Comparison of Enterococci and Coliform Microorganisms in

Transcription

1 Comparison of Enterococci and Coliform Microorganisms in Commercially Produced Pecan Nut Meats JAMES B. HYNDMAN U.S. Department of Health, Education, and Welfare, Food and Drug Admninistration, Dallas District, Dallas, Received for publication 21 January 1963 Texas ABSTRACT HYNDMAN, JAMES B. (Food and Drug Adminiistration, Dallas, Texas). Comparison of enterococci and coliform microorganisms in commercially produced pecan nut meats. Appl. Microbiol. 11: Pecan nut meats in the unbroken shell are sterile for enteric microorganisms. Recovery of coliform microorganisms or enterococci from finished pecan nut meats indicated contact contamination, assuming the tempering procedures to be satisfactory. Results of specific studies, designed toward developing background data on the sanitary significance of enterococci and coliform microorganisms in the production of pecan meats are reported. Unbroken pecan nuts or nut meats from various stages of shelling operations were diluted with a phosphate-buffered diluent. Serial dilutions were inoculated into Lactose Broth and Azide Dextrose Broth. The lactose fermentors were carried through indole, methyl red, Voges-Proskauer, and citrate reactions; the positive Azide Dextrose cultures were confirmed in Ethyl Violet Azide Broth and microscopically. Viable plate counts were obtained. Enterococci were found resistant to many deterrent factors affecting coliforms. Recoveries of enterococci were detected long after pollution had occurred. Little correlation was found between enterococcal recovery and observed insanitary practices in commercial shelling operations. Using the coliaerogenes group and, specifically, Escherichia coli as a sanitation index, microorganisms allowed accurate appraisal of tempering, personnel practices, and contact surface contaminating factors. It is felt this was due, in part, to the more delicate growth characteristics of E. coli. The fact that other pathogenic microorganisms, capable of causing gastrointestinal upsets, are associated with the presence of E. coli introduces a health factor which is important to regulatory agencies concerned with consumer protection. Ostrolenk and Hunter (1946) reported studies determining the frequency distribution of enteric streptococci in nature. They concluded, among other things, that, though generally outnumbered by Escherichia coli, the resistance of enterococci to chemical agents and possibly to other environmental factors makes them of sanitary significance as indices of fecal contamination and pollution. Many investigators have shown the presence of mnicroorganisms of sanitary significance in or on many foods. McCleskey and Boyd (1949) showed that coliform bacteria and enterococci were present in iced crabmeat. The numbers of coliform bacteria increased during storage, but the numbers of enterococci remained unchanged. Leininger and McCleskey (1953), in a study of coliform bacteria and enterococci in surface waters, found both died at a fairly rapid rate in stored samples. Burton (1949) found that, as indicators of fecal contamination, the coliform organisms appeared to be superior to the enterococci in unstored or briefly stored frozen foods, although the enterococci were a more reliable index in foods which had been held in freezer storage. Kereluk and Gunderson (1959) confirmed these findings, showing that after 481 days of frozen storage the coliforms decreased, whereas the enterococci remained relatively constant. Larkin, Litsky, and Fuller (1955), in a series of investigations of enterococci in frozen foods, felt that these organisms could be used advantageously in preference to coliform bacteria as indicators of contaminationi in frozen foods. It is an elementary principle that knowledge of a food product's source and the methods of its preparation and handling are fully as important as knowledge of the identity of the group of organisms detected in or on it (Hunter, 1939). Ostrolenk and Hunter (1939) and later Ostrolenk and Welch (1941) reported detailed studies on pecan meats, using the coli-aerogenes group of bacteria as index organisms. Essentially, their data revealed the fol- 268 lowing. (i) Nuts in the unbroken shell do not contain members of the coliform group. (ii) When large numbers of E. coli are present, correspondingly large numbers of other coliforms are found. (iii) Nut meats artificially contaminated with E. coli retain viability of that microorganism for approximately 68 days. Review of data from many plants has demonstrated clearly the absence of E. coli when proper sanitization of contact surfaces is practiced. The objective of this investigation was to compare enterococcal populations with the coliforms and to attempt to appraise the value of enterococci as sanitary index microorganisms in commercially produced pecan nut meats.

2 VOL. 11l ENTEROCOCCI AND COLIFORMS IN PECAN NUT MEATS 269 MATERIALS AND METHODS A 50-g amount of unbroken pecan nuts or pecan meats was aseptically weighed into an 8-oz screw-cap sample jar to which were added 50 g of Butterfield's bufferedphosphate diluent. The samples were then well-shaken, and serial dilutions of 1, 0.1, 0.01 g, etc. were obtained; portions were inoculated into Lactose Broth and Azide Dextrose Broth (Litsky, Mallman, and Fifield, 1953). Total aerobic plate counts were prepared. Lactose tubes, showing gas at 24 and 48 hr, or both, at 35-C incubation, were streaked on Eosin Methylene Blue Agar plates, and the coliform colonies were identified by their biochemical indole, methyl red, Voges-Proskauer, and citrate reactions. The positive Azide Dextrose Broth tubes were transferred to Ethyl Violet Azide Broth after 48 hr of incubation at 35 C. A purple button after 24 hr indicated a confirmed positive reaction. The purple button was additionally subjected to microscopic examination to confirm the presence of streptococci (Litsky, Mallman, and Fifield, 1953). RESULTS AND DISCUSSION Temnpering. Table 1 shows the results of the tempering procedure used in six different shelling plants. Most pecan orchards also serve as pasture land for cattle, sheep, goats, etc. When the nlits are ready to harvest, they fall or are knocked to the ground, thereby becoming contaminated exteriorly with soil and possibly fecal microorganisms. They are subjected to additional contaminating factors during transportation and storage prior to cracking and shelling. Upon receipt at a shelling plant, the whole nuts are generally subjected to an air-pressure cleaning procedure to remove bits of debris and loose dirt and are then Plant TABLE 1. graded for size at this point. They next undergo a "tempering" procedure which has a twofold purpose: (i) to moisten the shell, which allows the cracking machines to work efficiently, and (ii) to sanitize or sterilize the shell, thereby removing fecal and other filth and, of course, the index microorganisms. There are two methods of tempering in common use. In one, the nuts are tumbled in a boiling-water bath for various periods, depending on the size of the nuts, the quantity of nuts being tempered at one time, and whether the bath is heated from an outside source or by allowing steam to bubble through the water in the tank. The second and more common method in the South is to place the nuts in either tanks or metal drums. Water and a sanitizing agent, such as chlorine, are added at a concentration of 1,000 ppm. The nuts are then held in these containers for sufficient time to meet the twofold purposes mentioned above. The data in Table 1 indicate the recovery of both the coliforms and enterococci when appraising sanitization in tempering procedures. Plants 1, 2, 3, and 4 (Table 1) show satisfactory tempering procedures when using the coliaerogenes group, as evidenced by the absence of E. coli. Plants 5 and 6 show inadequate tempering procedures, since E. coli remained after the tempering procedure (Table 1). When using the enterococci as an index organism, plants 2, 3, 4, and 5 show satisfactory tempering procedures. Plants 1 and 6 show the continued presence of viable enterococci, indicating an inadequate tempering procedure (Table 1). Plant 1 shows satisfactory tempering when using the coli-aerogenes group, but inadequate Results of the tempering procedure in six different shelling pecan plants Enterococci Plant Type ofnutmatsexaminedimvic reactions* Confirmatory tests Type of nut meats Total aerobic plate examined-count per g Ii A, I1 A l0o- 1 10' lo-, 10-, 12 Is Ii 2,200, ,000 1,300,000 30,000 3,200, , I1 11 A2 600,000 80, , , ,000 40,000 * = typical E. coli (++ - -); I1 = typical intermediates (- + - A); 12 = atypical intermediates (++-+); Al = typical aerogenes (--+i); A2 = atypical aerogenes (4-++ ).

3 270 HYNDMAN APPL. MICROBIOL. sanitization when using the enterococci as index microorganisms. Plant 5 shows satisfactory tempering when using the enterococci, but inadequate sanitization when using the coli-aerogenes group as index microorganisms. Shelling. After the tempering procedure, the niuts are placed in the hoppers of cracking machines. From the cracking machines, the nut meats are carried by endless belts, elevator cups, or shaker troughs through a series of TABLE 2. Bacteriological recovery from various sizes of nut meats produced by six pecan plants Enterococci Plant examined Plant Type of nut meats examined IMViC. count per g reactlons* Confirmatory tests Total aerobic plate 1 lo-, 102 lo-, 1 lo- 102 io-3 1o-4 1 Halves A, A, ,000 I2 I1 + + _ 30,000 2 Halves 10,000 Large 20, 000 Medium _ - 160,000 Small 12 Al A ,000 3 Halves IL L + + _ - - Mold overgrowth L L L ,500,000 Ii + + _ - - 1,700,000 4 Halves _ ,000 Halves _ ,000 Large Ii L L L , 000 Medium Ii + + _ ,000 Medium L L ,000 Small Ii L ,000 5 Halves I I ,000 Halves I1 + _ , ,000 E ,000 6 Halves Il ,000 E E E ,000, ,200,000 Granules I2 Al ,000 * See footnote to Table 1 for explanation of symbols. TABLE 3. Bacteriological recoveries from 70 subsamples of pecan meats collected in interstate status from six pecan plants ~~~Enterococci Confirmatory tests No. of Plant samples Coliforms E coli Total plate range/aerobic count per g examinedcoiose.cl -I g 0 g 0. g I g 0.1 g 0.01 g ig 0.1 g 0.0O g ,000/130, ,000/6,100, ,000/920, ,000/400, lo ,000/2,000, ,000/2,100,000

4 VOL. 1 1) 1963 ENTEROCOCCI AND COLIFORMS IN PECAN NUT MEATS 271 operations which remove the shell and grade the nut meats. At various production stages, the nut meats are passed over inspection belts and shaker tables where bits of shell or decomposed meats are manually removed, occasionally with the aid of ultraviolet light. The packing operation may be either manual or mechanical. The nut meats in the unbroken shell are sterile for the coli-aerogenes group of microorganisms and the fecal enterococci. Therefore, contamination of the meats by these bacterial groups must come from a contaminated contact surface, provided the tempering procedure is satisfactory. These surfaces may include improper or inadequately sanitized belts, tables, containers, scoops, hands, or improper flotation procedures. Table 2 depicts the bacteriological recoveries obtained from various sizes of nut meats produced by the six plants shown in Table 1. The production of three of these plants (2, 5, and 6) shows the presence of E. coli, a microorganism indicative of fecal contamination, in their finished products. Two of these plants (5 and 6) had inadequate sanitization in their tempering procedure; therefore, subsequent contamination of the meats would be expected. Contamination of the production of plant 2 must necessarily have come from contact surfaces, since their tempering process was adequate. The production of all plants showed presence of fecal enterococci in varying concentrations. It is unfortunate that in-plant swabs of contact surfaces were not obtained in this study. Enterococci were recovered rather consistently in 1- and 0.1-g portions, probably owing to their greater resistance. M1ar ket samples. Table 3 depicts the bacteriological recoveries obtained from 70 subsamples of pecan meats collected in interstate status from the six pecan plants. It is interesting to note that the samples from which E. coli were recovered came from the three plants having improper manufacturing practices (plants 2, 5, and 6). Fecal enterococci were recovered from all 70 interstate samples. Procedures for recovering coliform and enterococci microorganisms are simple, effective, and rapid. The differentiation of coliforms through their biochemical reactions, however, is a time-consuming factor. There is general agreement among regulatory officials that the coliform group has merit as an index of sanitation, and it has been used as such for many years (Mossel, 1957). There is some controversy, however, as to the levels of coliform populations necessary to indicate pollution. The fecal streptococci, on one hand, do not originate from anywhere except the intestinal tract of man or warmblooded animals. These streptococci are very hardy microorganisms and maintain their viability over long periods. They are resistant to many deleterious influences, includinig cursory sanitization procedures. E. coli, on the other hand, is also a normal inhabitant of the mammalian intestinal tract; however, it is a relatively delicate microorganism. We used the recovery of this organism as substantiative evidence of inspectional observations within a given plant for confirmation of insanitary practices. From this standpoint, we feel that E. coli recoveries more clearly reflect an abnormal sanitary condition in a plant (Pederson and Skinner, 1955). The enterococci, although of some apparent significance in the tempering process, do not serve as well, since they were present in all market samples, even though in-plant sanitation was adequate. When there are marginal differences between good and bad plants, the presence of enterococci does not leave a regulatory agency with a clear-cut conclusion for interpretative purposes. Fecal enterococci are quite resistant to many deterrent factors affecting coliforms. Recoveries of enterococci are detected long after pollution has occurred; consequently, their use as a sanitation index to microorganisms in shelled pecan meats is greatly reduced. This study shows little correlation between enterococcal recovery and insanitary practices observed in the commercial shelling of pecan meats. The use of the coli-aerogenes group, and specifically E. coli, as a sanitation index microorganism allows accurate regulatory appraisal of tempering, personnel practices, and contact surface contaminating factors in the production and handling of shelled pecan meats. This is due, in part, to its more delicate growth characteristics. The fact that quite often other pathogenic microorganisms capable of causing gastrointestinal upsets are commonly associated with its presence introduces a health menace problem, which is advantageous to the regulatory agencies concerned with consumer protection. LITERATURE CITED BURTON, M Comparison of coliform and enterococcus organisms as indices of pollution in frozen foods. Food Res. 14: HUNTER, A. C Uses and limitations of the coliform group in sanitary control of food production. Food Res. 4: KERELUK, K., AND M. F. GUNDERSON Studies on the bacteriological quality of frozen meat pies. IV. Longevity studies on the coliform bacteria and enterococci at low temperature. Appl. Microbiol. 7: LARKIN, E. P., W. LITSKY, AND J. E. FULLER Fecal streptococci in frozen foods. I. A bacteriological survey of some commercially frozen foods. Appl. Microbiol. 3: LEININGER, H. V., AND C. S. MCCLESKEY Bacterial indicators of pollution in surface waters. Appl. Microbiol. 1: L1TSKY, W., W. L. MALLMAN, AND C. W. FIFIELD A new medium for the detection of enterococci in water. Am. J. Public Health 43: LITSKY, W., M. J. ROSENBAUM, AND R. L. FRANCE A comparison of the most probable numbers of coliform bacteria and enterococci in raw sewage. Appl. Microbiol. 1: MCCLESKEY, C. S., AND A. F. BOYD The longevity of coliform bacteria and enterococci in crabmeat. Food Technol. 3:

5 272 HYNDMAN APPL. MICROBIOL. MOSSEL, D. A. A The presumptive enumeration of lactose negative as well as lactose positive Enterobacteriaceae in foods. Appl. Microbiol. 5: OSTROLENK, M., AND A. C. HUNTER Bacteria of the colonaerogenes group of nut meats. Food Res. 4: OSTROLENK, M., AND A. C. HUNTER The distribution of enteric streptococci. J. Bacteriol. 51: OSTROLENK, M., AND H. WELCH Incidence and significance of the colon-aerogenes group on pecan meats. Food Res. 6: PEDERSON, H. T., JR., AND C. E. SKINNER A comparison of standard lactose broth with lauryl sulphate broth and with the Eijkman method for demonstrating fecal coliform bacteria. Appl. Microbiol. 3: ZABOROWSK H., D. A. HUBER, AND M. M. RAYMAN Evaluation of microbiological methods used for the examination of precooked frozen foods. Appl. Microbiol. 6: