J Sci Food Agric 1998, 78, 486È490 Fate of Escherichia coli O157 : H7 in Chinese-Style Sausage Subjected to Diþerent Packaging and Storage Conditions Chyang-Fuah Yu and Cheng-Chun Chou* Graduate Institute of Food Science and Technology, National Taiwan University 59, Lane 144, Keelung Rd, Sec 4, Taipei, Taiwan (Received 21 July 1997 ; revised version received 16 February 1998 ; accepted 20 March 1998) Abstract : In this study, Chinese-style sausages were subjected to air, vacuum or nitrogen packaging and stored at either 5 or 25¡C. The survival characteristics of Escherichia coli O157 : H7 during the storage period were determined. Results revealed that, when stored at 5¡C, the number of viable E coli O157 : H7 in sausages decreased slowly as the storage period extended, regardless of packaging methods. E coli O157 : H7 in sausages decreased from an initial population of ca 5É97 log CFU g~1 to ca 4É42È4É81 log CFU g~1 after 40 days of storage at 5¡C. It was also found that viable cells of E coli O157 : H7 declined more rapidly in sausage stored at 25¡C than at 5¡C. No viable E coli O157 : H7 was detected in either vacuum-packed or nitrogen-packed sausage after 40 days of storage at 25¡C. On the other hand, the population of E coli O157 : H7 reduced to non-detectable levels in air-packed sausages after 20 days of storage. Refrigerated storage and vacuum or nitrogen packaging provided conditions that slowed down the death rate of E coli O157 : H7 in sausage. Furthermore, it was noted that, among the curing agents tested, NaCl exerted the most signiÐcant lethal e†ect on E coli O157 : H7 in sausage during the storage period. ( 1998 Society of Chemical Industry. J Sci Food Agric 78, 486È490 (1998) Key words : Escherichia coli O157 : H7 ; Chinese-style sausage ; packaging and storage pork, beef and poultry (Beery et al 1985 ; Borczyk et al 1987 ; Doyle and Schoeni 1987). Foods of animal origin are regarded to be the primary sources of E coli O157 : H7 infection. Chinese-style sausage is made essentially from ground pork with a larger particle size than is normal in other types of sausage. It is cured and dried during the manufacturing process. The possible presence of E coli O157 : H7 in the raw meat used for the manufacture of Chinese-style sausage, mis-handling at processing facilities and the possibility of cross-contamination from other principal vectors for transmission to pork may all pose potential health risks. Previously, the authors studied the fate of E coli O157 : H7 during the drying steps of the manufacturing process and found that this pathogen might still survive under some of the drying INTRODUCTION Escherichia coli O157 : H7 has emerged as a recognised food-borne pathogen which causes haemorrhagic colitis and haemolytic ureamic syndrome (Riley et al 1983 ; Doyle and Schoeni 1987). Since the recognition of E coli O157 : H7 in 1982, numerous incidents of food-borne disease associated with this pathogen have occurred in the US, Canada and the UK (Doyle 1991 ; Anonymous 1995). Furthermore, it was reported recently that foodborne outbreaks caused by E coli O157 : H7, the largest in history, occurred in Japan, a†ecting more than 9000 individuals and resulting in seven deaths (Anonymous 1996). E coli O157 : H7 has been isolated from retail * To whom correspondence should be addressed. 486 ( 1998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain Fate of E coli in Chinese-style sausage 487 conditions currently employed in the sausage industry in Taiwan (Yu and Chou 1997). In the present study, they determined and compared the behavior of E coli O157 : H7 in sausage subjected in air, vacuum and nitrogen packaging and stored at 5 and 25¡C. In addition, the e†ect of curing agents on the survival characteristics of E coli O157 : H7 during the storage period were also identiÐed. MATERIALS AND METHODS Organisms and preparation of inoculum Four stains of E coli O157 : H7 including 933 (beef isolate), A8993-C32(human isolate), MF6707(veal isolate) and 18731A(veal isolate) were used as test organisms. They were all obtained from the Food Safety Inspection Service, Department of Agriculture, Washington, DC, USA. After two successive transfers of the test organism in tryptic soy broth (TSB, Difco, Detroit, MI, USA) at 37¡C for 24 h, the activated culture was inoculated into TSB and incubated statically at 37¡C for 18 h, when the population was ca 109 CFU ml~1. Equal volumes of the cultures for each strain were then combined to provide a four-strain mixture of E coli O157 : H7 and were properly diluted with TSB to serve as the inoculum. Preparation and storage of the sausage Detailed procedures for preparation of the sausage were described in a previous paper (Yu and Chou 1997). BrieÑy summarised, ground pork consisting of about three parts lean to one part fat was mixed with the TABLE 1 Curing agents for sausage curing ingredient shown in Table 1, unless otherwise speciÐed. After curing at refrigeration temperatures (5È 7¡C) for ca 12 h, the sausage batter was stu†ed into edible collagen casing and sectioned by hand. Each sausage was approximately 7 cm in length and 30 g in weight. The sausages were then subjected to 50¡C airblast drying for 6 h. The prepared sausage was inoculated with E coli O157 : H7 (ca 5É0È6É0 log CFU g~1) using a sterile syringe. They were inserted into either ordinary oxygen permeable polyethylene or nylon/ polyethylene laminate bags (Sun A Enterprise Co, Taichiung, Taiwan). The latter bags, having a gas transmission rate of 70 ml m~2 d~1 at 1 atm at 0% RH and 30¡C, were further vacuum-packed or nitrogenpacked. All the packages were stored at either 5 or 25¡C. Microbiological analysis Samples were assayed for the presence of E coli according to the methods described by Okrend et al (1990) and Abdul-Raouf et al (1993a). For enumeration of E coli O157 : H7, a 25-g meat sample was Ðrst homogenised with 225 ml sterile 0É1% peptone water. Serial dilutions were made with ButterÐeldÏs phosphate diluent. One-tenth of a ml of each dilution was spread on MacConkey sorbitol agar (Difco) with 0É2 g litre~1 4-methylumbelliferyl-b-D-glucuronide (Oxoid, UK) and incubated at 35¡C for 24 h. Typical colonies were picked and conÐrmed as E coli O157 : H7 using biochemical tests of IMViC reaction and lysine decarboxylase production as well as a serological test with O157 and H7 antisera (Difco). Aerobic plate counts (APC) were determined using procedures described in the Bacteriological Analytical Manual (FDA 1992). Determinations of pH, temperature and moisture content Ingredients g kg~1 meat Sucrose Sodium chloride Monosodium glutamate Soybean protein isolate Sodium tripolyphosphate Potassium sorbate Sodium ascorbate Sodium nitrite Rice wine Spices Wu-Shung powder White pepper powder Vanilla powder Cinnamon powder 100É00 20É00 10É00 5É00 2É00 2É50 0É50 0É12 10É00 2É10 1É25 1É25 0É08 The pH of the samples was analysed by blending the samples with equal amounts of distilled water and determining the pH using a digital pH meter (Jenco 6200, Jenco Electronic, Taipei, Taiwan). The moisture content of the sausage was determined by drying the samples at 105¡C (AOAC 1984). Statistical analysis The mean values and standard deviation were calculated from data obtained from three separate experiments. These data were then compared by DuncanÏs multiple range method (SAS 1989). C-F Y u, C-C Chou 488 RESULTS AND DISCUSSION Survival in sausage under di†erent packaging and storage conditions According to the Chinese National Standard, the wet type of Chinese-style sausage should have a water content of less than 40%. The sausages prepared in this study had a water content of about 39É0%, thus meeting the requirement. It is recommended that these sausages be stored at refrigeration temperature (Anonymous 1984). However, in traditional retail markets, sausages are often kept at room temperature for sale. Therefore, the behaviour of E coli O157 : H7 at 25¡C, in addition to that at 5¡C, was also examined in this study. Table 2 shows the survival of E coli O157 : H7 in sausages subjected to vacumm, nitrogen or air packaging and stored at 5 or 25¡C. Stored at 5¡C, regardless of the packaging method, the viable cells of the inoculated E coli O157 : H7 in all sausage showed a slight decrease during the storage period. A population reduction of only 1É16È1É55 log CFU g~1 was observed in sausage after 40 days storage at 5¡C. No signiÐcant di†erence (P [ 0É05) in the viable E coli O157 : H7 count was observed in the sausage with the three di†erent packaging methods. This is consistent with the report of Abul-Raouf et al (1993b), who indicated that no signiÐcant di†erence in the survival of E coli O157 : H7 in salad vegetables subjected to ambient atmosphere packaging and those subjected to packaging under an atmosphere containing 3% oxygen and 97% nitrogen. Regardless of packaging methods, a more rapid decrease in the viable cells of E coli O157 : H7 was noted in sausage kept at 25¡C than in those kept at 5¡C. For example, a 1É69È2É29 log CFU g~1 reduction was TABLE 2 Survival of Escherichia coli O157 : H7 in sausage with di†erent packaging methods and stored at 5¡C or 25¡Ca Packaging method 5¡C Air Vacuum N 2 25¡C Air Vacuum N 2 L og (CFU g~1) after days of storage 0 10 20 30 40 A5É97a A5É97a A5É97a AB5É62a B5É67a AB5É70a B5É20a B5É51a BC5É40a B5É03a C5É02a CD5É13a C4É42a C4É75a D4É81a A5É83a A5É83a A5É83a B3É54b B4É14a B4É00a Èb C3É67 C3É49 È D3É33 D3É11 È È È a Means in the same row with di†erent letters A, B, C, D or E and the same column for each storage temperature with di†erent letters a or b di†er signiÐcantly (P \ 0É05) according to DuncanÏs multiple range test. b No viable E coli O157 : H7 was detected when 0É1 ml of the lowest dilution represent 0É1 g samples were surface plated on the media used. observed in sausage held at 25¡C for 10 days, compared to a 1É16È1É55 log CFU g~1 in sausage held at 5¡C for 40 days. Additionally, a slightly higher number of viable E coli O157 : H7 was noted in the vacuum-packed and nitrogen-packed sausage than in the air-packed sausage. When held at 25¡C for 20 days, E coli O157 : H7 in the air-packed sausages decreased from an initial population of ca 5É83 log CFU g~1 to non-detectable level. At the same time, a higher viable E coli O157 : H7 count of 3É67 and 3É49 log CFU g~1 with only 2É16È 2É34 log CFU g~1 reduction was observed in the vacuum-packed and nitrogen-packed sausage, respectively. Both refrigeration and vacuum packaging have been generally regarded as beneÐcial for extending the shelflife of sausage (Anonymous 1984). Furthermore, a nitrogen gas atmosphere has been reported to be useful in controlling the microÑora and extending the shelf-life of lamb (Newton et al 1977), sandwiches (Kautter et al 1981 ; Bennett and Amos 1982), frankfurters (Simard et al 1983a,b) and pork (Hu†man 1974 ; Enfors et al 1979). However, results of the present study showed that refrigeration, vacuum packaging and nitrogen packaging are actually less detrimental to E coli O157 : H7 than ambient atmosphere packaging (air packaging). Yeh and Chou (1994), in a study of the fate of Campylobacter jejuni during the storage of Chinese-style sausage, found that refrigerated storage and vacuum storage provide conditions that slow down the death rate of C jejuni in sausage. As shown in Table 3, the APC in sausage held at 5¡C did not show any signiÐcant change (P [ 0É05) during the 40-day storage period. Packaging method caused no signiÐcant di†erence (P [ 0É05) in the change of APC in the sausage either. Held at 25¡C, the APC determined after 10 days storage in vacuum-packed or nitrogen-packed sausage showed a slight decrease. However, at the end of a TABLE 3 Changes of aerobic plate count in sausage with di†erent packaging methods and stored at 5 or 25¡Ca Packaging method 5¡C Air Vacuum N 2 25¡C Air Vacuum N 2 L og (CFU g~1) after days of storage 0 10 20 30 40 A6É12a A6É12a A6É12a AB5É85a A5É93a A6É05a AB5É72a A6É05a A5É95a AB5É90a A6É00a A5É92a B5É47a B5É52a B5É71a B6É10a C6É10a D6É10a A7É16a D5É27b E5É12b A7É42a A7É44a B7É69a A7É04b A7É70a A8É00a A7É29a B6É63b C6É84ab a Means in the same row with di†erent letters A, B, C, D or E and the same column for each storage temperature with di†erent letters a or b di†er signiÐcantly (P \ 0É05) according to DuncanÏs multiple range test. Fate of E coli in Chinese-style sausage 489 Fig 1. E†ect of sodium chloride on the survival of E coli O157 : H7 in sausage during storage at 25¡C in vacuum package : (…) 0 g kg~1 ; (L) 10 g kg~1 ; (@) 20 g kg~1 ; (È) 30 g kg~1 ; (=) 40 g kg~1 ; standard deviations are represented by the error bars. 40-day storage period, a slight increase of APC (0É94È 1É90 log CFU g~1) was found in all the sausages, regardless of the packaging method (Table 3). Acidity was regarded to be one of the principle factors which leads to the inactivation of E coli O157 : H7 during the storage of fermented dry sausage (Glass et al 1992). However, the pH of the sausages prepared, having an initial pH of 6É20È6É34, showed no signiÐcant change during the 40-day storage period, regardless of the packaging method and storage temperature (Table 4). Therefore, under the test condition, acidity should not exert any detrimental e†ect on the survival of E coli O157 : H7 and other microÑora during the storage of Chinese-style sausage. Survival in sausage containing various amount of NaCl, sorbate and nitrite during storage In order to identify and compare the antimicrobial activity of NaCl, sorbate and nitrite, sausages with various formula were prepared. Ground meat was Ðrst mixed with the curing ingredients listed in Table 1, with the exception of NaCl, sorbate and nitrite. Various amounts of either NaCl, sorbate or nitrite were then Fig 2. E†ect of sodium nitrite on the survival of E coli O157 : H7 in sausage during storage of 25¡C in vacuum package : (…) 0 g kg~1 ; (L) 0É07 g kg~1 ; (@) 0É10 g kg~1 ; (È) 1É20 g kg~1 ; (=) 1É50 g kg~1 ; standard deviations are represented by the error bars. added into the batter to prepare the sausage. Figures 1È3 show the survival of the inoculated E coli O157 : H7 in the prepared sausages subjected to vacuum packaging and held at 25¡C for a period of 15 days. A signiÐcantly higher reduction (P \ 0É05) in the viable E coli O157 : H7 was noted in sausages containing NaCl than that in sausage containing no NaCl (Fig 1). It was also found that, as the content of NaCl increased or storage period extended, the reduction of viable E coli O157 : H7 increased. After a 15-day storage period, the viable E coli O157 : H7 in sausage containing 20 g kg~1 NaCl, the level commonly employed, decreased from an initial population of ca 5É89 to 5É16 log CFU g~1. On the other hand, sausage with no added NaCl still had an E coli O157 : H7 population of ca 5É63 log CFU g~1, close to the initial population. Although a slight change in the population of E coli O157 : H7 was observed in sausages combined with the amount of sodium nitrite (0É07È0É15 g kg~1) during the storage period, the population in sausage containing sodium nitrite displayed no signiÐcant di†erence TABLE 4 Changes of pH in sausage with di†erent packaging methods and stored at 5 or 25¡C Packaging method 5¡C Air Vacuum N 2 25¡C Air Vacuum N 2 pH value after days of storage 0 10 20 30 40 6É20 6É20 6É20 6É16 6É21 6É16 6É22 6É20 6É17 6É18 6É24 6É21 6É19 6É19 6É24 6É34 6É34 6É34 6É36 6É41 6É38 6É43 6É41 6É41 6É35 6É36 6É45 6É39 6É37 6É41 Fig 3. E†ect of potassium sorbate on the survival of E coli O157 : H7 in sausage during storage at 25¡C in vacuum package ; (…) 0 g kg~1 ; (L) 1É0 g kg~1 ; (@) 1É5 g kg~1 ; (È) 2É0 g kg~1 ; (=) 2É5 g kg~1 ; standard deviations are represented by the error bars. C-F Y u, C-C Chou 490 (P [ 0É05) from that in sausage without sodium nitrite (Fig 2). Meanwhile, the Ðnal E coli O157 : H7 population found in sausage containing 2É0 g kg~1 or less sorbate was not signiÐcantly di†erent from that without sorbate (Fig 3). However, a signiÐcantly less E coli O157 : H7 population (P \ 0É05) was detected in sausage containing 2É5 g kg~1 sorbate. CONCLUSIONS Based on the data obtained in this study, it is concluded that, among the curing agents tested, NaCl exhibited the most profound lethal e†ect on E coli O157 : H7 in sausage during the storage period. If E coli O157 : H7 in the raw material was not completely inactivated during the sausage processing, as noted previously (Yu and Chou 1997), or if post-processing contamination occurs, the general practice of vacuum packaging or nitrogen packaging and refrigerated storage, while extending the shelf-life of the sausage, would also enable the contaminating E coli O157 : H7 to survive longer. ACKNOWLEDGEMENT This research was supported by the Department of Health, Executive Yuan (DOH 84-FS006), Taiwan, ROC. REFERENCES Abdul-Raouf U M, Beuchat L R, Ammar M S 1993a Survival and growth of Escherichia coli O157 : H7 in ground, roasted beef as a†ected by pH, acidulants, and temperature. Appl Environ Microbiol 59 2364È2368. Abdul-Raouf U M, Beuchat L R, Ammar M S 1993b Survival and growth of Escherichia coli O157 : H7 on salad vegetables. Appl Environ Microbiol 59 1999È2006. Anonymous 1984 Manufacture of Chinese style sausage. Meat T oday 2 2È6. Anonymous 1995 Escherichia coli O157 : H7 outbreak linked to commercially distributed dry-cured salami. Mortal. Weekly 44 (9) 157È160. Anonymous 1996 The latest news about O157. Up-to-Date Food Proc 31 (9) 31È42. AOAC 1984 Official Methods of Analysis (14th edn). Association of Analytical Chemists, Washington, DC, USA. Beery J T, Doyle M P, Schoeni J L 1985 Colonization of chicken cecae by Escherichia coli associated with hemorrhagic colitis. Appl Environ Microbiol 49 310È315. Bennett R W, Amos W T 1982 Staphylococcus aureus growth and toxin production in nitrogen-packed sandwiches. J Food Prot 45 157È161. Borczyk A A, Karmali M A, Lior H, Duncan L M C 1987 Bovine reservoir for verotoxin producing Escherichia coli O157 : H7. L ancet 1 98. Doyle M P 1991 Escherichia coli O157 : H7 and its signiÐcance in foods. Int J Food Microbiol 12 289È302. Doyle M P, Schoeni J L 1987 Isolation of Escherichia coli O157 : H7 from retail fresh meats and poultry. Appl Environ Microbiol 53 2394È2396. Enfors S O, Molin G, Ternstrom A 1979 E†ect of packaging under carbon dioxide, nitrogen or air on the microÑora of pork stored at 4¡C. J Appl Bacteriol 47 197È208. FDA 1992. Bacteriological Analytical Manual (7th edn). Food and Drug Administration, Washington, DC, USA. Glass K A, Loe†elholz J M, Ford J P, Doyle M P 1992 Fate of Escherichia coli O157 : H7 as a†ected by pH or sodium chloride and in fermented, dry sausage. Appl Environ Microbiol 58 2513È2516. Hu†man D L 1974 E†ect of gas atmospheres on microbial quality of pork. J Food Sci 39 723È725. Kautter D A, Lynt R K, Lilly T, Solomon H M 1981 Evaluation of the botulism hazard from nitrogen-packed sandwiches. J Food Prot 44 59È61. Newton K G, Harrison J C L, Smith K M 1977 The e†ect of storage in various gaseous atmospheres on the microÑora of lamb chops held at [ 1¡C. J Appl Bacteriol 43 53È59. Okrend A J G, Rose B E, Bennett B 1990 A screening method for the isolation Escherichia coli O157 : H7 from ground beef. J Food Prot 53 249È252. Riley L W, Remis R S, Helgerson S D, McGee H B, Wells J G, Davis B R, Hebert R J, Olcott E S, Johnson L M, Hargrett N T, Blake P A, Cohen M L 1983 Hemorrhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 308 681È685. SAS 1989 SAS UserÏs Guide : Statistics (Vers 6). SAS Institute, Cary, NC, USA. Simard R E, Lee B H, Laleye C L, Holley R A 1983a E†ects of temperature, light and storage time on the microÑora of vacuum- or nitrogen-packed frankfurters. J Food Prot 46 199È205. Simard R E, Lee B H, Laleye C L, Holley R A 1983b E†ects of temperature, light and storage time on the physicochemical and sensory characteristics of vacuum- or nitrogen-packed frankfurters. J Food Prot 46 188È195. Yeh C H, Chou C C 1994 Behavior of Campylobacter jejuni during the manufacture and storage of Chinese-style sausage. Food Microbiol 11 461È466. Yu C F, Chou C C 1997 Fate of Escherichia coli O157 : H7 in Chinese-style sausage during the drying step of the manufacturing process as a†ected by the drying condition and curing agent. J Sci Food Agric 74 551È556.