J. J. Sheridan TEAGASC, The National Food Centre, Dunsinea, Castleknock, Dublin 15
Abstract
The survival of S. kentucky and S. aureus in frozen minced beef with 5, 10, 20, 30 and 50% fat was determined over 10 weeks storage at -35°C. The survival of both pathogens improved as a result of the addition of fat up to a level of 20 or 30%, depending on the organism. At 50% fat there was a reduction in survival to the level of the lean meat.
Minced beef, pork and lamb were inoculated with the same pathogens and examined over 10 weeks storage at -35°C. The data showed that pathogen survival varied with the meat type being highest on pork and lowest on beef.
The factors altering pathogen survival in the fat experiments and those with different meat types are discussed. Data are presented in relation to the presence of cryoprotective or toxic substances in the mince.
Introduction
It is generally recognised that a number of factors will affect the survival of bacteria following freezing. These include:
- Bacterial type, Gram negative or positive
- Bacterial strains
- Cell age, i.e. exponential or stationary phase
- Growth conditions before freezing
- Nature of suspending medium. i.e. fish. meat or vegetables
- Temperature of freezing
- Storage time
- Thawing conditions.
The influences of these factors have been observed on meat in a number of studies. In general, Gram negative organisms survive less well than Gram positive (Sheridan, 1982a: Farrell and Upton, 1978; Speck and Ray, 1977). Differences due to bacterial strains, and the use of exponential or stationary phase cells, have been reported, with stationary phase cells showing a greater resistance to freezing (Gunderson and Rose, 1948; Mackey et al., 1980). Experiments have been carried out showing that the suspending medium can have a profound effect on bacterial survival, as can the temperature of freezing (Palumbo and Williams, 1991; El-Kest and Marth, 1992). The influence of storage time is well known and some studies have suggested that most cell death and injury occurs during the initial stages of storage, i.e. 1-2 weeks (Speck and Ray, 1977) although recently this view has been challenged (El-Kest and Marth, 1991). Finally, the effect of thawing on bacterial survival is greatly influenced by the thawing temperature. Very low thawing temperatures may decrease bacterial growth (Sheridan and Sherington, 1983) but generally there is an overall increase (Roberts, 1974).
Little information exists on the effect that different meat types have on the survival of pathogenic bacteria after freezing and storage (Mackey et al., 1980). The work carried out in the past suggests that pathogen survival could be influenced by meat type, a fact also observed in a preliminary study by Sheridan (1982b). A further examination of the literature showed that the levels of fat in meat could have a profound effect on the survival of pathogenic bacteria (Miller, 1955). The work reported in this study was carried out in an attempt to determine the influences of different meat species and varying lean/fat ratios on the survival of Gram positive and negative pathogenic bacteria after freezing and storage.
Materials and Methods
Beef ,pork and lamb were obtained from animals slaughtered in the on-site abattoir. The meat was trimmed of all visible fat and used either as lean or in the case of beef with added fat. The fat levels prepared were 5, 10, 20, 30 and 50%. Lean meat alone or lean meat and fat were minced together and an inoculum of stationary phase cultures Salmonella kentucky or Staphylococcus aureus added during mincing. Samples were double minced to ensure thorough dispersion of the inoculum.
Inoculated minced meat samples were dispensed in 100g lots in polythene bags, placed directly in a blast freezer, frozen to -35°C and stored for 10 weeks. At weekly intervals a sample was withdrawn from the freezer, placed in a waterbath at 37°C and thawed for 5 mins. To each bag 200mI of 0 1 % peptone water was added and the mixture macerated in a Colworth stomacher for 1 min. The survival of the two pathogens was detected using brilliant green agar or Baird Parker medium. The total counts were also monitored by plating on plate count agar. Plates were incubated at 37°C for S. kentucky and S. aureus and at 25°C for the total counts.
Results
Effect of varying lean fat ratios on pathogen survival
There was a decrease in the survival of S. kentucky during the storage period (Fig. 1) with the pattern of the decrease changing over time with the different fat levels (Table 1). In general, the lower the fat level the greater the decrease during the first week up to 20% fat. At 50% fat a higher decrease in the first week was again evident. The data also showed the overall decrease in viability over the storage period.
Figure 1
The relationship between counts of S. kentucky (log10
cfu/g) and time on frozen mince with varying levels of fat (%) |
|
Table 1
Decrease (%) in viability of S. kentucky in minced beef with
varying fat levels, frozen to -35°C and the relationship to storage time in
weeks |
Fat
level (%) |
Storage time in weeks |
|
| |
0-1 |
0-6 |
0-10 |
Overall |
5.0 |
70.0 |
17.0 |
4.0 |
91.0 |
10.0 |
44.0 |
31.0 |
8.0 |
83,0 |
20.0 |
5.0 |
75.0 |
0 |
80.0 |
30.0 |
5.0(+) |
61.0 |
10.0 |
76.0 |
50.0 |
66.0 |
12.0 |
11.0 |
89.0 |
The effect of time on the survival of S. aureus indicated that the decrease in
counts varied with fat level (Fig. 2). It was also noted that the pattern of decline of
this pathogen was different to that for S. kentucky (Table 2). The influence of 50%
fat was similar for both organisms.
Figure 2
The relationship between counts
of S. aureus (log10 cfu/g) and time on frozen mince with varying levels of fat
(%) |
 |
Table 2
Decrease (%) in viability of S. aureus in minced beef with
varying fat levels, frozen to -35°C and the relationship to storage time in
weeks |
Fat
level (%) |
Storage time in weeks |
|
| |
0-1 |
0-6 |
0-10 |
Overall |
5.0 |
29.0 |
41.0 |
8.0 |
78.0 |
10.0 |
40.0 |
20.0 |
17.0 |
77.0 |
20.0 |
62.0(+) |
47.0 (-) |
13.0 |
28.0 |
30.0 |
5.0(+) |
23.0 |
21.0 |
49.0 |
50.0 |
57.0 |
12.0
(-) |
22.0 |
67.0 |
The overall decline in pathogen numbers with time is shown in Table 3 (below). These
data show that for S. kentucky the decrease in counts with time was highly
significant, at each fat level (P<.001). For S. aureus significant decreases in
survival over time were again observed, except at 20 and 30% fat, where the change was
non-significant. When different fat levels were compared no significant differences were
found between the means for S. kentucky, while for S. aureus differences between 5,
10, and 20% were noted (P<.05).
The relationship of survival (%) to time is illustrated in Fig. 3. This shows he
overall effect of fat level on the survival of both organisms. It highlights the effect of
very high fat levels (50%) in decreasing the survival of both organisms and the difference
between Gram positive and negative organisms.
Table 3
Decrease in bacterial counts (log10
cfu/g) For S. kentucky and S. aureus in beef minced with varying fat levels
and stored for 10 weeks at -35°C |
Fat
level % |
S.kentucky |
S.
aureus |
5 |
0.88 |
0.66 |
10 |
0.76 |
0.64 |
20 |
0.70 |
0.15 |
30 |
0.62 |
0.29 |
50 |
0.98 |
0.47 |
Standard
error of
Differences (df=162) |
0.27 |
0.25 |
| Figure 3 The relationship between fat level % and survival
% of S. kentucky and S. aureus in minced beef frozen to -35°C and
stored for 10 weeks. |
 |
The Effect of Meat types of Pathogen Survival
The change in counts of S. kentucky and S. aureus over time, when
inoculated into beef, pork and lamb are shown in Figs. 4 and 5, respectively. Depending on
the organism, and the meat type used, the decline of the pathogen varied over time. For
beef the greatest decline was observed during the first week and this was also the case
for lamb inoculated with S. aureus (Table 4). For pork the decrease in viability
was not significant for either organism (results not shown).
Figure 4
The effect of freezing and
storage on the viability of S. kentucky on beef, pork and lamb mince stored at -35°C
for 10 weeks |
|
Figure 5
The effect of freezing and storage on the viability of S. aureus on
beef, pork and lamb mince stored at -35'C for 10 weeks |
|
Table 4
Decrease (%) in viability of S.
kentucky and S. aureus in beef, pork and lamb mince, frozen to -35°C
and the relationship to storage time. |
Meat
type |
Storage time in weeks |
|
| |
0-1 |
0-6 |
0-10 |
Overall |
| |
S.
kentucky |
Beef |
60.0 |
25.0 |
6.0 |
91.0 |
Lamb |
38.0 |
33.0 |
9.0(-) |
62.0 |
| |
S.
aureus |
Beef |
61.0 |
18.0 |
5.0 |
84.0 |
Lamb |
58.0(+) |
26.0(+) |
33.0 |
65.0 |
With beef and lamb, significant declines in counts with time were evident, particularly
for beef (Table 5).
Table 5
Decrease in bacterial counts (log10
cfu/g) for S. kentucky and S.aureus in minced meat frozen at -35°C
and stored for 10 weeks |
meat
type |
S.kentucky |
S.
aureus |
Beef |
1.06 P<0.001 |
0.79 P<0.001 |
Pork |
0.11 NS |
0.02 NS |
Lamb |
0.70 |
0.15 |
Standard
error of
Differences (df=162) |
0.42
P<0.001 |
0.45
P<0.001 |
An outstanding feature of the data was the significant differences between meat types
and the lack of any significant decline in viability when pork was used. The protective
effect of the three meat types is shown in Table 6 where the very high survival on pork
compared to the other types, is clearly demonstrated
Table 6
The relationship between meat
species and survival (%) of S. kentucky and S.aureus on minced meat frozen
at –35°C and stored for 10 weeks |
meat
type |
S.kentucky |
S. aureus |
Beef |
9.0 |
16.0 |
Pork |
78.0 |
96.0 |
Lamb |
39.0 |
35.0 |
Finally, the effect of the different meat types on the total counts after freezing are
shown in Table 7. The data indicated that the cryoprotective effects observed for the
inoculated pathogens were evident from the total microflora. The cryoprotective effects
observed for inoculated pathogens were evident, although a significant difference for lamb
could not be established. The large decrease in survival observed in beef suggested that
the majority of the decline involved the Gram negative portion of the microflora.
|
Table 7
Effect of meat species on the
decrease in total counts (log10 cfu/g) on mince inoculated with S. kentucky
and S.aureus and frozen at -35°C for 10 weeks |
meat type |
Meat
Inoculum |
| |
S.kentucky |
S. aureus |
Beef |
1.01 P<0.001 |
1.03 P<0.001 |
Pork |
0.36 P<0.001 |
0.15 NS |
Lamb |
0.29 P<0.05 |
0.43 P<0.001 |
Standard
error of
Differences (df=162) |
0.28 |
0.25 |
Discussion
In this study the temperature of freezing and storage was -35°C. This
temperature would have been conducive to the pathogens having a high level of survival,
since low freezing and storage temperatures aid microbial survival (Woodburn and Strong,
1960). Most of the data in the literature for pathogen survival on meat refer to
temperatures between - 18 and -26°C. The survival levels for Salmonella in
chickens and smoked bacon of 26°C and 10.0% respectively would not differ
greatly from that in the present study of 9.0 - 13% for lean beef (Gunderson and Rose,
1948: Farrell and Upton, 1978). The improved survival of 26% in chicken may be a
reflection of the suspending medium used (Gunderson and Rose, 1948).
For S. aureus there was an initial survival on beef of 16-22%. These values are
below those levels reported by others for beef mince (96.0%), bacon (42%) and minced pork
(41.0 and 79%) (Dempster, 1980; Farrell and Upton 1978; Miller, 1955). As will be
discussed later, such differences cannot reasonably be ascribed to meat types. Since the
freezing and storage temperatures were above (-18 to -22°C) those in the
present work (-35°C) this factor is also unlikely to explain these differences.
Differences in strains or cell age may be involved in explaining some of these (Mackey et
al., 1980, El-Kest and Marth, 1991).
In this study both pathogens showed an increased survival up to a level of 30% fat for S.
kentucky and 20% for S. aureus. Thereafter survival decreased and at 50% fat,
survival for both organisms had decreased to the level recorded for 5% fat (lean).
Increasing fat levels from 30 to 50% in minced pork decreased the survival of S. aureus
from 79 to 41 % (Miller, 1955). These results show that in the initial instance, fat had a
cryoprotective effect, but with increasing levels this changed and a reduction in pathogen
survival occurred. This suggests that at least two mechanism affecting pathogen survival
may be in operation.
Fat is not a cryoprotectant, since it does not penetrate into cells as can glycerol
(Meryman, 1966). On mincing and mixing however, fat cells may produce cryoprotective
substances that aid in the survival of the pathogens on freezing. One of the main
components of fats is glycerol, which is naturally present in small amounts (Frazier and
Westhoff, 1988). Some glycerol may also be produced by microbial and cell hydrolysis
(Frazier and Westhoff, 1988). For animal fats one of the major breakdown products is
glycerides and these may act as cryoprotectants under certain conditions (MacDonald and
Lanier, 1991).
The fat level at which a decrease occurs is thought to represent a concentration effect
in respect of the presence of free fatty acids in the lean/fat mixture. The antimicrobial
effect of free fatty acids is well known (Beuchat and Golden. 1989; Greenway and Dyke,
1979). In this study S. aureus survival was reduced at lower fat levels than S.
kentucky . In general it has been shown that free fatty acids are most effective
against Gram positive bacteria as a consequence of their penetration into these cells,
causing a change in the bacterial membrane (Greenway and Dyke. 1979: Sheu and Freese,
1973). Gram negative bacteria are less sensitive, due to cell wall lipids protecting the
membrane from the bactericidal action of the fatty acids (Beuchat and Golden, 1989). The
synergistic effect of damage to the cell wall, as occurs during freezing and the presence
of fatty acids may be the mechanism for entry to the cell membrane and death in Grain
negative cells.
Some of the most inhibitory fatty acids e.g. stearic (C18:0), are bactericidal to L.
monocytogenes a fat (Wang and Johnson, 1992) which is present in high concentrations
in beef. There may also be a relationship between the presence of free fatty acids and
triglycerides in the meat and the increase or decrease in survival may represent a balance
between these two substances (MacDonald and Lanier, 1991).
The effect of the different meat types on the survival of both pathogens showed that
pork gave a very high level of protection and that beef was least effective. Lamb being
intermediate between the two. The superior survival of these and other pathogens on pork
has been noted in the past (Sheridan, 1982a; 1982b; Mackey et al., 1980).
The improved cryoprotective properties of pork are difficult to explain. While proteins
have such properties, differences between pork, beef and lamb, in terms of protein
content, are not apparent (Lawrie, 1985). It may be however, that on mincing proteins from
pork become available in greater amounts than in the other species. A factor of some
importance may be the more rapid tenderisation of pork, compared to beef or lamb. This
could lead to a higher level of substances in the meat, such as amino acids which have
cryoprotective properties (Troy, 1987; Jiang et al., 1987).
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