870
Journal ofFood Protection, Vol. 44, No. II, Pages 870-873 (November I98I)
Copyright©, International Association of Milk, Food, and Environmental Sanitarians
Effect of Combinations of Fresh and Frozen Beef on
Microbial Flora of Ground Beef Patties
A. A. KRAFT 1* , K. V. REDDY 1, J. G. SEBRANEK 2, R. E. RUST 2 and D. K. HOTCHKISS 3
Departments ofFood Technology, Animal Science and Statistics, Iowa State University, Ames, Iowa 50011
(Received for publication December 14, 1980)
ABSTRACT
Beef patties composed of fresh beef, blast frozen beef or
combinations of fresh and frozen beef were then frozen by
liquid nitrogen (LN 2) or liquid carbon dioxide (LC0 2) and
stored at -20 C for 6 months. Analyses for various bacteria were
made at monthly intervals to evaluate effects of originally
combining fresh and frozen beef on the subsequent microbial
flora. Of the different combinations of fresh and frozen meat,
the mixture in a 50:50 ratio produced highest bacterial
numbers during frozen storage. Lowest bacterial counts
resulted from use of fresh beef with no blast frozen meat but
frozen subsequently with LN 2 or LC0 2 •
In recent years, hamburgers have been the meat items
consumed in greatest quantity by the U.S. population
eating meats away from home. Approximately 5.6 billion
"eater occasions" involved purchase of hamburger, with
a possible 6.7 billion hamburgers consumed in
restaurants in 1978 (6). Ground beef, therefore, is a
significant product for use in fast-food chains, which
make use of the beef in the form of frozen hamburger.
Because of these trends, ground beef has received
considerable attention with regard to its microbiology
and sanitary quality (3,4,9,16). Cryogenic freezing of
ground beef patties has been advocated for use with
centralized processing to provide increased sanitation in
handling (10). Meat frozen by cryogenic means has been
shown to have consistently good quality (7) and to
demonstrate reduced tissue damage as compared with
conventionally frozen meat (15).
Previous work in our laboratory (12) showed that
cryogenic freezing produced significantly greater reduc-
tion in counts of mesophiles and psychrotrophs in beef
patties than did mechanical freezing. Taste panel
evaluations also favored cryogenically frozen beef patties
compared with blast frozen patties (13).
Fast food restaurants use different proportions of fresh
'Department ofFood Technology.
'Department ofAnimal Science.
3
Department ofStatistics.
JOURNAL OF FOOD PROTECTION, VOL. 44, NOVEMBER 1981
and frozen meat for ground beef patties but there is no
information available on how this practice might affect
microbiological quality of the products. The primary
objective of this study was to determine the effects of
combining fresh and frozen beef in different proportions
on the microbial flora of ground beef patties. The
investigation also included effects of immediate cryo-
genic freezing and frozen storage in a blast freezer for 6
months.
MATERIALS AND METHODS
Ground beef patties were made from 600-800-lb (270-360 kg) "A"
maturity steer carcasses that were chilled to about 5 C for 24 to 72 h
before use. During boning, lean and fat tissues were combined into
batches composed of about 20% fat, which was confirmed by Any! Ray
analysis. Adjustments were made so that each batch had a fat content
of 20%. Meat to be used for frozen beef addition was packed in plastic-
lined boxes and frozen in a blast freezer at -30 C (air velocity 4040 cfm).
This beef was kept frozen for 1 week before combining with fresh beef.
Flaking of frozen beef was done with a Butcher Boy flaker after which
the flaked trim was ground through a 0.95-cm plate, mixed with
appropriate proportions of unfrozen (fresh) trim and ground through a
0.32-cm plate. Fresh and frozen meat combinations were as follows:
100% fresh; 80% fresh, 20% frozen; SO% fresh, SO% frozen; 100%
frozen. All products were blender-chilled until internal temperatures
reached 0.5 to 1.0 C. All formulations were made into patties in a
commercial patty forming machine (Hollymatic Model 500A), with four
patties per pound of meat. Patties were frozen cryogenically in a 20-ft
kwikfreeze tunnel (Airco Industrial Gases, Inc.) by liquid nitrogen
(LN 2) or liquid carbon dioxide (LC0 2). After freezing, patties were
packed in plastic-lined boxes and stored at about -20 C for periodic
evaluations. Samples were analyzed at monthly intervals up to 6
months of frozen storage for mesophiles, psychrotrophs, coliforms,
staphylococci. Clostridium perfringens and salmonellae. Analyses were
made of fresh ground beef, ground beef frozen for 1 week before
combining with fresh beef, and fresh-frozen patty combinations.
Patties were thawed in a refrigerator at about 5 C only long enough for
samples to be obtained for microbiological analyses. Bacteriological
procedures used are shown in Table 1. 1t is possible that
lactose-fermenting salmonellae might not have been detected because
Bismuth Sultite (BS) agar was not used. However, our previous work
indicated little difference between BS and BGS for recoveries of
salmonellae from meats.
To determine effects of combining different ratios of fresh and frozen
meat and the influence of cryogenic freezing methods on survival or
growth of various types of microorganisms, a taxonomic study was
done. A total of 418 cultures were characterized along with 14 known
cultures as reference organisms and grouped into genera by means of
 MICRO FLORA OF BEEF PATTIES 871

numerical taxonomy. Cultures purified for charaderization are listed
by treatment of patties in Table 2. Reference cultures included
Moraxella-Acinetobacter, Pseudomonas (several species), Flavobacte-
rium, Escherichia coli. Enterobacter aerogenes, Bacillus, Aeromonas,
Micrococcus and Staphylococcus. This characterization was done to
detect changes in the type of microflora before and after freezing and as
a result of frozen storage. Bergey's Manual of Determinative
Bacteriology (2) was followed for identification of isolates.
RESULTS AND DISCUSSION
Freezing caused reduction of all types of bacteria
tested before frozen meat was mixed with fresh beef
{fresh meat compared with blast frozen in Fig. 1). No
distinct differences were found in survival or growth of
different organisms because of method of cryogenic
freezing and counts were averaged for both methods, as
shown in Fig. 1. Combining frozen meat with fresh meat
resulted in increased bacterial counts when compared
with fresh beef alone. In general, microbial populations
decreased during frozen storage. Psychrotrophic popula-
tions paralleled those of mesophiles. Numbers of
coliforms decreased markedly during 6 months of frozen
storage; these organisms generally show a decline after
freezing and frozen storage. Populations of staphylococci
remained relatively constant, even throughout the
6-month holding period, similar to results obtained in
our previous study on composition of ground beef patties
(12).
Counts of C. peifringens were reduced considerably by
either freezing method and frozen storage, although
numbers initially were low in fresh beef. Vegetative cells
of C. peifringens were recovered from only 9 of 112
samples tested; but the organisms persisted throughout
frozen storage. While C. perfringens cells may be
expected to lose viability during frozen storage, three
isolations were made at the end of the storage period. No
salmonellae were recovered, regardless of type of meat
used or method of cryogenic freezing.
Effects of the different cryogenic freezing methods
were similar on numbers of organisms examined. Little
difference may be expected in counts of mesophiles,
psychrotrophs, coliforms or staphylococci when either
LN 2 or LC0 2 are used for freezing ground beef patties
with subsequent holding at freezing temperatures,
regardless of amount of previously frozen beef mixed

TABLE 1. Bacteriological procedures employed.

Quantitative
determinationsa Growth media Incubation tests
Psychrotrophs Trypticase soy agar Pour plates 5 C, 7 days
Mesophiles (BBL)b Pour plates 30 C, 36 h
Coliforms Violet red bile agar Pour plates 37 C, 24 h Gram stain
(Difco)c (overlayed) Levine EMB agar
(Difco)
Coagulase Staph 110 medium Tube coagulase test
positive with egg yolk
Staphylococcus (11)

Qualitative
determinationsd Enrichment Isolation Confirmatory tests
Salmonella Procedure for meats BGS agar (8)
(8)
Clostridium D-cycloserine Anaerobic pouches (1) Nitrate-motility
perfringens (17) medium
a30-gsample.
bBBL Division ofBioQuest, Cockeysville, Md.
cDifco Laboratories, Detroit, Mich.
d A swab technique was used for sampling about SO cm 2 of surface.

TABLE 2. Number characterized.

Product Treatment Number of cultures

Fresh beef Before 61
After 3 months
After freezing in frozen
100 o/o fresh beef patties Frozen by LN 2 33 27
Frozen by LC0 2 32 28
SO o/o fresh: SO o/o frozen Frozen by LN 2 30 28
Frozen by LC0 2 32 29
lOOo/o frozen beef patties Frozen by LN 2 32 28
Frozen LC0 2 32 26

JOURNAL OF FOOD PROTECTION, VOL. 44, NOVEMBER 1981

872 KRAFTETAL.

6.0 surviving frozen storage were very similar with both meat
0FRESH MEAT combinations (20% frozen: 80% fresh or 100% frozen).
r-
"'" fil BLAST FROZEN With the 50:50 combination, sufficient tissue-damaged
5.0
[:.
- ,-
0 MIXED FROZEN AND FRESH beef may have been provided to allow for maximum
r- ~~ OVERALL 6 MONTHS nutrient availability, hence bacterial numbers were
:Iii 4.0 , ..· FROZEN STORAGE
greatest with this combination. While no actual proof is
~
<!I
:·
r-
C":"
!·
- available from this work to conclusively explain the
...... 3.0
Q;
: results obtained, the possibilities described should be
<
ii'
I'·
[': k L considered. It is also possible that breaking of clumps of
~ organisms as a result of freezing may have added to the
!12.0 '· I r> counts in samples containing frozen meat in combination
0
z
I< with fresh beef. In the original unfrozen meat, fewer
8.... 1.0
f :l· ~.·· I•' injured cells might be expected to be present than in the

0
._t.JI__
MESOPHILES
;;;

PSYCHROTROPHS
t
COLIFORMS
L-L- '-
C. perfringens
STAPHYLOCOCCI
Figure 1. Bacterial counts of ground beefpatties before and
after freezing and frozen .~torage.
with fresh meat before cryogenic freezing.
Bacterial counts were subjected to a "t" test by pooled
variance and differences were expressed by calculating
least significant differences (14). Statistical analysis of
counts for various combinations of fresh and frozen beef
is given in Table 3. Numbers of all organisms were
significantly lower in fresh beef throughout storage than
in any of the mixtures of fresh and frozen beef.
Combination of fresh and frozen meat in a 50:50 ratio
was conducive to higher counts of spoilage organisms
than observed when lower percentages (0 or 20%) of
frozen meat were initially used for the patties.
Differences in numbers with this ratio were highly
significant (P < .01) for mesophiles, psychrotrophs and
coliforms, but significant at the 5% level for staphylo-
cocci. The combination of 20% frozen and 80% fresh
beef more closely resembled 100% frozen beef in
bacterial levels than it did fresh beef only. Possibly
greater availability of nutrients occurred when a
relatively small amount of frozen beef was combined with
fresh meat because of availability of tissue fluids as a
result of freezing. However, destruction of some bacterial
tissue and hence cells might similarly occur, as indicated
by Elliott and Michener (5). Both effects may have been
produced to the extent that numbers of organisms
A
frozen meat. These organisms, when given the available
nutrients resulting from tissue damage in the frozen
meat, might have readily grown to increase populations
in fresh-frozen combinations as compared with meat that
was not initially frozen, or with 100% frozen beef
containing more injured organisms.
It should also be recognized that plating procedures
may produce variability in counts. However, these
procedures were used consistently for all treatments and
statistical treatment of the data would also account for
inherent variation.
As indicated in Table 4, predominant genera or
groups recovered from beef patties at different treatment
stages were Moraxella-Acinetobacter and Pseudomonas,
with markedly lower levels of Flavobacterium, Staphy-
lococcus, Micrococcus and members of the Entero-
bacteriaceae. However, the predominant flora changed
in their proportions after freezing and frozen storage.
Moraxella-Acinetobacter increased considerably in per-
centage of total isolates after freezing, with a decline
during frozen storage. Pseudomonas was not greatly
affected in regard to proportion of the representative
population, but it showed a noteworthy increase as the
Moraxella-Acinetobacter group decreased in percentage
during frozen storage. The various types were more
uniformly distributed in proportion in fresh beef, but
after freezing, Moraxella-Acinetobacter and Pseu-
domonas dominated the bacterial population.
Changes in proportions of the dominant flora before
freezing, after freezing, and as a result of frozen storage
may be reflected in changes in the biochemical activity of

TABLE 3. Effect ofproportions offrozen and.fresh beef on bacterial counts of stored patties.

Percent Frozen Beef n Mesophiles Psychrotrophs Coliforms Staphylococci

0 28 4.31a 4.24a 2.24a 3.16a
20 28 4.57b 4.56b 2.42b 3.29b
so 28 4.72c 4.72c 2.ssc 3.3sbd
100 28 4.s8b 4.6ob 2.43b 3.26abe
For each column for the percent frozen be~f. counts having different letter superscripts are significantly (P<.Ol) different from each
other.
Note: Superscript "bd" differs from "b" significantly at the So/olevel but not at the 1 o/oleveL
Superscript "abe" differ§ from "a" significantly at the So/oievel but not at the 1o/o leveL
Superscript "b" does not differ signiticantly from "abe."

JOURNAL OF FOOD PROTECTION. VOL 44, NOVEMBER 1981

 MICROFLORA OF BEEF PATIIES
TABLE 4. Bacteria isolated from ground beefpatties.
Before
Kind of bacteria No. of isolates o/ooftotal
M oraxella-Acinetobacter 18 28.0
Pseudomonas 12 20.0
Flavobacterium 6 10.0
Staphylococcus 3 s.o
Micrococcus 7 13.0
Enterobacteriaceae 4 6.0
Other 11 18.0
Total 61
the organisms isolated at those stages. With regard to the
psychrotrophic nature of the isolates, about 75o/o grew
well at 5 C before freezing, with an increase to about 80%
after freezing and frozen storage. The percentage of
isolates capable of hydrolyzing beef fat increased from
about 18o/o before freezing to 35o/o after frozen storage,
probably due to the increase in proportion of
Pseudomonas. Tributyrin hydrolysis also was more
evident after freezing and frozen storage compared with
isolates from fresh beef. Proteolysis of casein and gelatin
was not greatly changed as a result of freezing and frozen
storage, but generally followed the pattern of alterations
in the proportions of Pseudomonas. The Moraxella·
Acinetobacter group is generally considered to consist of
metabolically inert organisms, although they are capable
of hydrolyzing tributyrin. In our previous study (12), all
isolates of this group hydrolyzed tributyrin.
In general, no health hazard was evident from any of
the combinations of fresh or frozen meat or from LN 2 or
LC0 2 freezing and frozen storage for as long as 6
months. The "mechanical" effects of freezing on meat
tissue may be important in making nutrients available
for uninjured organisms in the fresh meat additions. As a
result, the spoilage potential may be increased signifi-
cantly with use of frozen meat in combination with fresh
beef, particularly when fresh and frozen meat are mixed
in equal proportions. The practical implications of these
observations should be considered if fast food restau-
rants are to use frozen meat in combination with fresh
beef in formulation of ground beef patties.
ACKNOWLEDGMENTS
Journal Paper No. J-9911 of the Iowa Agriculture and Home
Economics Experiment Station, Ames, Iowa. Project No. 2252. The
authors thank Joan Andersen and Cynthia Lender for preparation of
the manuscript. Appreciation is also expressed to Airco Industrial
Gases, Inc .. Murray Hill. NY for technical assistance and partial
support of this work.
REFERENCES
I. Blade!. B. 0 .. and R. A. Greenberg. 1965. Pouch method for the
isolation and enumeration of clostridia. Appl. Microbiol.
13:281-285.
Erichsen and Molin, con't.fromp. 869
26. Taylor. A.A .. and D. B. MacDougalL 1973. Fresh beef packed in
mixtures of oxygen and carbon dioxide. J. Food Techno!.
8:453-461.
JOURNAL OF FOOD PROTECTION, VOL. 44. NOVEMBER 1981
873
After Freezing During Storage
No. of isolates %of total No. of isolates %oftotal
117 61.3 78 48.0
42 22.0 56 34.6
20 10.5 1 0.6
3 1.6 12 7.4
s 2.6 7 4.2
2 1.0 3 1.9
2 1.0 s 3.1
191 162
2. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's
manual of determinative bacteriology, 8th ed. The Williams and
Wilkins Co., Baltimore, MD.
3. Chestnut, C. M., B.S. Emswiler, A. W. Kotula, and E. P. Young.
1977. Bacteriological quality of ingredients used in ground beef
manufacture. J. Animal Sci. 44:213-217.
4. Duitschaever, C. L., D. H. Bullock, and D. R. Amott. 1977.
Bacteriological evaluation of retail ground beef, frozen beef patties
and cooked hamburger. J. Food Prot. 40:378-381.
5. Elliott, R. P., and H. D. Michener. 1965. Factors affecting the
growth of psychrophilic microorganisms in foods. A review. Tech.
Bull.1320. U.S. Dept. of Agriculture, Washington, DC.
6. Ernst, L. J. 1979. Changing demands for meat. Proc. Meat
Industry Research Conference. Am. Meat Sci. Assoc., Am. Meat
Ind. Found., Chicago, IL. pp. 1-14.
7. Fennema, 0. 1968. General principles of cryogenic processing.
Proc. Meat Industry Research Conference. Am. Meat Sci. Assoc.,
Am. Meat lnst. Found., Chicago, IL. pp. 109-118.
8. Galton, M. M., J. R. Boring, and W. T. Martin. 1968. Salmonellae
in foods. Center for Disease Control, Public Health Service, U.S.
Dept. of Health, Education, and Welfare, Atlanta, GA.
9. Goepfert, J. M. 1976. The aerobic plate count, coliform and
Escherichia coli content of raw ground beef at the retail level. J.
Milk Food Techno!. 39:175-178.
10. Harr, J. W., and M. E. Minard. 1977. Cryogenic meat
processing- cost and quality. Proc. Meat Industry Research
Conference. Am. Meat Sci. Assoc., Am. Meat Ind. Found.,
Chicago, IL. pp. 25-35.
11. Herman, L. G., and F. A. Morelli. 1960. The growth and isolation
of coagulase positive staphylococci on Medium No. 110 fortified
with egg yolk. Bacteriol. Proc. 102.
12. Kraft, A. A., K. V. Reddy, J. G. Sebranek, R. E. Rust, and D. K.
Hotchkiss. 1979. Effect of composition and method of freezing
on microbial flora of ground beef patties. J. Food Sci. 44:350-354.
13. Sebranek, J. G., P. N. Sang, R. E. Rust, D. G. Topel, and A. A.
Kraft. 1978. Influence ofliquid nitrogen, liquid carbon dioxide and
mechanical freezing on sensory properties of ground beef patties.
J. Food Sci. 43:842-844, 848.
14. Snedecor, G. W. 1950. Statistical methods, 4th ed. The Iowa State
University Press, Ames, lA.
15. Sills, J, T. 1969. The role of cryogenic freezing. Meats 35:26.
16. Surkiewicz, B. F., M. E. Harris, R. P. Elliott, J. F. Macaluso, and
M. M. Strand. 1975. Bacteriological survey of raw beef patties
produced at establishments under federal inspection. Appl.
Microbiol. 29:331-334.
17. Walker. H. W., and C. R. Rey. 1972. Methodology for Staphy-
lococcus aureus and Clostridium petfringens. Proc. 25th Ann.
Reciprocal Meats Conf. of Am. Meat Sci. Assoc.. Nat!.
Livestock and Meat Board, Chicago, IL.
27. Taylor. A. A., and B. G. Shaw. 1977. The etTect of meat pH and
package permeability on putrefaction and greening of vacuum
packed beef. J. Food Techno!. 12:515-521.