APPLIED MICROBIOLOGY, Mar. 1968, p. 445-449 Vol. 16, No.

3
Copyright 1968 American Society for Microbiology Printed in U.S.A.

Survival of Aerobic and Anaerobic Bacteria in

Chicken Meat During Freeze-Dehydration,
Rehydration, and Storage1
J. R. CHIPLEY AND K. N. MAY

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Departments of Food Science and Poultry Science, University of Georgia, Athens, Georgia 30601
Received for publication 18 September 1967

Total and anaerobic counts were ascertained on boneless, cooked, cubed, frozen
chicken meat. We determined survival of aerobes and anaerobes in the natural
flora after the meat was freeze-dehydrated and rehydrated at room temperature for
30 min and at 50, 85, and 100 C for 10 min. Total and anaerobic counts of bacteria
in the rehydrated meat were established during storage of samples at 4, 22, and 37 C
-until a spoilage odor was detected. Samples were also inoculated with Clostridium
sporogenes and were dried and rehydrated at 100 C and stored at 37 C. Approxi-
mately 21 % of the aerobes and 37 % of the anaerobes survived drying and rehydra-
tion at room temperature. Many genera of aerobes, anaerobes, and facultative
anaerobes survived drying and rehydration at 50 C; only sporeformers survived
rehydration at 85 or 100 C. Low-temperature (4 C) storage of rehydrated meat
produced ample shelf life (over 20 days), whereas storage at the higher temperature
resulted in a shelf life of less than 30 hr. Approximately 81% of the C. sporogenes
cells survived rehydration at 100 C and grew to over 107 cells within 40 hr. Our
study presents additional data for adequate microbiological control in processing of
freeze-dehydrated meat. Also, it points out the natural selection for sporeformers
at high temperature of rehydration, stressing the need for consumer education in
product handling for safety purposes.

Previous investigations in this laboratory (3)
have indicated that many aerobic bacteria, present
as natural flora on cooked, boned chicken meat,
survived freeze-dehydration and rehydration at
mild temperatures (up to 50 C). Sporeforming
bacteria survived rehydration at 85 and 100 C for
10 min; these bacteria were capable of rapid
growth when the rehydrated meat was main-
tained at high temperatures. Saleh and Goldblith
(4) reported similar results in their investigations
of freeze-dehydrated fish patties, shrimp, chicken,
and pork.
Wells (5) showed that spores of Clostridium
botulinum, inoculated on cooked chicken meat,
were reduced in number by 53% during freezing
and were further decreased by an additional 15%
during freeze-dehydration. Wells stated that there
was little doubt that C. botulinum spores would
persist in the dehydrated product for long periods
of storage. We found no reports of investigations
in which a wide variety of rehydration and storage
1 University of Georgia, College of Agriculture
Experiment Stations, Journal Series Paper No. 143, materials, 10-g meat samples (either nondried or dried
College Station, Athens, Ga. 30601.
temperatures were studied as to their effect on
survival of anaerobic bacteria in freeze-dehy-
drated meat.
This study was conducted to determine the
survival of natural aerobic and anaerobic bac-
teria, as well as inoculated C. sporogenes, in
chicken meat during freeze-dehydration, rehydra-
tion at various temperatures, and subsequent
storage at different temperatures.
MATERIALS AND METHODS
Source of meat. Cooked, cubed, and frozen meat
was purchased from a commercial processor for use in
our study. The meat had been boned from broilers
that had been cooked in water and then cubed into
small pieces, packaged in 5-lb polyethylene bags, and
frozen in a blast freezer. In the laboratory, frozen,
packaged meat was maintained at approximately
-30 C.
Freeze-dehydration. The frozen, cubed meatstored
was
freeze-dehydrated, and the dried product was
in the manner described by May and Kelly (3).
Bacteriology. By use of aseptic techniques and
and rehydrated) were weighed and were homogenized
445
446 CHIPLEY AND MAY
in an Omnimixer with 90 ml of 0.85% NaCl for 3 min.
Serial dilutions were made in saline, and duplicate
pour plates or shake tubes were prepared. We deter-
mined the aerobic counts using pour plates with
Tryptone Glucose Extract Agar (TGEA); anaerobic
counts were established by use of either shake culture
tubes (2) containing thioglycolate medium with
added agar (15 g/liter) or pour plates of the same
medium overlaid with 3% agar. (All media were
manufactured by either Difco or BBL.) Incubation
was at 37 C for 48 hr for aerobes and for 96 hr for
anaerobes; plates containing 30 to 300 colonies were
then counted and the results were recorded as count
per gram of meat. All colonies that developed in the
shake culture tubes or overlaid agar plates were re-
corded as anaerobes, although we realized that some
facultative anaerobic bacteria were included.
We selected colonies considered to be representa-
tive of the types of bacteria present from plates or
tubes used to determine counts and isolated on TGEA
or thioglycolate plates with agar overlay. These
colonies were then transferred to nutrient agar slants
or were stabbed in nutrient agar tubes, and they were
stored at 4 C until identification procedures were con-
ducted. Only the types of colonies that appeared pre-
dominant were selected, and no attempt was made
to identify all types of organisms present or to deter-
mine the numbers of each. We followed the outline of
Bergey's Manual for identification procedures.
Analyses performed. The frozen chicken meat as
received from the processor was sampled initially and
at eight additional times during the course of the study
(a period of about 4 months).
Samples of freeze-dehydrated meat were removed
from storage and were assigned rehydration and
storage treatments. Rehydration treatments were con-
ducted at room temperature for 30 min and at 50, 85,
and 100 C for 10 min. The samples rehydrated at room
temperature were not stored. The other samples were
stored at 4, 22, and 37 C. Temperatures of rehydration
and storage were the same as those employed by May
and Kelly (3); these temperatures were chosen to
cover the extremes that might be encountered in use
of such a product by a consumer. After rehydration,
the samples were transferred to clean, unsterilized
cheesecloth, allowed to drain for 3 min, and stored in
sterile 500-ml Erlenmeyer flasks. At intervals during
storage, 10-g samples were extracted, and aerobic and
anaerobic counts were determined. A panel of three
experienced persons judged the odor- and color-
acceptability of the meat during storage; it was con-
sidered spoiled when all three individuals inde-
pendently judged the meat to have an off-odor. In
some cases, storage was continued beyond the "spoil-
age" time to study further variations in numbers of
bacteria. Each rehydration and storage sequence was
replicated from five to eight times to determine
changes in numbers of microorganisms.
C. sporogenes was used as an inoculum in one part
of the study. The culture (supplied by M. K. Hamdy,
Food Science Department, University of Georgia)
was grown for 48 hr at 37 C in fluid thioglycolate
medium. Cells were harvested by centrifugation at
10,000 X g for 15 min and were washed twice with
APPL. MICROBIOL.
0.85%o NaCl. The cells were suspended in 0.85%
NaCl containing 0.1% tryptone, and 5-ml portions
were placed in 15-ml sterile tubes at -34.4 C. As
needed, these tubes were removed from the freezer
and thawed for 2 hr; a portion was extracted to deter-
mine the number of viable organisms, and the re-
mainder was used as an inoculum. To free the chicken
meat of competitive organisms, a piece of meat was
placed in a beaker of water and was autoclaved at
121 C for 15 min. By use of aseptic techniques, a
portion of the meat was transferred to sterile freeze-
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drying flasks, and a known number of cells of C.
sporogenes were added. The product was then freeze-
dried and rehydrated at 100 C, and serial dilutions
were prepared to determine changes in cell numbers.
We selected a storage temperature of 37 C so that
bacteria surviving the freeze-drying process would
begin to grow and subsequently multiply as quickly
as possible. Six replications were made for this part
of the study.
RESULTS
The precooked frozen chicken meat had a mean
bacterial count of 1.3 X 104 per g for aerobes
and 1.3 x 10 per g for anaerobes.
After freeze-drying and rehydration at room
temperature for 30 min, the meat had a mean
aerobic count of 2.8 X 103 bacteria per g. This
was a reduction of 1.1 X 104 bacteria per g during
the freeze-drying process or a survival rate of
approximately 21 %. However, the mean
10
9-
w
a.
7
CD
0
z
0
2
TIME
FIG. 1. Change in anaerobic bacteria per g offreeze-
dehydrated chicken after rehydration at 50 C for 30
min and storage at 4 C (0), 22 C (A), and 37 C (O).
Time represents hours for storage at 22 and 37 C and
days for storage at 4 C.
VOL. 16, 1968 BACTERIA IN FREEZE-DEHYDRATED CHICKEN 447
evident at 18 and 20 hr for the products stored
at 22 and 37 C, respectively.
Rehydration of meat at 100 C and storage at
4 C caused a rapid decline in numbers of anaero-
bic bacteria until the 9th day (Fig. 3). However,
from 9 to 30 days of storage no viable organisms
2 were recovered from the product rehydrated at
100 C and stored at 4 C; the product still had no
objectionable odors after 30 days of storage. The
7-,o anaerobes in the product rehydrated at 100 C

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and stored at 22 C grew rapidly for 14 hr, but
these anaerobes tended to increase slowly there-
m after, until spoilage odors became apparent at
48 hr of storage. In the product rehydrated at
0 100 C and stored at 37 C, the numbers of anaer-
obes remained relatively constant until about 15
hr; then they increased rapidly until spoilage
occurred at 31 hr.
Results of aerobic and anaerobic bacterial
counts obtained immediately after spoilage, as
c 5 10 15 20 25 30 35 well as the time required for a spoilage odor to
TIME develop in samples, are summarized in Table 1.
FIG. 2. Change in anaerobic bacteria per g offreeze- Decreasing storage temperature of rehydrated
dehydrated chicken after rehydration at 85 C for JO meat from 37 or 22 to 4 C extended shelf life,
min and storage at 4 C (0), 22 C (A\), and 37 C (Ol). but increasing rehydration temperatures from 50
Time represents hours for storage at 22 and 37 C and to 85 or 100 C increased shelf life to a lesser extent.
days for storage at 4 C. We identified both aerobic and anaerobic bac-
teria from meat rehydrated at each of the three
previously mentioned temperatures (Table 2).
anaerobic count was 4.9 X l02 bacteria per g, Many types of bacteria were recovered from
a reduction consisting of 8.5 X 102 bacteria per samples rehydrated at 50 C, but only sporeformers
g during the freeze-drying process or a survival
rate of approximately 37%.
Effects of rehydration of meat to 50 C and
storage at 4, 22, and 37 C on numbers of anaer- 10
obes are shown in Fig. 1. At 4 C the numbers
of anaerobic bacteria declined slightly for the 9
first 3 days and then increased rapidly until 21
days, when spoilage was noted. Growth was much 08
more rapid in meat stored at 22 and 37 C than
in that stored at 4 C, with growth beginning
immediately after rehydration. Spoilage occurred w6
at 24 and 16 hr for meat stored at 22 and 37 C,
respectively. Similar, but slightly greater, increases
in numbers of aerobic bacteria were obtained, 0
r4-
but these increases are not shown because they
resemble previously reported data (3).
Rehydration of chicken meat at 85 C and
storage at 4 C caused an extended lag phase (up 82
to approximately 13 days) for anaerobic bacteria
(Fig. 2). After the initial lag, growth was rapid
up to about 18 days after which it leveled off-
although the product was not considered spoiled TIME
until 24 days of storage. Growth curves for an- FIG. 3. Change in anaerobic bacteria per g offreeze-
aerobes in the product rehydrated at 85 C and dehydrated chicken after rehydration at 100 C for 10
stored at 22 or 37 C were almost identical (Fig. 2). min and storage at 4 C (0), 22 C (A), and 37 C (O).
Numbers remained almost stationary for about Time represents hours for storage at 22 and 37 C and
5 hr and then increased rapidly with spoilage odor days for storage at 4 C.
448 CHIPLEY AND MAY APPL.. MICROBIOL.

TABLE 1. Summary of shelf life and numbers of aerobic and anaerobic bacteria on freeze-dehydrated
chicken rehydrated and stored at various temperatures
Temp of Temp of Initial count per g Count at spoilage
rehydra- storage Time required to spoil
tion (C) (C) Aerobes Anae robes Aerobes Anaerobes

50 4 2.0 X 103 4.0 X 103 4.0 X 1011 4.0 X 101 21 days

50 22 1.6 X 103 6.3 X 103 1.0 X 109 1.0 X 108 24 hr
50 37 1.6 X 103 1.6 X 103 6.3 X 108 4.0 X 109 16 hr

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85 4 1.0 X 103 3.2 X 104 5.1 X 107 3.2 X 107 24 days
85 22 4.0 X 104 1.6 X 103 2.5 X 108 1.0 X 1011 20 hr
85 37 6.3 X 104 1.6 X 103 1.O X 108 4.0 X 109 18 hr
100 4 2.5 X 103 5.1 X 103 <1 at 30 day <1 at 30 day Terminated at 30 days
100 22 6.3 X 103 1.0 X 103 4.0 X 101 2.5 X 109 48 hr
100 37 1.0 X 103 2.5 X 103 4.0 X 109 1.6 X 108 31 hr

were isolated from samples rehydrated at 85 and
100C.
When we inoculated samples of meat with C.
sporogenes (58 cells per g) and subsequently
freeze-dehydrated and rehydrated these samples
at 100 C for 10 min, a mean survival of 81 % of
the organisms was observed. Counts increased
to a mean of 7.4 X 1007 C. sporogenes cells per
g of product within 40 hr when stored at 37 C.
DiscussIoN
The recovery of 21% of the aerobic and 37%
of the anaerobic bacteria present in the original
chicken meat after freeze-dehydration and rehy-
dration at room temperature is in agreement with
previous studies of May and Kelly (3); their data
showed a recovery of 32%. The variety of genera
surviving the freeze-dehydration and rehydration
at 50 C (Table 2) are also similar with the details
given by May and Kelly. Both Escherichia and
Salmonella spp. were identified in meat during
this study. In contrast, Saleh and Goldblith (4)
reported no salmonellae or coliform organisms, TABLE 2. Bacteria identified from representative
when they examined freeze-dehydrated chicken.
However, Gunderson et al. (1) revealed that E.
coli, Aerobacter aerogenes, and their variants are
frequently found in cooked, boned chicken meat.
May and Kelly (3) also identified E. coli in their
tests with freeze-dehydrated chicken meat.
Our study confirms the previous report from
this laboratory (3) that vegetative cells of many
types of bacteria can survive freeze-dehydration
and rehydration at low temperature (50 C). In
addition, these studies indicate that rehydration
of freeze-dried meat at high temperature (85 to
100 C) effectively selects for sporeforming species From anaerobic tubes and plates
of bacteria. Inoculated C. sporogenes had a sur-
vival rate of 81% after dehydration and rehydra-
tion at 100 C for 10 min; these results are in
accordance with the 81 % survival rate of C.
botulinum reported by Wells (5) in chicken meat
during freeze-dehydration. The rapid growth of
C. sporogenes (up to 7.4 X 107 cells per g within
40 hr) at 37 C suggests that surviving cells can
multiply to dangerous levels in a very short period
of time. Thus, the accidental or intentional delay
of use of rehydrated meat containing any spores
of pathogenic bacteria could lead to tragic results.
These studies give additional evidence of the
need for adequate microbiological control during
processing of freeze-dehydrated chicken. Further
more they point out that such control alone is
not sufficient for the protection of the ultimate
consumer. If the product is maintained at high
temperature after rehydration, pathogenic spore-
formers are capable of survival and rapid growth.
Thus, the consumer should be urged to refrigerate
any rehydrated product promptly if it is not used
immediately.
colonies isolated during the rehydration-
storage tests
Bacteria identified Rehydration
temp (C)
From aerobic plates
Lactobacillus spp ................ 50
Escherichia coli ................. 50
Aerobacter aerogenes ............ 50
Salmonella spp .................. 50
Streptococcus spp ............... 50
Staphylococcus aureus ........... 50
Bacillus cereus .................. 50, 85, 100
S. aureus ....................... 50
Clostridium sporogenes .......... 50, 85, 100
C. perfringens ................ 50, 85, 100
VOL. 16, 1968 BACTERIA IN FREEZE-DIEHYDRATED CHICKEN
LITERATURE CI1D
1. GUNDERSON, M. F., H. W. McFADDEN, AND
T. S. KYLE. 1954. The bacteriology of com-
mercial poultry processing. Burgess Publishing
Co., Minneapolis, Minn.
2. HAMDY, M. K., E. L. SHERRER, H. H. WEISER,
AND W. D. SHEETs. 1954. Microbiological
factors in the treatment of phenolic wastes.
Appl. Microbiol. 2:143-148.
3. MAY, K. N., AND L. E. KELLY. 1965. Fate of
449
bacteria in chicken meat during freeze-dehy-
dration, rehydration, and storage. Appl.
Microbiol. 13:340-344.
4. SALEH, B. A., AND S. A. GOLDBLITH. 1966. Micro-
bial evaluation of commercial freeze-dried
foods. Food Technol. 20:103-106.
5. WELLS, F. E. 1966. A study of the microbiology
of selected dehydrated food products. Tech.
Rept. 66-35-FD, U.S. Army Natick Labora-
tories, Natick, Mass.
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