Synergistic effect of ozone and microgard 300 for controlling Listeria monocytogenes in ready-to-eat cooked and cured ham.

1
Synergistic effect of ozone and microgard 300 for
controlling Listeria monocytogenes in
ready-to-eat cooked and cured ham. R. JHALA1,
K. Muthukumarappan, J. L. Julson, and R. I. Dave.
(1) Agricultural and Biosystems Engineering, AE -
02182120 South Dakota State University,,
Brookings, SD 57007
Abstract
Materials and Methods
Conclusions
The effectiveness of ozone and Microgard
(MG300) on the survival of known number of
Listeria monocytogenes in ham sample was
investigated in a closed system. The survival
rate of Listeria monocytogenes was studied as a
function of gaseous ozone concentration,
Microgard, temperature and storage period for a
fixed treatment period of 30 min and storage
temperature of 4ºC. The results indicate that a
synergistic effect of ozone, MG300 and storage
period can inactivate up to 99.94 of Listeria
monocytogenes on cooked and cured ham. The
temperature does not have any significant effect
on kill of Listeria monocytogenes. The
combination of ozone and MG300 can be an
effective hurdle to control Listeria
monocytogenes in cooked and cured ham.  

• As indicated in Table-1, ozone, MG300 Days have
  significant effect on mean kill of Listeria
  monocytogenes in cured ham. The table also
  indicates the significant interaction parameters
  of the experiment.
• The group 1 graphs shows that as ozone
  concentration increases from 0.0 to 0.5 ppm
  microbial inactivation significantly increased
  but above 0.5 ppm the inactivation was not
  significant.
• Increasing the MG300 level up to 2.0
  significantly increases the mean kill of
  Listeria monocytogenes.
• As the storage days increases from 1 to 5 and
  from 5 to 10 days the mean kill of Listeria
  monocytogenes increases exponentially.
• Temperature has no significant effect on mean
  kill of Listeria monocytogenes.
• Group 2 graphs show the effect of interaction
  of experimental parameters.
• The difference of least square means indicate
  that ozone MG300 has a synergistic effect in
  inactivating Listeria monocytogenes with ozone
  being a predominant inactivating parameter.
• Storage days have a predominant inactivating
  effect in the interaction of storage days and
  ozone. While MG300 has a predominant inactivating
  effect in the interaction of MG300 storage
  days.

• Isolation
• Listeria monocytogenes culture was obtained from
  Alfred Chairs Laboratory (SDSU, Brookings, SD).
• The culture was maintained at 80C in
  glycerol-nutrient broth.

• Propagation
• The cultures were given three transfers
  (16-16-18 hrs) before replications for the
  purpose of full activation.
• Media used for activation was trypticase soy
  broth with 0.6 yeast extract.
• Probable number of Listeria monocytogenes was
  determined by optical density measurement (at 603
  nm) using an UV spectrophotometer

• Statistical Analysis
• According to the experimental design a total of
  432 samples were treated. The whole experiment
  was replicated thrice (4 ozone level X 4 MG300
  level X 3 temperature X 1 exposure time X 3
  storage period X 3 replication).
• The mean kill data was analyzed using split-
  split- split-plot experimental design model with
  the help of statistical analysis software (SAS
  Institute, Cary, NC).

Results

• Inoculation
• About 0.1ml of the active culture of known number
  of Listeria monocytogenes is spread on 50.0-cm2
  area of Hyvee brand reduced fat (97 fat free)
  cooked cured ham slices.
• The inoculated samples are stored for 20 minutes
  under refrigerated condition, to allow the
  culture to absorb to the surface of the ham.

GROUP 1
Introduction

• In the recent years, Listeria monocytogenes has
  caused major listeriosis outbreaks in the U.S.
  associated with ready-to-eat meat products().
  Food pathogens like Listeria monocytogenes may
  survive the conventional food processing methods
  like pasteurization and cooking. Moreover, there
  is a demand for safe and judicious usage of
  sanitizers, bleaching agents, preservatives and
  chemicals in food processing (1). Thus, the food
  industry is currently in need of innovative
  processing technologies in order to meet
  consumers demand for fresher and safe
  ready-to-eat meat products (2). There are several
  methods available for inactivation of
  microorganisms in foods thermal, high pressure,
  pulsed electric field, oscillating magnetic
  field, irradiation and ozonation.
• In August 2001, Food and Drug Administration
  (FDA) has approved ozone as a direct food
  additive for the treatment, storage and
  processing of food in gaseous and aqueous phases
  (3). Ozones antimicrobial action is through the
  oxidation of bacterial cell wall components (4).
  The technologies used singly to ensure that a
  food is free of pathogens may cause changes in
  the sensory attributes of the food. Thus, in the
  food industry, the use of multiple parameter
  known as Hurdle Concept has been suggested (5).
  
• Microgard 300 is a bacteriocin like, low
  molecular weight metabolite material of
  Propionibacterium shermanii in a skim milk base.
  It has been used as a preservative in cottage
  cheese and other food products to inhibit
  psychrotrophic spoilage bacteria, yeast, mold and
  gram-positive organisms like Listeria
  monocytogenes (6).
• In this study we have hypothesized that
  application of ozone and Microgard300 hurdle
  concept can better control pathogens like
  Listeria monocytogenes in cooked and cured ham.
• Objectives 
• To evaluate the individual and synergistic
  effect of ozone and MG300 for controlling
  Listeria monocytogenes in cooked and cured ham.
• To Study the effect of treatment temperature in
  improving the synergy
• To Study the effectiveness of the synergy of
  ozone and MG300 for controlling Listeria
  monocytogenes in cooked and cured ham over the
  storage period.
•  
•  
•  

Experimental Design Ozone (ppm) - 0,
0.2, 0.5, 1.0 MG300 () - 0, 1.0, 2.0,
3.0 Temperature (C) - 10, 15, 20 Exposure time
- 30 min Storage days - 1, 5, 10

• Experimental set-up
• The inoculated samples were exposed to gaseous
  environment of ozone in a closed system housed in
  an incubation chamber to control the temperature.
• The percent solution of MG300 was prepared in pH
  7.0 phosphate buffer.
• 0.1 ml of MG300 () solution was spread after
  the ozone treatment to study the synergistic
  effect.
• The samples for the storage study were vacuum
  packed (Food saver) and stored in a refrigerator
  at 4C.

GROUP 2
INTERACTION OF STORAGE DAYS AND OZONE
INTERACTION OF STORAGE DAYS AND MG300

• Microbial Analysis
• Enumeration of the surviving Listeria
  monocytogenes was done according to the method
  suggested by murano et al, using the pour plate
  technique.
• The efficacy of the above treatment has been
  reported as percent kill of (Kill) Listeria
  monocytogenes.

OZONE LEVEL 0 PPM
MG300 0
INTERACTION OF OZONE AND MG300
O3 out
DAY 1
MG300 1
References
OZONE LEVEL 0.2 PPM
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Federal Register, 2001. Secondary direct food
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Murano, E.A., P.S Murano, R.E. Brennan, K.
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Ham sample
MG300 2
DAY 2
OZONE LEVEL 0.5 PPM
O3 in
MG300 3
DAY 3
OZONE LEVEL 1 PPM
Ozone Concentrator - Generator
Plexiglass Sample Treatment Chamber