16(12):1939.

1968,Appl. Microbiol. J M Goepfert and R A Biggie milk chocolate.senftenberg 775W inSalmonella typhimurium andSalmonella Heat resistance of

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APPUED MICROBIOLOGY, Dec. 1968, p. 1939-1940 Vol. 16, No. 12Copyright © 1968 American Society for Microbiology Printed in U.S.A.

Heat Resistance of Salmonella typhimurium andSalmonella senftenberg 775W in Milk Chocolate,

J. M. GOEPFERT AND R. A. BIGGIEFood Research Institute, andDepartment ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication 4 October 1968

The problem of Salmonella contamination indried foods has underscored our lack of knowl-edge concerning the heat resistance of entericmicroorganisms in dry and semidry environments.Confectionery manufacturing is one of the foodindustries concerned with dry-heat destruction ofSalmonella. The discovery of these organisms inseveral chocolate-containing products in therecent past has raised the question of whetherthey can be killed by heating the molten chocolateprior to casting. The results of experiments per-formed to determine the heat resistance of Salmo-nella in miLk and dark chocolate are presented inthis paper.

S. typhimurium and S. senftenberg 775W wereinoculated into milk chocolate in the followingmanner. The cells resulting from 24-hr growth in600 ml of Trypticase Soy Broth (BBL) incubatedstatically at 37 C were harvested centrifugally,resuspended in 20 ml of sterile skimmed milk, andlyophilized. Commercially formulated milkchocolate was dispensed in Sorvall Omnimixercups in 100-g quantities and warmed to the testtemperature in an oil bath. The lyophilized cellswere added to the molten chocolate and blendedfor 1 min at top speed on the Omnimixer. Thecups were returned to the oil bath and immersedso that the level of the chocolate was /4 (1.9 cm)inch below the surface of the oil. Agitation of theinoculated chocolate throughout the remainderof the experiment was accomplished by a me-chanical stirrer.

Periodically, 1-ml samples were taken by usingwide-bore pipettes and a vacuum-pressure pump.Dilutions were made in sterile 30% sucrose, andviable salmonellae were enumerated by the three-tube most probable number technique. Nutrientbroth was used as the recovery medium, andtubes showing turbidity after 48 hr at 37 C werestreaked on MacConkey Agar (Difco) and incu-bated at 37 C. Isolates were confirmed bio-chemically and serologically.The D values obtained in replicate trials at

70, 80, and 90 C are presented in Table 1. Theaverages of the D values are plotted against

I Publshed with the approval of the Director of theWisconsin Agricultural Experiment Station.

temperature in Fig. 1. The ZD values of 32.4 and34.2 are almost twice those generally obtained forspores exposed to moist heat. Ng (1) determinedthat the moist heat resistance (D values) of S.senftenburg 775W and S. typhimurium was 31 min

3

S TYPHIMURIUMZD= 34.2

z

zw12-

S. SENFTENBERG 775o ~~ZD= 32.4

LI

FIG. 1. Plot of log D value vs. temperature for S.senftenberg 775W and S. typhimurium heated in milkchocolate. Points represent averages of the times givenin Table 1.

and 1.2 min, respectively, at 57 C. Although adirect comparison of D values at 57 C is notpossible, it can be observed (Table 1) that anenormous increase in heat resistance occurs whensalmonellae are exposed to dry heat. These datastress the inadvisability of extrapolating heat-

1939

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APPL. MICROBIOL.

TABLE 1. D values of S. senftenberg 775W and S.typhimurium (the results of three trials

at each temperature)

D values (min)

Temp (C)S. sewfelberg S. typhimurium

775W

70 360 720480 678480 1050

80 144 22296 222108 222

90 36 7830 7242

resistance values obtained under one set of condi-tions to any other conditions.

S. typhimurium was more resistant to dryheating than S. senftenberg 775W. Ng (1) reportedthat the latter serotype was approximately 25times as resistant to moist heat as S. typhimurium,a value confirmed in our laboratory. Since weexamined only two serotypes, it is difficult topredict if strains will be uncovered that are moreresistant than S. typhimurium. These findingswarn against establishing time-temperature rela-tionships for dry-heat processes based on destruc-tion of S. senftenberg 775W.

The curves obtained in this study charac-teristically showed a rapid decline in numbers ofsurvivors (3-log cycles) during the first fewminutes of heating, followed by a slower rate ofdecrease thereafter. The rapid initial loss might beattributed to the death of cells injured during thelyophilization and inoculation processes. Thisproblem was obviated by taking the first sampleat least 30 min after the addition of the cells to thechocolate. Occasionally, curves were obtainedthat showed a "tail," but attempts to isolate moreresistant organisms from this portion of the curvewere unsuccessful. More often than not, astraight-line (logarithmic) relationship was ob-tained over the entire range of the test after theinitial delay in sampling.The heat resistance of Salmonella was essen-

tially the same in either milk or dark chocolate.The degree of heating required to kill Salmo-

nella in dry environments should lead to a re-assessment of the heat treatments given to manyfood products during manufacture. It now seemspossible that Salmonella may survive processingtreatments that formerly were assumed to beadequate to ensure their destruction.

This investigation was supported by the NationalConfectioners Association, Chicago, Ill.

LrrERATuRE Crrm1. Ng, H. 1966. The destruction of salmonellae. Agri-

cultural Research Service 74-37, p. 39-41.

1940 NOTES

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