FOOD

00474

Behavior of Listeria monocytogenes during processing and storage of experimentally contaminated hot-smoked trout

iiot-smoked fish like smoked trout is quite frequently contaminated with I_istc*rici f,lorn~~~t)~~*fle. in or&r to cstimnte the potential hcitlIh hitrid for the consumer ding such products. the behavior of L.. ,,to,tc~yrc~~~‘~~l’.s was studied during processing itnd st

sttrf;tcc inoculntcd

with

1.. trto~~~c~:\‘ro~:c’,rev. mnrittated.

65°C during 20 mitt), and stored nt 4 ;rnJ 8- i(!“C. respectivriy. for up to

20 duys. At diffcrcnt times during proaxing and storage. samples wc’rc taken and. by means of a MPN-method, qunntitativeiy analyscd for 1.. ,,lorrc~)‘fo~~~‘~tc:v. The initial Lbfcriu lev& in the trout were IO” MPN/g.

Until

smoking,

the concentrations

remained

ahout the same. After

the hot-smoking

process and during storage. however. f.. /,rt,nc~:vro~~~‘~tl’s could no longer hr: detected. in trials 3

and

4,

the trout were inoculated nflcr hot-smoking

at

;I

final ccmcenlrnlion of 4.5X IO

MPN/g nnd 3.1 x IO’ MPN/g. respectively. During htoragc at CC. neither itn increase nor a decrease of L. ar~~~~~~~r~~~~‘/t~~s was ohserved. At X- 10°C. however, ;I significant increase up to IO’ MPN/p occurred. By hot-smoking. climinatcd.

low

level contaminntions

Nevcrthelcss.

during storage al rcfrigeralion product temperature

of

it is of great importance

ahusc

lcmptxature~

raw fish with

L.

~0~~oc~r0~~~1~~~will

easily he

to prevent p)btpro$ f.ssing contamination.

hccause

growth of 1.. ,,rorrc~‘!‘ro~~~‘~r~~~ is possible. Therefore,

occurs. there might hr an incrcxd

if

risk for the consumer, when storing

such fish over :I long period of time. Key words: fxtwicc;

Smoked fish: Smoked trout: t icat resistance

Introduction Listericl tttotwcytogmes is a human pathogen, which is widely distributed in the environment. After four outbreaks of listcriosis, food-borne transmission has been recognized as the nxdjor source of infection. The implicated foods in these

T. Jemmi, Microbiology

Section. Federal

Veterinary

Office. CH-3097

epidemics wcrc coleslaw (Schlcch ct al., 1983). pastcurizcd milk (Fleming ct al., 19851, and soft cheese (James et al.. 1985; Bille and Glauscr, 1988). At present, thcrc arc no reports on outbreaks due to consumption of fish or seafood. Some sporadic casts of listcriosis, however, have been associated with the consumption of contaminated fish or shellfish (Lcnnon et al., 1984; Facinclli ct al., 1989). L. tnottocytogettes is frequently isolated from fish and seafood. especially smoked fish and cooked shrimp (Wcagant ct al., 1988; Caserio et al., 1989; Jcmmi, lYYOa, b). Jcmmi (IYYOb) showed that cold-smoked fish (e.g. salmon) with a contamination rate of 13.6% is more frcqucntly contaminated than hot-smoked fish (c.g., trout) with a corresponding rate of 8.9%. Guycr and Jcmmi (199 I) cxamincd the behavior of L. tttonocytogette.~ during the manufacture and storage of cold-smoked salmon. In two out of three trials, a significant increase in the lcvcls of Listeriu occurred during storage at 4 or IO”C, which implied an increasing risk of infection for the consumer by storing cold-smoked fish for a long period of time. In order to cstimatc the potential health hazard for the consumer eating contaminated hot-smoked fish, the behavior of L. tnottocytogettes W:IS studied during smoking and storage of cxpcrimcntally contaminated trout.

Material and Methods Freshly slaughtcrcd rainbow trout ((lrtcor/tytt~hlr.s tttykiss). weighing about 4(H) g each, were obtained from a Swiss trout farm. The guts and gills wcrc rcmovcd, the gut cavity cleaned out, and the trout washed. Four trials were conducted, with six trout being used per tiial. Ittoculutn preparation For all experiments, L. mottocytogettc scrotype l/2 b was used; in trials 1 and 3 a wild-type strain isolated from the intcstincs of a trout was used, while in trials 2 and 4 a serological rcfcrencc strain (SLCC 2755 from the Special Li.steriu Culture Collection, Wiirzburg, Germany) was used. lnocula were prepared by growing cultures in trypticasc soy broth (Becton-Dickinson, Basel, Switzerland) for IX h at 37°C. Enumeration was done by means of spread plating of serial dilutions t? : IO) on PALCAM agar (van Nctten ct al., 19891, incubated at 30°C for 48 h under microaerobic conditions. The targctcd levels in the inoculum broths were lOx L. tttotto~yro~~~tte.s/ml in trials I and 2, and 10” L. tnottocyrogette.s/mI in trials 3 and 4. Ittoculatiott atd preparutiott of .snwked trout In trials 1 and 2, the trout were transferred to the respective inoculum broth for 5 min, removed, and left to dry for 60 min at room temperature. Afterwards, they were kept for 12 h in a salt marinade containing 10% NaCl and 0.7% spices (pH 6.41, and smoked in a kiln as is usually done in industry: 30 min at 60°C (drying phase), then the kiln was heated up to 110°C (cooking phase). The fish was held in

341

this phase for 20 min after an internal temperature of W’C was rcachcd. Finaily the fish was smoked for 45 min at WC (smoking phase). The smoked trout were cooled.

first

to room

temperature

and then

packaged, and stored under refrigeration

in a chillroom

to 4°C.

vacuum

(one portion at 4°C the other at K-WC)

for up to 20 days. In trials 3 and 4 the trout wcrc cured and smoked as dcscribcd above, and then inoculated with the rcspcctivc inozulum broth. They wcrc Icft to dry for 60 min at room tcmpcraturc, vacuum packaged, and stored as described above.

In trials I and 2, trouts wcrc sampled in duplicate bcforc inoculation, after inoculation, after curing, after smoking. after cooling, and after 5, 10 and 2(1 days of rcfrigeratcd storage at 4 or 8- W’C. In trials 3 and 4. duplicate samples were taken after inoruration. and after 5, 10 and 20 days of rcfrigcratcd storage at 4 or 8- 1WC.

At each sampling, It1 g wcrc aseptically taken and, by means of the three-tube most probable number (MPN) technique, quantitatively analysed for L. motwcytogenes. The samples were maccratcd for 2 min in a stomacher with 90 ml of University of Vermont IUVMJ broth I IMcClain and Lee. 198X) and incubated for 24 h at 37°C (McClain and Lee, IYXS). Portions (0.1 ml) were spread-plated onto PALCAM agar (van Netten et al., 19X9), and then incubated at 30°C for 48 h conditions. Presumptive Lismin colonies were confirmed by means of Gram staining, cat&se test and beta-hemolysis (Guyer and Jemmi, 1991). From the MPN-values of the duplicate samples the arithmetic means were under microaerobic

calculated, the 95% confidcncc limits being taken from the Swiss Food Manual A significant increase or decrease of the Listmia levels was reached, when the confidence intcrvnls of the MPN-values did not intcrscct. Qualitative assays were also done, but only in trials 1 and 2 after 20 days of storage. The samples were mixed with 40 ml of UVM broth I and incubated for 24 h at WC, 0.1 ml of this broth was transfcrrcd into 10 ml of UVM broth II and incubated again for 24 h at 30°C (McClain and Let, IYSX). Then, 0.1 ml of UVM broth II was streaked onto PALCAM-agar tvan Nctten ct al., I9891 and Oxford-agar (Curtis et al., 19891. Both plates were incubated for 48 h at 30% Oxford in air and PALCAM microacrobically.

( 1988).

Aerobic plate cowzt Aerobic plate count (APC) was only performed in trials 1 and 2. Samples (IO.0 g) were macerated for 2 min in a stomacher with 90 ml of physiological N&l solution and diluted decimally. By means of the drop-plate method IBaumgart, 19901, 0.1 ml was placed on the surface of plate count agar (Merck, Darmstadt, Germany). The plates were incubated 2 days at 30°C and the coIonies counted.

pH and wateracrkity (a,,, ) demn~it~atiotu pH and aW determinations were only performed

in trials I and 2; pH values with an electronic pH meter (Type 691; Metrohm, Herisau, Switzerland) and a, values with a hygroscope (Type DT, Rotronic, Ziirich, Switzerland).

Results The behavior of L. nzonocyrogenes in smoked trout during trial 1 is shown in Table 1. Until smoking the inoculated concentrations of L. nzonocytogene.s remained about the same. After the hot-smoking process and during storage, however, this bacterium could no longer be detected. In fact, a 5.9 log,,, MPN/g reduction could be observed. As the results of trial 2 were very similar, these data are not shown. APCs are also shown in Table 1. Similar to L. nzonocyfognes, APCs decreased after smoking, and were not detected during storage except for the 20-day storage at 8- 10°C. The initial inoculum in trial 3 was 4.5 X IO’ MPN/g (Fig. 1). During storage at 4°C. neither a significant increase nor a decrease of L. monocyrogenes was observed. At 8- lO”C, however, the numbers of Listeria increased substantially; i.e., after 5 days, significant growth was observed, and at the end of storage (after 20 days) levels of about 10’ MPN/g were reached. In trial 4, the initial inoculum was 3.1 x 10’ MPN/g (Fig. 2). No significant change in the concentration of L. nronocymgenes was observed during storage at 4°C. As in trial 3, during storage at &lO”C, significant multiplication occurred. However, this increase proceeded somewhat slower than in trial 3; only after 10

TABLE

I

BehGsr

of L. r,~c,rloc:vro)ic(‘nev I/2

storage oi experimentidly Sampling; period

b (wild-type

contaminated

strain). and uerobic plate cmmt.

during processing and

smoked trout (trio1 I)

L. 1?101~OC1’fO~:(‘ll~:r

Aerobic plate courn

(MPN/gj

(CFU/g)

Inoculated

I .s x 10”

4.5 x IO5

After curing After smoking Storage 5 days at 4°C

x.0x IO’

4.9x IO5 5.0x I#

-

-

6.0X IO?

Raw fish

:’ days at H- 10°C I) Concentration L. moncrytogenes during storuge ilt X-IWC: gray area. 9.55 confidcncc limits of the MPN-dues.

MPNkt

maculated

5 days

10 days

20 days

stomge

storage

storage

Fig. 2. Growth of L. ~~wQw_J~~~~~s SLCC 2755 during storage of experimentally contaminated smoked trout (trial 4). inoculated L. monocytogeneh concentration; Concentration L. monocytogenes

(0)

(0)

during storage at 4°C; (0) Concentration

L. monocytogenes during storage at H-WC;

confidence limits of the MPN-values.

gray area, 95%

days the counts were significantly greater than those after cooling. After 20 days, concentrations reached 2.5 x 10' MPN/g. The pH values varied bctwccn 6.3 and 6.9 (data not shown): the uw values were in the range of 0.92-0.95, cxccpt one sample in trial I (after smoking) with an (I,,, of 0.91 (data not shown).

Discussion Until smoking, the lcvcls of L. tmmocytogcvm on artificially inoculated smoked trout remained about the same. In this regard, it has previously been shown that this bacterium can easily survive a salt concentration of 10% or grcatcr (Shahamat ct al., 1980). After the hot-smoking process, however, both strains of L. monocytogenes could no longer bc dctccted. The two parameters. heat and smoke, led to the elimination of this pathogen. During smoking, a tcmpcraturc of 110°C for 45 min was applied to obtain an internal tcmpcraturc of 65°C. Compared with other non-sporulating bacteria, Listeriu arc rclativcly heat-resistant, e.g., approximately four times greater than Sultttotd/~tspp. (Lcistncr ct al., 1989; Mackcy and Bratchcll, 1989). The latter authors stated that cooking food to an internal tcmperaturc of 70°C for 2 min ensures destruction of L. mottocytogettes. Lcistner ct al. (1989) recommended, for the manufacture of scalded sausage, a minimal internal tcmpcraturc of 72°C. In addition, some smoke components such as formaldehyde or phenols have antibacterial activity (Beltran et al., 1980; Mcssina ct al., 1988). In our cxperimcnts, hot-smoking to an internal tcmpcraturc of 65°C during 20 min led to a A log,,, reduction in numbers of L. ttrottocyto~ette.s.Also, during storage, L. muttocytogetre.scould not be detected by any method. The APCs wcrc almost always below the detection limit. Only after 20 days of storage at 8-10°C a lcvcl of 100 APC/g was obscrvcd. aw values below 0.92 arc prohibitive for growth of L. tttottocytot~ettes (Pctran and Zottola, 1989). In our study only one value was beneath this limit. Ncvcrthcless, this paramctcr can have an inhibitory cffcct on the growth of L. trtotwytogenes in smoked trout. pH values, howcvcr, had no significant influence on the behavior of L. tttotrocyto~~ettes. Kaw fish can be contaminated with L. mottucytogettes, but usually only in low numbers (Guycr and Jcmmi, 1990; Kcusch, 1991). Following the Good-Manufacturing-Practice-(GMP)-guidelines as described in the code of practice for smoked fish (Codcx Alimcntarius, 1979), this low lcvcl contamination can easily bc climinatcd. The reason why L. mottocytogettes is frequently found in hot-smoked fish (Jcmmi, 199Ob), must therefore be because of recontamination or postprocessing contamination at the filleting or packaging stage. The amounts of L. trwtwcytogettes inoculated onto the fish in trials 3 and 4 corresponded with those found in naturally contaminated hot-smoked trout (Keusch, 1991). During storage at 4”C, no significant growth of L. motrocytogetres was observed (Figs. 1 and 2). At 8-lO”C, however, significant multiplication of L. mottocytogetzes

occurred after IO days. The wild-type strain (Fig. I ) grew faster than L. ~~~tocytc~2755 (Fig. 2). It is probahlc that this wild-type strain was better adapted to grow on trout than the reference strain. After 20 days, both strains had grown to concentrations of > 10’ MPN/g.

gems SLCC

Guycr and Jcmmi (1001) did similar

inoculation studies with cold-smoked salmon,

and also obscrvcd that I,. r,lc~t~rtoc?,tu~~~ll’.s could multiply to lcvcls reaching IO’ MPN/g after 30 days of storage iIt 4°C as well as at 10°C. Unlike cold-smoked fish. hot-smoked fish has no compctitivc background florit, iis was observed by the APCs obtained in trials 1 and 2. Thus, in hot-smoked fish postprocessing contamination will thcrcforc more easily Icad to the multiplication of L. monocytopwcs during storage. The growth rate of Listerirr will depend on the storage temperaturc. At X- 10°C. its found in most household rcfrigcrators in Switzerland (Schmidt-Lorcnz. IWO). lcvcls of up to 105-IO’ L. nwrwcytogms/g arc possible within a few days. Thcsc ICVCISmny hc significant and can hc compared with those found in Vacherin hlont d’Or (it soft chccsc implicated in the Swiss listcriosis outbreak) (Billc and Glituscr, IWX). For these rcitsons it is ,)f great importance to prevent postprocessing contamination. As multiplication of I,. ,,forroc’?4ot~~s during storage at refrigeration temperaturcs is possihlc. there might bc an increasing risk of infection for the consumer by storing such fish over ii long period of time.

Conclusions An internal tcmperaturc of 65°C during 20 min in processing hot-smoked trout leads to iI 6 log,,, reduction of I.. tttottocyto~c’ttcs. Low level contaminations of raw fish will thcrcfore easily be eliminated. Positive findings in finished products are due to postprocessing contamination. This can be prevented by means of appropriate application of hygiene and technology, ix.. HACCP. Because a significant incrensc of L. ttzotwcytogcw.s during storage of hot-smoked trout at 8-10°C was obscrvcd. thcrc might be an increasing health hazard for the consumer by storing such fish over iI long period of time. It is thcrcforc of great importance to store hot-smoked fish at 4°C or lower. The refrigeration chain should be maintainLrl during the whole manufacturing and storage process until consumption.

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