au cours des premiers 25 jours d'entreposage a 5°C sur une pCriode totale de 64 jours. De la croissance a Ct6 observCe dans du jus de chou non-clarifiC et ...
Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice L. R. BEUCHAT, R. E. BRACKETT, D. Y.-Y. HAO,AND D. E. CONNER Department of Food Science, University of Georgia, Agricultural Experiment Station, Experiment, GA, U.S.A. 30212-5099
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Accepted July 2, 1986 BEUCHAT, L. R., R. E. BRACKETT, D. Y .-Y. HAO,and D. E. CONNER.1986. Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice. Can. J. Microbiol. 32: 791 -795. Studies were done to determine the interacting effects of pH, NaCI, temperature, and time on growth, survival, and death of two strains of Listeria monocytogenes. Viable population of the organism steadily declined in heat-sterilized cabbage stored at 5°C for 42 days. In contrast, the organism grew on raw cabbage during the first 25 days of a 64-day storage period at 5OC. Growth was observed in heat-sterilized unclarified cabbage juice containing 5 5 % NaCl and tryptic phosphate broth containing 5 1 0 % NaCI. Rates of thermal inactivation increased as pH of clarified cabbage juice heating medium was decreased from 5.6 to 4.0. At 58°C (pH 5.6), 4 x lo6 cells/mL were reduced to undetectable levels within 10 min. Thermal inactivation rates in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of up to 2% NaCI; however, heat-stressed cells had increased sensitivity to NaCl in tryptic soy agar recovery medium. Cold enrichment of heatstressed cells at 5OC for 21 days enhanced resuscitation. Results indicate that L. monocytogenes can proliferate on refrigerated (5°C) raw cabbage which, in turn, may represent a hazard to health of the consumer. Heat pasteurization treatments normally given to cabbage juice or sauerkraut would be expected to kill any L. monocytogenes cells which may be present. BEUCHAT, L. R., R. E. BRACKETT, D. Y.-Y. HAOet D. E. CONNER.1986. Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice. Can. J. Microbiol. 32: 791 -795. Des Ctudes ont CtC entreprises pour dkteminer les interactions du pH, du NaCI, de la tempkrature et du facteur temps sur la croissance, la survie et la mort de Listeria monocytogenes. Une population viable de cet organisme a rkgulikrement diminuC dans des choux stCrilisCs a la chaleur et entreposes A 5°C durant 42 jours. A I'opposC, I'organisme a pu croitre sur du chou c d au cours des premiers 25 jours d'entreposage a 5°C sur une pCriode totale de 64 jours. De la croissance a Ct6 observCe dans du jus de chou non-clarifiC et stCrilisC a la chaleur contenant 5% NaCI, ainsi que dans un bouillon de phosphate tryptique contenant 10%de NaCt. ies raux d'i~aaivationthermique ont augment6 lorsque le pH du milieu de jus de chou clarifiC soumis la chaleur a diminut de 5,6 i 4.0. A 58°C (pH 5,6), 4 X 106 cellules/mL ont kt6 rkduites 21 des niveaux non dCcelables en fiedans de 10 min. Les taux d'inactivation thermlque dans le jus de chou clarifiC (ph 5,6) n'ont pas CtC influences de faqon significative par la prPlsence de NaCI, jusqu'i 2 8 ; toutefois, les cellules stressCes par la chaleur ont prCsentC une augmentation de sensibilitt au NaCI dans un milieu de recauvrement d'agar tryptique a base de sauce de soya. Un traitement d'enrichissement au ffroid, i 5°C durant 21 jours, des cellules strcss&s par la chaleur a favorisk leur retour a la vie normale. Les rksultats indiquent que L . munocylogenes peel pmlifkrer sur du chou c d rCfrigCrC (5OC) et , donc, peut reprksenter un risque pour la santC des consomrnateurs. Les traitements de pasteurisation a chaleur du jus de chou ou de la choucroute devraient permettre de tuer les cellules de L . monocytogenes qul pourraient &treprksentes. [Traduit par la revue]
Introduction Confirmed outbreaks of human listeriosis in North America in recent years have incriminated foods from both plant and animal origin as causative vehicles. Schlech et al. (1983) identified coleslaw as the probable vehicle of transmission of Listeria monocytogenes which caused 7 adult cases and 34 perinatal cases of infection in the Maritime Provinces of Canada in 1981; there were 18 deaths. In 1985, Fleming et al. reported that pasteurized milk consumed in Massachusetts was the vehicle of infection which caused 14 of 49 patients who acquired listeriosis to die. More recently, a Mexican-style cheese processed in California caused 93 perinatal and 49 nonpregnant adult cases of listeriosis (Twedt 1985~). Listeria rnonocytogenes is known to be present on vegetation (Welshimer 1968) and in silage prepared from a variety of grasses (Gray 1960; Win 1969). Thus. the organism may exist on raw vegetables and fruits at the time of consumption. In addition, L. monaqtogenes apparently is tolerant to acid pH comrnonIy associated with anaerobically fermented vegetation. The presence of L. monorytogenes in commercial dairy products which had received pasteurization matment believed to be sufficient to inactivate human pathogens has raised concern about the organism's tolerance to heat. Viable cells 'R. M. Twedt. 1985. Listeria monocytogenes considered as a foodborne pathogen. Paper presented at the 10th Food Microbiology Research Conference, Chicago, IL, November 5-8, 1985. Pnnted In Canada / Irnpnme au Canada
of L . monocytogenes were recovered from heat-pasteurized (61.7"C for 35 min) fresh milk inoculated with 5 x lo4 cells/ml prior to heat treatment (Beams and Gerard 1958). Others (Bradshaw et al. 1985) observed that the decimal reduction time at 71.7"C (D71,7"C value) of L . monocytogenes associated with the Massachusetts milk-borne outbreak was 0.9 s. Because of the paucity of information concerning the behavior of L . monocytogenes in or on vegetation destined for human consumption, experiments were designed to determine patterns of growth and death of the organism in pasteurized cabbage and cabbage juice as well as in fresh cabbage stored at 5°C. Thermal inactivation rates in cabbage juice as influenced by NaCl concentration and pH were also determined. Sensitivity of heat-stressed cells to NaCl in recovery media was also investigated.
Materials and methods Organisms Two strains of L. monocytogenes (Scott A, a human isolate from the 1983 Massachusetts outbreak, and LCDC 81-861 from the Canadian outbreak caused by coleslaw) were evaluated. Both strains are serotype 4b. Organisms were cultured at 30°C in tryptic phosphate broth (TPB) (pH 7.3) or heat-sterilized, clarified cabbage juice (pH 5.6), depending upon the specific test to be performed. The formula for TPB consisted of 20 g of peptone, 2.0 g glucose, 5.0 g of NaCI, and 2.5 g of Na2HP04 per litre of distilled water.
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Preparation of cabbage and cabbage juice Heat-sterilized cabbage and cabbage juice as well as freshly shredded raw cabbage served as substrates forevaluation of growth and survival characteristics of L. monocytogenes. Fresh, raw cabbage was shredded for tests involving solid product. For tests requiring pasteurized cabbage, subsamples (350 g) were heated at 121°C for 20 min and cooled to ambient temperature before inoculating. Cabbage juice was prepared by chopping raw cabbage with a Hobart Silent Chopper (Hobart Mfg., Troy, OH; model 84142), grinding with a Morehouse stone mill (Morehouse Ind., Los Angeles, CA; model M-MS-3) set at 0.25 rnrn clearance and pressing with a Willmes Presser (Josef Willmes, Bensheim Hessen, Federal Republic of Germany; type 60) at 55 kg/cm2 pressure. Juice was collected through two layers of cheesecloth, dispensed into plastic containers, and stored at - 18OC until used. Juice was heat sterilized at 121°C for 15 min before use. In some experiments, clarified juice was used. Clarification was accomplished by aseptically centrifuging sterilized juice at 10 000 x g for 20 min to remove solid materials.
inwulated {I .O mL) into 100 rnL of sterile, clarified. cabbagejuice (pH 4.0,4.6, and 5.6) maintained at 50.52.54,56, and 58°C in a waterbath shaker. Sampies were withdrawn at I & d n intervals for up to 60 rnin. diluted in phosphate buffer, and plated (0.1 rnL) on TSA. Colonies were counted after 24 h of incubation at 30°C. Decimal reduction times (D values) and z values were calculated from the linear portions of thermal inactivation curves which transgressed at least three logcycles. The influence of NaCl (0, 1, and 2%) in clarified cabbage juice heating medium (pH 5.6) on rates of inactivation was determined as described above.
Survival on cabbage at 5°C Heat-sterilized cabbage (350 g) was inoculated with 10 mL of lo-' dilutions (0.1 M potassium phosphate buffer, pH 7.0) of 24-h cultures of L. monocytogenes LCDC 81-861 or Scott A grown in TPB at 30°C. The inoculated cabbage was thoroughly mixed and incubated at S°C. Initially and after 2, 5, 8, 14, 21, 28, 35, and 42 days of storage, samples were mixed and duplicate 25-g subsamples were analyzed for viable L. monocytogenes. Analysis consisted of homogenizing the samples with 225 mL of phosphate buffer in a Colworth stomacher for 2 min, serially diluting, and preparing pourplates with tryptic soy agar (TSA). Colonies were counted after 48 h of incubation at 30°C. The viability of only stran LCDC 81-861 was investigated on raw shredded cabbage which was not pasteurized. An active 24-h TPB culture was diluted in phosphate buffer (1500 mL) and combined with 1500 g of shredded cabbage. After thorough mixing, the cabbage was drained and adjusted to 5OC within 3 h. Samples were analyzed for selected microbial populations initially and after 9,25,39, and 64 days of storage. Before each analysis, cabbage was thoroughly mixed. Samples (100 g in duplicate) were combined with 200 mL of sterile phosphate buffer and homogenized in a stomacher for 1 min. The solid cabbage was allowed to settle over a 5-min period adn the fluid was analyzed in duplicate for populations of L. monocytogenes by applying 0.1-mL aliquots of the serial dilutions to the surface of Petri plates containing a basal agar medium prepared as described by Doyle and Schoeni (1986) but modified to omit acrillavin-HC1 and include fenic citrate (5 pg/mL) in the formulation. Colonies (1-2 rnm diameter, translucent to slightly opaque, grey-white to slightly blue; reverse with dark reddish brown center) were counted after plates were incubated for 48 h at 30°C. the total aerobic population was determined using plate count agar (30°C, 48 h); lactic acid bacteria were enumerated on MRS agar (Oxoid) supplemented with 0.1% potassium sorbate (MRSS) incubated at 42°C for 72 h.
Effect of cold enrichment on colony formation by heat-stressed cells After incubation and enumeration of colonies on TSA plates used to recover cells which had been subjected to various conditions of heat stress. plates containing 30-300 colonies as well as plates containing higher dilutions of heated cell suspensions were incubated at 5'C for 2 1 days. Following cold enrichment. plates were incubated once again (48 h) at 30°C Wore colonies were counted. This procedure was followed to determine if cells which may have been heat injured and not able to form colonies during the first incubation period (30°C, 24 h) could resuscitate at 5OC and form colonies during the second incubation period at 30°C.
Sensitivity of heat-stressed cells to NaCl To determine if L. monocytogenes is susceptible to injury upon sublethal heat treatment, the influence of NaCl(0, 2, and 4%) in TSA and uncIarified cabbage juice agar (2%) on colony formation by heat-stressed (5Z0C, up to 100 min) cells was investigated. Cells (40 to 46 h old) were healed in clarified cabbage juice (pH 5.6). diluted in phosphate buffer, and mixed with TSA recovery media in Petri dishes. Colonies were counted after 24 h of incubation at 30°C.
Results Survival on cabbage at 5°C The two test strains of L. monocytogenes exhibited similar patterns of survival on sterile cabbage stored at 5°C for 42 days (Fig. 1). Inactivation was most rapid during the first 8 days, and the LCDC 81-861 strain was less tolerant to refrigeration than was the Scott A strain. In contrast to the steady decline in viability of L. monocytugenes in heat-sterilized cabbage stored at 5°C. the organism (strain LCDC 8 1-561) actually increased from 1.6 X lo4 to 2.6 x lo8 (coiony-forming units (cfu)/g of raw shredded cabbage stored at 5°C for 25 days (Fig. 2)). Extended storage to 64 days resulted in only a slight decrease in viable population. The total aerobic microorganism popuiation in raw cabbage increased over the 64-day period at 5°C. whereas lactic acid bacteria increased in population during the first 39 days and then decreased at 64 days.
Tolerance to NaCl Heat-sterilized unclarified cabbage juice (100 mL) and TPB (100 mL) containing NaCl at concentrations of 0-12% in 1% increments were inoculated (1.0 mL) (5 X lo4 - 1 X 105/mL) with 40 to 46-h-old cells of L. monocytogenes cultured in clarified cabbage juice. Inoculated cabbage juice and TPB incubated at 30°C were monitored for growth at daily intervals for up to 2 weeks to determine the maximum NaCl concentration at which test strains would grow. Positive growth, i.e., an increase in cells per millilitre, was interpreted as development of turbidity in the juice and broth. At the end of the incubation period, cultures were streaked onto TSA. Plates incubated at 30°C were observed for colony development. Confirmation of L. monocytogenes was made by microscopic examination.
Tolerance to NaCl The tolerance of test strains to NaCl was similar but other constituents of the medium greatly influenced the maximum NaCl concentration at which L. monocytogenes would multiply. In unclarified cabbage juice, growth was observed only at concentrations of 5 5 % NaC1, whereas no growth was observed in TSB containing up to 10%NaCI. Since viable populations in media containing various concentrations of NaCl were not actually measured during the Zweek storage period, the number of viable cells per millilitre may have decreased with time. However, multiplication was concurrent in cabbagejuice (55% NaCl) and TSB (110% NaC1) to the extent that turbidity developed
Injuence of pH and NaCl on thermal inactivation Cells (40-46 h) grown in clarified cabbage juice at 30°C were
Influence of pH on thermal inactivation Thermal inactivation curves for L. monocytogenes LCDC
BEUCHAT ET AL.
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6
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0 2 7
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m
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A
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y
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Time (days)
FIG. 1 . Survival of Listeria monocytogenes LCDC 81-861 and Scott A on heat-sterilized shredded cabbage stored at 5OC for 42 days.
3
< ,.' 4 3 l o - U
2
0
0
10
lor
20 30 40 T ~ m e( m ~ n )
I
I
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FIG.3. Inactivation of Listeria monocytofenes LCDC 8 1-861 in 54 (A), and clarified cabbage juice (pH 5.6 and 4.6) at 50 (0),52 (a), 56°C ( 0 ) . e
-
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Time (days)
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FIG. 2. Populations of Listeria monocytogenes LCDC 8 1-861 (O), lactic acid bacteria (C!),and total aerobic microorganisms (A) in raw shredded cabbage stored at 5OC for 64 days.
81-861 and Scott A in clarified cabbage juice as influenced by pH are illustrated in Figs. 3 and 4, respectively. At pH 5.6, only slight decreases in population were noted after 60 min at 50°C. As the treatment temperature was increased to 56"C, the rates of inactivation also increased. No viable cells were detected in cabbage juice held at 58°C for 10 min. At pH 4.6, the viable population of L,monocylogenes was seduced by about 2 logs during a 60-min perid at 50°C. As in cabbage juice at pH 5.6, rates of inactivation progressively increased with increasing temperature of treatment. Experiments in which cabbage juice at H 4.0 was used as the heating medium revealed that 4 x 10 cfu/mL was reduced to nondetectable Ievels after 30 min at 50°C; complete inactivation withing 10 min was observed at r52°C. Decimal reduction times ( D values) and z values for test strains are listed in Table 1. At the same temperature, D values were higher at pH 5.6 than at pH 4.6. The Scott A strain exhibited higher resistance to thermal stress than did the LCDC
R
0:
Ib
,b
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0 a0 Time ( m i d
0
Q,
FIG.4. Inactivation of Listeria monocytogenes Scott A in clarified cabbage juice (pH 5.6 and 4.6) at 50 (O),52 (U), 54 (A), and 56°C (0).
8 1-861 strain. The z values ranged from 6.45 to 5.63OC at pH 5.6 to 7.01 to 7.54OC at pH 4.6. Injuence of NaCl on thermal inactivation
Rates of inactivation of both test strains in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of 1 and 2% NaCl. This observation was made at treatment temperatures of 50, 52, 54, 56, and 58°C. Sensitivity of heat-stressed cells to NaCl Heat-stressed cells of L. monocytogenes had increased
sensitivity to NaCl in TSA recovery medium (Fig. 5). The
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CAN. J. MICROBIIDL. VOL. 32, 1986
TABLE 1. Decimal reduction times (D values) and z values of L. monocytogenes LCDC 81-861 and Scott A heated at 50, 52, 54, and 56°C in cabbage juice at pH 4.6 and 5.6 D value (min) at:
Strain
pH'
50°C
52°C
54°C
56°C
LCDC 81-861
4.6 5.6 4.6 5.6
13.33 >60.00 25.00 >60.00
8.20 20.00 14.39 34.48
4.88 8.93 6.71 8.35
2.04 4.74 3.64 6.80
7.54 6.45 7.01 5.63
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z
value ("C)
NOTE:z value, degree Celcius change required to change D value by a factor of 10.
'r
LCDC 8 1-86 1
Scott A
0
I 20
I 40 60 Time ( m i d
I 80
1 100
FIG.5. Recovery of Listeria monocytogenes LCDC 81-861 and Scott A heated (52°C)in clarified cabbage juice (pH 5.6) and plated on 2 (a), and 4% NaCl (A). TSA containing 0 (O), percentage of cells capable of resuscitating and forming colonies on TSA supplemented with 2% NaCl increased as exposure to 52°C in clarified cabbage juice (pH 5.6) increased up to 100 min. This effect was even more pronounced when heated cell suspensions were plated on TSA containing 4% NaC1, indicating that sublethal injury is extensive as a result of thermal treatment. Unclarified cabbage juice agar served at a poor recovery medium for heat-stressed L. monocytogenes, regardless of the concentration of NaCl it contained. Effect of cold enrichment on colony formation by heat-treated cells After heat-treated cells had been plated on TSA and incubated at 30°C for 24 h, colonies were counted; plates were then incubated at 5°C for 21 days before once again incubating at 30°C for 48 h. this procedure resulted in increased numbers of colonies, e.g., up to 1 log in some instances. The enhanced effect of cold enrichment on subsequent colony development at 30°C was noted with greatest frequency in experiments in which cells had been exposed to the most severe conditions of acid pH and temperature during treatment or elevated NaCl concentration in TSA recovery medium.
Discussion The presence of L. monocytogenes in water, sewage, soil. and decaying organic materials and in the intestinal tracts of apparently healthy domestic and wild animals suggests that the organism is also present on raw vegetables, fruits, and other plant pats as they are delivered to the consumer. Results presented here indicate that L. monocytogenes grows at a temperature (5°C) and within a time period to which many types of raw produce are subjected during distribution and marketing. W i l e behavior of L, monocytogenes on produce other than cabbage cannot be projected from these results, it is reasonable to predict that growth and survival patterns may be similar. Further work is needed to confirm this hypothesis. The fact that acid pH enhances the rate of thermal inactivation of L. monocytogenes indicates that fermented vegetables which received minimal heat processing before marketing pose little threat to public health. However, the survival of L. monocytogenes in fermented plant products as influenced by various physical and chemical parameters has not been fully defined. The ability of L. monocytogenes to grow on raw cabbage but not on heat-sterilized cabbage at 5°C suggests that heat treatment either decreases the availability of certain nutrients or results in constituents which inhibit growth or are toxic. We have also observed that heat-sterilized cabbage juice has an apparent toxic effect on L. monocytogenes compared with the same juice which has been clarified by removing insoluble materials (Conner et al. 1986). Further investigations should be done to determine the reasons for these differences and whether the organism behaves similarly on other raw and heat-sterilized vegetables. There is great need to develop methodology for detecting and enumerating L,monocytogenes in plant products. In the present study, several direct isolation media were used to enumerate the organism, although only one medium is described. The ornission of acriflavin and the addition of ferric citrate to the formula described by Doyle and Schoeni (1986) aided in detecting L. monocytogenes colonies in the presence of other microflora. Visual characteristics of the colony surface together with dark reddish-brown centers of the reverse side of colonies formed on the iron-supplemented medium made presumptive identification of L. monocytogenes easier. Nevertheless, techniques for enumerating the organism, perhaps involving cold enrichment, must be improved if more accurate population estimations in plant products are to be achieved. Acknowledgments We are grateful to J. M. Farber, Sir Frederick G. Banting Research Centre, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ont., and E. H. Marth, Department of Food Science, University of Wisconsin, Madison, WI, for supplying the two strains of L. monocytogenes studied in this investigation. BEARNS, R. E., and K. F. GIRARD. 1958. The effect of pasteurization on Listeria Monocytogenes. Can. J . Microbiol. 4: 55-61. BRADSHAW, J. G., J. T. PEELER, J. J. CORWIN, J. M. HUNT,J. T. TIERNEY, E. P. LARKIN, and R. M. TWEDT.1985. Thermal resistance of Listeria monocytogenes in milk. J . Food Prot. 48: 743-745. CONNER, D. E., R. E. BRACKETT, andL. R. BEUCHAT.1986. Growth of Listeria monocytogenes in cabbage juice as affected by temperature, sodium chloride and pH. Appl. Environ. Microbiol. 52: 59-63. DOYLE,M. P., and J. L. SCHOENI. 1986. Selective enrichment
BEUCHAT ET AL.
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procedure for isolation of Listeria monocytogenes from fecal and biologic specimens. Appl. Environ. Microbial. 51: 1127- 1129. FLEMING, D. W., S. L. COCHI,K. L. MACDONALD, J. BRONDUM, P. S. HAYES,B. D. PLIKAYTIS, M. B. HOLMES,A. AUDURIER, C. V. BROOME,and A. L. REINGOLD.1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med. 312: 404-407. GRAY,M. L. 1960. Isolation of Listeria monocytogenes from oat silage. Science (Washington, D.C.), 132: 1767-1768. IRVIN,A. D. 1969. The inhibition of Listeria monocytogenes by an
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organism, resembling Bacillus rnycoides, present in normal silage. Res. Vet. Sci. 10: 106-108. SCKI.ECH, W.F..P. M. LAVIW, R. A. BORTOLUSSI, A. C.ALLEN, E. V. HALDANE. A. J. WORT,A. W. HIGHTOWER,S.E. JOHNSON. S. H. KING. E. S. NICHOLLS, and C. V. B~ooMe.1983. Epidemic listeriosis-evidence for uansmissjon by food. N. Engl. J. Med. 308: 203-206. WELSHIMER, H. J. 1968. Isolation of Listcia monocytoRenes from vegetation. J. Bactenol. 95: 300-303.
