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Loss of Viability by Listeria monocytogenes in Commercial Bovine Pepsin-Rennet Extract
L o s s of V i a b i l i t y b y Listeria monocytogenes in C o m m e r c i a l Bovine Pepsin-Rennet Extract
FATHY E. EL-GAZZAR and ELMER H. MARTH Department of Food Science and The Food Research Institute University of Wisconsin-Madison Madison 53706 ABSTRACT Loss of viability by Listeria monocytogenes strains California, V7, and Scott A in commercial bovine pepsin-rennet extract was determined during storage for 56 d at 7°C. Four levels (103 to 106/ml) of L. monocytogenes were added to the coagulant, and McBride listeria agar was used to determine numbers of survivors. Selected colonies thought to be L. monocytogenes were confirmed biochemically. Samples also were tested during and after completion of cold enrichment (up to 8 wk at 4°C). Coagulant inoculated with 103 to 104 L. monocytogenes/ml usually was free of viable cells of the pathogen alter 28 d and sometimes alter 14 d, as determined by direct plating and cold enrichment. When the inoculum was l05 to 106 cells/ml, samples of coagulant usually were free of viable L. monocytogenes after 42 d and sometimes after 28 d. The three strains of L. monocytogenes behaved similarly, although strain California was somewhat less hardy in the environment of the coagulant than were the other two strains. Bovine pepsin-rennet extract, berøre inoculation, was free of L. monocytogenes (direct plating and cold enrichment). INTRODUCTION Listeria monocytogenes is a bacterium that has become of considerable concem to the food
Received August 30, 1988, Accepted November 21, 1988. 1989 J Dairy Sci 72:1098-1102
industry in US, Europe, and perhaps elsewhere. Several outbreaks of listeriosis have implicated dairy products such as milk and cheese as vectors of the bacterium, which can cause an often fatal infection in susceptible individuals such as the elderly, immune-deficient adults, pregnant women, and newboms (1, 2, 3, 4, 5). Bovine and porcine pepsin are among the important substitutes for rennet extract as coagulants of milk. Use of bovine pepsin has increased, because it is readily available by extracting stomachs from older calves and adult cattle. Fox (7) reported that bovine pepsin is more similar in its properties to those of calf rennet than is porcine pepsin. Fox and Walley (8) found no significant difference in characteristics of Cheddar cheese made with bovine pepsin or rennet. Generally, liquid rennet and similar coagulants of animal origin are preserved by a high (14 to 20%) salt content and by addition of sodium propionate, sodium benzoate, and propylene glycol. Manufacture of ripened cheese requires use of a milk coagulant such as rennet or a rennet substimte. Some of the coagulants are of animal origin, and because L. monocytogenes is an animal pathogen, the coagulants could contain the pathogen at the time of manufacture. Hence, it is necessary to knowhow L. monocytogenes behaves in such products because such knowledge may make it possible to use a Listeria-free coagulant to make cheese. In view of this, the present study was undertaken to evaluate survival of L. monocytogenes in bovine pepsin-rennet extract during storage at 7"C. Also, this study focused on survival of different strains and initial levels of L. monocytogenes in the coagulant. Furthermore, we determined if our test samples of bovine pepsin-rennet were contaminatexl with L. monocytogenes before inoculation.
1098
LOSS OF LISTERJA VIABILITY MATERIALS AND METHODS Culturøs
Three strains of L. monocytogenes were used in this study, namely Scott A (clinical isolate, serotype 4b), V7 (milk isolate, serotype 1), and California (CA) (isolate from Mexican-style cheese, serotype 4b). These cultures were activated by three successive transfers. The first transfer was from tryptose agar (TA) slant to TA slant and incubation was at 35°C for 48 h, the second transfer was from TA to tryptose broth (TB), and the third was from TB to TB; broth cultures were incubated at 35°C for 24 h. Sufficient volume of the working culture was dispersed in 25 ml of coagulant to yield four initial levels of L. monocytogenes, ranging from ca. 103 to 106/ml. Levels of inoculum represented moderate to severe contamination of the product with the pathogen. Bovlne Pepsin-Rennet Extmct
Commercial bovine pepsin-rennet extract was obtained from Chr. Hansen's Laboratory, Inc., Milwaukee, WI. According to information on the label, the product we studied was a mixture of adult bovine and calf œnnets and porcine pepsin in salt brine. The mixture also contained 5% propylene glycol, 2% sodium propionate, and .1% sodium benzoate, used as preservatives, plus flavor and color. Inoculated samples of coagulant were incubated at 7°C, a temperature similar to those used during commercial handling of the product by the manufacturer, in distribution, and by the cheese maker. Enumøration of Listeria monocytogenes
Samples of the initial coagulant and decimal dilutions made with a sterile solution of .5% peptone in water were surface-plated onto McBride listeria agar (MLA) (10). All plates were incubated 48 h at 35°C. Colonies considered to be L. monocytogenes (small, low, convex, finely textured, bright blue green in color, watery consistency, wealdy 13-hemolytic, and an entire edge) were counted and randomly selected colonies were transferred to TA slants, incubated 24 h a t 35°C, and stored at 4°C for cønfirmation.
1099
Confirmatory Tests
Confirmatory tests done on isolates just mentioned included microscopic examination, presence of distinct blue-green colonies on TA when observed under obliquely transmitted light as described by Henry (9), observance of tumbling motility in TB cultures incubated 24 h at 22*C, and proper biochemical reactions on API 20S test strips (Analytab Products, Inc., Plainview, NY). Calculations
The percentage of løst viability after x days of storage was calculated as follows: % = -~
x 100,
where A = initial numbers of organisms (cfu/ ml) and B = number of survivors alter x days of incubation, (cfu/ml). To make this calculafion, samples receiving the highest levels of inoculum were chosen. Making the calculation overcame the difficulty associated with interpretation of results when initial numbers of L. monocytogenes were variable. RESULTS AND DISCUSSION
The coagulant was free (by direct plating and cold enrichment up to 8 wk) of L. monocytogenes before samples were inoculated with the bacterium. Results (Table 1) indicate that when four levels (103 to 106/ml) of L. monocytogenes strain CA were added to the coagulant, numbers of the pathogen were greatly reduced after 7 d of storage at 7°C. However, regardless of initial level of inoculum, alter 7 d viable cells of the organism were not completely eliminated from the coagulant. It is evident that increasing the inoculum to ca. 105 to 106/ml had a marked effect on the number of viable L. monocytogenes in the coagulant after 7 d of storage. After 14 d of storage, numbers of survivors in some samples were still decreasing, whereas nø viable L. monocytogenes cells were found in the 1:10 dilution of samples initially inoculated with ca. 103 or 104/ml. Survivors were reduced to numbers in the range of ca. 100 to 1000 cfu/ ml when high initial levels (more than 105/ml) Journal of Dairy Science Vol. 72, Nø. 5, 1989
1100
EL-GAZZAR AND MARTH
TABLE 1. Survival of L. monocytogenes (strain California) ha bovine-pepsin rennet during storage for 56 d at 7"C. Trial
0
A
8.0 1.4 9.0 1.4
Number per miUiliter arier days of storage 14 28 42
7 x x x x
B
5.0 x
C
1.1 5.5 1.0 9.5 2.0 7.5 1.0
× x x × x x x
103 104 10~ 106 10a 104 105 104 103 104 10s 106
10 10 1.0 1,0 20 20 1.9 7.0 10 30 1.0 2.0
x 104 x lœ x 103 x l0a
x 103 x 104
<10 (_)l <10 (-) 1000 100 <10 (-) <10(-) 300 500 <10 (-) <10 (-) 100 100
<10 (-) <10 (-) <10 (+) <10 (+) <10 (-) <10(-) <10 (+) <10 (+) <10 (-) <10 (-) <10 (+) <10 (+)
<10 (-) <10 (-) <10 (+) <10 (+) <10 (-) <10(-) <10 (+) <10 (+) <10 (-) <10 (-) <10 (+) <10 (+)
56
~(-) = Nø Listeria found by cold enfichment; (+) Listeria found by cold enrichment.
were used. Also, nø viable listeriae were found by cold enrichment at this point when we tested samples initially inoculated with low levels o f L. monocytogenes. Arier 28 d, viable listeriae were absent, either by direct plafing (.1 ml of sample) or cold enrichment, from coagulant initially inoculated with the two low levels of L. monocytogenes. With the two high levels of initial inoculum, nø viable listeriae were found by direct plating, but some cold enrichment tests gave positive results. Results obtained after 42 d of storage were similar to those noted after 28 d; after 56 d all but three cold-enrichment tests were negative. These results are similar to those observed by EI-Gazzar and Marth (6) when they studied behavior of L. monocytogenes strain C A in commercial calf rennet extract. Results obtalned with L. monocytogenes strain Scott A Œable 2) indicate small numbers o f survivors after 7 and 14 d of storage even when low (ca. 103 to 104/ml) initial numbers of the pathogen were present. After 28 d, nø L. monocytogenes could be found by direct plating of all samples receiving the two low levels of inoculum, but four of six samples remained positive when examined by cold enrichment. All samples receiving the two low levels of inoculum were free o f the pathogen by both tests after 42 and 56 d at 7°C. In contrast, five of six samples receiving the two high levels of inoculum had viable cells of L. monocytogenes arier 42 d as determined by direct plating, or Journal of Dairy Science Vol. 72, Nø. 5, 1989
cold enrichment, or both. Extending storage to 56 d reduced the number of positive samples from five o f six to one of six. It is evident from these data that straln Scott A was somewhat more resistant than strain CA to the detrimental effects of the coagulant. Tests with L. monocytogenes strain V7 (Table 3) indicate this strain of the pathogen behaved much as did stlaln Scott A dm'ing the first 28 d of storage in the coagulant. However, arier 42 and 56 d of storage essentially all samples were free o f L. monocytogenes. These data differ somewhat from those obtained with straln Scott A in that 5 of 12 samples inoculated with that strain remained positive arier 42 d. Results for both stralns (Table 2 and 3) were essentially comparable arier 56 d of storage. Calculated values in Table 4 summarize the behavior of the three sWains of L. monocytogenes in bovine pepsin-rennet extract during storage at 7"C for 14 and 28 d. It is evident that inactivadon of strain California was more nearly complete at these times than was inacUvation o f the other strains. These small differences tended to disappear arier 42 and 56 d of storage (Table 1, 2, and 3). Ryser and Marth (11) reported that L. monocytogenes survived in Cheddar cheese for more than 1 yr. Furthermore, they (12) demonstrated that, if present, L. monocytogenes can grow in Camembert cheese during ripening after the pH of cheese has increased sufficiently through action of Penicillium camemberti. Yousef and
LOSS OF LISTERIA VLABILITY
1101
TABLE 2. Survival of L. monocytogenes (strain Scott A) ha bovine-pepsin rennet during storage for 56 d at 7"C. Number per milliliter after days of storage Trial
0
A
76.0 8.4 50.0 10.0 48.0 8.1 36.0 13.0 69.0 10.0 46.0 19.0
B
C
x x x x × × x ×
103 lœ 103 106 103 104 105 10«
× 10 3
× 104 x 105 x 106
7
14
28
42
56
10 100 6.0 7.0 20 400 7.0 33.0 20 4.5 6.0 20.0
<10 (+)l 20 800 1.3 x <10 (+) 30 2.2 × 6.0 × 10 30 1.0 x 4.0 x
<10 (-) <10(+) 250 140 <10 (+) <10 (+) 240 730 <10 (-) <10 (+) 150 500
<10 (-) <10(-) <10 (+) 20 (+) <10 (-) <10 (-) <10 (-) <10 (+) <10 (-) <10 (-) <10 (+) 10 (-)
<10 (-) <10(-) <10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10 (+) <10 (-) <10 (-) <10 (-) <10 (-)
x 103 x 103
× 103 x 103 × 1Œ x 103 x l0 a
103
103 103
103 103
t(+) = Listeria fotmd by cold enrichment; (-) = nø Listeria found by cold enrichment.
Marth (13) reported that L. monocytogenes can remain viable in C o l b y cheese for a period that depends on initial i n o c u l u m , strain o f the pathogen, and c o m p o s i t i o n o f the cheese. In all instances, length o f survival e x c e e d e d the normal storage life o f C o l b y cheese. Hence, it is essential that nø ingredient, e.g., the coagulant, used in cheese m a k i n g contributes L. monocytogenes cells to the cheese. Results o f this study confmla those o f our p r e v i o u s w o r k (6). It is e v i d e n t f r o m our data that the coagulant, by g o i n g through shipping and distribution networks, can be held long e n o u g h before use to inactivate L. monocyto-
genes in the e v e n t the p a t h o g e n entered the fresh product as an inadvertent contaminant. Furthermore, if L. monocytogenes were naturally present in the coagulant, the numbers probably w o u l d be considerably smaller than e v e n our smallest inoculum. U n d e r such circumstances loss o f viability by the pathogen probably w o u l d be e v e n more rapid than was o b s e r v e d in our experiments. B e n z o i c acid, sod i u m propionate, p r o p y l e n e glycol, and salt in c o m b i n a t i o n are likely to be responsible for d e m i s e o f the b a c t e r i u m f r o m the coagulant. C o n s e q u e n t l y , c o m m e r c i a l b o v i n e pepsin-rennet extract with a preservative system o f the
TABLE 3. Survival of L. monocytogenes (strain V7) in bovine-pepsin rennet during storage for 56 d at 7"C. Number per miililiter after days of storage Trial
0
A
4.0 1.7 3.5 1.0
x x x x
B
3.5 x
C
8.0 4.5 3.5 3.5 2.0 4.5 1.0
x x x x × × x
103 104 105 104 103 103 103 10~ 103 104 103 106
7
14
28
42
56
100 180 2.0 2.2 100 200 10.0 3.2 300 300 24.0 7.4
100 50 700 1.1 x 103 10 10 400 840 20 30 1.9 x 103 4.2 × 103
<10 (_)1 <10 (-) 50 400 <10 (-) <10 (-) 60 100 <10 (-) <10 (+) 30 200
<10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10(-) <10(-) <10 (-) <10 (-) <10 (-) <10 (-)
<10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10(+) <10(-) <10 (-) <10 (-) <10 (-) <10 (-)
x 103 x 104
x 103 x 104
x 103 x 104
~(-) -- Nø Listeria found by cold enrichment; (+) = Listeria found by cold enrichment. Journal of Dairy Science Vol. 72, Nø. 5, 1989
1 102
EL-GAZZAR AND MARTH
TABLE 4. Percentage of viability lost by strains of L. monocytogenes in bovine-pepsin rennet during storage at 7*C. % Inactivation ~,2 Storage
California
(d)
X
14 28
99.98 99.999
SD .0196 0
Scott A
V7
X
SD
X
99.97 99.996
.0159 .0017
99.82 99.979
SD .1701 .0151
1High concentrations of L. monocytogenes; initially 106 or more/ml. :Average of three trials.
type in the product w e tested is unlikely to contaminate cheese with L. monocytogenes. In spite o f this conclusion, it w o u l d be prudent to m o n i t o r ingredients for the pathogen before they are used to manufacture cheese. E x c e p tions to the rule can occur, and these exception, if they go undetected, can cause problems.
ACKNOWLEDGMENTS R e s e a r c h supported by the C o l l e g e of Agricultural and Lire Sciences, U n i v e r s i t y o f W i s c o n s i n - M a d i s o n , the F o o d Research Institute o f the U n i v e r s i t y o f W i s c o n s i n - M a d i s o n , and Chr. H a n s e n ' s Laboratory, Inc., M i l w a u k e e , WI. W e thank R. L. Sellars for his helpful suggestions regarding this study.
REFERENCES 1 Anonymous. 1985. Listeriosis outbreak associated with Mexican- style cheese - California. Morbid. Mortal. Weekly Rep. 34:357. 2 Anonymous. 1985. Listeriosis transmitted by contaminated Jalisco-brand cheese. Calif. Morbid. No. 46, Nov. 22:1.
Journal of Dalry Science Vol. 72, Nø. 5, 1989
3 Anonymous. 1986. Brie cheese recalts extended by General Foods, US importer. Food Chem. News 27(52): 25. 4 Anonymous. 1986. FDA fmds listeria in Brie cheese from French-certified plant. Food Chem. News 27(50):35. 5 Anonymous. 1987. Listeria infectious dose calculated, Crawford says. Food Chem. News 29(17):25. 6 EI-Gazzar, F. E., and E. H. Marth. 1988. Loss of viability by Listeria monocytogenes in commercial calf rennet extract. J. Food Prot. 51:16. 7 Fox, P. F. 1969. Milk clotting and proteolytic activities of rennet and bøvine pepsin and porcine pepsin. J. Dairy Res. 36:427. 8 Fox, P. F., and B. F. Walley. 1971. lnfluence of sodium chloride on proteolysis of casein by rennet and by pepsin. J. Dalry Res. 38:165. 9 Henry, B. S. 1933. Dissociation in the genus Brucella. J. Infect. Dis. 52:374. 10 McBride, M. E., and K. F. Girard. 1960. A selective method for the isolation of Listeria monocytogenes from mixed bacterial populations. J. Lab. Clin. Med. 55:153. 11 Ryser, E. T., and E. H. Marth. 1987. Fate of Listeria monocytogenes during the manufacture and ripening of Cheddar cheese. J. Food Prot. 50:7. 12 Ryser, E. T., and E. H. Marth. 1987. Fate of Listeria monocytogenes during the manufacture and ripening of Camembert cheese. J. Food Prot. 50:372. 13 Yousef, A. E., and E. H. Marth. 1988. Behavior of Listeria monocytogenes during the manufacture and storage of Colby cheese. J. Food Prot. 51:12.
FATHY E. EL-GAZZAR and ELMER H. MARTH Department of Food Science and The Food Research Institute University of Wisconsin-Madison Madison 53706 ABSTRACT Loss of viability by Listeria monocytogenes strains California, V7, and Scott A in commercial bovine pepsin-rennet extract was determined during storage for 56 d at 7°C. Four levels (103 to 106/ml) of L. monocytogenes were added to the coagulant, and McBride listeria agar was used to determine numbers of survivors. Selected colonies thought to be L. monocytogenes were confirmed biochemically. Samples also were tested during and after completion of cold enrichment (up to 8 wk at 4°C). Coagulant inoculated with 103 to 104 L. monocytogenes/ml usually was free of viable cells of the pathogen alter 28 d and sometimes alter 14 d, as determined by direct plating and cold enrichment. When the inoculum was l05 to 106 cells/ml, samples of coagulant usually were free of viable L. monocytogenes after 42 d and sometimes after 28 d. The three strains of L. monocytogenes behaved similarly, although strain California was somewhat less hardy in the environment of the coagulant than were the other two strains. Bovine pepsin-rennet extract, berøre inoculation, was free of L. monocytogenes (direct plating and cold enrichment). INTRODUCTION Listeria monocytogenes is a bacterium that has become of considerable concem to the food
Received August 30, 1988, Accepted November 21, 1988. 1989 J Dairy Sci 72:1098-1102
industry in US, Europe, and perhaps elsewhere. Several outbreaks of listeriosis have implicated dairy products such as milk and cheese as vectors of the bacterium, which can cause an often fatal infection in susceptible individuals such as the elderly, immune-deficient adults, pregnant women, and newboms (1, 2, 3, 4, 5). Bovine and porcine pepsin are among the important substitutes for rennet extract as coagulants of milk. Use of bovine pepsin has increased, because it is readily available by extracting stomachs from older calves and adult cattle. Fox (7) reported that bovine pepsin is more similar in its properties to those of calf rennet than is porcine pepsin. Fox and Walley (8) found no significant difference in characteristics of Cheddar cheese made with bovine pepsin or rennet. Generally, liquid rennet and similar coagulants of animal origin are preserved by a high (14 to 20%) salt content and by addition of sodium propionate, sodium benzoate, and propylene glycol. Manufacture of ripened cheese requires use of a milk coagulant such as rennet or a rennet substimte. Some of the coagulants are of animal origin, and because L. monocytogenes is an animal pathogen, the coagulants could contain the pathogen at the time of manufacture. Hence, it is necessary to knowhow L. monocytogenes behaves in such products because such knowledge may make it possible to use a Listeria-free coagulant to make cheese. In view of this, the present study was undertaken to evaluate survival of L. monocytogenes in bovine pepsin-rennet extract during storage at 7"C. Also, this study focused on survival of different strains and initial levels of L. monocytogenes in the coagulant. Furthermore, we determined if our test samples of bovine pepsin-rennet were contaminatexl with L. monocytogenes before inoculation.
1098
LOSS OF LISTERJA VIABILITY MATERIALS AND METHODS Culturøs
Three strains of L. monocytogenes were used in this study, namely Scott A (clinical isolate, serotype 4b), V7 (milk isolate, serotype 1), and California (CA) (isolate from Mexican-style cheese, serotype 4b). These cultures were activated by three successive transfers. The first transfer was from tryptose agar (TA) slant to TA slant and incubation was at 35°C for 48 h, the second transfer was from TA to tryptose broth (TB), and the third was from TB to TB; broth cultures were incubated at 35°C for 24 h. Sufficient volume of the working culture was dispersed in 25 ml of coagulant to yield four initial levels of L. monocytogenes, ranging from ca. 103 to 106/ml. Levels of inoculum represented moderate to severe contamination of the product with the pathogen. Bovlne Pepsin-Rennet Extmct
Commercial bovine pepsin-rennet extract was obtained from Chr. Hansen's Laboratory, Inc., Milwaukee, WI. According to information on the label, the product we studied was a mixture of adult bovine and calf œnnets and porcine pepsin in salt brine. The mixture also contained 5% propylene glycol, 2% sodium propionate, and .1% sodium benzoate, used as preservatives, plus flavor and color. Inoculated samples of coagulant were incubated at 7°C, a temperature similar to those used during commercial handling of the product by the manufacturer, in distribution, and by the cheese maker. Enumøration of Listeria monocytogenes
Samples of the initial coagulant and decimal dilutions made with a sterile solution of .5% peptone in water were surface-plated onto McBride listeria agar (MLA) (10). All plates were incubated 48 h at 35°C. Colonies considered to be L. monocytogenes (small, low, convex, finely textured, bright blue green in color, watery consistency, wealdy 13-hemolytic, and an entire edge) were counted and randomly selected colonies were transferred to TA slants, incubated 24 h a t 35°C, and stored at 4°C for cønfirmation.
1099
Confirmatory Tests
Confirmatory tests done on isolates just mentioned included microscopic examination, presence of distinct blue-green colonies on TA when observed under obliquely transmitted light as described by Henry (9), observance of tumbling motility in TB cultures incubated 24 h at 22*C, and proper biochemical reactions on API 20S test strips (Analytab Products, Inc., Plainview, NY). Calculations
The percentage of løst viability after x days of storage was calculated as follows: % = -~
x 100,
where A = initial numbers of organisms (cfu/ ml) and B = number of survivors alter x days of incubation, (cfu/ml). To make this calculafion, samples receiving the highest levels of inoculum were chosen. Making the calculation overcame the difficulty associated with interpretation of results when initial numbers of L. monocytogenes were variable. RESULTS AND DISCUSSION
The coagulant was free (by direct plating and cold enrichment up to 8 wk) of L. monocytogenes before samples were inoculated with the bacterium. Results (Table 1) indicate that when four levels (103 to 106/ml) of L. monocytogenes strain CA were added to the coagulant, numbers of the pathogen were greatly reduced after 7 d of storage at 7°C. However, regardless of initial level of inoculum, alter 7 d viable cells of the organism were not completely eliminated from the coagulant. It is evident that increasing the inoculum to ca. 105 to 106/ml had a marked effect on the number of viable L. monocytogenes in the coagulant after 7 d of storage. After 14 d of storage, numbers of survivors in some samples were still decreasing, whereas nø viable L. monocytogenes cells were found in the 1:10 dilution of samples initially inoculated with ca. 103 or 104/ml. Survivors were reduced to numbers in the range of ca. 100 to 1000 cfu/ ml when high initial levels (more than 105/ml) Journal of Dairy Science Vol. 72, Nø. 5, 1989
1100
EL-GAZZAR AND MARTH
TABLE 1. Survival of L. monocytogenes (strain California) ha bovine-pepsin rennet during storage for 56 d at 7"C. Trial
0
A
8.0 1.4 9.0 1.4
Number per miUiliter arier days of storage 14 28 42
7 x x x x
B
5.0 x
C
1.1 5.5 1.0 9.5 2.0 7.5 1.0
× x x × x x x
103 104 10~ 106 10a 104 105 104 103 104 10s 106
10 10 1.0 1,0 20 20 1.9 7.0 10 30 1.0 2.0
x 104 x lœ x 103 x l0a
x 103 x 104
<10 (_)l <10 (-) 1000 100 <10 (-) <10(-) 300 500 <10 (-) <10 (-) 100 100
<10 (-) <10 (-) <10 (+) <10 (+) <10 (-) <10(-) <10 (+) <10 (+) <10 (-) <10 (-) <10 (+) <10 (+)
<10 (-) <10 (-) <10 (+) <10 (+) <10 (-) <10(-) <10 (+) <10 (+) <10 (-) <10 (-) <10 (+) <10 (+)
56
~(-) = Nø Listeria found by cold enfichment; (+) Listeria found by cold enrichment.
were used. Also, nø viable listeriae were found by cold enrichment at this point when we tested samples initially inoculated with low levels o f L. monocytogenes. Arier 28 d, viable listeriae were absent, either by direct plafing (.1 ml of sample) or cold enrichment, from coagulant initially inoculated with the two low levels of L. monocytogenes. With the two high levels of initial inoculum, nø viable listeriae were found by direct plating, but some cold enrichment tests gave positive results. Results obtained after 42 d of storage were similar to those noted after 28 d; after 56 d all but three cold-enrichment tests were negative. These results are similar to those observed by EI-Gazzar and Marth (6) when they studied behavior of L. monocytogenes strain C A in commercial calf rennet extract. Results obtalned with L. monocytogenes strain Scott A Œable 2) indicate small numbers o f survivors after 7 and 14 d of storage even when low (ca. 103 to 104/ml) initial numbers of the pathogen were present. After 28 d, nø L. monocytogenes could be found by direct plating of all samples receiving the two low levels of inoculum, but four of six samples remained positive when examined by cold enrichment. All samples receiving the two low levels of inoculum were free o f the pathogen by both tests after 42 and 56 d at 7°C. In contrast, five of six samples receiving the two high levels of inoculum had viable cells of L. monocytogenes arier 42 d as determined by direct plating, or Journal of Dairy Science Vol. 72, Nø. 5, 1989
cold enrichment, or both. Extending storage to 56 d reduced the number of positive samples from five o f six to one of six. It is evident from these data that straln Scott A was somewhat more resistant than strain CA to the detrimental effects of the coagulant. Tests with L. monocytogenes strain V7 (Table 3) indicate this strain of the pathogen behaved much as did stlaln Scott A dm'ing the first 28 d of storage in the coagulant. However, arier 42 and 56 d of storage essentially all samples were free o f L. monocytogenes. These data differ somewhat from those obtained with straln Scott A in that 5 of 12 samples inoculated with that strain remained positive arier 42 d. Results for both stralns (Table 2 and 3) were essentially comparable arier 56 d of storage. Calculated values in Table 4 summarize the behavior of the three sWains of L. monocytogenes in bovine pepsin-rennet extract during storage at 7"C for 14 and 28 d. It is evident that inactivadon of strain California was more nearly complete at these times than was inacUvation o f the other strains. These small differences tended to disappear arier 42 and 56 d of storage (Table 1, 2, and 3). Ryser and Marth (11) reported that L. monocytogenes survived in Cheddar cheese for more than 1 yr. Furthermore, they (12) demonstrated that, if present, L. monocytogenes can grow in Camembert cheese during ripening after the pH of cheese has increased sufficiently through action of Penicillium camemberti. Yousef and
LOSS OF LISTERIA VLABILITY
1101
TABLE 2. Survival of L. monocytogenes (strain Scott A) ha bovine-pepsin rennet during storage for 56 d at 7"C. Number per milliliter after days of storage Trial
0
A
76.0 8.4 50.0 10.0 48.0 8.1 36.0 13.0 69.0 10.0 46.0 19.0
B
C
x x x x × × x ×
103 lœ 103 106 103 104 105 10«
× 10 3
× 104 x 105 x 106
7
14
28
42
56
10 100 6.0 7.0 20 400 7.0 33.0 20 4.5 6.0 20.0
<10 (+)l 20 800 1.3 x <10 (+) 30 2.2 × 6.0 × 10 30 1.0 x 4.0 x
<10 (-) <10(+) 250 140 <10 (+) <10 (+) 240 730 <10 (-) <10 (+) 150 500
<10 (-) <10(-) <10 (+) 20 (+) <10 (-) <10 (-) <10 (-) <10 (+) <10 (-) <10 (-) <10 (+) 10 (-)
<10 (-) <10(-) <10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10 (+) <10 (-) <10 (-) <10 (-) <10 (-)
x 103 x 103
× 103 x 103 × 1Œ x 103 x l0 a
103
103 103
103 103
t(+) = Listeria fotmd by cold enrichment; (-) = nø Listeria found by cold enrichment.
Marth (13) reported that L. monocytogenes can remain viable in C o l b y cheese for a period that depends on initial i n o c u l u m , strain o f the pathogen, and c o m p o s i t i o n o f the cheese. In all instances, length o f survival e x c e e d e d the normal storage life o f C o l b y cheese. Hence, it is essential that nø ingredient, e.g., the coagulant, used in cheese m a k i n g contributes L. monocytogenes cells to the cheese. Results o f this study confmla those o f our p r e v i o u s w o r k (6). It is e v i d e n t f r o m our data that the coagulant, by g o i n g through shipping and distribution networks, can be held long e n o u g h before use to inactivate L. monocyto-
genes in the e v e n t the p a t h o g e n entered the fresh product as an inadvertent contaminant. Furthermore, if L. monocytogenes were naturally present in the coagulant, the numbers probably w o u l d be considerably smaller than e v e n our smallest inoculum. U n d e r such circumstances loss o f viability by the pathogen probably w o u l d be e v e n more rapid than was o b s e r v e d in our experiments. B e n z o i c acid, sod i u m propionate, p r o p y l e n e glycol, and salt in c o m b i n a t i o n are likely to be responsible for d e m i s e o f the b a c t e r i u m f r o m the coagulant. C o n s e q u e n t l y , c o m m e r c i a l b o v i n e pepsin-rennet extract with a preservative system o f the
TABLE 3. Survival of L. monocytogenes (strain V7) in bovine-pepsin rennet during storage for 56 d at 7"C. Number per miililiter after days of storage Trial
0
A
4.0 1.7 3.5 1.0
x x x x
B
3.5 x
C
8.0 4.5 3.5 3.5 2.0 4.5 1.0
x x x x × × x
103 104 105 104 103 103 103 10~ 103 104 103 106
7
14
28
42
56
100 180 2.0 2.2 100 200 10.0 3.2 300 300 24.0 7.4
100 50 700 1.1 x 103 10 10 400 840 20 30 1.9 x 103 4.2 × 103
<10 (_)1 <10 (-) 50 400 <10 (-) <10 (-) 60 100 <10 (-) <10 (+) 30 200
<10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10(-) <10(-) <10 (-) <10 (-) <10 (-) <10 (-)
<10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10 (-) <10(+) <10(-) <10 (-) <10 (-) <10 (-) <10 (-)
x 103 x 104
x 103 x 104
x 103 x 104
~(-) -- Nø Listeria found by cold enrichment; (+) = Listeria found by cold enrichment. Journal of Dairy Science Vol. 72, Nø. 5, 1989
1 102
EL-GAZZAR AND MARTH
TABLE 4. Percentage of viability lost by strains of L. monocytogenes in bovine-pepsin rennet during storage at 7*C. % Inactivation ~,2 Storage
California
(d)
X
14 28
99.98 99.999
SD .0196 0
Scott A
V7
X
SD
X
99.97 99.996
.0159 .0017
99.82 99.979
SD .1701 .0151
1High concentrations of L. monocytogenes; initially 106 or more/ml. :Average of three trials.
type in the product w e tested is unlikely to contaminate cheese with L. monocytogenes. In spite o f this conclusion, it w o u l d be prudent to m o n i t o r ingredients for the pathogen before they are used to manufacture cheese. E x c e p tions to the rule can occur, and these exception, if they go undetected, can cause problems.
ACKNOWLEDGMENTS R e s e a r c h supported by the C o l l e g e of Agricultural and Lire Sciences, U n i v e r s i t y o f W i s c o n s i n - M a d i s o n , the F o o d Research Institute o f the U n i v e r s i t y o f W i s c o n s i n - M a d i s o n , and Chr. H a n s e n ' s Laboratory, Inc., M i l w a u k e e , WI. W e thank R. L. Sellars for his helpful suggestions regarding this study.
REFERENCES 1 Anonymous. 1985. Listeriosis outbreak associated with Mexican- style cheese - California. Morbid. Mortal. Weekly Rep. 34:357. 2 Anonymous. 1985. Listeriosis transmitted by contaminated Jalisco-brand cheese. Calif. Morbid. No. 46, Nov. 22:1.
Journal of Dalry Science Vol. 72, Nø. 5, 1989
3 Anonymous. 1986. Brie cheese recalts extended by General Foods, US importer. Food Chem. News 27(52): 25. 4 Anonymous. 1986. FDA fmds listeria in Brie cheese from French-certified plant. Food Chem. News 27(50):35. 5 Anonymous. 1987. Listeria infectious dose calculated, Crawford says. Food Chem. News 29(17):25. 6 EI-Gazzar, F. E., and E. H. Marth. 1988. Loss of viability by Listeria monocytogenes in commercial calf rennet extract. J. Food Prot. 51:16. 7 Fox, P. F. 1969. Milk clotting and proteolytic activities of rennet and bøvine pepsin and porcine pepsin. J. Dairy Res. 36:427. 8 Fox, P. F., and B. F. Walley. 1971. lnfluence of sodium chloride on proteolysis of casein by rennet and by pepsin. J. Dalry Res. 38:165. 9 Henry, B. S. 1933. Dissociation in the genus Brucella. J. Infect. Dis. 52:374. 10 McBride, M. E., and K. F. Girard. 1960. A selective method for the isolation of Listeria monocytogenes from mixed bacterial populations. J. Lab. Clin. Med. 55:153. 11 Ryser, E. T., and E. H. Marth. 1987. Fate of Listeria monocytogenes during the manufacture and ripening of Cheddar cheese. J. Food Prot. 50:7. 12 Ryser, E. T., and E. H. Marth. 1987. Fate of Listeria monocytogenes during the manufacture and ripening of Camembert cheese. J. Food Prot. 50:372. 13 Yousef, A. E., and E. H. Marth. 1988. Behavior of Listeria monocytogenes during the manufacture and storage of Colby cheese. J. Food Prot. 51:12.