Survival of Aerobic and Anaerobic Bacteria in

APPLIED MICROBIOLOGY, Mar. 1968, p. 445-449 Copyright 1968 American Society for Microbiology Vol. 16, No. 3 Printed in U.S.A. Survival of Aerobic and Anaerobic Bacteria in Chicken Meat During Freeze-Dehydration, Rehydration, and Storage1 J. R. CHIPLEY AND K. N. MAY Departments of Food Science and Poultry Science, University of Georgia, Athens, Georgia 361 Received for publication 18 September 1967 Total and anaerobic counts were ascertained on boneless, cooked, cubed, frozen chicken meat. We determined survival of aerobes and anaerobes in the natural flora after the meat was freeze-dehydrated and rehydrated at room temperature for 3 min and at 5, 85, and 1 C for 1 min. Total and anaerobic counts of bacteria in the rehydrated meat were established during storage of samples at 4, 22, and 37 C -until a spoilage odor was detected. Samples were also inoculated with Clostridium sporogenes and were dried and rehydrated at 1 C and stored at 37 C. Approximately 21 % of the aerobes and 37% of the anaerobes survived drying and rehydration at room temperature. Many genera of aerobes, anaerobes, and facultative anaerobes survived drying and rehydration at 5 C; only sporeformers survived rehydration at 85 or 1 C. Low-temperature (4 C) storage of rehydrated meat produced ample shelf life (over 2 days), whereas storage at the higher temperature resulted in a shelf life of less than 3 hr. Approximately 81% of the C. sporogenes cells survived rehydration at 1 C and grew to over 17 cells within 4 hr. Our study presents additional data for adequate microbiological control in processing of freeze-dehydrated meat. Also, it points out the natural selection for sporeformers at high temperature of rehydration, stressing the need for consumer education in product handling for safety purposes. Previous investigations in this laboratory (3) have indicated that many aerobic bacteria, present as natural flora on cooked, boned chicken meat, survived freeze-dehydration and rehydration at mild temperatures (up to 5 C). Sporeforming bacteria survived rehydration at 85 and 1 C for 1 min; these bacteria were capable of rapid growth when the rehydrated meat was maintained at high temperatures. Saleh and Goldblith (4) reported similar results in their investigations of freeze-dehydrated fish patties, shrimp, chicken, and pork. Wells (5) showed that spores of Clostridium botulinum, inoculated on cooked chicken meat, were reduced in number by 53% during freezing and were further decreased by an additional 15% during freeze-dehydration. Wells stated that there was little doubt that C. botulinum spores would persist in the dehydrated product for long periods of storage. We found no reports of investigations in which a wide variety of rehydration and storage 1 University of Georgia, College of Agriculture Experiment Stations, Journal Series Paper No. 143, College Station, Athens, Ga. 361. temperatures were studied as to their effect on survival of anaerobic bacteria in freeze-dehydrated meat. This study was conducted to determine the survival of natural aerobic and anaerobic bacteria, as well as inoculated C. sporogenes, in chicken meat during freeze-dehydration, rehydration at various temperatures, and subsequent storage at different temperatures. MATERIALS AND METHODS Source of meat. Cooked, cubed, and frozen meat was purchased from a commercial processor for use in our study. The meat had been boned from broilers that had been cooked in water and then cubed into small pieces, packaged in 5-lb polyethylene bags, and frozen in a blast freezer. In the laboratory, frozen, packaged meat was maintained at approximately -3 C. Freeze-dehydration. The frozen, cubed meat was freeze-dehydrated, and the dried product was stored in the manner described by May and Kelly (3). Bacteriology. By use of aseptic techniques and materials, 1-g meat samples (either nondried or dried and rehydrated) were weighed and were homogenized 445

446 CHIPLEY AND MAY APPL. MICROBIOL. in an Omnimixer with 9 ml of.85% NaCl for 3 min. Serial dilutions were made in saline, and duplicate pour plates or shake tubes were prepared. We determined the aerobic counts using pour plates with Tryptone Glucose Extract Agar (TGEA); anaerobic counts were established by use of either shake culture tubes (2) containing thioglycolate medium with added agar (15 g/liter) or pour plates of the same medium overlaid with 3% agar. (All media were manufactured by either Difco or BBL.) Incubation was at 37 C for 48 hr for aerobes and for 96 hr for anaerobes; plates containing 3 to 3 colonies were then counted and the results were recorded as count per gram of meat. All colonies that developed in the shake culture tubes or overlaid agar plates were recorded as anaerobes, although we realized that some facultative anaerobic bacteria were included. We selected colonies considered to be representative of the types of bacteria present from plates or tubes used to determine counts and isolated on TGEA or thioglycolate plates with agar overlay. These colonies were then transferred to nutrient agar slants or were stabbed in nutrient agar tubes, and they were stored at 4 C until identification procedures were conducted. Only the types of colonies that appeared predominant were selected, and no attempt was made to identify all types of organisms present or to determine the numbers of each. We followed the outline of Bergey's Manual for identification procedures. Analyses performed. The frozen chicken meat as received from the processor was sampled initially and at eight additional times during the course of the study (a period of about 4 months). Samples of freeze-dehydrated meat were removed from storage and were assigned rehydration and storage treatments. Rehydration treatments were conducted at room temperature for 3 min and at 5, 85, and 1 C for 1 min. The samples rehydrated at room temperature were not stored. The other samples were stored at 4, 22, and 37 C. Temperatures of rehydration and storage were the same as those employed by May and Kelly (3); these temperatures were chosen to cover the extremes that might be encountered in use of such a product by a consumer. After rehydration, the samples were transferred to clean, unsterilized cheesecloth, allowed to drain for 3 min, and stored in sterile 5-ml Erlenmeyer flasks. At intervals during storage, 1-g samples were extracted, and aerobic and anaerobic counts were determined. A panel of three experienced persons judged the odor- and coloracceptability of the meat during storage; it was considered spoiled when all three individuals independently judged the meat to have an off-odor. In some cases, storage was continued beyond the "spoilage" time to study further variations in numbers of bacteria. Each rehydration and storage sequence was replicated from five to eight times to determine changes in numbers of microorganisms. C. sporogenes was used as an inoculum in one part of the study. The culture (supplied by M. K. Hamdy, Food Science Department, University of Georgia) was grown for 48 hr at 37 C in fluid thioglycolate medium. Cells were harvested by centrifugation at 1, X g for 15 min and were washed twice with.85%o NaCl. The cells were suspended in.85% NaCl containing.1% tryptone, and 5-ml portions were placed in 15-ml sterile tubes at -34.4 C. As needed, these tubes were removed from the freezer and thawed for 2 hr; a portion was extracted to determine the number of viable organisms, and the remainder was used as an inoculum. To free the chicken meat of competitive organisms, a piece of meat was placed in a beaker of water and was autoclaved at 121 C for 15 min. By use of aseptic techniques, a portion of the meat was transferred to sterile freezedrying flasks, and a known number of cells of C. sporogenes were added. The product was then freezedried and rehydrated at 1 C, and serial dilutions were prepared to determine changes in cell numbers. We selected a storage temperature of 37 C so that bacteria surviving the freeze-drying process would begin to grow and subsequently multiply as quickly as possible. Six replications were made for this part of the study. RESULTS The precooked frozen chicken meat had a mean bacterial count of 1.3 X 14 per g for aerobes and 1.3 x 1 per g for anaerobes. After freeze-drying and rehydration at room temperature for 3 min, the meat had a mean aerobic count of 2.8 X 13 bacteria per g. This was a reduction of 1.1 X 14 bacteria per g during the freeze-drying process or a survival rate of approximately 21 %. However, the mean 1 w 7 a. CD z 2 9- FIG. 1. Change in anaerobic bacteria per g offreezedehydrated chicken after rehydration at 5 C for 3 min and storage at 4 C (), 22 C (A), and 37 C (O).

VOL. 16, 1968 BACTERIA IN FREEZE-DEHYDRATED CHICKEN 447 m 2 7-, o c 5 1 15 2 25 3 35 FIG. 2. Change in anaerobic bacteria per g offreezedehydrated chicken after rehydration at 85 C for JO min and storage at 4 C (), 22 C (A\), and 37 C (Ol). anaerobic count was 4.9 X l2 bacteria per g, a reduction consisting of 8.5 X 12 bacteria per g during the freeze-drying process or a survival rate of approximately 37%. Effects of rehydration of meat to 5 C and storage at 4, 22, and 37 C on numbers of anaerobes are shown in Fig. 1. At 4 C the numbers of anaerobic bacteria declined slightly for the first 3 days and then increased rapidly until 21 days, when spoilage was noted. Growth was much more rapid in meat stored at 22 and 37 C than in that stored at 4 C, with growth beginning immediately after rehydration. Spoilage occurred at 24 and 16 hr for meat stored at 22 and 37 C, respectively. Similar, but slightly greater, increases in numbers of aerobic bacteria were obtained, but these increases are not shown because they resemble previously reported data (3). Rehydration of chicken meat at 85 C and storage at 4 C caused an extended lag phase (up to approximately 13 days) for anaerobic bacteria (Fig. 2). After the initial lag, growth was rapid up to about 18 days after which it leveled offalthough the product was not considered spoiled until 24 days of storage. Growth curves for anaerobes in the product rehydrated at 85 C and stored at 22 or 37 C were almost identical (Fig. 2). Numbers remained almost stationary for about 5 hr and then increased rapidly with spoilage odor evident at 18 and 2 hr for the products stored at 22 and 37 C, respectively. Rehydration of meat at 1 C and storage at 4 C caused a rapid decline in numbers of anaerobic bacteria until the 9th day (Fig. 3). However, from 9 to 3 days of storage no viable organisms were recovered from the product rehydrated at 1 C and stored at 4 C; the product still had no objectionable odors after 3 days of storage. The anaerobes in the product rehydrated at 1 C and stored at 22 C grew rapidly for 14 hr, but these anaerobes tended to increase slowly thereafter, until spoilage odors became apparent at 48 hr of storage. In the product rehydrated at 1 C and stored at 37 C, the numbers of anaerobes remained relatively constant until about 15 hr; then they increased rapidly until spoilage occurred at 31 hr. Results of aerobic and anaerobic bacterial counts obtained immediately after spoilage, as well as the time required for a spoilage odor to develop in samples, are summarized in Table 1. Decreasing storage temperature of rehydrated meat from 37 or 22 to 4 C extended shelf life, but increasing rehydration temperatures from 5 to 85 or 1 C increased shelf life to a lesser extent. We identified both aerobic and anaerobic bacteria from meat rehydrated at each of the three previously mentioned temperatures (Table 2). Many types of bacteria were recovered from samples rehydrated at 5 C, but only sporeformers 1 9 8 w6 r4-82 FIG. 3. Change in anaerobic bacteria per g offreezedehydrated chicken after rehydration at 1 C for 1 min and storage at 4 C (), 22 C (A), and 37 C (O).

448 CHIPLEY AND MAY APPL.. MICROBIOL. TABLE 1. Summary of shelf life and numbers of aerobic and anaerobic bacteria on freeze-dehydrated chicken rehydrated and stored at various temperatures Temp of Temp of Initial count per g Count at spoilage rehydra- storage Time required to spoil tion (C) (C) Aerobes Anae robes Aerobes Anaerobes 5 4 2. X 13 4. X 13 4. X 111 4. X 11 21 days 5 22 1.6 X 13 6.3 X 13 1. X 19 1. X 18 24 hr 5 37 1.6 X 13 1.6 X 13 6.3 X 18 4. X 19 16 hr 85 4 1. X 13 3.2 X 14 5.1 X 17 3.2 X 17 24 days 85 22 4. X 14 1.6 X 13 2.5 X 18 1. X 111 2 hr 85 37 6.3 X 14 1.6 X 13 1.O X 18 4. X 19 18 hr 1 4 2.5 X 13 5.1 X 13 <1 at 3 day <1 at 3 day Terminated at 3 days 1 22 6.3 X 13 1. X 13 4. X 11 2.5 X 19 48 hr 1 37 1. X 13 2.5 X 13 4. X 19 1.6 X 18 31 hr were isolated from samples rehydrated at 85 and 1C. When we inoculated samples of meat with C. sporogenes (58 cells per g) and subsequently freeze-dehydrated and rehydrated these samples at 1 C for 1 min, a mean survival of 81 % of the organisms was observed. Counts increased to a mean of 7.4 X 17 C. sporogenes cells per g of product within 4 hr when stored at 37 C. DiscussIoN The recovery of 21% of the aerobic and 37% of the anaerobic bacteria present in the original chicken meat after freeze-dehydration and rehydration at room temperature is in agreement with previous studies of May and Kelly (3); their data showed a recovery of 32%. The variety of genera surviving the freeze-dehydration and rehydration at 5 C (Table 2) are also similar with the details given by May and Kelly. Both Escherichia and Salmonella spp. were identified in meat during this study. In contrast, Saleh and Goldblith (4) reported no salmonellae or coliform organisms, when they examined freeze-dehydrated chicken. However, Gunderson et al. (1) revealed that E. coli, Aerobacter aerogenes, and their variants are frequently found in cooked, boned chicken meat. May and Kelly (3) also identified E. coli in their tests with freeze-dehydrated chicken meat. Our study confirms the previous report from this laboratory (3) that vegetative cells of many types of bacteria can survive freeze-dehydration and rehydration at low temperature (5 C). In addition, these studies indicate that rehydration of freeze-dried meat at high temperature (85 to 1 C) effectively selects for sporeforming species of bacteria. Inoculated C. sporogenes had a survival rate of 81% after dehydration and rehydration at 1 C for 1 min; these results are in accordance with the 81 % survival rate of C. botulinum reported by Wells (5) in chicken meat during freeze-dehydration. The rapid growth of C. sporogenes (up to 7.4 X 17 cells per g within 4 hr) at 37 C suggests that surviving cells can multiply to dangerous levels in a very short period of time. Thus, the accidental or intentional delay of use of rehydrated meat containing any spores of pathogenic bacteria could lead to tragic results. These studies give additional evidence of the need for adequate microbiological control during processing of freeze-dehydrated chicken. Further more they point out that such control alone is not sufficient for the protection of the ultimate consumer. If the product is maintained at high temperature after rehydration, pathogenic sporeformers are capable of survival and rapid growth. Thus, the consumer should be urged to refrigerate any rehydrated product promptly if it is not used immediately. TABLE 2. Bacteria identified from representative colonies isolated during the rehydrationstorage tests Bacteria identified Rehydration temp (C) From aerobic plates Lactobacillus spp... 5 Escherichia coli... 5 Aerobacter aerogenes... 5 Salmonella spp... 5 Streptococcus spp... 5 Staphylococcus aureus... 5 Bacillus cereus... 5, 85, 1 From anaerobic tubes and plates S. aureus... 5 Clostridium sporogenes... 5, 85, 1 C. perfringens... 5, 85, 1

VOL. 16, 1968 BACTERIA IN FREEZE-DIEHYDRATED CHICKEN 449 LITERATURE CI1D 1. GUNDERSON, M. F., H. W. McFADDEN, AND T. S. KYLE. 1954. The bacteriology of commercial poultry processing. Burgess Publishing Co., Minneapolis, Minn. 2. HAMDY, M. K., E. L. SHERRER, H. H. WEISER, AND W. D. SHEETs. 1954. Microbiological factors in the treatment of phenolic wastes. Appl. Microbiol. 2:143-148. 3. MAY, K. N., AND L. E. KELLY. 1965. Fate of bacteria in chicken meat during freeze-dehydration, rehydration, and storage. Appl. Microbiol. 13:34-344. 4. SALEH, B. A., AND S. A. GOLDBLITH. 1966. Microbial evaluation of commercial freeze-dried foods. Food Technol. 2:13-16. 5. WELLS, F. E. 1966. A study of the microbiology of selected dehydrated food products. Tech. Rept. 66-35-FD, U.S. Army Natick Laboratories, Natick, Mass.