(0N0)

 

HIGANS TSATE

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IIIIIIIIIIIIIIIIIIIIIlIIIIIIII

300917 3109

This is to certify that the

thesis entitled

EFFECT OF HICROBIAL LOAD 0N VACUUH PACKAGED FRESH
GROUND BEEP PA'ITIES SUBJECTED TO "IN PACKAGE"
THERMAL TREATMENT

presented by

PAKORN HANGIAIUIBOON

has been accepted towards fulfillment
of the requirements for

Jain—degree in ML

Major professor
Bruce R. Harte, Professor

Date _Se.p.f_emb_er_ll._1990

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

 

 

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PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

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ammo-9.1

MSU Is An Affirmative Action/Equal Opportunity Institution

“-.__._..__——-— ~— —- —-

 

EFFECT OF MICRoeIAL LOAD on VAC'UUM PACKAGED FRESH
GROUND BEEF PATTIES SUBJECTED To "IN PACKAGE" THERMAL
TREATMENT. '

BY

Pakorn Manomaiwiboon

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

School of Packaging

1990

IJ+ / '— 1)" UK,

ABSTRACT
EFFECT OF MICROBIAL LOAD ON VACUUM PACKAGED FRESH
GROUND BEEF PA'I'I'IES SUBJECTED TO "IN PACKAGE" THERMAL
TREATMENT.
By .

Pakorn Manomaiwiboon

 

The microbial load of fresh, vacuum packaged, cook-ln-bag uncured beef patties
was determined in two film stmctures, a commercial (PE/EVOH), and super barrier (Si02 coated
polyester) material. Packaged samples were cooked to internal temperatures of 71 and 82°C for
30 min., and stored in temperature abused (23:206) and refrigerated storage (4-60C). Barrier
properties had a significant effect (P<0.001) on aerobic and mesophilic growth in the abused
condition. Cooking temperatures had a statistically significant effect (P<0.05) on aerobic growth in
the refrigerated condition. The growth of anaerobes and psychrophiles were not significantly
effected by either variable. Storage time had the most significant effect (P<0.001) for all groups of
microorganism.

The model organism, C.sporogenes ATCC7955 was used to study the growth of
mesophilic anaerobic sporeforrners. Storage time had highly significant effects on its growth
(P<0.001) Cooking temperature had a significant effect on its growth In the abused condition
(P<0.05). in a spore profile test, an initial load of 102 spores or less per patty was reduced to a
nondetectable level for 60 days in refrigerated storage when samples were cooked at 71 or 82°C
for 30 minutes.

The physical properties of the commercial film (strength, thickness, and
shrinkage) were changed after exposure to thermal treatment, while the super barrier package

had virtually no change.

ACKNOWLEDGEMENTS

The author wish to thank Meat Laboratory and Food Microbiology
Laboratory, Department of Food Science and Human Nutrition. Michigan State
University for the supply of raw material and the assistant for using the

laboratory and equipment during this study.

TABLE OF CONTENTS

List of Tables

List of Figures

Introduction

Literature Review

Materials and Methods

Results and Discussions

Conclusions

Appendix: Standard Colony Count Methods
Bibliography

iv

vii

14
26
61
64
65

LIST OF TABLES

Table 1: Some physical characteristics of C. sporogenes
and C. botulinum.

Table 2: f-Test values for temperature abused samples
from 3 way ANOVA test.

Table 3: f-Test values for refrigerated storage samples
from 3 way ANOVA test. .

Table 4: Total count of aerobes in beef patties in
two type of packages, subjected to two
cooking conditions.

Table 5: Total count of anaerobes in beef patties in
two type of packages, subjected to two
cooking conditions.

Table 6: Total count of mesophiles in beef patties in
two type of packages, subjected to two
cooking conditions.

Table 7: Total count of psychrophiles in beef patties in
two type of packages, subjected to two
cooking conditions.

Table 8: Microbial guidelines and standards for cooked

meat products established by some states (1977).

10

28

28

29

34

38

42

46

Table 9: Total count of C. sporogenes in beef patties in 49
two type of packages, subjected to two
cooking conditions.

Table 10: pH of meat samples during 2 storage conditions 54
subjected to 2 cooking temperatures.

Table 11: Destruction of C. sporogenes and growth in 56
refrigerated storage in the commercial barrier material.

Table 12: Mechanical and physical properties of packaging 58

materials.

vi

LIST OF FIGURES

Figure 1: Sample preparation of cooked, uncured beef patties.

Figure 2: Microbial evaluation of noninoculated samples.

Figure 3: Microbial evaluation of C. sporogenes inoculated
samples.

Figure 4: Aerobic count of two films in the temperature abused
condition.

Figure 5: Aerobic count of two films in the refrigerated storage
condition.

Figure 6: Effect of cooking temp. on growth of aerobes in

refrigerated storage.

15

19

21

30

31

33

Figure 7: Anaerobic count of two films in the temperature abused 35

condition.

Figure 8: Anaerobic count of two films in the refrigerated storage. 36

Figure 9: Mesophilic count of two films in the temperature abused39

condition.

Figure 10: Mesophilic count of two films in the refrigerated
storage.

Figure 11: Psychrophilic count of two films in the temperature
abused condition.

Figure 12: Psychrophilic count of two films in the refrigerated
storage.

Figure 13: C. sporogenes count of two films in the temperature

vii

40

43

44

50

abused condition.

Figure 14: C. sporogenes coUnt of two films in the refrigerated 51
storage.

Figure 15: Effect of cooking temperatures on C. sporogenes 52

growth in the temperature abused condition.

viii

INTRODUCTION

In today’s market, there is potential demand for precooked,
refrigerated uncured meat products because of possible energy savings, ease
in preparation, and convenience in distribution, as compared to frozen products.
However, there are microbial concerns with regard to the products' quality and
safety. The initial microbial contamination of meat before processing is an
important factor affecting microbiological stability of products throughout storage
(Sutherland et al., 1982). Microorganisms from many sources such as the
animal itself, environment (soil and water), man, and processing equipment
(Johnston and Tompkin, 1984) may grow and cause quality changes, and
create an unsafe product. Several types of microorganisms are reported to grow
in precooked meats, if the products are cooked at inadequate thermal
processing conditions, temperature abused, mishandled or are post-processing
contaminated. For example, growth of psychrophilic microorganisms such as
Pseudomonas sp. can cause meat spoilage in prolonged refrigerated storage
condition. Some mesophilic microorganisms such asCIostridium sp. and
Staphylococcus sp. can cause outbreaks of food-borne diseases by growing
and producing toxin in these type of meat products.

Using vacuum packaging techniques for precooked meat products
results in longer shelf life and better product quality, because growth of aerobic
and some facultative anaerobe species are retarded by the low oxygen content
of the surrounding atmosphere inside the package. Many types of meat

products currently distributed in the US. market are packaged in vacuum form.

2

An important concern in vacuum packaged, precooked, uncured
meat products is the potential growth and subsequent toxin production
ofCIosfn'dium botulinum. Based on cultural and serological characteristics, there
are 4 groups of C. botulinum (group I - IV), which are also classified as 7
serotypes from type A to G. (Eklund, 1988). C. botulinum serotype E has been
used as a target group for creating thermal treatment conditions to destroy
spore of C. botulinum in processing. This serotype may not be suitable for use
as a target group because the study by Simunovic et al. (1985) reported that
spores from serotype E had considerably lower heat resistance than spores
from serotype B. Spores of C. botulinum which survive after thermal processing
of products are likely to grow and produce a fatal neurotoxin because
precooked, uncured meat has sufficient nutrients, favorable pH, little or no
added antimicrobial substances, and there will be less growth competition
because most other microorganisms are destroyed during processing. Vacuum
packaging also creates an oxygen-free environment (anaerobic condition),
which is required forC. botulinum to produce toxin (Mass et al., 1989).
Generally, growth and toxin production will occur if products are held at
temperatures higher than 10°C (Goldoni et al., 1980). There are some
serotypes (B, E, and F) that can grow and produce toxin at temperatures as low
as 3.300. (Genigeorgis, 1986). Based on its growth characteristics, and the
deadly nature of the neurotoxin produced by C. botulinum. it appears that the
hazard potential for C. botulinum growth in this type of meat products should be
high. However, the reported incidence of foodborne disease caused by
botulism toxin is low when compared to foodborne diseases caused by other
pathogens (Johnston and Tompkin, 1984).

There are two packaging techniques used to manufacture

vacuum, cook-in-package meat products. These are cook-in-strips, which use

3

different packages, one for processing and another for distribution; and cook-in-
ship, which uses the same package throughout processing and distribution
(Terlizzi et al.. 1984). Advantages of vacuum packaged, cook-in-ship meat
products with subsequent thermal treatment and refrigerated storage were
reported by Simunovic (1985) to be: 1) effectively eliminate post-processing
microbial contamination. 2) reduce moisture loss from product during
processing because it is sealed tightly in barrier materials and 3) extend
product shelf life by destroying or retarding growth of most pathogens and
spoilage microorganisms.

Today, the cook-in-package technique called ”Sous Vide", which
originated in France, is widely used in Europe for premium refrigerated entrees
(Swientek, 1989). In this technique, raw or precooked products are placed in
heat stable, high barrier plastic bags or pouches, packaged, sealed under
vacuum, and cooked to a time and temperature required for the individual
foods. The products are then chilled rapidly in an ice water bath, and stored in
controlled refrigerated conditions. Advantages for products prepared according
to the "Sous Vide" technique are; 1) product shelf life is generally acceptable up
to 2-3 weeks; 2) natural product flavor, aroma, and juice are sealed in; 3)
overcooking problems found with retort processing are avoided; 4) preparation
such as mixing ingredients is done at central locations, therefore, the quality
and taste of products will be consistent; and 5) convenient to consumer
because products need only to be reheated before consumption. (Raffael, 1984,
Light at al., 1988)

In the U.S., "Sous Vide" products are available only at the
foodservice level in some geographical areas. Products are manufactured
under the Hazard Analysis Critical Control Point (HACCP) program, which

requires strict control for processing and storage conditions (Swientek, 1989).

4

The Food and Drug Administration (FDA) has barred this product at the retail
level because of the potential microbiological safety concerns associated with
toxin production from C. botulinum if proper handling procedures are not strictly
followed, or products are temperature abused (Anon. 1988). In the UK,
distribution of Sous Vide products is not controlled. This can create false
confidence for consumers because this product/package system is believed to
be the perfect vessel to encourage growth of C. botulinum (Johnson and
Mitchell, 1989).

The present study is done with three specific objectives; First to
evaluate growth of microflora and C. sporogenes in two vacuum packaged,
cook-in-bag uncured meat systems (noninoculated and inoculated with C.
sporogenes PA 3679/ATCC 7955). The microbial load of each system is
determined as influenced by: a) The effect of commercial pasteurization (71°C)
versus severe pasteurization. (82°C). b) The barrier properties of a commercial
packaging material used for cook-In-bag meat products, versus a superior
barrier packaging material (Silicon Dioxide coated Polyester - Si02 coated
PET). and c) The effect of storage times in prolonged refrigerated storage and
room temperature abused storage. Second, to determine the level of
mesophilie anaerobic spores (using C. sporogenes PA 3679/ATCC 7955
spores as a model) initially present in raw meat which can be destroyed using
the vacuum, cook-in-bag technique and stored in a refrigerated condition. A
final objective is to determine changes in the physical and barrier properties of
cook-in-bag packaging materials after exposure to processing, handling and

storage.

LITERATURE REVIEW

There are three important factors which effect the shelf life of
vacuum packaged, cook-in-bag meat products system. These are: packaging
material selection, thermal processing conditions, and storage conditions. Many

studies have been done on each of these.

EII | I I . | . l i I I I I I

Taclindo et al. (1967) examined 113 prepared food samples in
plastic packages for growth of C. botulinum and found that the incidence of
growth in these food samples was extremely low. A further experiment was
conducted by inoculating a total of 64 food samples with C. botulinum type E
spores, with storage in an anaerobic condition at 30°C for 11 days. Growth and
toxin production were examined in turkey rolls and soybean cake samples
using conventional techniques. For the remaining 62 inoculated food samples,
60 samples showed the possibility of C. botulinum growth and toxin production
if a media enrichment technique was used. Borgstrom (1968) reported that
vacuum packaging was not able to inhibit microbial growth, but changed the
nature of microflora in meat products. Baren et al. (1969) observed that growth
of aerobes in vacuum packaged samples was retarded, while growth of
anaerobes occurred faster in vacuum packaged ground beef at 5°C storage.
Arafa and Chen (1975) determined growth of microorganisms in vacuum
packaged cut-up chickens. It was found that vacuum packaging did not confer

any protection to fresh poultry in refrigerated storage. Goepfert and Kim (1975)

6

used two types of packaging materials. cellophane and saran, to create aerobic
and anaerobic conditions for inoculated ground beef. The packaging materials
had little or no influence on behavior of microorganisms during 14 days storage
at temperatures ranging from 1 to 125°C.

Hanna et al. (1977), Blickstad and Molin (1983), and Simard et al.
(1984), reported that packaging materials and techniques strongly influenced
the type of microorganisms in meat, because they could alter the environment
surrounding the product. Seideman et al. (1976) vacuum packaged different
types of beef cuts (knuckles. ribs, chucks) in packages with oxygen transmission
rates ranging from 0.41-2.28 cc/1OO in2/24hr., with storage at 143°C for 0-35
days. Microbial counts were slightly higher at the higher oxygen transmission
rates, but the difference in number of psychrotrophic, mesophilic and
lactobacillus organisms in the different packaged products was not statistically
significant. Dainty (1979) reported that meat products vacuum packaged using
materials with oxygen permeabilities ranging from 5 to 90 cc/m2/d/atm had
extended shelf lives up to 8 weeks in refrigerated storage, due to retardation of
growth of some microorganisms, especially Pseudomonas sp.

It can be implied that barrier properties of packaging material
influence on types and numbers of microorganisms presented in meat products.
Therefore, the microbial load of precooked, uncured meat packaged in super
barrier material will be evaluated compared to the same products packaged in
commercial cook-in-bag material, which expected to have lower barrier
properties. Some mechanical and physical properties of both packaging
materials after exposed to processing will be tested because changes of these

properties may effect barrier and performance of final package.

Wt. ”tn”

7

Bag materials for cook-in-bag meat products must be heat
tolerant, impermeable to gas and moisture, and have sufficient strength and
toughness to withstand the severe conditions during vacuuming, cooking,
handling and shipping (Terlizzi et al.,1984). The authors suggested that the
oxygen permeability for this type of material in prolonged refrigerated storage
should be below 70 cc/mil/m2/24hr/atm at 20°C, 0%RH, to retard microbial
growth, as well as fat and light Induced color oxidation. Currently, these
materials are manufactured as multilayer composites using coextrusion and
lamination technology to combine two or more polymers together (Harte, 1987
and Rosinsky et al., 1989). Multilayer structures are designed to obtain specific
barrier, strength, machinability, sealability, and thermal stability properties
(Lundquist, 1987). Shrinkage of material after cooking at high temperature must
be controllable because it influences package appearance, product yield, and
adhesion properties between meat surface and the inner layer of the package.
(Rosinsky et al., 1989)

There are many type of polymers approved by the FDA for cook-in-
bag meat packaging. Multilayer packages are typically made by combining
barrier layer to sealant layer. For the barrier; layer, ethylene vinyl alcohol
(EVOH) copolymer is widely used because of its excellent oxygen barrier
properties. Bag material is made by sandwiching EVOH between two moisture
barn'er polymers (eg. polyethylene). Due to the hydrophilic nature of EVOH, loss
of barrier properties can occur when the moisture content of the film is high.
Polyvinylidene Chloride (Saran) copolymer has excellent moisture and oxygen
barrier properties. Bag material is made by coating Saran onto other base
polymers, such as polyester, polypropylene, and Nylon to increase physical

properties and machinability. For the sealant layer, polyethylene is used alone

8

or mixed with ethylene vinyl acetate (EVA) copolymer or linear low density
polyethylene (LLDPE) to improve mechanical and sealing properties. Surlyn',
which Is an lonomer, is the most commonly used sealant layer for vacuum
packaging of meat products because it has a broad range of sealing
temperatures (T erlizzi et al., 1984, Simms, 1985 and Lindquist, 1987).

:01 e 00‘... :“ozs : 0. so“. 0 o a" 0 ago 01'
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The United States Department of Agriculture (USDA) requires that
perishable, uncured meat must be cooked to a minimum internal temperature of
62.8-76.7°C (Tompkin, 1986). Sofos (1986) reported that many commercial
cook-in-bag meat products are cooked to an internal temperature of 71°C,
which is intended to destroy most Salmonella sp., but does not provide
sufficient assurance of microbiological stability for all meat products. Cook-in-
bag products are required by the USDA to be manufactured under HACCP
control to assure product quality and consumer safety. Refrigerated warning
labels are required on packages to warn retail outlets and consumers that
products need to be kept at temperatures of 4°C or lower to retard growth of
surviving microorganisms (Tompkin, 1986 and Prabhu et al.,1988). Many of
these products also have print expiration dates on packages to specify product
shelf life (NFPA, 1988 ).

The thermal destruction conditions necessary to destroy C.
botulinum in low acid foods were developed by the National Food Processors
Association - NFPA. (originally National Canners Association - NCA). Tuna in
oil (contained 20.5% fat) was reported to be the most protective medium tested.
For c. botulinum type E strain saratoga, the 03.22%; was 6.6 min (NCA 1973,
1976). Simunovic et al. (1985) reported that the 032,200 values for C. botulinum

9

type E were ranged from 0.15 min to greater than 4.9 min in reference media,
and from 0.74 min to 6.6 min in foodstuffs.

To determine actual thermal treatment in actual processing, the
use of C. botulinum as a tested organism is avoided, because of its hazardous
potential it in-plant contamination by spores of C.botulinum were to occur.
(Goldoni et al., 1980) An alternative microorganism, C.sporogenes PA 3679
IATCC 7955, is a nontoxic strain that has many physical and biochemical
characteristics similar to C. botulinum. This microorganism is used as a model
for thermal process evaluation of many food products. In table 1, a comparison
is made of physiological requirements for these two clostridium species. C.
sporogenes is a putrefactive, gram positive, straight-rod-shape-anaerobe. Its
spore is oval shaped with subterminal end location. Thermal death time curves
for both clostridium species were reported by Stumbo et al. (1950). They found
that C sporogenes PA 3679 spores had a higher heat resistance (as higher 2-
value) than C. botulinum spores, when suspended in various media prepared
from many types of food. Comparison of the D values (destruction time of spores
to 1 log cycle reduction) for many food products at temperatures ranging from
220-260°F, demonstrated that spores of C. sporogenes have higher D values
than spores of C. botulinum.

The microbial load of cook-in-bag meat products (noninoculated
study) and C. sporogenes inoculated study (which is used as a model for C.
botulinum) will be evaluated in this study using two cooking temperatures:
822°C for 30 min, which is used as a target temperature to destroy C.
botulinum; and 71°C for 30 min, which is previously reported to be used for
many cook-in-bag meat products (Sofos, 1986). For the effective of both
cooking conditions to the destruction of C. botulinum spores, different spore

concentration of model organism (C. sporogenes) will be evaluated.

TABLE1 :

10

Some physical characteristics of
C. sporogenes and C. botulinum

 

fireracteristlcs

C. sporogenes

C .botulinum

 

Casein digested
Lecithinase

lndole

Lipase

Glucose

Mannose

Maltose

Lactose

smmm

Produce H28 gas
Urease

Gelatin liquidfied
Nitrate reduced
Blood hemolyzed
Acetylmethylcarbinol
Toxin produced
Pathogenic to animals

 

 

yes
no
no
yes
yes
no
variable
no
n/d
yes
no
yes
no
yes
no
no
no

 

yes
no
no
yes
yes
no
variable
no
r variable
yes
no
yes
no
yes
no
yes
yes

 

n/d= need further tests to confirm

From: Bergey's Manual of Determinative Bacteriology (1974)

 

11

There have beenseveral studies dealing with the microbiological
stability of vacuum packaged fresh meat and poultry products. though studies
on vacuum packaged, precooked, in-package thermally treated uncured meat
and poultry products have been limited. Simunovic et al. (1985) tested
restructured beef formulated with 1.5% sodium chloride and 0.4% sodium
tripolyphosphate (STPP-antimicrobial agent), vacuum packaged, and
processed in pressurized water at 96°C and 1.38x105 Pa, until the center of the
meat was heated equivalent to 822°C for 15 min. No aerobic or anaerobic
colony-forming units were detected, and the microbial numbers in the product
remained stable for 16 weeks in a vacuum package, or for 2 weeks without
vacuum packaged at 4.400 storage. Prabhu et al. (1988) examined the shelf life
of vacuum packaged pork chops inoculated with C. sporogenes PA 3679 and
S. aureus 288. Product was cooked to internal temperatures of 66°C and 71 °C
in a temperature-controlled water bath set at 80°C. Product shelf life was 15-21
days at 2-4°C storage temperature and could be extended up to 60 days if
product was exposed to secondary in-bag cooking to 66°C. in prolonged
temperature abuse or mishandling, secondary cooking did not make the
product more microbiologically safe. Pork chops treated by dipping into
antimicrobial agents prior to packaging effectively prevented growth of
microorganisms on the surface under a simulated abuse condition (24-2500 for
48 hrs.).

Refrigerated, vacuumed. cook-in-bag uncured turkey breast rolls
were studied by Smith and Alvarez (1988). Turkey rolls were prepared, vacuum
packaged, and cooked to an internal temperature of 71 0C. No psychrotrophic

aerobic growth was detected during storage at 4°C for 87 days. Mesophilic

12

anaerobic microorganisms were detected, but the number did not change
significantly during storage. The implication was that these organisms can
survive at this cooking condition, and may grow when a product has been
temperature abused. Maas et al. (1989) studied the growth and toxin production
of C. botulinum in comminuted raw turkey formulated with sodium chloride,
sodium phosphate, and various levels of sodium lactate. Samples were
inoculated with a mixture of C. botulinum type A and B spores, vacuum
packaged. and cooked in hot water to an internal temperature of 71.100. C.
botulinum growth and toxin production were detected in samples treated with
0% sodium lactate after 2 days and 4 days storage at 27°C, for the 160 and 28
spores/ml. inoculated samples respectively. C. botulinum growth was delayed
to 4-5, 4-6, 7, and 7-8 days when the concentration of lactate present in the
meat was 2.0, 2.5, 3.0, and 3.5%.

"Sous Vide" products were evaluated by Schafheitle and Light
(1989) using chicken ballotine, as representative of a food product currently
manufactured by this method. All ingredients (boneless chicken, vegetables,
and seasonings) were placed into pouches, evacuated at a high setting, and
then sealed immediately. Pouches were cooked using a convection wet
steamer to an internal temperature at 80°C for 10 min., and then cooled in a
chilling cabinet for 90 min. prior to storage at 0-3°C. Microbiological evaluation
during 21 days storage found that no samples had mean spore counts
(aerobes, anaerobes, anaerobic spores, and anaerobic spores) higher than
1.3x102 g“. Carefully controlled processing and chilling were the most
important steps which maintained product shelf life for up to 21 days.

Cook-in-bag, smoked fish was processed using this technique
(Eklund et al.,1988) to determine the feasibility of the process to inactivate

growth and toxin production of C. botulinum types 8 and E. Smoked salmon

13

was inoculated with 106 spores of C. botulinum per sample, vacuum packaged
and cooked using different time and temperature conditions. Cooking at 922°C
for 45 min. and 55 min. prevented toxin production by type E during 120 days
storage at 10°C and 21 days storage at 25°C, respectively. To avoid toxin
production in products containing C. botulinum type B spores, longer cooking
times at all temperatures were required, when compared to the cooking times
used to destroy type E which is used as the target group for C. botulinum
studies.

According to these studies on vacuum, cook-in-bag uncured meat
products previously described, there are some variation on thermal treatment
and storage condition conditions which can provide adequate shelf life with
microbiologically safe for meat and poultry products. This study is intended to
provide additional information on microbial safety and shelf life for these type of

products, using the current thermal processing and storage conditions.

 

MATERIALS AND METHODS

II I I. [E II

Fresh lean trimmings from beef chucks (approximately 85-90%
lean) were obtained from the MSU Meat Laboratory. The beef was ground
using a coarse plate (0.63 cm. plate), and then finely ground (0.16 cm. plate)
using a Hobart grinder (Hobart Manufacturing, Troy, OH). The ground beef was
removed from the grinder and weighed equally into two batches; one was used
for the noninoculated study and the other for the C. sporogenes inoculation
study. Each batch of ground beef was mixed using a Hobart Laboratory Mixer
(Hobart Manufacturing, Troy, OH) for 5 minutes' to obtain uniform texture. The
noninoculated sample was mixed first , followed by the inoculated sample. Ten
ml. of sterile 0.1% peptone water was added to the noninoculated batch during
mixing. For the inoculated batch, a 10 ml. inoculum of Closfridium sporogenes
ATCC 7955 spores (prepared according to the methods described below) was
added to the beef instead of the peptone water. Each batch was removed from
the mixer and stuffed into a 4.4 cm. diameter cellulose casing to get uniform
patty diameter and portion control when sliced. Both samples were placed into

a freezer (-30°C) overnight before being sliced into patties.

Q . I I.
The spore inoculum of the model pathogen, Clostridium

sporogenes ATCC 7955/ NCA 3679,was prepared by propagation in a sterile

tube containing Beef Liver Medium (prepared according to the ATCC formula:

14

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16

ATCC, 1982). The tube was incubated under anaerobic conditions at 37°C for
24-48 hrs prior to use. Anaerobic conditions were achieved by adding sterile
mineral oil to the top of the liquid as a barrier layer to prevent oxygen contact.
An inoculum was made by transferring the spore suspension from the culture
media into the sterile tube, and 0.1 % sterile peptone water added to adjust total
inoculum to 10 ml. An inoculum concentration of approximately 105 spores per
meat patty was expected. The actual spore concentration was determined by
doing plate counts of C. sporogenes on the inoculated, vacuum packaged,

uncooked, fresh ground beef patties.

E I . I . I I I I_. -l I I

Two bag materials were used in this study, Both were supplied by
commercial film manufactures. The first material, a shrinkable, multi-ply
structure of polyolefin and ethylene vinyl alcohol, is currently used as a
commercial packaging material for cook-in-bag meat products, and precooked
meat products. The Oxygen Transmission rate of this material was reported by
the manufacturer to be 20 cc/m2 24 hrs atm at 23°C(73°F) 0% Relative
Humidity (Anon., 1989) The second bag material was a silicon dioxide (SiOz)
coated polyester (PET), laminated to a heat sealable polypropylene. This
material is currently used as a super barrier packaging material for some shelf
stable retortable food and pharmaceutical 'products in Japan. Oxygen
Transmission rate of this material was measured using the Oxtran-100 Oxygen
permeability tester (Modern Controls Inc., Elk River, MN.) to be approximately
0.4 cc/m2-24 hrs-atm at 23:1:2°C,100% RH. The SiOz coated layer is

responsible for the superior barrier properties of this material. (Anon. 1988)

17
E I . I .

The ground beef (in casings) was'removed from the freezer and
sliced into patties using a meat cutter (BIRO model 3334, BIRO Manufacturing
Co. Marblehead, OH). Each patty was about 0.5 cm. (0.2 in.) thick, 4.4 cm. (1.75
in). diameter and weighted approximately 10 grams. Patties were individually
placed into 10 x 15 cm. pouches made from both types of cook-in-bag
packaging materials. Each filled package was vacuum heat sealed using a
Multivac Vacuum Packaging Machine (Sepp Haggenmuller, W. Germany).
Packages were labelled, divided into two sets, one for each cooking condition,
and kept in a walk-in-cooler set at 0°C for approximately 30 minutes prior to be
thermal processed.

The first set of sample packages was taken from the cooler and
placed on racks inside the smokehouse with microprocessor controls (Drying
Systems, Inc.). Beef patties were cooked using a forced air technique at 100%
humidity until an lntemal patty temperature of 71 00 (commercial pasteurization
condition) was reached. The packaged products were then held at this
temperature for 30 minutes. The second set was cooked using the same
technique but to an internal temperature of 82°C (severe pasteurization).
Internal temperature was measured as duplicate values in two patty samples
placed in the center of the rack using two thermocouple probes, one connected
to the smokehouse microprocessor control unit (PC 5809 Butcher & Packer
Supply Co.), and the other to an external temperature reader (Honeywell Co.
Minneapolis, MN). Each probe was inserted into the center of the patty before
processing. lntemal temperature from each patty was read every 5 minutes
during processing. After cooking was completed, the packages were showered
with cold water in the smokehouse until an lntemal temperature of 23-25°C was

reached, and then immediately removed to cold storage (0°C) for 30 minutes to

18

thermally shock the remaining microorganisms and spores. Excess water on
each package surface was wiped off before placing samples into the two

different storage conditions.

SI I.I. .

Equal numbers of packages from the noninoculated and
inoculated study were kept in the two storage conditions; temperature abused
and refrigerated. For temperature abused, samples were stored at room
temperature (231:100) for 0, 12, 24, and 48 hours. Refrigerated storage samples
were stored in a refrigerated walk-in cooler for 0, 10, 20, and 40 days. The
temperature was chosen to fall within a range of 4-6°C to simulate refrigerated
storage at the retail level. Packages stored in both conditions were taken out
periodically as defined above and evaluated. For noninoculated samples, total
numbers of aerobic, anaerobic, mesophilic, and psychrophilic microorganisms
were determined. For inoculated samples, total numbers of C. sporogenes

were determined.

S I lit .III IE 2}

The total microbial count for each sample was determined using
the APHA Standard Plate Count method (Busta et.al., 1984). Each package was
torn open and a gram of meat was taken from the center of each patty. A 10'1
sample dilution was made by aseptically transferring meat from each sample
into a tube containing 9 ml. of sterile 0.1% peptone water and mixed well. Ten-
fold serial dilutions were made by aseptically transferring 1 ml. of the previous
dilution into a tube containing 9 ml. of sterile 0.1% peptone water and mixing
well. Serial dilutions for each sample were made from 10'1 to 104, and sample

dilutions from 10"2 to 10'4 were used for microbial enumeration. A one ml.

19

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20

aliquot was taken from each dilution to determine microbial numbers for the

four different groups of microorganisms (aerobes, anaerobes, mesophiles and

psychrophiles), using standard pour plate techniques (Busta et al., 1984).

Media and incubating conditions for each type of microbial group were as

follows:

- Aerobic Plate Count: Plate Count Agar (PCA, Difco 00., Detroit, MI), with
incubation at 37°C for 48-72 hours.

- Anaerobic Plate Count: Plate Count Agar with incubation at 37°C for 48-72
hours in anaerobic condition using GasPak 150 anaerobic jar with
generator and indicator system (BBL Microbiology Systems,
Cookeysville MD). Anaerobic condition was reached when the color of
anaerobic indicator changed to colorless.

- Mesophilic Plate Count: Trypticase Soy Agar (TSA, Difco Co.), with incubation
at 37°C for 48-72 hours. Plates were placed in an incubator for about an
hour until agar solidified, then plates were inverted face down.

- Psychrophilc Plate Count: Trypticase Soy Agar, with incubation at 7°C for 7-10
days.

Colonies from each plate were counted using the method
described in Appendix and calculated as the average colony forming units

(CFU) per gram meat. This number was calculated and reported as log1oCFU

for each sample.

(Inez: -. 2.0. o 0000:]: lo 3:. ,“o: .0
The model pathogen (Clostridium sporogenes) was enumerated
using the Standard Plate Count method as modified by Prabhu et.al. (1988).
Sample preparation and serial dilutions were done using the same procedures

as previously described for the noninoculated sample. The media used was

21

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22

Trypticase Soy Agar (T SA) + 1% Soluble starch (Difco). Plates were incubated
at 37°C for 72 hours in an anaerobic condition using a GasPak 150 anaerobic
jar with generator and indicator system. Colonies on each plate were counted
using the procedures described in Appendix. The CPU number (colonies per
gram meat) was reported as log1oCFU per gram sample.

C. sporogenes growth was confirmed by transferring colonies
from plate samples into a tube with an inverted vial containing sterile Trypticase
Soy Broth (T SB, Difco Co.) + 1% Soluble starch . A positive test is confirmed by
detecting gas production inside the vial after incubation in an anaerobic

condition for 48-72 hours at 37°C.

Sll'l'l I. III

A total of 4 replicates were done in each study (noninoculated and
C. sporogenes inoculated beef) to obtain sufficient data for statistical
analysis.Statistical analysis was evaluated based on three factors in the study.
These factors were; 1) type of package (commercial and super barrier); 2)
cooking temperatures (71 and 820C); and 3) length of storage time in
temperature abused (48 hours period), and refrigerated storage (40 days
period). Data for each type of microorganism (as log1oCFU value) was
analyzed using a 3-way Analysis of Variance (ANOVA) technique. Procedures
were obtained from ”Design and Analysis of Experiments" by Gill (1987).
Statistical results were calculated as mean square values of each single factor,
and all possible combined interactions of 2 and 3 factors to be tested for
significant. Significance differences of each factor was determined using the
Fisher's variance ratio test (f-test). The f-value was calculated from the mean
squares of the particular variable in question divided by the mean square error

(total mean square).

23

26mm

The pH of the meat was measured by blending meat in sterile
peptone water. An approximately 1 gram sample was taken from the package,
transferred into a tube containing 9 ml. of 0.1% sterile peptone water (pH 7.0i
0.1), and mixed well. The pH value was measured using a Corning pH Meter.

The pH was reported as an average from the four replicates.

W
A C. sporogenes (ATCC 7955) spore inoculum was prepared

using the procedures previously described to obtain six levels of spore
concentrations ranging from approximately 1 (10°) spore to 100,000 (105)
spores per patty. Six batches of ground beef were prepared and inoculated
with each level of spore dilution using the procedures described for the C.
sporogenes inoculation study. Packaging and processing procedures used
were described in the previous study except only one type of pouch
(commercial cook-in-bag) was used as the package material. After processing,
samples were kept at 4-6°C. The total number of viable C. sporogenes was
determined at 0, 10, 20, 40, and 60 days of storage for each sample, at each
level of spore inoculum. The procedures used were identical to the C.
sporogenes sample evaluation procedures previously described. Serial
dilutions for spore profile samples were made from 10'1 to 10:3. A one ml.
aliquot was obtained from each dilution to be microbially evaluated. The
procedures used for counting the numbers of sporogenes are followed the C.

sporogenes evaluation. Each test was repeated four times.

 

The commercial cook-in-bag and Si02 coated PET materials in
sheet and pouch forms were thermally processed in both cooking conditions
(71 and 82°C) for 30 minutes in the forced air smokehouse. The physical
properties of the film were evaluated as roll stock and thermally processed as
follows:

- Material thickness: Thickness of each film sample was determining using a
Testing Machines' Micrometer, model 549M. (Testing Machines Inc., Amityville
C.l., NY.). Thickness (in mil) was reported as an average from ten
measurements.

- Percent material shrinkage after thermal processing: Surface area of each
pouch before and after cooking was determined at both 71° and 82°C for 30
minutes using 5 sealed pouches per material type. Percent shrinkage was

calculated as:

Percent shrinkage = 100 x W
surface area before cook

Shrinkage was reported as average percent shrinkage for five pouches cooked
at each temperature.

- Package and seal leakage: An ARO non-porous package tester model F-099-
1080 (The ARO Corp., Buffalo NY.) equipped with vacuum pump was used to
detect material and seal integrity of the pouches. Five sealed pouches of each
material were tested before and after thermal processing at both temperatures.
Each pouch was submerged into the vacuum chamber, which was filled with
water, and vacuum slowly applied by adjusting the vacuum level to a maximum
level of 30 psi (2.18 kg/cmz). The package was then held at this vacuum level
continuously for 60 seconds. If there was no leakage (air bubbles) detected, the

result was recorded as non-detectable (N/D). Pouches from each sample

25

material were tested five times.

- Oxygen permeability of the materials before and after cooking: Three sheets
from each material (rollstock, 71°C cooked for 30 min., and 82°C cooked for 30
min.) were tested using the Oxtran-100 Oxygen permeability tester (Modern
Controls Inc., Elk River, MN). All permeability tests were done at 231200,
100%RH. Oxygen permeability was calculated and reported as an average from
three samples in units of cc/m2/24 hrs.atm.

- Strength of packaging material and seal (Tensile tests): The procedure
followed is described in ASTM Standard 0882-83. Film samples [Machine
Direction - MD, and Cross Direction -CD] were cut into 1" wide strips using a
precision sample cutter (T hwing-Albert Instrumental Co., PA). For sealed strips,
seals were made in both MD and CD directions using an Impulse Heat Sealer
(Sentinel Packaging Industries, Hyannis, MA) prior to cutting. The sealing
conditions used were the same as used for pouch sealing in the previous study.
Sample strips were tested using an INSTRONItensile testing machine model
4201 (lnstron Corp., Canton, MA.). Tensile strength was reported in kg/cm2

unit, and Strain (change in length of sample) in percent.

RESULTS AND DISCUSSION

II. I'I II. I. III llill'

Uncured beef patties were vacuum packaged in two different
cook-in-bag materials, processed at two temperatures, and stored in two
conditions; room temperature abused and refrigerated storage. The mean and
standard deviation values were reported to show some variations in microbial
count within the four replicates. These variations may have been due to meat
from different animals, and from unavoidable contamination during packaging,
processing, and microbial evaluation. The aerobic and anaerobic plate count of
vacuum, uncooked, fresh ground beef patties (raw material) were examined by
randomly selecting patties prior to thermal treatment from each batch. The initial
microbial population in the fresh patties ranged between nondetectable level
(log1oCFU less than 2.0) and log1oCFU 2.5 per gram meat.

In this study, the smallest dilution used for microbial evaluation
was 102, and the largest dilution used was 10“. If there was no growth
detected in the smallest dilution, the log1oCFU number was reported to be less
than 2.0 (<20). This value was substituted as (log1oCFU) 2.0 in the mean and
SD. calculation, and statistical analysis, corresponding to the minimum number
of microorganisms present in the meat. At the maximum dilution (104), if the
actual colony count in a plate was higher than 300 colonies, the log1oCFU
number was reported to be higher than 6.48 (log10300x104 = 6.48). This value
was substituted as (log1oCFU) 6.48 in the mean and SD. calculation, and

statistical analysis, corresponding to the maximum number of microorganisms

26

27

which could be detected using this evaluation technique. Both minimum and
maximum log1oCFU numbers were substituted to reduce deviation of data
caused by outlier values.

In table 2 and 3 are shown the f-values calculated for all four types
of microorganism in temperature abused and refrigerated storage conditions,
respectively. The calculated f-value was compared to standard f-test
distributions to obtain significance of the variabIes. Significance was reported
as a probability value for each factor. In this study, 95% probability (P<0.05) was
used as the minimum significant level. Several of the factors and their
interactions significantly influenced microbial growth in either room temperature
abused or refrigerated storage conditions.

a) Aerobes:

The average number of aerobic microorganisms in table 4 was
used to construct curves versus storage times for both types of package. The
plots are shown in figure 4 and 5 for room temperature abused and refrigerated
storage respectively. in the abused condition (figure 4), growth of aerobes was
higher in meat packaged in the commercial pouch than in the super barrier
pouch after 24 hours storage. In refrigerated storage (figure 5), there was an
approximately 1.5 log Increases in aerobic growth during a 40 day period of
refrigerated storage. The difference in barrier properties did not effect aerobic
growth in refrigerated storage because the growth pattern and number of
microorganisms in both packaging materials were not different.

Statistical analysis of factors effecting aerobic growth in the
temperature abused condition (table 2) indicated that type of package (barrier
properties) and storage time had strong influence on aerobic growth (P< 0.001 ).
The interaction of both factors was also significant (P<0.01). The rapid increase

in the number of aerobes in the commercial pouch was probably because the

28

 

 

 

 

 

 

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29

TABLE 4

Total count of aerobes in beef patties in two type of packages,
subjected to two cooking conditions.

(average from four replicates)

 

 

 

I lflCFU pggram of meat sample
Temp. | 71 C 82 C
I Mean SD. Mean SD.

 

Commercial pouch
Temp. Abused (hrs)

 

 

 

 

 

 

0 2.33 0.24 208* 0.15*
12 268* 0.46' 233* 0.29*
24 3.03 0.17 2.73 0.26
48 4.36 0.52 4.73 0.78
Refrigerated storage (days)
0 2.33 0.24 208‘ 0.15*
10 3.00 0.44 2.75 0.53
20 3.03 0.45 3.08 0.22
40 3.60 0.08 3.35 0.38
Super barrier pouch
Temp. abused (hrs)
0 2.33 0.24 208* 0.15*
12 22* 0.24“ 2.40 0.34
24 238* 0.43* 258* 0.40‘
48 3.50 0.53 3.60 0.68
Refrigerated storage (days)
0 2.33 0.24 208‘ 0.15*
10 3.00 0.24 2.70 0.34
20 3.08 0.32 3.10 0.18
40 3.53 0.21 3.28 0.19

 

 

*

value less than 2.0 was substituted by 2.0

 

30

 

 

 

 

6
q
s. 5‘
5 I Commercial
ll.
0
a 4 -
.l
o ..
u
g .
> 3
‘ Super barrier
2 T ' I fi l T r r T '
0 10 20 30 40 50

Time (hr.)
Figure 4: Aerobic count of two films in the temperature abused condition.

Avg. log CPU/gm.

31

 

A

 

Commercial

Super barrier

 

 

V l' V I V ' U I t

10 20 30 40

Time (deyc.)

Figure 5: Aerobic count of two films in the refrigerated condition.

50

32

abused temperature was close to the optimum growth temperature of many
aerobes. Ingram (1972) reported that growth of aerobic organisms is usually
higher on the meat surface, which is directly in contact with the package surface
when it is vacuumed. The amount of oxygen permeating through the
commercial material would be higher than the amount of oxygen permeating
through the super barrier material during the same period, because of the
difference in barrier properties. The amount of oxygen inside the commercial
pouch would support aerobic growth on the meat surface. Some of the growth
microorganisms found in the super barrier pouch may have been
microaerophiles or facultative anaerobes (eg. Lacfobacillus sp.), which require
only small amounts of oxygen to grow.

Statistical analysis of the samples exposed to refrigerated storage
are shown in table 3. Storage time had a strong influence (P<0.001) on growth
of aerobes. No differences were found dependent on package type. Cooking
temperature also had a significant effect (P<0.05), which may indicate that there
were different number of aerobes remaining after patties were cooked at 71 and
82°C. After the 71°C cook, the number of surviving aerobes was higher than
after the 82°C cook. Figure 6 showed the relationship between log1oCFU
numbers of samples and storage time in two cooking temperatures. The growth
of aerobes in refrigerated storage was small probably because the low
temperature can retarded growth.

b) Anaerobes:

From the average number of anaerobic growth in table 5, and the
statistical results of anaerobes in table 2 and 3, it was found that storage time
was the only factor that significantly influenced growth of anaerobes (P<0.001).
The average numbers in table 5 were plotted versus storage times for

temperature abused and refrigerated storage condition, and are shown in figure

33

 

 

 

 

4
'i . 71 c
E
5 82C
5?
E a
o
E
6 .
>
<
2 ' v I ' r r 1 V 1 v
0 10 20 30 40 50

time (deye)
Fig. 6: Effect of cooking temp. on growth of aerobes in the refrigerated storage

-J .' . A—JA.“.‘-.Lfll.“'

 

A} .

TABLE 5

Total count of anaerobes in beef patties in two type of packages,

subjected to two cooking conditions.

(average from four replicates)

l

Log FU per gram of meat sample

 

 

 

 

 

 

 

 

 

 

 

 

 

Temp. | 7 c_ 32 c:
| Mean SD. Mean SD.
Commercial pouch
Temp. Abused (hrs)
0 2.28 0.21 2.00‘ 0.00*
12 2.45 0.31 223* 0.29*
24 2.80 0.16 240* 0.27“
48 4.55 0.60 4.15 0.24
Refrigerated storage (days)
0 2.28 0.21 200‘ 0.00*
10 2.90 0.45 2.53 0.67
20 3.18 0.57 3.08 0.45
40 3.30 0.22 3.58 0.33
Super barrier pouch
Temp. abused (hrs)
0 2.28 0.21 200* 0.00‘
12 223' 0.45* 225* 050*
24 258* 0.51* 2.58 0.42
48 3.90 0.35 3.88 0.46
Refrigerated storage (days)
0 2.28 0.21 200* 0.00*
10 2.70 0.56 253‘ 0.56*
20 2.93 0.48 2.90 0.32
40 3.43 - 0.17 3.28 0.25

 

" value less than 2.0 was substituted by 2.0

 

Average log CPU/gm.

35

 

 

     
 

Commercial

Super barrier

 

 

Time (hr.)

50

Figure 7: Anaerobic count of two films in the temperature abused condition.

Avg. log CPU/gm

36

 

Commercial

    
  

Super barrier

 

 

 

Time (deye.)

50

Figure 8: Anaerobic count of two films in the refrigerated storage.

37

7 and 8 respectively. Growth of anaerobes in both storage conditions was not
significantly different for film type or cooking temperature in either storage
condition. The number of anaerobes enumerated may include some facultative
anaerobes since a small amount of oxygen probably remains inside the meat.
Their growth occurs at a short distance beneath the surface.

c) MeSOphiles:

Growth of some mesophilic sporeformers, such as Bacillus sp. and
Clostridium sp., have been reported as indicative of the sanitation quality of low
acid food products, packed in hermetically sealed containers (Lake and Lynt
1984). Loss of quality in these products due to mesophilic growth caused by
inadequate thermal processing condition and post-processing contamination.
Therefore, the mesophilic count was evaluated to check the possibilities of both
incidences during this study.

In figure 9 and 10, the relationship between average log1oCFU
numbers of mesophilic growth (table 6) and storage time were shown for both
storage conditions. Statistical data in table 2 and 3 indicated that storage time
was the most significant factor influencing mesophilic growth (P<0.001) in both
storage conditions. Type of packaging material (barrier properties) was another
factor that had significant influence (P<0.01) for temperature abused condition
(table 2). Barrier properties of the material effect growth of mesophilic aerobes
on the meat surface, because the oxygen permeating through the film allows
growth to occur. In refrigerated storage condition, there was slightly difference of
mesophilic growth for samples packaged in both films, which was not
statistically different (table 3).

The potential of either inadequate thermal processing, or post-
processing contamination caused by mesophilic microorganisms can be

observed from the rapidly increase of microbial at a short period of storage time,

TABLE 6

Total count of mesophiles in beef patties in two type of packages,

subjected to two cooking conditions.

(average from four replicates)

 

 

I

Log C—FU per gram of meat sample

 

 

 

 

 

 

 

 

 

 

 

Temp. | 71 C 82 C
I Mean SD. Mean SD.
Commercial pouch
Temp. Abused (hrs)
0 215" 0.17* 208* 0.15‘
12 248* 0.34* 235* 0.26‘
24 2.93 0.30 243* 0.35"
48 4.53 0.57 4.40 0.59
Refrigerated storage (days)
0 215* 0.17* 208* 0.15*
10 2.88 0.30 2.85 0.31
20 3.10 0.49 3.03 0.36
40 3.43 0.25 3.53 0.21
Super barrier pouch
Temp. abused (hrs)
0 215‘ 0.17‘ 215‘ 0.17“
12 2.43 0.38 208* 0.15*
24 2.43 0.49 “2.55 0.38
48 3.53 0.63 2.35 0.75
Refrigerated storage (days)
0 215* 0.17‘ 215' 0.17‘
10 2.73 0.29 253’ 0.51 "’
20 3.08 0.22 2.95 0.10
40 3.35 0.33 3.15 0.34

 

‘ value less than 2.0 was substituted by 2.0

 

39 '

 

     
 

Commercial

Average Log CPU/gm.
.5
1

Super barrier

 
 

 

 

 

2 I ' 1 fl r ' I ' l

0 10 20 30 40 50

Time (hr.)
Figure 9: Mesophilic count of two films in the temperature abused storage.

Avg. log CPU/gm

40

 

Commercial

  
 

Super barrier

 

 

 

2 I ' r ' l r I r I fi

0 10 20 30 40 50

Time (deye.)
Figure 10: Mesophilic count of two films in the refrigrated storage.

41

especially in temperature abused condition. From figure 9 and 10, growth
patterns of mesophiles for both type of packages did not indicate any rapidly
increased of microorganisms. Therefore, it can imply that neither inadequate
thermal processing, nor post-processing contamination was occurred in this
study.

d) Psychrophiles:

The presence of psychrophile in cooked, uncured meat products
can result in potential spoilage over prolonged storage for commercial
refrigerated meat products. Growth of psychrophile also implies inadequate
thermal treatment or post-processing contamination problems, since most
psychrophile can be destroyed by mild heat treatments. (Gilliland et.al. 1984).

In table 7, the average log1oCFU number of psychrophile at
immediately after processing (i=0) was nondetectable (log1oCFU <20). Plotting
the average log1oCFU of psychrophile versus storage time for both films (figure
11 and 12) showed that samples packed in commercial packages had slightly
higher psychrophilic growth than samples packaged in the super barrier
material after 24 hours in temperature abused (figure 11). Psychrophilic growth
was slowly increased in both type of packages after approximately 25 days in
refrigerated storage. At the end of both storage conditions, growth of
psychrophile had increased approximately 1 log. From the statistical analysis
(table 2 and 3), it can be seen that storage time was the main factor influencing
microbial growth (P<0.001).

The possibility of post-processing contamination or inadequate
thermal treatment caused by psychrophilic microorganisms can occurred in
refrigerated condition, which is closed to their optimum growth conditions. The
rapidly increase in psychrophilic numbers during storage period was observed

if either incidence occurred. In this study, the increase in psychrophile during

42

TABLE 7

Total count of psychrophiles in beef patties in two type of packages,
subjected to two cooking conditions.

(average from four replicates)

 

 

 

 

 

 

 

 

 

 

 

[ Log CT‘U per gram of meat sample
Temp. | '7—1_C_ 82 C_
| Mean SD. Mean SD.
Commercial pouch
Temp. Abused (hrs)
0 <2.00* 0.00* <2.00* 0.00*
12 213* 0.25* <200* 0.00*
24 238* 0.57* 220* 0.40*
48 3.28 0.33 275* 0.61 *
Refrigerated storage (days)
0 <2.00* 0.00* <2.00* 0.00*
10 2.63 0.58 2.55 0.45
20 2.60 0.26 2.60 0.35
40 3.22 0.45 3.08 0.49
Super barrier pouch
Temp. abused (hrs)
0 <2.00* 0.00* <2.00* 0.00*
12 200* 0.00* 200* 0.00*
24 213* 0.25* 200* 0.00*
48 285* 0.68* 2.55 0.48
Refrigerated storage (days)
0 <2.00* 0.00* <2.00* 0.00*
10 248* 0.59* 235* 0.43*
20 2.78 0.22 240* 0.49*
40 2.98 0.39 3.03 0.40

 

 

 

 

value less than 2.0 was substituted by 2.0

 

43

 

Average Log CPU/gm
.5
1

Commercial

 
 

Super barrier

 

 

   

 

0 1O 20 30 40 50

Time (hr.)
Figure 11 : Psychrophilic count of two films in the temperature abused storage

44

 

Commercial

 

  

a

   

Avg. log CPU/gm

Super barrier

 

 

 

0 1 0 2 0 3 0 4 0 5 0
Tlme (deye.)

Figure 12: Psychrophilic count of two films in the refrigerated storage.

45

storage in both conditions was small in refrigerated storage (figure 12), and
their growth patterns did not reveal any rapid increase. Therefore, neither
inadequate thermal processing, nor post-processing contamination of

psychrophile occurred.

1: : z .. ._ .. . aunt-o... to. . ._ :- . 0.10 oz: on =

A reasonable indicator of the basic quality of meat products can be
implied using the total microbial count (Sutherland 1982). The total plate count
on PCA at 35°C for fresh, prepared, cooked, uncured meat has been reported
by the USDA to be 104 colonies or less (log CFU 34.0) per gram sample
(Johnston and Tompkin, 1984). The unavoidable contamination during
processing, packaging and handling may increase the number of
microorganism on the product surface by levels of 101 to 102 per gram sample
(Johnston and Tompkin, 1984).

In recommendation pertaining to meat products by USDA
(T ompkin, 1986), and meat regulations defined by many states, the numbers of
aerobic plate count (APC) allowable in cooked, uncured meat products is used
as a controlling factor for product quality. Wehr (1978) reported that APC
numbers ranged from less than 5.0x104 to 1.0x106 colonies per gram meat
(log1oCFU=4.7-6.0) in most regulations established by individual US. States
(table 8).

The purpose of temperature abusing samples was to create a
situation where meat was mishandled by holding at temperatures higher than in
normal refrigerated conditions. Such temperatures have been reported to result
in the majority of food poisoning outbreaks, and are common to food handling
(Buchanan, 1986). By examining the aerobic total count (table 4), the microbial

shelf life of this vacuum packaged, oook-in-bag meat product subjected to

46

TABLE 8: Microbial guidelines and standards for cooked meat products
established by some states (1977)

 

State Product(s) APC per gram meat log1oAPC
Idaho cooked meat <1x106 6-0
Massachusetts cooked meat <5x104 4-7
Nabraska delicatessen <1 x105 5-0
smoked or heat treated <1x106 5-0
North Dakota cooked or smoked meat <1x106 5-0
Oregon cooked meat <1x106 5-0
Tennessee processed meat <5x106 6-7
East Virginia delicatessen <1x104 4-0

 

Source: Wehr (1978) Food Technology Vol. 31 (1): 64

47

temperature abuse (23120C) can be considered. The growth of aerobes in
cooked uncured meat, packaged in the commercial pouch increased
approximately 2.5-3 log (figure 4) to log1oCFU 5.0 after 48 hours abuse, while
samples packaged in the high barrier material increased about 1.5 log. Average
number of aerobes in commercial package was exceed the allowable APC
number indicated in table 8. Therefore, if these types of products are left at
abusive temperatures longer than 24 hours, their microbial shelf life may be not
acceptable.

Refrigerated storage was used to create retail and conventional
storage conditions. The temperature used in this study (4-60C) retarded the
growth of the surviving microorganisms. The average APC number was slightly
increased from log1oCFU 2.0 to log1oCFU 3.5 after 40 days of storage.
Refrigerated storage also eliminated the difference in barrier properties of the
packaging materials In refrigerated storage (fig. 5). Using the regulations stated
in table 8 as the basis, the microbial shelf life of products in refrigerated storage
was still acceptable, after 40 days at 4-6°C storage.

Growth of microbial in cook-in-bag uncured meat products was
caused by some groups of microorganisms, mainly thermophiles and
sporeformer groups, which can survive after products are thermally treated.
They adjust to the new environment during the first period of storage. and will
use nutrients from meat to grow and produce biochemical substances such as
acids or toxin. The process can occur quickly at room temperature, which is
close to the optimum growth temperature for these microorganisms. Some of
the substances produced can cause food poisoning and loss of meat quality

resulting from odor, discoloration, flavor change etc.

48
U ..._ : . ._'... ee‘ee:l: no ::eoe_|e_e:: nu:

The actuai concentration of C. sporogenes inoculated into each
batch was counted from vacuum, uncooked, inoculated, fresh ground beef
patties to be in range of 5x104-1x105 spores (log1oCFU 4.7-5.0) per patty. This
concentration was chosen to insure that the model organism was the dominant
species in the meat, and to avoid the competitive growth of other
microorganisms present in the meat. The confirmatory test for C. sporogenes
(detection of gas in liquid media) was done on all plates which containing
suspected C. sporogenes colonies (visual observation).

The average log1oCFU number of C. sporogenes after thermal
processing is shown in table 9. Number of C. sporogenes was reduced by both
thermal treatments conditions by 2.5-3.0 log immediately after samples were
thermal processed (ta-0). Figure 13 and 14 to show the relationship between
average log1oCFU number of C. sporogenes and storage time in both storage
conditions. In the temperature abused condition, number of C. sporogenes
increased rapidly after 24 hours (figure 13). In refrigerated storage (figure 14),
growth of C. sporogenes was increased less than 1 log. Therefore, refrigerated
storage effectively retarded growth of surviving C. sporogenes after cooking.

Growth of C. sporogenes (table 6) was statistically analyzed using
the same procedures as described for the noninoculated study. Storage time
was the most significant factor (table 2 and 3) effecting growth of C. sporogenes
(P<0.001). Cooking temperature was another factor that showed significant
difference for the temperature abused samples (P<0.05), but not for the
refrigerated storage samples. The numbers of C. sporogenes after cooking
versus storage time at the two temperatures in the abuse condition is shown in
figure 15. At the time immediately after cooking (€=0), the average number of C.

sporogenes in sample cooking at 71°C was reduced from log1oCFU 5.0 to 2.4,

 

49

TABLE 9 .

Total count of C. sporogenes in beef patties in two type of packages,
subjected to two cooking conditions.

(average from four replicates)

 

 

| Log $0 per gram of meat sample

 

 

 

 

 

 

 

 

 

 

 

 

Temp. | 716 82 ('3
| Mean ST). Mean fl.
Commercial pouch
Temp. Abused (hrs)
0 2.40 0.11 2.10 0.14
12 2.43 0.29 2.08 0.15
24 3.28 0.43 2.75 0.52
48 5.60 0.25 5.60 0.41
Refrigerated storage (days)
0 2.40 0.11 203* 0.05*
10 2.68 0.24 2.78 0.22
20 2.73 0.57 2.65 0.47
40- 2.90 0.08 2.70 0.08
Super barrier pouch
Temp. abused (hrs)
0 2.40 0.11 2.10 0.14
12 233* 0.25* 22* 0.24*
24 3.03 0.55 2.65 0.62
48 5.73 0.32 5.78" 0.66“
Refrigerated storage (days)
0 2.40 0.11 2.10 0.14
10 2.43 0.34 2.35 0.44
20 2.83 0.57 2.68 0.59
40 3.00 0.32 2.90 0.54

 

Q.

value less than 2.0 was substituted by 2.0

value higher than 6.48 was substitdted by 6.48

 

5O

 

  
   

‘ Super barrier

Commercial

Average Log CFU/gm.
h
l

 

 

 

 

1O 20 30 4O 50

Tlme (hr.)
Figure 13: C. sporogenes count of two films in the temperature abused storage.

Avg. log CFU/gm.

51

 

 

 

 

 

4
Super barrier
3-
fl
Commercial
2 l ' I ' l ‘ T ' I '
10 20 30 40 50

Time (deye.)

Figure 14: C. sporogenes count of two films in the refrigerated storage.

Avg. log CFU/gm. sample

52

 

 

 

 

 

 

6
71 C
4-
820
2 I ' f r f V I I 1 a
0 10 20 30 40 50

Storage tlme (hre.)
Fig. 15 Effect of cooking temperatures on sporogenes growth
in the temperature abused condition

53

while cooking at 82°C reduced it to 2.1. At 48 hours of abusive temperature
storage, the number of C. sporogenes was not different in either cooking
condition. This may be do to the fact that the spores subjected to 82°C cooking
could grow with less competition from other microorganisms.

Shelf life of the product based on growth of C. sporogenes in a
retail, refrigerated condition (4-6°C) would probably be acceptable for 40 days
storage, slnce most of the inoculated spores (approximately 25-3 log) were
destroyed after cooking and the growth of the surviving spores was very limited
at low temperature. Although the microbial shelf life of this products based on
model organism would be acceptable, growth and toxin production of C.
botulinum could be a problem at this refrigerated temperature, because some
serotypes (B, E, F) have previously been reported to grow and produce toxin at
temperatures as low as 3.300. Other factors also effect shelf life, such as

change in physical properties, appearance, and/or flavor.

W

The pH of noninoculated and inoculated meat samples were
measured during storage in both conditions. The data shown in table 10 were
averaged from all four replicates tested. The pH of noninoculated and C.
sporogenes inoculated samples showed small difference in both storage

conditions within range of 5.95-6.22 and 5.92-6.21, respectively.

WW

Different spore levels of the model organism (C. sporogenes
ATCC 7955) were used as inoculum to study the number of mesophilic
anaerobic sporeformers (such as C. botulinum) initially present in meat, which

could be destroyed using both thermal treatments conditions to the

54

Table 10: pH of meat sample during 2 storage conditions
subjected to 2 cooking temperatures.
(measured in 0.1% sterile peptone water)

 

 

 

 

 

Temp. abused pH
Time (hrs.)' Material Cooking temp.(C) Noninoculated'l Inoculated
0 Commercial 71 6.22 6.17
82 6.21 6.19
12 Commercial 71 . 6.15 6.16
82 6.18 6.18
Super barrier 71 6.20 6.15
82 6.18 6.18
24 Commercial 71 5.95 5.99
82 5.98 6.02
Super barrier 71 5.98 6.03
82 6.00 6.06
48 Commercial 71 5.96 5.89
82 5.98 5.96
Super barrier 71 5.99 5.97
82 6.00 5.92
Refrigerated storage
Time (days)
0 Commercial 71 6.22 6.17
82 6.21 6.19
10 Commercial 71 6.14 6.15
82 6.17 6.19
Super barrier 71 6.15 6.16
82 6.18 6.16
20 Commercial 71 6.1 1 6.09
82 6.13 6.13
Super barrier 71 6.13 6.14
82 6.13 6.14
40 Commercial 71 6.19 6.18
82 6.20 6.21
Super barrier 71 6.20 6.19
82 6.21 6.20

 

 

 

 

 

 

 

 

55 .

nondetectable level (in this study, log1oCFU <1.0 was the minimum sensitivity
level). Growth of the remaining spores of C. sporogenes in refrigerated
conditions (4-6°C) was evaluated for 60 days period. No growth of
microorganism could be detected in samples which had less than 100 spores
per patty initially during 60 days of storage (table 11). Growth of C. sporogenes
was detected in samples which had an initial number of 1000 spores per patty
at 60 days of refrigerated storage for both 71 and 82°C cooking. The destructive
effect of different cooking temperatures on C. sporogenes spore reduction was
evident for samples having an initial number of 10,000 spores per patty and
above. The number of spores remaining in patties cooked at 82°C was initially
lower than in patties cooked at 71°C.

For samples immediately evaluated after thermal processing (t=0),
it was observed that both cooking conditions reduced the number of spores by 3
log. Growth of the surviving spores in samples cooked at 71°C increased
approximately 1 log cycle during 60 days of refrigerated storage at 4-6°C.
Growth of C. sporogenes was increased in samples cooked at 82°C to be
closed to samples cooked at 71°C after 40 days of storage, probably due to the
lack of competition from other microorganisms.

The possibility of contamination by anaerobic sporeformers from
C. botulinum in a conventional processing plant (for cook-in-bag meat products)
is probably low due to; 1) the use of strict regulations and inspection of meat in
the HACCP program for vacuum packaged, cook-in-package uncured meat
products; 2) the lack of anaerobic conditions in most equipment in food
processing plant cause the nonconductive conditions for growth and
sporulation of anaerobes (Lake and Lynt, 1984); and 3) the occurrence of C.
botulinum spores in meat and meat products has been reported to be very low,

approximately 1-2 spores per 10 kg. of meat (Simunovic et al., 1985). When

56

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6:39: BEE 365858 9.: 5
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”Z. m..m<._.

 

57

compare the spore profile study of model mesophilic sporeformer (C.
sporogenes) to C. botulinum , thermal processing to internal temperatures of
71°C or 82°C for 30 minutes would be enough to reduce spores of C.
botulinum to an undetectable level if the raw meat has an initial number of
spores less than 100 per gram. Outbreaks caused by botulism in cook-in-bag

meat products would be at low risk, unless samples are abused or mishandled.

 

The mechanical and physical properties of the commercial and
super barrier packaging materials were tested after 3 treatments: rollstock, heat
treated at 71°C and heat treated at 82°C for 30 minutes to observe changes
after exposure to thermal treatments. The data are shown in table 12, and are
discussed as follows:

a) Material thickness: Thickness of the commercial package material increased
proportionally with increasing cooking temperature, which may also due to
shrinkage following cooking. There were no changes in thickness of the super
barrier package material. Change in thickness effects many of the mechanical
and physical properties of packaging materials.

b) Shrinkage of pouch surface area: The percent shrinkage of the commercial
package increased proportionally with increasing cooking temperature, while
the super barrier package showed no change in dimensions at both cooking
temperatures. Shrinkage of cook-in-bag material must be controllable because
it influences final package appearance and cost of material. In more severe
cooking conditions such as retorting, the commercial cook-in-bag package
might not suitable.

c) Package and seal leakage test: All tested pouches from both packaging

materials withstood pressure up to 30 psi for 60 seconds with no leakage

 

58

 

.5 8 58 58 no 8 2 8588 8; 5.5858 No .8828 a: 8; 88 .

 

 

8.2 m 52 3. 8.8 9. a m .8 no
8.8 8.8 8. z 8.8 58 8.8 as. 558$
38 8.83. 3.8 3.8. 8.89 8.88
a. E8 «.88 3.8» 8.89 «.28 988 a: .88 .8» «.59.. 8:83
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8.8 8.8 8.8 $8 «.5 8. E as. 558*
$8 $8 $8 «.8» 88 m. .8 so
P. .8 m. .8 ~88 8.85 mm 3. 8.88 a: $559.. 283
885:
£888. 0 8 © 55 58 ~58.
885 . 92 88.5 8.8 8.8 8.8 $8258 58:0
92 Qz Qz Qz Qz Qz 355885585.
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3 m.» 3 e 8 ma 2....5 8858:
888 o 8 888 o E 8.82: 8.58 o 8 858 o E 888:: 8:858 .85

 

 

 

mmmxoma EEmeaam

 

mmmxowa _w_emEEoo

 

 

.mfinmfie 968.03 B moEmaoa 8083.. 2a 82:28.2

up m..m<._.

59

occurring. This test evaluated the ability of material and seal to withstand the
severe conditions which pouches were subjected to, in vacuum packaging, at
pressures of 20-25 psi. This test can be used to check pinholes in the seal and
package surface caused when packages are subjected to severe cooking
conditions or material and seal defects. Leakage of seal and package can
cause loss of vacuum and post-processing contamination.

d) Oxygen barrier properties: Due to the amount of aerobic growth, it could be
predicted that the amount of oxygen permeating through the commercial
package material would be higher than that permeating through the super
barrier package material. This can influence the number of aerobes in beef
patties packaged in the commercial material. The oxygen transmission rate of
the commercial packaging material in rollstock form was 82.1 oc/m2 atm 24 hrs.
at 2312°C 100%RH. as determined using the Oxtran-100 Oxygen Permeability
tester. An oxygen transmission rate of 20 cc/m2~24 hrs atm at 23°C (73°F), 0%
RH was provided by the manufacture of this commercial packaging material
(Anon, 1989). The barrier properties of the commercial package were
considerably less at the high humidity condition. This may be because the
barrier layer of the commercial package is ethylene vinyl alcohol, which losses
some of its barrier properties at high humidities. For the thermally treated
commercial package at both cooking temperatures, barrier measurements were
difficult to obtain because of the change in film thickness and surface area due
to shrinkage. For the super barrier package, there was almost no change in the
oxygen barrier properties after thermally processing at both cooking
temperatures. The oxygen transmission rate of this material was very low ( 0.4-
0.5 cc/m2 atm 24 hrs. at 231200 100%RH) , which is approximately 150-180
times lower than the commercial package. The excellent barrier properties of

this material are due to the SiOz coated layer.

60

e) Tensile tests: Tensile tests were conducted to observe changes in the
material and seal strength of the materials before and after heat treatment. For
the commercial package, tensile values were reported (table 12) at the
breakage point of the material or the separation point of seal strips. For the
super barrier package material, it was observed that the film separated into two
layers during the test. One layer (polypropylene sealant layer) continue to
elongate until breakage, while the other layer (Si02 coated PET) stretched
slowly without breakage. Tensile values were reported (table 12) at the point
where the fracture completely occurred at the center of sealant layer.
Commercial package material showed loss of tensile strength in both machine
and cross direction after cooking. Changes in material and seal strength of the
commercial package material was difficult to measure for the heat treated
samples, due to material shrinkage and change of thickness. The super barrier
package material had a higher tensile strength than the commercial package
material. Changes of tensile strength of samples after heat treated were
fluctuated, which may cause by some stnictural changes of the sealent layer.
Low strain of this material was observed because of the inability of the barrier

layer (Si02 coated PET) to expand.

 

CONCLUSIONS

The results of microbial evaluation on vacuum packaged cook-in-
bag uncured beef patties for noninoculated and C. sporogenes inoculated
samples were statistically analyzed. Also, the changes of packages and
packaging materials after thermal processing was evaluated. It can be
concluded that:

1) The effect of two different cooking temperatures (71 and 82°C) was
demonstrated by the reduction of microorganisms in the meat after cooking. For
aerobic growth in refrigerated storage, and for C. sporogenes growth in
temperature abused condition, a significant difference (P<0.05) was found. The
internal cooking temperatures at 82°C resulted in lower numbers of
microorganism or spores remaining in the products after thermally processing
than cooking at 71°C.

2) The effect of different cook-in-bag packaging materials was significant at the
level of P<0.001 for aerobic growth in temperature abused condition. This factor
was also significant for mesophilic aerobes. Growth of aerobes in the patties
rapidly increased in samples packaged in the commercial pouch (PE/EVOH),
due to a high amount of oxygen permeating through the package surface.
Comparing the oxygen transmission rates (OTR) showed that the commercial
package had OTR approximately 150-180 times higher than the super barrier
package (SiOz coated PET), before and after exposure to the defined thermal
processing conditions used in this study. In refrigerated storage, the barrier

properties of material showed no significant effect on aerobic growth.

61

62

3) Storage time was the most significant factor effecting the number of
microorganism for all types of microbial groups, at both temperature abused
and refrigerated storage conditions (P<0.001). This could be used to predict
microbial shelf life for the vacuum packaged, cook-in-bag meat products by
comparing the number of microorganisms at the defined storage times to the
allowable number of microorganisms defined in meat products regulations by
USDA or many states. If the microbial load is close to or exceeds the defined
number, this product would not be acceptable. The microbial load of meat
products subjected to temperature abuse is of concern because growth of the
remaining microorganisms (after cooking) increases rapidly within a short
period of time (24 hr.). At refrigerated storage (4-6o C), growth of
microorganisms was low, so, products might be considered acceptable for 40
days of storage.

4) The spore profile test with the model organism, C. sporogenes, showed that
different temperatures used for thermal processing had different capacity to
destroy the same number of meSOphilic anaerobic spores in the same cooking
period. An initial concentration of 102 C. sporogenes spores per patty or less
can be reduced to nondetectable levels for 60 days of refrigerated storage (4-
6°C) when the meat patties are thermally processed at 71 or 82°C for 30
minutes.

5) The commercial package (PEIEVOH) showed changes in most physical
properties after exposure to thermal processing. Loss of mechanical strength
after cooking was observed. Oxygen permeability of this material was
inconsistent after cooking because of the surface area changes (shrinkage) and
increased thickness. There were small changes in both mechanical and barrier
properties of the super barrier package after cooking. Therefore, change in

material properties must be considered when selecting the appropriate

63

packaging materials to be used for cook-in-package meat products.

APPENDIX

W

Each sample in the study contained 3 plates form 10'2 to 10'4
dilution. Plates from the same sample were individually counted and calculated
as units of CFU per gram meat sample following these steps. Reported number
of CFU per gram sample was averaged from the CFU value calculated from
each plate of the same sample.

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

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

3) If colony growth in each plate ranged between 25-300 colonies - the number
of colonies on plate was counted and reported as CFU per gram sample.

4) If the growth in all plates was more than 300 colonies - then it was reported
as more than 3.0x106 CFU per gram. An estimated number of colonies in plate
was obtained from the average count of 4 (four) 1x1 cm squares selected
randomly in a 10'4 dilution, then multiplying this number by the plate surface

area.

64

BIBLIOGRAPHY

BIBLIOGRAPHY

1) ATCC 1982 The ATCC Catalog of strains: Vol 1: bacteria, phages, and rDNA
vectors. 12-15 ed. American Type Culture Colection Rockville, MD.

2) ASTM 1983 Designation D 882-83 Standard test methods for tensile
properties of thin plastic sheeting. ln: Selected ASTM standards on
packaging, 1st ed. American Society for Testing and Materials,
Philadelphia, PA. p. 49-60.

3) Anonymous 1988 Vacuum pouch sous vide cooking barred by FDA retail
code In: Food Chemical News, January 18, p.57

4) Anonymous 1988 Super-barriers from glass make new packages safer. In:
Modern Plastics, Vol. 65 (8):19.

5) Anonymous 1989 Cryovac CN-Series cook-in materials. Cryovac , Duncan
SC.

6) Arafa, AS, and Chen, TC. 1975 Effect of vacuum packaging on micro-
organisms on cut-up chickens and in chicken products etc. J.of Food
Science 40:50

7) Baren W.L., Kraft AA, and Walker H.W. 1969 Effects of carbon dioxide and
vacuum packaging on color and bacterial count of meat. J. Milk Food
Tech. 33:77.

8) Blickstad,E. and Molin, G. 1983 Carbon dioxide as a controller of the
spoilage of pork, with special reference to temperature and sodium
chloride. J. Food Protect. 46:756

9) Borgstrom, G. 1968 Principle of Food Science. Vol.1. MacMillan Co., NY. p:
- 136.

10) Buchanan, RE. and Gibbons, NE. 1974 Bergey's Manual of determinative
bacteriology vol.1. Krieg, N.R. ed. The Williams & Wilkins 00., Baltimore,
MD. p. 556.559

11) Buchanan, R.L. 1986 Processed meats as a microbial environment. Food
TechnologY. Vol. 40(4):134

65

66

12) Busta, F.F., Peterson, E.H. Adams, 0M. and Johnson, MG. 1984 Colony
count methods. In: Compendium of Methods For the Microbiological
Examination of Foods. American Public Health Assoc. (APHA)
Washington 00., p. 63-83.

13) Dainty, R.H., Shaw, B.G., Charmaigne, D., Harding and Silvia Michanie
1979 The spoilage of vacuum-packed beef by psychrophiles. In: Cold
Tolerent Microbes in Spollage and the Environment. A.D. Russell and R.
Fuller (editors). Academic Press, N.Y. p. 83-100.

14) Eklund, M.W., Peterson, M.E., Paranjpye, R. and Pelroy GA. 1988
Feasibility of a heat pasteurization process for the inactivation of
nonproteolytic Clostridium botulinum types B and E in vacuum-
packaged, hot-process (smoked) fish. J. of Food Protect. 51 (9):720

15) Genigeorgis, C. 1987 The risk of transmission of zoonotic and human
diseases by meat and meat products. In: Elimination of Pathogenic
Organisms From Meat And Poultry Smoulders, F.J.M. (editor). Elsevier
Science Publishers B.V. Amsterdam, Netherlands. p:111-145

16) Gill, J.L. 1987 Design and analysis of experiments. (in the animal and
medical sciences) 4th ed., Vol.1 and 3. The Iowa State University Press,
Ames, Iowa. pp. 228-235 (Vol.1), 21-50 (Vol.3).

17) Gilliland, S.E., Michener, H.D. and Kraft, A.A. 1984 Psychroyrophic micro-
organisms. ln: Compendium of Methods For the Microbiological
Examination of Foods. American Public Health Assoc.(APHA)
Washington DC. p. 135-141

18) Goepfert, J.M. and Kim, H.U. 1975 Behavior of selected food-borne
pathogens in raw ground beef. J. Milk Food Technol. 38(8):449.

19) Goldoni, J.S., Kojima, S., Leonard, S., and Heil, JR 1980 Growing spores
of PA 3679 in formulations of beef heart infusion broth. J. of Food Sci.
45:467

20) Hanna, M.O., Stewart. J.C., Carpenter, Z.L. and Vanderzant, C. 1977 Effect
of packaging methods on the development of Yarsinia enterocolitica on
beef stakes. J. Food Safety 1:29

21) Harte, BR. 1987 Packaging of restructured meats. In: Advances in Meat
Research Vol. 3. Pearson, A.M. (editor) AVI Publishing Co. Westport, CT.
p.433.

22) Ingram, M. 1972 Meat preservation- past, present and future. Royal Soc. of
Health Journal 92(3):121.

23) Johnson M., and Mitchell A. 1989 Crisis in food. In: Marketing (UK). Oct. 26
p. 24-29.

67

24) Johnston, R.W. and Tompkin, RB. 1984 Meat and poultry products. In:
Compendium of Methods For the Microbiological Examination of Foods.
American Public Health Assoc. (APHA) Washington DC. p.611-623.

25) Lake, DE. and Lynt, R.K. 1984 Mesophilic anaerobic sporeformers. In:
Compendium of Methods For the Microbiological Examination of Foods.
American Public Health Assoc. (APHA) Washington DC. p. 221-233

26) Light N., Williams R., Barrett J., Hudson P., and Schafheitle J.M. 1988 A
pilot study on the use of vacuum cooking as a production system for high
quality foods in catering and retail. Int. J. of Hospitality Management 7:21.

27) Lundquist , BR. 1987 Protective packaging of meat and meat rpoducts. In:
The Science of Meat and Meat Products 3rd edition. Price, J.F. and
Schweigert,B.S. (editors). Food and Nutrition Press Inc. Westport, CT. p.
487-506

28) Maas M.R., Glass K.A., and Doyle MP. 1989 Sodium Lactate delays toxin
production by Clostridium botulinum in cook-in-bag turkey products.
Appl. and Environ. Microbio. 55 (9)2226 ,

29) National Canners Association (currently NFPA) 1973 Thermal destruction
of type E Closfn'dium botulinum. NCA Reseach Found. Washington DC.

30) National Canners Association (currently NFPA) 1976 Processes for low acid
canned foods in metal containers. Research Laboratory Bull. No. 26-L.

31) National Food Processors Association (NFPA) 1988 Factors to be
considered in establishing good manufacturimg practices for the
production of refrigerated foods. Dairy and Food Sanitation 8 (6)288.

32) Prabhu G.A., Molins R.A., Kraft A.A., Sebranek J.G., and Walker H.W.1988
Effect of heat treatment and selected antimicrobials on the shelf-life and
safety of cooked, vacuum packaged, refrigerated pork chops. J. of Food
Science 53 (5):1270

33) Raffael, M. 1984 Revolution in the kitchen. In: Caterer and Hotelkeeper, 16
August.

34) Rosinsky M.J., Barmore C.R., Dick R.L. and Acton J.C. 1989 Film sealant
and vacuum effects on two measures of adhesion at the sealant-meat
interface in a cook-in packaging system for processed meat. J. of Food
Science 54 (4):863.

35) Schafheitle J.M. and Light ND. 1989 Technical note: Sous-vide preparation
and chilled storage of chicken ballotine. Int. J. of Food Science and
Tech. 24:199.

36) Seideman, S.C., Vanderzant, C., Hanna, M.O., Carperter, Z.L., and Smith,
GO. 1976 Effect of various types of vacuum packages and length of

68

storage on the microbial flora of wholesale and retail cuts of beef. J. Milk
Food Technol. 39(11):745.

37) Simard, R.E., Zee, J., and L'Heureux, L. 1984 Microbial growth in carcasses
and boxed beef during storage. J. Food Protect. 47:773.

38) Simms, W.C. (editor) 1985 Packaging Encyclopedia and Yearbook Vol. 30
(4). Cahners Publishing Denver, CO. p.52-67.

39) Simunovic J., West R.L., and Adums JP 1985 Formulation of a pasteurized
restructured beef product. J. of Food Science 50:693.

40) Simunovic. J., Oblinger. J.L., and Adams, JP 1985 Potential for growth of
nonproteolytic types of Clostridium botulinum in pasteurized restructured
meat products: a review. J. of Food Protect. 48(3)265.

41) Smith D.M. and Alvarez VB. 1988 Stability of vacuum cook-in-bag turkey
breast rolls during refrigerated storage. J. of Food Science 53 (1):46

42) Sofos, J.N. 1986 Antimicrobial activity and functionability of reduced
sodium chloride and potassium sorbate in uncured poultry products. J. of
Food Science 51 (1):16

43) Stumbo, C.R., Murphy, JR and Cochran, J. 1950 Nature of thermal death
time curves for PA 3679 and Clostridium botulinum. Food Technology,
Vol. 4(8):321

44) Sutherland, JP. and Varnam, A. 1982 Fresh meat proceessing. In: Meat
Microbiology. M.H. Brown (editor) Applied Science Puiblishers Ltd.,
London, UK. p. 103-128.

45) Swientek R.J. 1989 Sous Vide- refrigerated upscale food in a vacuum
pouch. Food Processing, Vol. 50(6):34

46) Taclindo C. JR., Midura T., Nygaard S. and Bodily H.L. 1967 Examination of
prepared foods in plastic packages for Closfridium botulism Applied
Microbiology 15 (2): 426

47) Terlizzi, F.M., Perdue, R.R., and Young LL. 1984 Processing and
distributing cooked meats in flexible films Food Technology, Vol. 61 (3):67

48) Tompkin, RB. 1986 Microbiology of ready-to-eat meat and poultry products.
In: Advances in Meat Research Vol.2, Pearson, A.M. (ed.) AVI Publishing
Co. Westport, CT. p.89

49) Townsend, C.T., Somers, LL, Lamb, EC. and Olson, NA 1956 A laboratory
manual for the canning industry. 2nd ed. NCA Research Laboratories,
Washington DC.

69

50) Wehr, M.H. 1978 Attitudes and policies of state governments. Food
Technology. Vol. 31(January):63.

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