International Journal of Food MIcrobwlogy, 12 (1991) 235-246 © 1991 Elsevier Science Pubhshers B.V. 0168-1605/91/$03.50
235
F O O D 00383
Effect of temperature history on the growth of Listeria monocytogenes Scott A at refrigeration temperatures * R.L. Buchanan and L.A. Klawitter Mtcrobtal Food Safety Research Umt, U.S. Department of Agrtculture, A RS Eastern Regional Research Center, Phdadelphla, PA, U.S.A. (Received 17 April 1990; accepted 19 November 1990)
The effect of pre-inoculation temperature on the subsequent growth of l.asterta monocytogenes Scott A at 5 ° C was examined in microbiological medium. U H T milk. canned dog food, and raw ground beef (untreated and irradlatton-sterilized). In microbiological medium, the duration of the lag phase was decreased when aerobic and anaerobic cultures were initially grown at < 28 and < 130C, respectively. Subsequent exponenual growth rates and m a x i m u m population densities of the 5 ° C cultures were not affected by temperature history. Differences in lag phase durations were also observed when L. monocytogenes imtially cultured at 19 and 3 7 0 C were grown at 5 0 C in U H T milk and some of the canned dog food varieties. Growth of L. monocytogenes was not observed m either untreated or irradiation-sterilized raw ground beef. While temperature history can affect the growth klneucs of L. monocytogenes at 5 * C. ~t did not account for the lack of growth in raw meat, suggesting that there ts an inhibitory condiuon or c o m p o n e n t in ground beef that is lost upon cooking. Key words: Meat; Milk; Lag phase; Competition
Introduction While Lssteria monocytogenes can grow in a variety of refrigerated foods including processed meat products, several teams of investigators have reported that the microorganism grows poorly, if at all, in refrigerated raw meat, particularly ground beef (Kahn et al. 1972, 1973, 1975; Gouet et al,, 1978; Buchanan et al., 1987; Johnson et al., 1988a,b; Glass and Doyle, 1989; Shelef, 1989; Kaya and Schmidt, 1989). However, Kahn et al. (1972, 1973) demonstrated that L. monocvtogenes grew on the surface of sterile lamb stored at 8°C. Gill and Reichel (1989) observed Correspondence address" R.L. Buchanan, Microbial Food Safety Research Umt, U.S. Department of Agriculture, ARS Eastern Regional Research Center, 600 East Mermaid Lane, Philadelphia, PA 19118. U,S.A. • Mention of brand or firm n a m e s does not constitute an endorsement by the U.S. Department of Agriculture over others of a stmilar nature not mentioned.
236
growth on vacuum-packed beef strips at temperatures as low as 0 ° C. but did not detect growth when the meat was stored at < 10 ° C in a CO2-enriched atmosphere. Grau and Vanderlinde (1988) reported that L. monocytogenes grew readily at 5.3°C on strips of beef, particularly on the adipose tissue. They hypothesized that other investigators did not observe growth at refrigeration temperatures due to the use of inocula grown at elevated temperatures (30-37 ° C), resulting in extremely extended lag phases when the microorganism was transfered to refrigerated meat. Grau and Vanderlinde (1988) used an inocuhim grown at 1 0 ° C and observed a minimal lag phase. The ob3ective of the current study was to test this hypothesis by examining the effect of temperature history on the growth kinetics of L. monocvtogenes incubated at 5 ° C in microbiological medium, U H T milk, sterile cooked meat products, raw ground beef and irradmtion-sterilized raw ground beef.
Materials and Methods
Microorgamsm Listeria monocytogenes Scott A was used throughout the study. Stock cultures were maintained in Brain Heart Infusion Broth (Difco) at 4 ° C , and transferred monthly. Inocula were grown in 50 ml Tryptose Phosphate Broth (TPB) (Difco) in 250-mi Erlenmeyer flasks incubated for 144 h at 5 ° C , 96 h at 1 0 ° C and 13°C, 48 h at 1 9 ° C , or 24 h at 28°C, 37°C, and 4 2 0 C on a rotary shaker (150 rpm). Mtcrobtologtcal medtum The microorganism was cultured aerobically and anaerobically in TPB according to the method of Buchanan et al. (1989). TPB was adjusted to p H 6.75 with concentrated HCI, and 50-ml portions were transferred to either 250-ml Erlenmeyer flasks (aerobic cultures) or 250-ml trypsinizing flasks (anaerobic cultures). Erlenmeyer flasks were capped with foam plugs, and trypsinizing flasks were sealed with screw caps and rubber septa. All flasks were sterilized by autoclaving and then prechilled to 5 ° C . Starter cultures were diluted and apportioned to achieve an inoculum of approximately 103 c f u / m l in each flask. Immediately after inoculation, the trypslnizing flasks were flushed for 10 rain with N 2 sterilized by filtration and sealed tightly to maintain anaerobiosis. All flasks were incubated at 5 o C on a rotary shaker (150 rpm). At least two replicate cultures were studied for each t e m p e r a t u r e / atmosphere combination. Periodically, 2.5-ml samples were removed from the Erlenmeyer and trypsimzing flasks using a pipette or hypodermic needle + syringe, respectively. Flasks were kept in an ice-water bath during sampling to ensure that the 5 ° C incubation temperature was maintained continuously. Samples were diluted appropriately with sterile 0.1% peptone water and plated in duplicate on Brain Heart Infusion Agar (BHIA) (Difco) plates using a Spiral Plater (Model D, Spiral Systems, Bethesda, MD). All plates were incubated for 24 h at 3 7 ° C and enumerated. It was checked that extended incubation did not increase count significantly. The pH of 0-h samples was measured using a p H meter (Model 601A, Orion) to ensure the initial pH had been sustained.
237 Milk
U H T milk was p u r c h a s e d f r o m a local s u p e r m a r k e t a n d p r e c h i l l e d to 5 ° C. T h e milk was a s e p t i c a l l y t r a n s f e r r e d to p r e s t e r i l i z e d 250-ml E r l e n m e y e r flasks a n d i n o c u l a t e d to achieve a n initial level o f 103 c f u / m l . A l l flasks were i n c u b a t e d at 5 ° C w i t h o u t agitation, with five r e p l i c a t e cultures b e i n g e x a m i n e d for each p r e - i n o c u l a tion t e m p e r a t u r e . D u r i n g s a m p l i n g , flasks were k e p t in an ice w a t e r b a t h a n d swirled to a t t a i n a h o m o g e n e o u s sample. S a m p l e s were r e m o v e d , d i l u t e d , p l a t e d . i n c u b a t e d , a n d c o u n t e d as d e s c r i b e d above. C a n n e d dog f o o d
' A l l - m e a t ' c a n n e d d o g f o o d was used as a m o d e l for c o o k e d m e a t p r o d u c t s . T h r e e varieties (all beef, beef a n d liver, a n d m e a t a n d gravy) in 4-oz cans were p u r c h a s e d at a local s u p e r m a r k e t a n d p r e c h i l l e d to 5 ° C a l o n g with sterile 500-ml beakers. T h e c o n t e n t s of the cans were t r a n s f e r r e d a s e p t i c a l l y to i n d i v i d u a l beaekrs. P r e - i n o c u l a g r o w n at 19 o r 37 ° C were d i l u t e d a n d used to i n o c u l a t e the s a m p l e s to a target level of 103 c f u / g . T h e final d i l u t i o n of the i n o c u l u m was m a d e using d i l u e n t (9.9 ml) c o n t a i n i n g a p p r o x i m a t e l y 0.1 ml of a green f o o d d y e ( D u r k e e , W a y n e , N J). T h e m e a t s a m p l e s were b l e n d e d until the d y e was u n i f o r m l y s p r e a d t h r o u g h o u t the sample. All s a m p l e s were i n c u b a t e d at 5 ° C. A t least three r e p l i c a t e s a m p l e s were s t u d i e d for each p r e - i n o c u l a t i o n t e m p e r a t u r e a n d p r o d u c t v a r i e t y c o m b i n a t i o n . Periodically, d u p l i c a t e 1.0-g p o r t i o n s o f each s a m p l e were t r a n s f e r r e d to screw c a p tubes c o n t a i n i n g 9.0 ml o f sterile 0.1% p e p t o n e water. A f t e r a g i t a t i n g to d i s p e r s e the m e a t in the diluent, a p p r o p r i a t e d i l u t i o n s were m a d e , a n d 0.2-ml p o r t i o n s were d i s t r i b u t e d evenly across the surface o f p r e p o u r e d B H I A plates using a sterile glass s p r e a d e r . A l l p l a t e s were i n c u b a t e d for 24 h at 37 ° C a n d e n u m e r a t e d . A g a i n . it was d e t e r m i n e d that e x t e n d e d i n c u b a t i o n d i d n o t increase c o u n t s significantly.
TABLE I Equations for Gompertz funcuon and derived growth kinetics values Gompertz's function: L( t ) = A -e Ce -eB''-~'
where: L(t) = Log count of bacteria at time (m hours) t (Log(cfu/ml)). A= Asymptouc log count of bacteria as ume decreases indefinitely (t.e., mmal level of bacteria) (Loglcfu/ml)). C = Asymptotm amount of growth that occurs as t increases indefimtely (t.e.. number of log cycles of growth) (Log(cfu/ml)) M = Time at whtch the absolute growth rate is maxtmal (h). B= Relauve growth rate at M (Log(cfu/ml))/h). Derived growth kinetics equations: Exponenual growth rate (EGR) = B C / e ((Log(cfu/ml))/h) Generauon time (GT) = (Log(2))e/BC (h) Lag phase duratson (LPD) = M - ( l / B ) (h) Maximum population density (MPD) = A + C (Log(cfu/ml))
238
Raw ground beef Ground beef was purchased at a local supermarket, and 100-g portions were transferred to sterile 500-ml beakers. The samples were then inoculated and incubated as described above for canned dog food. Five replicate samples were examined for each pre-inoculation temperature. Periodically, duplicate 1.0-g portions of each sample were transferred to screw cap tubes containing 9.0 ml of 0.1% sterile peptone water. After mixing thoroughly and diluting, 0.2 ml of each sample was spread evenly across prepoured plates of Modified Vogel Johnson Agar (MVJ') (Buchanan et al., 1987) containing 200 # g / m l sodium arsenite. This extremely selective medium took advantage of the high level of arsenite resistance inherent in strain Scott A. Preliminary studies indicated that this medium could quantitatively recover the organism from meat samples. This proved a very convenient system for following the growth of L. monocytogenes in raw meat since the test strain was essentially the only organism that grew on the MVJ + arsenite plates. All plates were incubated for 48-72 h at 37"C and enumerated. The pH of meat samples was measured at 216 h. Irradianon-sterilized, raw ground beef
Ground beef purchased locally was divided into 100-g portions, vacuum packed in individual low permeability plastic bags (All-Vak :~13, International Kenfield Distributing Co., Rosemont, IL), and then frozen and maintained at - 7 0 °C before and during irradiation. The frozen samples were irradiated with 48.5 kGy of gamma irradiation using a CS137 source. After radiation sterilization, the meat samples were equilibrated to 5°C. The samples were then transferred aseptically to sterile, prechilled beakers and inoculated, incubated, sampled, and counted as described above for the canned dog food samples. Growth curves
Where appropriate, growth curves were generated using the Gompertz function (Table I) (Buchanan et al., 1989). The four Gompertz parameters were used subsequently to calculate exponential growth rates (EGR), generation times (GT), lag phase durations (LPD), and maximum population densities (MPD).
Results L. monocytogenes was initially grown at various temperatures (5-42°C) to determine the effect that incubation temperature of the inoculum had on the subsequent lonetics of aerobic and anaerobic growth at 5°C in TPB (Table II). Temperature history of the inoculum had relatively tittle effect on either EGR, GT, or MPD, whereas more distinct differences in LPDs as a function of temperature history were observed in both the aerobic and anaerobic cultures. Aerobically, the LPD was increased 10-20 h when the inocutum was grown at > 3 7 ° C , as compared to the lower incubation temperatures. This effect appeared to be more pronounced with the anaerobic cultures, with an approximate 2-fold increase in
239 TABLE II Effect of initially culturing Li$lerm monocywgenes Scott A at various temperatures on its subsequent growth kineucs at 5 ° C in tryptose phosphate broth, a (For explanation of abbrevsations, see Table l) Premcubadon temperature t°C) n
Gompertz values
A
C
B
M
2.0 (0.0) 3.4 (0.6) 3.5
7.4 (0.2) 6.1 (0.6) 6.1
0.008 (0.001) 0.013 (0.006) 0.013
159.8 (4.6) 125.9 (17.9) 104.2
(0.0)
(0.3)
(0.001)
3.7 tO.l) 3.5 (o.1) 3.8
6.1 (0.2) 6.2 (o.1) 6.1
0.017 (0.003) 0.014 (o.ool) 0.013
(0.2)
(0.2)
3.7 (0.1)
6.3 (0.2) 7.5 (0.1) 6.9
LPD (h)
EGR (Log(cfu /ml)/h)
GT (h)
MPD (Log(cfu /ml))
36.8 (12.0) 39.3 (18.0) 29.1
0.023 (0.001) 0.029 (0.013) 0.030
13.6 (0.5) 11.7 (3.4) 10.1
9.5 (0.2) 9.5 (0.4) 9.6"
Aerobic cultures
5
2
10
7
13
2
19
5
28
2
37
6
42
2
(3.0)
(0.002)
(0.7)
(0.3)
95.9 (10.4) 108.2 (5.1) 128.3
(5.7)
36.7 (3.7) 36.7 (8.7) 50.6
0.038 (0.005) 0.032 (0.001) 0.030
8.0 (1.1) 9.5 (0.4) 10.5
9.7 (0.1) 9.7 (0.0) 9.8
(0.002)
O1.7)
(15.6)
(0.005)
(1.8)
(0.2)
0.013 (0.000)
125.9 (4.0)
49.6 (4.0)
0.031 (0.001)
10.0 (0.3)
10.0 (0.1)
0.010 (0.001) 0.009 (0.001)
132.2 (10.0) 135.4
27.4 (4.6) 27.8
0.027 (0.002) 0.023
11.4 (0.7) 12.9
(8.3) 24.7
(0.002) 0.034
(1.1) 9.0
9.6 (0.1) 9.5 (0.4)
(0.1) 49.1
(0.001) 0.036
(0.1) 8.5
(0.1) 9.6
(5.9)
(0.002)
(0.5)
(0.I)
0.033 (0.001) 0.032 (0.005) 0.034
9.2 (0.1) 9.6 (1.7) 8.8
9.9 (0.0) 9.7 (0.1) 9.9
(0.000)
t0.1)
t0.1)
Anaerobic cultures
5
2
10
5
2.0 (0.1) 2.6
13
2
(1.1) 3.6
(1.5) 5.9
19
2
(0.0) 3.7
28
2
37
6
42
2
0.015
(8.8) 89.4
(0.1) 5.9
(0.000) 0.016
(1.6) 109.4
(0.0)
(0.I)
(0.001)
3.7 (0.0) 3.7 (0.2) 3.6
6.2 (0.0) 6.0 (0.1) 6.2
0.014 (0.000) 0.015 (0.002) 0.015
(0.0)
t0.0)
(0.000)
(1.6) 121.4 (11.7) 121.0 (12.6) 112.3
51.7 (10.8) 50.0 (8.5) 45.4
(2.8)
(3.1)
9.6
Values are means of n replicatecultures. Values in parentheses are standard dev~auons.
LPD
being observed with the higher inoculum
cultivation temperature. The effect
w a s a l s o m o r e p r o n o u n c e d in t h a t i n c r e a s e d L P D ' s w e r e o b s e r v e d at p r e - i n o c u l a t i o n t e m p e r a t u r e s as l o w as 1 9 ° C in t h e a n a e r o b i c c u l t u r e s , c o m p a r e d to t h e 3 7 ° C c u t o f f in t h e a e r o b i c c u l t u r e s . A s i m i l a r p a t t e r n was o b s e r v e d w h e n U H T m i l k was i n o c u l a t e d w i t h L. monocytogenes cells g r o w n at 19 o r 3 7 ° C (Fig. 1). A n a p p r o x i m a t e 3 - f o l d i n c r e a s e in t h e
240
40
0
GT
LPD
MPD
(hr)
(hr)
(Log[cfu/ml])
re ,,, o. ~-
F~g. 1. Effect of initially cultunng L. monoqywgenes Scott A aerobically at 19 and 3 7 ° C on its subsequent growth at 5 ° C m UHT-pasteurized milk. G T ffi generation ume, L P D = lag phase duration, M P D ffi m a x i m u m population density. Average of five determinations.
mean L P D (16.2 + 12.1 vs. 51.9 _+ 18.7 h) was observed with the higher pre-inoculation temperature, whereas the mean GTs (19.3 ± 4.0 vs. 17.7 + 3.3 h) and MPDs (7.4 ± 0.1 vs. 7.6 ± 0.2 log(cfu/ml)) of the cultures were similar. Commercially available 'all meat' dog food was used as a model for cooked meat products. The 4-oz cans of this type of product proved to be a convenient test
T A B L E 111 Effect of initially culturing l~ster~a monocytogenes Scott A aerobically at 19 vs. 37 ° C on its subsequent growth kineucs at 5 ° C m canned dog food " (For explanation of abbrevmuons, see Table I) Product vanety b
1
Premcubauon temperature (°C)
Gompertz values
A
C
B
M
19
1.6 (0.4) 2.6 (0.1)
6.7 (0.3) 5.5 (0.2)
0.0063 (0.0014) 0.0059 (0.0013)
260.9 (63.5) 249.6 (22.5)
1.7 (0.2) 2.7 (0.1)
6.8 (0.2) 5.6 (0.1)
0.0068 (0.0007) 0.0108 (0.0004)
1.7 (0.2) 2.7 (0.1)
6.5 (0.2) 6.3 (0.2)
0.0051 (0.0007) 0.0054 (0.0003)
37 2
19 37
3
19 37
LPD (h)
EGR (Log(cfu /roll/h)
GT (h)
MPD (Log(cfu /mill
61.2 (5.8) 54.3 (7.7)
0.015 (0.004) 0.012 (0.003)
25.0 (8.0) 28.9 (4.4)
8.3 (0.3) 8.2 (0.2)
207.3 (19.3) 166.9 (9.2)
52.0 (2.3) 73.7 (10.7)
0.017 (0.002) 0.022 (0.001)
18 7 (2 0l 13.6 (0.4)
8.5 (0.1) 8.3 (0.1)
254.3 (10.2) 274.2 (22.6)
46.0 (16.9) 88.8 (31.4)
0.012 (0.002) 0.012 (0.001)
26.4 (2.8) 24.1 (0.5)
8.2 (0.3) 8.9 (0.3)
" Values are m e a n s of at least three independent determinations. Values m parentheses are standard devlat,ons b 1 = ' a l l beef'. 2 • ' b e e f and hver': 3 = ' m e a t and gravy'.
241 10
IJl --
8 7
.j
">"
II:~ ~
19 37 19 37
C/sterile C/sterile C/non-sterile , C/non-sterile ~U
6
f
~ Z
I=
0
4
_o
3
m
"5
2
L
0
260
4oo
600-
#60
5 C Incubation Time (hr) Fig. 2. Effect of iniually culturing 1_ monoo, togenes Scott A aerobically at 19 and 3 7 ° C on ~ts subsequent survwal at 50C in raw ('non-sterile') ground beef and gamma irradiated ('sterile') raw ground beef. Average of three determinations.
system,
providing
product.
Three
pre-inoculation
an
adequately
different
product
temperatures
sterilized, varieties
o f 19 a n d 3 7 ° C
reasonably were
homogeneous
evaluated
(Table
III).
in
meat-based
conjunction
The MPDs
with
were roughly
TABLE IV Taxonormc characterisucs of zsolates from ground beef that possessed activ]ty against l_d$1eraa monoc_wogenes Scott A Shape Gram staan Motility Catalase Growth at 12 o C Tellunte reductton H2S production
Medium length rods +
Carbohydrate
3 Days
Glucose Sucrose Maltose Mannose Lactose Manmtol Xylose Ribose Rhamnose Arabinose Melibiose
+
7 Days
28°C
120C
280C
12°C
+ + + + + + ~-4± ± +
+ + + + +
+ + + + + +
-
-l-
+ -
+ +
+ + + + + + -
242
equivalent regardless of product type or pre-inoculation temperature. E G R / G T was unaffected by pre-inoculation temperatures in two of the product varieties (No. 1 and No. 3), but an approximate 30% decrease in G T was observed with the 3 7 ° C / v a r i e t y No. 2 cultures. The effect of pre-inoculation temperature on LPD was also product-dependent. An approximate 50% increase in LPD was observed with the higher pre-inoculation temperature in variety No. 2, and a 2-fold increase occurred with variety No. 3. However, the LPD of variety No. 1 was unaffected b 3 pre-inoculation temperature. Raw ground beef sterilized by gamma irradiation did not support significant growth at 5 ° C of L. monocytogenes regardless of pre-inoculation temperature (Fig. 2); however, the microorganism did survive for extended periods. When unsterilized raw ground beef was inoculated with cells initially incubated at 19 and 37 o C, the microorganism died off at a relatively rapid rate. The pH of the meat at 216 h, the time when inactivation was complete was 7.2-7.4. Representative members of the meat's microflora were isolated and screened for activity against L. monocytogenes using BHIA plates seeded with a lawn of L. monocytogenes Scott A. Two isolates produced distinct zones of inhibition. Both isolates had similar taxonomic characteristics and were tentatively classified (Sneath et al., 1986) as Lactobacillus plantarurn, based on the criteria list in Table IV.
Discussion
The lag phase is an adjustment period during which bacterial cells modify themselves and their surroundings in order to take advantage of a new environment and initiate exponential growth. This implies that the smaller the degree of difference between the new and old environments, the smaller the durauon of the lag phase. The results of the current study on the effects of temperature history on the 5 ° C growth kmeucs support this in a general manner; however, they indicate that the response does not follow a continuum. Instead, there were critical pre-inoculation temperatures above which extended LPD's were observed (Table IIL This suggests that the phenomenon involves an underlying o n / o f f physiological process and not a gradual increase or decrease in a cellular component(s). The differential response between aerobic and anaerobic cultures (Table II) suggests that components involved in the microorganism's oxidative metabolite processes play a role in determining the impact of temperature history on subsequent low temperature growth. Identificauon of the factors underlying the altered sensitivity of anaerobic cultures to temperature history will require additional research. Demonstrauon that incubation temperature of the inoculum can influence the subsequent low temperature growth of L. rnonocytogenes in U H T milk (Fig. 1) and model products for cooked meats (Table IlI) indicates that temperature history may have a small but measurable impact on the growth of the microorganism in refrigerated foods. The results indicate that cells of L. monocytogenes adapted to a cooler environment (e.g., chill rooms, refrigerated ingredients, etc) would initiate growth in a refrigerated food somewhat sooner than cells coming from a warm
243 environment (e.g., food handlers). The differing response among canned dog food varieties (Table II) indicates further that there may be nutritional/formulation factors that influence the response of the microorganism to temperature history. The ingredient labels on these products were not detailed enough to allow comparison of the formulations of the varieties, and further investigations will be needed to identify the cause of the differential responses observed among the product varieties. The increase in /_, monocytogenes levels of less than a log cycle in irradiationsterilized, raw ground beef after more than a month of storage at 5 ° C, regardless of pre-inoculation temperatures of 19 and 37 ° C, does not support the hypothesis of Grau and Vanderline (1988) that the lack of growth of the pathogen in refrigerated meat reported by various investigators is due to the use of elevated temperatures for culturing inocula. The small differences noted between the cultures initially incubated at 19 and 37 ° C toward the end of the 5 ° C storage period were too small to be distinguished from the normal variation associated with plating microbial samples. It is possible that the levels of L. monocytogenes in the irradiation-sterilized meat would have continued to increase if the incubation period were extended further; however, the time frames are substantially different from those of Grau and Vanderlinde (1988) who reported that the levels of L. monocytogenes increased from 103 to 10 ~ c f u / c m 2 within 16 and 21 days of incubation at 5.3°C on the fat and lean surfaces of beef strips, respectively. Similarly, Gill and Reichel (1989) found that L. monocytogenes reached levels of 10 ? c f u / s a m p l e within one month on beef strips incubated at 5 ° C. It is worth noting that these time frames are similar to those observed with cooked meat products (i.e., canned dog food) in the current study. The use of irradiation-sterilized meat eliminated the possibility that the lack of growth in raw ground beef was due to a competing microflora. This suggests that the failure of L. monocytogenes to grow was due to either an inhibitory condition or component in raw ground beef that is eliminated upon cooking. It is possible that the differences between the current study and that of Grau and Vanderlinde (1988) and Gill and Reichel (1989) were due to different strains of L. monocytogenes being employed; however, this seems unlikely since a variety of strains have been used by other investigators (Kahn et al. 1972, 1973, 1975; Gouet et al., 1978; Buchanan et al., 1987: Johnson et al., 1988a,b; Shelef, 1989; Kaya and Schmidt, 1989) who have reported no growth in raw meat. An explanation worth investigating is that the observed differences among the reports were due to the physical form of the meat. The investigators reporting a lack of growth at refrigerated temperatures used raw comminuted meats whereas those reporting growth employed meat strips. This suggests that some attribute of the surface of meat favors the growth of the organism or inactivates the hypothesized inhibitory c o n d i t i o n / c o m p o n e n t . One possibility is that comminution and dispersal of the inoculum throughout the meat sample results in high CO 2 microenvironments. Gill and Reichel (1989) reported that, while L. monocytogenes grew at temperatures as low as 0 ° C on vacuumpackaged beef strips, the organism did not grow below 10 ° C when incubated in an elevated CO2 atmosphere.
244 In a d d i t i o n to e s t a b l i s h i n g that the lack of g r o w t h was n o t d u e to a c o m p e t i n g m i c r o f l o r a , the use of i r r a d i a t i o n - s t e r i l i z e d g r o u n d b e e f h e l p e d d e m o n s t r a t e t h a t the i n a c t i v a t i o n of L. m o n o c y t o g e n e s in u n t r e a t e d g r o u n d b e e f was a f u n c t i o n o f the microflora. T h e relatively r a p i d rate of inactivation, in c o n j u n c t i o n w i t h the n e u t r a l p H o f the m e a n s a m p l e s after 200 h of i n c u b a t i o n , suggest the p r e s e n c e o f c o m p e t i n g m i c r o o r g a n i s m s c a p a b l e of p r o d u c i n g an a n t i m i c r o b i a l agent(s) t h a t affects the p a t h o g e n . G o u e t et al. (1978) r e p o r t e d that the level of L. m o n o c y t o g e n e s d e c r e a s e d when it was c o - c u l t u r e d at 8 ° C w i t h L. p l a n t a r u m in sterile g r o u n d beef. A n u m b e r o f b a c t e r i o c i n s p r o d u c e d b y v a r i o u s b a c t e r i a l species have been r e p o r t e d to have activity a g a i n s t 1_, m o n o c y t o g e n e s ( H o o v e r et al., 1988; Pucci et al., 1988; B h u n i a et al., 1988; B e n k e r r o u m a n d Sandine, 1988; C a r m i n a t i et al., 1989; C h u n g et al., 1989; Ortel, 1989). T h e c u r r e n t i s o l a t i o n of a L. p l a n t a r u m strain with activity a g a i n s t L. m o n o c y t o g e n e s as a m a j o r c o m p o n e n t of the m i c r o f l o r a of g r o u n d b e e f suggests that the lactic a c i d b a c t e r i u m m a y have p l a y e d a role in the i n a c t i v a t i o n of the p a t h o g e n . This suggests f u r t h e r that u t i l i z a t i o n o f selected c u l t u r e s with a c t i v i t y a g a i n s t L m o n o c y t o g e n e s has p o t e n t i a l as a m e a n s o f c o n t r o l l i n g the p a t h o g e n in m e a n p r o d u c t s . I n v e s t i g a t i o n s to c h a r a c t e r i z e the a c t i v i t y o f the L p l a n t a r u m isolates a r e c u r r e n t l y u n d e r w a y .
References Benkerroum, N. and Santhne, W.E. (1988) Inhibitory action of nisin against Dstena monocvtogenes. J. Dairy Sci. 71, 3237-3245. Bhunia, A.K., Johnson, M.C. and Ray, B. (1988) Purification, characterization and antlrmcrobial spectrum of a bactenoan produced by Pediococcus ac~dilacnci. J. Appl. Bacteriol. 65, 261-268. Buchanan, R.L., Stahl, H.G. and Archer, D.A. (1987) Improved plating media for simplified, quantitative detection of l.~stena monocytogenes in foods. Food Microbiol. 4, 269-275. Buchanan. R.L., Stahl, H.G. and Whiting. R.C. (1989) Effects and interactions of temperature, pH, atmosphere, sodium chloride, and sodium nitrite on the growth of Listerm monocytogenes. J. Food Protect. 52, 844-851. Carminatx. D., Giraffa, G. and Bossi, M.G. (1989) Bacteriocin-like inhibitors of Streptococcus lactls against lastena monocytogenes. J. Food Protect. 52, 614-617. Chung. K.-T., Dickson. J.S. and Crouse, .I.D. (1989) Effects of nisin on growth of bacteria attached to meat. Appl. Envwon. Microbiol. 55, 1329-1333. Gdl, C.O. and Reichel, M.P. (1989) Growth of the cold-tolerant pathogens Yersima enterocohnca, Aeromonas hydrophda, and 1.zsterm monocytogenes on high-pH beef packaged under vacuum or carbon dioxide. Food Microbiol. 6, 223-240. Glass. K.A. and Doyle, M.P. (1989) Fate of Ltsteria monocytogenes in processed meat products dunng refngerated storage. Appl. Environ. Microbtol. 55, 1565-1569. Gouet, P., Labadle. J. and Serratore, C. (1978) Development of LJsterm monocytogenes m monoxenic and polyxeruc beef minces. Zbl. Bacteriol. Hyg. I. Abt. Orig. B. 166, 87-94. Gran, F.H. and Vanderhnde, P.B. (1988) Growth of Listeria monocytogenes on vacuum packaged beef. Proc. 34th Int. Cong. Meat Sci. Technol. 34, 518-519. Hoover. D.G., Walsh, P.M., Kolaetis, K.M. and Daly, M.M. (1988) A bactenoem produced by Pedmcoccus species assocmted with a 5.5-megadalton plasmid. J. Food Protect. 51.29-31. Johnson, J.L., Doyle, M.P., Cassens, R.G. and Sehoeni, J.L. (1988a) Fate of Ltsterm monocytogenes in ussues of experimentally infected cattle and in hard salami. Appl. Environ. Mierobiol. 54, 497-501.
245 Johnson, J.L.. Doyle, M.P. and Cassens, R.G. (1988b) Survaval of Ltsterza monocytogenes in ground beef. Int. J. Food Mlcroblol. 6. 243-247. Kaya, M. and Schmidt, U. (1989) Verhahen yon L,sterla monocytogenes im Hackflelsch bel Kutd- und Gefrierlagerung. Fleischwirtschaft 69, 617-620. Khan, M.A., Palmas, C.V., Seaman, A. and Woodbine. M. (1972) Survival versus growth of a facultatwe psychotroph. Acta Mierobiol. Aead. Sci. Hung. 19, 357-362. Khan. M.A., Palmas, C.V.. Seaman. A. and Woodbine, M. (1973) Survival versus growth of a facultative psychotroph: Meat and products of meat. Zbl. Baktenol. Hyg. Abt. Ong. B 157, 277-282. Khan, M.A., Newton, J.A., Seaman, A. and Woodbine, M. (1975) Survival of l_zsterJa monocytogenes inside and outside its host. In: M. Woodbine (Ed.), Problems m Llstenosls, Leicester Umverslty Press, Leicester, pp. 75-83. Ortel, S. (1889) Listenocins (monocins). Int. J. Food Microbiol. 8. 249-250. Puccl, M.J., Vedamuthu, E.K., Kunka, B.S. and Vandenbergh, P.A. (1988) Inhibition of Ltstena monocytogenes by using bacteriocin PA-1 produced by Ped~ococcus actdllacttct PAC 1.0. Appl. Environ. Microbiol. 54. 2349-2353. Shelef, L.A. (1989) Survival of l_dsterla monocytogenes in ground beef or liver during storage at 4 and 25 * C. J. Food Protect. 52, 379-383. Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G. (1986) Bergey's Manual of Systematic Bacteriology, Vol. 2, Williams and Wilkins, Baltimore, MD.
235
F O O D 00383
Effect of temperature history on the growth of Listeria monocytogenes Scott A at refrigeration temperatures * R.L. Buchanan and L.A. Klawitter Mtcrobtal Food Safety Research Umt, U.S. Department of Agrtculture, A RS Eastern Regional Research Center, Phdadelphla, PA, U.S.A. (Received 17 April 1990; accepted 19 November 1990)
The effect of pre-inoculation temperature on the subsequent growth of l.asterta monocytogenes Scott A at 5 ° C was examined in microbiological medium. U H T milk. canned dog food, and raw ground beef (untreated and irradlatton-sterilized). In microbiological medium, the duration of the lag phase was decreased when aerobic and anaerobic cultures were initially grown at < 28 and < 130C, respectively. Subsequent exponenual growth rates and m a x i m u m population densities of the 5 ° C cultures were not affected by temperature history. Differences in lag phase durations were also observed when L. monocytogenes imtially cultured at 19 and 3 7 0 C were grown at 5 0 C in U H T milk and some of the canned dog food varieties. Growth of L. monocytogenes was not observed m either untreated or irradiation-sterilized raw ground beef. While temperature history can affect the growth klneucs of L. monocytogenes at 5 * C. ~t did not account for the lack of growth in raw meat, suggesting that there ts an inhibitory condiuon or c o m p o n e n t in ground beef that is lost upon cooking. Key words: Meat; Milk; Lag phase; Competition
Introduction While Lssteria monocytogenes can grow in a variety of refrigerated foods including processed meat products, several teams of investigators have reported that the microorganism grows poorly, if at all, in refrigerated raw meat, particularly ground beef (Kahn et al. 1972, 1973, 1975; Gouet et al,, 1978; Buchanan et al., 1987; Johnson et al., 1988a,b; Glass and Doyle, 1989; Shelef, 1989; Kaya and Schmidt, 1989). However, Kahn et al. (1972, 1973) demonstrated that L. monocvtogenes grew on the surface of sterile lamb stored at 8°C. Gill and Reichel (1989) observed Correspondence address" R.L. Buchanan, Microbial Food Safety Research Umt, U.S. Department of Agriculture, ARS Eastern Regional Research Center, 600 East Mermaid Lane, Philadelphia, PA 19118. U,S.A. • Mention of brand or firm n a m e s does not constitute an endorsement by the U.S. Department of Agriculture over others of a stmilar nature not mentioned.
236
growth on vacuum-packed beef strips at temperatures as low as 0 ° C. but did not detect growth when the meat was stored at < 10 ° C in a CO2-enriched atmosphere. Grau and Vanderlinde (1988) reported that L. monocytogenes grew readily at 5.3°C on strips of beef, particularly on the adipose tissue. They hypothesized that other investigators did not observe growth at refrigeration temperatures due to the use of inocula grown at elevated temperatures (30-37 ° C), resulting in extremely extended lag phases when the microorganism was transfered to refrigerated meat. Grau and Vanderlinde (1988) used an inocuhim grown at 1 0 ° C and observed a minimal lag phase. The ob3ective of the current study was to test this hypothesis by examining the effect of temperature history on the growth kinetics of L. monocvtogenes incubated at 5 ° C in microbiological medium, U H T milk, sterile cooked meat products, raw ground beef and irradmtion-sterilized raw ground beef.
Materials and Methods
Microorgamsm Listeria monocytogenes Scott A was used throughout the study. Stock cultures were maintained in Brain Heart Infusion Broth (Difco) at 4 ° C , and transferred monthly. Inocula were grown in 50 ml Tryptose Phosphate Broth (TPB) (Difco) in 250-mi Erlenmeyer flasks incubated for 144 h at 5 ° C , 96 h at 1 0 ° C and 13°C, 48 h at 1 9 ° C , or 24 h at 28°C, 37°C, and 4 2 0 C on a rotary shaker (150 rpm). Mtcrobtologtcal medtum The microorganism was cultured aerobically and anaerobically in TPB according to the method of Buchanan et al. (1989). TPB was adjusted to p H 6.75 with concentrated HCI, and 50-ml portions were transferred to either 250-ml Erlenmeyer flasks (aerobic cultures) or 250-ml trypsinizing flasks (anaerobic cultures). Erlenmeyer flasks were capped with foam plugs, and trypsinizing flasks were sealed with screw caps and rubber septa. All flasks were sterilized by autoclaving and then prechilled to 5 ° C . Starter cultures were diluted and apportioned to achieve an inoculum of approximately 103 c f u / m l in each flask. Immediately after inoculation, the trypslnizing flasks were flushed for 10 rain with N 2 sterilized by filtration and sealed tightly to maintain anaerobiosis. All flasks were incubated at 5 o C on a rotary shaker (150 rpm). At least two replicate cultures were studied for each t e m p e r a t u r e / atmosphere combination. Periodically, 2.5-ml samples were removed from the Erlenmeyer and trypsimzing flasks using a pipette or hypodermic needle + syringe, respectively. Flasks were kept in an ice-water bath during sampling to ensure that the 5 ° C incubation temperature was maintained continuously. Samples were diluted appropriately with sterile 0.1% peptone water and plated in duplicate on Brain Heart Infusion Agar (BHIA) (Difco) plates using a Spiral Plater (Model D, Spiral Systems, Bethesda, MD). All plates were incubated for 24 h at 3 7 ° C and enumerated. It was checked that extended incubation did not increase count significantly. The pH of 0-h samples was measured using a p H meter (Model 601A, Orion) to ensure the initial pH had been sustained.
237 Milk
U H T milk was p u r c h a s e d f r o m a local s u p e r m a r k e t a n d p r e c h i l l e d to 5 ° C. T h e milk was a s e p t i c a l l y t r a n s f e r r e d to p r e s t e r i l i z e d 250-ml E r l e n m e y e r flasks a n d i n o c u l a t e d to achieve a n initial level o f 103 c f u / m l . A l l flasks were i n c u b a t e d at 5 ° C w i t h o u t agitation, with five r e p l i c a t e cultures b e i n g e x a m i n e d for each p r e - i n o c u l a tion t e m p e r a t u r e . D u r i n g s a m p l i n g , flasks were k e p t in an ice w a t e r b a t h a n d swirled to a t t a i n a h o m o g e n e o u s sample. S a m p l e s were r e m o v e d , d i l u t e d , p l a t e d . i n c u b a t e d , a n d c o u n t e d as d e s c r i b e d above. C a n n e d dog f o o d
' A l l - m e a t ' c a n n e d d o g f o o d was used as a m o d e l for c o o k e d m e a t p r o d u c t s . T h r e e varieties (all beef, beef a n d liver, a n d m e a t a n d gravy) in 4-oz cans were p u r c h a s e d at a local s u p e r m a r k e t a n d p r e c h i l l e d to 5 ° C a l o n g with sterile 500-ml beakers. T h e c o n t e n t s of the cans were t r a n s f e r r e d a s e p t i c a l l y to i n d i v i d u a l beaekrs. P r e - i n o c u l a g r o w n at 19 o r 37 ° C were d i l u t e d a n d used to i n o c u l a t e the s a m p l e s to a target level of 103 c f u / g . T h e final d i l u t i o n of the i n o c u l u m was m a d e using d i l u e n t (9.9 ml) c o n t a i n i n g a p p r o x i m a t e l y 0.1 ml of a green f o o d d y e ( D u r k e e , W a y n e , N J). T h e m e a t s a m p l e s were b l e n d e d until the d y e was u n i f o r m l y s p r e a d t h r o u g h o u t the sample. All s a m p l e s were i n c u b a t e d at 5 ° C. A t least three r e p l i c a t e s a m p l e s were s t u d i e d for each p r e - i n o c u l a t i o n t e m p e r a t u r e a n d p r o d u c t v a r i e t y c o m b i n a t i o n . Periodically, d u p l i c a t e 1.0-g p o r t i o n s o f each s a m p l e were t r a n s f e r r e d to screw c a p tubes c o n t a i n i n g 9.0 ml o f sterile 0.1% p e p t o n e water. A f t e r a g i t a t i n g to d i s p e r s e the m e a t in the diluent, a p p r o p r i a t e d i l u t i o n s were m a d e , a n d 0.2-ml p o r t i o n s were d i s t r i b u t e d evenly across the surface o f p r e p o u r e d B H I A plates using a sterile glass s p r e a d e r . A l l p l a t e s were i n c u b a t e d for 24 h at 37 ° C a n d e n u m e r a t e d . A g a i n . it was d e t e r m i n e d that e x t e n d e d i n c u b a t i o n d i d n o t increase c o u n t s significantly.
TABLE I Equations for Gompertz funcuon and derived growth kinetics values Gompertz's function: L( t ) = A -e Ce -eB''-~'
where: L(t) = Log count of bacteria at time (m hours) t (Log(cfu/ml)). A= Asymptouc log count of bacteria as ume decreases indefinitely (t.e., mmal level of bacteria) (Loglcfu/ml)). C = Asymptotm amount of growth that occurs as t increases indefimtely (t.e.. number of log cycles of growth) (Log(cfu/ml)) M = Time at whtch the absolute growth rate is maxtmal (h). B= Relauve growth rate at M (Log(cfu/ml))/h). Derived growth kinetics equations: Exponenual growth rate (EGR) = B C / e ((Log(cfu/ml))/h) Generauon time (GT) = (Log(2))e/BC (h) Lag phase duratson (LPD) = M - ( l / B ) (h) Maximum population density (MPD) = A + C (Log(cfu/ml))
238
Raw ground beef Ground beef was purchased at a local supermarket, and 100-g portions were transferred to sterile 500-ml beakers. The samples were then inoculated and incubated as described above for canned dog food. Five replicate samples were examined for each pre-inoculation temperature. Periodically, duplicate 1.0-g portions of each sample were transferred to screw cap tubes containing 9.0 ml of 0.1% sterile peptone water. After mixing thoroughly and diluting, 0.2 ml of each sample was spread evenly across prepoured plates of Modified Vogel Johnson Agar (MVJ') (Buchanan et al., 1987) containing 200 # g / m l sodium arsenite. This extremely selective medium took advantage of the high level of arsenite resistance inherent in strain Scott A. Preliminary studies indicated that this medium could quantitatively recover the organism from meat samples. This proved a very convenient system for following the growth of L. monocytogenes in raw meat since the test strain was essentially the only organism that grew on the MVJ + arsenite plates. All plates were incubated for 48-72 h at 37"C and enumerated. The pH of meat samples was measured at 216 h. Irradianon-sterilized, raw ground beef
Ground beef purchased locally was divided into 100-g portions, vacuum packed in individual low permeability plastic bags (All-Vak :~13, International Kenfield Distributing Co., Rosemont, IL), and then frozen and maintained at - 7 0 °C before and during irradiation. The frozen samples were irradiated with 48.5 kGy of gamma irradiation using a CS137 source. After radiation sterilization, the meat samples were equilibrated to 5°C. The samples were then transferred aseptically to sterile, prechilled beakers and inoculated, incubated, sampled, and counted as described above for the canned dog food samples. Growth curves
Where appropriate, growth curves were generated using the Gompertz function (Table I) (Buchanan et al., 1989). The four Gompertz parameters were used subsequently to calculate exponential growth rates (EGR), generation times (GT), lag phase durations (LPD), and maximum population densities (MPD).
Results L. monocytogenes was initially grown at various temperatures (5-42°C) to determine the effect that incubation temperature of the inoculum had on the subsequent lonetics of aerobic and anaerobic growth at 5°C in TPB (Table II). Temperature history of the inoculum had relatively tittle effect on either EGR, GT, or MPD, whereas more distinct differences in LPDs as a function of temperature history were observed in both the aerobic and anaerobic cultures. Aerobically, the LPD was increased 10-20 h when the inocutum was grown at > 3 7 ° C , as compared to the lower incubation temperatures. This effect appeared to be more pronounced with the anaerobic cultures, with an approximate 2-fold increase in
239 TABLE II Effect of initially culturing Li$lerm monocywgenes Scott A at various temperatures on its subsequent growth kineucs at 5 ° C in tryptose phosphate broth, a (For explanation of abbrevsations, see Table l) Premcubadon temperature t°C) n
Gompertz values
A
C
B
M
2.0 (0.0) 3.4 (0.6) 3.5
7.4 (0.2) 6.1 (0.6) 6.1
0.008 (0.001) 0.013 (0.006) 0.013
159.8 (4.6) 125.9 (17.9) 104.2
(0.0)
(0.3)
(0.001)
3.7 tO.l) 3.5 (o.1) 3.8
6.1 (0.2) 6.2 (o.1) 6.1
0.017 (0.003) 0.014 (o.ool) 0.013
(0.2)
(0.2)
3.7 (0.1)
6.3 (0.2) 7.5 (0.1) 6.9
LPD (h)
EGR (Log(cfu /ml)/h)
GT (h)
MPD (Log(cfu /ml))
36.8 (12.0) 39.3 (18.0) 29.1
0.023 (0.001) 0.029 (0.013) 0.030
13.6 (0.5) 11.7 (3.4) 10.1
9.5 (0.2) 9.5 (0.4) 9.6"
Aerobic cultures
5
2
10
7
13
2
19
5
28
2
37
6
42
2
(3.0)
(0.002)
(0.7)
(0.3)
95.9 (10.4) 108.2 (5.1) 128.3
(5.7)
36.7 (3.7) 36.7 (8.7) 50.6
0.038 (0.005) 0.032 (0.001) 0.030
8.0 (1.1) 9.5 (0.4) 10.5
9.7 (0.1) 9.7 (0.0) 9.8
(0.002)
O1.7)
(15.6)
(0.005)
(1.8)
(0.2)
0.013 (0.000)
125.9 (4.0)
49.6 (4.0)
0.031 (0.001)
10.0 (0.3)
10.0 (0.1)
0.010 (0.001) 0.009 (0.001)
132.2 (10.0) 135.4
27.4 (4.6) 27.8
0.027 (0.002) 0.023
11.4 (0.7) 12.9
(8.3) 24.7
(0.002) 0.034
(1.1) 9.0
9.6 (0.1) 9.5 (0.4)
(0.1) 49.1
(0.001) 0.036
(0.1) 8.5
(0.1) 9.6
(5.9)
(0.002)
(0.5)
(0.I)
0.033 (0.001) 0.032 (0.005) 0.034
9.2 (0.1) 9.6 (1.7) 8.8
9.9 (0.0) 9.7 (0.1) 9.9
(0.000)
t0.1)
t0.1)
Anaerobic cultures
5
2
10
5
2.0 (0.1) 2.6
13
2
(1.1) 3.6
(1.5) 5.9
19
2
(0.0) 3.7
28
2
37
6
42
2
0.015
(8.8) 89.4
(0.1) 5.9
(0.000) 0.016
(1.6) 109.4
(0.0)
(0.I)
(0.001)
3.7 (0.0) 3.7 (0.2) 3.6
6.2 (0.0) 6.0 (0.1) 6.2
0.014 (0.000) 0.015 (0.002) 0.015
(0.0)
t0.0)
(0.000)
(1.6) 121.4 (11.7) 121.0 (12.6) 112.3
51.7 (10.8) 50.0 (8.5) 45.4
(2.8)
(3.1)
9.6
Values are means of n replicatecultures. Values in parentheses are standard dev~auons.
LPD
being observed with the higher inoculum
cultivation temperature. The effect
w a s a l s o m o r e p r o n o u n c e d in t h a t i n c r e a s e d L P D ' s w e r e o b s e r v e d at p r e - i n o c u l a t i o n t e m p e r a t u r e s as l o w as 1 9 ° C in t h e a n a e r o b i c c u l t u r e s , c o m p a r e d to t h e 3 7 ° C c u t o f f in t h e a e r o b i c c u l t u r e s . A s i m i l a r p a t t e r n was o b s e r v e d w h e n U H T m i l k was i n o c u l a t e d w i t h L. monocytogenes cells g r o w n at 19 o r 3 7 ° C (Fig. 1). A n a p p r o x i m a t e 3 - f o l d i n c r e a s e in t h e
240
40
0
GT
LPD
MPD
(hr)
(hr)
(Log[cfu/ml])
re ,,, o. ~-
F~g. 1. Effect of initially cultunng L. monoqywgenes Scott A aerobically at 19 and 3 7 ° C on its subsequent growth at 5 ° C m UHT-pasteurized milk. G T ffi generation ume, L P D = lag phase duration, M P D ffi m a x i m u m population density. Average of five determinations.
mean L P D (16.2 + 12.1 vs. 51.9 _+ 18.7 h) was observed with the higher pre-inoculation temperature, whereas the mean GTs (19.3 ± 4.0 vs. 17.7 + 3.3 h) and MPDs (7.4 ± 0.1 vs. 7.6 ± 0.2 log(cfu/ml)) of the cultures were similar. Commercially available 'all meat' dog food was used as a model for cooked meat products. The 4-oz cans of this type of product proved to be a convenient test
T A B L E 111 Effect of initially culturing l~ster~a monocytogenes Scott A aerobically at 19 vs. 37 ° C on its subsequent growth kineucs at 5 ° C m canned dog food " (For explanation of abbrevmuons, see Table I) Product vanety b
1
Premcubauon temperature (°C)
Gompertz values
A
C
B
M
19
1.6 (0.4) 2.6 (0.1)
6.7 (0.3) 5.5 (0.2)
0.0063 (0.0014) 0.0059 (0.0013)
260.9 (63.5) 249.6 (22.5)
1.7 (0.2) 2.7 (0.1)
6.8 (0.2) 5.6 (0.1)
0.0068 (0.0007) 0.0108 (0.0004)
1.7 (0.2) 2.7 (0.1)
6.5 (0.2) 6.3 (0.2)
0.0051 (0.0007) 0.0054 (0.0003)
37 2
19 37
3
19 37
LPD (h)
EGR (Log(cfu /roll/h)
GT (h)
MPD (Log(cfu /mill
61.2 (5.8) 54.3 (7.7)
0.015 (0.004) 0.012 (0.003)
25.0 (8.0) 28.9 (4.4)
8.3 (0.3) 8.2 (0.2)
207.3 (19.3) 166.9 (9.2)
52.0 (2.3) 73.7 (10.7)
0.017 (0.002) 0.022 (0.001)
18 7 (2 0l 13.6 (0.4)
8.5 (0.1) 8.3 (0.1)
254.3 (10.2) 274.2 (22.6)
46.0 (16.9) 88.8 (31.4)
0.012 (0.002) 0.012 (0.001)
26.4 (2.8) 24.1 (0.5)
8.2 (0.3) 8.9 (0.3)
" Values are m e a n s of at least three independent determinations. Values m parentheses are standard devlat,ons b 1 = ' a l l beef'. 2 • ' b e e f and hver': 3 = ' m e a t and gravy'.
241 10
IJl --
8 7
.j
">"
II:~ ~
19 37 19 37
C/sterile C/sterile C/non-sterile , C/non-sterile ~U
6
f
~ Z
I=
0
4
_o
3
m
"5
2
L
0
260
4oo
600-
#60
5 C Incubation Time (hr) Fig. 2. Effect of iniually culturing 1_ monoo, togenes Scott A aerobically at 19 and 3 7 ° C on ~ts subsequent survwal at 50C in raw ('non-sterile') ground beef and gamma irradiated ('sterile') raw ground beef. Average of three determinations.
system,
providing
product.
Three
pre-inoculation
an
adequately
different
product
temperatures
sterilized, varieties
o f 19 a n d 3 7 ° C
reasonably were
homogeneous
evaluated
(Table
III).
in
meat-based
conjunction
The MPDs
with
were roughly
TABLE IV Taxonormc characterisucs of zsolates from ground beef that possessed activ]ty against l_d$1eraa monoc_wogenes Scott A Shape Gram staan Motility Catalase Growth at 12 o C Tellunte reductton H2S production
Medium length rods +
Carbohydrate
3 Days
Glucose Sucrose Maltose Mannose Lactose Manmtol Xylose Ribose Rhamnose Arabinose Melibiose
+
7 Days
28°C
120C
280C
12°C
+ + + + + + ~-4± ± +
+ + + + +
+ + + + + +
-
-l-
+ -
+ +
+ + + + + + -
242
equivalent regardless of product type or pre-inoculation temperature. E G R / G T was unaffected by pre-inoculation temperatures in two of the product varieties (No. 1 and No. 3), but an approximate 30% decrease in G T was observed with the 3 7 ° C / v a r i e t y No. 2 cultures. The effect of pre-inoculation temperature on LPD was also product-dependent. An approximate 50% increase in LPD was observed with the higher pre-inoculation temperature in variety No. 2, and a 2-fold increase occurred with variety No. 3. However, the LPD of variety No. 1 was unaffected b 3 pre-inoculation temperature. Raw ground beef sterilized by gamma irradiation did not support significant growth at 5 ° C of L. monocytogenes regardless of pre-inoculation temperature (Fig. 2); however, the microorganism did survive for extended periods. When unsterilized raw ground beef was inoculated with cells initially incubated at 19 and 37 o C, the microorganism died off at a relatively rapid rate. The pH of the meat at 216 h, the time when inactivation was complete was 7.2-7.4. Representative members of the meat's microflora were isolated and screened for activity against L. monocytogenes using BHIA plates seeded with a lawn of L. monocytogenes Scott A. Two isolates produced distinct zones of inhibition. Both isolates had similar taxonomic characteristics and were tentatively classified (Sneath et al., 1986) as Lactobacillus plantarurn, based on the criteria list in Table IV.
Discussion
The lag phase is an adjustment period during which bacterial cells modify themselves and their surroundings in order to take advantage of a new environment and initiate exponential growth. This implies that the smaller the degree of difference between the new and old environments, the smaller the durauon of the lag phase. The results of the current study on the effects of temperature history on the 5 ° C growth kmeucs support this in a general manner; however, they indicate that the response does not follow a continuum. Instead, there were critical pre-inoculation temperatures above which extended LPD's were observed (Table IIL This suggests that the phenomenon involves an underlying o n / o f f physiological process and not a gradual increase or decrease in a cellular component(s). The differential response between aerobic and anaerobic cultures (Table II) suggests that components involved in the microorganism's oxidative metabolite processes play a role in determining the impact of temperature history on subsequent low temperature growth. Identificauon of the factors underlying the altered sensitivity of anaerobic cultures to temperature history will require additional research. Demonstrauon that incubation temperature of the inoculum can influence the subsequent low temperature growth of L. rnonocytogenes in U H T milk (Fig. 1) and model products for cooked meats (Table IlI) indicates that temperature history may have a small but measurable impact on the growth of the microorganism in refrigerated foods. The results indicate that cells of L. monocytogenes adapted to a cooler environment (e.g., chill rooms, refrigerated ingredients, etc) would initiate growth in a refrigerated food somewhat sooner than cells coming from a warm
243 environment (e.g., food handlers). The differing response among canned dog food varieties (Table II) indicates further that there may be nutritional/formulation factors that influence the response of the microorganism to temperature history. The ingredient labels on these products were not detailed enough to allow comparison of the formulations of the varieties, and further investigations will be needed to identify the cause of the differential responses observed among the product varieties. The increase in /_, monocytogenes levels of less than a log cycle in irradiationsterilized, raw ground beef after more than a month of storage at 5 ° C, regardless of pre-inoculation temperatures of 19 and 37 ° C, does not support the hypothesis of Grau and Vanderline (1988) that the lack of growth of the pathogen in refrigerated meat reported by various investigators is due to the use of elevated temperatures for culturing inocula. The small differences noted between the cultures initially incubated at 19 and 37 ° C toward the end of the 5 ° C storage period were too small to be distinguished from the normal variation associated with plating microbial samples. It is possible that the levels of L. monocytogenes in the irradiation-sterilized meat would have continued to increase if the incubation period were extended further; however, the time frames are substantially different from those of Grau and Vanderlinde (1988) who reported that the levels of L. monocytogenes increased from 103 to 10 ~ c f u / c m 2 within 16 and 21 days of incubation at 5.3°C on the fat and lean surfaces of beef strips, respectively. Similarly, Gill and Reichel (1989) found that L. monocytogenes reached levels of 10 ? c f u / s a m p l e within one month on beef strips incubated at 5 ° C. It is worth noting that these time frames are similar to those observed with cooked meat products (i.e., canned dog food) in the current study. The use of irradiation-sterilized meat eliminated the possibility that the lack of growth in raw ground beef was due to a competing microflora. This suggests that the failure of L. monocytogenes to grow was due to either an inhibitory condition or component in raw ground beef that is eliminated upon cooking. It is possible that the differences between the current study and that of Grau and Vanderlinde (1988) and Gill and Reichel (1989) were due to different strains of L. monocytogenes being employed; however, this seems unlikely since a variety of strains have been used by other investigators (Kahn et al. 1972, 1973, 1975; Gouet et al., 1978; Buchanan et al., 1987: Johnson et al., 1988a,b; Shelef, 1989; Kaya and Schmidt, 1989) who have reported no growth in raw meat. An explanation worth investigating is that the observed differences among the reports were due to the physical form of the meat. The investigators reporting a lack of growth at refrigerated temperatures used raw comminuted meats whereas those reporting growth employed meat strips. This suggests that some attribute of the surface of meat favors the growth of the organism or inactivates the hypothesized inhibitory c o n d i t i o n / c o m p o n e n t . One possibility is that comminution and dispersal of the inoculum throughout the meat sample results in high CO 2 microenvironments. Gill and Reichel (1989) reported that, while L. monocytogenes grew at temperatures as low as 0 ° C on vacuumpackaged beef strips, the organism did not grow below 10 ° C when incubated in an elevated CO2 atmosphere.
244 In a d d i t i o n to e s t a b l i s h i n g that the lack of g r o w t h was n o t d u e to a c o m p e t i n g m i c r o f l o r a , the use of i r r a d i a t i o n - s t e r i l i z e d g r o u n d b e e f h e l p e d d e m o n s t r a t e t h a t the i n a c t i v a t i o n of L. m o n o c y t o g e n e s in u n t r e a t e d g r o u n d b e e f was a f u n c t i o n o f the microflora. T h e relatively r a p i d rate of inactivation, in c o n j u n c t i o n w i t h the n e u t r a l p H o f the m e a n s a m p l e s after 200 h of i n c u b a t i o n , suggest the p r e s e n c e o f c o m p e t i n g m i c r o o r g a n i s m s c a p a b l e of p r o d u c i n g an a n t i m i c r o b i a l agent(s) t h a t affects the p a t h o g e n . G o u e t et al. (1978) r e p o r t e d that the level of L. m o n o c y t o g e n e s d e c r e a s e d when it was c o - c u l t u r e d at 8 ° C w i t h L. p l a n t a r u m in sterile g r o u n d beef. A n u m b e r o f b a c t e r i o c i n s p r o d u c e d b y v a r i o u s b a c t e r i a l species have been r e p o r t e d to have activity a g a i n s t 1_, m o n o c y t o g e n e s ( H o o v e r et al., 1988; Pucci et al., 1988; B h u n i a et al., 1988; B e n k e r r o u m a n d Sandine, 1988; C a r m i n a t i et al., 1989; C h u n g et al., 1989; Ortel, 1989). T h e c u r r e n t i s o l a t i o n of a L. p l a n t a r u m strain with activity a g a i n s t L. m o n o c y t o g e n e s as a m a j o r c o m p o n e n t of the m i c r o f l o r a of g r o u n d b e e f suggests that the lactic a c i d b a c t e r i u m m a y have p l a y e d a role in the i n a c t i v a t i o n of the p a t h o g e n . This suggests f u r t h e r that u t i l i z a t i o n o f selected c u l t u r e s with a c t i v i t y a g a i n s t L m o n o c y t o g e n e s has p o t e n t i a l as a m e a n s o f c o n t r o l l i n g the p a t h o g e n in m e a n p r o d u c t s . I n v e s t i g a t i o n s to c h a r a c t e r i z e the a c t i v i t y o f the L p l a n t a r u m isolates a r e c u r r e n t l y u n d e r w a y .
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