Title:
Effect of Ozone on Listeria monocytogenes and Shelf-Life of Ready-to-Eat
Meat Products (Hot Dogs/Ham) - NPB# 01-081
Investigator:
Siobhan S. Reilly
Institution:
Oklahoma State University
Date Received:
8/28/2002
Abstract
Numbers of Listeria monocytogenes were reduced on hot dogs when exposed to
ozone. The most effective treatment was exposure for 30 seconds (99% and 93 %
reductions for 106 and 103 inoculums respectively).
The 10-second exposure did
reduce L. monocytogenes but not as effectively (60% and 30% reductions for 106 and
103 inoculums, respectively). L. monocytogenes was also reduced on ham slices when
exposed to ozone. After a 30-second exposure, L. monocytogenes declined 42.4 and
56.1% for the 106 and 104 inoculum levels, respectively. A 10-second exposure
resulted in only a 10.9 and 22.2% reduction for the same two inoculum levels.
No differences were apparent between the control and ozone-treated hot dogs or
ham slices with respect to microbial shelf life. The bacterial counts (total aerobic and
lactic acid) remained similar for the treated/control samples when stored for 28 days
under refrigeration conditions (5 oC).
Although in most cases there was not a statistically significant difference in the
TBARS-values and color attributes between the control and ozone-treated hot dogs,
there was a trend towards an increased formation of oxidation products and changes in
color as storage time progressed. This suggested that the ozone treatment may
negatively affect some sensory attributes of hot dogs. However, sensory panelists
could not detect a difference between ozonated hot dogs and non-ozonated hot dogs.
For ham slices, the 10 and 30 second ozone treatments led to increased production of
oxidation products when compared to the control (no ozone) but this did not adversely
affect color. Sensory panelists were able to detect differences for both treated samples
(10 and 30 seconds) when compared to the control.
Ozone gas may be an effective post-process treatment for controlling or
eliminating L. monocytogenes on certain ready to eat meat products. In this study,
ozone is clearly more effective in reducing numbers of L. monocytogenes on hot dogs
compared to ham slices. In addition, sensory characteristics for ozone treated hot dogs
remained unaffected; however, ham slices were negatively affected by the ozone
treatment.
Introduction
Producing safe animal food products is an increasing challenge. Uses of
chemicals to kill microorganisms on food products may be restricted by governing
agencies (e.g., chlorine). This is not the case for ozone, which has been approved by
the FDA and denoted “Generally Recognized as Safe (GRAS) (3,4). Ozone has been
used to eliminate microorganisms in drinking water, and is deemed safe to use directly
on food products.
Listeria monocytogenes can cause serious and sometimes fatal infections in
children, elderly people, and others with weakened immune systems. Healthy people
may suffer only short-term symptoms (i.e., high fever, headache, nausea, abdominal
pain, and diarrhea), but it may cause miscarriages and stillbirths. As of 1999, the
largest number of food recalls (25 of 55) has been due to contamination with L.
monocytogenes. Food Safety and Inspection Service (FSIS) microbiological monitoring
data from 1993-99 suggest that hot dogs and luncheon meats are two vehicles for
foodborne Listeria monocytogenes. According to FSIS, the prevalence of L.
monocytogenes was highest in ham and pork.
Ozone in air is effective in killing Escherichia coli 0157:H7 (8). Restaino et al.
(1995) (9) found that among the pathogens studied, Listeria monocytogenes was the
most sensitive to ozone. Numbers of L. monocytogenes were instantaneously
decreased by four log cycles when exposed to ozonated water. Other organisms that
were decreased (P< .05) included Salmonella typhimurium, E. coli, Pseudomonas
aeruginosa, Yersinia enterocolitica, Staphylococcus aureus, and Enterococcus faecalis.
Also, ozone was effective in decreasing Candidia albicans and Zygosaccharomyces
bailii (yeast cells) up to five log cycles instantaneously; however, ozone was only slightly
effective against fungal spores (Aspergillus niger) (6).
Ozone is effective in killing certain pathogens and spoilage organisms in water,
on beef, and on poultry (6,7,9). The effectiveness of ozone on ready-to-eat meat
products (i.e., ham, hot dogs, etc.) has not been explored thoroughly or reported in the
literature. Therefore, this research is intended to investigate the effectiveness of ozone
on L. monocytogenes found in ready-to-eat meat products. In addition, if it is to be used
as a bacteriocidal agent, it is important to learn if there are deleterious effects of ozone
on various food quality attributes (e.g., oxidative rancidity, color). Because ozone is a
powerful oxidizing agent, its use to extend shelf-life may have negative effects that lead
to off-flavors and odors not commonly associated with these products (5).
Objectives
1) To determine the effectiveness of an ozone gas flush (at various flushing
times) in reducing or eliminating L. monocytogenes on ready-to-eat meat products
(ham/hot dogs). 2) To determine the shelf-stability of these products when exposed to
ozone treatment. 3) To determine what effect (if any) ozone treatment has on the
sensory attributes of the hot dog and ham products.
2
Procedures
Bacterial strains and concentration preparation
A cocktail of Listeria monocytogenes was used to make dip solutions containing
approximately 1.0x105 and 1.0x108 CFU/ml. The strains used were: L. monocytogenes
Scott A, V7-2, 39-2 and 383-2. (Note: these strains are streptomycin resistant). The
four strains were grown in 100mls of tryptic soy broth and incubated at 300C for 24
hours. The cultures were then centrifuged using pre-weighed, sterile centrifuge bottles
for 20 minutes at 7000rpm. Cells were then re-suspended in 20ml of sterile phosphate
buffer and washed several times. Dip solutions were then made using the appropriate
amount of Listeria concentrate and sterile phosphate buffer.
Seeding of hot dogs and exposure to ozone
Hot dogs/Ham slices were placed into the Listeria dip solutions for 1 minute,
placed into vacuum package bags (four hot dogs/bag or six ham slices/bag) and either
sealed immediately (control) or treated with 10 seconds or 30 seconds of ozone gas.
While hot dogs/ham slices were exposed to ozone, the bag was agitated to allow all
surfaces to be adequately exposed to the ozone gas. After each treatment, samples
were vacuum packaged and place in an ice bath until plated.
Ozone Standard Curve
Ozone concentration was determined using the potassium iodide method as
referenced in the Standard Methods for the Examination of Water and Wastewater (18th
edition). Ozone gas for specific time periods was passed into a solution of potassium
iodide followed by titration with sodium thiosulfate. The standard curve data are as follows:
r2 =0.99, y=0.3041x + 1.2279. (10 second treatment = 4.27 mg O3, 30 second treatment =
10.35 mg O3).
Evaluation of microbial shelf-stability
Hot dogs/Ham slices were aseptically placed (4 hot dogs/bag) in vacuum
packages and either sealed immediately or ozonated for 10 or 30 seconds. Each week
samples were pulled from the 50C cooler, plated in duplicate, and analyzed for total
microbial count and lactic acid bacteria. Three replications were preformed within each
week.
Plating
Listeria enumeration: Composite samples of the hot dogs/ham were spiralsurface plated in duplicate on tryptic soy agar containing streptomycin. Plates were
then incubated for 24-36 hours at 30 oC. After incubation, plates were counted on spiral
plate counter and recorded.
Microbial shelf-stability enumeration: Control and treated samples were stored at
o
5 C and sampled weekly for 28 day period. Samples were plated in duplicate for total
aerobic bacteria and lactic acid bacteria.
3
Analysis of lipid oxidation products (TBARS)
On each day, specimens were taken from the surface and interior in addition to a
composite specimen from treated and non-treated hot dogs. For the ham slices, a
composite sample was made by chopping slices for 30 sec in a food processor, and
specimens taken from the composite for chemical analysis. TBARS values of samples
were determined spectrophotometrically according to the method of Lemon et al.
(Lemon, 1975) as used by Richards & Hultin (Richards and Hultin, 2000) with slight
modifications to account for presence of interfering sugars (dextrose a component in
hotdog formulation). Moisture content of hotdog and ham samples was determined
according to AOAC method 983.18. TBARS values were expressed as moles/kg
sample.
AOAC Official Methods of Analysis. Association of Official Analytical Chemists:
Washington, DC, 1995.
Lemon, D. W. “An improved TBA test for rancidity,” Halifax Laboratory, 1975.
Richards, M. P. and Hultin, H. O. Effect of pH on lipid oxidation using trout
hemolysate as a catalyst: Possible role for deoxyhemoglobin. J. Agric. Food
Chem. 2000, 48, 3141-3147.
Color analysis
On each day, the color of three treated and non-treated hot dogs from each
package were determined using a Hunter colorimeter. For the ham samples, the color
of the first two slices from each package was determined. The attributes determined
were L-value (refers to lightness or darkness of a sample; 0 = black, 100 = white), avalue (refers to the yellowness-blueness) and b-value (refers to the rednessgreenness).
Sensory evaluation
Ozone treated and non-ozone treated hot dogs were stored refrigerated at 5° C
for a total of 28 days. Samples were pulled on 0, 7, 14, 21, and 28 days. On each day,
treated and non-treated samples were cooked in separate containers of boiling water for
7 minutes. The containers were removed from the heat, and the samples were placed
on separate cutting boards and cut into three equal portions. They were then returned to
their original containers and covered.
Ozone treated and non-ozone treated slices of ham were stored refrigerated at
5° C for a total of 28 days. Samples were pulled on 0, 7, 14, 21, and 28 days and
immediately provided to the sensory panel.
A group of 12 untrained panelists then evaluated the hot dogs or ham slices via a
triangle test under red filtered lights. Each panelist received two sets of samples, and
each sample was identified by a random three digit code. The first set of samples
consisted of either two controls and one 10 second treatment, or two 10 second
treatments and one control. The second set of samples consisted of either two controls
and one 30 second treatment, or one control and two 30 second treatments. All
samples were approximately 10 grams each. Panelists were asked if they could
determine a difference between the samples based on flavor, odor, or texture.
4
Results (Objective 1)
Effect of ozone on L. monocytogenes on hot dogs
Numbers of L. monocytogenes seeded onto hot-dogs at a level of 1.6 x 106
CFU/g were reduced over 2 log cycles, representing more than a 99% reduction (1.2 x
104 CFU/g) when exposed to ozone for 30 seconds. Reduction of L. monocytogenes
was greater than 60 % (6.3 x 105 CFU/g) when exposed to ozone gas for only 10
seconds. Hot dogs inoculated with 2.0 x 103 CFU/gram and treated for 30 seconds
showed a 93 % reduction (1.4 x 102 CFU/g). When hot-dogs were ozonated for 10
seconds there was a 30 % reduction (1.4 x 103 CFU/g) in L. monocytogenes. (See
Figure 1).
a)
6
Effect of ozone on L. monocytogenes (10 )on hot dogs.
1.8E+06
1.6E+06
1.4E+06
CFU/g
1.2E+06
1.0E+06
8.0E+05
60.6% Reduction
6.0E+05
4.0E+05
2.0E+05
99.3% Reduction
0.0E+00
Control (No ozone)
10 second (ozone)
30 second (ozone)
Listeria monocytogenes
b)
3
Effect of ozone on L. monocytogenes (10 ) on hotdogs
2.50E+03
2.00E+03
30.0% Reduction
CFU/g
1.50E+03
1.00E+03
5.00E+02
93.0% Reduction
0.00E+00
Control (No ozone)
10 second (ozone)
30 second (ozone)
Listeria monocytogenes
Figure 1. Effect of ozone gas on L. monocytogenes seeded onto hot dogs; a) 1.6 x 106
inoculum and b) 2.0 x 103 inoculum. Values reported are averages of three replicate
measurements.
5
Results (Objective 1)
Effect of ozone on L. monocytogenes on ham slices
Numbers of L. monocytogenes seeded onto ham slices at a level of 9.2 x 106
CFU/g decreased 42.4% (5.3 x 106 CFU/g) when exposed to ozone for 30 seconds.
Reduction of L. monocytogenes was 10.9 % (8.2 x 106 CFU/g) when exposed to ozone
gas for 10 seconds. Ham slices inoculated with 1.8 x 104 CFU/gram and treated for 30
seconds showed a 56.1% (7.7 x 103 CFU/g) reduction. When ham slices were
ozonated for 10 seconds there was a 22 % (1.4 x 104 CFU/g) reduction in L.
monocytogenes. (See Figure 2).
Effect of ozone on L. monocytogenes (106) on ham slices
a)
1.0E+07
9.0E+06
10.9% Reduction
8.0E+06
7.0E+06
CFU/g
6.0E+06
42.4% Reduction
5.0E+06
4.0E+06
3.0E+06
2.0E+06
1.0E+06
0.0E+00
Control (No Ozone)
10 second (Ozone)
30 second (Ozone)
Listeria monocytogenes
4
Effect of ozone on L. monocytogenes (10 ) on ham slices.
b)
2.0E+04
1.8E+04
1.6E+04
22.2% Reduction
1.4E+04
CFU/g
1.2E+04
1.0E+04
56.1% Reduction
8.0E+03
6.0E+03
4.0E+03
2.0E+03
0.0E+00
Control (No Ozone)
10 second (Ozone)
30 second (Ozone)
Listeria monocytogenes
Figure 2. Effect of ozone gas on L. monocytogenes seeded onto ham slices; a) 9.2 x 106
inoculum and b) 1.8 x 104 inoculum. Values reported are averages of three replicate
measurements.
6
Results (Objective 2)
Evaluation of microbial shelf-stability of ozone treated hot dogs
There was very little microbial growth observed over 28 days storage at
refrigeration temperatures. There appears to be little difference between the ozone
treated hot dogs (10 or 30 seconds) and the control (no ozone). See Table 1.
Storage
(days)
0
7
14
21
28
Total Aerobic Plate Counts (CFU/g sample)
Time Control
Ozone
Ozone
(no ozone)
(10 second)
(30 second)
<10
<10
<10
<10
<10
<10
1
1.3 x 10
<10
<10
<10
<10
<10
2
<10
1.6 x10
<10
Storage
(days)
0
7
14
21
28
Lactic Acid Bacterial Counts (CFU/g sample)
Time Control
Ozone
Ozone
(no ozone)
(10 second)
(30 second)
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
2
<10
1.6 x 10
<10
Table1. Total Aerobic and Lactic Acid bacteria counts for hot dogs stored at 5 oC for 28
days.
7
Results (Objective 2)
Evaluation of microbial shelf-stability of ozone treated ham slices
Microbial populations (total aerobic microorganisms and lactic acid bacteria) of
ham slices over 28 days of storage at refrigeration temperatures did not decline,
indicating that there was little effect of ozone on the natural microflora existing on the
ham slices.
There appears to be little difference between the ozone treated ham slices (10 or 30
seconds) and the control (no ozone). See Table 2.
Storage
(days)
0
7
14
21
28
Total Aerobic Plate Counts (CFU/g sample)
Time Control
Ozone
Ozone
(no ozone)
(10 second)
(30 second)
4
4
5.0 x 10
4.4 x 10
4.3 x 104
5
6
7.6 x 10
2.1 x 10
1.3 x 106
9.9 x 105
8.0 x 105
9.6 x 105
7
7
3.3 x 10
2.7 x 10
1.6 x 107
2.3 x 107
3.2 x 107
5.2 x 107
Storage
(days)
0
7
14
21
28
Lactic Acid Bacterial Counts (CFU/g sample)
Time Control
Ozone
Ozone
(no ozone)
(10 second)
(30 second)
4
4
7.9 x 10
8.3 x 10
5.4 x 104
6
6
1.8 x 10
2.2 x 10
1.8 x 106
5.5 x 105
4.4 x 105
5.4 x105
7
7
3.8 x 10
5.9 x 10
4.8 x 107
7
7
4.7 x 10
3.9 x 10
7.2 x 107
Table 2. Total Aerobic and Lactic Acid bacteria counts for ham slices stored at 5 oC for
28 days.
8
Results (Objective 2)
Evaluation of oxidative/color stability of ozone-treated hot dogs
The effect of ozone treatment (10 sec vs. 30 sec) of hot dogs was evaluated by
determining the formation of lipid oxidation products (malondialdehyde) in addition to
changes in hotdog color. Ozone-treated and non-treated hot dogs were stored at
refrigerated temperatures (5°C) for a total of 28 days. At 0, 7, 14, 21 and 28 days,
samples were taken for analysis of Thiobarbituric Acid Reactive Substances (TBARS;
malondialdehyde lipid oxidation products). Sample color was also determined.
Treatment of hotdog surfaces with the highly reactive ozone may be expected to
result in higher rate of chemical reactions at the surface as compared to the interior of
the hot dogs. However, there was no significant difference (p < 0.05) between TBARS
values of specimens taken from the surface and interior of the hot dogs, suggesting that
this was not the case. Therefore, the TBARS results for the surface/interior/composite
specimens analyzed were averaged and used for statistical analysis.
The TBARS results revealed no significant (p < 0.05) difference between the
control and the ozone treated samples (Figure 3). However, the TBARS values tended
to be higher in the ozone treated hot dogs stored for more than 14 days. This indicates
that ozone treatment results in higher formation of TBARS compounds which may affect
overall product sensory quality.
The effect of ozone treatment on hotdog color (L-, a- and b-values) is shown in
Figure 4a-c. On average, all the samples tended to become lighter (higher L-value) with
storage time (Figure 4a). However, although not significantly different in many cases,
the ozone-treated hot dogs were generally slightly lighter in color than the non-treated
control. The yellowness of the ozone-treated hot dogs also tended to increase with
storage time as measured by the decrease in the a-value (Figure 4b), especially at the
longer storage times. This directly reflects the trend observed for the hotdog lightness.
The ozone treatment had no significant effect (p < 0.05) on the blueness-greenness (bvalue) of hot dogs (Figure 4c).
9
14
TBARS [µmoles/kg tissue]
12
Control
10 sec ozone
30 sec ozone
10
8
6
4
2
0
0
7
14
21
28
Storage time [days]
Figure 3. Changes in TBARS upon storage of hot dogs for up to 4 weeks at
refrigerated temperatures. Values are the averages of quadruple measurements on
composite samples (n = 4). Data from three replicate experiments are shown.
10
51
17
a)
50
b)
16
a- value
48
47
15
14
13
46
12
45
0
7
14
21
11
28
0
7
Storage time [days]
14
21
28
Storage time [days]
42
40
Control
10 sec ozone
30 sec ozone
b- value
L- value
49
c)
38
36
34
32
0
7
14
21
28
Storage time [days]
Figure 4. Changes in color upon storage of hot dogs; a) lightness (L-value), b)
redness-yellowness (a-value), c) blueness-greenness (b-value). Values reported are
averages of triplicate measurements on surface of the top two slices (n = 6).
11
Results (Objective 2)
Evaluation of oxidative/color stability of ozone-treated ham slices
For ham, TBARS values of the ozone treated samples were generally higher
than that of the control (Figure 5). The samples treated with ozone for 30 sec were
consistently higher than the control, whereas those treated for 10 sec were in some
cases not different.
The data followed the same trend for the three replicate
experiments performed.
The effect of ozone treatment on the color (L-, a- and b-values) of ham slices is
shown in Figures 6a-c. Each point on the graph represents the average of duplicate
measurements on the top two ham slices from a single package with a total of four
measurements. There was no apparent change in the L-values of the samples over the
storage time (Figure 6a). The large variation in the data can be in part explained due to
the fact that ham slices are not uniform in color because of the different muscles used
(dark and white). The average a-values of ham tended to increase with time (Figure
6b), suggesting an increased redness, whereas the b-values tended to decrease
(Figure 6c). No apparent differences were observed between the control and ozonetreated samples.
14
TBARS [µmoles/kg tissue]
12
Control
10 sec ozone
30 sec ozones
10
8
6
4
2
0
0
7
14
21
28
Storage time [days]
Figure 5. Changes in TBARS upon storage of ham slices. Values reported are
averages of measurements on surface, interior and composite specimens (n = 12).
12
63
62
18
a)
16
b)
14
a- value
60
59
58
12
10
57
8
56
55
0
7
14
21
6
28
0
7
Storage time [days]
14
21
28
Storage time [days]
10.0
9.5
c)
9.0
Control
10 sec ozone
30 sec ozone
a- value
L- value
61
8.5
8.0
7.5
7.0
6.5
0
7
14
21
28
Storage time [days]
Figure 6. Changes in color upon storage of ham slices; a) lightness (L-value), b)
redness-yellowness (a-value), c) blueness-greenness (b-value). Values reported are
averages of at least quadruple measurements on surface of hotdog specimens (n = 4).
13
Results (Objective 3)
Sensory evaluation of ozone treated hot dogs
Analysis of the sensory data showed that there were no significant differences
(p<.05) between the control and the treated samples (10 sec and 30 sec) during the 28
days of storage at 5 oC. However, the data suggested an increase in the number of
panelists able to differentiate between the control and the samples treated for 30
seconds as storage time progressed (See Figure 7). Further testing beyond 28 days
may be necessary to evaluate this trend.
12
# of panelist responses
a)
Correct answers
Incorrect answers
10
8
6
4
2
0
0
7
14
21
28
Storage time (days)
12
b)
# of panelist responses
10
Correct answers
Incorrect answers
8
6
4
2
0
0
7
14
21
28
Storage time (days)
14
Figure 7. Sensory response for hot dogs stored at refrigeration temperatures for 28
days; a) 10 second ozone treatment and b) 30 second ozone treatment. Values
reported are averages of three replications.
15
Results (Objective 3)
Sensory evaluation of ozone treated ham slices
Analysis of the sensory data for the ozonated ham showed an apparent
difference between the 10 second treated ham and the control on all days except 0 and
14. However, an apparent difference was seen between the 30 second treated ham
and the control on all days (See Figure 8).
12
# of panelist responses
a)
Correct answers
Incorrect answers
10
8
6
4
2
0
0
7
14
21
28
Storage time (days)
12
Correct answers
Incorrect answers
# of panelist responses
10
b)
8
6
4
2
0
0
7
14
21
28
Storage time (days)
Figure 8. Sensory response for ham slices stored at refrigeration temperatures for 28
days; a) 10 second ozone treatment and b) 30 second ozone treatment. Values
reported are averages of three replications.
16
Literature cited
1. APHA, AWWA and WPCF (1992) Standard Methods for the Examination of Water
and Wastewater. 18th edition. American Public Health Association. Washington DC.
2.
Bader, H. and Hoigne, J. (1981) Determination of Ozone in Water by the Indigo
Method. Water Res. 15:449-456.
3. Byun MW, Kwon OJ, Yook HS, Kim KS. (1998) Gamma Irradiation and Ozone
Treatment for Inactivation of Escherichia coli O157:H7 in Culture Media. J. Food
Prot. 61:728-730
4. Code Federal Regulations. “Direct Food Substances Affirmed as Generally
Recognized as Safe (GRAS).” Title 21, Vol. 3, Part 184.
5. Graham DM. (1997). Use of Ozone for Food Processing. Food Tech. 51:72-75.
6. Jindal V, Waldroup AL, Forsythe RH. (1995) Ozone and Improvement of Quality and
Shelf-life of Poultry Products. J. Appl. Poultry Res. 4:239-248.
7. Majchrowicz A. (1998) Food Safety Technology: A Potential Role for Ozone? Agric.
Outlook. 252:13-15
8. Reagan JO, Acuff GR, Buege DR, Buyck MJ, Dickson JS, Kastner CL, Marsden JL,
Morgan JB, Nickelson R, Smith GC, Sofos JN. (1996) Trimming and Washing of
Beef Carcasses as a Method of Improving the Microbiological Quality of Meat. J.
Food Prot. 59:751-756
9. Restaino L, Frampton EW, Hemphill JB, and Palnikar P. (1995) Efficacy of Ozonated
Water Against Various Food-Related Microorganisms.
Appl. Environ Microb.
61:3471-3475
17