusa (1996) Food Microbiology, 13, 23-33

Adipose tissues Lean tissues

56789

s (lo

g C

FU/c

m

4567

s (lo

g C

FU/c

1-2 log reduction

after 28 days

1234

iste

ria c

ount

s

untreatedwater treatednisin treated 1

23

ster

ia c

ount

suntreatedwater treatednisin treated

after 28 days

00 10 20 30

Time (days)

L 00 10 20 30

Time (days)

Linisin treated

14


Inactivation of Salmonella Typhimurium with l t i l t l


carvacrol on stainless steel

risTe

ch

sTec

h

Biofilmadherent cells8

ou Decontamination with 5 mM carvacrol

F.D

B -

Agr

oPar

F.D

B -

Agr

oPar

i

Biofilm3D-structure

5

6

7

log

CFU

/ml o

Adherent cells

2

3

4

ella

cou

nts

(/c

m2

2D-structure

Planktonic cells0

1

2

Salm

one

Detection threshold

15

0 5 10 15 20 25 30Time (min)


Activity on biofilms


Observation with Confocal Laser Scanning Microscopy (CLSM)

Biofilmadherent cells

tBridier et al, 2009 – Eurobiofilms Roma

Pseudomonas aeruginosa biofilm

t0

BAC, 1%

t5min

16Green : Syto 9 (all the bacteria) and Red/ yellow : Propidium Iodide (Membrane altered cells)


III N t l ti i bi l i tiIII- Natural antimicrobials in active-packaging

Surface coating with natural antimicrobials

Active packaging

Environment

Pack

Food product

Environment

17

Canadian Meat Council’s Technical Symposium October 1-2, 2009 Active-packaging

EVAPORATION

Release of natural antimicrobials from a sachet

EVAPORATION

Incorporation of natural antimicrobials in package

EVAPORATION

DIFFUSION

Coating of the packaging with natural antimicrobials

EVAPORATION

18

DIFFUSION


Inactivation of Salmonellali d t t b 6

7

8

ts

n si

te)

Active-packaging

on sliced tomatoes by carvacrol in vapor-phase

Decontaminated tomatoes were sliced,cut into 4 pieces and inoculated with

3

4

5

6

mon

ella

cou

nFU

/inoc

ulat

ion

4°C for 10 days

Carvacrol in vapor-phase

cut into 4 pieces and inoculated withSalmonella.

0

1

2

0 2 4 6 8 10

Salm

(log

CF

controlcarvacrol 41.5 µL/Lcarvacrol 83.3 µL/Lcarvacrol 125 µL/L

4 C for 10 days

789

nts

n si

te)

789

10

nts

site

)

Tomatoes slices0 2 4 6 8 10

Time (days)

3456

mon

ella

cou

nU

/inoc

ulat

ion

34567

mon

ella

cou

nU

/inoc

ulat

ion

10°C pd 10 jours 25°C pd 10 jours

0123

0 2 4 6 8 10

Salm

(logC

FU controlcarvacrol 41.5 µL/Lcarvacrol 83.3 µL/Lcarvacrol 125 µL/L

0123

0 2 4 6 8 10

Salm

(log

CFU control

carvacrol 41.5 µL/Lcarvacrol 83.3 µL/Lcarvacrol 125 µL/L

19

0 2 4 6 8 10Time (days)

0 2 4 6 8 10Time (days)

Carvacrol in vapor phase is more active when temperature is lowObaidat and Frank (2009) JFP, 72,315-324


Active packaging containing nisin

Active-packaging

Active packaging containing nisinSlices of cheddar were inoculated with Listeria innocua or Staphylococcus aureusat 2-4 105 CFU/gBioactive inserts were prepared allowing adsorption of nisin on cellulose packaging

(7650 UI/ 2) d l d th li f h

8LAB

Cheese were stored at 4°C in MAP

paper (7650 UI/cm2) and placed over the slices of cheese.

567

C/c

m²)

LAB

Listeria innocua

234

og N

(UFC

Listeria: 3 units log dropS aureus: 2 log units drop

Listeria innocua

Staphylococcus aureus

01

0 5 10 15 20 25

lo S.aureus: 2 log units drop

20

0 5 10 15 20 25Time (h)

Filled symbols: untreated inserts Empty symbols: films prepared with nisin Scannell et al. (2000) IJFM, 60,241-249


Active packaging containing essential oils

Active-packaging

Bologna slices were inoculated with Listeria monocytogenes at 103 CFU/cm2.

Active packaging containing essential oils

Alginate-based edible films immerged in 20% calcium chloride andcontaining 1% essential oils of oregano, cinnamon or savory were

3 5 no filmcontrol film

containing 1% essential oils of oregano, cinnamon or savory wereapplied to slices to prevent pathogen growth.

2,5

3

3,5

s (lo

gCFU

/g) control film

oreganocinnamonsavory

1

1,5

2

onoc

ytog

enes

2.5 log more reduction in 4 dayswith cinnamon and in 5 days fororegano and savory

0

0,5

1

List

eria

mo

Cinnamon-based films were the most effective on

Listeria growth inactivation

21

1 2 3 4 5 6Days

Oussalah et al (2007) JFP, 70, 4, 901-908


IV C bi ti f t t tThere is some eventual smelly and tasty effects of antimicrobials when used alone

IV- Combinations of treatments

« Hurdle Technology »

at efficient levels

M ltif t i l P tiGould (1985)

… or « Multifactorial Preservation »

Roller (2000)

1 2 3st d th3 i lt h dlhurdle hurdle hurdle

Combination of antimicrobials

3 simultaneous hurdles

Combination of antimicrobials

22

with non-thermal processes Combination of antimicrobials


Numerous potentialities of combinations

Combinations

pTreatment 1 Treatment 2 Examples Micro-organism Reference

Bacteriocin Essential oil Nisin-carvacrol B.cereus Periago et Moezelaar, 2001, IFFM, 68, 141-148

Bacteriocin Essential oil Nisin-thymol L.monocytogenes, B.subtilis Ettayebi et al, 2000, FEMS Microbiology Letters, 183, 191-195

Bacteriocin Organic acid Nisin-lactic, acetic acid or salts L.monocytogenes Samelis, 2005, Lebensm. –Wiss. U.-Technol.

38, 21-28

Bacteriocin EDTA Nisin-EDTA Pseudomonas, Brochothrix Economou et al, 2009, Food Chemistry, 114, 1470-1476

Bacteriocin Irradiation Pediocin-irradiation 2kGy L monocytogenes Chen et al 2004 JFP 67 1866-1875Bacteriocin Irradiation Pediocin-irradiation 2kGy L. monocytogenes Chen et al, 2004, JFP, 67, 1866-1875

Essential oil Essential oil 20 plant aromatic extracts S.Typhimurium Nazer et al, 2005. Food Microbiology, 22, 391-398

Essential oil Essential oil Cymene-thymol B.cereus Delgado et al, 2004, Food Microbiology, 21, 327-334

Th l l tiEssential oil Organic acid Thymol, carvacrol-acetic, lactic citric acids S. Typhimurium Zhou et al, 2007, JFP, 70, 1704-1709

Essential oil MAP Eugenol or thymol - MAP Spoilage flora Valero et al, 2006, Postharvest Biology and Technology, 41, 317-327

Essential oil refrigeration Carvacrol, cinnamaldhyde or thymol - refrigeration Bacillus cereus Valero et al, 2006, Food Microbiology, 23, 68-73y g

Essential oil HHP Carvacrol-HHP L. monocytogenes Karatzas et al, 2001, JAM, 90, 463-469

Organic acids HHP Lactate-HHP L. monocytogenes and

Salmonella Aymeric et al, 2005 – JFP, 68, 173-177

23

Most of these studies analyze cumulative effects of treatments but not really synergistic effects


Combination with two antimicrobials

Combinations

Compound 1Compound 2 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% CMI du composé 1

0% x x10% x x x20% x x x30% x x x

MIC of compound 1

40% x x x50% x x x60% x x x70% x x x80% x x x90% x x x100% x x

CMI duMIC of

80

100

))

Antagonistic effect

CMI du composé 2

MIC of compound 2

Isobolographic analysis

40

60

80

osé

2 (%

CM

I)nd

2 (%

of M

IC)

Additive effect

Isobole = curve connectingequivalent doses of bothcompounds that lead to totalbacterial inhibition

Isobolographic analysis

0

20

40

Com

poC

ompo

un

Synergistic effect

Additive effectbacterial inhibition

24

0 20 40 60 80 100Composé 1 (% CMI)Compound 1 (% of MIC)


Numerous potentialities of combinations

Combinations

Numerous potentialities of combinationsWhatever the application, nisin is often used in combination with other treatments: organic acids, EDTA, hexametaphosphate, lysosyme, thermal treatment…

Nisin Heat stablehypoallergenic,Degraded by proteolytic enzymes in the human intestinal tract

Combinations of nisin and High Hydrostatic Pressure (HHP) on mechanically recovered poultry meat

Example of hurdle technology:

68

10

mes

ophi

le

g C

FU/g

)

Control350 Mpa-5min350 Mpa-15min450 Mpa-5min450 Mpa-15min

0

24

Aer

ophi

lic m

coun

ts (l

og

25

0 ppm 12.5 ppm 100 ppm 200 ppmNisin concentration

Yuste et al (2002) Food Control, 13, 451-455


Multifactorial preservation consequences

Combinations

Multifactorial preservation consequences

S l l f b i lSame level of bacterial growth inhibition

Dos

e

Sensorial threshold

2 compounds1 compound

Sensorial threshold

Regulation limits

2 compounds4 compounds

8 compounds…

bacterial STRESS ?Sub-inhibitory doses of each antimicrobial can cause:

26

increasing of tolerance to bactericidal activity ?


Bacterial reactivity in presence of sub-inhibitory doses f i id

Combinations

50

60

otal

eof organic acids

Saturation of membrane in presence of organic acids

area

)

30

40

rapp

ort à

l'ai

re to in Salmonella Typhimurium

ds (%

of t

otal

a

10

20

% A

G p

ar

Unsaturated Fatty Acids

Saturated Fatty Acids

Cyclopropanoïc Fatty Acids

Unsaturated Fatty Acids

Fatty

aci

UFACFA

0Témoin Ac.citrique 4 mM Ac.citrique 22 mM

SFAControl Citric acid 22 mMCitric acid 4 mM

y

Stress proteins synthesisBi-dimensional electrophoresis

kDa

27At acidic pH in comparison with

neutral pH

electrophoresis

PI


Bacterial reactivity in presence of sub-inhibitory doses f i id

Combinations

Acid Tolerance Response (ATR)

of organic acids

Bacterial adaptation to organic acids

Increasing of toleranceto acidic environnemental conditionsorganic acids to acidic environnemental conditions

01e

de

L

pH 7 culturesto disinfectants

U/m

L) DDAB= didecyl dimethyl ammonium bromide (quaternary ammonium compound)

1234ga

rithm

ique

ion

CFU

/mL

pH 5.5 culturesNaïtali et al (2009) IJFM, 130, 101-107

ts (l

og C

FU

4567éd

uctio

n lo

gla

pop

ulat

Scott A L. monocytogenesCIP 7839 L. monocytogenes

iste

riaco

un

28

70 0,5 1 1,5 2 2,5 3 3,5

Concentration en BDDA (x 10-2 mg/L)

Ré

Li

DDAB concentration


Bacterial reactivity in presence of sub-inhibitory d f ti d

Combinations

80a)

SFA expoS

Fatty acids membrane composition of Salmonella Typhimurium

doses of aromatic compounds

4050607080

of t

otal

are

a SFA statUFA expoUFA statCFA expoCFA stat

010203040

atty

aci

ds (%

When cells enter stationary phase

CFA th t5

1020

0Control 0.3 mM Thymol 0.6 mM Thymol 0.9 mM Thymol

Fa

In presence of thymol

UFA CFACFA synthetase

S- adenosyl methionine

S- adenosyl homocysteine

3

4

0

5

10 15

CFA

Similar results for carvacrol, citral and

eugenol

29

methionine(SAH)(SAM)

y

10 15 20 25 30 35 40

20

Temperature (°C)

Induction of antimicrobials tolerance ?


ConclusionConclusion

The interest for natural antimicrobials has expend in recent years inresponse to consumer demand for greener additives.

Their applications are likely to increase in the future:

as food preservativesas food preservativesfor surfaces decontamination in active-packaging

In all cases, we have underlined the interest of combined systems

Further studies are still necessary to validate and optimize thesesystems

30


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Th k f tt ti !Thank you for your attention !

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Documents

Natural antimicrobials in sanitationin sanitation… · 4. Canadian Meat Council’s Technical Symposium October 1-2, 2009 Natural antimicrobials Introduction Antimicrobials from