DEFINITION FUNCTIONS

Outline
O portunidades y de safíosen elcontrolde
patógenos de origen alim entario con
antim icrobianos naturales
P.M ichaelD avidson
D epartm entofFood Science and Technology
U niversity ofTennessee
Knoxville,Tennessee,U SA
pm davidson@ utk.edu
•D efinition
•Function
•N atural
•Im proving Effectiveness
•Com m ents on the use of“food
preservatives”
Antimicrobials in Food Processing
DEFINITION
Chem icalCom pounds Presentin or
Added to FO O D S,FO O D
PACKAG IN G ,FO O D CO N TACT
SU RFACES,orFO O D PRO CESSIN G
EN VIRO N M EN TS thatInhibitthe
G row th of,or Inactivate,
PATH O G EN IC or SPO ILAG E
M icroorganism s
Functions
FUNCTIONS
•Inhibit/inactivate spoilage and
pathogenic m icroorganism s in
food products or via food
packaging
•Prolong Shelflife/Preserve Q uality
•Im prove safety by inhibiting
pathogenic m icroorganism s
Use of an Antimicrobial Compound
as a Primary Barrier Against a
Foodborne Pathogen
N itrite – Clostridium botulinum
N isin & Lysozym e – C.botulinum
Sodium lactate & Sodium diacetate –
Listeria m onocytogenes
Lactoferrin – E.coliO 157:H 7
O thers… .
IS NATURAL BETTER?
Traditional or RegulatoryApproved Antimicrobials in the US
• Acetates
• Benzoates
• D im ethyl
D icarbonate
• Lactates
• Lactoferrin
• Lauric Arginate
• Lysozym e
• N atam ycin
• N isin
• N itrites
• Parabens
• Phosphates
• Propionates
• Sorbates
• Sulfites
Why Naturally Occurring
Antimicrobials???
•N o new “synthetic” com pounds
•Expanded antim icrobialactivity
•Labeling
•Potentialhealth benefits
•Especially plant-based
•N otbecause w e are questioning the
safety ofexisting antim icrobials!
What is Natural?
•Acetic – Vinegar
•Benzoic – Cranberries
•Lactic -Lactic Acid
Bacteria
•Propionic -Sw iss
Cheese
•Sorbic Row anberries
Evolution
• 1930s
• Food Research -G arlic,onion,
horseradish
• 1966
• Elm er M arth
• Residue Review s
• 1980
• Baranow skiJ.D .,P.M .D avidson,C.W .
N ageland A.L.Branen.1980.Inhibition
ofSaccharom yces cerevisiae by naturally
occurring hydroxycinnam ates.Journalof
Food Science 45:592-594.
Animal Sources
Naturally Occurring
Antimicrobials
•Lysozym e
•Lactoferrin
•Lactoperoxidase System
•Chitosan
Anim alSources
PlantSources
M icrobialSources
•Chitin hydrolysis
•G ram negative bacteria
•Packaging
•Peptides– N aturally occurring,
enzym atically produced
Plant Sources
Thai Spices
•Spices
• Fingerroot
• Boesenbergia pandurata
• U sed in Thaicooking;m edicinal
•EssentialO ils,EO com ponents
•Isolated Phenolic Com pounds
•Allium
•Isothiocyanates
•O thers
uses
• Extracts
• M ethods
• Thongson,C.,P.M .D avidson,W .
M ahakarnchanakul,P.Vibulsresth.2004.
Letters in Applied M icrobiology.39:401406.
• Com m ercialEssentialO ils
Microbial Sources
Salmonella Typhimurium in whole milk
with EDTA (ppm) and Fingerroot EO (%)
Strain 2308
10.0
10.0
•Pediococcus
8.0
8.0
Log CFU/mL
Log CFU/mL
•N isin
•O ther Bacteriocins
Strain 2486
6.0
4.0
2.0
•Pediocin
6.0
4.0
•Carnobacterium
2.0
0.0
0
1
Control
10000 ppm
2
3
Time (Days)
5
7
0.3%
0.3%+10000 ppm
0.0
0
1
Control
10000 ppm
2
3
Time (Days)
5
7
0.3%
0.3%+10000 ppm
Thongson,C.,P.M .D avidson,W .M ahakarnchanakul,P.Vibulsresth.2005.
Antim icrobialeffectofThaispices againstListeria m onocytogenes and Salm onella
Typhim urium D T104.JournalofFood Protection 68:2054-2058.
•Piscicolin
Microbial Sources
•Protective Cultures
•ActualCultures
•Cultured M edium w ith Inhibitors
•M icroG ARD TM
•M icrobially-Produced Antim icrobials
•Propionates
Microbial Sources
•Bacteriophage – Concerns
•Consum er reaction
•Concentrated extraneous D N A
•M utationsto create resistantstrains
•Effectofreduced com petition
Microbial Sources
• Bacteriophage
• Intralytix
• LM P-102™
•6 bacteriophages isolated from the environm ent
•“Signficantly reduces (usually by 100-1000-fold)
… L.m onocytogenes”
•“all-naturalproduct”
•Approved by FD A and U SD A
Natural/Synthetic
•Lauric arginate
•Lauram ide arginine ethylester
•< 200 ppm in RTE m eatproducts
•M irenat® -N
•Vedeqsa Lam irsa G roup – Spain
•M inim um Inhibitory Concentrations
•10-70 μg/m lfor G ram negative and
G ram positive bacteria
•Com pany research on effectiveness
•H am ,beef,poultry
Natural Antimicrobials
Challenges to Application
• Activity
• M any are new ly discovered
• Lim ited spectrum data
• Application Studies
• Early research
•D iscovery w ithoutapplication
•Purification,identification
• Purification m ay notbe bestroute
Natural Antimicrobials
Challenges to Application
•ToxicologicalD ata
•Lacking on m ostnovelcom pounds
and system s
•Regulatory Approval
Natural Antimicrobials
Challenges to Application
• Cost
• Purification – synthesis?
• Activity Validation M ethods
Natural Antimicrobials
Challenges to Application
• Liability for Claim s
• Sensory Effects
• ProofofActivity
• Storage Stability
• CO M PATABILITY
•O regano w illnotw ork in fruitjuice or
• Acquired -m utations
• Biofilm s
• Concentrations
•Activity too low to be used
• Resistance D evelopm ent
• Influence on N aturalM icroflora
CAN WE MAKE
THESE THINGS
WORK BETTER?
ice cream
individually
•“H urdle concept”
Application Challenges
(Least to Most Difficult)
• Carbohydrate-based Beverages
• Positives -pH ,com position,dispersion
• Bakery Products
• N egative -pH
• Fruits and Vegetables
• N egatives – pH (vegetables),Targeting
• D airy Products
• N egatives – interference ofprotein,fat,
m inerals;pH
• Positive over m eat– som ew hathom ogenous
• M eatProducts
Why Are Meats So Difficult?
• N eutralpH
• Com position
• Interaction ofantim icrobialsw ith
com ponents
• Lipids
• Proteins
• Cations
• Structure
• Location ofm icroorganism s
• N on-hom ogeneity ofcontam ination
Factors Affecting Activity
• Food com ponentinteraction
•Polarity
•M ostare am phiphilic – partially
hydrophobic,partially hydrophilic
•Interaction w ith proteins,fats*
• Solubility
•Bestare notparticularly w ater soluble
*Rico-M unoz,E.and P.M .D avidson.1983.Effectofcorn
oiland casein on the antim icrobialactivity ofphenolic
antioxidants.JournalofFood Science.48:1284-1288.
Novel Delivery Systems –
Targeted Application
•M olecular-based targets
•M echanism based
•Antibody-linked com pounds
•Specific for particular m icroorganism
•Physiochem ical
Novel Delivery Systems –
Targeted Application
•Solution:Encapsulate Antim icrobials
•Surfactant-based M icelles
•Liposom es
•Polym eric N anoparticles
•O vercom e problem sofinteraction
•Targeted to m icrobialsurfaces
Surfactant-Based Micelles
• O bjectives
Nanoparticle Components
Eugenol
Carvacrol
• D eterm ine the antim icrobialactivity of
tw o phytophenols encapsulated in
surfactant-based nanoparticles against
strains ofEscherichia coliO 157:H 7 and
Listeria m onocytogenes
Surfynol
465 m,n=10
485W m,n=30
Minimum Inhibitory Concentration –
E. coli O157:H7
Minimum Inhibitory Concentration – Listeria monocytogenes
Conclusions
Nanoparticle Predicted Structure - NMR
• Encapsulation in em ulsifier nanoparticlescan
im prove the antim icrobialactivity and
applicability ofphytophenols in foods
Groups predicted to be in contact to the formation of antimicrobial
nanoparticles
Surfynol 485W
7nm in diameter
Surfynol 485W (5wt%) with
Eugenol (0.6wt%)
• G aysinsky,S., etal.2007.Antim icrobialefficacy of
eugenolm icroem ulsions in m ilk against Listeria
m onocytogenes and Escherichia coliO 157:H 7.Journalof
Food Protection 70:(in press)
• G aysinsky,S.,etal.2005.G row th inhibition ofEscherichia
coliO 157 :H 7 and Listeria m onocytogenes by carvacrol
and eugenolencapsulated in surfactantm icelles.Journal
ofFood Protection 68:2559-2566.
• G aysinsky,S.,etal.2005.Stability and antim icrobial
efficiency ofeugenolencapsulated in surfactantm icelles as
affected by tem perature and pH .JournalofFood
Protection 68:1359-1366.
8nm in diameter
Liposomes
Listeria monocytogenes Growth in Milk in the
Presence of Free Nisin and Liposome-Encapsulated Nisin
• Sphericallam ellar bilayers
• Charged or neutral
• Phosphatidylcholine,-glycerol
Conclusions
8
6
Log CFU/ml
com posed ofphospholipid
m olecules in aqueous fluids
• Encapsulate partially
hydrophobic or hydrophilic
com pounds
• Phospholipids
4
Positive Control
Free Nisin
PC Nisin
PC/PG 8/2 Nisin
PC/PG 6/4 Nisin
2
0
0
10
20
30
40
50
Time (hr)
Antimicrobial Encapsulation
• Liposom es
•Encapsulation in
liposom es m ay
im prove the
antim icrobialactivity
and applicability of
partially hydrophobic
com pounds in foods
• Taylor,T.M .,etal.2008.Listeria m onocytogenes
•
•
•
•
and Escherichia coliO 157:H 7 inhibition in vitro by
liposom e-encapsulated nisin and ED TA.J.Food
Safety (in press)
Taylor,T.M .,etal.2007.Characterization of
antim icrobial-bearing liposom es by zeta-potential,
vesicle size,and encapsulation efficiency.Food
Biophysics 2(1):1-9.
Taylor,T.M .,etal.Liposom alnanocapsules in food
science and agriculture.2005.Crit.Rev.in Food
Science and N utrition 45:587-605.
Taylor,T.M .,etal.2005.U ltrasonic spectroscopy
and differentialscanning calorim etry ofliposom alencapsulated nisin.J.Agriculturaland Food
Chem istry 53:8722-8728.
W ere,L.M .,et al.2004.Encapsulation ofnisin and
lysozym e in liposom es enhances efficacy against
Listeria m onocytogenes.J.Food Protection 67:922927.
Other Strategies
•U tilization ofcasein m icelles as
encapsulants for nanoscale
particles
•U tilization ofspray drying
technologies to produce
nanoscale particles
T he E nd
Overall Summary
• M any unknow ns asfar asapplying
food antim icrobials
• Effective research requiresa
know ledge of:
• Targetm icroorganism s
• Interfering food com ponents,
physicochem icalfactors,storage
conditions