Radicidation for Elimination of Salmonellae in Frog Legs
Total Page：16
File Type：pdf, Size：1020Kb
820 Journal ofFood Protection, Vol. 45, No. 9, Pages 820·8231/uly 1982) Copyright ©,International Association of Milk, Food, and Environmental Sanitarians Radicidation for Elimination of Salmonellae in Frog Legs D.P. NERKAR* and N. F. LEWIS Biochemistry and Food Technology Division, Bhabha Atomic Research Centre, Bombay· 400 085, India (Received for publication September 22, 1981) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/9/820/1655352/0362-028x-45_9_820.pdf by guest on 25 September 2021 ABSTRACT 7.0, and suspended in the same buffer at levels of approx. 108 cells/mi. Preparation ojfrog leg homogenates The D10 values of three Salmonella spp. often encountered in frog legs, i.e., S. typhimurium, S. enteritidis and S. newport, Portions (25 g) consisting of muscle were removed from at least 4-5 were found to be between 18 to 30 krad when cells were frog leg samples and placed in 225 ml of lactose broth. The samples were blended for 90s at high speed (approx. 14,000 rpm) in a Waring irradiated at 0-2°C in 0.1 M phosphate buffer, pH 7.0. The Blendor and the homogenates were poured in sterile flasks. The radiation sensitivities of these Salmonella spp. increased only Salmonella count of these homogenates was assessed by the most marginally when cells were irradiated in frog leg homogenate. probable number (MPN) method (J). The doses of radiation required for eradication ofthis pathogen in fresh and frozen frog legs were 300 and 400 krad, respectively. Radiation sensitivity studies Buffered cell suspensions of S. typhimurium, S. newport and S. enteritidis were irradiated in ice in a Gamma Cell 220 (AECL, Canada) at doses ranging from SO to 2SO krad and a dose rate of 180 krad/h. In India is the world's largest producer of frog legs, the another set of experiments. cell suspensions of the above three annual production being about 3,500 tons. Most of the Salmonella spp. were added to heat-sterilized frog leg homogenate 7 production is exported to the U.S.A. and France. In (10o/o) at a level of 1 x 10 cells/ml, dispensed in test tubes (15 X 2 em), placed in a beaker containing crushed ice and irradiated at 0-2°C at recent years, a major problem facing the frog leg industry doses ranging from SO to 300 krad. In a third set of experiments, is the frequent contamination of these samples with heat-sterilized frog leg homogenate inoculated with S. typhimurium Salmonella, owing to inadequacy of the conventional and S. enteritidis (l X 107 cells/ml) were frozen in liquid nitrogen and method (treatment with 500 ppm chlorine water) used for then irradiated under frozen (dry ice) conditions (-40° C) at doses elimination of this enteric pathogen. Studies in our ranging from SO to 500 krad. In the above three sets of experiments, the survivors were recovered on plate count agar after incubating plates at laboratory have revealed the efficacy of utilizing gamma 37° C for 48 h. radiation doses in the range of 0.1 · 0.25 Mrad for extending the shelf-life of seafoods through selective Irradiation offrog legs destruction of spoilage bacteria (3-5). Other investigators Frog legs were packed in sterile polyethylene bags (gauge 200) and have reported (6,10) the use of radiation doses in the irradiated in either a fresh condition (26-28°C) or a frozen condition range of 0.3 - 1.0 Mrad for eradicating salmonellae in egg (using dry ice) at doses ranging from 100 to 400 krad. After irradiation, products, horse meat, desiccated coconut and animal samples were stored at ·20°C for 4 months and evaluated for aerobic plate count, Salmonella count and organoleptic score at 4-week feeds. Attempts were, therefore, made to develop a intervals. radicidation process for eliminating salmonellae from Organoleptic evaluation fresh and frozen frog legs. The present communication Sensory scores were assessed by five trained taste panelists using the reports on the sensitivity of three species of Salmonella to hedonic scale of Miyauchi eta!. (7). gamma radiation in buffer and in frog leg homogenate, and the development of a radicidation process for Anijicial contamination offrog legs destruction of salmonellae in fresh and frozen frog legs. Frog legs were placed four at a time in a sterilized beaker containing 1 L of a lxlOS cells/ml suspension of S. typhimurium in phosphate buffer. After 3 h the frog legs, which contained 10"- 105 cells/g. were removed MATERIALS AND METHODS aseptically, packed in sterile polyethylene bags, and irradiated at doses ranging from 100 to 400 krad as described above. Fresh S. Microorganisms typhimurium cell suspensions were used for each dip treatment. To confirm that S. typhimurium was completely eliminated from the frog Salmonella typhimurium, Salmonella newpon and Salmonella enteritidis originally isolated from frog legs. were used in this study. legs after irradiation offresh or frozen samples at 300 or 400 krad, the samples were preenriched in lactose broth for 48 h, followed by selective Preparation of cell suspensions enrichment in selenite-cystine broth for 24 h. Portions from the The three Salmonella spp. were grown individually in nutrient broth selective enrichment medium were plated on bismuth sulfite agar, (Difco) for 18 h, centrifuged, washed with 0.1 M phosphate buffer, pH brilliant green agar and Salmonella-Shigella agar. JOURNAL OF FOOD PROTECTION, VOL. 4S,JULY 1982 RADICIDATION OF SALMONELLAE IN FROG LEGS 821 Aerobic plate count marginal protection to the salmonellae during irradia For determination of aerobic plate counts, portions of the sample tion. that were blended in lactose broth were serially diluted in saline and 0.1 Irradiation in the frozen condition offered distinct ml of the appropriate serial dilutions were streaked on prepoured plates of plate count agar. These plates were incubated at 37°C for48 h. protection to S. typhimurium and S. enteritidis as can be For quantitation of salmonellae by the most probable number seen by the increase in the D10 value of S. typhimurium (MPN) method, 1-ml portions of frog leg homogenates in lactose broth from 35 to SO krad and of S. enteritidis from 40 to SO were serially diluted and 1 ml each of the decimal dilutions were added krad (Fig. 2). to each of three separate tubes of selenite-cystine broth. This medium The effect of various doses of radiation on the aerobic was incubated for 48 hat 37°C, then streaked on brilliant green agar to determine the number of positive tubes. The Salmonella count was plate count as well as the Salmonella count in fresh estimated from a table of most probable numbers U). samples of frog legs is shown in Table 1. In five different experiments, it was observed that the initial counts of RESULTS AND DISCUSSION total bacteria and Salmonella ranged between 4.8 x 1QS- 7 2 4 x 10 and 2.3 x 10 - 1.5 x 103, respectively. In general, Data on the radiation sensitivity of S. typhimurium, S. higher aerobic plate counts correlated with higher Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/9/820/1655352/0362-028x-45_9_820.pdf by guest on 25 September 2021 newport and S. enteritidis in phosphate buffer are shown Salmonella counts. In all cases it was observed that a in Fig. 1. The survival curves of all three isolates are dose of 300 krad was effective in totally eliminating Salmonella, although the aerobic plate counts were still exponential in nature with D 10 values, i.e., the doses required for 90% inactivation, ranging from 18 to 30 quite significant (102 to 104/g}. krad. None of the survival curves shows the 'tailing effect' Results of studies on the effect of irradiation of frog reported by Quinn et al. (8) for several Salmonella spp. legs in the frozen condition are also shown in Table 1. The D 10 values of these isolates in frog leg homogenate Frozen samples had a similar degree of Salmonella varied from 22 to 38 krad, the homogenate offering only contamination (initial counts) as fresh frog legs, but the aerobic plate counts of the former were less by about two orders of magnitude. It was observed that complete elimination of Salmonella in the frozen samples required l:::r----t:, ~·enteritidis a dose of 400 krad. No growth was observed on any of the o---o §.. typhimurium selective agar plates. Similar results were also obtained D---0 §. newport from inoculation studies indicating that the high levels of S. typhimurium (104 - 105 cells/g) used in these -o- _2. eonteoritidis -a- .2_. typhimurium IN FROZEN FROG LEG HOMOGENATE --' ·1 <{ ~ IN FRESH FROG LEG > HOMOGENATE 0:: :::> Vl ·01 ·1 ;;! ·001 ·0001 ·00 001 L---:!:;--~-::---:-'::1-::-----,J__,_~_.,)._j__~ 50 100 150 300 3 50 DOSE, krad DOSE, k rod Figure 1. Salmonella spp. cultures were suspended (1 x 107 Figure 2. S. enteritidis and S. typhimurium were suspended in cellslm[) in 0.1 M phosphate buffer, pH 7.2, and irradiated frog leg homogenate (1 x 107 cellslml in 10% homogenate) and with gamme rays. Survivors were enumerated on plate count irradiated in the fresh or frozen condition. Survivors were agar. enumerated on plate count agar. JOURNAL OF FOOD PROTECTION. VOL. 45, JULY 1982 822 NERKAR AND LEWIS TABLE 1. Irradiation offrog legs in the fresh and frozen state. a Treatment Aerobic plate Salmonella count (krad) count (per g)b (per g) Fresh 6.7 X 1Q6 ± 0.826 4.3 X 102 ± 1.127 Control Frozen 4.3 X 104 ±0.73 1.5 X 102 ± 0.56 1 Fresh 5.6 X 105 ± 1.00 3.9 X 10 ± 0.740 100 Frozen 3.8 X 103 ± 1.32 7.5 X 101 ± 1.50 Fresh 3.7 X 104 ± 1.33 f X 101 ± 1.240 200 Frozen 1.2 X 102 ± 1.68 3.9 X 101 ± 1.43 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/45/9/820/1655352/0362-028x-45_9_820.pdf by guest on 25 September 2021 Fresh 2.8 X 10 3 ± 1.03 nil 300 Frozen 3X101 ±0.94 4 ± 1.22 400 Frozen nil nil a Frog legs were packaged in presterilized polyethylene bags and irradiated in the fresh or frozen condition at different doses.