Dehydration, Rehydration, and Storagel

APPLIED MIROBIOLOGY, May, 1965 opyright 1965 American Society for Microbiology Vol. 13, No. 3 Printed in U.S.A. Fate of Bacteria in hicken Meat During Freeze- Dehydration, Rehydration, and Storagel K. N. MAY AND L. E. KELLY Food Science Department and Poultry Science Department, University of Georgia, Athens, Georgia Received for publication 4 December 1964 ABSTRAT MAY, K. N. (University of Georgia, Athens), AND L. E. KELLY. Fate of bacteria in chicken meat during freeze-dehydration, rehydration, and storage. Appl. Microbiol. 13:340-344. 1965.-Total plate counts were determined on boneless cooked, cubed chicken meat obtained from a commercial processor. Survival of the natural flora was determined after the meat was freeze-dehydrated and rehydrated at room temperature for 30 min and 50, 85, and 100 for 10 min. Total counts of bacteria in the rehydrated samples were determined during storage of the meat at 4, 22, and 37 until spoilage odor was detectable. Meat samples were inoculated with Staphylococcus aureus, then dried, rehydrated, and stored at the same temperatures. Numbers of surviving organisms in the inoculated samples were determined with use of both selective and nonselective media. Representative genera surviving the various rehydration treatments were determined. Approximately 32% of the bacteria in the meat survived during dehydration and rehydration at room temperature. Many numbers and types of vegetative bacteria also survived rehydration at 50. When meat was rehydrated at 85 or 100, the initial count was less than one per gram. The only organisms isolated from samples rehydrated at 85 or 100 were of the genus Bacillus. S. aureus in inoculated samples survived dehydration and rehydration at 60. Storage of all rehydrated samples at 4 gave a good shelf life (18 or more days). The study indicates that freeze-dehydrated meat should be produced with adequate microbiological control and that such meat should be rehydrated in very hot water. Freeze-dehydration is now a commercially accepted method for preservation of meat and other food products. Freeze-dehydrated chicken meat is used extensively in soup mixes and other retail food items, as well as in military rations. Although much research has been done on freeze-dehydrated meat, it has been confined largely to factors affecting physical and organoleptic properties of the product (Sosebee, May, and Powers, 1964a; Sosebee, May, and Schmittle, 1964b; Wells, May, and Powers, 1962; Wang et al., 1954, 1958; Weir et al., 1958). Goldblith, Karil, and Lusk. (1963) pointed out the need for information on microbiological aspects of freeze-dehydrated meat. Since lyophilization has long been used as a method of preserving bacteria, it would appear logical that bacteria might survive freeze-dehydration in a meat product. Davis (1963) reported that all but 3 of 277 cultures of lyophilized organisms under study were viable after storage for 21 years. onditions under which the product was rehy- 1 Journal Paper No. 406, ollege Experiment Station, ollege of Agriculture Experiment Stations, University of Georgia, Athens. drated, and subsequent holding conditions, would also be pertinent in determining the microbial quality of dried meat. The present study was conducted to determine the fate of the natural microbial flora, as well as inoculated Staphylococcus aureus, in chicken meat during freeze-dehydration, rehydration at various temperatures, and subsequent storage at various temperatures. MATERIALS AND METHODS Source of meat. The chicken meat used in this study was purchased from a commercial processor. The meat had been boned from White Leghorn fowl which had previously been cooked in boiling water in open steam-heated kettles. The meat had been diced into small cubes and frozen in a Os freezer and, after arriving at the laboratory, was held at -34.4 until used in the experiment. Freeze-dehydration. The meat was dried as needed in a VirTis 40 port freeze dryer (The Vir- Tis o., Inc., Gardiner, N.Y.) as previously described by Sosebee et al. (1964a). lean containers were used, but no attempt was made to maintain aseptic conditions during the drying process. The dried samples were placed in unsterilized new 340

VOL. 13, 1965 BATERIA IN FREEZE-DEHYDRATED HIKEN 341 polyvinylidene bags which were evacuated and stored at -34.4 until analyses were performed. Bacteriology. With use of aseptic techniques and materials, 10-g meat samples (either nondried or dried and rehydrated) were weighed and placed in a Waring Blendor (Waring Products orp., New York, N.Y.) with 90 ml of 0.85% Nal. The blender was operated at high speed for 3 min, samples were removed immediately, and serial dilutions were made in saline. Duplicate pour plates were made of each dilution with Trypticase Glucose Extract Agar (TGEA). (All media used were manufactured by BBL.) Incubation was at 20 or 37, depending on the analyses being conducted. After incubation, plates containing 30 to 300 colonies were counted and results were recorded as count per gram of meat. ounts on inoculated samples were determined in the same way, except that pour plates were made with use of both TGEA and Staphylococcus 110 (S-110) media. olonies considered to be representative of types of bacteria present were selected from plates used to determine counts and streaked for purity on TGEA plates. olonies were then transferred to nutrient agar slants and stored at 4 until identification procedures were conducted. No attempt was made to identify all types of microorganisms present or to determine relative numbers of each. Identification procedures followed the outline of Bergey's Manual. Analyses performed. The cooked chicken meat was sampled upon receipt and at 16 other times during the study (a period of about 12 months). Random samples of freeze-dehydrated meat were removed from storage and assigned rehydration and storage conditions as shown in Table 1. The mildest rehydration treatment consisted of rehydration for 30 min at room temperature. This treatment was found to cause apparent complete rehydration without danger of destruction of bacteria by heat. The 10-min time at the elevated temperatures (50, 60, 85, and 100 ) used had been previously selected during preliminary studies as giving apparent complete rehydration. These temperatures were chosen as the possible range of rehydration temperatures which might be used by consumers. After rehydration, the samples were TABLE 1. Experimental conditions under which chicken was rehydrated and stored Temp Rehydration Time Storage temp in Room 30 Not stored 50 10 4, 22, and 37 60* 10 4, 22, and 37 85 10 4, 22, and 37 100 10 4, 22, and 37 * Used only for samples Staphylococcus aureus. inoculated with transferred to clean, unsterilized cheesecloth, allowed to drain for 3 min, and then were placed in sterile 500-ml Erlenmeyer flasks for storage. Storage temperatures were selected as possible extremes to which the product might be exposed after rehydration. At intervals during storage, 10-g samples were removed and bacterial numbers were determined. Three persons judged the color and odor acceptability of the meat during storage. The product remained in storage until judged unacceptable by all three individuals. In some cases storage was continued past the "spoilage" time to study further changes in numbers of bacteria. Each rehydration temperature and storage temperature condition was replicated at least twice. In one phase of the study, samples were inoculated with S. aureus (culture supplied by M. K. Hamdy, Food Science Department, University of Georgia) before dehydration. A 24-hr nutrient broth culture was centrifuged for 15 min at 10,000 X g, and the cells were suspended in 100 ml of sterile 0.85% Nal containing 0.1% Phytone (BBL). The 100-ml suspension of S. aureus was divided into 4-ml samples, placed in 15-ml test tubes, and held at -34.4 until used for inoculation. When needed, a 4-ml fraction was removed and the numbers of viable bacteria present were determined by plate count. Ten different samples of 75 g each were inoculated with 20 ml (five 4-ml fractions) of the S. aureus suspension (4.2 X 109 viable cells as determined with TGEA or 3.2 X 108 with S-110 medium). The inoculum and meat were placed in a freeze-dehydration flask which was stoppered and agitated by shaking before being dried. RESULTS The total bacterial count of the frozen precooked chicken meat ranged from 8.7 X 103 to 4.8 X 105 per gram, with a mean count of 2.0 X 104. The count did not appear to be related to time in storage, and the variation in counts was attributed to variation in contamination during processing. When freeze-dehydrated chicken was rehydrated at room temperature for 30 min, the mean number of bacteria per gram was 6.5 X 103. This represented a mean survival of about 32% of the original number of bacteria present before dehydration. Results of rehydration of samples at 50 and subsequent storage at 4, 22, and 37 on bacterial numbers are shown in Fig. 1. Immediately after rehydration, the mean total count was 1.0 X 103 to 1.4 X 103 bacteria per gram, representing a mean survival of about 5% of the number of bacteria present prior to dehydration. The total count in the rehydrated sample stored at 4 declined slightly after 2 days, then slowly increased thereafter, with spoilage odor noted on the 19th day. Total counts in samples stored at

342 MAY AND KELLY APPL. MIROBIOL. 10 10 9 9 8 8 2 6 / / L5 10 15 20 25 Time FIG. 1. hange in bacteria per gram of freeze-dehydrated chicken after rehydrationat 50 for 10 min and storage at 4 (0), 22 (X), and 37 (O). Times represent hours for storage at 22 and 37 and days for storage at 4. 22 and 37 increased rapidly, with spoilage odor occurring in less than 24 hr. Total counts of samples rehydrated at 85 and subsequently stored at 22 and 37 are shown in Fig. 2. No viable organisms were found immediately after rehydration. In samples stored at 37, the count increased rapidly for about 12 hr, then leveled off. In samples held at 22, the count went up less rapidly, but was still increasing at 35 hr. When samples were stored at 4, no viable organisms were found for 30 days, at which time the tests were terminated. When samples were rehydrated at 100, no viable cells were found immediately after rehydration or after storage for 30 days at 4 or 7 days at 22, at which time tests were termninated. Growth did occur in samples held at 37, but results were so variable in six separate trials that a growth curve could not be prepared. Results of bacterial counts obtained immediately after rehydration and at spoilage, as well as time required for spoilage odor to develop in samples, are summ-arized in Table 2. Increasing the tempemtatre of rehydration or reducing the.0 0 5 E 4 0 X1 2 10 20 30 40 Time, hr hange in bacteria per gram in freeze-de- FIG. 2. hydrated chicken after rehydration at 85 for 10 min and storage at 22 (0) and 37 (X). holding temperature of meat samples resulted in extended shelf life. Bacteria identified from representative colonies taken from total count plates of the rehydrationstorage temperature studies are given in Table 3. A number of genera of bacteria were identified from samples rehydrated at 50. The only organisms identified from samples rehydrated at 85 or 100 were of the genus Bacillus, which was repeatedly isolated. When samples inoculated with S. aureus were freeze-dried, then rehydrated for 30 min at room temperature, the count was reduced from 5.6 X 108 to 1.4 X 104 per gram with TGEA and from 4.3 X 106 to 5.4 X 102 per gram with S-110 medium. When inoculated samples were rehydrated at 60 for 10 min, the mean count was zero on S-110 and 1.85 X 102 on TGEA. Inoculated samples stored at 22 or 37 had rapidly increasing counts on both TGEA and S-110, with spoilage odor becoming evident by 19 and 10 hr, respectively. When inoculated samples were rehydrated at either 85 or 100 and then stored at 22 or 37,

VOL. 13, 1965 BATERIA IN FREEZE-DEHYDRATED HIKEN 343 TABLE 2. Summary of shelf life and bacterial numbers on freeze-dried chicken rehydrated and stored at various temperatures Temp of Temp of Initial counit ount at spoilage Time required to spoil rehydration storage per gram 50 4 1.0 X 103 1.4 X 105 19 days 50 22 1.0 X 103 1.5 X 109 23 hr 50 37 1.4 X 103 2.0 X 10' 16 hr 85 4 <1 <1 at 30 days Terminated without spoilage at 30 days 85 22 <1 5.4 X 108 35 hr 85 37 <1 2.5 X 108 17 hr 100 4 <1 <1 at 30 days Terminated without spoilage at 30 days 100 22 <1 <1 at 7 days Terminated without spoilage at 7 days 100 37 <1 Variable* Variable but less than 40 hr * Attempts to obtain reproducible counts were unsuccessful during six replications. rable 3. Bacteria identified from representative colonies isolated during the rehydrationstorage tests Rehydra- Storage Bacteria identified ton temp temp 50 40 Achromobacter eurydice, Lactobacillus, Streptococcus 50 22 A. eurydice, Micrococcus, Bacillus, Escherichia coli, Lactobacillus, Arthrobacter 50 37 B. cereus, E. coli, Streptococcus 85 4 No isolates 85 22 Bacillus 85 37 Bacillus 100 4 No isolates 100 22 No isolates 100 37 Bacillus steadily increasing counts were obtained on TGEA, with spoilage occurring in less than 28 hr. However, no growth was obtained at any time on S-110 medium from samples rehydrated at 85 or 100 for 10 min. DISUSSION The recovery of 32% of the original number of bacteria from chicken meat after freeze-dehydration and rehydration at room temperature indicates that microbiological control of such a process is necessary. Rehydration of the dried meat at 50, either deliberate or accidental, would allow the survival of many numbers and types of vegetative bacteria (Tables 2 and 3). Low-temperature storage (4 ) of meat rehydrated at 50 was an effective method of delaying spoilage, since the meat had good odor and appearance for 18 days. Holding at high temperatures (22 and 37 ), however, resulted in spoilage in less than 24 hr. Rehydration of samples inoculated with S. aureus at 60 for 10 min allowed survival of the organisms. Thus, survival of vegetative cells of potential food poisoning or food infection organisms is a distinct possibility in freeze-dehydrated meat. Rehydration of inoculated samples at 60 apparently reduced the ability of S. aureus to tolerate Nal. This was evidenced by no growth on S-110 immediately after rehydration. However, after sample storage at 22 or 37, the organisms regained the ability to grow on S-110. Raising the rehydration temperature to 85 or 100 for 10 min effectively reduced the initial count of organisms in the meat to less than one per gram (Table 2). However, when the rehydrated meat was subsequently held at high temperatures, growth did occur, indicating the survival of sporeformers. The survival of sporeformers only at these high rehydration temperatures is supported by the fact that Bacillus was the only genus identified in meat rehydrated at 85 or 100 (Table 3). These studies indicate that freeze-dehydrated meat should be produced with adequate microbiological control. In addition, the ultimate consumer should be cautioned to rehydrate the meat only in very hot, preferably boiling, water and to store the rehydrated product at refrigerator temperatures only. AKNOWLEDGMENTS This investigation was supported by the Thomas J. Lipton Foundation. We are grateful for the technical assistance of Nona Saunders.

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