The Bactericidal Effect of Wild Blueberries on Listeria monocytogenes
Kristin Ahrens Russell, Aileen Artus, Anne Nillo and Carrie M. H. Ferstl; Covance Food Solutions, Livermore, California
Listeria monocytogenes is a bacterial pathogen that has been associated with
cases of foodborne illness linked to dairy, seafood, fruit, and vegetable products.
This microorganism can be particularly problematic for the food industry due to its
ubiquity in the environment, its ability to persist and grow at low temperatures,
and the current U.S. ‘zero tolerance’ policy for ready-to-eat foods. Jasper Wyman
& Son requested the assistance of The National Food Laboratory (now Covance
Food Solutions) to design and execute a study in order to determine the fate of L.
monocytogenes on individually quick frozen (IQF) wild blueberries stored under
frozen conditions and under conditions simulating a thaw step at refrigeration
temperatures.
Results
Initial Bacterial Effect Observed
Extended Bacterial Effect Observed
▶ A substantial level of L. monocytogenes died-off immediately after inoculation of the wild
blueberries prior to or during enumeration of the Time 0 samples for Trials 1 and 2
▶ IQF wild blueberries did not support the growth of L. monocytogenes at any time during
28 days of storage
0.2 mL
(B)
▶ Under frozen conditions, the population level remained stable (1.06-1.65 Log CFU/g)
▶ Under refrigerated conditions, the population level decreased to undetectable levels
25 g wild blueberries (C)
L. monocytogenes
inoculum
6
Actual Population Recovered at Time 0
<10 CFU/g or <1.00 Log CFU/g, N=3 (Trial 1)
35 CFU/g or 1.44 Log CFU/g, N=3 (Trial 2)
Objective
To evaluate the behavior of Listeria monocytogenes on artificially inoculated wild
blueberries during storage under frozen and refrigeration conditions.
6.05 x 105 CFU/mL (Trial 1)
(A)
1.82 x 107 CFU/mL (Trial 2)
𝐀𝐀 𝐱𝐱 𝐁𝐁
= Calculated Population Added
𝐂𝐂
4.84 x 103 CFU/g or 3.68 Log CFU/g (Trial 1)
1.45 x 105 CFU/g or 5.16 Log CFU/g (Trial 2)
Materials & Methods
Products
VS.
▶ IQF peas, purchased at commercial retailer (pH 6.7, aw 0.99)
▶ A third trial was conducted where IQF peas (neutral pH, not known for antimicrobial
activity) was tested alongside wild blueberries to ensure accuracy of inoculation and
recovery methods
Listeria monocytogenes 5-strain cocktail
▶ The population of L. monocytogenes recovered from artificially-inoculated peas was very
similar to the calculated population added
▶ Wyman’s IQF Wild Blueberries (pH 3.4, aw 0.98)
▶ NFPA 7064, NFPA 7077, NFPA 7085, NFPA 7092, ATCC 51772
Actual Population Recovered at Time 0
▶ Acid-adapted in Brain Heart Infusion broth with 1% glucose
3.95 x 105 CFU/g or 5.60 Log CFU/g, n=3 (Trial 3)
VS.
Target Inoculation Level
▶ 105 to 106 colony forming units (CFU) per gram
Calculated Population Added
3.60 x 105 CFU/g or 5.56 Log CFU/g (Trial 3)
Storage Conditions
▶ Sterile Whirl-Pak® bags (Nasco Industries)
▶ Refrigeration temperature 4 ± 0.5°C (39.2°F)
Sampling Times
▶ Trial 1: Time 0 (initial), 3 days
▶ Trial 2: Time 0 (initial), 3, 7, 10, 14, 21, 28 days
▶ Trial 3: Time 0 (initial), 1 hour, 1, 3 days
Enumeration
▶ Diluted with 100 mM phosphate buffer to neutralize pH of blueberries
▶ Plated in duplicate onto Modified Oxford Agar (MOX)
▶ Incubated plates at 35°C for 2 days
Presented at Berry Health Benefits Symposium 2017
5
4
3
2
1
0
0
7
14
21
28
Time (days)
Frozen conditions (-18°C)
Refrigerated conditions (4°C)
Figure 2. Average population level of L. monocytogenes recovered from artificially
inoculated wild blueberries after storage at frozen (-18°C) and refrigerated (4°C)
conditions. Vertical bars indicate standard deviation (n=3), dotted line indicates the
limit of detection by plating (1 Log CFU/g).
Conclusions
▶ For all trials, a substantial level of L. monocytogenes died-off immediately after inoculation
of the IQF wild blueberries. This phenomena was not observed when the study was
performed using frozen peas, thus suggesting that the wild blueberries exerted some
bactericidal effect against L. monocytogenes.
▶ The remaining population of L. monocytogenes survived on the wild blueberries stored
under frozen conditions but continued to die-off on wild blueberries stored under
refrigeration conditions, thus indicating that storage temperature plays a role in the
bactericidal effects observed.
6
L. monocytogenes (Log CFU/g)
▶ Frozen temperature -18 ± 1°C (-0.4°F)
L. monocytogenes (Log CFU/g)
Introduction
5
4
3.47
3.72
3
Future
▶ Other researchers have reported that the low pH, sugars, acids, and salts of blueberries
were not, alone, responsible for antimicrobial effects observed, however, limited
information is currently available and additional investigation is needed in order to
determine the mechanism and antimicrobial compound(s) responsible for the bactericidal
effects observed against L. monocytogenes.
2.68
2
1
0
1
2
3
Trial
Blueberries- population inoculated
Peas- population inoculated
Blueberries- population recovered
Peas- population recovered
Figure 1. Average population level of L. monocytogenes recovered from wild
blueberries and peas immediately after inoculation compared to calculated
inoculation level. Vertical bars indicate standard deviation (n=3).
Acknowledgements
Financial support for this research was provided by Jasper Wyman & Son.