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dissertation entitled
MICROBIAL SAFETY OF A MODIFIED ATMOSPHERE
PACKAGED MINIMALLY PROCESSED MEAT-

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presented by

C hong Hyun Lee

has been accepted towards fulfillment
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MSU IoAn Affirmative Action/Ema! Opponunlly Intuition

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MICROBIAL SAFETY OF A MODIFIED ATMOSPHERE PACKAGED
MINIMALLY PROCESSED MEAT-VEGETABLE PRODUCT

By
Chong Hyun Lee

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition
1993

ABSTRACT

Microbial Safety of a Modified Atmosphere Packaged Minimally
Processed Meat-Vegetable Product

By
Chong Hyun Lee

The effects of varying modified atmospheres and temperatures on the
growth of Pseudomonas fragi, Salmonella typhimurium, and Clostridium
sporogenes on cooked ground beef and peas (two different internal

temperatures of 55 and 71°C) were studied. An atmosphere containing
75% C02 and 0% 02 (MA4) was the most effective in inhibition of P.

fragi and S. typhimurium and MAI containing 75% C02 and 25% 02 was
the most effective in inhibition of C. sporogenes. The sample of higher
cooking temperature of 71°C delayed the outgrowth of C. sporogenes for
2-4 days than that of cooking temperature of 55°C at abusive temperature
of 25°C. However, the modified atmosphere had less or no inhibitory
effect on the growth of microorganisms at abusive temperatures.
Therefore, processors must ensure the safety of a minimally processed
MAP product throughout the shelf-life and under condition of
temperature abuse. Predictive models can be extremely valuable in
assessing the safety of extended shelf-life refrigerated foods.

In the development of predictive growth models, sigmoid growth

curves were fitted to the growth data using non-linear regression analysis

in conjunction with the Gompertz function and the polynomial growth rate
model was developed to aid in making decisions about microbiological
safety of minimally processed/MAP product. The predictive models
provide a cost-effective approach to understanding the microbial growth
response in MAP foods, to predict expected time for a microorganism to
reach target population levels, and is a powerful tool which can be used to
ensure the safety and stability of MAP foods. From the test models, the
results between experimental data and predicted growth data from the

predictive models had good agreements.

This work is dedicated to my father and mother, In Gyu Lee and Gum
Joon Joo, who have instilled in me the courage and determination to
accomplish my personal goals; to my loving wife, Kyung J. Jeon, a source
of support and encouragement to me; and to my daughters, Nanmi and
Yoonjin Lee, who are too young to read this now, but will someday

hopefully forgive me for all those forgotten bedtime stories.

ACKNOWLEDGMENT

The author wish to express my appreciation to the many individuals
who have provided guidance, support, and encouragement through the
course of my doctoral studies and during the completion of this
dissertation. To Dr. Jerry Cash, my advisor and chairperson of guidance
committee, my heartfelt thanks for his guidance direction and
encouragement to me.

The author would also like to thank my committee members, Dr. Jack
Giacin, Dr. Bruce Harte, Dr. John Linz, and Dr. Mark Uebersax, for their
support and concern in my research efforts.

To my wife Kyung Ia and daughters Nanmi and Yoonjin, especial
thanks for their patience, support and encouragement.

Finally, to my parents, In Gyu Lee and Gum Icon 100, kept me going
with their love, moral support, and financial assistance. Their

contribution to my education could never be fully expressed.

TABLE OF CONTENTS

Page
List of Tables ............................................................................................ ii
List of Figures ........................................................................................... V
Introduction ........................................................................................... 1
Literature Review .................................................................................... 4
Materials and Methods ............................................................................ 20
Bacterial cultures ............................................................................. 20
Sample preparation and inoculation ................................................ 20
Packaging of samples ...................................................................... 21
Analysis of headsapce composition ................................................. 22
Enumeration of bacteria .................................................................. 22
Experimental design ........................................................................ 23
Statistical analysis ........................................................................... 26
Statistical predictive modeling ........................................................ 26
Results and Discussion ........................................................................... 30
Experiment I ................................................................................... 3O
Experiment 11 .................................................................................. 47
Predictive modeling ........................................................................ 58
Model predictions ........................................................................... 67
Experiment III .................................................................................. 69
Conclusion .............................................................................................. 78
Appendix I SC-10A Thermocouple Psychrometer ................................ 80
Appendix 11 Standard Colony Count Methods ...................................... 81
Appendix III SYSTAT Statistics ............................................................ 82
Appendix IV Preliminary growth test ................................................... 83
Appendix V Gas Composition Change for Experiment I ----------------------- 88
Appendix VI Gas Composition Change for Experiment 11 --------------------- 93
Appendix VII Gas Composition Change for Experiment III ------------------- 98
Bibliography ........................................................................................... 1m

LIST OF TABLES

Table Page
1. Growth of Pseudomonasfragl in pure culture -------------------------------- 32
2. Growth of Pseudomonasfragi in mixed cultures ---------------------------- 34
3. Growth of Salmonella typhimurium in pure culture ----------------------- 38
4. Growth of Salmonella typhimurium in mixed cultures ------------------- 4O
5. Growth of C lostridium sporogenes in pure culture ------------------------ 45
6. Growth of C lostridium sporogenes in mixed cultures -------------------- 45
7. Growth of P. fragi in pure culture at abusive temperatures ------------- 48
8. Growth of P. fragi in mixed cultures at abusive temperatures --------- 48
9. Growth of S. typhimurium in pure culture at abusive temperatures 52

10. Growth of S. typhimurium in mixed cultures at abusive temperatures 52

11. Growth of C. sporogenes in pure culture at abusive temperatures ----- 56

12. Growth of C. sporogenes in mixed cultures at abusive temperatures 56

13. Gompertz equation parameters and calculated growth curve values

for P. fragi with various combination of temperature and

atmosphere in pure culture .............................................................. 60
14. Gompertz equation parameters and calculated growth curve values

for P. fragi with various combination of temperature and

atmosphere in mixed cultures .......................................................... 6O
15. Gompertz equation parameters and calculated growth curve values

for S. typhimurium with various combination of temperature and

atmosphere in pure culture ............................................................... 61

Table Page

16.

17.

18.

19.

20.

21.

22.

Gompertz equation parameters and calculated growth curve values

for S. typhimurium with various combination of temperature and
atmosphere in mixed cultures ........................................................... 61
Gompertz equation parameters and calculated growth curve values

for C. sporogenes with various combination of temperature and
atmosphere in pure culture ............................................................... 62
Gompertz equation parameters and calculated growth curve values

for C. sporogenes with various combination of temperature and
atmosphere in mixed cultures ........................................................... 62
Estimated time (day) for S. typhimurium to increase from 1 to

10,000 CFU/g ................................................................................... 63
Comparison of selected examples of experimentally observed vs.
predicted values for the polynomial model of the growth of

s. typhimurium in pure culture ........................................................ 65
Comparison of selected examples of experimentally observed vs.
predicted values for the polynomial model of the growth of

S. typhimurium in mixed cultures .................................................... 66
Mean values for Pseudomonas fragi counts (loglo CFU/ g) of

cooked samples to an internal temperature of 71°C in mixed culture

at abusive temperature of 25°C. ........................................................ 70
Mean values for Salmonella typhimurium counts (loglo CFU/ g) of
cooked samples to an internal temperature of 71°C in mixed culture

at abusive temperature of 250C. ........................................................ 7O

24. Mean values for C lostridium sporogenes counts (loglo CFU/ g) of

cooked samples to an internal temperature of 71°C in pure culture

at abusive temperature of 250C. ......................................................... 71

Table Page

25. Mean values for C lostridium sporogenes counts (loglo CFU/ g) of

cooked samples to an internal temperature of 71°C in mixed culture

at abusive temperature of 25°C. ......................................................... 71
26. Mean values for aerobic counts (loglo CFU/ g) of the samples in pure

culture of C. sporogenes at temperatures of 8, 20, and 25°C. ---------- 72
27. The change in pH of samples in pure culture of C. sporogenes at

temperatures of 8, 20, and 250C ...................................................... 73
28. Comparison of selected examples of experimental counts (Loglo

CFU/ g) versus predicted counts (Log10 CFU/ g) from the polynomial

models for the growth of P. fragi in mixed culture at 8°C. ------------- 76
29. Comparison of selected examples of experimental counts (Loglo

CFU/ g) versus predicted counts (Loglo CFU/ g) from the polynomial

models for the growth of P. fiagi in mixed culture at 20°C. ---------- 76
30. Comparison of selected examples of experimental counts (Log10

CFU/ g) versus predicted counts (Loglo CFU/ g) from the polynomial

models for the growth of S. typhimurium in pure culture at 20°C 77
31. Comparison of selected examples of experimental counts (Loglo

CFU/g) versus predicted counts (Loglo CFU/ g) from the polynomial
models for the growth of S. typhimurium in mixed culture at 20°C -- 77

iv

Figure Page
1. Sequence used to prepare samples for testing for growth of three
organisms at various atmospheres and temperatures in both
pure and mixed cultures .................................................................... 24
2. Selected modified atmospheres used under varying abusive
temperature situations in both pure and mixed cultures with
different inoculum levels .................................................................. 25
3. Growth of p. fragi at 5°C in pure culture ....................................... 33
4. Growth of p. fragi at 12. 5°C in pure culture ................................... 33
5, Growth of p. fragi at 25°C in pure culture ...................................... 33
6. Growth of p. fragi at 5°C in mixed cultures ................................... 35
7. Growth of P. fragi at 125°C in mixed cultures ------------------------------ 35
8. Growth of p. fiagi at 25°C in mixed cultures ................................. 35
9. Growth of S. typhimurium at 125°C in pure culture ---------------------- 39
10. Growth of S. typhimurium at 25°C in pure culture ------------------------- 39
11. Growth of S. typhimurium at 125°C in mixed cultures ------------------ 41
12. Growth of S. typhimurium at 25°C in mixed cultures --------------------- 41
13. Growth of C. sporogenes at 25°C in pure culture --------------------------- 46
14. Growth of C. sporogenes at 25°C in mixed cultures ---------------------- 46
15. Growth of P. fiagi with 102 CFU/g at abusive temperature
of 12. 5°C ......................................................................................... 49
16. Growth of P. fragi with 102 CFU/ g at abusive temperature
of 2 5°C ............................................................................................ 49

LIST OF FIGURES

Figure Page

17.

18.

19.

20.

21.

22.

24.

25.
26.

Growth of P. fiagi with 104 CFU/ g at abusive temperature

of 125°C ......................................................................................... 50
Growth of P. fragi with 104 CFU/ g at abusive temperature

of 25°C ............................................................................................ 50
Growth of S. typhimurium with 102 CFU/ g at abusive temperature

of 125°C ......................................................................................... 53
Growth of S. typhimurium with 104 CFU/ g at abusive temperature

of 125°C ......................................................................................... 53
Growth of S. typhimurium with 102 CFU/ g at abusive temperature

of 2 5°C ............................................................................................ 54
Growth of S. typhimurium with 104 CPU g at abusive temperature

of 2 5°C ............................................................................................ 54
Growth of C. sporogenes with 104 CFU/ g at abusive temperature

of 125°C ......................................................................................... 57
Growth of C. sporogenes with 104 CFU/ g at abusive temperature

of 2 5°C ............................................................................................ 57
Growth curve fitting of S. typhimurium at 125°C -------------------------- 59

The square root model plot for S. typhimurz'um growth rate
at range of 10 to 25°C ...................................................................... 68

vi

INTRODUCTION

Although various forms of modified atmosphere packaging (MAP) have
been used in the past, the advent of new packaging technologies and
increased consumer interest in "fresh like" foods has created a strong
demand for minimally processed products that are convenient, nutritious,
and of high quality; that have little or no preservatives; and that require
minimal preparation time. MAP has a great deal of promise as a processing
technology which can provide consumers with minimally processed food
products. The concept of extending shelf life of fresh prepared foods utilizes
the technology of high barrier packages in which a normal air has been
partially or wholly replaced by other gases (usually combinations of carbon

dioxide, oxygen and nitrogen). The modification usually results in a

reduction of the 02 content of the air in the package head space while
increasing the level of C02. This results in an extension of a product's shelf
life via inhibition of aerobic spoilage bacteria without using chemical or
physical treatments such as preservatives, freezing, and drying. Although a
great deal has been written about controlled atmosphere (CA) and MA
technology during the recent past, there are still many questions which are
unanswered. Chief among these is concern for the microbiological safety of
MAP products.

There is very little known about what effect MAP and low oxygen
atmospheres will have on growth patterns of microorganisms which
contaminate minimally processed/ MAP foods. The most obvious concern

deals with whether MAP suppresses the growth of normal spoilage

1

2

microorganisms while allowing pathogens to grow and flourish. It is likely
that MAP conditions may favor pathogen growth, which could create unsafe
products without causing detectable organoleptic changes. This situation
may be further exacerbated by the emergence of several new pathogens that
may grow under refrigerated conditions (Palumbo, 1987). In addition to
these factors, there is always the possibility that temperature abuse can
occur. Although most food pathogens do not grow well at refrigeration
temperatures the lack of refrigeration at any time during the life of a product
can allow for rapid growth of pathogens. Under non MAP conditions loss of
refrigeration usually causes spoilage organisms to proliferate and develop
obvious undesirable changes (i.e. flavor, texture and appearance). This acts
as a safety factor by alerting consumers and preventing consumption of the
temperature abused product. However, if MAP suppresses the growth of
spoilage organisms while allowing the growth of pathogens then temperature
abuse may produce some very toxic minimally processed foods without any
obvious organoleptic changes.

MAP foods could pose a potential public health hazard if prOper
controls are not in effect. These public safety issues have forced significant
regulatory activity related to MAP technologies and, in general, refrigerated
and minimally processed foods that are marketed in a less than commercially
sterile condition. Present regulatory guidelines do not specifically cover
minimally processed, refrigerated foods so the food industry must take full
responsibility for determining the parameters that must be met to deliver
products which have good quality and are safe under a wide variety of
conditions. Loss of quality may result from changes that are essentially
physical, chemical, enzymatic or microbiological and a variety of

complementary preservation techniques are applied that aim to prevent or

3

retard such changes. Microorganisms may play a key role in quality loss.
Therefore, microorganisms are the principal targets for the major
preservation techniques that are used to improve the keepability and safety
of foods (Gould, 1989). Control of microbial growth and activity is key to
extension of product shelf life during transit and subsequent storage in stores
or in homes. Understanding the various factors that combine to control
microbial growth (pH, water activity, temperature, atmosphere, etc.) through
data bases for specific organisms will lead to mathematical models that
predict their growth responses from knowledge of the physical and chemical
properties of the product. To develop the means of predicting the stability
and safety of foods, there is a need of predictive mathematical models for a
better understanding of the effects on microbial growth of combination of
factors relevant to food products. A manufacturer could use these models to
estimate the possible extent of microbial growth in foods stored under a
variety of MAP conditions.

The primary objectives of this study were to determine the effects of
varying modified atmospheres and temperatures on co-inocula of a spoilage
organism, a pathogen, and an organism which is physically and
biochemically similar to proteolytic strains of Clostridium botulinum, to
compare the outgrowth of these organisms in MA at abusive temperatures,
to generate growth data which can be used to develop an empirical
mathematical model(s) that effectively describes the effects and interactions
of temperature and modified atmosphere on the growth kinetics of those
three organisms in a minimally processed/MAP product, and to test
predictive models at different storage temperatures within the temperature

range studied.

LITERATURE REVIEW

A multitude of articles have been published on the benefits of the MAP
technology and the dramatic extension of shelf life for various foods
(Anonymous, 1985; Fierheller, 1984; Genigeorgis, 1985; Wolf, 1980).
However, very few publications exist dealing with the microbiological safety
or with the total quality control needed for successful MAP implementation
(Bernard, 1987; Hintlian and Hotchkiss, 1986; Hotchkiss, 1988; Farber,
1991).

Gases Used in MAP
The MAP technique involves packaging of product in an impermeable
film with the appropriate gas mixtures and heat sealing of the package.

Gases commonly used in MAP are nitrogen, oxygen, and carbon dioxide.

Each gas plays a distinct and specific role in MAP foods. N2 is an inert gas

which has no effect on the food and it has no antimicrobial properties on its

own. It is used mainly as a filler gas to prevent packaging collapse in

products that can absorb C02. In addition, N2, by displacing oxygen in the
pack, can delay oxidative rancidity and also inhibit the growth of aerobic
microorganisms.

02 is generally avoided in gas packaging mixtures unless it is used to fulfill
one of the three functions: 1) it might be used with gas packaging of red
meats in order to maintain color or "bloom", 2) it is used in low

concentrations in packaging of products which respire, such as fruits and

4

5

vegetables, and 3) it may prevent anaerobic conditions and limit the growth
of potentially harmful anaerobes, especially C. botulinum. C02 is the most
important gas in the gas mixture. C02 is both bacteriostatic and fungistatic,
i.e., it inhibits bacterial and mold growth. Although the inhibitory effect of
C02 in foods has been known for many years, its mechanism of
antimicrobial action has not been fully determined. In an excellent review
on the effects of C02 on microbial growth and food quality by Daniels et al.
(1985) the following appears to be the salient points of all previous
investigations: 1) The exclusion of oxygen by replacement with carbon
dioxide may contribute slightly to its overall antimicrobial effect by slowing
the growth of aerobic spoilage microorganisms, 2) The carbon
dioxide/bicarbonate ion has an observed effect on the permeability of cell
membranes, 3) C02 is able to produce a rapid acidification of the internal pH
of the microbial cell with possible ramifications relating to metabolic
activities, and 4) C02 appears to exert an effect on certain enzyme systems.
Therefore, C02 is effective for extending the shelf life of perishable foods by
retarding microbial growth.

It has been shown by several researchers that elevating carbon dioxide
in MAP foods has an inhibitory effect on the growth of aerobic spoilage
microflora (Clark and Lentz, 1969, 1973; Enfors and Molin, 1980; Gill and
Newton, 1978; King and Nagel, 1967, 1975; Paradis and Stiles, 1978; Baily
et. al., 1979; Eklund and Jarmund, 1983; Baker et. al., 1985). King and
Nagel (1967) controlled the various growth factors for pure cultures of P.
aeruginosa, and found a linear relationship between generation time and
C02 level. This relationship was more recently confirmed by Blickstad et
al.(1981), who concluded that the bacteriostatic/preservative effect of C02

increases with increasing concentration. The overall effect of C02, in

6

conjunction with refrigeration, is to increase both the lag phase and
generation time of spoilage microorganisms. However, the higher C02
levels raise the possibility of establishing conditions where pathogenic

organisms such as C. botulinum may survive (Daniels et al., 1985).

Several factors influence the inhibitory effects of C02 on

microorganisms in a culture medium or food system, specifically, types and
numbers of microorganisms, gas concentration, temperature, and packaging

film permeability.

Types of microorganisms

Microorganisms differ considerably in their sensitivity to C02 and this
sensitivity is related to the oxygen requirements of microorganisms. It has
been shown that C02 is most effective against aerobic spoilage
microorganisms. Common aerobic spoilage organisms of meat, fish and
poultry (Pseudomonads, Acinetobacter/Moraxella ) are inhibited by low
concentrations of C02, a fact that is exploited in the MAP of muscle foods.
Molds, which also require oxygen for growth, are similarly inhibited by
C02. Several studies have shown that low concentrations of C02 (10%) can
be used to suppress mold growth. However, as with bacteria, mold species
may vary in their sensitivity to the inhibitory effects of C02.

Several studies have shown that C02 has little or no effect on the
growth of facultative organisms belonging to the genus Enterobacteriaceae
or Brocothrix thermosphacta or microaerophilic lactic acid bacteria. These
organisms have been reported capable of growth in high concentrations of
C02 (75-100%) (Gill & Tan, 1980; Brody, 1990). Silliker and Wolfe (1980)

inoculated ground beef with Salmonella and stored the product under 60%
Goa/25% 02. Results indicated only minor growth changes in 7 days at

7

10°C except in air which demonstrated 3 log cycle increases. Salmonella
count increases occurred under all atmospheres at 20°C, but inhibition by
CO; was observed. Ground chicken inoculated with Salmonella
typhimurium (Baker et al., 1986) and stored 2, 7 and 13°C for 18 days
showed lower counts in packages containing 80% C02 than in air. Growth
retardation has been demonstrated on cooked, sliced roast beef inoculated
with P. fragi, S. aureus, and S. typhimurium under 75% C02 and 0-25% 02
at 128°C (Hintlian and Hotchkiss, 19873). Counts increased 5 log cycles in
air, but by less than 3 log cycles in 75% C02 and 25% 02. Counts were even
lower under less than 25% 02. All of the studies reported that C02 has an
inhibitory effect on Salmonella with the degree of inhibition increasing with
decrease in temperature.

Anaerobic bacteria, such as food poisoning organisms C lostridium
botulinum and Clostridium perfringens, are not affected by the presence of
C02 and the anaerobic conditions inside MAP foods may be conducive to
their growth. There is concern that these organisms represent a potential
public health hazard if present in MAP foods, particularly if packaged under
completely anaerobic conditions and stored under temperature abuse
conditions. Recently, concerns have been expressed about the
microbiological safety of MAP foods, particularly with regard to the
possible growth of C. botulinum. These concerns are justified in view of 1)
the ability of C. botulinum to grow under anaerobic conditions, 2) the
stimulatory effect of C02 on spore gemtination (Enfors and Molin, 1978;
Fodgeding and Busta, 1983), 3) the inhibition of normal aerobic spoilage
microorganisms of meat which are indicators of incipient spoilage, and 4)
the potential for temperature abuse. Thus, it has been recommended that 02

be included in the package headspace of high risk foods such as meat

8

(Hintlian and Hotchkiss, 1987). However, the addition of 02 should not be
regarded as an additional safety factor, particularly under temperature abuse
storage conditions. In a recent challenge study with C. botulinum in pork,
Lambert et al. (1991a) reported that in pork packaged with 20% 02, the level
of Oz rapidly decreased and headspace C02 increased to 20-40% due to the
respiratory activity of meat and microorganisms, and appeared to enhance
toxin production by C. botulinum under temperature abuse conditions.
C. botulinum strains can be grouped into seven different types A to G, each
type producing a serologically distinct neurotoxin. The most common types
involved in human illness are A, B, and E. There are major differences
among the strains, with the most important ones being temperature ranges
for growth and spore heat resistance (Hauschild, 1989). The proteolytic type
A, B, and F strains produce very heat-resistant spores which are a major
concern in the processing of canned foods. C. sporogenes is physically and
biochemically indistinguishable from these strains and is a convenient
surrogate organism for use in laboratories which are not equipped to work
with C. botulinum because it does not produce toxin (Sperber, 1982). The
nonproteolytic type B, E, and F strains can grow at refrigerated
temperatures, but produce spores of very low heat-resistance. Of this group,
type B is the most prevalent in both marine and freshwater environments and
has been responsible for most of the botulism outbreaks from fishery
products (Eklund, 1982).
Influence of Temperature

The proteolytic stains of C. botulinum do not grow under condition of
good refrigeration, i.e., at temperatures below 10°C. The optimum growth
temperature is 37°C, with very slow growth at 125°C and fairly rapid

growth at 45°C. The nonproteolytic strains of C. botulinum grow at

9

refrigerator temperatures. Therefore, adequate refrigeration cannot be relied
upon as a complete safeguard against botulism.
Microbial competition '

Certain strains of lactic acid bacteria produce antimicrobial substances
including nisin (Spelhaug and Harlander, 1989). Scott and Taylor (1981)
showed that nisin prevented the outgrowth of C. botulinum types A, B, and
E spores in media. Tanaka et a1. (1985), in a challenge study involving
bacon stored at 27°C, reported the antibotulinal effect of inoculation with
lactic acid bacteria when used in conjunction with sucrose and lower
concentrations of sodium nitrite. C. perfringens and other pathogens are
inhibited by bacteriocins produced by lactic acid bacteria (Spelhaug and
Harlander, 1989). Ahn and Stiles (1990) observed that lactic acid bacteria
isolated from vacuum-packaged meats have an antibacterial activity against
L. monocytogenes and some Enterobacteriaceae. Co—inoculation with lactic
acid bacteria has also been suggested as a means to control C. botulinum
outgrowth in minimally processed products such as sous vide products
(Lambert et al., 1991).

Influence of oxidation-reduction potential (Eh) & Headspace composition

C. botulinum is an anaerobe with a low tolerance for 02. However, as
Sperber (1982) has pointed out, even though C. botulinum is an anaerobe
and maximal growth occurs at an Eh of -350 mV (Smoot and Pierson, 1979),
it still may grow in the presence of low amounts of 02. It is not the
composition of the headspace gas that affects growth but rather the Eh of the
food. The Eh of many foods stored in atmospheres containing 1-5% 02 may
still be low enough to allow toxin production. Huss et a1. (1980) found the
maximum Eh permitting growth in smoked herring to be +250 mV, while

the maximum value in fresh herring was +100 mV (Huss et al., 1979).

10

Concentration of CO;

It has been established that the success in controlling aerobic spoilage
deterioration of food was not simply dependent on the elimination of 02;
rather there was a definite requirement for C02 in the gas atmosphere.
Coyne (1932) reported that the growth of Achromobacter, F lavobacterium,
Micrococcus, Bacillus and Pseudomonas was markedly inhibited by 25%
C02 and completely inhibited by 50% C02. In a later study, Coyne (1933)
reported that the optimal concentration for inhibition of aerobic spoilage
microorganisms was 40-60% C02. No additional extension of shelf life was

obtained by using higher concentration of C02 and bacterial growth was less

inhibited below these concentrations.
More recently, Gill and Tan (1980) examined the effects of various

concentrations of C02, equivalent pressures of 100-300 mm Hg, i.e., l3-39%
C02 in air, on the respiration rates of Pseudomonas species, Acinetobacter,
Alteromonas putrefaciens, Yersinia enterocolitica, Enterobacter and
Brocothrix thermosphacta (formerly Microbacterium thermosphactum).
They reported that the respiration rates of most of the common spoilage

organisms under investigation, with the exception of Enterobacter and

Brocothrix thermosphacta, were affected by elevated levels of C02 in air.
The level of C02 that resulted in maximum inhibition of the common
spoilage organisms was approximately 200 mm Hg, or 26% C02 in air.
However, the level of C02 investigated in this study had no antimicrobial
effect on B. thermosphacta which requires concentrations of 75% or more
C02 for complete inhibition.

Several studies have shown that mold growth is also inhibited by low

concentration of C02. For example, many Aspergillus, Rhizopus and

11

Cladosporium species are completely inhibited by 5-10% C02 at 1°C.
Other studies have shown that 20-30% C02 was sufficient to prevent the
growth of meat-associated molds while 30-50% C02 was found to
completely inhibit all mold species associated with the spoilage of bread and

cakes (Seiler, 1978).
It is evident from these studies that the concentration of C02 in the gas

mixture is very important to obtain the desired extension of microbiological
shelf life of the product. For example, a concentration of 60 to 80% C02
appears most practical for extended storage (i.e., 28 days) for ground

chicken meat (Baker et al., 1985).

Storage Temperature

Storage temperature also affects the antimicrobial activity of C02. It
has been shown that the C02 is a very effective antimicrobial agent at low
storage temperatures, but less effective at higher temperatures. This
increased inhibitory effect has been attributed to the greater dissolution of
C02 in the aqueous phase of products at lower storage temperatures (Fine,
1982) and resultant changes in intra-cellular pH and enzymatic activities of
microorganisms. Therefore, any decrease in inhibition of spoilage/
extension of shelf life at higher storage temperatures results from the lower
solubility of C02 in the aqueous phase of the product. MAP should not be
regarded as a substitute for proper storage temperature. While MAP slows
the deterioration of a food product, it never totally arrests this deterioration;
Proper refrigeration is essential in order to assure the effectiveness of C02 as
an antimicrobial agent and to prevent potential growth of pathogenic

organisms.

12
For maximum antimicrobial effect, the storage temperature of a MAP
product should be kept as low as possible because the solubility of C02
decreases dramatically with increasing temperature (Daniels et al., 1985).

Thus, imprOper temperature control will usually eliminate the beneficial
effects of elevated C02.

Packaging materials

One of the most important factors influencing the antimicrobial effect
of C02 is packaging film permeability. The success or failure of MAP for
respiring and non-respiring foods depends on both the 02 and C02
impermeability of packaging materials in order to maintain the correct gas
mixture in the package headspace. In addition to controlling the gas
mixture, packaging materials should also have low water vapor transmission
rates to prevent moisture loss or moisture gain. Polymers commonly used
for MAP of food include polyester (nylon), polypropylene (PP),
poly(vinylidene chloride) (PVDC), ethylenevinyl alcohol (EVOH) and
polyethlene (PE). Since all the desired characteristics of a packaging film
i.e., strength, impermeability and heat scalability are seldom found in one
polymer, individual polymers are laminated to one another to produce films
with the desired characteristics for MAP of both nonrespiring and respiring
products. Examples of laminated structure for gas packaging of non-
respiring products include nylon/PE, nylon/PVDC/PE or nylon/EVOH/PE.
These composite structures have all the desired characteristics for gas
packaging of non-respiring product, specifically, strength, provided by the
outermost layer of nylon; gas and moisture vapor impermeability, provided
by EVOH or PVDC; and heat scalability, provided by PE (Goodbum and
Halligan, 1988).

13

Microbiological Safety of MAP

A major concern about MAP foods is that they may be a public health
risk, particularly if subjected to temperature abuse during distribution and
retail storage. A number of individuals have reviewed the food safety
problem with MA storage of foods (Farber, 1991; Genigeorgis, 1985;
Hintlian and Hotchkiss, 1986; Hotchkiss, 1988; Palumbo, 1986). However,
no relationship has been established between storage life and safety and the
relationship between storage life, consumer acceptance, and microbiology of
modified atmosphere packaged foods is not known.

There is very little known about what effect modified atmospheres and
low 02 atmospheres will have on growth patterns of microorganisms which
contaminate minimally processed/MAP foods. A minimally processed food,
to attain the desired shelf-life, will need to have not only pathogens
controlled, but also the variety of spoilage microflora that are able to
multiply under refrigerated storage (Palumbo, 1986). The most obvious
concern with MA storage of fresh prepared foods is whether pathogenic
bacteria, such as S. typhimurium or C. botulinum, can grow to substantial
levels or produce toxin before the MA-inhibited spoilage bacteria have
grown enough to provide sensory evidence that the food is spoiled.

Stier et a]. (1981) and Post et a1. (1985) conducted studies of fish fillets
inoculated with C. botulinum and packaged in modified atmospheres to
determine if organoleptic evidence of product spoilage would cause the
product to be rejected before the fillets became toxic. However, their
conclusions appeared contradictory: Stier et al. found that spoilage usually

was evident before toxigenesis, but Post et al. found that toxigenesis often

14

preceded spoilage. This concern is especially important during storage at
less-than-optimal refrigeration temperatures.

Other MAP safety concerns which have not been properly addressed
product safety under temperature abuse of refrigerated foods during
processing, storage, distribution, retailing, or in the hands of the consumer.
Although most food pathogens do not grow well at refrigeration
temperatures, pathogens can grow in almost any atmosphere (Silliker and
Wolfe, 1980; Hintlian and Hotchkiss, 1987a) under conditions of product
temperature abuse and this may result in unsafe products. The presence of
air in non-MAP conditions with lack of refrigeration usually supports the
growth of aerobic spoilage organisms which causes development of
undesirable odors, colors, or slime. In refrigerated products, this acts as a
critical safety factor by alerting consumers and preventing consumption of
the temperature abused product. If MAP suppresses the growth of spoilage
organisms while allowing the growth of pathogens (facultative and/or
obligate anaerobes) under oxygen-excluded MAP conditions with
temperature abuse, packaging of minimally processed foods could result in
the loss of this safety factor.

In addition to these factors, the psychrotrophic pathogens (including
Yersinia enterocolitica, Listeria monocytogenes, and Aeromonas hydrophila)
which may grow at refrigeration temperatures as low as 3.3°C(NFPA, 1988);
it is very likely that MA conditions will favor pathogen growth, which could
create unsafe products without causing detectable organoleptic changes.
Listeria monocytogenes is of key concern in MAP foods because of its
persistence in food and the use of conditions to inhibit normal spoilage
organisms. It has a minimum growth temperature of approximately 25°C,

and its growth under refrigeration is also stimulated by carbon dioxide (Gray

15
& Killinger, 1966). However, Gill and Reichel (1989) reported that, while
L. monocytogenes grew at temperatures as low as 0°C on vacuum—packaged
beef strips, the organisms did not grow below 10°C when incubated in an
elevated C02 atmosphere.

Understanding the growth interactions and competition between
spoilage and pathogenic bacteria on modified atmosphere stored foods has
been identified as a vitally important area of food safety research (Hotchkiss,
1988). Although the relationship between spoilage and pathogenic
organisms can be better understood for a given product/storage combination,
it does not provide sufficient data for absolute safety decisions. Hotchkiss
(1988) suggested that a determination of the relationships between
pathogenicity and spoilage combined with a statistically predictive approach
to risk assessment would be an appropriate microbial safety of food

products.

16

Predictive Modeling of Microbial Growth

Predictive modeling is a promising field of food microbiology. Models
are used to describe the growth of microorganisms under different
environmental conditions such as temperature, pH, aw, and atmosphere.
These models allow the prediction of microbial safety or shelf life of
products, the detection of critical parts of the production and distribution
process, and the optimization of production and distribution chains
(Zwietering et al., 1990).

In predictive modeling applications, models based upon lag time are
most appropriate for pathogens with zero growth tolerance like C.
botulinum, Salmonella, Listeria, etc. (Baker and Genigeorgis, 1990).
Growth rate models are best for pathogens which must have significant
increase in numbers to reach an infective/toxic dose or prediction of
microbial food spoilage (Baird-Parker and Kilsby, 1987).

Models for proteolytic strains of C. botulinum types A and B grown in
a simulated meat product have been studied (Gibson et al., 1982; 1984;
Roberts and Gibson, 1986; Roberts et al., 1981a;b;c; 1982). The resultant
mathematical model predicted the probability of toxin production (i.e.,
growth) in the model system as a function of NaCl, nitrite, heat treatment,
the presence or absence of other preservatives such as isoascorbate,
polyphosphate or nitrate, and incubation (storage) temperature (Robinson et
al., 1982). A similar logistic regression analysis has been used to calculate
the probability of toxin production from one spore of C. botulinum in fish
homogenate, as a function of temperature and inoculum size (Lindroth and
Genigeorgis, 1986). Regression analysis was also used by Jensen et al.

(1987) to model growth of C. botulinum in laboratory medium and by Ikawa

l7
and Genigeorgis (1987) for fish fillets stored under modified atmospheres.

These probabilistic models only give an indication of the probability of
growth or toxin production, not how quickly, or how much, growth takes
place.

Models which relate generation times of a wide range of organisms to
temperature have been proposed (Ratkowsky et al., 1982; 1983) and are
relevant to foods (Gill, 1984; Smith, 1985). The square-root model has
recently been extended to take account of the effect of aw/sodium chloride
concentration on growth rate of a strain of Staphylococcus xylosus
(McMackin et al., 1987). Given knowledge of the growth response of
microbes of concern with respect to physical and chemical properties of
foods i.e. pH, aw, level of preservatives, storage temperature, models can be
constructed which predict the likelihood and extent of growth. An example
is available for one strain of S. aureus and one strain of S. typhimurium
grown in UHT milk at a range of temperatures and pH levels, with aW
adjusted with glucose (Broughall et al., 1983; Broughall and Brown, 1984).

In order to build models to describe the growth of microorganisms in
foods, it is necessary to measure growth curves. To reduce the measured
data to interesting parameters such as the growth rate, it is recommended
that the data be described with a model instead of by using linear regression '
over a subset of the data. Si gmoidal models to describe the growth data can
be constructed growth curves with three or four parameters.

Several nonlinear regression models have been proposed to describe the
whole microbial growth curve. The most currently popular model is the
Gompertz function, which with parameter modification (Zwietering et al.,
1990), gives the lag time, the maximum growth rate constant, and the

maximum microbial load (corresponding to the stationary phase) directly

18

from nonlinear regression of the numbers versus time data. Buchanan
(1990) has been developing a pathogen growth modeling program, which is
applicable to five pathogens, Salmonella spp., L. monocytogenes, Shigella
flexneri, S. aureus, and Aeromonas hydrophila. The equations were derived
by response surface analysis of the growth curve data "fitted" using the
Gompertz function in conjunction with a nonlinear regression analysis
(Buchanan & Phillips, 1990). Evaluation of the models has indicated that
they provide useful "first round estimates" of the microorganism growth in a
food system for a given specific condition. A simple modification of this
program using an interpolation method for any condition between the test
values could be used to predict growth for a fluctuating condition if it were
broken into a series of constant condition time segments.

Most MAP studies in the literature have little data that can be used for
pathogen growth rate prediction. Usually comparisons were done on the
difference in growth or organoleptic shelf life under air and MAP conditions.
The effect of different gaseous environments on microbial spoilage of MAP
foods is less well understood compared to factors such as temperature, pH,
and water activity. MAP technology must be addressed to how the gas
composition affects both the lag time and growth rate (generation time) in
the log phase for spoilage organisms and pathogens, and whether the effect
is beneficial or detrimental to the shelf life and safety of the product (Labuza
et al., 1992).

Therefore, by determining the effects of modified atmosphere storage
on the relative growth rates of spoilage and pathogenic organisms at a
particular time, temperature, and atmosphere, a model may be developed to
predict the growth rate of organisms under specific conditions using the

Gompertz function with an appropriate curve fitting software. If such a

19

model can be devised, it may be utilized to predict the storage life of
minimally processed MAP products and it may aid in making decisions

about microbiological safety.

MATERIALS AND METHODS

Bacterial cultures

Freeze-dried cultures of the following organisms were obtained from
the American Type Culture Collection (ATCC), Rockville, MD:
Pseudomonas fragi (ATCC #27363); Salmonella typhimurium (ATCC
#13311); Clostridium sporogenes (ATCC #3584). Pseudomonasfragi was
used as a spoilage organism and Salmonella typhimurium was used as a
pathogen. The growth of C. sporogenes was used as an indicator of

potential growth of a botulism organism.

Sample preparation and inoculation

Ground beef and peas were purchased from a local grocery on the day they
were to be cooked. The ground beef/peas were cooked with corn starch in a
conventional oven at 150°C for 1.5 min. to an internal temperature of 55°C
and for 2.5 min. to an internal temperature of 71°C. Cooked ground
beef/peas were cooled overnight at 44°C in a home-type refrigerator. The
pH value of the sample was measured using a Corning pH Meter and the
average pH for the four replicates was found to be 6.25. The water activity

(aw) of the product was measured using SC-10A Thermocouple
Psychrometer (Decagon Devices Inc., Pullman, WA) and aw=0.982 was the
average for the four replicates.

The instructions for rehydration of each culture were followed

according to the recommendation that accompanied the bacteria (ATCC,

20

21

1989). The rehydrated each culture in tube was incubated prior to use under
the following conditions: P. fiagi was grown for 48 hr at 26°C in trypticase
soy broth (TSB; DIFCO). S. typhimurium was grown for 48 hr at 37°C in
nutrient broth (DIFCO). C. sporogenes was grown for 48 hr at 37°C in
reinforced clostridial medium (RCM; DIFCO) under anaerobic condition.
Anaerobic conditions were achieved by adding sterile mineral oil to the top
of the liquid as a barrier to prevent oxygen contact.

Serial dilution of the working cultures were made in sterile peptone
water (0.1%) to obtain the desired inocula. One ml of a diluted culture
(103) was transferred into a sterile tube containing 39 ml sterile peptone
water to obtain an inoculum of approximately 2.5x103 CFU/ml. Colony
forming units (CFU) of the inoculum were confirmed by the pour plate
method. For sample inoculation, the 1.0 ml inoculum was distributed onto
the surface of each sample which has approximately 100 CFU/ g of each
culture. 25.010.1g of sample was put into the sterile jars.

Packaging of samples

Sterilized l-pint glass jars with sealed lids fitted with inlet and outlet
rubber septa for gas distribution were used as sample chambers for varying
the gas atmospheres. Gas mixtures were obtained by blending the different
gases (C02, 02, NZ) to the desired ratios using a gas flowmeter (DWYER).
Standard curves were prepared by flushing empty jars with combinations of
gases and analyzing the mixture by gas chromatography (SERVOMEX)
against a standard mixture of gases. Prepared samples were placed in
sterilized l-pint glass jars (25.010.1g). After sealing, each jar was
continuously flushed for 1.5 to 3.0 min with the appropriate gas mixture by

inserting inlet and outlet needles through the rubber septa. Jars containing

22
samples to be stored were analyzed by gas chromatography to insure proper

gas mixtures.

Analysis of headspace composition

Composition of the gaseous headspace was evaluated on each sampling
period. Prior to opening each package for microbiological evaluation, an
analysis of the headspace gas composition was performed in terms of the 02
and C02 content of the package atmosphere. Samples (20 fit) of the gas
were injected into a Servomex series 1100 gas chromatograph (London,

England) using a Hamilton Gastight series 1705 Syringe (50W).

Enumeration of bacteria

Bacteria were enumerated using standard microbial plate count methods
(Messer et al., 1985). Each sample was aseptically removed from the
sterilized glass jars and placed individually into a sterile stomacher bag to
which 225 ml of sterile 0.1% peptone water was added to achieve an initial
dilution of 1: 10. The diluted sample was homogenized for 2.0 min using the
Stomacher (Tekmar Model STO 400, Cincinnati, OH) and additional
decimal dilutions were performed as needed using peptone water. Duplicate
platings were made by removing 1.0 ml from the diluted samples.

Appropriate dilutions were plated out on the following selective and/or
differential media: P. fragi on Pseudomonas agar base plus a supplement
containing centrimide, fucidin and cephaloridine (Oxoid); S. typhimurium on
MacConkey agar and Triple sugar iron agar; and C. sporogenes on RCM and
SP8 agar. Colony forming units (CFU) were counted after incubation at 26
or 37°C for 48 hr, and 8 to 10 selected colonies were confirmed on API 20E
and An-IDENT(Analytab Products) strips at each sampling period. For each

23

species, logloCFU/g was calculated using the guidelines of the American

Public Health Association (Richardson, 1985).

Experimental design

Experiment I: The experimental design used in this study was 3x5 factorial

with 3 replicate experiments in both pure and mixed cultures. The cooked

 

ground beef/pea product (55°C internal temperature) was inoculated with
Pseudomonas fragi, Salmonella typhimurium and Clostridium sporogenes.

Each replicate experiment consisted of three temperatures (5, 12.5 and 25°C)
and 5 atmospheres (Air, MA containing 75% C02 with 25% 02 (MAl),

10% 02(MA2), 5% 02 (MA3), or 0% 02 (MA4) (balance N2)). At each
sampling period (0, 2, 4, 7, 14, 21, 28, and 35-day intervals), two samples
were used to calculate microbial populations for each replicate. Mean values
were reported as the average of duplicate plating of each of 6 samples per

sampling time. The number of bacteria present on the sample was

determined for each sampling and expressed as log 10CFU/ g.

24

Figure 1. Sequence used to prepare samples for testing for growth of three

organisms at various atmospheres and temperatures in both pure and mixed

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

cultures.
Cooked beef/pea
Inoculated Samples
I l l |
Air Control MA 1 MA2 MA3 MA4

(75% C02 (75% CO2 (75% C02 (75% CO2

+ + + +

25% 02 10% O2 5% 02 0% 02

+ + + +

0% NZ) 15% N2) 20% b5) 25% bk)

 

 

 

 

 

 

 

 

 

 

 

 

l l l |
1

Hold each MA's samples for 35 days at:

g | s
I I I

5°C 125°C 25°C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1). Data to be generated for growth curves of the individual organisms at
various atmospheres and temperatures in both single and mixed cultures.
2). Select the MA's which are the most effective on the growth control for

each individual organism.

25

Experiment II: Temperature abuse study

Figure 2. Selected modified atmospheres used under varying abusive

temperature situations in both pure and mixed cultures with different

inoculum levels.

 

Inoculated samples

1

 

 

 

 

 

 

 

 

 

 

F I . l . e l .

Air control MA MA MA
(P. fragi ) (S. typhimurium) C. sporogenes)

(75% C02 (75% C02 (75% C02
4» + +
0% c2 0% c2 25% 02,

+ + +

25% N2) t 25% 13) r 0% N2) J

 

 

 

 

 

 

 

 

 

1“] [‘1

s'c 5'C s'c 5C
(3 wk) (3 wk) (3 wk) (3 wk)

I

12.5? 25c 12.5C 25c
(1 wk) (1 d) (1 wk) (1 d)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SC so SC SC
(1 wk) (1 wk) (1 wk) (1 wk)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

- Data to be generated for growth curves of the individual organisms at
selected MA's with abusive temperatures in both pure and mixed cultures

with different inoculum levels of 102 CFU/g and 104 CFU/ g.

 

26

Statistical analysis

Microbiological count data were analyzed using one-way analysis of
variance comparing air-packaging (control) to each of the other treatments,
independently, at each of the seven storage intervals. One-way analysis of
variance was also done on data within each packaging treatment over the
seven storage intervals. When significant (P < .05) main effects were
observed in the analysis of variance, mean separation was accomplished by
use of the Duncan's new multiple range test (Steele & Torrie, 1980).
Variance analysis in a "split plot" design was used to establish differences

among the various treatments at different storage temperatures.

Statistical Predictive Modeling
A number of approaches have been used to develop predictive models that
describe the effects of various cultural factors on the growth of selected
foodbome pathogens. One that appears particularly promising is response
surface regression analysis of data generated is the "Gompertz function" to
quantitatively describe growth kinetics. This approach has been used
successfully to empirically model the effects and interaction of temperature,
pH, and aw on the growth of Salmonella (Bratchell et al., 1989; Gibson et
al., 1988; Roberts, 1989).
Modeling was canied out in two stages:
- The first stage involved modeling the bacterial growth curve by a
Gompertz function.
- The second stage of modeling concentrated on describing the variation of
the parameters of the growth curve as a function of growth conditions

(i.e., temperature and atmosphere).

27
At each combination of temperature and atmosphere, the bacterial count was
modeled as a function of time, using the Gompertz growth curve given by:
L(t) = A + C exp{-exp [-B(t-M)]}
where:
L(t) = Log count of bacteria at time (in days) t
A = Asymptotic log count of bacteria as time decreases indefinitely
(i.e., initial level of bacteria)
C = Asymptotic amount of growth that occurs as t increases
indefinitely (i.e., number of log cycles of growth)
B = Relative growth rate at M

M = Time at which the absolute growth rate is maximum.

Derived Growth Kinetics Equations (Gibson et al., 1987):
Exponential growth rate = BC/e
Generation time = Log(2)e/BC
Lag time = M - (1/B)

Maximum population density = A + C

The function chosen to model the parameters was a polynomial of the form:
Y=a+brT+b2A+b3TQ+ b4A2+b5TA+e
where Y is the response variable, i.e., the parameter to be modeled; T and A

represent temperature and modified atmosphere respectively, and e

represents a random error.

28

Experiment III:

1. Effect of higher cooking temperature of sample (71°C) on the outgrowth
of C. sporogenes at the same conditions (i.e., modified atmospheres,
inoculum level, and cultures) in Experiment I for 7 day-storage period at
abusive temperature of 25°C.

2. Test predictive models at storage temperatures of 8 and 20°C for 7 day-
storage period for the same conditions (i.e., cooking temperature,

modified atmospheres, inoculum level, and cultures) in Experiment I .

pH of the stored sample

The pH of the sample was measured by blending the sample in sterile
peptone water. A 25 gram sample was taken from the glass jar, placed into a
sterile stomacher bag containing 225 ml of 0.1% sterile peptone water (pH
7.0:i:0.1), and homogenized for 2.0 min using the Stomacher (Tekmar Model
STO 400, Cincinnati, OH). The pH value was measured using a Corning pH

Meter and was reported as an average from the duplicate.

Aerobic count

Each sample was aseptically removed from the sterilized glass jars and
placed individually into a sterile stomacher bag to which 225 ml of sterile
0.1% peptone water was added to achieve an initial dilution of 1:10. The
diluted sample was homogenized for 2.0 min using the Stomacher (Tekmar
Model STO 400, Cincinnati, OH) and additional decimal dilutions were
performed as needed using peptone water. Duplicate platings were made by
removing 1.0 mi from the diluted samples. Appropriate dilutions were

plated out on Plate Count Agar (Difco) with plate incubation at 37°C for 48-

29

72 hours. Colonies from each plate were counted using the method

described in Appendix II and calculated as the average colony forming units

(CFU) per gram sample. This number was calculated and reported as logro
CPU for each sample.

RESULTS AND DISCUSSION

Experiment I:

The results of growth changes in average log10 CFU/ g for Pseudomonas

fragi, Salmonella Iyphimurium, and Clostridium sporogenes are listed in
Tables 1 - 6 and shown in Figures 3 - 14, respectively.

At 5°C, Pseudomonas fragi grew rapidly under air. However, the
growth of P. fragi under MA's was significantly less (P<0.05) than under
air, with slightly less than 2 logs increase in numbers under MA-storage,
compared with an increase of more than 7 logs under air-storage of 14 days
in both pure and mixed cultures (Figures 3 and 6). The modified
atmospheres with high 75% C02 had an inhibitory effect on the organism
and had more inhibitory effect with decreased levels of 02 in MA's at 5°C.
Therefore, use of any MA-storage could substantially decrease growth of
pseudomonads and thus extend the product shelf life at 5°C.

At 125°C, the growth of P. fragi under MA's was also signicantly less
(P<0.05) than under air: with less than 3 logs increase under MA-storage,
compared with an increase of more than 7 logs under air-storage of 7 days in
both pure and mixed cultures (Figures 4 and 7). The MA had an inhibitory
effect on the organism and had more inhibitory effect with decreased levels
of 02 in MA's at 125°C. Enfors and Molin (1980) have previously
demonstrated the inhibitory effect of C02 on P. fiagi. Ingham et al. (1990)
reported that MA (80% C02/0%N2) was more inhibitory to growth of P.
fiagi than was MA (50% C02/10% 02) on cooked chicked loaf at 3, 7, and
1 1°C.

30

 

31
At 25°C, P. fragi grew rapidly and reached maximum growth with an

increase of more than 7 logs in pure culture under air-storage in the first 2
days, whereas in MA's reached maximum growth with an increase of 1-4
logs during the first 4 days (Figures 5 and 8). In mixed cultures, the growth
of P. fragi was slightly slower than in pure culture at all atmospheres,
especially, there was 1-2 logs different growth of P. fragi at MA-storage. It
may be due to competition of growth with other organisms such as S.
typhimurium which increased number of CFU in 5-65 logs at MA's in the
first 2 days. The results indicated that MA's inhibitory effect was decreased

with increased storage temperature.

 

32

TABLE 1. Mean values for Pseudomonas fragi counts (loglo CFU/g)a in

pure culture.

Storage Storage
temp.

time
da 5
5.0

35

Air
2.20:.05°
3.78:. 1
6.10:.23e
8.75:52
9.95:.348
9.59:.36g
9.59:.258
9.20:.41 g

2.20:.05°
8. 1
8.60:.
9.95:.39e
9.93:.41e
9.48:.26e
9.60:.52e
8.70:. 1

2.20:.05°
9.48:.33
9.08:. 1
8.54:.

6.

6
4.20:.238
2. 28:. 120

MAI
2.20:.05C
3.04:.

2.20:.05c

2.20:.05c

6

MA4
2.20:.05c

MAZ
2.20:.05°

2.20:.05c

2.20:.05c

e

 

aAverage of triplicate samples : standard error of the mean.
bMeans within a common storage interval that are underlined are significantly
oddifferent (P < .05) from means obtained from air packaged samples.
0‘19 f'gMeans 1n the same column bearing a common superscript do not differ

(P> ..05)

 

33

 

Figure 3. Growth of P. tragi at 5°C in pure culture.
1 - , _. .

 

 

 

 

8* i -——a—— )uR
or .. —+— MA 1
\ —I— MA2
if 6- " -——e—— Mus

+ MA4

U .1
g' 4" ' .— c c
.J - -4

C 1 l r I ' I '

0 10 20 30 40
11ME(DAY)

 

Figure 4. Growth of P. fragi at 12.5°C in pure culture
10 - _

Log CPU/g

 

0
0

 

 

 

 

 

 

 

 

' I ' I v
0 10 20 30 4O
11ME(DAY)

34

TABLE 2. Mean values for Pseudomonas fragi counts (log10 CFU/g)a in

mixed cultures.

Storage Storage ' treatmen
temp. time

Air MA2 MA3
2.20:.05C 2.20:.05c 2.20:.05C
3.68:3- C
5.30:.48e . C

9.70:.248
9.65:.358
9.60:.438
9.30:.178

 

2.20:.05c
7
8.54:.16e
9.1 l

9.81
9.48:.
8.76:.546
8.60:.30e

2.20:.05c

8.92:.3

8.48:.2

8.18:.4

3. - - -

1.30:.048 - - - -

35 - - - - -
21Average of triplicate samples : standard error of the mean.
bMeans within a common storage interval that are underlined are significantly
different (P < .05) from means obtained from air packaged samples.

CdefgMeans in the same column bearing a common superscript do not differ
(P > .05).

 

35

 

 

  
  
  

 

 

 

 

 

 

 

 

 

 

 

Figure 6. Growth of P. fragi at 5°C in mixed cultures
1 J ——r M
8‘ .MR
3‘ ' 11.1155
2 .. m:
U ..
In 4-1
O
—l
2
C ‘ l f I T 1
o 1o 20 30 4o
TIME(DAY)
Figure 7. Growth of P. fragi at 12.5°C in mixed cultures
10 —
6i
\
3
lb
U
51
O
_l
C I I T I u I '
o 10 20 3o 40
TIME(DAY)

Figure 8. Growth of P. fragi at 25°C in mixed cultures

 

  

10
‘ Au:
8‘ MAI
. MA2
MA3
MA4

 

Log CPU/g
‘1’

 

 

 

O 10
TIME(DAY)

36

Salmonella typhimurium did not grow under either air or MA's at 5°C.
The minimum growth temperature reported for S. typhimurium is 62°C on
trypticase soy agar (Matches and Liston, 1968).

At the slightly abusive storage temperature of 12.5°C, S. typhimurium
grew under air and all MA's, but there is a significantly different (P<0.05)
growth of the organism between in air and in MA storage. Although the
number of S. typhimurium increased by 25 log cycles in all atmospheres

with reached maximum growth in 14 days(Figures 9 and 10), the MA had an
inhibitory effect on the organism due to high concentration (75%) of C02

and decreased levels of 02. However, the MA had a less effect on the

growth of S. typhimurium than that of P. fragi. Interestingly, although the
rate of growth of S. typhimurium was almost the same in both pure and
mixed cultures, numbers of CFU were lower with lower 02 levels of
modified atmosphere in MA-storage. This organism has a preference for
oxidative metabolism and a competition for 02 with P. fragi. Partial
inhibition of Salmonella spp. by C02 on other substrates has been observed
by others (Baker et al., 1986; Eklund and Jarmund, 1983; Gray et al., 1984;
Hintlian and Hotchkiss, 1987a; Luiten et al., 1982). Silliker and Wolfe
(1980) inoculated ground beef with six Salmonella strains and then stored
the meat under different gas atmospheres at either 10 or 20°C. After 10 days
at 10°C, the number of Salmonella in samples stored in air had increased by
more than 3 logs, while in MA-stored samples (60% C02, 25% Oz, 15% N2)
the number had only increased by approximately 0.5 logs.

At 25°C, Growth of S. typhimurium was rapidly increased by about 5 to
7 logs during the first 4 days in all atmospheres and in both pure and mixed
cultures (Figures 11 and 12). At 25°C, the atmosphere composition had no

37

effect on S. typhimurium which flourished in all atmospheres and cultures.
These results reiterate the fact that MAP cannot be used effectively at room
temperature and cannot be used as a substitute for refrigeration
(Genigeorgis, 1985). Whether MA—storage minimally processed meat-
vegetable product could be a salmonellosis hazard prior to spoilage would
depend upon such factors as the initial levels of pseudomonads and S.
typhimurium strains, and the resistance of persons ingesting the product.
Numbers of S. typhimurium considerably lower than 105-106 cells/g may
cause salmonellosis in some individuals (Banwart, 1989). In this study, the
growth of S. typhimurium , although slowed by MA storage still occurred at
a rate that could result in a risk of salmonellosis at abusive temperatures.
Data indicated that the presence of P. fragi has no real effect on the growth

of S. typhimurium in mixed cultures.

 

38

TABLE 3. Mean values for Salmonella typhimurium counts (log10 CPU/g)a
in pure culture.

treatmen

Storage Storage

temp.

5

time
da 3

35

Air

2.20:.03C
3.57:.13
5.43:.22e

7.92:.388
7.48:.248

7.30:.1 g

6.26:34

2.20:.03c
8.60:.
8.95:.1

9.

8. 15:.3
6.94:.24e
2.84:.10c

MAI

2.20:.03C

4

2.20:i:.03c
8.32:.1
8.85:.1
8.7

6.48:.23e
2.00:.04c

MA2

2.20:.03c

2.20:.O3°

8.61:.28e
8.48:.426
8.34:.33
6.
3.34:.168

MA3

4

 

 

*NG = no growth

21Average of triplicate samples : standard error of the mean.

bMeans within a common storage interval that are underlined are significantly
different (P < .05) from means obtained from air packaged samples.

OdefgMeans in the same column bearing a common superscript do not differ

(P > .05).

39

Figure 9. Growth of S. typhimurium at 12.5°C in pure culture.
1

 

 

 

 

 

8.4
e .1
= 6‘ k A A
b v
at ///
o 4" /
..I
’ -——e—- MA]
2 + MA2
+ MA4
O r 1 . r . 1 r
O 10 20 30 4O
TIME(DAY)

Figure 10. Growth of S. typhimurium at 25°C in pure culture.
10

 

 

Log CFU/g

 

 

 

O ' ' I ' I r
10 20 3O
TIME(DAY)

.IEEF—-Ha

40

TABLE 4. Mean values for Salmonella typhimurium counts (log10 CFU/g)a

in mixed cultures.

Storage Storage ' treatmen

temp. time
da 5 Air MAZ MA3

5

0 2.2%.03C 2.20:.03c
2 3.45:.1

4 5.34:.26e . C
7 6.48:32
14 7.40:.208
21

28

35

 

7.40:.258
6.78:.32 8
6. 18: 12

O 2.20:.03c 2.20:.03°
2 8.65:.1 8.32:.23
4 8. 8.71:.11
7 9.
14 7.70:.10e 7.36:.16e
21 6.67:.338 6.15:3
28 2.60:.13C 2.30:.10c 4
35 - - 1.30:.
*NG = no growth
aAverage of triplicate samples : standard error of the mean.
bMeans within a common storage interval that are underlined are significantly
different (P < .05) from means obtained from air packaged samples.
CdefghMeans in the same column bearing a common superscript do not differ

(P > .05).

 

41

Figure 11. Growth of S. typhimurium at 12.5°C in mixed cultures.

 

1

 

 

 

 

 

Bl
\
3
L
U
5|
0
..l
l -—+—- MA3
+ MA4
G T I l v 1 1
O 10 20 30 40
TIME(DAY)

Figure 12. Growth of S. typhimurium at 25°C in mixed cultures.

 

1

Log CFUlg

 

 

 

 

TIME(DAY)

42

Clostridium sporogenes did not grow at 5°C because its minimum
growth temperature was about 10°C on pure media (Billy, 1962). At 12.5°C,
MA's containing moderate 02 concentrations (5-25%) prevent growth of C.
sporogenes on samples in both pure and mixed cultures except MA4 (0%
02) in mixed cultures in which its numbers increased by only 1 log during
35-day storage period. This may be due to the very slow growth rate of C.
sporogenes at 12.5°C (Sperber, 1982) and the residual 02 remaining in the
packages of MA4 in single culture. Silliker and Wolfe (1980) reported that
C. sporogenes failed to grow in any atmosphere at 10°C. Baker et al. (1986)
showed that C. sporogenes failed to grow above inoculum levels in ground
chicken meat under MA storage of 80% C02 (balance air) at 13°C during
28-day storage time.

At 25°C,C. sporogenes grew rapidly at MA4 (0% Oz) in both pure and
mixed cultures. In air and MAI (25% 02) storage,C. sporogenes failed to
grow above the inoculum levels during the first 2 days in pure culture due to
the oxygen content of headspace (Figure 12 in Appendix V). However, C.
sporogenes can grow after the oxygen in the headspace was consumed by P.
fragi and S. typhimurium. in mixed cultures. It is likely that 02 consumption
by P. fragi and S. typhimurium created a more favorable anaerobic
atmosphere in mixed cultures, whereas, in pure culture, 02 was consumed by
ground beef/pea product and by any bacteria on it. This was confirmed by
total aerobic plate count in pure culture, which reached total counts of 106 -
103CFU/ g in Day 2 at 25°C. Headspace analysis by gas chromatography
confirmed that 02 levels in 25°C samples on Day 2 were less than 1% in

MA2 and MA3 of pure culture. Lambert et al. (1991) showed that the

oxygen level decreased to less than 1% within 2 days and COzlevel

43
increased to 33% for inoculated C. botulinum on fresh pork packaged
initially with 10% 02 (balance N2) at 25°C and detected C. botulinum toxin
in 2 days.

Ingram (1962) showed that all the oxygen was consumed in 2-4 days at
15°C or 1-2 days at 37°C in cans of bacon under the air pack. The indicated
rates of oxygen consumption were of the order of 0.1 ml of 02/ g of bacon in
1 day at 37°C or in 2 days at 15°C. It was suggested that the oxygen
consumed from the joint respiration of the meat tissue and any bacteria on it.
It was observed a rate of 15x10'5 ml of Oz/min/g of lean bacon tissue, which
corresponds roughly to 0.9 ml/ g in 4 days. To respiration of the bacteria, for
example, the average uptake of oxygen from air by a single cell of
Escherichia coli at 37°C was approximately 5x10‘12 ml/min. During the
first 2 days the bacon stored in air at 37°C carried about 107 bacteria of
various kinds/ g. It means an oxygen uptake by the bacteria of the order of
5x10'5 ml/min/g of bacon, i.e., about 0.5 ml/g in 2 days. Therefore, it was
suggested that the influence of both the product tissue itself and of its
bacteria must be equally considered in interpreting atmospheric changes in
the package and also in other biochemical changes.

Lovitte et al. (1987) reported that C. sporogenes is a proteolytic
organism, capable of growth in complex media containing no carbohydrate,
the addition of glucose to such media causes a significant enhancement of
growth. In this study, besides ground beef meat tissue, peas had consumed
oxygen and corn starch may be utilized by C. sporogenes as a source of
carbohydrate even after cooking temperature of 55°C.

The results of this study indicate that the key to preventing growth of S.
typhimurium and C. sporogenes on the product is proper refrigeration. P.

fragi was inhibited by the high C02 concentration, S. typhimurium by the

44
elevated C02 and reduced 02 levels, and C. sporogenes by the presence of
02. For this reason, the inclusion of 02 in MAP is recommended as a safety
precaution (Hintlian and Hotchkiss, 1987a).

Presence of P. fiagi did not prevent the outgrowth of C. sporogenes in
the sample cooked to 55°C and total aerobic plate count reached to 106 -
103CFU/ g in Day 2 at 25°C in pure culture. Therefore, it is necessary for an
additional experiment to effect of higher cooking temperature (71°C) on the

outgrowth of C. sporogenes.

 

45

TABLE 5. Mean values for Clostridium sporogenes counts (logro CFU/g)al

 

 

 

 

 

 

 

 

 

 

 

 

 

 

in pure culture.
Storage Storage Packaging treatmentb
temp. time
(°C) (days) Air MAI MAZ MA3 MA4
5 NC NC NG N6 NO
125 NO N0 N6 NO NO
25 0 2.20:.06° 2.20:.O6C 2.20:.06c 2.20:.06c 2.20:.06c
2 2.20:.05C 2.20:.04C 2.48:.12C 2.50:.10c 4.40;;3‘1
4 4.67:.23d 4.86:.24d 4.60:.15d 5.00:.25d 7.483.223 ,
7 7.15:.356 7.11:.196 8.00:206 8.50:326 8781-241
14 8.78:32f 9.04:25f 9.18:32i 8.84:.266 9.26:19
21 9.18:12f 9.11:31r 9.20125f 8.95:.426 9.23127f
28 8.78:27f 9.18:12f 9.18:.27f 9.001.306 9.23:31f
35 9.00:.10f 8.88:15f 9.15:191’ 9.00:.276 9.10:20T

 

 

 

 

 

 

 

NG = no growth

TABLE 6. Mean values for C lostridium sporogenes counts (logro CFU/g)a

in mixed cultures.

 

 

 

 

 

 

 

 

 

 

 

 

Storage Storage Packaging treatmentb

temp. time

(0C) (days) Air MAI MAZ m3 MA4

5 NO NO NO NG N6

125 N6 N0 NG NG :1:

25 0 2.20:.06C 2.20:.06° 2.20:.06c 2.20:.06° 2.20:.06"
2 2.201.106 2.50:.216 2.90120d 3.60:.12d 6.23:.27d
4 5.23:.276I 5.40:.30d 6.00:__.1_5_e 6.90:.24" 7.54:.263
7 7.48:.4ze 7.84:.226 8.26:2;4} 8.48:22 8.78:12I
14 9.28:23f 9.23117f 9.30:.308 9.08:.36lg 9.28:33
21 9.08:32f 9.32:18f 9.40:.528 9.23:4rg 9.32:29f
28 9.23:47f 9.20:25r 9.30:.428 9.30:.25f8 9.34:35f
35 8.78:52f 9.10:30f 9.28:.338 9.151.388 9.30:4of

 

 

 

 

 

 

 

 

 

NG = no growth
*— - numbers increased by about 1 Log CFU/ g during 35 days.

2‘Average of triplicate samples : standard error of the mean.
bMeans within a common storage interval that are underlined are significantly

different (P < 0.5) from means obtained from air packaged samples.

°°° f'gMeans 1n the same column bearing a common superscript do not differ
(P > ..05)

Figure 13. Growth of C. sporogenes at 25°C in pure culture.

 

Log CFU/g

 

 

I

C . 1 . .
20 3O
TIME(DAY)

 

 

40

Figure 14. Growth of C. sporogenes at 25°C in mixed cultures

 

Log CFUlg

 

 

40

 

TIME(DAY)

47

Experiment 11: Temperature abuse study.

In experiment I, an atmosphere containing 75% C02 and 0% Oz (MA4)
was identified as the most effective in inhibition of P. fragi and S.
typhimurium, whereas MAI (75% C02 and 25% 02) was the most effective
in inhibition ofC. sporogenes.

The effects of l-week storage at 12.5°C and 1-day storage at 25°C
following optimum refrigerated 3-week storage (5°C) on the growth of P.
fragi, S. typhimurium , and C. sporogenes in air and selected MA's (MA4
and MAI) with different inoculum levels (approximately 102 and 104
CFU/ g) are listed in Tables 7-12 and shown in Figures 15-24, respectively.
P. fiagi (102 CFU/g) grew rapidly and reached maximum growth with an
increase of more than 7 logs in both pure and mixed cultures under air-
storage in 14 days. The growth of P. fiagi (104 CFU/g) reached maximum
within 7 days in pure culture, but 14 days in mixed cultures. The MA4 had
significant inhibitory effect (P<0.05) on the growth of P. fragi than that of

in air-storage and even after the abusive temperatures.

43

TABLE 7. Mean values for Pseudomonas fragi counts (loglo CFU/g)a in
single culture at abusive temperatures.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inoculum levels 102 CFU/ g I 104 CFU/ g

Storage Storage Packaging treatmentb

$521)" (33:) Air MA4 Air MA4
5 0 2.15.4046 2.15:.046 4.18:.O6C 4.18:.O6C
5 7 8.76:.27d 2.183.056 10.04:.246 4.26;.09C
5 14 9.93:.336 220-5076 9.84:.17de 4.11:.11
5 21 9.64:.196 ;.26:066 9.84:.20de 4.15:.056
12.5 22-28 9.70:.206 2.40:0566 10.04:.36e 4.3062336
5 29-35 9.52:.286 2.19:.036 9.30125d 4.15:.08°
25 22 9.90:.316 =2.6729096 9.95:.19d6 4.343ch
5 23-29 9.54:.26e 245-50566 9.61:.2366 4.19:.076

 

 

 

 

 

 

 

 

 

TABLE 8. Mean values for Pseudomonas fragi counts (log10 CFU/g)a in
mixed cultures at abusive temperatures.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inoculum levels 102 CFU/ g I 104 CFU/ g
Storage Storage Packaging treatmentb
‘33" Egg) Air MA4 Air MA4
5 0 2.15:i:.04c 2.15:.04c 4. 1821:.06c 4.18:i:.06c
5 7 8.26:.14‘I 2.20:.05c 9.84:.266 4.28:.04c
5 14 9.66:.23e 24:38:06: 9.95:30e 4.17;;93c
5 21 9.65:.25° 2.234.046 9.78:.416 4.15;_0_36
12.5 22-28 9.70:.306f 2.26:.OSC 9.80:.226 4.17:.08C
5 29-35 9.34:.16° 2.1 1:.03C 8.84:1:.l7d 4.17:.05c
25 22 9.78:326T 2.60:.09“ 9.49:.246 4.30:_.07_6
5 23-29 9.1 1:.18° 2.23:.05c 8.78:.31d 4.09:05C

 

 

 

 

 

 

 

 

 

aAverage of triplicate samples : standard error of the mean.

bMeans within a common storage interval that are underlined are significantly
different (P < .05) from means obtained from air packaged samples.

Cd”Means in the same column bearing a common superscript do not differ

(P > .05).

 

49

Figure 15. Growth of P. fragi with 102 cells/g at abusive
temperature of 12.5°C.

 

Log CFU/g

 

 

 

 

, . , .
4O

 

TIME (DAY)

Figure 16. Growth of P. fragi with 102 cells/g at abusive
temperature of 25°C.

 

Log CPU/g

 

 

 

 

 

20 30

TIME (DAY)

50

Figure 17. Growth of P. fragi with 10‘ cells/g at abusive
temperature of 12.5°C.

 

Log CFU/g

 

 

 

 

 

1 . r . r r .
10 20 30 40
TIME (DAY)

Figure 18. Growth of P. fragi with 10‘ cells/g at abusive
temperature of 25°C.

 

11

Log CPU/g

 

 

 

 

 

I ' I u
30

TIME (DAY)

51

During storage at 5°C, the numbers of S. typhimurium decreased in all
atmospheres because 5°C is lower than the minimum growth temperature
(62°C) for this organism. However, some cells remained viable at this low
temperature until 21 day-storage period and were able to multiply during
holding at 12.5 and 25°C.

In abusive temperature of 12.5°C for 7 days, S. typhimurium grew by 3
logs increased in air storage at both different cultures and inoculum levels.
However, the numbers of S. typhimurium was increased in less than 1 log
with 102 CFU/g and 2 log increased with 104 CFU/g in MA-storage. It was
observed similar results at abusive temperature of 25°C, but time to
maximum numbers is shorter at 25°C than that of 12.5°C.

Although MA-storage consistently had lower counts than air-stored
samples for P. fragi and S. typhimurium even at the abusive temperatures,
the MA's had not much of an inhibitory effect on the growth of S.
typhimurium than that of P. fiagi.

In this study, the results indicated that viable cells of S. typhimurium
which can cause health problem if injested after abusive temperature even

though they did not grow under refrigeration temperature.

TABLE 9. Mean values for S. typhimurium counts (loglo CFU/g)a in single
culture at abusive temperatures.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inoculum levels 102 CFU/ g I 104 CPU g
Storage Storage Packaging treatmentb
Tong“ (2:38) Air MA4 Air MA4
5 0 2.151.056 2.151.056 4.181.056 4.181.056d
5 7 2.041.066 1.841.046 4.081.046 3.941.046
5 14 1.951.046 1.651.056 3.901.076 3.701.086
5 21 1.951.046 1.601.066 3.601.096 3.401.036
12.5 22-28 4.901.156 2.501.106d 6.65122f 5.321.176
5 2935 3.901096 2.111.076 5.601.146 4.201.066d
‘ 25 22 4.601.126 2451,0863 5.781.326 5.521.276
5 23-29 3.901.14d 2.041.046 4.601.156d 4.431.076d

 

 

TABLE 10. Mean values for S. typhimurium counts (loglo CFU/g)‘il in
mixed cultures at abusive temperatures.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inoculum levels 102 CFU/ g I 104 CFU/ g
Storage Storage Packaging treatmentb
$3" (23:) Air MA4 Air MA4
5 0 2.151.05c 2.15105d 4.181.056 4.181.056T
5 7 2.081046 1.701.11Cd 4.001.106 3.801.126d
5 14 1.951.056 1.481.126 3.901.156 3.521.186d
5 21 1.901076 1.40108‘3 3.54-1.06c 3.30109c
12.5 22-28 4.781.126 ;351.055 6.49116e 5.301%;e
5 2985 3.601096] 2.081136 5.481236 4.111.05d
25 22 4.481.116 2. 231 07d 5.521.17a 5.401.106
5 23 29 3 ..481 086I 2. 041. 0561 4.231. 226 4.111.19d

 

 

abAverage of triplicate samples 1 standard error of the mean.

bMeans within a common storage interval that are underlined are significantly

different (P < .05) from means obtained from air packaged samples.
Cde f'Means 1n the same column bearing a common superscript do not differ

(P> ..05)

 

53

Figure 19. Growth of S. typhimurium with 102 cells/g at
abusive temperature of 12.5°C.

 

8‘
+ Air-mixed
—'*— MA4—mixed

Log CPU/g
T

 

 

 

 

c . , . , . , .
0 10 20 3O 40

TIME (DAY)

Figure 20. Growth of S. typhimurium with 102 cells/g at
abusive temperature of 25°C.

 

IC

 

 

 

3' —o— MA4-mixed
: 5" .
u-
U 1
8' 4-
_l
2
c . . . , .
o 10 20 30

TIME (DAY)

54

Figure 21. Growth of S. typhimurium with 10‘ cells/g at
abusive temperature of 12.5°C.

 

 

 

 

10
q + Air-pure
——°— h'1A4-pure
8- + Air-mixed
"—"'- MA4-mixed
5|
3
u. 6"
U
3'
_. 4
Z-I
O f T V I I r I
O 10 20 3O 40

TIME (DAY)

Figure 22. Growth of S. typhimurium with 10‘ cells/g at
abusive temperature of 25°C.

 

10

8‘ —-— Air-mixed
—.— MA4-mixed

Log CPU/g

 

 

l ' I

0 10 20 30
TIME (DAY)

 

55

During storage at 5°C, C. sporogenes (ca. 102 CFU/g) decreased to
undetectable levels and did not recover following the transfer of samples to
12.5 and 25°C after Day 21. The C. sporogenes with ca. 104 CFU/g
inoculum also failed to grow above the initial inoculum level in any
combination of temperature and atmosphere, but the decline in CFU of C.
sporogenes was greater in the air samples than in MA samples. It appears
that the level of C02 used in these experiments has some protective effect on
C. sporogenes but there was still remained oxygen in headspace until 21
days at air and MAI (25% O7) storage. Silliker and Wolfe (1980) reported
similar findings in ground beef. They observed a declined in the CFU of C.
sporogenes at 10°C with a greater decline in air control compared to C02.
Baker et al. (1986) also reported that C. sporogenes failed to grow above
initial inoculum levels (>104 cells/ g) under air or MA of 80% C02 (balance
air) at 13°C. Although oxygen was consumed by P. fragi and S.
typhimurium in mixed cultures during abusive temperatures, the samples
were returned to 5°C-storage which is lower than minimum growth
temperature of C. sporogenes. It is indicated that MAP can prevent growth

of C. sporogenes in the presence 02 with proper refrigeration temperature.

56

TABLE 11. Mean values for C. sporogenes counts (log10 CPU/g)a in single
culture at abusive temperatures.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inoculum levels 102 CFU/ g I 104 CFU/ g
Storage Storage Packaging treatmentb
‘fg' (32;) Air MA4 Air MA1
5 0 N0 N0 4.181.066 4.181.066
5 7 N0 N0 3.181.066 3.411.066
5 14 N0 N0 3.041.066 3.1 11.066
5 21 NO NO 2.84:.06C 2.90106c
12.5 22-28 N0 N6 2.74-1.06C 2.771206c
5 29-35 No NG 2.50106C 2.72106C
25 22 N6 N6 2.831.066 2.901.066
5 23-29 NG NG 2.621.060 2.84:i:.06c

 

 

 

 

TABLE 12. Mean values for C. sporogenes counts (log10 CFU/g)a in mixed
cultures at abusive temperatures.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Inoculum levels 102 CFU/ g I 104 CFU/ g
Storage Storage Packaging treatmentP
:33" (32;) Air MA4 Air MA1
5 O NO NO 4.18:i:.08e 4.18:.08d
5 7 N0 N0 3,301.106d 3.401.066
5 14 N0 N9 3.111.16c 3231.120
5 21 NG NG 2.901.096 3.041.266
12.5 22-28 NG NG 2.7411 1c 3.()4:1:.08c
5 2935 NO NG 2.7l:i:.05c 2.94106c
25 22 N6 NG 3.04106‘2 3.1 121:. 19c
5 2329 N9 N6 2.781.046 2.901.076 |

 

 

 

NG=No Growth

 

abAverage of triplicate samples 1 standard error of the mean.

bMeans within a common storage interval that are underlined are significantly

different (P < .05) from means obtained from air packaged samples.
Cd'eMeans 1n the same column bearing a common superscript do not differ

(P> ..05)

“.3

.71—

57

Figure 23. Growth of C. sporogenes with 10‘ cells/g at
abusive temperature of 12.5°C.

 

 

 

 

10
8.1
“—3— Air-pure
2' —6— MA1-pure
:1 6- -—I—- Air-mixed
: —.— MA1-mixed
3‘ 4
_l
2..
c . r r r
0 10 20 3O 40

TIME (DAY)

Figure 24. Growth of C. sporogenes with 10‘ oeIls/g at
abusive temperature of 25°C.

 

 

 

 

10
8—
+ Air-pure
'3 —'°'—' MA1-pure
=1 6~ ——-— Air-mixed
t . ——+— MA1-mixed
g‘ 4N“
..1
2-
f I fl l
0 10 20 30

TIME (DAY)

58

Predictive Modeling

Growth curve fitting

All growth curves were generated from the experimental data using the
Gompertz equation in conjunction with SYSTAT statistics package, Version
5.2, a nonlinear regression program that employs a Gauss-Newton
integration procedure. Growth curve data were used to derive "best fit"
Gompertz values for each of the variable combinations of modified
atmosphere and storage temperature for P. fragi, S. typhimurium, and C.
sporogenes (Tables 13-18). It was found that growth curves could be fitted
well with growth data points and all of the Gompertz values were derived
from the growth curves.

As examples of the types of growth curves obtained, Fig 25 illustrates
the effects of MA's decreasing oxygen concentration on the growth of S.
typhimurium during storage at 12.5°C. From those fitted curves the
parameters B, M, C and A were derived. An indication of the goodness of
fit of the Gompertz function to the data is given by the Residual Sum of
Squares (RSS): the smaller the tabulated value, the closer the fit. Tables
(13-18) list values for growth rate, lag time and generation time calculated
from the parameters B, M, and C obtained by fitting the Gompertz curves to
the data.

59

Figure 25. Growth curve fitting of S. typhimurium at 12.5°C

 

 

 

 

 

 

 

8 I I

7 — -

6 — ..
= v 0
LL [—l
U S " 1.1 "
c1 _
o n
d I

4 _. 4 . ..

u/
e/
3 -- in "
.1"
Z ' l .l
0 10 20 30

TIHECDAY)

60

TABLE 13. Gompertz equation parameters and calculated growth curve
values for P. fragi with various combinations of temperature and
atmosphere in pure culture.

 

Growth Generation Lag Max. Pop. Residual

 

Temp. MA's Gompertz Parameters Rate Time Time Density sums of
1°C) A C B M (Log #/d) (day) (day) (Log cf u/g) sguares
5. 0 Air 2.20 7.61 0.44 2.93 1.24 0.24 9. 61 0.206
5.0 MA1 2.20 1.28 0.95 1.22 0.45 0.67 0.17 3. 48 0.040
5.0 MA2 2.20 1.00 0.94 1.14 0.34 0.87 0.08 3.20 0.040
5.0 MA3 2.20 0.28 1.35 2.32 0.14 2.16 1.58 2.48 0.015
5.0 MA4 2.20 0.10 0.97 2.21 0.04 8.43 1.18 2.30 0.001
12.5 Air 2.20 7.45 1.47 1.00 4.03 0.08 0.32 9.65 0.413
12.5 MA 1 2.20 2.03 1.07 1.16 0.80 0.38 0.23 4.23 0.036
12.5 MA2 2.20 1.33 1.40 l. 12 0.69 0.44 0.40 3.53 0.095
12.5 MA3 2.20 0.69 0.62 2.08 0. 16 2.34 0.47 2.89 0.004
12.5 MA4 2.20 0.39 0.66 1.61 0.09 3.24 0.09 2.59 0.006
25.0 Air 2.20 7.23 5. 19 0.92 13.79 0.02 0.72 9.43 0.004
25.0 MA1 2.20 4.06 2.32 0.91 3.46 0.09 0.48 6.26 0.011
25.0 MA2 2.20 3.34 1.80 0.93 2.22 0.14 0.38 5.54 0.541
25. 0 MA3 2.20 1.81 2.08 0.90 1.38 0.22 0.42 4.01 0.003
25. 0 MA4 2.20 1.18 2.45 0.94 1.06 0.28 0.53 3.38 0.001

 

TABLE 14. Gompertz equation parameters and calculated growth curve
values for P. fragi with various combinations of temperature and
atmosphere in mixed cultures.

 

Growth Generation Lag Max. Pop. Residual

 

Temp. MA's Gompertz Parameters Rate Time Time Density sums of
1°C) A C B M (Log#/d) (day) (day) (Log cfu/g) guares
5.0 Air 2.20 7.58 0.37 3.69 1.04 0.29 1.00 9.78 0.151
5.0 MA1 2.20 1.25 0.62 1.42 0.28 1.06 0.19 3.45 0.064
5.0 MA2 2.20 0.91 0.66 1.34 0.22 1.37 0.19 3.11 0.052
5.0 MA3 2.20 0.22 0.81 2.93 0.07 4.57 1.70 2.42 0.003
5.0 MA4 2.20 0.10 0.74 2.53 0.03 10.66 1.18 2.30 0.003
12.5 Air 2.20 7.13 1.14 1.12 2.99 0.10 0.24 9.33 0.640
12.5 MA1 2.20 1.76 1.18 1.22 0.77 0.39 0.37 3.96 0.055
12.5 MA2 2.20 1.28 1.37 1.06 0.64 0.47 0.33 3.48 0.072
12.5 MA3 2.20 0.68 0.60 2.32 0.15 2.01 0.65 2.88 0.003
12.5 MA4 2.20 0.30 0.75 1.54 0.08 3.63 0.21 2.50 0.004
25.0 Air 2.20 6.80 4.06 0.91 10.17 0.03 0.66 9.00 0.001
25.0 MA1 2.20 2.32 2.59 0.84 2.21 0.14 0.45 4.52 0.002
25.0 MA2 2.20 1.40 2.27 0.93 1.17 0.26 0.49 3.60 0.020
25.0 MA3 2.20 0.82 1.86 1.00 0.56 0.54 0.46 3.02 0.001
25.0 MA4 2.20 0.09 1.92 0.74 0.06 4.74 0.21 2.29 0.002

 

61

TABLE 15. Gompertz equation parameters and calculated growth curve
values for S. typhimurium with various combinations of temperature and
atmosphere in pure culture.

 

Growth Generation Lag Max. Pop. Residual
Temp. MA's Gompertz Parameters Rate Time Time Density sums of

_(°C) A C B M (Log#/d) (dav) (dayHLog cfu/g) squares

5.0 ---- ---- ---- ---- ---- ---- ---- ---- ----

12.5 Air 2.20 5.45 0.44 2.74 0.88 0.34 0.46 7.45 0.273
12.5 MA 1 2.20 3.42 0.39 4.29 0.49 0.62 1.71 5.62 0.060
12.5 MA2 2.20 2.93 0.30 5.07 0.32 0.94 1.69 5.13 0.020
12.5 MA3 2.20 2.64 0.32 5.42 0.31 0.98 2.26 4.84 0.036
12.5 MA4 2.20 2.54 0.32 5.77 0.30 1.02 2.61 4.74 0.045
25.0 Air 2.20 7.04 2.66 0.87 6.87 0.044 0.50 9.24 0.018
25.0 MA 1 2.20 6.55 2.82 0.89 6.80 0.044 0.54 8.75 0.023
25.0 MA2 2.20 6.02 2.81 0.90 6.23 0.048 0.55 8.22 0.010
25.0 MA3 2.20 5.52 3.05 0.91 6.20 0.049 0.58 7.72 0. 128
25.0 MA4 2.20 4.93 3.31 0.92 6.00 0.050 0.62 7.13 0.009

TABLE 16. Gompertz equation parameters and calculated growth curve
values for S. typhimurium with various combinations of temperature and
atmosphere in mixed cultures.

 

Growth Generation Lag Max. Pop. Residual
Temp. MA's Gompertz Parameters Rate Time Time Density sums of

_(°C) A C B M (Log #ld) (day) (day) (Log cfu/g) sguares

5.0 ---- ---- ---- ---- ---- ---- ---- ---- ----

12.5 Air 2.20 5.15 0.47 2.71 0.89 0.30 0.58 7.15 0.106
12.5 MA 1 2.20 3.55 0.28 5.06 0.36 0.74 1.46 5.75 0.059
12.5 MA2 2.20 2.86 0.32 5.55 0.33 0.82 2.37 5.06 0.006
12.5 MA3 2.20 2.52 0.31 5.89 0.29 0.93 2.70 4.72 0.01 1
12.5 MA4 2.20 2.36 0.34 6.06 0.29 0.93 3 .08 4.56 0.028
25.0 Air 2.20 6.74 2.77 0.88 6.86 0.039 0.52 8.94 0.009
25.0 MA 1 2.20 6.23 2.97 0.89 6.81 0.040 0.55 8.43 0.089
25.0 MA2 2.20 5.76 2.89 0. 89 6. 14 0.044 0.54 7.96 0. 188
25.0 MA3 2.20 5.27 3.15 0.90 6.10 0.044 0.58 7.47 0.044
25.0 MA4 2.20 4.83 3.30 0.91 5.85 0.046 0.61 7.03 0.004

62

TABLE 17. Gompertz equation parameters and calculated growth curve
values for C. sporogenes with various combinations of temperature and
atmosphere in pure culture.

 

Growth Generation Lag Max. Pop. Residual

 

Temp. MA's Gompertz Parameters Rate Time Time Density sums of
_(_°C) A C B M (Log #ld) (dav) (dayHLog cfu/g) squares
5.0 --- --- --- --- ---- ---- ---- --—- ----

12.5 —-- --- --- --- ---- ---- ---- ---- ----

25.0 Air 2.20 6.80 0.45 4.33 1.14 0.265 2.13 9.00 0.301
25.0 MA1 2.20 6.88 0.45 4.32 1.15 0.262 2.11 9.08 0.292
25.0 MA2 2.20 6.99 0.56 3.96 1.45 0.207 2.19 9.19 0.007
25.0 MA3 2.20 7.04 0.68 3.86 1.75 0.172 2.37 9.24 0.014
25.0 MA4 2.20 6.99 0.69 2.21 1.78 0.169 0.76 9.19 0.040

 

TABLE 18. Gompertz equation parameters and calculated growth curve
values for C. sporogenes with various combinations of temperature and
atmosphere in mixed cultures.

 

Growth Generation Lag Max. Pop. Residual

 

Temp. MA's Gompertz Parameters Rate Time Time Density sums of
_(_°C) A C B M (Logj/d) (dgy) (dayHLog cfu/g) gguares
5.0 --- --- --- --- ---- ---- ---- --- ----

12.5 --- --- --- --- ---- ---- ---- ~--- «~-

25.0 Air 2.20 6.96 0.51 3.94 1.30 0.232 1.96 9.16 0.516
25.0 MA1 2.20 7.00 0.56 3.77 1.46 0.207 2.00 9.20 0.334
25.0 MA2 2.20 7.09 0.58 3.31 1.50 0.200 1.58 9.29 0.120
25.0 MA3 2.20 6.94 0.66 2.67 1.67 0.180 0.17 9.14 0.130
25.0 MA4 2.20 6.96 0.69 1.43 1.76 0.171 0.02 9.16 0.874

 

63

The Gompertz equation was used also to estimate the combined impact
of changes to lag times and generation times. This was accomplished by
using the values for B, M, and maximum population density (MPD) from
Tables 9 and 10, and substituting arbitrary set values for L(t). The equation
was rearranged and solved for t. For example, Table 19 shows the estimated
time for S. typhimurium to a 104-fold increase (e. g., from 1 CFU/g to 10,000
CFU/ g). This assumed that there was no change in B, M, and MPD as a
result of the decreased initial level (A=0), and that C=MPD - A.

TABLE 19. Estimated time (day) for S. typhimurium to increase from 1 to
10,000 CFU/ g.

 

 

Culture Temp. MA's
(0C) Air MA1 MA2 MA3 MA4
Pure 12.5 3.72 7.14 10.04 10.70 1 1.35
25.0 0.94 0.98 1.02 1.05 1.09
Mixed 12.5 3.86 8.99 10.02 11.69 12.19

25.0 0.96 0.99 1.0; 1.05 1.08

Modeling the parameters

The polynomial growth models for S. typhimurium in pure culture, for
example, took the following forms using the SYSTAT General Linear
Model procedure:
Growth rate (Log #/d) = 1.2188 — 0.344T - 0.0163A + 0.0214T2 + 0.0003A2

+ 0.0016TA
Ln(Generation time) = 9.0638 - 0.9803T — 0.0001A + 0.02T2 - 0.0003A2
- 0.0001TA
Ln(Lag time) = 8.6108 - 0.884T - 0.0225A + 0.0209r2+ 0.0007A2
- 0.0001TA

Ln(M)=12.2438 - 1.1986T - 0.0059A + 0.0283T2 + 0.00004A2 + 0.00003TA

The polynomial growth models for S. typhimurium in mixed cultures,
for example, took the following forms using the SYSTAT General Linear
Model procedure:

Growth rate (Log #/d) = 1.2837 - 0.35331" — 0.0177A + 0.02151‘2 + 0.0001A2
+ 0.002TA

Ln(Generation time) = 9.0478 - 0.9812T - 0.0028A + 0.0199r2+ 0.00002A2
- 0.000er

Ln(Lag time) = 8.46 - 0.8436T - 0.0143A + 0.01951“ 2+ 0.00009A2
+ 0.00005'1‘ A

Ln(M) 212.095 - 1.1668'1‘ - 0.0043A + 0.0272T2 + 0.00005A2 + 0.00002TA
where T=temperature (°C), A=atmosphere composition (i.e., % 02

levels), Ln=natural logarithm, and M=time to reach maximum growth rate.
In modeling the parameters it was necessary to impose the constraint

that the predicted values could not be negative. Fitting the polynomial

growth model directly to the parameter values could lead to negative

65

predicted values, so modeling was performed after taking the natural

logarithm of generation time, lag time and M which had the further

advantage of stabilizing the variance. The M value (time to reach maximum

growth rate) is an appropriate parameter for modeling because it takes into

account both lag time and growth rate.

Comparisons of selected examples of experimentally observed vs.

predicted values for the polynomial growth models of the growth of S.

typhimurium in both pure culture and mixed cultures are listed in Tables 20

and 21, respectively.

TABLE 20. Comparison of selected examples of experimentally observed

vs. predicted values for the polynomial model of the growth of S.

typhimurium in pure culture.

 

 

 

Experimental Values Polynomin Models

Growth Generation Lag Growth Generation Lag
Temp. MA's Rate Time Time Rate Time Time
10C) (Log #ld) (dag Ldav) (Log #ld) ((13);) (day)
12.5 MA1 0.49 0.62 1.71 0.54 0.75 1.95
12.5 MA2 0.32 0.94 1.69 0.33 0.90 1.93
12.5 MA3 0.3 1 0.98 2.26 0.29 0.92 2.07
12.5 MA4 0.30 1.02 2.61 0.26 0.94 2.29
25.0 MA 1 6.80 0.044 0.54 6.77 0.041 0.54
25.0 MA2 6.23 0.048 0.55 6.26 0.050 0.55
25.0 MA3 6.20 0.049 0.58 6. 19 0.052 0.59
25.0 MA4 6.00 0.050 0.62 5.99 0.053 0.65

 

TABLE 21. Comparison of selected examples of experimentally observed

vs. predicted values for the polynomial model of the growth of S.

typhimurium in mixed cultures.

 

Exoen'mentaiyflL

 

Polynomial Models

 

 

Growth Generation Lag Growthi Generation Lag
Temp. MA 's Rate Time Time Rate Time Time
10C) (Log #/d) (day) (day) (Log #ld) (day) mm
12.5 MA 1 0.36 0.74 1.46 0.47 0.81 1.97
12.5 MA2 0.33 0.82 2.37 0.31 0.86 2.30
12.5 MA3 0.29 0.93 2.70 0.27 0.88 2.45
12.5 MA4 0.29 0.93 3.08 0.23 0.91 2.62
25.0 MA 1 6.81 0.040 0.55 6.76 0.040 0.49
25.0 MA2 6. 14 0.044 0.54 6.22 0.044 0.57
25.0 MA3 6.10 0.044 0.58 6.05 0.046 0.60
25.0 MA4 5.85 0.046 0.61 5.89 0.048 0.64

 

This approach was motivated by objectivity, simplicity, and utility. The
Gompertz curve was used because it does not assume a constant growth rate;
it has been established that the specific growth rate of an organism is not
constant over the growth period, but increases to a maximum, then decrease
(Jason, 1983; Broughall and Brown, 1984; Gibson et al., 1987).

Providing that sufficient data are available, fitting a sigmoid curve of the
Gompertz type provides a more objective characterization of the growth
curve than calculating generation and lag times from a slope "judged by eye"

to be in the exponential phase of growth (Broughall et al., 1983).

67

Model predictions

Ratkowsky et al. (1982, 1983) proposed a linear relationship between
the square root of growth rate and temperature to describe the effect of
temperature on the growth of bacteria. The growth rates of salmonellae in
minced beef at temperatures between 10 and 35°C conformed to the simple
square root relationship by Mackey and Kerridge (1988). In Figure 26, the
square root model plot for S. typhimurium gives a straight line with good
agreement. Therefore, this model can be used to predict the growth rate for

any temperature condition within the limits of temperature tested.

68

FIGURE 26. The square root model plot for S. typhimurium
growth rate at range of 10 to 25°C.

 

 

 

 

 

3.0
y = - 1.0813 + 0.14519x R42 = 0.995

g 2.5"
«H
B
L 4
.8
'3' 2 0-
° .
L
U!
h
0
a 1.5-
O
O
L
2
u 1.0'
3
U'
‘0 4

0.5-

0.0 - r . . . , 1 1 1 I .

0 5 10 15 20 25 30

Temperature (C)

69

Experiment III:

1. Effect of higher cooking temperature (71°C) of the sample on the
outgrowth of C. sporogenes for 7 day-storage period at abusive
temperature of 25°C.

There was little difference in the growth of P. fragi and S. typhimurium
on the sample cooked to different temperatures between 55 and 71°C (Tables
22 and 23). However, C. sporogenes failed to grow above the inoculum
level in pure culture within 7 day-storage at air and MA1 and 4 day-storage
at MA2 and MA3 (Table 24). It was also failed to grow above the inoculum
level in mixed culture in the first 2 day-storage at air, MA1 and MA2 (Table
25). It may be due to the higher cooked temperature.

Many commercial pasteurized meat products are cooked to an internal
temperature of 71°C (Sofos, 1986). Processing meat to an internal
temperature of 71°C is usually adequate to destory the psychrotrophic
aerobic organisms as they do not form spores (APHA, 1984). In this study,
initial aerobic counts was 240 CFU/ g for cooked to an internal temperature
of 55°C and 30 CFU/ g for cooked to an internal temperature of 71°C (Table
26). The pH values of the samples were 6.25 for cooked to an intemal
temperature of 55°C and 6.24 for cooked to an internal temperature of 71°C.
The pH values were decreased during 7-day storage time with greater pH
change in MA4. It may be due to the growth of bacteria (i.e., aerobes) and
the solubility of carbon dioxide in the product. Huffman (1975) suggested

that pH changes may be a result of aerobic growth on the meat product. The
solubility of C02 in muscle tissue of pH 5.5 at 0°C was approximately 960

ml at STP/kg of tissue (Gill, 1988). The solubility of C02 decreased with

increasing temperature.

70

TABLE 22. Mean values for Pseudomonas fragi counts (loglo CFU/g)a of

cooked samples to an internal temperature of 71°C in mixed culture at
abusive temperature of 25°C.

 

 

 

 

 

 

 

Storage Storage Packaging treatmentb
temp. time
(6(1) (days) Air MAI 114.42 MA3 MA4
25 0 2.261046 2.261.046 2.261.046 2.261.046 2.261.04
2 8.461.226 4.841.116 3.5011566 2911.186 2341.16
4 8.3211866 4.131.236 3.511.216 3.001.153 2.151.08Ea
7 7. 861. 246 3. 211.113— 2.601.186 2. 001.266 1.8011

 

 

 

 

 

 

aAverage of triplicate samples 1 standard error of the mean.
bMeans within a common storage interval that are underlined are significantly

different (P < .05) from means obtained from air packaged samples.

 

Cd"iMeans 1n the same column bearing a common superscript do not differ
(P > .0.5)

TABLE 23. Mean values for Salmonella typhimurium counts (log10 CFU/g)a
of cooked samples to an internal temperature of 71°C in mixed culture at
abusive temperature of 25°C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Storage Storage Packaging treatmentb
temp. time
(6C) (days) Air MAI MAZ MA3 MA4
25 0 2.231.03c 2.23103c 2.23103c 2.231.03° 2.231036
2 7.981.166 7.80123d 7.271.14d 6.781.226 6.701306
4 8.841.24e 8.541.266 8.36-1.34e 7 631.276 7. 141.1
7 8. 691 316 8.161.14d6 7. 941. 286 7.131.173d 7.121.21

 

aAverage of triplicate samples 1 standard error of the mean.
bMeans within a common storage interval that are underlined are significantly

different (P < .05) from means obtained from air packaged samples.

OdeMeans 1n the same column bearing a common superscript do not differ
(P > .0.5)

 

71

TABLE 24. Mean values for Clostridium sporogenes counts (log10 CFU/g)a
of cooked samples to an internal temperature of 71°C in pure culture at

abusive temperature of 25°C.

 

 

 

 

 

 

 

Storage Storage Packaging treatmentb

temp. time

(0% (day s) Air MA] 11442 MA3 MA4

25 0 2.231.05c 2.23105c 2.231.050 2.23105C 2.231.05C
2 :1: :1: :1: :1: 3.23:1:30d
4 r1: :1: :1: :1: 5.70i.ISe
7 .1 4 2.301.286 3.60119d 8.08121f

 

 

 

 

 

 

 

 

*= failed to growth above the inoculum level.
aAverage of triplicate samples 1 standard error of the mean.
bMeans within a common storage interval that are underlined are significantly

different (P < .05) from means obtained from air packaged samples.

CdefMeans in the same column bearing a common superscript do not differ
(P > .05).

TABLE 25. Mean values for Closm'dium sporogenes counts (loglo CFU/g)a
of cooked samples to an internal temperature of 71°C in mixed culture at

abusive temperature of 25°C.

 

 

 

 

 

 

 

Storage Storage Packaging treatmentb
temp. time
(0C) (days) Air MAI MAZ MA3 MA4
25 0 2.23105c 2.231.05c 2.231.05c 2.231.05° 2.23105c
2 * * * 2.30-1.10c 4281:1661
4 3.261.24d 3.48112r 3.901156 4.02129(1 7.041.226
7 5261.326 5.30120e 5.481.23e 6.1 11.18e 8.90131I

 

 

 

 

 

 

*2 failed to growth above the inoculum level.
3Average of triplicate samples 1 standard error of the mean.
bMeans within a common storage interval that are underlined are significantly

different (P < .05) from means obtained from air packaged samples.

 

CdefMeans in the same column bearing a common superscript do not differ
(P > .05).

 

 

72

TABLE 26. Mean values for aerobic counts (log10 CPU/g)a of the samples in
which inoculated onlyC. sporogenes at temperatures of 8, 20, and 25°C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Storage Storage Packaging treatmentb
temp. time
(°C) (days) Air M41 MAZ MA3 M44
8 0 2.381.06C 2.381.06c 2,381.06c 2.381.06c 2.381.06c
2 2.26122C 2.201.11C 2.181.15c 2.151.18c 2.081.16d
4 2.181.186 1.951.236d 2.001.216 1.901.156d 1.781.086
7 2.201.246 1.65119d 1,601.18d 1.481.266 1.30110i
Storage Storage Packaging treatmentb
temp. time
(°C) (days) Air MAI MAZ MA3 MA4
20 0 2.381.06C 2.381.06c 2.381.06C 2.38-1.06C 2.381.06C
2 - 5.651.226I 4.601.11d 4301.15d 4.201.18d 3.761.16d
4 7.66118T 6.50123f 6.181.216 5.951.156 3.951086
7 7.001.24e 6.95119e 6.721.186 6.60126e 3.801.107i
Storage Storage Packaging treatmentb
temp. time
(°C) (days) Air MAI M42 MA3 MA4
25 0 1.48115C 1.481.15c 1.481.15c 1.481.15c 1.48115c
2 3.541.22d 2931.11d 2561.15d 2.431.18d 2.261.16d
4 4.901.186 4.481.236 4.40121e 4.001.15e 3.54108"
7 5. 97124f 5. 541 19I 5. 481 18I 5. 451261 3.341.106

 

 

 

 

 

 

2‘Average of tn plicate samples 1 standard error of the mean.
bMeans within a common storage interval that are underlined are significantly

different (P < .05) from means obtained from air packaged samples.

 

 

0‘13 f'Means 1n the same column bearing a common superscript do not differ
(P > ..05)

73

TABLE 27. The change in pH of samples in pure culture of C. sporogenes
at temperatures of 8, 20, and 25°C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Storage Storage Packaging treatment
temp. time
(°C) (days) Air MAI M42 MA3 MA4
8 0 6.25 6.25 6.25 6.25 6.25
2 6.23 6.03 6.00 6.1 1 5.92
4 6.25 5.96 5.94 5.95 5.88
7 6.25 5.96 5.93 5.93 5.88
Storage Storage Packaging treatment
temp. time
(°C) (days) Air MAI MAZ MA3 MA4
20 0 6.25 6.25 6.25 6.25 6.25
2 6.25 6.14 5.96 5.87 5.72
4 6.1 1 5.93 5.92 5.76 5.69
7 6.07 5.76 5.77 5.67 5.61
Storage Storage Packaging treatment
temp. time
(°C) (days) Air MAI MA2 MA3 MA4
25 0 6.24 6.24 6.24 6.24 6.24
2 6.19 6.14 6.12 6.07 6.05
4 6.19 6.14 6.1 1 6.08 6.03
7 6.21 6.07 6.05 6.01 5.90

 

 

 

 

 

 

 

 

 

 

74

In Experiment III:

2. Test predictive models at storage temperatures of 8 and 20°C for 7 day-

storage period .

The polynomial predictive models for P. fragi in mixed culture, for
example, took the following forms using the SYSTAT General Linear
Model procedure:

Ln(B)= -0.44105 -0.00008T+0.01165A+0.00155T2 -0.00059A2+0.00092TA
Ln(C)= -3.10854 -0.18492T+0.27743A-0.0055T2 - 0.00756A2+0.00084TA
Ln(M) =1.24777— 0.04933T -0.06691A+0.00064T2+0.00165A2 + 0.0001TA

The polynomial predictive models for S. typhimurium in mixed culture,
for example, took the following forms using the SYSTAT General Linear
Model procedure:

Ln(B)= 1.86413 -O.45011T-0.00634A+0.01692T2 +0.00018A2-0.00015T A
Ln(C)= -0.99001+0.20498T +0.00865A-0.00412T2 - 0.0002A2+0.00038TA
Ln(M) =12.094781.16679'1‘-0.00433A+0.02719r2+0.00005A2 +0.00002TA

The polynomial predictive models for S. typhimurium in pure culture,
for example, took the following forms using the SYSTAT General Linear
Model procedure:

Ln(B)= 1.72482 -0.42693T-0.0069A+0.01618T2 +0.0005A2-0.00037TA
Ln(C)= -0.9939+0.20988T+0.00689A-0.00424TQ - 0.00022A2+0.00046T A
Ln(M) =12.24335-1.19851T—0.00583A+0.02826T2+0.00004A2 +0.00003TA

75

Comparisons of selected examples of experimental counts (Loglo
CFU/ g) versus predicted counts (Loglo CFU/ g) from the polynomial models
for the growth of P. fragi and S. typhimurium in pure and mixed culture at
8 and 20°C are listed in Tables 28 to 31. The results between experimental
data and predicted growth counts from the predictive models had good
agreements. It was assumed that the predicted initial counts at storage time
0 were the same as the inoculum levels of each organism in experimental

counts.

76

TABLE 28. Comparison of selected examples of experimental counts (Loglo
CFU/ g) versus predicted counts (Loglo CFU/ g) from the polynomial models for the
growth of P. fragi in mixed culture at 8°C.

 

 

 

 

Experimental counts (LogloCFU/ g) Predicted counts (Loglo CFU/ g)
Time MA 1 MA2 MA3 MA4 MA 1 MA2 MA3 MA4
(daJS)
0 2.26 2.26 2.26 2.26 2.26 2.26 2.26 2.26
2 3.49 3.17 2.42 2.30 3.31 2.95 2.46 2.28
4 3.92 3 .31 2.59 2.56 3.89 3.41 2.69 2.32
7 4.08 3.50 2.74 2.51 4.03 3.53 2.76 2.36

 

 

 

 

TABLE 29. Comparison of selected examples of experimental counts (Loglo
CFU/ g) versus predicted counts (Loglo CFU/ g) from the polynomial models for the
growth of P. fragi in mixed culture at 20°C.

 

 

 

 

Experimental counts (LogloCFU/ g) Predicted counts (LogIOCFU/g)
Time MA1 MA2 MA3 MA4 MA1 MA2 MA3 MA4
(days)
0 2.26 2.26 2.26 2.26 2.26 2.26 2.26 2.26
2 4.52 3.40 2.60 2.49 4.64 3.67 2.76 2.36
4 4.93 3.97 2.81 2.36 5.02 4.02 2.96 2.45
7 5.05 4.07 2.72 2.00 5.04 4.04 2.98 2.46

 

 

 

 

77

TABLE 30. Comparison of selected examples of experimental counts (Loglo
CFU/ g) versus predicted counts (Loglo CFU/ g) from the polynomial models for the

growth of S. typhimurium in pure culture at 20°C.

 

 

 

 

Experimental counts (Log IOCFU/ g) Predicted counts (Loglo CFU/ g)
Time MA 1 MA2 MA3 MA4 MA1 MA2 MA3 MA4
(dayS)
0 2.23 2.23 2.23 2.23 2.23 2.23 2.23 2.23
2 6.10 5.74 5.40 5.20 6.24 5.67 5.49 5.30
4 7.40 6.92 6.51 6.43 7.56 6.87 6.61 6.35
7 7.94 7.51 7.12 6.80 8.04 7.34 7 .03 6.70

 

 

 

 

TABLE 31. Comparison of selected examples of experimental counts (Loglo
CFU/ g) versus predicted counts (Loglo CFU/ g) from the polynomial models for the

growth of S. typhimurium in mixed culture at 20°C.

 

 

 

 

Experimental counts (LogloCFU/ g) Predicted counts (Loglo CFU/ g)
Time MA1 MA2 MA3 MA4 MA1 MA2 MA3 MA4
@208)
0 2.23 2.23 2.23 2.23 2.23 2.23 f 2.23 2.23
2 5.78 5.66 5.34 5.14 5.92 5.47 5.29 5.1 1
4 7.32 6.51 6.43 6.04 7.25 6.65 6.39 6.13
7 7.81 7.22 6.98 6.28 7.94 7.12 6.81 6.49

 

 

 

 

CONCLUSION

The success of MAP depends on many factors including good initial product
quality, good hygiene from harvest/slaughter on, correct packaging material
selection, the appropriate gas mixture for the product, reliable packaging
equipment, and maintenance of controlled temperatures. The results of this
study indicate that the key to preventing growth of pathogens such as S.
typhimurium on the minimally processed/MAP product is proper
refrigeration. Although MA-storage does effectively reduce the growth rate
of S. typhimurium , it cannot, in the absence of proper refrigeration, be relied
upon to prevent salmonellosis. Since MAP selectively inhibits spoilage
bacteria such as P. fragi over pathogens like S. typhimurium or C.
botulinum, obvious signs of spoilage may not be present even though
pathogenic organisms can be in dangerously high concentrations at abusive
temperatures. Therefore, additional control measures are needed to insure
the microbiological safety of minimally processed products. In addition to
MAP, other barriers include the use of films of higher gas permeability, the
use of time-temperature indicators and co-inoculation with lactic acid
bacteria.

A packaging film with selective permeability to permit ingress of 02
into the package headspace may prevent the growth of C. botulinum while
allowing C02 produced by respiration of food tissue and spoilage bacteria to
permeate out of the package. Time-temperature indicators may also be a
useful inclusion as they would indicate to the consumer that the product has

been subjected to temperature abuse and may possibly be of public health

78

79

concern. Competitive inhibition of C. botulinum through co-inoculation
with lactic acid bacteria can be used in conjunction with MAP and
refrigeration to prevent growth and toxin production by C. botulinum .

By combining inhibitory factors at subinhitory levels processors may
achieve a safe product without sbustantially changing the character of the
product. The use of predictive models can be extremely valuable in
assessing the safety of extended shelf-life refrigerated MAP foods.

Since current growth rate models have been based on constant
temperature studies, they seem to be adequate to predict growth rates at any
other constant temperature within the temperature range studied. There is a
need for studies under cycling temperatures with interaction of other factors

in the future.

APPENDIX I

SC-10A Thermocouple Psychrometer

DECAGON DEVICES INC.
FD. BOX 835
NW 115 STATE ST.#211
PULLMAN, WA 99163

PROGRAMS TO COMPUTE WATER ACTIVITY Using the BASIC
language:

5 INPUT"PSYCHROMET ER CONSTANT”;G

10 INPUT"MICROVOLTS";V

20 INPUT”TEMPERATURE—C";TA

30 TW=TA-V/60

40 T=TA:GOSUB 500:EA=E

50 T=TW2GOSUB 500:EW=E

60 INPUT"ENTER A FOR AW, P FOR POTENTIAL, OR C FOR CALIBRATE";A$
70 IF LEFT $(A$,l)="A" THEN GOT O 200

80 IF LEFT$(A$,1)=”P" THEN GOTO 300

90 IF LEFT $(A$,1)=”C” THEN GOTO 400

100 GOTO 60

200 AW=(EW-G*(TA-TW))/EA

210 PRINT"AW=”,AW

220 GOTO 10

290 REM***IN BASIC LOG IS THE DESIGNATION FOR NATURAL LOG (LN)
300 WP=8.3141*(TA+273)*LOG((EW-G*(TA-TW))/EA)/.018

310 PRINT ”WP (J/KG)=",INT(WP)

320 GOTO 10

400 INPUT"WATER ACTIVITY OF STANDARD SOLUTION” ;AS

410 G=(EW-AS*EA)/(TA-TW)

420 PRINT”PSYCHROMETER CONSTANT="G

430 GOTO 10

500 REM *** SUBROUTINE TO CALCULATE SATURATION VAPOR PRESSURE
510 E=EXP(52.5763-6790.4985#/(T+273. 16)-5.02808*LOG(T+273. 16))

520 RETURN

80

APPENDIX II

Standard Colony Count Methods

Based on Busta et al. (1984), each sample in the study contained 3 plates
form 10'2 to 10'4 dilution. Plates from the same sample were individually
counted and calculated as units of CFU per gram sample following these
steps. Reported number of CFU per gram sample was average from the
CFU value calculated from each plate of the same sample.

1. If there was no colony growth on all plates at the same dilution - the CFU
number was reported as less than 100 CFU per gram.

2. If colony growth in each plate was less than 25 colonies in all dilution -
the actual number of colonies appearing on each plate was counted and
reported as estimated CFU per gram sample.

3. If colony growth in each plate range between 25-250 colonies - the
number of colonies on plate was counted and reported as CFU per gram
sample.

4. If the growth in all plates was more than 250 colonies - then it was
reported as more than 2.5x10° CFU per gram. An estimated number of
colonies in plate was obtained from the average count of 4 (four) 1x1 cm
squares selected randomly in a 10'4 dilution, then multiplying this number

by the plate surface area (in cm2)

81

APPENDIX III

SYSTAT: Statistics. Version 5.2 Edition

SYSTAT, Inc., 1992. 724pp.
1800 Sherman Avenue
Evanston, IL 60201-3793

CONTENTS

Introduction to statistics
Cluster

Correlation

Factor

Multidimensional scaling
Multivariate General Linear Hypothesis
Nonlinear models
Nonparametric

Series

10. Stats

1 1. Tables

PWHP‘V‘PP’P!‘

82

APPENDIX I V

Preliminarv growth test at abusive temperature of 24 and 35°C to detennine

inoculum levels

83

34

Figure 1. Growth of P. fragi in liquid culture (TSB) stored under air at
abusive temperatgre of 24 and 35°C.
1

 

 

 

 

 

 

:
x.
3
LI.
0
D1
O
..l
0 10 20 30 4O 50
TIME (hour)

Figure 2. Growth of S. typhimurium in liquid culture (TSB) stored under air

at abusive temperature of 24 and 35°C.
10

 

 

____j§;_____:3

-iF—- 24C
--O-- 35C

Log CFUIII
05
l

 

 

 

 

2 T ' I ‘ T ' I
0 10 20 30 40 50
TIME (hour)

Figure 3. Growth of C. sporogenes in liquid culture (RCM) stored under
anaerobic condition at abusive temperature of 24 and 35°C.

1 I W

 

-—-G-- 24C
--+-- 35C

Log CFUIII

 

 

 

 

. , . , .
0 10 20 3o 40 50
TIME (hour)

85

Figure 4. Growth of P. fiagi on ground beef/pea homogenate in pure
culture stored under air at abusive temperature of 24 and 35°C.

10

 

     
   

24 C w/Shaking
—O— 24 C w/o Shaking
—-II— 35 C w/Shaking

-—°-—- 35C w/o Shaking

Log CFU/g
O"
l

 

 

 

4 .-
2 - . , . , . 1 .
0 10 20 30 40 50
TIME (hour)

Figure 5. Growth of P. fragi on ground beef/pea homogenate in mixed
culture stored under air at abusive temperature of 24 and 35°C.

 

 

 

    

 

10
J
8 ..
51
x
E
o 6 _ 24 C w/ Shaking
3' 24 C w/o Shaking
-' 35 C w/ Shaking
4 _ 35 C w/o Shaking
. - r .

 

 

r ' r
20 30 40 50
TIME (hour)

86

Figure 6. Growth of S. typhimurium on ground beef/pea homogenate in pure
culture stored under air at abusive temperature of 24 and 35°C.

 

 

 

 

   

 

 

10
. :9
3 -
O
‘5 -4
3
IL
(.1 5 -
g‘ 24 C w/ Shaking
" —0—— 24 C w/o Shaking
4 '- --l—- 35 C w/ Shaking
—+— 35 C w/o Shaking
2 ~ , I 1
30 40
TIME (hour)

 

50

Figure 7. Growth of S. typhimurium on ground beef/pea homogenate in
mixed culture stored under air at abusive temperature of 24 and 35°C.

 

10

Log CFUIg

 

 

   

30

TIME (hour)

24 C w/ Shaking
24 C w/o Shaking
35 C w/ Shaking

35 C w/o Shaking

I

40

 

 

SO

87

Figure 8. Growth of C. sporogenes on ground beef/pea homogenate in pure
culture stored under air at abusive temperature of 24 and 35°C.

 

 

 

 

 

10
3 L

a —EI-- 24 C w/o Shaking
E —o—— 35c w/o Shaking
u 6 j
a d
o a:
d 1

4 -t

0 40 SO

10 20 30
TIME (hour)

Figure 9. Growth of C. sporogenes on ground beef/pea homogenate in
mixed culture stored under air at abusive temperature of 24 and 35°C.

 

10
8 -1
a
3 —G— 24 C w/o Shaking
b 6 1 —.— 35c w/o Shaking
a
O
.J

 

 

 

 

TIME (hour)

APPENDIX V

Gas Composition Change for Experiment I

89

Figure 10 Changes in headspace gas composition: 02 and C02 of P. fragi

in pure culture at 5, 12.5 and 25°C.

At 5°C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

40
TIME (DAY)
At 12.5°C
100 ’
80
a 60
0’
fi 40
20
O
0 10 20 30 40
TIME (DAY)
At 25°C
100 . - —— ¢ ; c
30 l-..'—-',-_-.-—_.__—'==—=—"—°::———_'._‘—=_'
8 60
U
3‘ 40
201:, N
o V
O 10 20 30 40

TIME (DAY)

AIR (02)
AIR (002)
MA1 (02)
MA1 (C02)
MA2 (02)
MA2 (002)
MA3 (02)
MA3 (C02)
MA4 (OZ)
MA4 (002)

AIR (02)
AIR (002)
MA1 (02)
MAI (002)
MA2 (02)
MA2 (002)
MA3 (02)
MA3 (002)
MA4 (02)
MA4 (002)

AIR (02)
AIR (002)
MA1 (02)
MAI (002)
MA2 (02)
MA2 (002)
MA3 (OZ)
MA3 (002)
MA4 (02)
MA4 (002)

90

Figure 11.
typhimurium in pure culture at 12.5 and 25°C.

At 12.5°C

Changes in headspace gas composition:

 

100

 

 

 

 

0 10 20 30
TIME (DAY)

40

 

 

 

 

 

 

TIME (DAY)

 

 

02 and C02 Of S.

AIR (02)
AIR (002)
MA1 (02)
MA1 (002)
MA2(02)
MA2 (002)
MA3 (02)
MA3 (002)
MA4 (02)
MA4 (002)

AIR (02)
AIR (002)
MA1 (02)
MA1 (002)
MA2 (02)
MA2 (002)
MA3 (02)
MA3 (002)
MA4 (02)
MA4 (002)

91

Figure 12. Changes in headspace gas composition: 02 and C02 of C.

sporogenes in pure culture at 12.5 and 25°C.

At 12.5°C

 

 

 

 

 

 

 

 

  

 

TIME (DAY)
At 25°C
100

. .... a”...
80 " _._
1 ..... .....

60 "
a ..... .....
:9 _D_

a -
4O ..... kn“
. —*—
20 ° ..... I”...
O. +

0 ' . r! 1 4 . 4

 

 

0 1 0 20 30 40
TIME (DAY)

AIR (02)
AIR (002)
MA1 (02)
MA1 (002)
MA2 (02)
MA2 (002)
MA3 (02)
MA3 (002)
MA4 (02)
MA4 (002)

AIR (02)

AIR (COZ)
MA1 (02)
MAI (C02)
MA2 (OZ)
MA2 (C02)
MA3 (OZ)
MA3 (COZ)
MA4 (OZ)
MA4 (C02)

92

Figure 13. Changes in headspace gas composition: 02 and C02 of P. fiagi,
S. typhimurium andC. sporogenes in mixed culture at 12.5 and 25°C.

At 12.5°C

 

100

AIR (02)
AIR (002)
MA1 (02)
MA1 (002)
MA2 (02)
MA2 (C02)
MA3 (02)
MA3 (C02)
MA4 (02)
MA4 (C02)

 

 

 

 

 

 

 

 

----- Ian-- AIR (02)
—9— AIR (C02)
----- l----- MA1 (02)
—°— MA1 (C02)
----- I----- MA2 (02)
—-n— MA2 (C02)
----- Am" MA3 (02)
—a— MA3 (C02)
----- I----- MA4 (02)
—-+-— MA4 (C02)

 

 

 

 

 

40

TIME (DAY)

APPENDIX VI

Gas Composition Change for Experiment 11

Figure 14. Changes in headspace gas composition: 02 and C02 of P. fragi

with 102 CFU/ g at abusive temperature of 12.5°C.

 

100

80”

$6113

 

 

 

TIME (DAY)

 

 

AIR (02)-pure
AIR (C02)-pure
MA4 (02)-pure
MA4 (C02)-pure
AIR(02)-mixed
AlR(COZ)-mixed
MA4(OZ)-mixed
MA4(C02)-mixed

Figure 15. Changes in headspace gas composition: 02 and C02 of P. fragi

with 102 CFU/ g at abusive temperature of 25°C.

 

100

 

TIME (DAY)

93

 

 

AIR (02)-pure

AIR (C02)-pure
MA4 (OZ)-pure
MA4 (C02)-pm
AlR(02)-mixed
AlR(C02)-mixet
MA4(OZ)-mixer
MA4(COZ)-mix:

94

Figure 16. Changes in headspace gas composition: 02 and C02 of P. fragi

with 104CFU/g at abusive temperature of 12.5°C.

 

  

 

  

 

 

 

 

100

60 7
3
G 1
a: 40 -

20 7"...-

-'"i'M-’::c:: --
0 v I I .1 11""?“W
o 10 20 30 4O

-- AIR (02)-pure
—e—— AIR (C02)-pure
-- MA4 (OZ)-pure
—o-— MA4 (C02)-pure
~~ AlR(OZ)-mixed
——-u— AlR(COZ)-mixed
-- MA4(OZ)-mixed
—¢1— MA4(C02)-mixed

Figure 17. Changes in headspace gas composition: 02 and C02 of P. fragi

with 104 CFU/ g at abusive temperature of 25°C.

 

100

BOP/”f

 

 

TIME (DAY)

 

 

AIR (02)-pure
AIR (C02)-pure
MA4 (02)-pure
MA4 (C02)-pure
AlR(OZ)-mixed
AlR(COZ)-mixed
MA4(02)-mixed
MA4(C02)-mixed

Figure 18. Changes in headspace gas composition: 02 and C02 of S.

typhimurium with 102CFU/g at abusive temperature of 12.5°C.

 

 

 

 

TIME (DAY)

AIR (OZ)-pure
AIR (C02)-pure
MA4 (OZ)-pure
MA4 (C02)-pure
AlR(02)-mixed
AlR(C02)-mixed
MA4(02)-mixed
MA4(COZ)-mixed

Figure 19. Changes in headspace gas composition: 02 and C02 of S.
typhimurium with 102 CFU/ g at abusive temperature of 25°C.

100

 

801M

 

 

 

 

TIME (DAY)

AIR (02)-pure
AIR (C02)-pure
MA4 (OZ)-pure
MA4 (C02)-pure
AlR(OZ)-mixed
AlR(C02)-mixed
MA4(OZ)-mixed
MA4(COZ)-mixed

96

Figure 20. Changes in headspace gas composition: 02 and C02 of S.
typhimurium with 104CFU/g at abusive temperature of 12.5°C.

 

..... 13.-m AIR (OZ)-pure
——e— AIR (C02)-pure
..... 5..-. MA4 (02)-pure
_¢_ MA4 (C02)-pure
..... ..... AlR(02)-mixed
—o— AlR(C02)-mixed
..... A...“ MA4(02)-mixed
—-—¢—— MA4(COZ)-mixed

 

 

 

 

 

TIME (DAY)

Figure 21. Changes in headspace gas composition: 02 and C02 of S.
typhimurium with 104 CFU/ g at abusive temperature of 25°C.

 

 

 

100 ’
30 ----- lam-- AIR (02)-pure
__..._ AIR (C02)-pure
..... 1.... MA4 (ozwure
8 50 q —o—— MA4 (C02)-pure
G . ----- u-u- AlR(02)-mixed
* 40 . —a— AlR(COZ)-mixed
MA4(OZ)-mixed
MA4(C02)-mixed

 

 

 

TIME (DAY)

Figure 22. Changes in headspace gas composition: 02 and C02 of C.
sporogenes with 104 CFU/g at abusive temperature of 12.5°C.

100

XGu

 

 

 

 

 

 

 

 

TIME (DAY)

AIR (02)-pure
AIR (C02)-pure
MA1 (02)-pure
MA1 (C02)-pure
AlR(02)-mixed
AlR(COZ)-mixed
MA1 (OZ)-mixed
MA1(COZ)-mixed

Figure 23. Changes in headspace gas composition: 02 and C02 of C.
sporogenes with 104 CFU/ g at abusive temperature of 25°C.

100 ’

 

80

$6113

 

 

 

 

0h

 

 

TIME (DAY)

 

AIR (OZ)-pure
AIR (C02)-pure
MA1 (02)-pure
MA1 (C02)-pure
AlR(02)—mixed
AlR(C02)-mixed
MA1 (02)-mixed
MA1 (C02)-mixed

APPENDIX VII

Gas Composition Change for Emiment III

Figure 24. Changes in headspace gas composition: 02 and C02 of C.
sporogenes in pure culture at 25°C.

 

AIR (02)
AIR (002)
MA1 (02)
MA1 (002)
MA2 (02)
MA2 (002)
MA3 (02)
MA3 (C02)
MA4 (02)
MA4 (002)

 

 

 

 

TIME (DAY)

Figure 25. Changes in headspace gas composition: 02 and C02 of C.
sporogenes in mixed culture at 25°C.

 

1 00
AIR (02)

AIR (C02)
MA1 (02)
MAI (C02)
MA2 (02)
MA2 (C02)
MA3 (OZ)
MA3 (C02)
MA4 (OZ)
MA4 (C02)

 

 

 

 

 

TIME (DAY)

98

99

Figure 26. Changes in headspace gas composition:

typhimurium in pure culture at 20°C.
100

80 4T”
60

 

 

 

 

 

 

 

TIME (DAY)

Figure 27. Changes in headspace gas composition:
sporoggges in pure culture at 20°C.

sci—M

60-

 

x60:

401

 

 

 

 

 

TIME (DAY)

02 and 002 of s.

  

AIR (02)
AIR (002)
MA1 (02)
MAI (002)
MA2 (OZ)
MA2 (C02)
MA3 (02)
MA3 (002)
MA4 (02)
MA4 (C02)

02 and 002 of C.

- .... 9....-

AIR (02)
AIR (002)
MA1 (02)
MAI (002)
MA2 (02)
MA2 (C02)
MA3 (02)
MA3 (002)
MA4 (02)
MA4 (002)

Figure 28. Changes in headspace gas composition: 02 and C02 of P. fragi,

S. typhimurium and C. sporogenes in mixed culture at 20°C.
, ..... 9....

 

100

 

 

80
60

$6113

40

 

20

 

 

TIME (DAY)

AIR (02)
AIR (C02)
MA1 (OZ)
MA1 (C02)
MA2 (OZ)
MA2 (C02)
MA3 (OZ)
MA3 (C02)
MA4 (OZ)
MA4 (C02)

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