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This is to certify that the
thesis entitled
EFFECT OF GAS CONCENTRATION ON THE GROWTH OF AEROBIC AND
ANAEROBIC MICROORGANISMS IN A FOOD-PACKAGE MODEL SYSTEM

presented by

Luis Martin Rayas

has been accepted towards fulfillment
of the requirements for

M.S. . Food Science
degree in

 

 

flm fl /7%‘

Major professor

Date July 9, 1992

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

LIBRARY
Michigan State .
University

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PLACE IN RETURN BOX to remove this checkout from your record.
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I IT—TI I

MSU le An Affirmative ActiorVEquel Opportunlty Inetttution
ammo-pt

EFFECT OF GAS CONCENTRATION ON THE GROWTH OF AEROBIC AND
ANAEROBIC MICROORGANISMS IN A FOOD-PACKAGE MODEL SYSTEM.

BY

Luis Martin Rayas

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science

1 992

ABSTRACT
EFFECT OF GAS CONCENTRATION ON THE GROWTH OF AEROBIC AND
ANAEROBIC MICROORGANISMS IN A FOOD-PACKAGE MODEL SYSTEM.
By .
Luis Martin Rayas

 

Growth of both 535mg; gyms-(aerobic) and Wm; M (anaerobic)
was determined individually and simultaneously using the same model system. Oxygen
concentration in the head space was also monitored over the storage time. The parameters studied
were: a) Initial oxygen content in the head space (1%, 5%, 21% oxygen and 21% oxygen with
oxygen absorber <FreshPax'“>); b) Bacterial growth in the model system (aerobic vs. anaerobic
and mixture of both); c) Water activity <Aw> (0.96 and >039); and (1) storage temperature (10 and
25°C).

Storage time, initial oxygen content in the head space, and abusive temperature
(25°C) had a significant effect on bacterial growth (P<0.01). An oxygen absorber was effective in
reducing oxygen content and preventing growth of aerobic microorganisms (gagLus M5) at
25°C. Anaerobic growth was detected at 25°C with oxygen concentrations of 1% and <0.5% in the
head space. Anaerobic growth was prevented at 10°C in jars at all oxygen head space
concentrations including 1% oxygen and <0.5% oxygen. Aerobic growth was observed at 10°C in
jars with initial oxygen concentrations of 1%, 5%, 21% with oxygen absorber, and 21% in the mixed
culture. No growth of aerobic organisms was observed at 10°C for the samples containing pure
culture and 21% initial oxygen in the head space. Oxygen level was reduced from 21% to about
0.1% in about 5 hours at 25°C and in approximately 12 hours at 10°C with an oxygen absorber,

regardless of the water activity in the media.

To:

my wife, Ana Teresa; for her infinite love, help, and understanding;
my son, Luis Alberto; and

my parents, Luis and Irma; for their unconditional support and love

ACKNOWLEDGMENTS

The author wish to thank Food Microbiology Laboratory, Department
of Food Science and Human Nutrition, Food and Pharmaceutical Laboratory,
School of Packaging, and Post harvest Laboratory, Department of Horticulture,
Michigan State University for the supply of raw material and the assistant for using
the laboratory and equipment during this study.

The author also want to thank Dr. Bruce R. Harte, Dr. John E. Linz,

and Roscoe Warner for their guidance and help through this research.

TABLE OF CONTENTS

List of Tables

List of Figures
Introduction

Literature Review

Effect of package barrier on the shelf life of a food-package
system _

Effect of head space gas content in a food-package system
Effect of thermal processing and the shelf life of food-
package products

Effect of food composition and water activity in the shelf

life of a food-package system

Effect of storage temperature and time on the shelf life of

a food-package system

Oxygen absorbers and oxidation in food systems

Materials and Methods

Package system preparation

Model preparation

g sporogenes inoculum preparation
_B_. _sybtflis inoculum preparation
Experimental design

Oxygen absorbers used for study

Incubation (storage) conditions

vi

vii

12

15

17

23
27
27
29
29
31
32

35

Sampling
Statistical analysis of data
Effect of temperature and water activity on activity of oxygen

absorbers

Results and Discussions

Growth of §_a_ci_l1g§ m in samples stored at abusive
temperature of 25°C and water activity >0.99
Qiostridium sporogenes evaluation of samples stored at
25°C and water activity >0.99

Growth of Bacillus subtilis in samples stored at abusive

 

 

temperature of 25°C and water activity 0.96
Change in head space oxygen content in media with

Bacillus subtilis evaluation of samples stored at 10°C and

 

 

water activity >0.99
Growth of glostridum sporogenes in samples stored at 10°C
and water activity >0.99 I

Temperature effect on oxygen absorber

Conclusions
Appendix I: Standard Colony Count Methods
Appendix II: Spectrophotometric method

Bibliography

35
38

4O

4O

45

5O

61

7O

78

89

98

LIST OF TABLES

Table 1: Some physical characteristics of C_. sporogenes and
Q, botulinum

Table 2: Some differential characters of Bacillus and glostridium

 

genera

Table 3: Typical cultural response of B, subtilis and C_. sporogenes

 

at 35°C in Thioglycollate media
Table 4: Analysis of Variance for ; subtilis at 25°C and Aw>0.99

 

Table 5: Analysis of Variance for _C_. sporogenes at 25°C and
Aw>0.99

Table 6: Analysis of Variance for B. subtilis at 25°C and Aw=0.96

Table 7: Analysis of Variance for % oxygen change in the head
space (Aw>0.99) ‘

Table 8: Analysis of Variance for B_. subtilis at 10°C and Aw>0.99

 

Table 9: Analysis of Variance for Q_. sporogenes at 10°C and
Aw>0.99

Table 10: Two-way Analysis of Variance for % oxygen in the head
space over time and over presence of FreshPaxT"

Table 11: Two-way Analysis of Variance for % oxygen in the head
space over time and over water activity

Table 12: Two-way Analysis of Variance for % oxygen in the head

space over time and over temperature

vi

13

21

72

79

80

81

LIST OF FIGURES

Figure 1: Comparison of normal vs. MAP shelf life
Figure 2: Food model system design
Figure 3: Experimental design used for model system setup

Figure 4: Bacillus subtilis: Pure culture; T=25°C; Aw>0.99

 

Figure 5: Bacillus subtilis: Mixed culture; T=25°C; Aw>0.99

 

 

Figure 6: Clostridium sporogenes: Pure culture; T=25°C;
Aw>0.99

Figure 7: Clostridium sporogenes: Mixed culture; T=25°C;
Aw>0.99

Figure 8: Bacillus subtilis: Pure culture; T=25°C; Aw=0.96

 

Figure 9: Bacillus subtilis: Mixed culture; T=25°C; Aw=0.96

 

 

Figure 10: % oxygen change (1% initial oxygen in the head I

space)

Figure 11: % oxygen change (5% initial oxygen in the head
space)

Figure 12: % oxygen change (21 % initial oxygen in the head
space)

Figure 13: % oxygen change (21% initial oxygen in the head
space + FreshPaxT”)

Figure 14: % oxygen change: mixed culture; 1% and 5%

initial oxygen in the head space)

vii

11

28

33

43

47

48

52

53

57

59

60

62

Figure 15: % oxygen change: mixed culture; 21 % initial
oxygen and 21 % oxygen + FreshPaxT"

Figure 16: Baily; supine; (CFUlml) and oxygen change (%)
at 10°C; 21% initial oxygen + FreshPaxTM

Figure 17: Clostridium sporogenes (CF Ulml) and oxygen
change at 10°C; 21% initial oxygen + FreshPaxT"

Figure 18: Bacillus subihs (CFUlml) and oxygen change (%)

 

at 10°C; 21% initial oxygen

Figure 19: Clostridium sporogenes (CF Ulml) and oxygen
change at 10°C; 21 % initial oxygen

Figure 20: Bacillus subtilis (CFU/ml) and oxygen change (%)

 

 

at 10°C; 5% initial oxygen
Figure 21: Clostridium sporogenes (CF U/ml) and oxygen
change at 10°C; 5% initial oxygen

 

 

Figure 22: Bacillus subtilis (CF Ulml) and oxygen change (%)
at 10°C; 1% initial oxygen ‘

Figure 23: Clostridium sporogenes (CF Ulml) and oxygen
change at 10°C; 1% initial oxygen

Figure 24: % oxygen change in the head space for control
samples at 10°C

Figure 25: Temperature effect on oxygen absorbers. Aw>0.99

Figure 26: Temperature effect on oxygen absorbers. Aw=0.88

Figure 27: Absorbance vs. time for ; subtilis and g; sporogenes

in thioglycollate medium at 37°C

Figure 28: Log CFUlml vs time for .B_. subtilis and _C_. sporogenes

in thioglycollate medium at 37°C

viii

63

67

69

71

73

74

75

76

77

83

91

92

Figure 29: Log CF Ulml vs. absorbance for ; subtilis at 37°C
Figure 30: Log CF Ulml vs. absorbance for Q_. sporogenes at 37°C
Figure 31: Standard curve for Bacillus subtilis

Figure 32: Standard curve for Clostridium botulinum

93

95

INTRODUCTION

To control oxygen in a packaged product, vacuum, steam
condensation, oxygen absorbers and gas flushing can be used. Oxygen leads to
the growth of aerobic spoilage organisms and also is necessary for various
oxidative reactions. Use of oxygen absorbers in concert with barrier packaging can
be used to obtain oxygen levels of 0.01 % in a sealed package. However, growth
of anaerobes must also be considered relative to food safety.

The market demand for prepared fOods in optimal conditions has
lead packaging and food scientists to work to increase the shelf life of perishable
food products. The role of oxygen in degradation of perishable foods is well
documented (Labuza, 1987; Brody, 1987). Although the kind of spoilage to be
expected can often be predicted from the composition of the food (Ayres et al.,
1980), microorganisms from many sources such as the product itself, environment
(soil and water), man, and processing equipment may grow and cause quality
changes, and create an unsafe product (Manomaiwiboon, 1990). Oxygen leads to
the growth of aerobic spoilage organisms such as .BfliLIU—S bacteria and Penicillium
and Asgergillus molds. By controlling the gases in the package the growth of
surface molds is slowed when the oxygen content is below 1% (Brody, 1987). The
use of carbon dioxide at high concentrations (>25 %) provides good control over
the growth of aerobic organisms but may impart a flavor to the product (Brody,
1987) or when used with ground-roasted coffee, increases the chance that the
package will burst (Labuza, 1987). Although carbon dioxide is bacterostatic and

mycostatic, chalk-like yeast growth can occur on bread in storage (Brody, 1987).

Oxygen is also necessary for various oxidative reactions such as lipid oxidation
and color change (Fennema, 1985), which can occur in potato chips, dried fish,
beef jerky, semi-moist cookies, and other products. These reactions are important
in food degradation and have economic importance. Thus far, the most common
ways of controlling oxidative deterioration of lipids in foods are through either
removal of the oxygen and gas-tight packaging (i.e., vacuum packing or replacing
the air in the package with an inert gas, such as nitrogen); or the incorporation of a
suitable antioxidant into the food (Stewart et al., 1982).

The current utilization of oxygen absorbers in the food
industry is limited. "With respect to US. regulations, the Food and Drug Adminis-
tration requires that the film used for oxygen absorber sachets be made of
materials approved for use under 21CFR 175-178. In addition, any printing inks
have to be from approved substances or shown not to be able to migrate into the
food" (Labuza, 1987). Oxygen can be reduced to approximately 0.5 % by vacuum
packaging or gas flushing. However, oxygen absorbers are capable of reducing
residual oxygen in the package head-space to less than 0.01 % (Labuza, 1987). At
0.5% there is short term control of growth of aerobic organisms, but less control of
oxidative chemical reactions. And in bread, because of the increase in packaging
material barrier requirements, the cost of packaging for controlled atmosphere is
approximately triple that of the chemicals and double that of pasteurization in
Germany (Brody, 1987). There are data available to show that with use of oxygen
scavenger sachets, a significant shelf-life improvement is attained (Labuza, 1987).
The technology seems to work well for bakery products. At 0.01 % oxygen there is
complete control of aerobic growth and effective control of oxidative reactions.
This enhanced control is, in many cases, economically significant. However,

whether such very low residual oxygen levels (0.01%) enhance the risk of the

outgrowth and toxin production of anaerobic organisms of food safety concern,
such as Clostridium botulinum, has not been studied in the US. Koski (1988)
suggested that there may be a question as to whether controlled atmosphere
packaging and modified atmosphere packaging will gain acceptance in the US. not
because there is a question whether the technology will be established, but, rather
to what extent it can safely capture substantial segments of the US. food industry.
Oxygen-free atmosphere at a product water activity greater than 0.92 can be con-
ducive to the growth of many microbial pathogens, including Clostridium botulinum
(Labuza, 1987), thus, use of the sachet under certain conditions can be
dangerous. Furthermore, there are some Clostridia serotypes (B, E, and F) that
can grow and produce toxin at temperatures as low as 33°C (Genigeorgis, 1987).
In general, growth and toxin production will occur if products are held at
temperature higher than 10°C (Goldoni et al., 1980). To date, most companies
have relied on pH, water activity, temperature, and additives such as nitrite to
ensure that there is no food safety concern when using oxygen absorbers. A
variety of well known chemical additives ranging from sorbic acid to the benzoates
can exert beneficial microbiostatic effects (Brody, 1987). However in Japan, where
oxygen absorbers have had longer use, suppliers report that there is no greater
danger at the lower residual oxygen levels (0.01%) generated by an oxygen
absorber than at the higher levels (0.5%) generated by gas flushing. Reportedly,
this opinion is based on the concept of oxidative reduction potential and findings
that this factor is not substantially changed whether at 0.5% or 0.01% residual
oxygen. However, this has not yet been substantiated in the US. under inoculum
levels acceptable and customary to the US. food industry and academia. Also,
there may be consumer resistance to the use of sachets in a food package. A

concern may be preoccupation of consumers due to accidental ingestion and

potential toxicity of too much iron. This is minimized by use of the sachet concept
as well as printing the words "Do not eat" on the sachet. The largest sachet
contains only 79 of iron, so this would amount to an accidental consumption of
only 0.1 glkg for the standard 70-kg person, about 200 times less than the L050
value of 16 glkg body weight for rats (Labuza, 1987). When-these negatives are
overcome, use of this technology, (perhaps in other forms as well), will have
significant impact for the US. food industry.

The hypothesis in which this study was based consisted on testing
the microbial food safety concern existed in using oxygen absorbers in packages
containing food products with high water activity and abused temperatures. To
acomplish this, the present study had three specific objectives; First to evaluate
growth of Q_. sporogenes and g m in a food-package model system
inoculated with Q, sporogenes (ATCC 11437) and/or ; subtilis (ATCC 9372) both

 

individual and in a mixed system. These organisms were used to determine
relative growth of aerobeslanaerobes in a perishable food product packaged with
oxygen absorbers and modified atmospheres. The microbial load of each system
was determined as influenced by: a) Storage temperature. b) Presence of oxygen
absorber. c) Initial head space atmosphere. and d) Water activity. The second
objective was to determine the effect of oxygen concentration and temperature on
the growth of these organisms individually and in a mixed culture. A final objective
was to determine the efficiency of the oxygen absorber utilized in this study as
provided by Multifonn Desiccants, Inc. This oxygen absorber used was

"FreshPaxW" type B-300, and tested under the influence of temperature and water

activity.

LITERATURE REVIEW

There are at least five important factors which have an effect on the
shelf life of a packaged food-product. These are: package barrier, head space gas
content, thermal processing conditions, food composition and water activity (Aw),

and storage temperature.

Effect of package barrier on the shelf life of a food-package system.
When specifying packaging materials, the following should be

considered in terms of shelf life: Gas and water permeability; heat seal ability and
adhesion; UV transmission properties; and chemical interactions (food I packaging
interactions). Lewis (1989) defines packaging as. the process of forming the
packaging material into the desired shape, adding the product, establishing the
gas atmosphere and sealing the package. Saunders (1988) reported that the use
of suitable equipment, protective film, and suitable gas mixture, integrated with
clean and appropriate handling, have allowed the food industry to achieve longer
shelf life of food products. MAP packaging machines have been designed
(Anonymous, 1978; Rice, 1991) to obtain optimum packaged food products.
Examples of such machines include "Neutrafill" gas flushing system for packing
potato products; 452 Flowpak system for packing bakery, tobacco, dairy/cheese,
meat products, coffee/beverages, biscuit, snack food and vending packs; and SLS-
VGF series fill/vacuumizelgas flush/seal machines for deli salads and vegetarian
cuisine. Pinto (1979) reported that gas flush and vacuum lines are strikingly similar

and the same equipment is often used for both systems.

According to Lundquist (1969) "a good vacuum package should have
a minimum of 24-26 in Hg of vacuum in the packages and be formed from a film
having a maximum oxygen permeability of 0.8 cc oxygen/100 sq. in124 hr/73°F -
50 % relative humidity". He indicated that a decrease in package vacuum below 24
in Hg will permit spoilage organisms to grow and as the oxygen permeability
increases above 0.8 cc, the opportunity for yeast and mold to develop increases.
Sacharow (1968) indicated that package permeability to gas and moisture has a
major effect on shelf life of candies.

Morrison et al. (1977) reported that Owen-Illinois Co. developed a
system for replacing vacuum-packing in metal cans with gas-flushing in composite
cans with application to noodles, peanuts, soybeans, nuts, chips, and similar
products. This system required low-initial investment, nitrogen usage was
moderate, and the packed products had shelf lives equivalent to vacuum-packed
products. More recently, use of wide mouth PET (polyester), polypropylene and
polycarbonate jars has increased dramatically and will become more significant in
the 1990's (Pack alimentaire '90, 1990).

Cabral et al. (1979) observed that metallized polyethylene
terephthalate used as a protective barrier for potato chips yielded 33 days of shelf
life under different conditions and had less oxidation, less moisture pick-up and
better sensory grades than other potato chips. Labuza (1982) reported that potato
chips having a moisture content of 3.57% were rejected by a taste panel because
of their texture deterioration. These changes can be prevented by using of various
moisture barriers.

Barnett et al. (1987) studied the effect on extended refrigerated
storage on the shelf life of treated and non treated (2.3% potassium sorbate dip)

boned trout (Salmo gairdneri) packaged in laminated high/low density "semi-

 

permeable" polyethylene bags in the presence of a COz-enriched modified
atmosphere (MA). They found that the combination of packaging, refrigeration, and
MA effectively doubled the shelf life of the fresh trout product. Potassium sorbate
limited bacterial growth but did not extend shelf life beyond what was obtained with
MA alone. The carbon dioxide content in the trout flesh appeared to be a function
of 002 in the head space of the bags which was reduced by about 50 % during
the first 20 days of storage.

Kautter et al. (1978) evaluated the botulism hazard in mushrooms
packaged in commercial polyvinylchloride (PVC) film. They found of 1,078

packages examined that all were free of botulinum toxin.

Effect of head space gas content in a food-package system.

In all industrialized countries, today the demand for food products is
generally oriented to the ready-to—eat or the ready-to-cook packaged foods, fresh
or with fresh appearance, low processed and additives-free (Castelvetri, 1991 ).
Modified or controlled atmospheres have been in existence since the 1920s
(Smock, 1979). Modified atmosphere packaging (MAP) "is the enclosure of food
products in gas-barrier materials, in which the gaseous environment has been
changed to slow respiration rates, reduce microbiological growth, and retard
enzymatic spoilage -with the final effect of lengthening shelf life" (Koski, 1988).
Many studies (Hotchkiss, 1988; Silliker et al., 1980; Ginigeorgis, 1985; Hintlian et
al., 1986; and Hobbs, 1979) confirm that the behavior of microorganisms in the
presence of a MA is variable, in that: (1) not all microbial species are inhibited by a
definitive atmosphere and, if inhibited, not with the same rate; (2) the effect of a
definitive atmosphere depends on the nature of the food being packaged; and (3)

particularly with non-heat processed foods, the possibility that MA supports

pathogens while delaying spoilage is still a matter of concern. Rothwell ( 1988) and
Louis (1990) reported that these systems have been in use for a longer time and
developed faster in Western Europe (Primarily The United Kingdom, Germany,
Denmark, Sweden, Italy, Switzerland, Norway, Finland, and France), Japan, and
Australia than the US. Lioutas (1988) explained that MAP systems have been in
European markets for longer times due to (1) Geographically smaller
countries/distribution systems; (2) European buyers shop more frequently; (3)
Greater refrigerated food awareness/acceptance by the European consumer“, (4)
Total system/quantity approach for the MA technology; (5) Higher prices of food in
European marketplace as compared to US. made MAP economically feasible; (6)
Different driving forces for MA and refrigerated foods in Europe and US.
(European market is retail driven while the US. market is packer/consumer driven);
and (7) the European presence of large food chains dedicated to high-quality
refrigerated foods.

More than half of all food products are perishable (Woods, 1987).
There is evidence that MAP can extend the shelf life of many perishable products
including meat, fish, poultry, and some forms of produce by 50-400 % (Hotchkiss,
1988). Zagory et al. (1988), Labell (1985), Anonymous (1990),. Feldheim et al.
(1979), Ohkata (1990), Schulz (1979), Anonymous (1979), Transfresh (1979),
Rovema (1979), Anonymous (1977), observed extension in the shelf life of various
food products such as snacks, cut chilled fruit, bread, fresh produce, shredded
produce, and other foodstuffs by the use of MAP. Wolfe (1980), determined that in
the case of fresh meat, fish, and poultry, the major components of concern
resulting from dynamic changes are enzymatic aging processes, microbial
spoilage, fat oxidation, and the various oxidation states of the myoglobin pigment.

These changes can be prevented by using modified atmospheres (MA). Another

broad area of application is in packaging oxygen-sensitive powdered foods in gas-
flushed composite cans since this contributes to increased shelf stability
comparable to products in metal cans but at a more economical cost (Anonymous,
1980). Also for packaging raw and processed peanuts, Container Corp. of
America’s "Containervac" system (bag-in-box container and a machine that pulls a
vacuum and then heat seals the bag) was tested and it was found that cold
storage was unnecessary since it provides excellent sanitation and protection
(Food and Drug Packaging, 1979). This system is in use for frozen fruits, ice
cream flavorings and hops and has potential use for rice, beans, pecans, corn,
and other foodstuffs.

Rice (1990), reported that modified atmosphere packaging has had
success in marketing prepared, refrigerated potato products. The potato products
were included in two varieties, hash browns and mashed potatoes and were first
introduced in foodservice sizes, 20-lb. MAP gas-flushed barrier bags packed in
corrugated cartons. Northern Star test marketed 20-oz, consumer-size versions of
the hash browns in MAP bags and found that consumers responded well. Both
products now are moving into national distribution. Rice (1990) also reported that
Chinese entrees in CPET barrier trays (6" x 5" x 1 5/ " deep) and barrier film (anti-
fog multi-layer structure with a nylon base) are being packaged in a carbon
dioxide/nitrogen gas blend. This is accomplished manually by depositing the
batch-cooked and cooled product into the trays, evacuating air and backflushing
with the gas blend and the lidding film heat-sealed to the trays. The oxygen
permeability exhibited was 0.04 ccltraylday at 72°F and 21% (ambient level)
oxygen. The shelf life of the refrigerated trays was 21-day without use of MSG or
freezing at refrigeration temperature. Foley (1984) demonstrated that for an

oxygen-permeable air-evacuated packaging system of fresh high quality fish fillets

.10

stored at 32°F, shelf life was 16-17 days. No significant odor buildup occurred.
Flavor, texture and appearance were retained which translated to increased
retailer sales and profits. The "Veryfrais" process was developed in France and
combines MA and a new regenerating system. for packing of fish and meat
products and is available to the United States under licensing agreement (Louis,
1987)

Rothwell (1988) reported that the principal packaging systems for
MAP are: horizontal form-fill-seal, rigid and semi-rigid "Atmospack"; horizontal &
vertical form-fill-seal, flexible "Pillow-Pack," FFS pouches; pre-formed plastic tray
or bag; form-fill-seal composite board/plastic tray or pre-formed board/plastic tray;
pre-formed coated pulp tray; bag in box; darfresh vacuum skin packaging; Cryovac
shrink vacuum bags; bivac vacuum shrink system; isopak vacuum skin system;
and trayvac systems.

MAP is specially important in extending the shelf life of perishables
at the retail level (Fig. 1). Sakamaki et al. (1988) studied the stability of oat cereal
as influenced by selected barriers packaged with and without oxygen absorbers.
They placed 50 g of oat cereal in plastic pouches made from either polyethylene
(PE) with thickness of 84 pm or polyvinylidene chloride (PVDC)-coated
poIypropylenelpolyethylene (PP/PE) with thickness of 86 pm. An oxygen absorber
(0A) was introduced (consisting of iron powder, water and catalysts) into the
package. The initial oxygen concentrations were 1, 5, or 21% for the second film,
and lipid oxidation was monitored using a modified TBA test. They observed that
since the oxygen absorbing ability of the scavenger (160 ml of oxygen/day at 21°C
and 40% RH.) was low compared to the oxygen permeability of the PE pouch
(220m llpouch/day), the 0A was quickly overwhelmed by oxygen permeating

through the poor barrier material. They concluded that in general, in the good

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barrier material (PVDC-coated PPIPE), oxygen absorber retarded or delayed lipid
oxidation in the oat product during storage. A nitrogen gas flushed sample (1%
initial head space oxygen concentration) showed deterioration after one month
compared to samples with OA that prevented oxidative changes for up to three
months in the PVDC-coated PPIPE barrier. Finally, they reported that there was a
five-fold increase in the TBA index for product in the PE pouch without absorber
compared to product in the PVDC-coated PPIPE package with absorber at 41°C.

Effect of thermal processing and the shelf life of food-package products.
Concern has been expressed by regulatory authorities (Gill, 1988),

food industry groups (NFPA, 1988) and others (Eklund, 1982) that MAP may
create an undue safety hazard. This increased concern for safety is due to the
increased knowledge concerning the survival and growth of pathogenic bacteria at
refrigeration temperatures (Palumbo, 1987).

In research studies the use of ; botulinum as a test organism is
often avoided, because of hazard potential if in-plant contamination by spores of
§_. botulinum were to occur (Manomaiwiboon, 1990). An alternative microorganism
is g; sporogenes, which is a non toxic strain that has many physical and
biochemical characteristics similar to Q_. botulinum. Table 1 shows a comparison of
physiological requirements for these two clostridium species.

Elkund et al. (1988) demonstrated the feasibility of a heat-
pasteurization process for certain vacuum-packaged hot-smoked fishery products
to inactivate the spores of the non-proteolytic Group II C_. botulinum types B, E,
and F. They injected 106 spores to already smoked salmon and then heat-
pasteurized in a water bath held at a constant temperature. A total of 85, 65, and

55 min in the 185°F (85°C), 192°F (889°C), and 198°F (922°C) baths,

 

13

Table 1: Some physical characteristics of c_. sporogenes and §_. botulinum

 

Characteristics C. sporogenes C— botulinum
' (T was A. B. 8- F We)

4

 

.5.

H2 produced

lndole produced

Lecithinase produced

Lipase produced

Nitrate reduced

Milk reaction

Meat digested

Acid produced from
Amygdalin - -
Arabinose - -
Cellobiose - -
Fructose -w -w
Galactose - -
Glycogen - -
lnositol - -
lnulin - -
Lactose - -
Maltose -w -w
Mannitol - -
Mannose - ~ -
Melezitose - -
Melibiose - -
Raffinose - -
Rhamnose - -
Ribose - -
Salicin - -
Sorbitol -w -
Starch - -
Sucrose - -
Trehalose - -
Xylose - -

+Ql+r
+Ql+t

 

 

 

 

 

Symbols: +, reaction positive for 90-100% of strains; -, reaction negative for 90-100% of
strains; 4, represents abundant ('-" to ”4+" scale); -w, weak reaction (pH of sugars 5.5-5.9)

From: Buchanan (1974)

14

respectively, prevented toxin production by type E during 21 day of incubation at
25°C. Longer times, 175, 85, and 65 min. respectively, were required to prevent
toxin production by non-proteolytic type B. Toxin production by type E during 120
day of storage at 10°C was prevented by a 45 minute treatment in the 198°C
(922°C) bath. They also found that outgrowth and toxin production by type E was
prevented by a 55 minute process at 198°F (922°C) and type B was prevented by
a 65 minute process. This process does not, however, inactivate the more heat-
resistant proteolytic strains of g; botulinum Group I or necessarily ether spore-
former.

A basic principle to preserve perishable foods (Labuza, 1982) is to
control or destroy microorganisms by: (1) lowering temperature to slow growth; (2)
raise temperature to kill microorganisms; (3) remove or bind water to slow or
prevent their growth; (4) lower pH to slow or stop their growth by adding acid or
through fermentation; (5) Control 02 or C02 level to control population; or (6)
manipulate food composition to remove nutrients needed by the microbes. The use
of heat to preserve foods is based on: (1) destruction of pathogens; (2) destruction
of spoilage microbes; (3) denaturation of enzymes; and (4) softening of tissues to
make them more digestible.

Different thermal processes used include: a) blanching: application of
heat under mild conditions, or at high temperature for a short time, to achieve
enzyme degradation and soften food tissues; b) pasteurization: mild heat
treatment used to reduce the number of live microorganisms in the food so as to
extend its shelf-life and destroy important pathogens (the product is not sterile); c)
commercial sterilization: condition achieved by application of heat which renders
food free of viable forms of microorganisms having public health significance, as

well as any microorganisms of non-health significance capable of reproducing in

15

the food under normal non-refrigerated conditions of storage and distribution

(Lopez, 1987); d) hot filling; and e) water bath cook.

Effect of food composition and water activig in the shelf life of a food-package
system. '

Most chemical reactions decrease in rate as the water content
decreases (Labuza, 1982). Although various chemical treatments increase the
shelf life of food products, there are some cases in which chemicals have
deleterious effects on sensory or physical qualities of foods (Golden et al., 1987).

Both moisture and enzymes cause lipolysis of the triglyceride
molecule into glycerol and free fatty acids (Labuza, 1982).

The microbiological load or state of the prepackaged food is a critical
variable compounded in the term quality (Lioutas, 1988). Glass et al. (1991)
examined the relationship between water activity (Aw) of fresh pasta and toxin
production by proteolytic Q botulinum. They inoculated 4 types of fresh pasta (4
different Aw) with Q, botulinum spores, packaged under modified atmosphere, and
stored at either 4 or 30°C for 8 to 10 weeks. They found that no toxin was
produced in any fresh pasta held at 4°C for up to 8 weeks. However, toxin was
detected in meat tortellini with Aw of 0.99 and 0.95 at 2 and 6 weeks, respectively,
when held at 30°C. Toxin was not detected in tortellini with an Aw of 0.94 or below
when held at 30°C for 10 weeks. Also toxin was produced at 2 weeks in linguine at
Aw 0.96 at 30°C. No toxin was found in linguine or fettucine (Aw 0.93 or 0.95) and
held at 30°C during 10 or 8 weeks, respectively. Toxin was not detected in either
filled or flat noodle pasta at any water activity, including the highest Aw (0.99 for
filled pasta and 0.96 for flat noodles) held at 4°C for up to 10 weeks. They

concluded that Aw of fresh pasta is a principal factor in preventing formation of

16

botulinan toxin for type A and B and minimum Aw for growth and toxin production
of Q, botulinum type A and type B is 0.95 and 0.94, respectively.

Shirazi et al. (1989) studied the feasibility of controlling in-package
relative humidity (IPRH) using compounds possessing Type lll sorption isotherm
behavior on green tomatos. They placed sorbitol, xylitol, NaCl, KCI and CaClz with
one mature green tomato at 20°C in sealed packages for 48 days. By using
different quantities of each compound they observed that IPRH was a function of
the ratio of chemical to fruit mass. They found that relative humidities within control
packages were in the range of 96 to 100% throughout the experiment, thus the
humidity control system provided a method, for stabilizing package humidities
which could improve the efficacy of modified atmosphere packaging.

Sugar is used in the preservation of several foods, including
sweetened condensed milk, jams, jellies, and other foodstuffs. Banwart (1989)
reported that low levels of sucrose are utilized by many microorganisms as a
source of energy, but high level of this chemical can have inhibitory properties.
Thus, the preservative effect of sucrose is due to the development of osmotic
forces and a lowered water activity. The control of microbial growth in acid medium
products is currently helped by filling these products while still hot, by heating the
surface by steam injection, spraying the surface with a chemical fungicide, or using
a cover paper impregnated with a fungicide (Banwart, 1989).

Esteban et al. (1990), studied the water activities (Aw) of culture
media used in food microbiology and found that fluid thioglycollate medium (from
Difco) had a final Aw of 0.995 (pH=7.2), thus they considered it as a very high Aw
medium.

Kuo et al. (1985) manufactured a chinese dried pork product with
combinations of 15, 22.5, and 30% sugar and 0.5, 1.5, and 2.5% salt (9

 

17

treatments), and evaluated for microbiological, chemical and sensory properties at
0, 7, 14, and 21 days of storage at 4°C. They found that aerobic and anaerobic
plate counts increased during storage, with the highest sugar and salt combination
resulting in the least microbial growth. They obtained values for total anaerobes of
2.59 and 4.88 log CFUlg at 0 and 21 days respectively for pork product with
Aw=0.81 (highest sugar and salt combinations), and values of 2.39 and 7.18 log
CFU/g at 0 and 21 days respectively for product with Aw=0.88 (lowest sugar and
salt combinations). For aerobes the values obtained were 3.55 and 6.11 log CF U/g
at 0 and 21 days for the lowest Aw product (0.81) and 3.84 and 8.95 CFUlg at 0
and 21 days for product with the highest Aw (0.88).

Effect of storage temperature and time on the shelf life of a food-package system.

Labuza (1982) concluded that one of the major environmental factors
which results in increased loss of quality and nutrition for most foods is exposure
to increased temperature. Therefore, in order to predict shelf life, and to be able to
put a shelf life date on a product, knowledge of the rate of deterioration as a
function of environmental conditions he temperature) is necessary. Several
authors (Mohr et al., 1980; Reichardt et al. 1982; Ratkowski et al.,1983) have
studied microbe activity as a function of temperature, but there is little agreement
of what is the best model to apply. Besides temperature, other environmental
parameters affect the lag phase and the rate of growth of microorganisms, and
include oxidation reduction potential of the food and the composition of the
atmosphere in which the food is kept.

Labuza (1982) suggested that microorganisms constitute a major
mechanism by which many foods, especially fresh ones, lose quality. This is

because microbes are ubiquitous in the environment, and grow rapidly at ambient

18

temperature. In predictive modeling applications, models based upon lag time are
most appropriate for pathogens with zero growth tolerance like 9_. botulinum,

Salmonella, Listeria, etc. (Baker et al., 1990). Growth rate models are best for

 

pathogens which must have significant increase in numbers to reach an
infective/toxic dose or cause food spoilage (Baird-Parker et al., 1987).

Burdett et al. (1986) studied the growth kinetics of _B_. Mi; and
found a doubling time of 120 min. at 35°C. They used a spectrophotometer at 600
nm to control growth of subcultures, so that cultures were inoculated from a single
colony and periodically subcultured over 3 days to maintain an absorbency of
<03. They also found an exponential pattern of increase.

Various authors have shown (Clark and Lentz, 1969, 1973; Enfors
and Molin, 1980; King and Nage, 1967, 1975; Paradis and Stiles, 1978; Eklund
and Jarmund, 1983; Baker et al., 1985) that a high head space concentration of
carbon dioxide in packaged foods has an inhibitory effect on the growth of aerobic
spoilage microflora. However, elevated C02 levels can establish conditions where
pathogenic organisms such as C_. botulinum may survive (Daniels et al., 1985).

Molins et al. (1987) studied ham-type product made from soy-
extended cured beef. The product was inoculated with Q_. sporogenes PA3679 and
stored under wholesale (2-4°C) and retail (5°C) refrigerated storage conditions,
during abuse-temperature holding for 24 and 48 hr at 24—25°C. They used
Trypticase soy agar and BBL® Anaerobic GasPak Systems to enumerate C_.
sporpgenes (30°C, 48 hr) and total viable spores (heat-shocked at 80°C for 20
min. followed by 30°C, 48 hr anaerobic incubation). Vegetative cells and viable
spore counts were determined by counting typical rhizoid colonies characteristic of
the culture. Samples held at 2-4°C did not exceed 104 CFU/g after 4 wk for

mesophilic, psychrotrophic, anaerobic and lactic bacterial counts. Inoculated

19

PA3679 did not grow in the product during 24 or 48 hr of simulated mishandling
(24-25°C). Molins et al. (1985) reported that the culture characteristic rhizoid
colony formation of Q sporogenes had been selected for and was determined to
be stable.

Silliker and Wolfe (1980) inoculated ground beef with enterococci,
Salmonella, and S_. aureus 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 increased by more than 3 log cycles, whereas
in MA stored samples (60% C02, 25% 02, 15% N2) the number increased by
approximately 0.5 log cycles. At 10°C there was no evidence that elevated levels

of 002 increased the hazard from S_._ aureus, or enterococci. At 20°C, however,

 

Salmonella grew almost equally as well in air or in MA stored samples. They also
studied pork cubes into which Q botulinum spores had been inoculated and
reported that elevated 002 level did not increase hazard from botulism. They
reported that chicken cubes inoculated with Q botulinum spores grew in C02 and
concluded that the 002 atmosphere did not enhance the potential hazard from
botulism, but did not reduce it either. A recent study (Nelson et al., 1989) indicated
that spores of Q subtilis (ATCC 9372) and Q smrpgenes (ATCC 7955) were

 

inactivated by exposure to oxygen gas plasma. Spores of Q sporogenes were
grown under anaerobic conditions at 32-37°C in beef heart infusion media,

whereas spores of Q subtilis were cultured aerobically on nutrient agar containing

 

CaCl2 and MnSO4 at 32-37°C. Exposures of cultures at 50 and 200 watts plasma
power settings, for 3 exposure periods, 5, 30, and 60 min. were studied. The
results showed that greater than 3.4 spore logarithmic reductions (SLR) for a 30
min. exposure and greater than 3.5 SLR for a 60 min exposure were achieved in a

50 watt plasma for both microorganisms. Greater than 3.4 SLR was achieved for

20

both microorganisms at 200 watts for all exposure periods. They concluded that
oxygen gas plasma may be feasible for inactivation of microorganisms that are
resistant to other sterilization methods. Table 2 shows differential characteristics of

both endospore-forming bacteria Bacillus and Slostridium.
Stier et al. (1981) inoculated salmon fillets with Q botulinum A, B,

 

and E spores. They compared those fillets stored in air with those stored in MA for
outgrowth and found that toxin production occurred in products stored in air and
modified atmosphere at 272°C within 3 days. Outgrowth and toxin production did
not occur at 44°C. They found that organoleptic spoilage generally preceded toxin
formation. The time interval between toxigenesis and spoilage apparently was
shortened with increased storage temperature. Luiten et al. (1982) observed that
S_. uphimurium was unable to grow on beef steaks stored at 10°C in an
atmosphere containing 60% C02 and 40% 02, although there was no die-off of
the organism during the 9 day storage period. This is in contrast to air stored beef
samples in which an approximately 3-log increase in the numbers of Salmonella
was observed.

Dixon et al. (1987) found that there was no influence of carbon
dioxide (pCOz) partial pressure on the growth of Q sporogenes (NClB 8053) when
grown in a culture containing pancreatic digest of casein medium. Bacto fluid
thioglycollate medium contains also pancreatic digest of casein (Difco, 1984). In
media where optimal p002 was observed for growth of the bacteria it was
approximately 0.45 atm.

Gray et al. (1984) studied the combined effect of potassium sorbate
and C02 on growth of S_. aureus and Salmonella on chicken thighs. They noted

that at 10°C, 100% carbon dioxide flushing resulted in S. aureus counts of 2 logs

 

lower than on thighs packaged in air. Growth of Salmonella on chicken thighs

21

Table 2: Some differential characteristics of Bacillus and Slostridium genera

 

 

 

 

 

 

 

Characteristics Bacillus Clostridium
Rod-shaped + 4-
Diameter over 2.5 pm - -
Endospore produced + +
Motile ’ + +
Stain Gram-positive at + W
least in young cultures
Strict aerobes D -
Strict anaerobes - 1'
Catalase + -

 

Symbols: +, 90 % or more of strains positive; -, 10% or less of strains positive; 0,
substantial proportion of species differ; “, rarely Gram negative.

From: Buchanan (1974)

22

stored in high-barrier bags at 10°C was inhibited by 100% carbon dioxide but was

not affected by vacuum packaging.
Baker et al. (1986) inoculated minced chicken meat with Q Fragi, S_.
typhimurium, S_. aureus, or Q sporogenes and stored samples at 2, 7, and 13°C.

Throughout an 18 day storage period, numbers of S_. aureus were lower in 80%

 

002 MA than in air controls. Counts of Q sporogenes, which is culturally similar to
proteolytic strains of Q botulinum, decreased under all conditions, but number in
air-packaged samples declined more than in 002 indicating that MA had a
protective effect on the organism. Hintliand and Hotchkiss (1987) noted that while
modified atmospheres containing 5 or 10% oxygen inhibited the outgrowth of Q
perfringes in sliced and cooked roast beef co-inoculated with gP_. fr_agj, S_.
gphimurium, S, aureus, and Q perfringes, the numbers were the greatest in air

 

controls. It is probable that Q perfringes was able to grow following consumption
of oxygen by E_. fiagj whichgrew to high numbers. The growth of Q perfringes was
inhibited 1 log by a MA blend containing 75% C02 and 25% 02 and by several log
cycles by MA containing 2, 5, or 10% 02, compared to growth in air.

The physiology of Q sporogenes (NCIB 8053) was studied by Lovitt
et al. (1987a). They found that when batch cultures of the microorganism were
incubated at 37°C with stirring at 250 rev/min, H2 production was detectable in the
cultures only under conditions of nutrient excess. They determined the microbial
concentration by measuring absorbance at 520 nm and relating this to a standard
curve. They also studied the growth and nutritional needs of Q sporogenes NCIB
8053 (Lovitt et al., 1987b). They incubated at 37°C with no shaking in Buchner
flasks sealed with a rubber bung and fitted with a sample port, bladder and gas
filters to allow flushing and evacuation of the flask head space. They used

thioglycollate as a reducing agent in the media, and found that this media

23

supported the growth of this and several other strains of this species. The flasks
were autoclaved at 121°C for 15 min, and after cooling, sterile supplements were
added and the head space was filled with N21002 (95:5 vlv). Growth in flasks was
estimated by depositing samples in cuvettes and measuring optical density at 680
nm. They confirmed that the microorganism required 10 amino acids and one
vitamin to grow, and was capable of growing in media containing no carbohydrates
(although these stimulate growth). They also measured change in gas
concentration in the head space using gas chromatography to estimate

fermentation products.

Qxygen absorbers and oxidation in food systems.

Labuza (1982) reported that all fats are subject to deterioration by
oxidative and hydrolytic rancidity leading to the formation of objectionable odors
and flavors. Hydrolytic rancidity is also responsible for "soapy" flavors and
facilitates deterioration by direct oxidation. Oxidative rancidity results in food
spoilage associated with fat deterioration, is. presence of pungent or acrid odors.
He also reported that in the case of potato chips, which are dried to a finished
moisture level of 3% or less (which relates to an Aw of about 0.2), moisture gain
leads to texture changes (sogginess) increased rate of oxidation, and loss of
consumer acceptance. Moisture gain is a function of the package type and its
permeability characteristics (Paine, 1991).

Labuza (1987) mentioned that only type E "Agelessm" sachets
(oxygen scavenger) were in use in the United States (at that time). The patent that
supports this product is US. patent 4,421,235 held by Mitsubishi (Moriya, 1983).
Watanabe (1988) reported that sales of this oxygen scavenger have increased (up

to 20% in 1987 over 1986), and reached 15 billion yen ($120 million) for some 350

 

24

million pieces. Several more patents have been issued in the US. that are related
directly to oxygen scavengers (Takahashi et al., 1985; Farrel et al., 1985; and
Otsuka et al., 1986). These more recently issued patents cover the oxygen
absorbent, oxygen scavengers that can be included in a multilayer construction in
which the scavenger remains passive until such time needed, and the oxygen
absorbent packet.

In addition to Ageless” (ferrous oxide that is oxidized to ferric oxide,
-used in packaging of bakery items, precooked pasta, potato chips, nuts and
coffee, etc.-), there are currently other oxygen absorber products in the market
including "Oxysorbm" (absorbs oxygen by converting iron into iron oxide and
hydroxide <0.01% 02, -used with beef jerky, salami, peanut butter), "SmartCap
Absorberm" (artificial gill for extracting oxygen from liquid, -for use in beverage
packaging-), ’Toppan°" (non-ferrous metal particles that replaces 02 absorbed
with 002); "Mega Mold" (oxygen scavenger plus mold/microbe prevention -e.g.
yeast fungus and _B_acflus, s_r.rpti_li_s_-); and "Keplon°" (oxygen absorber -for use in
beef jerky-). In Japan, the companies that produce oxygen absorbers (January
1986) were: Mitsubishi Gas Chemical Co., Inc (75.6% of market); Toa Synthetic
Chemical Co., Ltd (5.9% ); Toppan printing Co., Ltd (5.7 %); Nippon Sohda Co.,
Ltd (4.3%); Kepron (3.1%); Ohji Kakoh (2.6%); and Hakuyoh (1.25%). The price of
the oxygen absorber depends on the amount of oxygen it absorbs.

Multiform Desiccants, Inc. (Buffalo, NY.) is assignee of a recent US.
patent (Cullen et al. 1991). This patent allows the company to produce and sell
"FreshPaxTW' (oxygen scavenger sachets), and is recommended for use in:
breads, cookies, cakes, pizza, pastries, croissants, buns, powdered milk, sponge
cakes, pastas (fresh and pre-cooked), cheeses, meats, quiche, Chinese fried

noodles, dry seaweed products, wines, snacks, coffees, dry tea,

25

candies/confectioneries, beans, dried eggs, spices, dried fruits, dry baby foods,
potato chips, nuts, fruits and vegetables (tomatoes, avocados, lettuce), chocolates,
flour, dehydrated foods, and fish. The "FreshPaxTM" components are on the FDA's
Generally Recognized As Safe (GRAS) list. Criteria to be considered when using
this products include: (1) plastic film must be checked for its oxygen permeability
(<20 chMZ atmosphere per day or less is recommended), and (2) hermetic seals
are essential. These sachets can reduce oxygen content to less than 0.1%. The
oxidizing system made up of ferrous salts and an oxidation modifier conveniently
delays primary oxidation for several hours until the absorbing packet is introduced
into the food package (Anonymous, 1989). Therefore, the user does not have to
be concerned with premature oxidation during handling or packaging of the
absorbing system. In a recent publication (Anonymous, 1991) Mr. Ronald Idol,
director of technology for Multiform Desiccants, Inc. described the newest
innovation: "FreshMaxW", "as one which will duplicate the oxygen scavenging
performance of sachets currently placed in food packages". He referred to
"FreshMaxW" as a multi-functional gas scavenging technology that can be
adhesive/backed with various adhesives,.for numerous applications. This makes it
far less likely to be ingested compared to sachets currently in use. The
FreshMaxT" (like other oxygen absorbing technologies), protects food from mold
growth and rancidity that can result from oxidation, by absorbing available oxygen
within a 24 hour period. It can also eliminate the need for costly food additives
and/or preservatives. FreshMaxT" can cost less than gas flushing or vacuum
packaging which usually require a substantial capital investment (Anonymous,
1989).

Based on this review and by following the objectives stated in the

introduction, the oxygen absorbers, temperature and high water activity influences

 

 

26

on the microbial growth were tested over time. Time intervals were established for
sampling of head space percent oxygen and microbial load, so that a relation of all
factors was obtained. The hypotesis was fulfilled in the parameters studied when

final results were obtained and relations among the factors were analyzed.

 

MATERIALS AND METHODS

Package system preparation
One pint, wide mouth mason jars were obtained from the General

Stores at M.S.U. The jars were prepared as follows (see figure 2): Three holes
were made in each metal lid using a drill with a 13/64" diameter bit. One hole was
positioned in the center of the lid and two at 3l4" from the center hole and opposite
from each other. The holes were slightly enlarged using a metal shaft so that the
edges left by the drill were dulled. Three rubber stopper septums, size 7 mm
(Sigma Chemical Co., St. Louis, M0) were placed in the holes. Two borosilicate
disposable pipettes (VWR Scientific Inc, San Francisco, CA) of 1 mm3 were
obtained and the cotton in the pipettes drawn down into the narrow part (using a
wire). The pipettes were then cut from the narrow part so that about 2% inch
segments were obtained. All cuts were made using a triangular file and dulled
using sandpaper. The pipette segments were then placed into the 2 outside
septums which left the center septum free.

The lids were placed on a table and the inside (facing the jar when
assembled) sprayed with an enamel to prevent corrosion by high relative
humidities. Special care was taken to cover the pipette hole segments with tape to
prevent enamel clogging. Two hours between each enamel application were
allowed. After the third application, the enamel was allowed to dry for 72 hours at
room temperature.

After drying, the assembled lids were placed on the jars. The

package model system was stored until used.

27

 

 

28

 

Rubber
, septum

 

 

 

 

 

 

Metal
Lid

 

 

 

 

 

 

 

 

 

 

I Cotton I
i _ filter

 

\ Oxygen

absorber
[when rocked]

 

 

 

150ml—

 

Glass I
jar

 

 

 

 

Figure 2: Package model system design.

29

Model re aration.
The model system utilized for this study was Bacto Fluid

Thioglycollate Media (from Difco Laboratories, Detroit, MI). This media was chosen
because it allows the growth of both bacteria used in this experiment (table 3). The
media is formulated with Bacto Yeast Extract, Bacto Casitone and resazurin.
Resazurin is used to indicate the status of oxidation or aerobiosis (OR indicator) in
the media. Dextrose (glucose) is included in the Bacto Fluid Thioglycollate media
because it has been shown (Difco manual, 1984; Lovit et al. 1987b) that most
organisms grow earlier and have more vigorous growth in the presence of a
carbohydrate.

The preparation of the media was performed according to the method
suggested in the Difco manual (1984), and had a pH of 7.2.

In one series of samples the Aw was adjusted to 0.96 by addition of
sucrose -measure of Aw was obtained using an electric hygrometer indicator
(Hygrodynamics, lnc., Silver Spring, MD)- to the media before preparation (as
powder). This yielded a final concentration of 40% (wt/vol) sugar with respect to
the water. Once the media and sugar were mixed, the mixture was added to the
water with constant agitation to prevent clumping. In the other series of samples

the water activity was not adjusted and was >0.99.

Q sppgpggnes inoculum preparation
Slostridium sporogenes (ATCC 11437) was obtained as a freeze-

dried culture from American Type Culture Collection (Rockville, MD). This
microorganism was chosen because it closely resembles phenotypically to Q
botulinum types A, B, and F, which are toxin producer proteolytic strains. The

culture was activated using Bacto Fluid Thioglycollate (Difco) and the instructions

 

 

30

Table 3: Typical Cultural Response After" 18-48 Hours at 35°C

 

Organism Bacto Fluid

Thioglycollate Medium

 

 

Bacillus subtilis good to excellent
(ATCC 6633)
Clostridium sporogenes good to excellent

 

 

 

(ATCC 1 1437)

 

'theeecultureemaybeincubetedat30-35°Cfor2—7days,ifpreferred,perUSP.

From: DIFCO (1984)

 

31

for rehydration were followed according to the recommendation that accompanied
the bacteria (ATCC, 1989). Once the culture was activated, an agar plate was
prepared and inoculated with the microorganism and placed in anaerobic
conditions at 37°C (:1°C) to allow grow of the. organisms. From this plate, one
single colony was taken using a platinum wire and deposited into fresh media to
assure production of cells genetically similar for use during the study. Once the
new batch was obtained, a series of stock cultures were prepared by aseptically
(in front of a bunsen burner and using dispdsable sterile 1-ml pipettes -Coming
Laboratory Science 00., Corning, NY-) mixing one part culture with 50% glycerol.
The mixture was distributed into cryo-vials and closed so that 0.5 ml. per vial was
obtained. All vials 'were stored at -80°F in a bio-freezer (Forrna Scientific, Marietta,

OH.) until needed in the experiment.

8. sgptilis inoculum preparation
Sacillus subtilis (ATCC 9372) was obtained as a freeze-dried culture

 

from American Type Culture Collection. The culture was taken and started using
Bacto Fluid Thioglycollate (Difco Laboratoires, Detroit, MI) media by following the
instructions for rehydration accompaning the bacteria (ATCC, 1989). Once the
culture was obtained, an agar plate was prepared and inoculated with the
microorganism and placed in aerobic conditions at 37°C (:1’C) to allow growth of
the organism. From this plate one single colony was taken and deposited into
fresh media to assure production of cells genetically similar for use in the study.
Once the new batch was obtained, a series of stock cultures were prepared by
aseptically (in front of a bunsen burner and by using disposable sterile 1-ml
pipettes -Corning Laboratory Science 00., Corning, NY) mixing one part culture

with 50% glycerol. The mixture was distributed into cryo-vials and closed so that

 

 

 

32

0.5 ml. per vial was obtained. All vials were stored at -80°F in a bio-freezer (Forma

Scientific, Marietta, OH.) until needed in the experiment.

Experimental design (figure 3)

The package-model systems were labelled with the bacteria
designation, water activity, initial oxygen in head space and storage temperature.

Approximately 150 ml of prepared media was placed into each jar.
This was accomplished by transfering media from the erlenmeyer where it was
prepared using a 150-ml beaker and gloves. Half of the jars were filled with media
having a Aw of 0.96 and the other half a Aw of >0.99 for samples stored at 25°C.
All jars stored at 10°C were filled with 150 ml of media with a Aw of >0.99.

A fresh package containing the oxygen absorbers (Multiform
Desiccants, Inc. Buffalo, NY.) was opened (stored under vacuum and high barrier
film) and one sachet per container was placed in a total of one quarter of the lot
(32 and 16 jars for storage temperatures of 25° and 10°C, respectively). The
sachet was attached using sterilizing condition-resistant tape between the two
glass tubes so that the oxygen absorber was in the center of the lid (see figure 2).
The food-package model systems were sterilized at 250°F for 15 minutes
(commercial sterilization). After sterilization, the jars were allowed to cool at room
temperature for 12 hours and then placed in the designated storage temperature
rooms. Once the jars were sterilized they remained closed during the rest of the
experiment.

Once the jars reached the storage temperature specified (by allowing
sample jars a 12 hour period in the storage temperature rooms), the initial oxygen
head space contents were obtained by mixing nitrogen and air from standardized

tanks and flushing the mixed gas into the jars containing the media for the time

33

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34

required to remove atmospheric gases to obtain the mixture specified. Four
different oxygen systems were used: 1% Oz, 5% 02, 21% 02, and 21% 02 with
oxygen absorber.

The microorganisms were inoculated into the jars as follows: The jars
were divided into four lots. The middle rubber septum was rubbed using a cotton
swab with 70% ethanol, and then used to introduce the bacteria into the jars using
1cc tuberculin disposable syringes (Becton Dickinson & Co., Rutherford, NJ) and
20G1 Precision Glide" needles (Becton Dickinson and Co., Rutherford, NJ)
during the inoculation process. Inoculation of the microorganisms into the media
and gas exchange was accomplished hermetically through the rubber septums.
The first lot (32 jars) were used as controls, no microorganisms were added, a
second lot was inoculated with Bacillus subtilis (ATCC 9372) at a concentration of
approximately 102 CFUIml. The third lot was inoculated with Slostridium
sporogenes (ATCC 11437) in the same concentration of about 102 CFUIml. The

 

 

final lot was inoculated with a mixed culture containing approximately equal
amounts (102 CFUlml each) of both bacteria, Q subtilis and Q sporogenes. This
was accomplished by adding first the aerobic and then the anaerobic

microorganisms.

Qflgen absorbers used for study

Multiform Desiccants, Inc. supplied the appropriate size and formula
of oxygen absorber for the study. The oxygen absorbers used were "FreshPaxm"
type B-300 and have the capability of absorbing 200 cc. of oxygen. The jars used
after adding 150 ml of media had an approximate head space of 325 cc. (about 68

cc oxygen).

35

The oxygen absorber contains ferrous powder (with a 70-80%
efficiency), salt (an electrolite), and water. Theoretically, the amount of ferrous
powder contained in the B-300 type oxygen absorber (FreshPaxm) is capable of
absorbing 400 cc. of oxygen. The efficiency and reactions before and during
packaging of the ferrous powder with the atmosphere makes necessary to lower
the specification to assure 200 cc. of oxygen absorbed.

The basic reaction that takes place in the oxidation process is:

4 Fe + 3 02 + 6 H20 --> 2 Fe203-3H20

Incubation (storage) conditions

In storage, all jars were agitated by constant rotation at. 100 RPM by
placing them in an incubator shaker (Lab-Line instruments, Inc. Melrose Park, IL.)
at room temperature (25 :1°C) and in an open shaker (Eberbach, Co., Ann Arbor,
MI.) at a temperature of 10 :1°C. For the 25°C jars, samples were taken at 0, 24,
.48, and 72 hr. for product at Aw>0.99; and at 0, 120, 240, 288, 336, 384 and 432
hr. for jars stored at Aw=0.96. For jars stored at 10°C samples were taken at 0, 1,

2, 3, and 4 weeks

We

The jars containing the different media, microorganisms, and gas
concentration were sampled in the same order every sampling period and the
oxygen level in the head space measured at the specific time intervals cited
above. This was performed utilizing a 0.5 cc series A Gas Syringe "Pressure-lok°"
(Fisher Scientific, Pittsburgh, PA) and injecting the 0.5 cc head space gas sample
into a Servomex series 1100 gas chromatograph (London, England). After the

oxygen concentration had been determined, anaerobic and aerobic CFU's per

36

milliliter were determined under aerobic and anaerobic conditions, respectively, as
follows: the jars were taken to the food microbiology laboratory and at least 2-4
samples of one ml each were taken from the media using a 20x4" long non-coring
stainless steel needle (Popper & Sons, Inc., New Hyde Park, NY); the needles
were pre-conditioned according to the following procedure: twelve needles were
contained in a beaker with ethanol at 70% (v/v) near the flame of a bunsen burner.
One needle was attached to a disposable 1 cc sterile tuberculin syringe (Becton
Dickinson & Co., Rutherford, NJ.). The needle was then placed into 9 ml sterile
peptone water (0.1% v/v) contained in 16x125 culture tubes (Corning Laboratory
Science Co., Corning, NY) that were previously flamed using a bunsen burner to
assure sterile conditions and then flushed 5 times to remove any 70% ethanol in
the needle, the peptone water was then disposed of. One of the samples was
placed in a disposable cuvette in an Spectronic 601 spectrophotometer (Milton
Roy, Rochester, NY) and absorbance was measured at 620 nm, zeroing with one
ml of pure media (no microorganims) stored under the same conditions as used in
the study. This value was then compared to the values obtained for the standard
curves (see appendix ll) under optimal growth (37°C for Q w and Q
sporpgenes, aerobically and anaerobically, respectively; absorbance value and
CFU/ml were determined at specific time intervals) to determine the approximate
number of total CFUlml expected for the bacteria tested. A second 1 ml sample
was taken and diluted according to the CFU/ml desired for the plate counts. One
ml of the dilution media was taken and used to inoculate the agar used in the plate
count. For samples in which the dilution was not needed additional 2 ml samples
(1 ml each) were taken from the jars and used for inoculating the agar. Two
replications were performed for each dilution. Dilutions were performed as

necessary to obtain between 25 and 250 CFU's per milliliter (see appendix I). To

37

accomplish anaerobic conditions, BBL® microbiology generator (envelope with

reagents and palladium catalyst for producing H2 and C02) and BBL® GasPak

disposable anaerobic indicator (Becton Dickinson and Co., Cockeysville, MD)

systems were used. Media and incubating conditions for each type of microbial

group were as follows:

9 Aerobic Plate Count: Trypticase Soy Agar (DlFCO Laboratories, Detroit, MI),
with aerobic incubation at 37°C for 48-72 hours.

9 Anaerobic Plate Count: Trypticase Soy Agar with incubation at 37°C for 48-72
hours in anaerobic condition using petri desiccators and high vacuum silicone
stopcock grease (American Scientific Products, McGraw Park, IL) with BBL®
Microbiology System anaerobic condition generator (Becton Dickinson and Co.,
Cockeysville, MD) and BBL® GasPak disposable anaerobic indicator (Becton
Dickinson and Co., Cockeysville, MD). Anaerobic condition was reached when
the color of anaerobic indicator changed from blue to colorless.

Colonies from each plate were determined as described in appendix

l, and calculated as the average colony forming units (CFU) per milliliter of culture

medium. This number was calculated and reported as log1oCFU for each sample.

Strains of Q spl;t_i1i_s_ and Q sporpgenes were strictly aerobic and

anaerobic, respectively. Because of this, growth of Q s_u_bt_ili§ was used as a

microbial indicator of presence of oxygen at any time in the closed system, and

inversely, Q sporogenes was used to detect the absence of oxygen in the food-
package system at the time of sampling.

At the end of the experiment, the final pH was measured using a pH
meter model 240 (Coming Co., Corning, NY). pH in the media for jars containing

Q subtilis as a pure culture had a final pH of 5.11 while for media in jars

 

containing Q sporogenes as a pure culture had a final pH of 5.79.

 

 

38

Statistical analysis of data

To deterrnlne significant differences three factors were taken into
account: a) Bacterial inoculation with 2 levels for determining growth of microbes
(either pure Q .mtjis and mixed culture with assay for Q sgb_tili_s_ or pure Q
smrogenes and mixed culture with assay for Q sporogenes); and 4 bacterial
inoculation conditions determining change in oxygen concentration in the head
space (Control -no bacteria inoculated-, pure Q su_t_3_t_i_l_i§ culture, pure Q
spprpgenes culture, and mixed culture), b) Initial Atmosphere with 4 levels (1%,
5%, and 21% Oz in head space and 21% oxygen plus oxygen absorber), and c)
Time (depending of number of time intervals sampled for each analysis). Two
replications for each possible combination were performed so that the statistical
test performed was a Completely Randomized Design with 3 factor factorial
treatments for microbial growth analysis. Completely Randomized Design with 4
factor factorial was performed for analysis of change in oxygen level in the head
space. The factors analyzed for the change in oxygen level in the head space
were: a) Initial atmosphere concentration (1%, 5%, 21% and 21% + oxygen
absorber); b) Bacterial inoculation (pure cultures of Q subtilis and Q sporogenes

and mixed culture of these two bacteria); c) Temperature (10° and 25°C); and d)

 

Time (0 and 24 hours). MSTAT-C statistical program, version 1.4 (1990) from the
Department of Crop and Soil Sciences and Department of Agricultural Economics,
Michigan State University was used as an aid for all statistical calculations in this

study.

ffect of tern erature and water activi on activit of 0 en absorbers
This study consisted of placing samples with different designations in

constant temperature chambers for 24 hours. Sampling of the head space was

39

performed every hour for 12 hours and at 16 and 24 hours. The characteristics
studied were: (1) Water activity <Aw> with values of 0.88 and >0.99, and (2)
Temperature with two values: 10°C and 25°C. All jars contained media prepared
as suggested by DIFCO (1984). The water activity was adjusted to 0.88 by adding
sugar. The low water activity (0.88) was chosen because the water activity of 0.96
is not very different from the medium not adjusted (Aw >0.99) and still can support
growth of many bacteria (only the more tolerant microorganisms would grow at
0.88); also, the difference of water activity was larger and therefore the evaluation
of the water activity influence was more significant. At 10°C, a final concentration
of 69.02% sugar (wlv) to water was needed and at 25°C, a final concentration of
65.51% sugar (wlv) to water was needed to reach this Aw.

Two replications were used for each designation. Half of the total jars
included an oxygen absorber (FreshPaxW) and the other half were used as the
controls. This study was performed using constant temperature rooms located in
the post harvest laboratory, Department of Horticulture at Michigan State
University. Agitation was not provided to samples. Evaluation of % oxygen in the
head space was performed as described for the microbial study.

The statistical analysis of this study was done using a two-way
analysis of variance for % oxygen in the headspace over time and for each factor
studied, such as: a) Presence or absence of FreshPaxT"; b) Water activity of the
media (0.88 and >0.99); and c) Temperature (10°C and 25°C). MSTAT-C
statistical program, version 1.4 (1990) from the Department of Crop and Soil
Sciences and Department of Agricultural Economics, Michigan State University

was used as an aid for the statistical calculations in this study.

RESULTS AND DISCUSSIONS

From preliminary studies (see appendix II), a Spectrophotometric
method was developed and used as a complementary aid to approximate the
number of bacteria present at the moment of sampling. A growth curve of number
of microorganisms (log CF Ulml) was compared to the absorbance level of samples
over time using a spectrophotometer at 620 nm (this value was observed to give
highest absorbance value for both bacteria in a range of 400-650 nm). Optimal

conditions of growth for Bacillus subtilis and Slostridium sporogenes (37°C and

 

 

aerobic and anaerobic conditions, respectively) were used to obtain a standard
curve for each microorganism. Using the equation corresponding to each bacteria:

. Log Q subtilis CF Ulml = 1.775 (Absorbance) + 6.243138 and

 

. Log Q sporogenes CFU/ml = 1.65392 (Absorbance) + 6.583369
relating the absorbance value with the CFU/ml value from the standard curve an
approximate CFU/ml number was obtained. Dilution corresponding to the CFU/ml
value obtained from the equation was performed and dilutions at a level lower and
higher of this value were also analyzed. Duplicate plate samples were prepared for
the 3 dilutions selected. If absorbance value for the CFU/ml was low (<0.25), the
calibration curve was not used and dilutions of 10°, 10“, 102, 103 and 104 were
prepared, since it was observed that initial growth of bacteria was not detected by

the spectrophotometer (very low absorbance value).

rowth ofB ill u ill in sam les stored at abusive tem erature of 25° and

water activity >0.99
Sample jars stored at 25 :1°C were agitated at 100 RPM at all times.

Samples of 1 ml were taken and aerobic plate count (APC) method was applied

(see appendix l). The statistical results for samples cultured in media with a water

40

 

41

activity of >0.99 are shown in table 4. The calculated F-value was compared to
standard F-test distributions to obtain significance. Significance was reported as a
probability value for each factor. In this study, 99% probability (P<0.01) was used
as the minimum significant level. The model used to analyze the data was a
Completely Randomized Design using a three factor factorial. Storage time (0, 24,
48 and 72 hours); inoculum (whether Q subtfls was inoculated as a pure culture
or combined with Q sporogenes as a mixed culture), and initial oxygen level (1%,
5%, 21%, and 21% + FreshPaxT") in the head space were the factors statistically
evaluated.

In this part of the study, the smallest dilution used was 10°, and the
largest dilution used was 10‘8. If no growth was detected in the smallest dilution,
the log1o CFU number was reported to be less than 2.0 (<2.0). This value was
substituted as (log1o CF U) 2.0 in the mean and statistical analysis corresponding
to the approximate minimum number of microorganisms inoculated in the sample.

All factors and their interactions significantly influenced growth of

Bacillus subtilis (ATCC 9372) at 25°C (P<.01). Time, oxygen, and their interaction

 

were the primary factors that affected the growth of this aerobic bacteria. The
maximum values for the number of microorganisms obtained for this and all other
observations may not represent the real maximum value (the absolute maximum
value could be in a specific instant between the sampling times stablished in this
study). Figures 4 and 5 show the curves obtained for growth of Q ENE at 25°C
in media of Aw >0.99. The log1o number corresponding to the CFU/ml of Q
su_bti_l_i§ at the time of sampling was used to construct growth curves . Numbers of
Q _sztjfls were about the same in the jars containing 5 and 21% oxygen (initially)
in the head space. The media used as the food model was capable of supporting

the growth of both bacteria (table 3). In the presence of Q sporogenes in the

 

 

 

 

42

Table 4: Analysis of Variance for Q subtilis (log CFU/ml) at 25°C and Aw >0.99

 

 

 

 

 

 

 

 

DEGREES OF SUM OF MEAN

FACTOR FREEDOM SQUARES SQUARE
Time 3 142.380 47.460”
lnoculum 1 4.265 4.265“
Time x inoculum 3 6.696 2.232”
Oxygen 3 78.667 26.222”
Time x oxygen 9 90.549 10.061 **
lnoculum x oxygen 3 5.198 ~ 1.733”
Time x inoc. x oxygen 9 19.737 2.193“
Error 32 1.144 0.036
Total 63 348.634

 

 

Coefficient of Variation = 5.41 %

 

 

 

 

Log CFU/ml

 

 

+ 1 % oxygen

—0— 5% oxygen

 

—'— 21% oxygen

\ —<>— 21 % 02 + O.A.

 

 

 

 

 

 

 

 

 

 

 

.\
\‘L
.\ .. q
\ \
X \
\.\
..\\‘,\\
\T..\.
\l
\
\x
\
l l L . I
r T T v 1
1o 20 30 40 50 60 7o 80

Time (hr)
Figure 5: Bacillus subtilis. Mixed culture; 25°C; Aw>0.99

45

mixed culture, Q subtilis did not grow at 1% oxygen but grew when inoculated as a

 

pure culture (figure 4). This may have occurred because Q sporogenes consumed
the nutrients necessary for growth or build up of waste products occurred. Oxygen
was shown to be a very important factor influencing the growth of both Q s_ubt_ll_l_s_
(p<0.01) and Q sporogenes (tables 4 and 5, respectively). No growth was
observed in samples inoculated in the 21% initial oxygen in the head space 4-
FreshPaxT". This is attributed to the anaerobic atmosphere generated in the head
space within 5 hours by the oxygen absorber that did not allow the growth of the

aerobic bacteria.

I ri i m n evaluation of sam les stored at 5° and wat ractivi
>0.99

For Q sporogenes, statistical analysis of factors effecting growth is
presented in table 5 for temperature of 25°C and water activity >0.99. The value of
the mean square presented showed that as in the case of Q sgtLilis, all factors
tested: time (0, 24, 48, and 72 hr); inoculum (either pure or mixed culture); oxygen
level in the head space (1, 5, 21% oxygen or 21 % 02 plus oxygen absorber) were
highly significant, and greatly influenced the growth of Q sporogenes. Figures 6
and 7 show the growth pattern of Q sporogenes as influenced by the various
factors. In the pure culture the bacteria grew in sample jars containing
atmospheres with an initial oxygen content of 1 and 5% 02, and 21% 02 plus
"FreshPaxW". Growth in jars containing 5% initial oxygen had total lower numbers
compared to jars with 1% initial oxygen and 21 % initial oxygen + oxygen absorber.
it is sugested that the redox potential (Eh) in the media was the factor that
influenced the growth of the bacteria. Banwart (1989) sugested that many

anaerobic clostridia would grow at Eh levels from +85 to +160 even with presence

_46

Table 5: Analysis of Variance for Q sporogenes (log CF Ulml) at 25°C, Aw >0.99

 

 

 

DEGREES OF SUM OF MEAN

FACTOR FREEDOM SQUARES SQUARE
Time . 3 129.478 43.159“
lnoculum 1 4.378 4.378“
Time x lnoculum 3 4.823 1608*“
Oxygen 3 51.953 17.318”
Time x oxygen 9 93.350 ’ 10.372"
lnoculum x oxygen 3 16.549 5.516"
Time x inoc. x oxygen 9 19.411 2.157“
Error 32 0.782 0.024
Total 63 320.723

 

 

 

 

 

Coefficient of Variation = 4.65 %

 

Log CFU/ml

47

 

 

._._ 1% oxygen

 

—°— 21 % oxygen

—°“" 21% 02 + O.A.

 

 

 

—U— 5% oxygen _ ——

 

 

 

 

 

Time (hr)

Figure 6: Clostridium sporogenes. Pure culture; 25°C; Aw>0.99

 

Log CFU/ml

OI

48

 

 

 

 

 

 

 

 

 

 

. —+— 1% oxygen
Z\\\ —0— 5 % oxygen __
\ —*— 21 % oxygen
/ —«>— 21% 02 + O.A.
/ \ \i i
\ w
\
\\ \\
\
b
\ \
\
/ \ \
1”,, \‘t x
j/ \,
\ \\

 

 

lL i j % i I 1

—-o-—
P—
—<i——

E?”

0 1 0 20 30 40 5O 60 7O 80

Time (hr)

F lgure 7: Clostridium sporogenes. Mixed culture; 25°C; Aw>0.99

49

of oxygen. The Eh value for the media at the end of the study for samples
containing Q sporogenes as a pure culture was +86. Bacterial growth in jars with
1% oxygen in the head space reached an upper value of log1o CFU/ml of almost
8, which was similar to that found for the 21% oxygen + FreshPaxTM samples. At
48 hours the value was less than 2. Values obtained were similar to those
obtained by Molins et al. (1987) in a study with Q smrogenes PA 3679. One
percent oxygen in the head space was found to be favorable for growth of Q
s r enes (figure 6). The level of bacteria after 24 hours corresponded to a
spoilage level of 106 also used by Elkund et al. (1988) for Q botulinum. Since Q
sporogenes resembles Q botulinum phenotiplcally (Buchanan, 1974), it can be
differentiated only by toxin neutralization in mice, by polyacrylamide gel
electrophoretic examination of soluble cellular proteins, or by gas chromatography
of trimethylsilyl derivatives of whole cell hydrolysates. The toxin produced by Q
botulinum is a protein with a characteristic neurotoxiclty (Kautter et al., 1984).
Kautter et al., (1984) reported that in the US, 766 botulism outbreaks were
recorded from 1899 through 1977. These involved 1961 cases and caused 999
deaths. Of outbreaks in which the toxin type was determined, 199 were due to type
A, 60 to type B, 32 to E, and 1 to type F.

A lower log1o CFU/ml value was obtained for Q sporogenes in jars with 5%
oxygen but high numbers were held for one more day than at 1% initial oxygen
(value of less than 2 reached at 72 hr). This is opposite to that observed for Q
m under the same incubation conditions. Q sporogenes did not grow at 5 %
oxygen (figures 7 and 5, respectively) though Bacillus subtilis did (figure 5) in the
mixed culture. This may have been because Q su_btlll_s_ grew faster initially and
consumed the nutrients required for growth of Q sporogenes since the system was

closed and fresh media was not provided. Another possibility was the build up of

50

waste products by the bacteria that grew first, so that unfavorable conditions were

achieved in the media and the other bacteria could not start their growth.

rowth of 8 oil! b ilis in sam les stored at abusive tern erature of 25° and

water activlg 0.96
For media with water activity of 0.96 (sugar level = 40%) and

temperature of 25°C, only Bacillus subtilis grew. Table 6 shows the statistical

 

results of this evaluation, for Q _s_L_i_b_t_lLs only. Factors considered in the calculations
included: time (0, 0.21, 5, 10, 12, 14, 16, and 18 days); inoculum (Q s_u_b_til_ls in
pure versus mixed culture); and oxygen level initially in the head space where
growth was observed (5 and 21%). All factors excluding initial oxygen level and all
interactions such as time-inoculum, time-oxygen, inoculum-oxygen, and time-
inoculum-oxygen were highly significant and influenced the growth of the bacteria.

Growth of Q subtilis was not significantly effected at 5 or 21% oxygen initial

 

atmosphere at the statistical level of P<0.01. Time and whether or not the bacteria
was inoculated as a pure culture or as a mixed culture with Q sporogenes were
the most influential factors. The difference in time required to produce the same
growth response was approximately 5 days: 5 and 10 days for the mixed and the
pure culture, respectively (figures 8 and 9). A prolonged growth curve was
observed with the reduced Aw (0.96). However, water activity was probably not the
factor that influenced extension of the growth curve since Busta et al. (1984)
reported that the approximate minimum Aw permitting growth (not optimum) of Q
su_gtijjs was 0.90. This number is far lower than the Aw in the media (0.96). Q

subtilis did grow well in the media without sugar (figures 4 and 5). Therefore,

 

extension in the growth response was probably because the metabolic rate (Q

 

 

51

Table 6: Analysis of Variance for Q subtilis (log CF Ulml) at 25°C and Aw = 0.96

 

 

 

 

DEGREES OF SUM OF MEAN

FACTOR FREEDOM SQUARES SQUARE
Time 8 273.464 34.183”
lnoculum 1 19.301 19.301“
Time x inoculum 8 70.497 8.812”
Oxygen 1 0.249 0249*
Time x oxygen 8 21.694 ' 2.712“
lnoculum x oxygen 1 4.813 4.813“
Time x inoc. x oxygen 8 ‘ 16.887 2111'"
Error 36 1.369 0.038
Total 71 408.274

 

 

 

 

 

 

Coefficient of Variation = 4.78 %

 

Log CFU/ml

52

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

9

+ 1 % oxygen

—0— 5% oxygen
8

-—’— 21% oxygen

—<>— 21 % 02 + 0A.
7 /

//
//// /
/ /
6 9
_/
¢//
5 / fl \\
//’/ \
4 // l\ j
l
,x/ \
«)6/ i \
2 —i ¢ ¢ G c i all 8 l
0 5 10 15
Time (days)

Figure 8: Bacillus subtilis. Pure culture; 25°C; Aw=0.96

 

 

 

 

Log CF Ulml

53

 

 

+ 1 % oxygen

_Cl— 5% oxygen

 

—’— 21 % oxygen

/ —<>— 21 %02+O.A.

 

 

 

 

 

 

 

 

 

 

 

 

 

\
\
\. \
¢ ¢ 4 H912 a l
5 1o 15 20

Time (days)
Figure 9: Bacillus subtilis. Mixed culture; 25°C; Aw=0.96

 

 

 

54

subtilis) was lowered due to the osmotic pressure created by the amount of sugar,

as suggested by Pelczar et al. (1983) and Banwart (1989).

Change in head space oxygen content in media with Aw >0.99

Statistical evaluation of the change in oxygen level was based on
analysis taken at 0 and 24 hours, in order to include more factors in the statistical
analysis. Four factors were considered: Head space oxygen content (1%, 5%,
21%, and 21% plus oxygen absorber); inoculum (pure and mixed culture);
temperature (10 and 25°C); and time (0 and 24 hour). Table 7 shows the analysis
of variance for the factors considered and their interactions. From the statistical
results, once comparison of the F values obtained versus the percent points in the
F distribution table, it was found that all factors and interactions had a highy
significant effect on the oxygen content within a 24 hour period.

Two factors and an interaction influenced predominantly in the
change of oxygen concentration: Initial head space oxygen, time, and their
interaction. The initial head space oxygen was important because of consumption

of oxygen by Q subtilis. Figure 10 shows the change in the oxygen present in the

 

head space at 0 and 24 hours for samples with 1% initial oxygen. The change in
oxygen level was about the same magnitude for these samples and the final
oxygen concentrations were below 0.2%, exept for the samples at 25°C containing
Q sporogenes (this samples also had the highest final % oxygen of all, of about
1%). This reduction in oxygen may be due to oxygenation of the liquid media by a
diffusion process, as suggested by Karel (1975). Q fltilfi might have used some
of the oxygen since it grew at the 1% level in the pure culture. The oxygen change
in the 5% 02 jars is shown in figure 11. Initially the media was in an anaerobic

status as indicated by the resazurin indicator (which remained colorless) and with

55

Table 7: Analysis of Variance for % 02 change in heaspace. Aw >0.99

 

 

 

 

 

 

 

DEGREES OF SUM OF MEAN

FACTOR FREEDOM SQUARES SQUARE
Headspace (A) 3 5250.062 1 750.021 **
lnoculum (B) 3 57.152 19.051 **
headspace x inoculum 9 106.424 11.825“
Temperature (C) 1 36.643 36.643”
A x C interaction 3 107.852 35.951 **
B x C interaction 3 55.552 18.517“
A x B x C interaction 9 109.733 12.193”
Time (D) 1 1876.478 1 876.478”
A x D interaction 3 1886.054 , 628.685”
B x D interaction 3 55.681 18.560”
A x B x D interaction 9 109.988 12.21“
C x D interaction 1 61.138 61 .138”
A x C x D interaction 3 97.419 32.473“
B x C x D interaction 3 49.728 16.576“
A x B x C x D interaction 9 110.230 12.248"
Error 64 1 .033 0.016
Total 127 9971.167

 

 

Coefficient of Variation = 1.59 %

 

 

 

 

 

 

 

 

 

 

 

 

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/
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............. .u... n... . [a .5... ....” ......“ .....m..r_..v.ta........T...1sw..~3a.V

. r.
r .n
h 4
0 2
= =
t t

/ fl / .V
_ _ a _ A d
6 5 4 3 2 4| 0

woman one: o5 E cogxo .x.

 

mocoaolaw d ”o mmuh

 

2:53 d ”o mm»;

.2250 no own...

mocomocomm d ”o o _.u._.

 

2:53. d no Sup

85:00 ”0 Drug.

Figure 11: Oxygen change (%); 5% initial oxygen in the head space

58

introduction of the modified atmosphere and agitation, oxygen may have become
dissolved into the media (indicator turned red, which indicates aerobiosis). Within
24 hours for all samples at 10°C and at 25°C for the control or in Q sporogenes

samples, there was no significant difference in the percent oxygen change. There

 

was a large change in the samples inoculated with Q subtilis at 25°C. This was

probably because the oxygen was consumed by Q'subtilis that grew to a value of

 

>8 log CFU/ml (figure 4). The 21% initial oxygen present in the head space was

lowered significatively in the jars containing Q subtilis at 25°C (figure 12). This

 

was also found in the jars with 5% initial oxygen (figure 11). The oxygen in the
21% jars was reduced to less than 0.5%. At 25°C, in jars with an initial oxygen
concentration of 21% the concentration was lowered to about 0.1% for some

samples (figure 12). Q subtilis reduced the oxygen from 21% to levels similar to

 

those obtained by vacuum or gas flushing systems (approximately 0.5%). This

suggests that even if there was no initial growth of Q sporogenes in these jars, Q

 

smrogenes could start growing. Q subtilis produces a toxin called "subtilin", which
is a polypeptide with marked action against a wide range of Gram positive
bacteria, and depending on concentration, it can be bacteriostatic or bactericidal.
This antibiotic is known to prevent outgrowth of bacteria after gerrnlnation.
However, Ayres et al. (1980) reported that this toxin does not halt growth of Q
smrggenes PA 3679. Thus, it is unlikely that this inhibited Q sporogenes in the

jars containing both bacteria and where only Q subtilis was observed to grow. It is

 

probable that in jars with 5% oxygen initially in the head space Q subtilis stopped
growing because the oxygen was reduced and Q sporogenes did not grow

because there was a lack of nutrients in the media.

in figure 13 is shown the change observed in the oxygen level for

sample jars initially containing 21% oxygen + FreshPaxT". In all samples, oxygen

 

 

 

 

59

 

25-

 

Clt=0hr. Bt=24hr

 

{—7

   
  
  

20-

_.l
0'
I

% Oxygen in the head space
a
l

 

 

 

 

 

 

 

 

 

 

 

01 A
‘6 en U) B (I) m
a E 8 t: 3.5.. 8
C .0 o C .D Q
3 3
3. "a e 3. "f e
0 ml 0 0 ml 0
9 (5 "f “.3 6 "’.
.1 2 0' .l :3 0'
u ('5 u ('5
1- 2 1- 3
II II
1- 1—

Flgure 12: Oxygen change (%); 21% initial oxygen in the head space

60

 

 

’Dmom. 4: t=24hrj

 

25 —/

 

 

 

 

 

 

 

 

 

W mocmmouomm d ”o mm"...

W 1w.

.11 l - .l 1 2:53 m Mo mmuh

 

1.111 l l .l 1 ill. _ozcoouo mmnh

.. i ll:. 1 l mloco. mouomm 1o. no 9n:

l
H
i
‘1
‘l
l

 

 

__1

2:33 ..m no 9":

 

/
a
0
1

woman cum; on. E coo>xo _x.

15—K

6:50 no 09”.:

 

 

mm
P
h
s
e
r
F
+
n
e
9
V.
x
o
...Ila
.6“
.m
%
1
2
voh
o/
(
e
9
n
m
n
e
9
y
x
O
3.
1
m
u
.nluv
F

61

level fell to below 0.5%. Variations of 0.4 to 0.5 % oxygen were found in samples
where Q sporggenes grew (figs. 6 and 7). All other jars had oxygen values less
than 0.2% in the head space after only 5 hours at 25°C, and 12 hours at 10°C. No

growth was observed for Q subtilis at any temperature when the oxygen absorber

 

was used. Q sporogenes was found to grow rapidly at the 25°C storage
temperature in the presence of the oxygen absorber (figures 6 and 7). Figure 14
shows the oxygen change in jars containing a mixed culture and either 1 or 5%
initial oxygen in the head space. After 24 hours, initial oxygen in the head space
was reduced to about the same level in samples with 1% initial oxygen, with
growth observed for Q sporogenes (figures 6 and 7). Q subtilis grew in samples at
25°C and 5% oxygen (figures 4 and 5). in figure 15 is shown oxygen content in jars
inoculated with the mixed culture and either 21% initial oxygen or 21% oxygen +
FreshPaxT". The oxygen (%) changed significantly in all jars at 25°C, and in jars
stored at 10°C with oxygen scavenger. The only jars that maintained about the
same oxygen levels as the controls were samples with bacteria and no
FreshPaxT'" stored at 10°C. The decrease in oxygen from 21% to <1% in jars
stored at 25°C without FreshPaxT" is attributed to consumption of oxygen by Q

flIb—tik-

Bagillgs subtilis evaluation of samples stored at 10°C and water activig >0.99

All samples stored at 10°C were studied at a water activity of >0.99.
In table 8 is shown the analysis of variance for the results collected. Time (0 to 4
weeks) and the oxygen level (1%, 5%, 21% and 21% plus oxygen absorber)
affected significantly (P<0.01) the growth of Q subtilis _a_t 1Q°C. The other factor,

 

inoculum method and interactions of all possible combinations were non-

 

 

 

 

% oxygen in the head space

62

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6 _
[:lt=0hr. Et=24hr g
5 _
4 .—
3 ._
2 _
/

1 ~/

T=10C; T=25C; T=10C; T=25C;

1% 1% 5% 5%
oxygen oxygen oxygen oxygen

Figure 14: Oxygen change (%); mixed culture

% oxygen in the head space

63

 

 

[51:01“. It=24fl

 

£7

 

 

 

 

 

 

T=1 0C;
21 %
oxygen

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T=2sc; T=10C; T=25C;

21 % 21 % + 21 % +
oxygen CA. GA

Figure 15: Oxygen change (%); mixed culture

Table 8: Analysis of Variance for Q subtilis (log CFU/ml) at 10°C and Aw >0.99

 

 

 

 

 

 

 

DEGREES OF SUM OF MEAN

FACTOR FREEDOM SQUARES SQUARE
Time 4 83.230 20.808"
lnoculum 1 0.018 0.018
Time x inoculum 4 0.200 0.050
Oxygen 3 42.835 14.278”
Time x oxygen 12 20.661 1.722
lnoculum x oxygen 3 12.660 4220*
Time x inoc. x oxygen 12 13.937 1.161
Error 40 29.866 0.747
Total 79 203.407

 

Coefficient of Variation = 24.63 %

 

65

significant, thus, they did not influence significantly (P<0.01) the growth of the
bacteria at 10°C.

Figure 16 shows a continuous pattern for growth of Q subtilis (pure

 

culture) at levels of about 105 CFU/ml through the fourth week in jar samples
containing the oxygen absorber. Growth under anaerobic conditions was observed
to yield large numbers; this was not expected since the bacteria was aerobic. This
may be due to the presence of glucose in the media, and some authors, as Ayres
et al. (1980) reported that under anaerobic conditions, species in the Mug
group fermented sugars; and Buchanan (1974) reported that glucose permit a
much restricted anaerobic growth of Q Ms in complex media. Bacterial growth
in the mixed culture was not detected until the third week, with levels attained of
about 104 CFU/ml and at the fourth week about 3 x 104 CF Ulml. In these samples
the oxygen was depleted to less than 0.1% in about 12 hours. By comparing
figures 4 and 16, in which Q _sgbtjljs was inoculated as a pure culture, no growth
was observed at 25°C and growth was observed with values of almost 105 for
samples incubated at 10°C. This was attributed to various possible reasons: 1) pH
change from 7.2 to 5.11. Banwart (1989) reported that the optimum pH for Q

subtilis was markedly changed by altering the incubation temperature, so that the

 

optimum pH was 7.4 at 37°C and 5.4 at 10°C; 2) Incubation time was longer at
10°C compared to 25°C. This probably allowed the bacteria to grow slowly initially
in samples incubated at 10°C and lower the pH so that it generated a more
favorable condition for growing and generating large number of cells.

Figure 17 shows growth of Q subtilis after week 2 for samples

 

containing a mixed culture, with values of about 4 x 105 CFU/ml at the fourth
week; these sample jars had 21 % initial oxygen in the head space. No growth was

observed for samples containing Q subtilis at any time when it was inoculated as a

 

 

 

 

66

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5 f 25
——°— Pure culture /
4.5 ‘
—<>— Mixed culture / O
a + Oxygen (p.c.) I
4 1:7 20
—1:— oxygen (m.c.) My
3.5
§
3 15 3
— 3
E .8
E o
o 2 5 5
0’ .E
3 5
U)
2 1o 3‘
33
1.5
1 .\ 5
0.5 ‘
0 . Via-3’53 0
0 0.071 1 2 3 4

. . _ . Time (weeks)
Figure 16: Bacullus subtilis CFU/ml and oxygen change (%). 10°C; 21 %

oxygen in the head space + FreshPaxTM

Log CF Ulml

Time
Figure 17: Bacillus subtilis CFU/ml a

(.0

67

 

 

 

 

 

 

 

 

 

 

 

 

 

(weeks)

 

25
‘—'— Pure culture
—<>-'— Mixed culture
- —'— Oxygen (p.c.)
—0— Oxygen (m.c.) * 20
4/
/ .- .5
/
2x\ ,1/
/ -- 1o
\
\k
l.
\\ /
\ /
\\ / ~~ 5
\\ fi/
/.
\ /
\\ /
1 1 1 o
0 1 2

% oxygen in the head space

nd oxygen change (%). 10°C; 21% initial
oxygen in the head space

68

pure culture over the same time period. This is sugested to dissolved oxygen had

an inhibitory effect on growth of Q subtilis. Banwart (1989) reported that dissolved

 

oxygen had an inhibitory effect on Bacillus fragilis, and showed that was

 

independent of the oxidation-reduction potential. The oxido-reduction potential in

the media with Q subtilis as a pure culture at the end of the study was of +99 mv,

 

which is in the range of growth of +85 to +160 mv as reported by Banwart (1989).
Oxygen level was about the same for samples containing pure culture and mixed
culture during this period. Oxygen in the head space was reduced from 21% to
about 16%. This is attributed to oxygen dilution in the liquid media which was also
reported by Piper et al. (1989), who found that the amount of gas which partitions
between water and the atmosphere is in equilibrium and inversely proportional to
the temperature. Temperature was the most important factor affecting the oxygen
concentration in water. The oxygen level in the head space of samples at 10°C at
equilibrium was 16% while in samples stored at 25°C it was 18%. This indicates
that more oxygen dissolved in the media at 10°C since all samples had about 21%
initial oxygen in the jar head space.

Figure 18 shows the relationship between the log CFU/ml and time,
and also the relationship between % oxygen and time for Q am in samples
with 5% initial oxygen in the head space. The most bacterial growth was obtained
in sample jars with 5% initial oxygen in the head space as compared to the other
initial oxygen concentrations at 10°C. Logaritmic growth was detected during the
first 3 week period, with about 5.0 x 107 CFU/ml for samples containing pure
culture and about 4.8 x 106 CFU/ml in the samples containing a mixed culture. By
the fourth week this had decreased to 1 x 107 CFU/ml and 5.8 x 105 CFU/ml,
respectively. Oxygen in the head space decreased from about 4.5% in both pure

and mixed culture to about 2%. For the bacteria cultured at 10°C a decrease in

69

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

8 5
—~ 4.5
7
-- 4
6
—- 3.5
5 §
1 3 g-
— a
E 2
D
... 4 2.5 2
o E
5 '3
o:
-_ 2 5‘
3 32
+ 1.5
2 —*—- Pure culture
—<>— Mixed culture + 1
1 + Oxygen (p.c.)
—D—- Oxygen (m.c.) “ 0'5
0 1 1 1 1 0

 

O 1 2 3 4

__ Time (weeks) . ..
Figure 18: Bacillus subtilis CFU/ml and oxygen change (%). 10°C; 5% initial

oxygen in the head space

70

oxygen was observed but not to the low levels (0.1%) found for the samples stored
at 25°C (more bacterial growth) (see figures 4, 5, 11, and 12).

In figure 19 is shown the growth of Q su_btifls at a concentration of
1% oxygen in the jar head space and at 10°C. Growth was observed to be
logarltmlc for bacteria inoculated as a pure culture while a slower rate was found
for bacteria inoculated as a mixed culture. The levels attained at the fourth week
were 3.2 x 106 and 2.3 x 104 CFU/ml for pure and mixed culture, respectively.
Oxygen in the head space was lowered from about 0.55% to about 0.1% for both

the pure and mixed culture.

Growth of los ridium 5 en in sam les stored at 10° and water activi
>0.99

Analysis of variance for factors effecting growth of Q sporogenes at
10°C is presented in table 9. There was no single factor or interaction between
factors that significantly influenced growth of this bacteria. Figures 20, 21, 22, and
23 present the growth curves for Q sporogenes (Log CF U/ml vs. time), and %
oxygen change in the head space vs. time for samples stored at 10°C. No growth
was observed in samples with initial oxygen in the head space of 21% and 5%
(figures 21 and 22). Decreased log CFU/ml values from about 200 to 10 CFU/ml
during the 4 week period was observed for samples containing either FreshPaxT"
or 1% initial oxygen.

The amount of oxygen in samples containing Q sporogenes was
very similar to that in the controls (compare oxygen levels of figures 20-23 with
figure 24). The oxygen levels in samples containing 21% initial oxygen was

reduced to about 16%; for samples of 5% initial oxygen, oxygen levels were about

 

71

 

0.7

 

 

 

+ Pure culture
—<>— Mixed culture /
0.6

+ Oxygen (p.c.) /

 

 

—0— Oxygen (m.c.)

 

 

// o...

 

Log CFU/ml

 

 

/ .
‘\
\\\
% oxygen in the head space

0.3

 

 

0.2

 

 

O

 

‘1
r
O

1 2 3 4
Time (w

eeks
Figure 19: Bacillus subtilis CF U/ml and oxyg)en change (%). 10°C; 1% initial

oxygen in the head space

72

Table 9: Analysis of Variance for C.sporogenes (log CF Ulml) at 10°C and

 

 

 

Aw >0.99
DEGREES OF SUM OF MEAN
FACTOR FREEDOM SQUARES SQUARE

Time 4 0.002 0.001
lnoculum 1 0.000 0.000
Time x inoculum 4 0.001 0.000
Oxygen 3 0.001 0.000
Time x oxygen 12 0.005 0.000
lnoculum x oxygen 3 0.001 0.000
Time x inoc. x oxygen 12 0.006 0.001
Error 40 0.018 0.000
Total 79 0.034

 

 

 

 

 

Coefficient of Variation = 1.05 %

 

73

 

 

 

 

 

 

 

 

 

 

 

 

 

2 fl xx 25
H—fi\
\
\
1.8 \ fl
\
1.6 [1 \ \ //;?X 20
\ / \
1.4 V ‘ 1
\\ 8
co
9.
m.
, , 'o
_ \\ g
E \1 5
E t. .e
o 1 / 1. o
C» 5:”
o
_, . m
1' 5
0.8 1 1o 8
\ 1 °’
1 5
l 1 5
0.6 \ I
1
1’
0.4 \(1 5
11, —°— Pure culture
I“ —>—- Mixed culture
0.2 + Oxygen (9.0-)
—0— Oxygen (m.c.)
0 1 1 426% 0
O 0.071 1 2 3 4

 

 

 

 

Time weeks)

Figure 20: Clostridium sporogenes C Iml

and oxygen change (%). 10°C;

21 % initial oxygen in the head space + FreshPaxTM

Log CFU/ml

74

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2
1.8 X?
1.6 \\
\.
\
1.4 \
1.2
1
0,3 —*— Pure culture
“‘3‘" Mixed culture
0.6 + Oxygen (p.c.)
—0— Oxygen (m.c.)
0.4
0.2
0 1 1 s 1 '2 1 1'

 

 

 

0

1 2 3 4
Time weeks)

25

20

_s
U"

8
% oxygen in the head space

Figure 21: Clostridium sporogenes CF Iml and oxygen change (%). 10°C;

21 % initial oxygen in the head space

75

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2 5
1.8 k , 4.5
\\ —°—- Pure culture
\ ‘ —<>— Mixed culture
1.6 .. 4
+ Oxygen (p.c.)
—0— Oxygen (m.c.)
1.4 \ 3.5
\ §
1.2 \ \ 5 3 ‘9,
E 3
5 .5
:3 1 ‘1 \ 2 5 g
5 - e
U)
0.8 2 5‘
32
0.6 1.5
0.4 1
0.2 0.5
0 1 1.: 1: 4o» 1 o 1 a o
0 1 2 3 4

. . Time weeks)
Figure 22: Clostridium sporogenes C Ulml and oxygen change (%). 10°C; 5%

initial oxygen in the head space

76

2.5 0.8

 

 

—*— Pure culture

'l
—- 0.7
\% —<>—- Mixed culture
2

—+— Oxygen (p.c.)
K —G—— Oxygen (m.c.) ‘” 0-6

 

 

 

 

 

O
01

 

 

Log CF Ulml
_t E;
> \\/o
/ //
O
J:
% oxygen In the head space

6
/ .
\\.

 

 

 

 

 

~~ 0.2
0.5 \\ f
A
—~ 0.1
0 l l l l 0
0 1 2 3 4
Time weeks)

Figure 23: Clostridium sporogenes C Ulml and oxygen change (%). 10°C; 1%
initial oxygen in the head space

77

 

 

25
§
cm; 20 ‘K
'0
3
O
5
E 10 /
C
Q
3 5
f; M 21% 02 + O.A.
° N 21 % oxygen
-.~ 5% oxygen
00 0.071 1 2 / 1% oxygen
3
Time (weeks) 4

Figure 24: Oxygen change (%) in the head space for control samples. 10°C

78

2%; for samples with 1% initial oxygen, oxygen levels were about 0.3%; and for all
samples containing FreshPaxT", the oxygen level was reduced to less than 0.1%.

Altough no growth of Q_. sporogenes was observed, a comparison
with 9_. botulinum strains in terms of growth and toxin production can be performed
as follows: Kautter et al. (1984) reported that the optimum temperature for C_.
botulinum growth and toxin production of the proteolytics (all A, and some B and F
strains) is close to 35°C, while that of the nonproteolytics (all E, and remaining of
B and F strains) is approximately 26°C. They reported that nonproteolytic types 3,
E, and F are able to produce toxin at refrigeration temperature (3°C to 4°C), but
toxins of the nonproteolytic do not manifest maximum potential toxicity until they
are activated with trypsin; and toxins of the proteolytics generally occur in fully, or
close to fully, activated form.

Kautter et al. (1984) concluded that a food can contain viable C_.
botulinum and still not be capable of causing botulism, and as long as the
organisms do not grow, toxin is not produced. The usual vehicles of botulism are
those foods which are processed to prevent sopilage, and which are not usually
refrigerated. Also, unless the temperature is very precisely controlled and kept
below 3°C, refrigeration will not prevent growth and toxin formation by

nonproteolytic strains.

Temperature effect on ongen absorber

Table 10 shows the two-way analysis of variance for % oxygen in
sample head space as a function of time and in the presence of FreshPaxT". The
statistical analyses presented in tables 10, 11, and 12 were calculated for the first

12 hours (0, 1, 2, 12 hours). The oxygen absorber decreased the % oxygen

79

Table 10: Two-way Analysis of Variance for % oxygen in the head space over time

and in presence of F reshPaxT"

 

 

 

 

DEGREES OF SUM OF MEAN
SOURCE FREEDOM SQUARES SQUARE
Time 12 235.96 . . 19.664
Oxygen absorber 1 1624.91 1624. 908“
Error 12 217.77 , 18.147
Total 25 2078.64

 

 

 

 

 

Coefficient of Variation = 34.40 %

80

Table 11: Two-way Analysis of Variance for % oxygen in the head space over time

and as affected by water activity (Aw)

 

 

 

 

 

 

 

 

DEGREES OF SUM OF MEAN
SOURCE FREEDOM SQUARES SQUARE
Time 12 235.96 19.664“
Water activity 1 0.32 0318*
Error 12 0.68 - 0.057
Total - 25 236.96

 

Coefficient of Variation = 1.92 %

 

 

81

Table 12: Two-way Analysis of Variance for % oxygen in the head space over time

and as affected by Temperature

 

 

 

DEGREES OF SUM OF MEAN
SOURCE FREEDOM SQUARES SQUARE
Time 12 235.96 19.664“
Temperature 1 1 8.23 1 8.226“
Error 12 12.17 ' 1.014
Total 25 266.36

 

 

 

 

 

 

Coefficient of Variation = 8.13 %

82

change in the jar head space with a significance at the 1% level compared to
sample jars without FreshPaxT". Further orthogonal contrast for presence vs. no
presence of FreshPaxTM showed an F value of 89.539 with effect of -7.905 and a
probability of 0.0%, which confirmed the highly significance effect of the
FreshPaxT“ on the % oxygen in the head space. Figure 25 shows the % oxygen
for samples stored at 10°C and 25°C containing media with a Aw of >0.99.
Controls % oxygen did not change over time. Jars containing FreshPaxT" had
oxygen levels in the head space of less than 0.1% in about 7 hours for samples
stored at 25°C and in about 12 hours for samples stored at 10°C.

In table 11 is shown the statistical evaluation for the influence of
analysis of variance for % oxygen head space as affected by time and water
activity (Aw). Water activity did not influence significantly (P<0.01) the % oxygen in
the head space of the samples. Orthogonal contrast was performed on the water
activity factor and an effect of 0.111 with an F value of 5.616 and probability of
3.5% was found. This indicates that at a 1% level of significance, water activity did
not influence the % oxygen in the head space of the samples studied. Results for
samples with water activity of 0.88 are shown in figure 26. By comparing figures 25
and 26 it is shown that values were very similar under the same conditions.
Controls had about the same level of oxygen over the time period studied. For
samples containing oxygen absorber and stored at 25°C % oxygen in the head
space decreased to less than 0.1% in about 5 hours while for samples stored at
10°C the % oxygen in the head space decreased to the same amount in about 12
hours.

Table 12 shows the two-way analysis of variance for % oxygen in
the head space as a function of time and temperature. Temperature was observed

to significantly (P<0.01) affect the activity of the oxygen absorber. Orthogonal

83

 

 

 

 

 

 

 

 

 

 

 

 

25
20
4:.

8 15

to

‘O

as

.C

Q

5

.E

C

m

s \

o 10 X

32 1
—'— Control, 25°C
_Cl— FreshPax'“, 25°C
—’— Control, 10°C

5 ——<>— FreshPax'”, 10°C —
0 M53:— ; :2 f 9—1

 

 

 

Time (hr)

Figure 25: Temperature effect on oxygen absorber. Aw>0.99

25

20

_s
01

_x
O

% oxygen in the head space

 

 

 

l
\ 1 ——I— Control, 25°C
1 _Cl— FreshPax’“, 25°C

——°—— Control, 10‘C

 

 

" J‘ ——<#*— FreshPax‘". 10°C

 

 

 

Time (hr)

Figure 26: Temperature effect on oxygen absorber. Aw=0.88

85

contrast for the temperature effect yielded a statistical effect of 0.837 with and F
value of 17.971 and a probability of 0.1%, which confirmed that it had a significant
effect at the 1% level of significance. By comparing figures 25 and 26 a difference

was observed at 10 and 25°C for samples containing F reshPaxT".

CONCLUSIONS

A food-package model system was inoculated with _B_._ subtilis ATCC

 

9372, C_. sporogenes ATCC 11437, and a mixture of both. Growth of the
microorganisms as influenced by several factors (time, inoculum, and oxygen
level) was determined. Change in percent oxygen in the head space of the
samples was also determined. It can be concluded that:

1) The effect of different inoculum (pure vs. mixed cultures) was significant at the
level of P<0.01 for both ; Mg and Q sporogenes in the temperature abusive
condition (25°C). Growth of aerobes rapidly increased with oxygen head space
levels of 1, 5, and 21 % for the pure culture and at 5 and 21 % for the mixed culture.
Anaerobes increased rapidly at 1 and 5% and 21% + oxygen scavenger for pure
culture samples. Anaerobes also increased rapidly at oxygen levels of 1% and
21 % + oxygen absorber for the mixed culture.

2) The effect of two Aw (40% sugar -Aw=0.96- and Aw>0.99) influenced the growth
of ; gubLlls. At the low Aw, growth was extended to about 16 to 18 days. This Aw
also prevented the growth of Q sporogenes.

3) Time was found to have a highly significant effect (P<0.01) for all inoculums for
samples stored at 25°C. Bacterial numbers increased with time and values of
about 107 CFU/ml were obtained as a maximum.

4) Oxygen level (1, 5, 21, and 21% + FreshPaxT") was found to have a highly
significant effect on bacterial growth at the level of P<0.01. _B_. m did not grow
at 25°C even at high initial values of oxygen (21%) when the oxygen absorber was

present because anaerobic conditions were obtained very rapidly (approximately 5

86

87

hours) which prevented aerobic growth. Anaerobic bacteria were able to grow in
media containing high initial oxygen content (21%) after the oxygen was
scavenged. Interactions of time-inoculum and time-oxygen level were also found to
be highly significant. .

5) The effect of F reshPaxT" in jars containing 21% initial oxygen was found to be
highly significant (P<0.01). Oxygen was lowered to less than 0.5% in the
headspace after 5 hours at 25°C and after 12 hours at 10°C.

6) At 25°C and media with Aw of 0.96 (40% sugar) the oxygen level was not
significant on growth of Q ML”! in sample jars with 5% and 21 % initial oxygen in
the head space.

7) At 25°C and media of Aw 0.96, growth of Q sub_tiis_ was delayed and CFU/ml
values of 1 x 107 for samples containing 21% and 5% initial oxygen in the head
space were not obtained until 12 days had passed.

8) Samples stored at 10°C (media Aw >0.99) did not support the growth of Q
sporogenes even under anaerobic conditions. Numbers of Q sporogenes cells
decreased for sample jars with initial oxygen concentrations of 1% and 21% with
the oxygen absorber. For samples where the initial oxygen concentrations were
5% and 21 %, no growth was observed at any time during weeks one to four.

9) At 10°C Q m growth was detected through the 4 week incubation for
samples containing 1% and 5% initial oxygen and reached >106 CFU/ml for the
pure culture and for the mixed culture with 5% initial oxygen. Samples inoculated
with mixed culture and 1% initial oxygen had values of about 2.3 x 104 CFU/ml for
_B_. sgbtjjs at the fourth week. In samples containing 21% initial oxygen growth of
Q Mills was detected for the mixed culture after the second week. No growth

was observed in samples containing pure culture and 21% initial oxygen. Growth

 

88

was observed (3.1 x 104 CFU/ml at the fourth week) for samples containing
FreshPaxT".

10) No water activity -Aw- effect was observed to influence the FreshPaxT"
effectiveness in the values studied (0.88 and >0.99).

11) Temperature was observed to effect the effectiveness of the oxygen absorber
but only in the short time (the first 12 hours), since after 12 hours the oxygen
levels are similar.

12) FreshPaxT" (oxygen absorbers) could be used in foods to remove the oxygen
in the head space only if refrigeration below 3°C is always maintained once the
oxygen absorber is introduced and the package is closed. Also in products held
above 3°C if the food, naturally or by design, is acid, has low water activity, a high
sodium chloride concentration, an inhibitory sodium nitrite concentration, or two or

more of these in combination.

APPENDIX

APPENDIX |

Standard Colony Count Methods

Based on Busta et al (1984), each sample in the study was taken and
diluted according to the dilution recommended by the use of spectrophotometer
technique in a range of 10"1 to 101 (3 dilutions total). The dilution was related to
the absorbance value for each specimen analyzed. Duplicate plates from the
sample were individually counted and calculated as units of CFU per milliliter
sample following these steps. Reported number of CFU per milliliter sample was
averaged from the CF U value calculated from each plate of the same.

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

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

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

4) If the growth in all plates was more than 250 colonies - then it was reported as
more than 2.5x106 CF U per milliliter. An estimated number of colonies in plate was
obtained from the average count of 4 (four) 1x1 cm squares selected randomly in
the most diluted solution, then multiplying this number by the plate surface area (in

cm2).

89

APPENDIX II

Spectrophotometric Method

A spectrophotometric method was developed and used as a
complementary aid to approximate the number of bacteria present at the moment
of sampling. This was performed to avoid utilization of extra of petri dishes and to

save time at the moment of sampling. Growth of Q subtilis and Q sporogenes was

 

monitored by using an absorbance method (figure 27) and a plate count method
(figure 28). The relationship between log CFU/ml and absorbance for Q subtilis
and Q sporogenes is shown in figures 29 and 30 as obtained in thioglycollate

medium at 37°C. Aerobic and anaerobic conditions were set for Q subtilis and Q

 

sporogenes, respectively (optimal growth conditions). A growth curve of number of
microorganisms as log1o CFU/ml was compared to the absorbance level of
samples over time using a spectrophotometer at 620 nm (this value was observed
to give highest absorbance value for both bacteria in a range of 400-650 nm).

Optimal conditions of growth for Bacillus subtilis and Clostridium sporogenes

 

 

(37°C, aerobic and anaerobic conditions, respectively) were used to obtain a
standard curve for each microorganism (see figures 31 and 32).
Using the equation corresponding to each bacteria:

. Log Q subtilis CFU/ml = 1.775 (Absorbance) + 6.243138 and

 

. Log Q sporogenes CFU/ml = 1.65392 (Absorbance) + 6.583369
relating the absorbance with the CFU/ml, from the standard curve an approximate

CFU/ml for the bacteria analyzed was obtained. This value was then used as a

90

 

91

 

1.6

 

1.4

1.2

 

a—l

 

Absorbance (620 nm)
0
Q)

 

 

 

 

 

 

 

 

 

0.6 /
/
0.4 Pr’
,1” + Q subtilis
,/
0.2 ‘ :1 ._
/ /
///
o 1; r + 1 + 1 1
o 4 6 8 1o 12 14

Time

hr
Figure 27: Absorbance vs. time forIQ )subtilis and Q sporogenes.

Thioglycollate medium; 37°C

8.5

7.5

Log CFU/ml
O)
01

5.5

4.5

92

 

 

 

,r’i/w

 

' /
/ /
/ /

/
1

 

 

 

 

 

 

 

 

 

 

 

/
/
f
f/ + Q subtilis
/)1{ —U— Q sporogenes
l l l l l l l
0 2 4 6 8 10 12 14

. _Time (hr) . .
Figure 28: Log CF Ulml vs. trme for Q subtilis and Q sporogenes.

Thioglycollate medium; 37°C

Log CFU/ml

.5

01

93

 

 

 

 

 

 

 

l
I

0.2

l
I

o4

I J
I I

0.6 0.8
Absorbance (620 nm)

“‘1"

1

I
r

1.2

l
I

1.4

Figure 29: Log CF Ulml vs. absorbance for Bacillus subtilis. 37°C

 

 

Log CFU/ml

 

 

8.5

 

 

7.5

6.5

 

5.5 —'

 

 

 

Ll
l

O

I I I I I I I I
I I I I I I 7 I

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Absorbance (620 nm)

Figure 30: Log CF Ulml vs. absorbance for Q sporogenes. 37°C

 

Log CFU/ml

95

 

 

 

 

\.

/ //

 

 

 

 

 

 

7 // ///
/

 

 

/ Log CF Ulml = 1.775 (Absorbance) + 6.243138

6.5
1/ R = 0.1991

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

o 0.2 0.4 0.6 0.8 1 1.2 1.4
Absorbance (620 nm)

Figure 31: Standard curve for Bacillus subtilis

 

Log CFU/ml

8.5

.‘1
on

6.5

96

 

 

K

 

 

//

 

,7

 

 

 

 

 

 

 

Log CF Ulml = 1.65392 (Absorbance) + 6.583369

 

 

I

 

 

R = 0.984

 

 

 

 

 

 

 

 

 

 

 

0.2 0.4 0.6 0.8 1 1 .2
Absorbance (620 nm)

Figure 32: Standard curve for Clostridium sporogenes

 

1.4

 

97

base idea for making the respective dilutions as follows: 1) Absorbance value was
put in the equation depending on the bacteria tested; 2) A log CF Ulml value was
obtained; 3) Inverse of the log CFU/ml value was performed (10") so that a
CFU/ml value was obtained for the bacteria; 4) From this number, depending on
the exponent, a dilution corresponding to yield 100 CFU/ml was performed (log
CFU/ml = 2), also a dilution of 10 CFU/ml and 1000 CFU/ml (log CFU/ml = 1 and

3, respectively), to assure plates with 25-250 colonies counts.

 

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