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Use of Enterococcus Faecium NRRL B-2354 as a Surrogate
December 8, 2015
Speakers
Linda Harris, UC Davis (Moderator)
Linda Harris, UC Davis
Maria Marco, UC Davis
Shirin Abd & Carrie Ferstl, The National Food Laboratory
Linda Harris, University of California, Davis
Validating the use of Enterococcus faecium NRRL B-2354 for almond processes
Linda J. Harris, Ph.D. Specialist in Cooperative Extension Food Science and Technology Western Center for Food Safety
Surrogates: when appropriate or necessary?
• In-plant validation studies – usually almost always involve use of surrogates – introduction of the pathogen usually considered an unacceptable risk
• E.g., continuous flat bed dry roaster
Pathogens: when appropriate or necessary?
• When parameters can be mimicked in the laboratory or on pilot scale equipment – E.g., oil roast, water blanch
• In very rare cases pathogens may be used for in-plant validation where: – No surrogate exists – Contamination of equipment/facility is unlikely – Precautions are taken/planned in advance – Experiments are planned/executed with
guidance of experts
What makes an ideal surrogate?
• Non-pathogenic
• In low moisture foods (e.g., almonds) – Similar desiccation tolerance
• Represents pathogen of concern or “target organism” – For almonds and other tree nuts: Salmonella
• Similar inactivation characteristics and kinetics – Predict target organism
• Easy preparation – Stable high-density populations – Easy to enumerate
Salmonella
See: http://www.fda.gov/Food/ScienceResearch/ResearchAreas/SafePracticesforFoodProcesses/ucm094141.htm
http://www.fda.gov/Food/ScienceResearch/
Almond Board Microbial Surrogate Development 2004-2007
Potential Surrogates for Salmonella Culture Names and Source ID
Salmonella culture Salmonella Enteritidis Phage Type 30 (ATCC BAA-1045) (SE PT 30)
Surrogate cultures
Pantoea agglomerans SPS2F1 (PA) Pediococcus spp. NRRL B-2354
(Reclassified as Enterococcus faecium) (PD) Enterococcus faecalis (ATCC 49452) (EF) Lactobacillus plantarum (ATCC 14917) (LP) Lactobacillus fermentum (ATCC 9338) (LF) Streptococcus faecalis (ATCC 33186) (SF)
Potential Surrogates Screened (Dry Heat)
Salmonella Enteritidis PT30
Pantoea agglomerans
Enterococcus faecium
0.300.400.500.600.700.800.901.001.10
260 270 280 290 300 310
Log
D
Hot Air Temperature (°F)
Pantoea agglomerans and Enterococcus faecium were chosen for further study
E. faecium NRRL B-2354/ATCC 8459
• Originally Micrococcus freudenreichii Guillebeau – Isolated from milk and dairy utensils – Used as a surrogate for thermal processing of dairy products (1947)
• Renamed Pediococcus sp.
• Reclassified as Enterococcus faecium (Ma et al., 2007) – Evaluating use as a surrogate for meat
• Deposited to – American Type Culture Collection ATCC 8459 – Northern Regional Research Laboratory NRRL B-2354
• USDA National Center for Agricultural Utilization Research
http://atcc.org/Products/All/8459.aspx Annous and Kozempel. 1998. J. of Food Protection 61(5):578
Ma et al. 2007. J. of Food Protection 70(4):952 Kornacki, J. L. 2012. Food Safety Magazine.
https://commons.wikimedia.org/wiki/File:Entercoccus_sp2_lores.jpg
PresenterPresentation NotesIsolated from cheese
1970 renamed pedio
2007 renamed faecium (work from the Doyle lab)
Long history of safe use as surrogate organism in almonds, ground beef, and cider, amongst other products.
Reduction of Salmonella Enteritidis PT30 and E. faecium NRRL B-2354 (laboratory ovens, 2007)
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6 7 8
Redu
ctio
n (lo
g CF
U/g
)
265 310 295 280
S E 24
S E 15
S E 10
S E 25
S E 15
S E 45
S E 6
S E 12
Deibel - Salmonella
NFL - Enterococcus
NFL - Salmonella
Deibel - Enterococcus
Time (min) Temperature (°F)
PresenterPresentation NotesWater activity and moisture content not measured post-inoculation
Thermal treatment: 15 min, 280°F; Fisher Ovens
Influence of storage time at 4°C on thermal tolerance of E. faecium NRRL B-2354 and Salmonella Enteritidis PT30
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
0 1 2 3 4 5 6 7 8 9 10
Redu
ctio
n (lo
g CF
U/g
)
Week(s) of storage at 4°C/39°F 0 10 8 6 4 2 1 12 0 0
UCD 2012; (625G) NFL
2007; (851F)
Deibel 2007; (851F)
Salmonella significantly greater reduction at all time points No
sig.diff No
sig.diff
Thermal treatment: 15 min, 280°F; Fisher Ovens
Influence of storage time at 4°C on thermal tolerance of E. faecium NRRL B-2354 and Salmonella Enteritidis PT30
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
0 1 2 3 4 5 6 7 8 9 10
Redu
ctio
n (lo
g CF
U/g
)
Week(s) of storage at 4°C/39°F 0 10 8 6 4 2 1 12 0 0
UCD 2012; (625G) NFL
2007; (851F)
Deibel 2007; (851F)
Salmonella significantly greater reduction at all time points No
sig.diff No
sig.diff
0
1
2
3
4
1 4 8 12
Redu
ctio
n (lo
g CF
U/g
)
Weeks
Gravity Feed Oven Forced Air Oven
15 min, 280°F in gravity feed (Fisher 625 G) or forced air (Fisher 725 F) oven
Ovens are not equal - Enterococcus faecium NRRL B-2354
Hot oil 121°C/250°F
0
1
2
3
4
5
6
7
8
9
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Log
CFU
/g
Treatment time (min)
Salmonella Enteriditis PT 30
E. faecium NRRL B-2354
Kaur and Harris, unpublished
PresenterPresentation NotesE. faecium NRRL B-2354 has been used for many years as a successful surrogate organism for Salmonella
In this example, work done by the lab of Dr. Linda Harris, *describe graph and medium and oil*, this thermal tolerance curve measures the effect this treatment has on each organism, almost the same, successful surrogate for this medium and this method of pasteurization
E. faecium NRRL B-2354 a useful surrogate in moist air heating
• Conclusions • NRRL B-2354 is a conservative
surrogate for SE PT30 in the targeted reduction range of 4 to 5 log
• a potentially useful surrogate
for steam heating
Jeong et al. 2011. Journal of Food Protection 74(4):603.
Log
redu
ctio
n, E
. fae
cium
NR
RL
2354
Comparisons to other pathogens
0
1
2
3
4
Red
uctio
n (L
og C
FU/g
)
A BC
C C
D
B B B B B B
AB
Reductions during desiccation on almonds
Moussavi and Harris, unpublished
PresenterPresentation NotesMean ± SD reduction of E. faecium, Salmonella Enteritidis PT30 and different �E. coli O157:H7 or L. monocytogenes strains on inoculated almonds before exposure to heat treatments (n = 6). Columns denoted with different letters are significantly different (P < 0.05).
Reduction of in hot water (176°F/80°C) E. faecium NRRL B-2354, E. coli O157:H7, Listeria monocytogenes, and S. Enteritidis PT30
01234567
0 0.5 1 2 3Treatment time (min)
Moussavi and Harris, unpublished
Reduction in oil (250°F/121°C) Listeria monocytogenes, E. coli O157:H7, E. faecium NRRL B-2354 and S. Enteritidis PT30 on almonds
0
1
2
3
4
5
6
7
0 30 60 90 120Time (s)
Moussavi and Harris, unpublished
E. faecium NRRL B-2354 is an appropriate surrogate for Salmonella in almonds • Jeong et al., 2011 Journal of Food Protection
74(4):603 – Moist air or steam processes (ambient or vacuum)
• NFL and Deibel Laboratories, 2007 – Dry-heat processes:
• dry roast, • brine and pre-wet dry roast, • dry roast flavoring, • dry plasticizing
Jeong et al. 2011.
ABC, 2014
http://www.almondboard.com/Handlers/Documents/Enterococcus-Validation-Guidelines.pdf
Acknowledgements
• Gordon Davidson
• Lillian Khan
• Harbir Kaur
• Mahta Moussavi
• Chris Theofel
Funding Provided By:
Maria Marco, University of California, Davis
Biosafety of Enterococcus faecium NRRL B-2354
Maria L Marco
Associate Professor Department of Food Science & Technology
University of California, Davis
E. faecium: impact on human health
Used in some food fermentations
Sold as a probiotic (humans and animals)
Antibiotic-resistant, opportunistic pathogen
Each bacterial strain is genetically distinct Thermal tolerance
Antibiotic resistance Virulence
Strain specificity of E. faecium
78% genetically related 70% genetically related
Staley 1997 Cur Op Biotechnol
How to evaluate bacterial strains for safety
Phenotype Genotype
Complete genome: Chromosome (2.6 Mbp) One plasmid (214 kbp) Genome of E. faecium ATCC 8459 is over 99% identical
Genome sequence of NRRL B-2354
Kopit et al (2014) Appl Environ Microbiol Kim et al (2013) Appl Environ Microbiol
Clinical Community
Hierarchical clustering based on 128 E. faecium genomes
NRRL B-2354 is a “community” strain
NRRL B-2354 is distinct from other strains
• 45 genes not found in >100 other E. faecium genomes. Contained in loci (SPs)
• Lacks cas –CRISPRs for viral recognition
• Fewer mobile elements, virulence genes, and genes for antibiotic resistance than clinical strains
Cultures grown on tryptic soy agar with 5% defibrinated horse blood
Negative control: E. coli DH5α
Positive Control: E. faecalis ATCC
29212
cyl genes are not present in NRRL B-2354
NRRL B-2354 does not produce hemolysin NRRL B-2354
PresenterPresentation NotesThinking back to the EFSA document, the next step was Antibiotic sensitivity
acm E. faecium NRRL B-2354
acm E. faecium TX0082 – Clinical
E. faecium NRRL B-2354 contains: - scm (collagen I and IV adhesin) - sagA (fibrinogen, fibronectin, collagen I adhesin) - acm (collagen I adhesion)
100% coverage, 99% nucleotide identity
Virulence Factor: adhesion to human proteins
PresenterPresentation Notes1.1 kbp insertion at the 5’ end when compared to clinical strain and …
NRRL B-2354 does not bind collagen
Adherent cells were stained with crystal violet, positive control: clinical strain E. faecium TX0082, Tukey’s HSD, P < 0.05
n=3
Clinical
PresenterPresentation NotesBound to collagen type I, adhered cells stained with crystal violet and measured absorbance at 595nmResults agree with genome, non-functional, adhesion barely above background levelsWe also looked at the production of a hemolysin, a trait commonly seen in clinical strains
NRRL B-2354 does not form biofilms
2000 bp
NRRL B-2354 does not make pili
Transmission electron microscopy B-2354 contains pil operon and efaA but lacks ebp genes
NRRL B-2354 TX0082
Minimum inhibitory concentration, BD Phoenix system, UC Davis Medical Center
NRRL B-2354 is sensitive to antibiotics
PresenterPresentation NotesAs you can see, all were sensitive, but wanted to call attention to
Aminoglycosides Binds to 30S (some to 50S) ribosomal subunit, inhibit protein synthesis Cephalosporins (first generation) Similar to beta-lactams, inhibit peptidoglycan synthesis Cephalosporins (second generation) Similar to beta-lactams, inhibit peptidoglycan synthesis Glycopeptides Inhibit peptidoglycan synthesis Macrolides Binds to 50S ribosomal subunit, inhibit protein synthesis Nitrofurans Damages bacterial DNA (reduced form is very reactive) Penicillins (and combinations) Similar to beta-lactams, inhibit peptidoglycan synthesis Quinolones Inhibit DNA replication/transcription by inhibiting DNA gyrase Tetracyclines Bind to 30S ribosomal subunit and inhibit binding of aminoacyl-tRNA to mRNA-ribosome complex
Incubated in physiological saline, pH 2.4
Clinical
Limit of detection
NRRL B-2354 is acid tolerant
NRRL B-2354 is thermal tolerant
50 °C
60 °C
Incubated in physiological saline, LOD = 20 CFU/mL
Clinical
Limit of detection
Regulatory advice: E. faecium strain biosafety
European Food Safety Authority (2012)* Absence of esp, hyl, IS16 (marker of hospital-
associated strains) Sensitive to ampicillin
* Safety evaluation of E. faecium strains intended as additives in animal feed
Conclusions
E. faecium NRRL B-2354 (ATCC 8459) lacks phenotypic and genotype features of clinical strains According to our assessments E. faecium NRRL B-2354 (ATCC 8459) is safe for use in thermal process validation tests
PresenterPresentation NotesBased on our findings and combined with the long history of safe use of this organism, this strain could continue to be used as a surrogate for the validation of thermal processing.
Acknowledgements Mary Moore Sandra Appelt Zach Bendiks Ben Golomb Xiaochen Yin Dustin Heeney Annabelle Yu Zhengyao Xue Brendan McCarthy-Sinclair Yanin Srisengfa Irene Yim Zach Quart Elissa Goldberg
Lauren Kopit
Eun Bae Kim
Collaborator: Linda Harris, UC Davis
Shirin Abd & Carrie Ferstl The National Food Laboratory
Validation of Thermal Processes Applied to Almonds Continuing Investigations to Support the Use of E. faecium NRRL B-2354 as a Surrogate
December 8, 2015
Overview
• Review criteria to select a surrogate organism • Review ABC guidelines for preparation and evaluation of surrogate heat
resistance • Summary of ongoing research at The National Food Laboratory to evaluate the
appropriateness of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella Enteritidis PT30 on almonds
Extended storage of surrogate-inoculated almonds Thermal resistance of surrogate on different almond types
PresenterPresentation NotesEvaluation of Thermal Resistance of Surrogate on Four Different Almond TypesImpact of Extended Storage on Inoculated Almonds with Surrogate Organism What is the impact of almond types on thermal resistance of the surrogate?
Selection of a Surrogate
• Identify the target organism Based upon food safety plan Outbreaks in similar ingredients/products
• Food matrix composition/parameters Worst case scenario
• Thermal process parameters • Select potential surrogate(s) Non-pathogenic Stable Easy to grow to high levels Similar thermal resistance to the target organism
How is the appropriateness of a surrogate organism determined?
Enterococcus faecium
Pediococcus acidilactici
PresenterPresentation NotesEvaluation of Thermal Resistance of Surrogate on Four Different Almond TypesImpact of Extended Storage on Inoculated Almonds with Surrogate Organism What is the impact of almond types on thermal resistance of the surrogate?
Selection of a Surrogate
• Understand the thermal resistance of target organisms in the selected food matrix
• Understand the thermal resistance of the surrogate organism to the same conditions
Main goals when evaluating a surrogate:
Surrogate organism should demonstrate similar or greater
resistance to heat
PresenterPresentation NotesEvaluation of Thermal Resistance of Surrogate on Four Different Almond TypesImpact of Extended Storage on Inoculated Almonds with Surrogate Organism What is the impact of almond types on thermal resistance of the surrogate?
Selection of a Surrogate for Almonds
Consideration Identify the target organism
Food matrix
Process parameters
Selection of a surrogate
Almonds S. Enteritidis PT30
Whole kernel almonds
Dry roasting
E. faecium NRRL B-2354
PresenterPresentation NotesEvaluation of Thermal Resistance of Surrogate on Four Different Almond TypesImpact of Extended Storage on Inoculated Almonds with Surrogate Organism What is the impact of almond types on thermal resistance of the surrogate?
ABC Surrogate Evaluation Guidelines
ABC published a protocol, “Guidelines for Using Enterococcus faecium NRRL B-2354 as a Surrogate Microorganism in Almond Process Validation”, to provide guidance to the industry on the following points: Preparation of E. faecium NRRL B-2354 culture for inoculation
Inoculation of almonds with the prepared E. faecium NRRL B-2354 culture
Heat resistance verification for E. faecium NRRL B-2354 on the inoculated almonds
Storage of the inoculated almonds prior to validation testing
Can the protocols be refined further to provide more flexibility in their use for the validation of almond processes?
PresenterPresentation NotesEvaluation of Thermal Resistance of Surrogate on Four Different Almond TypesImpact of Extended Storage on Inoculated Almonds with Surrogate Organism What is the impact of almond types on thermal resistance of the surrogate?
Reevaluate ABC Surrogate Evaluation Guidelines
Reevaluate the following limitations of the protocol:
The almonds inoculated with the surrogate should be used within two weeks of preparation and stored at 38-40°F (refrigeration temperatures). Can inoculated almonds be stored beyond 14 days without sacrificing heat
resistance? Do inoculated almonds need to be stored at refrigeration temperatures?
The heat resistance of E. faecium NRRL B-2354 and its correlation to S. Enteritidis PT30 was completed using whole intact almonds. Can whole kernel inoculated almonds be used in plant validation studies for other
almond types?
Thermal Resistance of Surrogate on Almonds -Recent Studies at The NFL
Study Design Overview
S. Enteritidis PT30 E. faecium NRRL B-2354
Refrigeration Room temperature
Up to 60 days Forced Air Oven
280°F for 15 minutes
Calculate the log reduction to evaluate the thermal resistance of organisms on almonds
PresenterPresentation NotesTo develop a study protocol to evaluate the following:Stability over the extended shelf life of inoculated almonds (60 days)Storage temperatures: 4°C and 25°CComparative heat resistance of E. faecium and SEPT30Forced Air Oven vs. Gravity Oven
Extended Storage of Surrogate-Inoculated Almonds
The current shelf life of almonds inoculated with E. faecium NRRL B-2354 can be extended from 14 to 60 days when stored at 4°C.
Extended Storage of Surrogate-Inoculated Almonds
E. faecium NRRL B-2354 is an appropriate surrogate for S. Enteritidis PT30 when the inoculated almonds are stored at 4°C.
PresenterPresentation NotesThe heat resistance of E. faecium NRRL B-2354 on inoculated almonds is similar to heat resistance of S. Enteritidis PT30 when the inoculated almonds are stored at 4°C, but not at 25°C.
Extended Storage of Surrogate-Inoculated Almonds
The almonds inoculated with the surrogate should be used within two weeks of preparation and stored at 38-40°F (refrigeration temperatures). Can inoculated almonds be stored beyond 14 days without sacrificing heat
resistance? Do inoculated almonds need to be stored at refrigeration temperatures?
Thermal Resistance of Surrogate on Different Almond Types
Natural Dice 12-8 Natural Split
NonPl 27/30 (Nonpareil Whole Kernels without chip of scratch
27/30)
Monterey Whole/Broken
(Monterey natural whole and broken,
36aos)
Thermal Resistance of Surrogate on Different Almond Types
Almond type has an impact on the heat resistance of E. faecium NRRL B-2354
PresenterPresentation NotesHeat resistance of E. faecium NRRL B-2354 or S. Enteritidis PT30 at each sampling time was significantly different (pThermal Resistance of Surrogate on Different Almond Types
Heat resistance of S. Enteritidis PT30 is comparable to E. faecium NRRL B-2354 on all four almond types.
PresenterPresentation NotesHeat resistance of E. faecium NRRL B-2354 or S. Enteritidis PT30 at each sampling time was significantly different (pThermal Resistance of Surrogate on Different Almond Types
The current shelf life of almonds inoculated with the surrogate can be extended beyond 14 days at refrigeration temperature.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Log R
educ
tion
E. faecium NRRL B-2354 Day 0Day 14
Day 28
A
AA
AA
AA
AB
B
A A
PresenterPresentation NotesThe heat resistance of E. faecium NRRL B-2354 and S. Enteritidis PT30 on Natural Dice 12-8, Natural Split, and NonPI 27/30 almonds did not change over 28 days of storage at 4°C.
Chart1
Natural Dice 12-8Natural Dice 12-80.3956864160.20161765080.20161765080.22902434340.22902434340.33455496440.3345549644
Natural SplitNatural Split1.27520857870.11526738630.11526738630.34550857540.34550857540.55142445370.5514244537
NonPl 27/30NonPl 27/302.27857056050.59932831210.59932831210.29521903010.29521903011.15645049361.1564504936
Monterey BrokenMonterey Broken2.36511999140.61679869110.61679869110.55410950320.55410950321.08797662561.0879766256
A
A
A
A
A
A
A
A
B
B
A
A
Day 0
Day 14
Day 28
Log Reduction
E. faecium NRRL B-2354
0.2927502849
0.4537626428
1.0315400027
0.885367817
2.0118809622
2.1486092375
1.4499724326
1.0817828768
Natural Dice 12-8
Natural Dice 12/8
Trial 1Trial 2Trial 3
Day 0Day 14Day 28Day 0Day 14Day 28Day 0Day 14Day 28
UninoculatedSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faecium
AwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoisture
0.46030.44460.43130.45680.47860.47650.4839
0.45580.4241
0.45020.4277
Trial 1
E. faeciumSalmonella
wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevLog reductionwet samples-3-3-4-4-5-5-6-6averagecfu/glog cfu/gavgstdevLog reduction
92117104.51045000008.027.894045084564560000008.668.67
516256.5565000007.754405364884880000008.69
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
564249490000007.697.720.10505552.55250000008.728.720.01
563143.5435000007.64564550.55050000008.70
696567670000007.83624553.55350000008.73
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
212190201201000007.307.250.070.420.4775657070000006.856.850.171.871.86
112181146.5146500007.170.550.07100116108108000007.031.680.17
195184189.5189500007.280.44524548.548500006.692.03
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
1101021061060000008.038.070.054042414100000008.618.650.03
126105115.51155000008.06464344.54450000008.65
1501201351350000008.135046484800000008.68
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
492939390000007.597.750.230.480.32292828.5285000007.457.430.021.191.21
100109104.51045000008.020.050.23262525.5255000007.411.240.02
553344440000007.640.43272827.5275000007.441.21
Day 28Day 28
Positive Control-5-5-6-6-7-7-8-8Positive Control-4-4-5-5-6-6-7-7
919894.5945000007.987.580.55414241.54150000008.628.600.07
528166.5665000007.82464545.54550000008.66
90909090000006.953234333300000008.52
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
110809595000006.987.100.140.610.48148128138138000007.147.480.301.461.12
180180180180000007.260.330.14335443.5435000007.640.960.30
130108119119000007.080.51464646460000007.660.94
Trial 2
E. faeciumSalmonella
wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevwet samples-4-4-5-5-6-6-7-7averagecfu/glog cfu/gavgstdev
685159.5595000007.777.80485149.54950000008.698.64
726066660000007.824236393900000008.59
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
265138.5385000007.597.560.037351626200000008.798.620.17
403437370000007.573545404000000008.60
373033.5335000007.532929292900000008.46
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
403035350000007.547.500.110.020.0635454040000006.606.780.172.021.84
247234240.5240500007.380.180.11112668989000006.951.670.17
443238380000007.58-0.0269556262000006.791.83
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
395547470000007.677.770.08585958.55850000008.778.780.07
508668680000007.837072717100000008.85
685762.5625000007.80436051.55150000008.71
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
302728.5285000007.457.370.080.310.401011071041040000008.027.870.230.760.91
241203222222000007.350.420.08453439.5395000007.601.180.23
200195197.5197500007.300.471157896.5965000007.980.79
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
252525250000007.407.430.04144157150.51505000008.188.140.06
183526.5265000007.421481481481480000008.17
352530300000007.48127112119.51195000008.08
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
354439.5395000007.607.420.24-0.160.01133165149149000007.177.430.390.970.71
133145139139000007.140.290.24846876760000007.880.260.39
254133330000007.52-0.09165182173.5173500007.240.90
Trial 3
E. faeciumSalmonella
wet samples-4-4-5-5-6-6-7-7averagecfu/glof cfu/gavgstdevwet samples-4-4-5-5-6-6-7-7averagecfu/glog cfu/gavgstdev
9310699.59950000009.009.031664218419241924000000010.2810.15
13210411811800000009.071056105610561056000000010.02
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
585456560000007.757.720.042737323200000008.518.360.20
555454.5545000007.741351371361360000008.13
613347470000007.672826272700000008.43
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
189172180.5180500007.267.370.110.460.35475450.5505000007.707.640.090.650.72
259218238.5238500007.380.340.11445047470000007.670.680.09
352429.5295000007.470.25373134340000007.530.83
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
619276.5765000007.887.880.00523342.54250000008.638.630.01
000.00463741.54150000008.62
000.004145434300000008.63
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
78144111111000007.057.250.260.840.64694456.5565000007.757.680.100.870.95
151133142142000007.150.730.26564852520000007.720.910.10
432634.5345000007.540.35353937370000007.571.06
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
708075750000007.887.770.09315844.54450000008.658.730.08
307251510000007.715375646400000008.81
476053.5535000007.73555454.55450000008.74
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
175122148.5148500007.177.080.080.600.6912099109.51095000008.047.820.270.690.91
96111103.5103500007.010.760.08273933330000007.521.210.27
120103111.5111500007.050.72808281810000007.910.82
Natural Splits
Natural Splits
Trial 1Trial 2Trial 3
Day 0Day 14Day 28Day 0Day 14Day 28Day 0Day 14Day 28
UninoculatedSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faecium
AwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoisture
0.60660.57630.57150.59570.58360.58790.5827
0.57090.5751
0.57360.5733
Trial 1
E. faeciumSalmonella
wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevLog reductionwet samples-3-3-4-4-5-5-6-6averagecfu/glog cfu/gavgstdevLog reduction
737674.5745000007.877.963864904384380000008.648.67
116103109.51095000008.04614401507.55075000008.71
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
796974740000007.877.630.21110109109.51095000008.048.380.30
352731310000007.494426353500000008.54
392833.5335000007.534331373700000008.57
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
343333.533500006.536.550.031.101.07282727.5275000007.447.420.230.940.97
43333838000006.581.050.03149152150.5150500007.181.210.23
44283636000006.561.07483843430000007.630.75
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
1137091.5915000007.967.900.08424141.54150000008.628.470.13
1007587.5875000007.943119252500000008.40
705964.5645000007.81242524.52450000008.39
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
867580.580500006.917.080.391.000.82404040400000007.607.241.000.871.23
422533.5335000007.530.380.39120841021020000008.010.461.00
86446565000006.811.0912014013013000006.112.35
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
703653530000007.727.800.14202924.52450000008.398.420.05
969093930000007.972525252500000008.40
663751.5515000007.71253630.53050000008.48
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
19020019519500006.296.610.391.511.19303733.5335000007.537.360.240.901.07
114109111.5111500007.050.750.391212120000007.081.340.24
342730.530500006.481.32293029.5295000007.470.95
Trial 2
E. faeciumSalmonella
wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevwet samples-4-4-5-5-6-6-7-7averagecfu/glog cfu/gavgstdev
674656.5565000007.757.784244434300000008.638.66
706065650000007.81504949.54950000008.69
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
413739390000007.597.650.095545505000000008.708.700.02
473641.5415000007.62535252.55250000008.72
674757570000007.764850494900000008.69
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
57595858000006.766.660.140.890.99303130.5305000007.487.630.181.221.07
283531.531500006.501.160.14607567.5675000007.830.870.18
525553.553500006.730.93483039390000007.591.11
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
333735350000007.547.600.09180199189.51895000008.288.150.14
416151510000007.711601341471470000008.17
432936360000007.5610499101.51015000008.01
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
1126588.588500006.956.980.160.660.62574852.5525000007.727.810.110.430.34
128158143143000007.160.450.16744459590000007.770.380.11
716869.569500006.840.76948087870000007.940.21
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
145159152152000007.187.330.14113134123.51235000008.098.160.07
212250231231000007.361332031681680000008.23
292728280000007.45153138145.51455000008.16
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
18019818918900006.286.580.271.050.7592115103.5103500007.017.110.091.151.05
46444545000006.650.680.27190130160160000007.200.960.09
52766464000006.810.52130121125.5125500007.101.06
Trial 3
E. faeciumSalmonella
wet samples-4-4-5-5-6-6-7-7averagecfu/glof cfu/gavgstdevwet samples-4-4-5-5-6-6-7-7averagecfu/glog cfu/gavgstdev
10666868600000008.938.96960206415121512000000010.1810.21
9793959500000008.981888163217601760000000010.25
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
537765650000007.817.800.111351311331330000008.128.080.07
798079.5795000007.90119134126.51265000008.10
435348480000007.68971031001000000008.00
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
73837878000006.896.770.160.911.03178170174174000007.247.160.110.830.91
56806868000006.830.970.1612694110110000007.041.030.11
374038.538500006.591.21175144159.5159500007.200.87
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
706567.5675000007.837.630.20363736.53650000008.568.600.04
292627.5275000007.444337404000000008.60
592542420000007.62474043.54350000008.64
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
303934.534500006.546.420.181.091.2197899393000006.977.130.291.631.48
173154163.516350006.211.420.18681088888000006.941.660.29
38263232000006.511.13322629290000007.461.14
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
781401091090000008.048.000.04303532.53250000008.518.600.08
5712591910000007.963260464600000008.66
9310699.5995000008.004242424200000008.62
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
16818617717700006.256.120.141.751.88424543.5435000007.647.630.080.960.97
7511092.59250005.972.030.14425850500000007.700.900.08
12015813913900006.141.86343635350000007.541.06
NonP XI 2730 FPO
NonP XI 27/30 FPO
Trial 1Trial 2Trial 3
Day 0Day 14Day 28Day 0Day 14Day 28Day 0Day 14Day 28
UninoculatedSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faecium
AwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoisture
0.53690.55430.53930.54270.54370.53040.5468
0.55820.5534
0.556250.5464
Trial 1
E. faeciumSalmonella
wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevLog reductionwet samples-3-3-4-4-5-5-6-6averagecfu/glog cfu/gavgstdevLog reduction
314437.5375000007.577.652192312252250000008.358.40
466053530000007.722533012772770000008.44
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
73636868000006.836.860.02444142.5425000007.637.730.11
88627575000006.88766570.5705000007.85
76747575000006.88674053.5535000007.73
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
7212096960004.984.910.361.881.955969646400005.815.920.271.931.82
180155167.51675005.221.640.36174159166.516650006.221.510.27
372832.5325004.512.35624553.55350005.732.01
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
93758484000006.927.050.13617668.5685000007.847.740.09
101130115.5115500007.06454746460000007.66
160140150150000007.18406954.5545000007.74
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
160181170.51705005.235.060.281.821.995242474700005.675.780.322.071.96
2101241671670005.221.830.2810617013813800006.141.610.32
496054.5545004.742.324424343400005.532.21
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
1049599.599500007.006.970.12847680800000007.907.740.25
90466868000006.83263128.5285000007.45
130108119119000007.08865671710000007.85
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
9411210310300006.015.880.180.961.091845812112100006.085.810.241.651.92
4254484800005.681.290.18505351.55150005.712.020.24
809989.58950005.951.024840444400005.642.09
Trial 2
E. faeciumSalmonella
wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevwet samples-3-3-4-4-5-5-6-6averagecfu/glog cfu/gavgstdev
432835.5355000007.557.6423695165.51655000008.228.11
505954.5545000007.74901101001000000008.00
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
64686666000006.826.720.15292024.5245000007.397.450.09
30403535000006.54173425.5255000007.41
84426363000006.80333835.5355000007.55
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
183210196.51965005.295.270.351.431.45374440.54050005.615.410.371.842.04
5262764014010005.601.120.354739434300005.631.820.37
719181810004.911.818910597970004.992.46
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
14197119119000007.087.030.04534951510000007.717.720.05
81134107.5107500007.03563847470000007.67
1099099.599500007.00675058.5585000007.77
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
2221782002000005.305.030.241.732.018561737300005.865.920.181.851.79
1175385850004.932.110.245765616100005.791.930.18
865872720004.862.1814811613213200006.121.60
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
253.535000006.546.690.2111293102.5102500007.017.080.08
71440000006.60138155146.5146500007.17
988.585000006.9397128112.5112500007.05
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
799084.5845004.934.640.261.762.05433740400004.604.690.093.113.03
143725.5255004.412.290.26455148480004.683.030.09
542539.5395004.602.10566560.5605004.782.93
Trial 3
E. faeciumSalmonella
wet samples-4-4-5-5-6-6-7-7averagecfu/glof cfu/gavgstdevwet samples-4-4-5-5-6-6-7-7averagecfu/glog cfu/gavgstdev
647569.56950000008.848.99380423401.540150000009.609.51
8019413713700000009.14241276258.525850000009.41
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
353635.535500006.556.650.19583044440000007.647.710.06
254233.533500006.53575154540000007.73
271197373000006.86536458.5585000007.77
Treated 280F/15min-2-2-3-3-4-4-5-5treated at 280F/15min-3-3-4-4-5-5-6-6
193276234.5234504.374.020.382.282.6316314115215200006.186.050.121.531.66
38464242003.623.020.388811210010000006.001.710.12
114116115115004.062.5911670939300005.971.75
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
25889173.5173500007.247.010.21392833.5335000007.537.520.01
65656565000006.81293632.5325000007.51
118749696000006.98392733330000007.52
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
283933.5335004.534.560.092.492.456094777700005.896.120.271.631.39
514146460004.662.350.0910013211611600006.061.450.27
253831.5315004.502.51282526.526500006.421.10
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
126148137137000007.147.160.077210086860000007.937.940.02
175166170.5170500007.23699582820000007.91
107148127.5127500007.11839991910000007.96
Treated 280F/15min-2-2-3-3-4-4-5-5treated at 280F/15min-3-3-4-4-5-5-6-6
20282424003.383.310.083.633.701109610310300006.016.100.091.921.83
10322121003.323.690.08144171157.515750006.201.740.09
15191717003.233.7812512912712700006.101.83
Monterey whole broken
Monterey natural whole and broken, 36aos
Trial 1Trial 2Trial 3
Day 1Day 14Day 28Day 0Day 14Day 28Day 0Day 14Day 28
UninoculatedSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faeciumSalmonellaE. faeciumUninoculatedE. faeciumUninoculatedE. faecium
AwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoistureAwMoisture
0.53830.57180.56600.54290.56710.54360.5605
0.56690.5571
0.569350.56155
Trial 1
E. faeciumSalmonella
wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevLog reductionwet samples-3-3-4-4-5-5-6-6averagecfu/glog cfu/gavgstdevLog reduction
206205205.5205500007.317.522482782632630000008.428.50
574852.5525000007.72372383377.53775000008.58
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
233179206206000007.317.060.28635760600000007.787.890.09
58565757000006.76879289.5895000007.95
144120132132000007.12927885850000007.93
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
11396104.510450006.026.010.181.041.058250666600005.825.880.302.072.01
150161155.515550006.190.870.1817015216116100006.211.680.30
7165686800005.831.234042414100005.612.27
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
523342.542500006.636.620.01999999990000008.007.970.02
57254141000006.611018894.5945000007.98
562540.540500006.611007989.5895000007.95
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
1607611811800006.076.050.430.540.5710989999900006.006.520.831.981.46
433840.54050005.611.010.43121120120.512050006.081.890.83
362329.529500006.470.15273330300000007.480.50
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
452836.5365000007.567.600.07966480800000007.908.000.08
574149490000007.69110115112.51125000008.05
324136.5365000007.561101101101100000008.04
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
43293636000006.566.480.071.051.1312110111111100006.056.160.211.951.83
25312828000006.451.160.071309011011000006.041.960.21
242926.526500006.421.18262525.525500006.411.59
Trial 2
E. faeciumSalmonella
wet samples-3-3-4-4-5-5-6-6averagecfu/glof cfu/gavgstdevwet samples-3-3-4-4-5-5-6-6averagecfu/glog cfu/gavgstdev
555856.5565000007.757.8111899108.51085000008.048.04
856173730000007.861111151131130000008.05
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
74204139139000007.147.170.03469369.5695000007.847.780.05
134169151.5151500007.18506457570000007.76
137175156156000007.19436855.5555000007.74
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
99132115.511550006.066.080.311.111.099710098.59850005.995.920.131.791.86
497059.55950005.771.400.315565606000005.782.000.13
162341251.525150006.400.771079299.59950006.001.78
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
190263226.5226500007.367.370.061442001721720000008.248.030.19
169245207207000007.32130721011010000008.00
253027.5275000007.44796371710000007.85
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
13812413113100006.126.370.231.251.00817678.578500006.896.620.241.141.41
322528.528500006.450.920.2336263131000006.491.540.24
254635.535500006.550.8225353030000006.481.55
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
124127125.5125500007.107.200.10292125250000007.407.460.05
91300195.5195500007.29273430.5305000007.48
170150160160000007.20293331310000007.49
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
898.585003.934.560.573.272.631468611611600006.066.320.271.391.14
123100111.51115005.052.150.5743374040000006.600.860.27
356952520004.722.4811760020202020000006.301.16
Trial 3
E. faeciumSalmonella
wet samples-4-4-5-5-6-6-7-7averagecfu/glof cfu/gavgstdevwet samples-4-4-5-5-6-6-7-7averagecfu/glog cfu/gavgstdev
56116868600000008.938.8027325726526500000009.429.47
474445.54550000008.66317334325.532550000009.51
Day 0Day 0
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
121143132132000007.127.160.04845971.5715000007.857.920.09
160136148148000007.17100109104.51045000008.02
159159159159000007.20797878.5785000007.89
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
107132119.51195005.084.960.312.092.2122021021521500006.336.390.421.591.53
354540400004.602.560.3166706868000006.831.090.42
189120154.51545005.191.981139110210200006.011.91
Day 14Day 14
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
163162162.5162500007.217.420.21324136.53650000008.568.590.04
252726260000007.414838434300000008.63
513342420000007.62334036.53650000008.56
Treated 280F/15min-3-3-4-4-5-5-6-6treated at 280F/15min-3-3-4-4-5-5-6-6
3789636300005.805.730.201.621.68282928.528500006.456.620.272.131.96
787777.57750005.891.530.20362530.530500006.482.100.27
353032.53250005.511.9096748585000006.931.66
Day 28Day 28
Positive Control-3-3-4-4-5-5-6-6Positive Control-4-4-5-5-6-6-7-7
18083131.51315000008.127.920.18167166166.51665000008.228.220.08
556158580000007.761981901941940000008.29
807577.5775000007.891381361371370000008.14
Treated 280F/15min-2-2-3-3-4-4-5-5treated at 280F/15min-3-3-4-4-5-5-6-6
40625151003.714.590.774.223.3355816868000006.836.590.391.381.63
828684840004.923.000.7755696262000006.791.420.39
1181601391390005.142.78165110137.513750006.142.08
Summary
Enterococcus faecium NRRL B-2354Salmonella Enteritidis PT30
Natural Dice 12-8Natural SplitNonPl 27/30Monterey Whole BrokenNatural Dice 12-8Natural SplitNonPl 27/30Monterey Whole BrokenEntrococcusSalmonella PT30
Day 0Trial 1Rep 10.421.101.881.04Day 0Trial 1Rep 11.870.941.932.07Day 0Day 14Day 28Day 0Day 14Day 28EntrococcusSalmonella PT30
Rep 20.551.051.640.87Rep 21.681.211.511.68Natural Dice 12-80.290.450.401.471.020.91Day 0Day 14Day 0Day 14
Rep 30.441.072.351.23Rep 32.030.752.012.27Natural Split1.030.891.280.981.011.03Natural Dice 12-80.470.321.861.21
Avg0.471.071.951.05Avg1.860.971.822.01NonPl 27/302.012.152.281.841.722.26Natural Split1.070.820.971.23
Stdev0.070.030.360.18stdev0.170.230.270.30Monterey Broken1.451.082.371.801.611.53NonPl 27/301.951.991.821.96
Trial 2Rep 10.020.891.431.11Trial 2Rep 12.021.221.841.79Monterey1.050.572.011.46
Rep 20.181.161.121.40Rep 21.670.871.822.00Stdev0.200.230.330.580.190.33
Rep 3-0.020.931.810.77Rep 31.831.112.461.780.120.350.550.170.740.140.070.230.170.02
Avg0.060.991.451.09Avg1.841.072.041.860.600.301.160.280.340.590.030.390.231
Stdev0.110.140.350.31Stdev0.170.180.370.130.620.551.090.340.520.400.360.280.270.32
Trial 3Rep 10.460.912.282.09Trial 3Rep 10.650.831.531.590.180.430.30.83
Rep 20.340.973.022.56Rep 20.681.031.711.09
Rep 30.251.212.591.98Rep 30.830.871.751.91
Avg0.351.032.632.21Avg0.720.911.661.53E. faecium NRRL B-2354S. Enteritidis PT30EntrococcusSalmonella PT30
0.110.160.380.31stdev0.090.110.120.42Day 0Day 0Day 14Day 14
Avg of 9 samples0.291.032.011.45Avg of 9 samples1.470.981.841.80
Stdev of 9 samples0.200.120.600.62Stdev of 9 samples0.580.170.280.34
Day 14Trial 1Rep 10.481.001.820.54Day 14Trial 1Rep 11.190.872.071.98Natural Dice 12-80.471.860.321.21
Rep 20.050.381.831.01Rep 21.240.461.611.89Natural Split1.070.970.821.23
Rep 30.431.092.320.15Rep 31.212.352.210.50NonPl 27/301.951.821.991.96
Avg0.320.821.990.57Avg1.211.231.961.46Monterey1.052.010.571.46
Stdev0.230.390.280.43stdev0.021.000.320.83
Trial 2Rep 10.310.661.731.25Trial 2Rep 10.760.431.851.140.070.170.230.02
Rep 20.420.452.110.92Rep 21.180.381.931.540.030.230.391
Rep 30.470.762.180.82Rep 30.790.211.601.550.360.270.280.32
Avg0.400.622.011.00Avg0.910.341.791.410.180.30.430.83
0.080.160.240.230.230.110.180.24
Trial 3Rep 10.841.092.491.62Trial 3Rep 10.871.631.632.13
Rep 20.731.422.351.53Rep 20.911.661.452.10
Rep 30.351.132.511.90Rep 31.061.141.101.66
Avg0.641.212.451.68Avg0.951.481.391.96
0.260.180.090.200.100.290.270.27
Avg of 9 samples0.450.892.151.08Avg of 9 samples1.021.011.721.61
Stdev of 9 samples0.230.350.300.55Stdev of 9 samples0.190.740.340.52
Day 28Trial 1Rep 10.6061.510.961.05Day 28Trial 1Rep 11.460.901.651.95
Rep 20.3290.751.291.16Rep 20.961.342.021.96
Rep 30.5091.321.021.18Rep 30.940.952.091.59E. faecium NRRL B-2354S. Enteritidis PT30EntrococcusSalmonella PT30EntrococcusSalmonella PT30
Avg0.481.191.091.13Avg1.121.071.921.83Day 0Day 0Day 14Day 14Day 28Day 2800.291.032.011.45
0.140.390.180.070.300.240.240.21140.450.892.151.08
Trial 2Rep 1-0.161.051.763.27Trial 2Rep 10.971.153.111.39280.401.282.282.37
Rep 20.290.682.292.15Rep 20.260.963.030.86Natural Dice 12-80.291.470.451.020.400.91
Rep 3-0.090.522.102.48Rep 30.901.062.931.16Natural Split1.030.980.891.011.281.03
Avg0.010.752.052.63Avg0.711.053.031.14NonPl 27/302.011.842.151.722.282.26
0.240.270.260.570.390.090.090.27Monterey Broken1.451.801.081.612.371.53
Trial 3Rep 10.601.753.634.22Trial 3Rep 10.690.961.921.38
Rep 20.762.033.693.00Rep 21.210.901.741.42Stdev0.200.580.230.190.330.33
Rep 30.721.863.782.78Rep 30.821.061.832.080.120.170.350.740.550.14
Avg0.691.883.703.33Avg0.910.971.831.630.600.280.300.341.160.59
0.080.140.080.770.270.080.090.390.620.340.550.521.090.40
Avg of 9 samples0.401.282.282.37Avg of 9 samples0.911.032.261.53
Stdev of 9 samples0.330.551.161.09Stdev of 9 samples0.330.140.590.40
E. faecium NRRL B-2354
Natural Dice 12-8Natural SplitNonPl 27/30Monterey Broken
Day 00.291.032.011.45
Day 140.450.892.151.08
Day 280.401.282.282.37
Stdev0.200.120.600.62
0.230.350.300.55E. faecium NRRL B-2354
Natural Dice 12-8Natural SplitNonPl 27/30Monterey Broken
0.330.551.161.09Day 00.471.071.951.05
Day 140.320.821.990.57
S. Enteritidis PT30
Natural Dice 12-8Natural SplitNonPl 27/30Monterey0.070.030.360.18
Day 01.470.981.841.80
0.230.390.280.43
Day 141.021.011.721.61
S. Enteritidis PT30
Day 280.911.032.261.53Natural Dice 12-8Natural SplitNonPl 27/30Monterey
Day 01.860.971.822.01
Stdev0.580.170.280.34Day 141.211.231.961.46
0.190.740.340.520.170.230.270.3
0.330.140.590.400.021.00.320.83
Summary
0.07304669040.07304669040.23348658220.2334865822
0.02746130720.02746130720.38713210470.3871321047
0.36213163130.36213163130.28341311550.2834131155
0.17965615960.17965615960.4316104540.431610454
Day 0
Day 14
Log Reduction
E. faecium NRRL B-2354
Summay-Analysis Results
0.170.170.020.02
0.230.2311
0.270.270.320.32
0.30.30.830.83
Day 0
Day 14
Log Reduction
S. Enteritidis PT30
0.07304669040.07304669040.170.17
0.02746130720.02746130720.230.23
0.36213163130.36213163130.270.27
0.17965615960.17965615960.30.3
E. faecium NRRL B-2354
S. Enteritidis PT30
Log Reduction
Day 0
0.23348658220.23348658220.020.02
0.38713210470.387132104711
0.28341311550.28341311550.320.32
0.4316104540.4316104540.830.83
E. faecium NRRL B-2354
S. Enteritidis PT30
Log Reduction
Day 14
00.07304669040.07304669040.02746130720.02746130720.36213163130.36213163130.17965615960.1796561596
��������
0.23348658220.23348658220.38713210470.38713210470.28341311550.28341311550.4316104540.431610454
Natural Dice 12-8
Natural Split
NonPl 27/30
Monterey Broken
Log Reduction
E. faecium NRRL B-2354
0.170.170.230.230.270.270.30.3
��������
0.020.02110.320.320.830.83
Natural Dice 12-8
Natural Split
NonPl 27/30
Monterey Broken
Log Reduction
S. Enteritidis PT30
0.20161765080.20161765080.22902434340.22902434340.33455496440.3345549644
0.11526738630.11526738630.34550857540.34550857540.55142445370.5514244537
0.59932831210.59932831210.29521903010.29521903011.15645049361.1564504936
0.61679869110.61679869110.55410950320.55410950321.08797662561.0879766256
A
A
A
A
A
A
A
A
A
A
A
B
Day 0
Day 14
Day 28
Log Reduction
E. faecium NRRL B-2354
0.57967493250.57967493250.19160793590.19160793590.33079175920.3307917592
0.1682274030.1682274030.73523481560.73523481560.14314909540.1431490954
0.28477431260.28477431260.34045558490.34045558490.59182500940.5918250094
0.33769661260.33769661260.52482660330.52482660330.40468609290.4046860929
A
A
A
A
A
A
A
A
A
A
A
A
Day 0
Day 14
Day 28
Log Reduction
S. Enteritidis PT30
0.20161765080.20161765080.57967493250.5796749325
0.11526738630.11526738630.1682274030.168227403
0.59932831210.59932831210.28477431260.2847743126
0.61679869110.61679869110.33769661260.3376966126
A
A
A
A
A
A
A
A
E. faecium NRRL B-2354
S. Enteritidis PT30
Log Reduction
Day 0
0.22902434340.22902434340.19160793590.1916079359
0.34550857540.34550857540.73523481560.7352348156
0.29521903010.29521903010.34045558490.3404555849
0.55410950320.55410950320.52482660330.5248266033
A
A
A
A
A
A
A
A
E. faecium NRRL B-2354
S. Enteritidis PT30
Log Reduction
Day 14
0.33455496440.33455496440.33079175920.3307917592
0.55142445370.55142445370.14314909540.1431490954
1.15645049361.15645049360.59182500940.5918250094
1.08797662561.08797662560.40468609290.4046860929
A
A
A
A
A
A
A
A
E. faecium NRRL B-2354
S. Enteritidis PT30
Log Reduction
Day 28
Natural Dice 12-8
Natural Split
NonPl 27/30
Monterey Broken
00.20161765080.20161765080.11526738630.11526738630.59932831210.59932831210.61679869110.6167986911
��������
0.22902434340.22902434340.34550857540.34550857540.29521903010.29521903010.55410950320.5541095032
��������
0.33455496440.33455496440.55142445370.55142445371.15645049361.15645049361.08797662561.0879766256
A
B
C
D
A
B
B
C
A
A
B
C
Natural Dice 12-8
Natural Split
NonPl 27/30
Monterey Broken
Log Reduction
E. faecium NRRL B-2354
00.57967493250.57967493250.1682274030.1682274030.28477431260.28477431260.33769661260.3376966126
��������
0.19160793590.19160793590.73523481560.73523481560.34045558490.34045558490.52482660330.5248266033
��������
0.33079175920.33079175920.14314909540.14314909540.59182500940.59182500940.40468609290.4046860929
A
A
B
C
A
A
A
A
A
B
C
C
Natural Dice 12-8
Natural Split
NonPl 27/30
Monterey Broken
Log Reduction
S. Enteritidis PT30
EntrococcusDay 0Day 14Day 28Conf. Level
Natural Dice 12-80.290.450.40NSD
Natural Split1.030.891.28NSD
NonPl 27/302.012.152.28NSD
bba
Monterey Broken1.451.082.3795%
Salmonella PT30
Natural Dice 12-81.471.020.91NSD
Natural Split0.981.011.03NSD
NonPl 27/301.841.722.26NSD
Monterey Broken1.801.611.53NSD
E. faecium NRRL B-2354S. Enteritidis PT30
Day 0Day 0Conf. Level
Natural Dice 12-80.291.47NSD
Natural Split1.030.98NSD
NonPl 27/302.011.84NSD
Monterey Broken1.451.80NSD
E. faecium NRRL B-2354S. Enteritidis PT30
Day 14Day 14Conf. Level
Natural Dice 12-80.451.02NSD
Natural Split0.891.01NSD
NonPl 27/302.151.72NSD
Monterey Broken1.081.61NSD
E. faecium NRRL B-2354S. Enteritidis PT30
Day 28Day 28Conf. Level
Natural Dice 12-80.400.91NSD
Natural Split1.281.03NSD
NonPl 27/302.282.26NSD
Monterey Broken2.371.53NSD
E. faecium NRRL B-2354Natural Dice 12-8Natural SplitNonPl 27/30Monterey BrokenConf. Level
dcab
Day 00.291.032.011.4595%
cbab
Day 140.450.892.151.0895%
cbaa
Day 280.401.282.282.3795%
S. Enteritidis PT30
bcaa
Day 01.470.981.841.8095%
Day 141.021.011.721.61NSD
ccab
Day 280.911.032.261.5395%
Analysis Method: Analysis of Variance
Software used: SPSS
NSD: Not significantly different at the confidence level of 95%.
Mean ratings with different superscripts differ significantly at the 95% confidence level (Duncan's Multiple Comparison Test).
Thermal Resistance of Surrogate on Different Almond Types
The heat resistance of E. faecium NRRL B-2354 and its correlation to S. Enteritidis PT30 was completed using whole intact almonds. Can whole kernel inoculated almonds be used in plant validation studies for other
almond types?
Conclusions
• Shelf life of almonds (whole kernel) inoculated with E. faecium NRRL B-2354 can be extended from 14 to 60 days when stored at 4°C.
• Almonds inoculated with E. faecium NRRL B-2354 should not be stored at 25°C, or heat resistance will change.
• Shelf life of the Natural Dice 12-8 and Natural Split almonds inoculated with E. faecium NRRL B-2354 can be extended to 28 days at 4°C.
• Heat resistance of E. faecium NRRL B-2354 or S. Enteritidis PT30 is significantly different depending on almond type.
PresenterPresentation NotesThe inoculated almonds with E. faecium NRRL B-2354 should not be stored at 25°C as the heat resistance of the surrogate will change, and will no longer have comparable resistance to S. Enteritidis PT30.
Contributors
Shirin J. Abd, M.S.
Research Microbiologist, Food Safety [email protected]
925-556-4812
Carrie Ferstl, Ph.D. Director, Food Safety
[email protected] 925-551-4293
Questions?
Use of Enterococcus Faecium NRRL �B-2354 as a SurrogateSpeakersLinda Harris, �University of California, DavisValidating the use of �Enterococcus faecium NRRL B-2354 �for almond processesSurrogates: when appropriate or necessary?Pathogens: when appropriate or necessary?What makes an ideal surrogate?Almond Board Microbial Surrogate Development 2004-2007Potential Surrogates Screened (Dry Heat)E. faecium NRRL B-2354/ATCC 8459Reduction of Salmonella Enteritidis PT30 and E. faecium NRRL B-2354 �(laboratory ovens, 2007)Influence of storage time at 4°C on thermal tolerance of �E. faecium NRRL B-2354 and Salmonella Enteritidis PT30Influence of storage time at 4°C on thermal tolerance of �E. faecium NRRL B-2354 and Salmonella Enteritidis PT30Ovens are not equal - Enterococcus faecium NRRL B-2354 Hot oil 121°C/250°FE. faecium NRRL B-2354 a useful surrogate in moist air heatingComparisons to other pathogensReduction of in hot water (176°F/80°C) E. faecium NRRL B-2354, �E. coli O157:H7, Listeria monocytogenes, and S. Enteritidis PT30Reduction in oil (250°F/121°C) Listeria monocytogenes, E. coli O157:H7, �E. faecium NRRL B-2354 and S. Enteritidis PT30 on almondsE. faecium NRRL B-2354 is an �appropriate surrogate for Salmonella �in almondsAcknowledgementsMaria Marco, �University of California, DavisSlide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Shirin Abd & Carrie Ferstl�The National Food LaboratoryValidation of Thermal Processes Applied to AlmondsOverviewSelection of a SurrogateSelection of a SurrogateSelection of a Surrogate for AlmondsABC Surrogate Evaluation GuidelinesReevaluate ABC Surrogate Evaluation GuidelinesThermal Resistance of Surrogate on Almonds�-Recent Studies at The NFL�Study Design OverviewExtended Storage of Surrogate-Inoculated AlmondsExtended Storage of Surrogate-Inoculated AlmondsExtended Storage of Surrogate-Inoculated AlmondsThermal Resistance of Surrogate on Different Almond TypesThermal Resistance of Surrogate on Different Almond TypesThermal Resistance of Surrogate on Different Almond TypesThermal Resistance of Surrogate on Different Almond TypesThermal Resistance of Surrogate on Different Almond TypesConclusionsContributorsQuestions?