a‘. SURVWAL 0F SELECTED PATHOGENIC ORGANESMS DURtNG‘ PROCESSING OF A FERMENTED TURKEY SAUSAGE PRODUCT Thesis for the Degree of Ph. D. MlCHiGAN STATE UNEVERSITY. WILLEAM LEE BARAN 1973 SURVIVAL OF SELECTED PATHDGENIC ORGANISMS DURING PROCESSING OF A FERMENTED TURKEY SAUSAGE PRODUCT Thesis for the Degree of Ph. D. 'MICHTGAN STATE UNIVERSITY. WILLLAM LEE BARAN 197 3 This is to certify that the thesis entitled SURVIVAL OF SEIEC‘I'ED PATHOGENIC ORGANISIVB DURING PROCESSING OF A FERMEN‘I'ED TURKEY SAUSAGE PRODUCT presented by William Lee Baran has been accepted towards fulfillment of the requirements for Wares: in —Feeé—Se—ie&ee v Major professor Date 7/24/ /g{ ”757 // , ‘D ‘ p‘.. fill ‘ | .5 amnme av "' i ROAR 8: SUNS‘ 5 BOOK BINDERY THE. 3 LIBRARY BINDERS SPRINGPORT. MICHIGI] \— ’— -"‘-"fi‘ 3..st A v fi"'\'\‘ U'bns‘.‘ 0—.“ “IKE." ‘ . : l « \‘., :‘ “‘50 Q" “V "“A‘ u '7... 4b.. . i the bl ::;sa~ l U ‘. 2"‘s ‘IQ: ‘ "w d o *D. .'-. '~:.; v.: ‘A ”y A s § C» ABSTRACT SURVIVAL OF SELECTED PATHOGENIC ORGANISMS DURING PROCESSING OF A FERMENTED TURKEY SAUSAGE PRODUCT BY William Lee Baran The potential growth or survival of selected pathogens in a fermented turkey sausage process was determined. A fermented sausage was developed using 45% dark turkey meat, 45% light turkey meat, and 10% turkey fat. Sorbitol was used as a humectant and improved the binding properties of the sausage mixture. The sausage mixture was stuffed into 14 mm casing and heated for 3 hrs. at 27 C, 4 hrs. at 32 C, and 5 hrs. at 46 C. The product could be developed further for commercial use. The complete time for processing was reduced to 8 days. Pathogens such as Salmonella EEEf' Staphylococcus aureus, Clostridium perfringens, and enteropathogenic strains of Escherichia coli were inoculated into the sausage at various concentrations from 14 organisms/g to 2.2 X 106 organisms/g. Most of these selected pathogens have been responsible for food- borne disease outbreaks associated with turkey products. .‘ "huh” 4 3" 'IITT. J'i-WIW ‘ j 1:. TI-Il' ens . ,- .uv ‘- . . £25 382823.“. 31:3; ens . '.'"A" “a ’Load SH; . :’o. . ‘ “ ‘ s an ""“O: ~$§Joo 9'»- Lecreased > - z: “‘0' R ‘. “vooe-‘ae . D ~ 2‘s ....._ h“ a / J 'L’ O :33 823 V “.3118. A x :.A use: N. h: ‘c in; 'x‘os ‘5 he: ‘8 ,;.. o.~ ‘n 5‘et ’1: T .x \l.." \'e ‘Hv. \ William Lee Baran Standard methods were used to enumerate the pathogens. Aerobic plate counts, lactic acid bacteria counts, and counts of coliforms and yeasts and molds were determined along with the counts of the selected pathogens. Aerobic plate counts were reported for the various spices, freshly processed turkey meat, and meat formulation before and after processing. The results of this investigation indicated that most pathogens were not completely destroyed by the pro- t cess at the inocula used. Salmonellae populations 9 decreased 1 5.1 to 1.3 logs depending on strain of Salmonellae and initial inoculum used. Clostridium per- fringens populations were reduced 0.55 to 3.4 logs. There was very little difference in destruction of C. perfringens between heat-sensitive and heat-resistant strains. A reduction of 0.83 to 3.0 logs occurred with enteropathogenic Escherichia coli. E. coli strain 0128 was relatively resistant to the fermented turkey sausage process. Staphylococcus aureus strain 243 multiplied during the processing of the sausage even with as low an inoculum as 5500 organisms/g meat. However, no detectable enterotoxin was found in any of the sausages sampled. The highest g. aureus cell cound in the finished product was 2.3 x 107 cells/g meat. These results indi- cate that most pathogens, if present in high.numbers, survive the process used for fermented turkey sausage. in De SURVIVAL OF SELECTED PATHOGENIC ORGANISMS DURING PROCESSING OF A FERMENTED TURKEY SAUSAGE PRODUCT BY William Lee Baran A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1973 DED ICAT ION To my wife, Sara ii excuse f c : rw ‘c. . :rcfess ::;:se of t :‘e "V A F\ *Utao To t}: ”'8‘ .‘oi‘bg \MS of e:- Q, . “SeCla .: u. ‘ A ACKNOWLEDGMENTS This author wishes to express appreciation and gratitude for the guidance and interest given by his major professor, Dr. Kenneth E. Stevenson, during the course of the investigation and preparation of this thesis. To the graduate guidance committee which con- sisted of Dr. L. E. Dawson, Dr. L. G. Harmon, Dr. J. F. Price, and Dr. D. E. Ullrey appreciation is expressed for their contribution of time, equipment, and suggestions concerning this project. Special appreciation is given to Mrs. Marguerite Dynnik for her assistance and patience in preparing materials for this study. Many thanks are given to Dr. E. H. Marth, Department of Food Science, University of Wisconsin, and Dr. F. L. Bryan, U.S. Public Health Service, Center for Disease Control, Atlanta, Georgia, for supplying cultures of enteropathogenic Escherichia coli strains. Special thanks is given to Mr. Gary Gann for his help in making quality slides and Mr. Allen Kirleis iii .o. uaob -. y H l ‘-A n...“ ~§o;‘~: ..~‘ I ' . . 0... “ . c ....--.-€S a is: :3 the I;':#- . ‘ :5*~a.ci rue". d1 for his suggestions concerning graphs for this thesis. Also, special thanks to my fellow graduate students who encouraged me and provided assistance when it was needed. This author is indebted to his wife and family for their support, encouragement, and interest throughout his school years. Appreciation is given to the Department of Food Science and Human Nutrition for providing excellent facilities and the funds to make this study possible. Also to the Veterans Administration for providing personal monthly funds through the G.I. Bill. iv r21- ”en-“an. Al‘v c‘u‘qvvyo‘go‘ . nog‘u..:‘ “1‘"? 1'. l ' ‘ ”‘Ottnov.\~ ‘u, C: “An ‘ ‘- UncuuV.5 “ fl‘ - . fia.’ ’4“ VOUbb‘lu‘ a "‘ Vfln & "" ivvau‘ h I h ‘ ‘ '5'!on A. :vavolv‘0\'r ' J (‘1 "C‘ Y' ' A .‘ V.va. lul 7‘ a .CX3;;357.t : y n a, “‘ ‘16:-rr0Cl \‘n ”I “t" pro-d... herotioll :Ezented Sa' NI. ‘ ~be 0‘ St: rOCESSin: I U C I flu. ““599 IRCI! - d ‘:§u‘ TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . Poultry Carcass Quality . . . . . . . Initial contamination . . . . . . . Salmonellae . . . . . . . . . . Clostridium perfringens . . . Enteropathogenic Escherichia coli . Staphylococcus aureus . . . . . . Clostridium botulinum . . . . . . Toxoplasma gondii . . . . . . . Further-Processed Products . . . . . . New products and legislation. . . . . Microbiology of new products. . . . . Fermented Sausages . . . . . . . . . Use of starter cultures . . . . . . Microbiology of fermented sausage . . . Processing of fermented sausage. . . . MATERIALS AND METHODS o o o O o o 0 Q o Sausage Ingredients and Spices . . . . . Sausage Process . . . . . Sample Preparation for Microbial Evaluation. Preparation of Culture Inocula . . . . . Enmmeration of Selected Pathogens . . . . Salmonellae . . . . . . . . . Clostridium perfringens . . . . . . Enteropathogenic Escherichia coli . . . Coagulase-Positive Staphylococcus aureus. Page 0» H haw N c>owo~aoxpwe FJH new I-" 03 HPHFJ xooxm N H NNNNN \anfile‘" 27 29 32 .- n 9. ‘ha no»--. \ ,1 -I-.~ . 0"- s. “Ti" ’9. “ N... “K H Detection of Staphylococcus Aureus Entero— toxin . . Enumeration of Nonpathogenic Microorganisms Aerobic plate counts . Lactic acid bacteria . Coliform . . . . . Yeast and molds. . . pH and Total Acidity Determinations RESULTS AND DISCUSSION . . Spice Contamination . . Processing Parameters. . Microbial Contamination During Processing Studies of Selected Pathogens Salmonellae . . . . Clostridium perfringens Enteropathogenic Escherichia coli. Staphylococcus aureus. CONCLUSIONS . . . . . . BIBLIOGRAPHY. . . . . . APPENDIX Appendix A. Tables . . . . . vi 0 O O O Page 32 33 33 33 34 34 36 36 36 40 42 42 46 49 51 59 61 71 DO| n“. “ST, . (I) '1'! J Table II. III. IA. IIA. IIIA. IVA. LIST OF TABLES Spices and ingredients used in the turkey sausage formulation . . . . . . . Microbial counts of spices and ingredients added to the turkey meat mixture. . . Processing results for a fermented turkey sausage product inoculated with four different pathogenic organisms . . . Aerobic plate counts of turkey sausage at various processing steps . . . . . Determination of total acidity, pH, salmon- ellae counts, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Salmon- ella pullorum . . . . . . . . . Determination of total acidity, pH, salmon- ellae counts, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Salmon- ella senftenberg 775 W . . . . . . Determination of total acidity, pH, counts of Clostridium perfringens, and percent weightTISSS’ifi four experimental batches of fermented turkey sausage inoculated with Clostridium perfringens ATCC 3624. Determination of total acidity, pH, counts of Clostridium perfringens, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Clostridium perfringens NCTC 8238. vii Page 23 37 38 41 71 72 73 74 Inn. «y‘ ‘ n one! We. - Nun ”n l q “‘00 VI ‘ “'- o Table Page VA. Determination of total acidity, pH, counts of Escherichia coli, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Escherichia coli 026 . . . . . . . . 75 VIA. Determination of total acidity, pH, counts of Escherichia coli, and percent weight loss in three experimental batches of fer- mented turkey sausage inoculated with Escherichia coli 0128. . . . . . . . 76 VIIA. Determination of total acidity, pH, counts of Escherichia coli, and percent weight loss in three experimental batches of fer- mented turkey sausage inoculated with Escherichia coli 0125. . . . . . . . 77 VIIIA. Determination of total acidity, pH, counts of Staphylococcus aureus, and percent weigfit Toss in {Bree experimental batches of fermented turkey sausages inoculated with Staphylococcus aureus strain 243 (unwashed cells) . . . . . . . . . 78 IXA. Determination of total acidity, pH, counts of Staphylococcus aureus, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Staphylococcus aureus strain 243 (washed celIs) . . I. . . . . . . . 79 XA. Various nonpathogenic microorganisms enumerated in processed sausages inocu- lated with Salmonella pullorum and Salmonella senftenberg 775 W . . . . . 80 XIA. Various nonpathogenic microorganisms enumerated in processed sausages inocu- lated with Clostridium perfringens ATCC 3624 . . . . . . . . . . . . . 81 XIIA. Various nonpathogenic microorganisms enumerated in processed sausages inocu- lated with Escherichia coli strain 026 . . 82 XIIIA. Various nonpathogenic microorganisms enumerated in processed sausages inocu- 1ated with Staphylococcus aureus strain 243 (washed cells). . . . . . . . . 83 viii '1'! l 0 M "1 n v-zv n n- ('1 0') LIST OF FIGURES Figure Page 1. Effect of fermented turkey sausage processing on the survival of Salmonella pullorum inoculated at three different concen- trations and enumerated by the three tube MPN technique . . . . . . . . . 43 2. Effect of fermented turkey sausage processing ~5' on the survival of Salmonella senftenberg, 3 775 W inoculated at three different concen- _ _ trations and enumerated by the three tube ‘;4“ MPN technique. . . . . . . . . . . 44 3. Effect of fermented turkey sausage processing on the survival of Clostridium perfringens ATCC 3624 inoculated at four different con- centrations and enumerated by the SPS pouch method . . . . . . . . . . . 47 4. Effect of fermented turkey sausage processing on the survival of Clostridium perfringens NCTC 8238 inoculated at three different concentrations and enumerated by the SPS pouch method . . . . . . . . . . . 48 5. Effect of fermented turkey sausage processing on the survival of Escherichia coli strain 026 inoculated at three different concen- trations and enumerated by the three tube MPN technique. . . . . . . . . . . so 6. Effect of fermented turkey sausage processing on the survival of Escherichia coli strain 0128 inoculated at three different concen- trations and enumerated by the three tube MPN technique. . . . . . . . . . . 52 Figure Page 7. Effect of fermented turkey sausage processing on the survival of Escherichia coli strain 0125 inoculated at three different concen- trations and enumerated by the three tube MPN technique . . . . . . . . . . . 53 8. Effect of fermented turkey sausage processing on the survival of unwashed cells of Staphylococcus aureus strain 243 inoculated at Ehree’different concentrations and enumerated by the Vogel-Johnson plate count method . . . . . . . . . . . 54 9. Effect of fermented turkey sausage processing on the survival of washed cells of Staphylococcus aureus strain 243 inocu- lated at three different concentrations and enumerated by the Vogel-Johnson plate count method . . . . . . . . . . . . . 55 INT RODU CT ION walker and Ayres (1959) investigated the micro- organisms present on carcasses of commercially processed turkeys, but few investigations have been reported on the microbiology of new further-processed turkey products such as turkey frankfurters, turkey bologna, or turkey— fermented sausage. Turkey carcasses and turkey rolls have been examined for the presence of salmonellae, Clostridium perfringens, and Spaphylococcus aureus. However, the quantitation of salmonellae in further- processed turkey products has been omitted in most of the research to date. Turkey meat has been implicated as a vehicle of foodborne disease. An averaging of data from yearly foodborne disease reports indicates that turkey products were the vehicles in 8.4% of the outbreaks [United States Department of Health, Education, and Welfare (USDHEW) , 1967-1971]. In the same five-year period, C, perfringens was the most prevalent pathogen present in turkey-related outbreaks of foodborne disease, accounting for 37%, followed by Salmonella spp. 27%, Staphylococcus aureus 23%, and unknown sources 12%. “1'5" ' ' ‘u’o'. .|‘ I 'I - . -. v 4 . m we" I I; n . . . , - .o. ' . ‘.‘.'§.b 0“" s“. .l, ...o‘.' ., ' . a. .o O o -_ .~_' ransmva «r ~ - 9“ . \ _._..-qv ' "R, . .fl-n' ,5. air"!!! _. .v‘.‘ eon-O A ’DV'. ': F ‘. 9 {yo tn 0 u... UV .' a O D.- .‘Dq F at Wu...“ . ~01... oil-b. . I . ' “Ann,— _ Fl ng~s.bao O "O Foodborne disease has been grossly under-reported (USDHEW, 1971) . In addition, the problem of determining the source of the pathogenic organisms has been neglected in most cases which have been reported. The parasite, Toxoplasma gondii, may be of con- cern to food processors if new turkey products are developed which do not rely on heating to assure a safe product. In order to destroy T. gondii, turkey meat used in fermented sausage formulations should be processed to a temperature of at least 137 F or should be frozen for 15 to 20 days prior to use. In this study a small diameter fermented turkey Sausage was developed and microbiological tests were made to determine if such a product presents any potential public health hazard. The sausage was inoculated with various initial numbers of C. perfringens, Salmonella gpp., enteropathogenic Escherichia coli, and Staphylo- cLoccus aureus. The effect of processing on the survival and possible growth of the inocula was determined. The implications of the results with respect to health were evaluated . L ITERATURE REVIEW Poultry CarcassLQuality Initial contamination. Ayres (1951) stated that the type of flora encountered as well as the initial rmmber of microorganisms has a definite influence on the ultimate storage life of meat. Studies have deter- nuned the generic distribution of microorganisms isolated from the surfaces of beef sausage and chickens. Jensen Q945) found that the surface of beef sausage normally contained eight genera of bacteria. Ayres g£_al, (1950) reported that surfaces of chicken carcasses contained 14 genera of bacteria and Gunderson §E_al. (1947) reported an additional 7 genera as being present on chicken carcasses. Although there have not been definite studies concerning the generic distribution of microorganisms isolated from the surfaces of cured meats or turkey carcasses, Pseudomonas spp. and Achromobacter gpp, have been suggested as the organisms primarily responsible fiu'the spoilage of turkey meat (Mast and Mountney, 19“”. Lactobacillus was the predominant bacterial if ~. . ‘ J {Anonr 5.: .~ ‘005 .-.-FA~"": d.-V‘uv"' nou- on - In; 2' 5 00-. . q . ‘. "at-cu... 3; :Aj-FJ 'P ~vlisoH ”'34-“; “We..." . . .I : 1" He.» . C: Uu _ "s. ‘ "~:., {’5‘ ‘9Vo wfi' Ir.~" ‘ "w... 3.0 '- ."‘ t 'v‘: :P, ' 84“ genus found in cecal feces of turkeys (Harrison and Hansen, 1950), and Corynebacterium, Pseudomonas, Sarcina, Micrococcus, and Brevibacterium species were prevalent in turkey giblets (Salzer 35 Sin 1967) . The numbers and types of microorganisms encountered in the evisceration and dressing of poultry were estab- lished by Gunderson it. a_l_. (1954) and Walker and Ayres (1959). Walker and Ayres (1959) investigated the micro- organisms associated with commercially processed turkeys and found the bacterial population on the turkey skin increased ten-fold during processing. The final product contained a median count of 44,000 bacteria/cm2 of skin surface . Salmonellae. Since Salmonella spp. were reported as the prevalent species in poultry products, emphasis has been placed on research investigations of salmonellae in turkey products. Salmonella spp. were isolated more often from turkeys than from chickens, probably due to lower numbers or more fastidious types of salmonellae present on chickens (Walker and Ayres, 1959) . A survey of market poultry by Sadler and Corstvet (1965) showed that 4.16% of the turkeys, 1.42% of the chicken fryers, and 0.65% of the chicken hens being com- mercially slaughtered carried salmonellae in their intestinal tracts. Their results also indicated that -n‘-‘ ." .X ”ali' O - ~ 3 .UI' “. ~a...” “‘vu C H ‘ ' V. ‘Vf‘ I. .‘.‘ 'I... ‘ 03;. ‘~. . . I e l (I '9' birds contaminated with salmonellae were slaughtered on 56% of the days for turkey, 32% for chicken fryers, and 14% for chicken hens. Bryan 2E _al. (1968) evaluated "further-processed" turkey products and found that swab samples from chilled, eviscerated turkey carcasses, and finished products, most frequently contained serotypes of Salmonella san— diego and Salmonella anatum. S. anatum was the most common of 7 serotypes of Salmonella spp. isolated from turkey giblets by Salzer g _a_l_.' (1967) . Baran 23. El. (1973) failed to recover Salmonella gpp. from turkey carcass meat samples which were chilled in three rocker chiller tanks containing chlorinated water. Thus, the number of Salmonella _s_pp_. found on turkey products may be related to the sanitation pro- cedures practiced in the various plants. Surveys conducted at turkey processing plants indicated that both equipment and plant workers were associated with the transfer of salmonellae to finished products (Brobst gp a_l., 1958; Bryan _e_t; _a_l;., 1968; Dixon and Pooley, 1961, 1962; Galton 23 E” 1955). In one survey of a turkey processing plant, the defeathering equipment was contaminated with salmonellae 76% of the time; also, 63% of the carcasses leaving the defeathering machine were positive for salmonellae (Bryan, 1965). However, airborne contamination by I?! ~ 0 3>--:EL¢E b... (r) In..." "'.~~~ ‘ sv'vuunob U _y..-~ I ' -...~ I to. 4' l ' . \u'c‘ “ I l'un-U‘. ‘ .V ‘ "'p-n ,. n H ‘ “Cow“. . .y '0"! “"u. N " ”I" “‘1‘ ' w. .. .‘ 1.53.3.9! o.:.. . ‘ 4 7‘~-~~at fits». . 'u C a l - r k M ‘ b. _J N R) salmonellae in turkey processing plants was found only in the live-bird and picking room areas, whereas the pro- cessing areas were free from airborne salmonellae con- tamination (Zottola e_t_ a_]_._., 1970). Surveys of dressed poultry collected from poultry processing plants or from retail markets have shown varying, but frequently high, levels of salmonellae (Schneider and Gunderson, 1949; Thatcher and Loit, 1961; Felsenfeld g3 _a_l., 1950; Wilson _e_t; 3.1." 1961; Woodburn, 1964; Bryan e_t_ _al” 1968; and Wilder and MacCready, 1966). No studies were found in which Salmonella spp. were quantitated for turkey products. Clostridium perfringens. In studies by Yamamoto E a]: (1961), 110 turkeys were sampled using Ellners' medium and 28 samples contained (_2_. perfringens. However, Baran g2 all. (1973) using direct plating methods found low numbers of C. perfringens ( < 10 organisms/g to 435 organisms/g) from turkeys chilled in rocker chillers containing chlorinated water. Commercial turkey pro- ducts contained 110 to 500 viable cells of C. perfringens/g (Shahidi and Ferguson, 1971) . Frozen further-processed turkey products contained _C_. perfringens in 20% of the 35 samples examined. Lillard (1971) recovered _C_. perfripgens from chicken carcasses before processing in the range of 5 < 10 to 10 cells/g, but found < 10 cells/g after the 0'. .'-' -"..“' “=5 '61 III .‘I OCI no. u. '1 nurhgl II tl~ 1.; ~o..nr ‘.f.| 0' “OJ u n...,‘_ I- I e-o'uvan .:‘; up< "v ax, :‘r‘ A “on! IICZE ~ -I I chill tank treatment. However, she indicated that using enrichment techniques, 10% of the turkey breasts sur- faces were contaminated with C. perfringens after chilling in a chill tank which did not contain chlorinated water. This result may show a spreading of C. perfringens from contaminated birds to relatively clean birds during the chilling process. In another study, Hall and Angelotti (1965) reported that 58% of 26 commercially processed chickens contained (_2. perfringens. Although large populations of C. perfringens were not found using direct counting methods, the use of enrichment techniques did show frequent contamination of frozen turkey products by C. perfringens (Zottola and Busta, 1971). Roast turkey involved in a food poisoning incident contained 3.3 X 105 g. perfringens/g (Shahidi and Ferguson, 1971). Thus, the numbers of g, perfringens present on turkey products initially is low, but improper handling and storage may cause suf- ficient growth of the organism to produce a food-poison- ing outbreak . Enterppathogenic Escherichia coli. For years, enteropathogenic strains of Escherichia coli (EEC) have been associated with gastroenteritis occurring mostly in infants. Recently, food has been implicated as a source in a few cases of EEC infections in adults. I'. '05 I ..p .0. ~ I'A- I..\ \.' Adults can be asymptomatic carriers of EEC and transfer the organisms to children. The identification and survival of EEC in dairy pmoducts have been the subjects of several investigations (Charter, 1965; Jones g£_al., 1967; Read 32 al., 1961; Wilson and Weiser, 1949; and Yang and Jones, 1969). Yang and Jones (1969) studied the physiological char- acteristics of EEC and nonenteropathogenic E, ggli (NEEC) isolated from pasteurized dairy products produced in Canada. They were interested in finding a simple biochemical test to differentiate between the pathogenic and nonpathogenic _E. coli. They determined that some EEC were capable of surviving a laboratory batch pas- teurization of 145 F for 30 minutes. Strain PE 616 (serotype 0128:812) and strain PE 815 (serotype 0119:B14) had 0147 values of 20 and 10 minutes, respectively. Storage tests indicated some EEC strains could survive and multiply at 7 C whereas the NEEC strains did not sllrvive. One strain, PE 712 (serotype 026:36), was able to increase 1,000-fold in 6 days at 7 C. Recently, imported French cheese was shown to ccDntain EEC ("serogroup" 0124) . The organism was isolated from the cheese and from stools of several patients who acquired EEC infections as a result of ingesting the cheese. This was the first well—documented u v“ u't - v o'. I.' u u u.- food-poisoning outbreak caused by enteropathogenic E. c_o_l_i in the United States [Center for Disease Con- trol (CDC), 1971]. Research to determine if EEC is present in poultry products has been limited. However, Tamura spill. (1971) found 9.3% of 188 frozen broilers con- taminated with EEC. Staphylococcus aureus. Contamination of poultry Products by Staphylococcus aureus is usually a result Of human negligence. The organism does not grow when high numbers of Streptococcus spp. are present (Gilliland and Speck, 1972) . Contamination of poultry products by §Eaphylococcus aureus is potentially significant and uSually occurs after processing. Walker and Ayres (1959) found Staphylococcus app. Present on turkey skin surfaces in the range of < 10 to 3 o 100 cells/cmz, whereas counts from the scald and chill water tanks did not exceed 500 cells/ml. However, the skin surface counts reported were high since only 38% of 260 colonies tested were coagulase-positive staphylo- roci. The quantities of Staphylococcus _s_pp. isolated fJi‘om turkey products were no higher than those reported for chickens (Walker and Ayres, 1959) . Ostovar it; 2.]: (1971) did not find Staphylococcus gpp. present on TIA 10 deboned turkey meat. However, Zottola and Busta (1971) found 71% of the frozen raw turkey products they sampled contained staphylococci. Clostridium botulinum. The presence of Clostridium botulinum spores in raw meats in the United States and Canada is rare (Ingram and Roberts, 1966) . Botulism resulting from the ingestion of poultry is also rare. From 1899-1968 only 2 of 647 reported botulism outbreaks were associated with poultry products (CDC, 1968) . Abrahamsson and Riemann (1971) sampled 372 meat products and found 5 samples contained type A and 1 sample con- tained type B Q. botulinum. Of 41 samples of smoked tturkey examined, only 1 contained type B toxin (Abrahams- Son and Riemann, 1971) . Toxgalasma gondii. A sausage mixture which con- tains pork is required by law to be heated to an internal te-l'uperature of 137 F or held frozen for 20 days prior to use in order to destroy the organisms which cause trichi- n<>sis. Turkey sausage which contains no pork would be exempt from this law. However, Toxoplasma gondii, an intracellular protozoan which is widely distributed gecagraphically, could possibly be a problem in under- ccDoked poultry. Jacobs 35 El. (1962) studied the amount of T gondii infections which occurred naturally in poultry \. ‘- ‘u ll originating along the eastern seaboard of the United States and processed in a Baltimore poultry plant. They tested 124 pooled samples each consisting of 10 oviducts or ovaries from hens that appeared normal on macroscopic inspection of their viscera. They found 9 ovary pools and 10 oviduct pools contained T. gondii. Forty—six individual birds were also examined using the ovary, shelled eggs, leg muscle, and brain from each bird. Four ovaries and 1 leg muscle sampled contained 3. gondii While the other tissues of these birds were negative for E. gondii. Jacobs and Melton (1966) using the digestion- inoculation technique examined 108 individual hens and discovered 4 hens with chronic toxoplasmosis; none of the 108 shelled eggs taken from these birds contained 2- gondii. One egg was found to contain T. gondii when 327 eggs were examined from 16 birds with experimentally induced chronic infections. When sacrificed 3 to 10 mOnths after inoculation with T. gondii, all inoculated birds had T. gondii cysts in l or more of the following QJfi‘gans or tissues: brain, ovary, oviduct, kidney, gizzard, and intestines. Bickford and Saunders (1966) examined chickens 1:hiat received intramuscular inoculations of T. gondii. They found the parasite present in the heart, testicles, liver, pancreas, and brain. Zarde e_E _a_]_.. (1968) also Ami tell 4 'G 'N C. 12 detected toxoplasmic infections in 176 chickens. Simitch e_t_ §_1_. (1965) determined that turkeys are susceptible to infections by '_I'_. gondii, especially the cystic form. The chance of acquiring toxoplasmosis from infected meat can be minimized either by thorough cooking or by freezing and thawing of the meat as recommended for destruction r1111 of trichinosis . Further-Processed Products .11 Newproducts and legislatign. For many years whole carcasses have been the primary way of merchandis— ing poultry meat. Recently, new products have been mer- chandised using poultry meat. Dawson (1970) emphasized that products containing combinations of poultry and beef could be developed if federal and state regulations did nOt prohibit this practice. If products containing Poultry and beef were permitted, the processors could tiake advantage of the "generally lower" price of poultry and provide the consumer with lower priced meats which have good nutritional value. The development of poultry sausage products has been reported by some researchers. Baker 23'; 31;. (1966) reported the use of 100% fowl meat to produce "chicken franks" and ”chicken bologna." Other products have been developed such as: "chicken sausage" (Mori and Zambo- l"ini, 1968; Fujita, 1968), cooked poultry "hamburgers" I. l 5., ‘.n 13 (Gorizontova E _a_1_., 1962), and fresh turkey sausage (Dawson, 1970). Kebede (1969) produced fermented poultry sausage from 100, 75, and 50% poultry meat mixed with 0, 25, and 50% beef, respectively, using 7 different binders. Sodium chloride was the best meat binder and the optimum concentration was 1-3%. Microbiology of new products. Recently, microbial studies have been conducted on some of the new poultry products such as turkey rolls, deboned poultry meat, frankfurters containing 15% deboned turkey meat, and further-processed frozenturkey products. Wilkinson £13 a; (1965) determined the relationship between the internal temperature at the end of cooking and the destruction of specific species of Salmonella, Staphy- 1Ococcus, and Streptococcus which had been inoculated \——— into uncooked turkey rolls. They suggested that non- fIt‘ozen turkey rolls should be cooked to an internal tem- Perature of 71 C to assure a safe product for the con- sElmer. da Silva (33 E}: (1967) reported that Staphylo- ~°\°ccus aureus was present on 60% of the uncooked turkey r011 surfaces, but not on the cooked turkey roll sur- faces. Fewer than 300 aerobes/cm2 were found on the co(bleed turkey roll surfaces, whereas raw rolls had QC>unts of approximately 100,000/cm2. v'u No.4 .z RIO: -r‘ 0.. an 1") ‘1' 14 Eastern and western types of cooked turkey rolls, differing by the methods of cooking, packaging, and sub- sequent storage, are produced in the United States. Kinner g5 11;. (1968) microbiologically examined the ingredients of eastern-type turkey rolls. They also examined the skin, "meat jelly," and meat of the eastern- type, “ready-to-eat," turkey rolls for total aerobes, coliforms, and enterococci. The results indicated that the hot flowing meat jelly contaminated the skin and internal parts of the turkey rolls. Mercuri e_t_ _a_l_. (1970) studied the microbial flora Of eastern-type turkey rolls immediately after processing and after various periods of refrigerated storage. After 2 weeks at 5 C the concentrations of aerobes on the sur- face of sliced and whole rolls was 106 to 107/cm2. In 8tiered whole rolls, counts of coliforms and enterococci, 2 reapectively, ranged from 104 to > 106/g and from < 10 to 106/9. Post-cooking operations did not significantly affect the total count of turkey rolls. Out of 28 finished rolls sampled, 8 contained coagulase-positive No salmonellae or g. perfripgens were The initial bac- staphylococci . detc-acted in any of the rolls sampled. teria counts for eastern-type turkey rolls and counts from rolls stored in a laboratory refrigerator for 4 days were relatively low. ~“" ‘ It: fl .I-AI‘ I . .vdv“ Iii: l . u 'v‘. ., Huge. 0."! "owe I. H I“ ! I. ' '. ~ . .i l I I a, "A 0 'v 15 Recently poultry meat from deboning machines has been evaluated microbiologically. Studies of processed deboned meat, when stored at 3 and ~15 C, have shown the presence of salmonellae in 8.3 and 16.6% of the samples examined from 2 different poultry plants. C. perfringens was detected in 2.7 and 16.6% of the poultry samples examined (Ostovar e_t; _a_1_., 1971). The majority of the psychro-tolerant organisms isolated from deboned turkey meat were Pseudomonas, Flavobacterium and Achromobacter species (Ostovar (_e_t; a_l_., 1971) . Investigations by Froning pp al. (1971) compared frankfurters containing 15% fresh, mechanically deboned turkey meat against red meat frankfurters and found no difference in flavor or stability of the products. Both tYpes of frankfurters showed some increase in total Connts during refrigerated storage. The use of mechani- c=ally deboned turkey meat which was frozen for 90 days, resulted in inferior products with low flavor acceptance. Zottola and Busta (1971) investigated the micro- biological quality of further-processed turkey products and suggested microbial counts for raw and cooked pro- ducts which would indicate unsanitary preparation of these products. They analyzed 35 samples of raw turkey p~‘l’-‘oducts and found all contained coliforms, 19 contained \. %_s_, and 3 contained salmonellae. In the 38 cooked E coli, 25 contained S. aureus, 7 contained (_2. perfrin— ‘i'L‘II' ,. R .". n A ".Mu‘v ..uaF' \ not-V” . . .-. ”"Ia' n.»W' a I ‘ .h‘!‘ avoth’ ,- nay -. :’:H u ‘0‘! . . 1"» N‘.\ "n ‘ "‘i a h.. L ’l r“ ‘4': 16 products which were analyzed, the incidence of these food- poisoning organisms was lower than the incidences in raw turkey products. Neither salmonellae nor _E_J. coli was found, whereas, 16 contained coliform types, 6 contained C. perfringens, and only 1 contained S. aureus. Fermented Sausages Use of starter cultures. The use of pure cultures for fermented meat products was introduced in the United States in 1921 (Kurk, 1921). New concepts were intro- duced in 1955 that led to the development of Pediococcus cerevisiae as a strain suitable for use in the fermen- tation of meat products (Niven e_t_ 9.2-.” 1955) . Other organisms have been suggested for use as starter cultures, 8I-ilczh as Micrococcus strain M53 (Niinivaara, 1955), wergillus oryzae, Bacillus mesentericus, and Biggi- %terium linens (Redel 3E El” 1969) . Everson 233 E- (1970) developed a frozen Pediococcus cerevisiae starter Q\Il.‘.l.ture which is reconunended for use in semi-dry fish and poultry sausages. Microbiology of fermented sausage. The micro- biology of sausage products is complicated because of Variations in types of meats used, processing methods used, and types of products produced. Steinke and Foster (1951) studied the microbial flora present in liver aausage and bologna and the change in organisms during .‘ I e_rv‘rownmj ' ll,- u.':~ \- ' a .vvo ' '3: RV .-. b. \ .6!" I II... A] ' J: on... :J' Is'b. uq~.:~ 4 \ :o‘uui a n. A‘ 1' "l .. '.: H Iodu u . v:"" "Viv. u :On'a I“.v .._ o-‘a N - I‘. ‘ v... I ‘5 "h. a u. ‘ .- l7 storage at various temperatures when packaged in "Saran." The predominant organisms were micrococci in both pro- ducts when they were stored at 30 C for 20 days. The only organism isolated from bologna was M. candidus, but both L4. candidus and M. epidermidis were present in liver sausage. Bacillus _spp. were also present in liver sausage at the two higher storage temperatures (16 C and 30 C) and these organisms eventually became predominant in the sausage after prolonged storage at 30 C. The bacterial genera present in fresh pork sausage were determined by Sulzbacher and McLean (1951) . The authors believed that Microbacterium spp. may be responsible for the development of the acid taste in stored sausage. They reported that the prevalent genera Were Bacterium, 20.6% of the total isolates, Achromo- ‘bacter, 12.7%, and Pseudomonas, 10.8%. The microbial f:l-ora of raw sausages were determined by Maleszewski % El. (1969). They examined 370 samples of "Metka" a~t1d 88 samples of "Kielbasa polska surowa," two types of Polish sausage, and reported that 96% of the samples Q<>ntained coliforms, 73% contained enterococci, and 18% QOntained coagulase-positive staphylococci. No salmon- ellae were detected in the sausages. Sidorenko _e_p E}: (1969) examined 1,000 samples Qf raw meat and found 30-50% contained (_2. perfringens. Before stuffing, 275 samples of sausage mixtures were \i 30 :v. I. IA ‘1 I- ‘I 'I p. 18 examined and 45-100% contained C. perfringens. They examined ingredients added to the sausage mixture and found 79.6% of the flour samples and 66.6% of the starch samples contained _C_. perfringens. The microbial content of the Spices was not reported. Aroma-producing bacterial strains were isolated from raw sausage during the ripening process by Zanguchi ] and Debindate (1956) . The bacteria found belonged to the ! genera Alcaligenes, Achromobacteg, Escherichia, Aerobacter, and Pseudomonas. E Deibel 31; all. (1961) determined that total viable counts in processed sausage mix were relatively low and the predominant flora were lactobacilli. The authors mentioned that ruptured casings were observed, probably due to the formation of gas, and that large numbers of h~eterofermentative lactobacilli were detected in the Sausage. The rupturing of sausage casings during fer- Inentation was attributed by Kebede (1968) to over-stuffing of the sausage. The microbial content of summer sausage, thurin- 96:, and some types of fermented sausages such as salami, eervelat, genoa, goteborg, and lebanon was described by Deibel _ei _e_l. (1961) . The predominant flora of the :EQrmented sausages consisted of lactobacilli. Although Qfiusage mixes contained small numbers of pseudomonads Lhd other gram—negative rods, coagulase-positive v o r“ ”- ". .‘l ”A. b,‘ E l9 staphylococci were not detected in any of the samples. The predominant flora of the finished thuringer sausage were Pediococcus gpp” even though these bacteria were not detected in the initial sausage mixture. No expla- nation was given for this finding by the authors. Goepfert and Chung (1970) studied the behavior of salmonellae during the production and storage of a fermented semi-dry sausage product. The authors found that salmonellae were able to grow at pH 5.2 while the sausage was being heated at 46 C. Although the finished thuringer sausage contained lower numbers of salmonellae than the sausage mixture, the process did not insure Complete destruction of salmonellae when the sausage was 3 inoculated with 10 to S X 105 cells/g. Processing of fermented sausage. The manufactur- ing of fermented sausage is sometimes considered an art more than a science. Most industrial sausage formu- lations are trade secrets and new methods of production are jealously guarded. Niinivaana gt _a_l_. (1964) reported that the pro- Qeasing time for European dry sausages varied from 10 to 100 days or more. For example, Hungarian salami can be 6 months old when marketed. Some sausages are cooked Qt smoked while others are not. Sometimes mold growth 3‘ a allowed to develop on the sausage exterior, imparting characteristic flavor to the product. Meat formulations 1" : .Q'.. I- U“ v-LU a u b. to us I “~- 9 "I... ~...._ \l ‘ ' I “V 20 and processing conditions for the various type of semi— dry and dry sausages were described by Adajian and Gartner (1946). The classification of fermented sausages was explained by Price and Schweigert (1971) as being based on meat formulations, particle size, spicing, degree of tang, intensity of smoked flavor, finished temper- ature, and type of casing. They mentioned the pH of fermented sausages ranged from 4.8 to 5.4 and semi-dry sausages differ from dry sausages by having a more tangy flavor, and softer less chewy texture. In addition, semi- dry sausages contain approximately 50% moisture whereas dry sausages contain about 35%. The majority of the dry sausages in the United States have diameters of 3 to 4 inches and are dried for 50 to 70 days. Two common tl’pes are genoa and "b.c." salamis. The only recent major change in the production of semi-dry and dry sausages has been the use of starter cultures of Pediococcus cerevisiae and Micrococcus. Deibel 22 3;. (1961) demonstrated that holding the curing Inli—a'zture for 48 to 72 hours before stuffing was not t~equired for an acceptable product. The production 1:zdi-Ine can also be reduced from the traditional 150 hours “Q 12-15 hours with the use of a frozen starter culture (hVerson gt 9.1., 1970). MATERIALS AND METHODS Sausage Ingredients and Spices A flock of broad-breasted white-breeder tom tur- keys, approximately 1 1/2 years old, was obtained from the Department of Poultry Science, Michigan State Uni- versity, slaughtered, and processed. The carcasses were wrapped in "cryovac" bags and placed in a freezer (-29 C) lintil used. The carcasses were defrosted in a walk-in refrigerator (3 C) and cut into strips. The dark meat, white meat, and fat were divided into separate portions which were frozen and then ground through a plate con- taining l/4-inch holes attached to a model 5010 meat Silrinder (Toledo Scales Co., Toledo, Ohio). The ground meat and fat were then distributed into lS-lb. batches <=<>nsisting of 45% dark.meat, 45% white meat, and 10% fat including skin which were mixed by hand, placed jLIIto "cryovac“ bags, frozen, and stored at -29 C until used. The ground mixture was defrosted at 3 C for 2 ‘Eiwfilys and placed through a meat grinder containing a 9late with 3/16-inch holes. The 15 lbs of formulated Jut‘4ithure were divided into 3 5-1b batches and placed into 21 ¥ ,c.‘ .09 u «1‘ ll! 'fl. 22 shallow aluminum pans. Spices and other ingredients (Table I) were added and mixed into the formulation by hand. For inoculation of the mixture with selected pathogens, the appropriate pathogen was sprayed on the meat using a Sprayon sprayer (Sprayon Products Inc., Cleveland, Ohio) and mixed thoroughly by hand. The pans of formulated mixture were incubated for 24 hours at 10 C and 72% relative humidity. The incubation period provided time for the salt to extract out the myosin which is necessary for prOper binding of the meat. The 24-hour period also allows the inoculated pathogen to acclimate to the new environmental conditions. The third reason for this incubation period was to simulate the greening room procedure which is used in some pro- cessing operations. After the incubation period, 3 1 .4l-g portions of Accel (Merck & Co., Rahway, N.J., 07 065) were each placed into 45 ml of water and mixed by hand into S-lb batches of the meat mixture. Sausage Process A 4-liter hand stuffer (F. Dick) was used to pack 14 mm artificial "collagen" casing (Brechteen Co., Mount Clemens, Mi., 48043) . The stuffed sausage was bILaced into a smoking chamber and heated in air with a relative humidity of 80-90% according to the following §Qhedule= 27 c for 3 hours, 32 c for 4 hours, and 46 c E Qr 5 hours. During the heating process, no smoke was .- 23 TABLE I. Spices and ingredients used in the turkey sausage formulation. Grams/5 lbs Ingredient Sausage Source Allspice 1.41 Archibald & Kendall, Inc. Chicago, Ill. Black pepper 8.49 The Frank Tea & Spice Co. Cincinnati, Ohio Red pepper 5.66 The Frank Tea & Spice Co. Cincinnati, Ohio .Paprika 5.66 B. Heller Co. Chicago, Ill. Garlic powder 0.80 Meisel Co. Detroit, Mich. D“Sorbitol 15.0 Pfanstiehl Lab. Inc. waukegan, Ill. Salt 45.4 Hardy Salt Co. St. Louis, Mo. Glucose 16.95 Fisher Scientific Co. Fairlawn, N.J. Sodium Nitrate 0.177 J. T. Baker Chem. Co. Phillipsburg, N.J. Sodium Nitrite 0.177 Mallinckrodt Chem. Works \ St. Louis, MO. 24 used. The heated sausage was cooled by spraying with cold water until an internal temperature of 16-18 C was obtained. The sausage was dried in a chamber at 10 C and 72% relative humidity for 8 days. Sample Preparation for Microbial Evaluation The first sample for enumeration of the selected pathogens was taken just prior to stuffing the sausage into the casing. The second sample was examined for the selected pathogens after 8 days of storage. For each sample, 5 separate 100-g portions of meat were each .Placed into 900 ml 0.1% peptone solution and blended for ‘2 ndnutes in waring blendors. Duplicate samples from each of 5 blenders were then placed into the appropriate huroth.or agar medium for the enumeration of the selected Pathogens . Preparation of Culture Inocula All pathogenic cultures were treated similarly chunting preparation of the inocula. Each.culture was tzransferred into duplicate nutrient agar (Difco Labora- ‘Dories, Detroit, Michigan) slants before each new ‘3Xperiment. One tube was used for culture maintenance “Vhile the other was used for preparing the inoculum. Strains of Salmonella pullorum and Salmonella .§3enftenberg 775 W were obtained from the culture col- ‘3Lection of the Department of Food Science and Human z! V: ‘I 25 Nutrition, Michigan State University. The cultures were thawed at 37 C and transferred into lactose broth (Difco). The cells were streaked onto brilliant green sulfadiazine (BGS) agar (Difco) and incubated for 24 hours at 37 C. A typical salmonellae colony from each culture was transferred to both nutrient agar slants and triple sugar iron (TSI) agar (Difco) slants which were incubated for 24 hours at 37 C. TSI agar slants show- ing an alkaline slant, acid butt, and H28 production were considered positive for salmonellae. Inocula were obtained from nutrient agar slants which contained coaonies that had given typical reactions for salmon- tallae on TSI agar slants. A 5-ml aliquot of lactose lxroth was added to each nutrient agar slant. The tube ‘mas shaken and transferred to a lOO-ml flask of pre— heated lactose broth (37 C). The culture broth was iJacubated at 37 C on a Gyrotory Shaker (New Brunswick Scientific Co., New Brunswick, N.J.) and used to iJnoculate the sausage product. A heat-sensitive Clostridium perfringens strain ZVTCC 3624 and a heat—resistant strain NCTC 8238 were <>btained from the Department of Food Science and Human 1Nutrition, Michigan State University. The C. perfringens <=ultures were maintained in cooked meat medium (Difco) Eat 5 C. Thioglycollate medium (Difco) with resazurin 'Eis an oxidation-reduction indicator was used as the .,o- '0" 0‘ q VI. . v'a IOI 1.1 26 growth medium prior to inoculation. The medium was incubated at 37 C without agitation. Three strains of enteropathogenic Escherichia coli cultures were used in these studies. E} coli strains 0128:B12 and 0125:B12-HIC were obtained from the Uni- versity of'Wisconsin and strain 026:86 was obtained h??fl1‘ frmm the Center for Disease Control, Atlanta, Ga. Hereafter these strains will be referred to as 0128, The cultures were received 0125, and 026, respectively. The on agar slants and transferred to lactose broth. broth was incubated at 45.5 C for 24 hours. A loopful of'the lactose broth was streaked on eosin methylene kitue (EMB) agar (Difco) and incubated at 35 C for 24 hkiurs. A colony with.a metallic sheen was transferred 't&> a nutrient agar slant, a tube of EC medium (Difco), Eirnd a slant of veal infusion agar (Difco). The EC “neudium was incubated at 45.5 C. Both strains 026 and 0125 did not grow well in EC medium when incubated at ‘lllis temperature. The growth on the veal infusion agar (IDifco) slants was used for the slide agglutination method to determine the serological identity of the QIllture . Staphylococcus aureus strain 243 ATCC 14458 “°as obtained from the Department of Food Science and Iiluman Nutrition, Michigan State University. A typical 5§E, aureus colony was fished from a mannitol salt agar I‘- 27 (Difco) plate and inoculated into brain heart infusion (BHI) broth (Difco). After 24 hours at 37 C the BHI culture was streaked onto a Vogel-Johnson agar (Difco) plate and incubated at 37 C for 24 hours. A typical S. aureus (black) colony from the Vogel-Johnson agar plate was transferred to a tryptose agar (Difco) slant and maintained at 5 C. Prior to use, the culture was inoculated into BHI broth and incubated at 37 C for 24 hours . Enumeration of Selected Pathogens Salmonellae. The sausage samples were prepared ias described previously. The procedure for the detection of Salmonella spa. is described by Galton _e_t; _a__l_._. (1968) . Etilution of 10-4, 10-5, and 10"6 were made from each of ‘tlae duplicate samples from the 5 blenders and ltml aliquots of each dilution were placed into three tubes Of selenite-cysteine broth (Difco) and incubated for 48 hours at 37 C. The most probable number (MPN) technique was used to quantitate the salmonellae pre- Sent in the sausage. BGS agar plates were streaked Vvith inocula from the selenite-cysteine broth tubes Eand incubated for 24 hours at 37 C. Colonies from ‘the BGS agar plates were transferred to TSI agar slants Ifor presumptive identification. Further identification cf the colonies from the BGS agar plates was accomplished .1. d! u. 28 with the use of the microcolony indirect fluorescent antibody technique (Thomason, 1971). For samples taken after processing a pre- enrichment lactose broth step was used prior to the selenite-cysteine broth. The pre-enrichment tubes were incubated at 37 c for 36 hours. Dilutions of lo'l, lo’2 and 10"3 were used for the processed sausage samples. The microcolony indirect fluorescent antibody technique employed a 1:8 dilution of specific salmon- ellae antisera (Thomason, 1971). The specific serotype antiserum used for Salmonella pullorum was Salmonella 0 group D, factor 9 (Difco), and for Salmonella senften- EEEEE 775 W the specific antiserum was Salmonella 0 group 134, factor 19 (Difco). Anti-rabbit globulin labeled Vtith.fluorescein isothiocyanate (FI) [Baltimore Biologi- cal Laboratory (BBL), Div. Becton, Dickinson & Company, Cockeysville, Maryland, 21030] was used as the indicator of the antigen-antibody reaction. The Leitz-Ortholux fluorescence microscope system used consisted of a I5380-200 mercury lamp for a light source with an optics SYStem consisting of a 1:20 dark field condensor, IENG-38 and BG-lZ exciter filters, and a K 430 barrier 1filter. Three loopfuls of a 48-hour selenite-cysteine lbroth culture were placed on a BGS agar plate at three ‘CSifferent points and incubated for three hours at 37 C. I :- C'- n o" .0. 0" d an. ‘- w l. 29 A slide cleaned with 95% ethanol was placed on the micro- colonies and removed with forceps. The slide was air- dried and fixed in a solution of 6 parts 95% ethanol:4 parts chloroform:1 part formaldehyde. The slides were then washed in 95% ethanol and dried (Thomason, 1971) . A 1:8 conjugate, diluted with phosphate buffered saline (PBS), was placed on each smear, and the slide was held at 37 C for 30 minutes. The slides were washed in PBS for three minutes, rinsed with distilled water, and dried. A drop of anti-rabbit globulin labeled with FI was placed on each, smear and the slides were incubated at 37 C for 20 minutes. After washing the slides in PBS, a drop of 9:1 glycerol-PBS solution was placed on the Smear and covered with a cover glass (18 RD # l, E. H. Sargent & Co., Chicago, 111.). The slide was viewed under the microscope and the presence of fluorescent cells was considered a positive reaction and indicated that the specific serotype of salmonellae was present. Clostridium perfringens. Sulfite-polymyxin— Sulfadiazine (SPS) basal medium (Angelotti gt .a_l_., 1962) with 0.4 pg/g D-cycloserine added was used for the recovery of C. perfringens from inoculated turkey sausages. The basal medium contained 1.5% tryptone (Difco), 3.0% agar (Difco), 1.0% yeast extract (Difco), 0.05% ferric citrate (K a K Laboratory Inc., Plainview, ..- .a\ .- Q I n C I I u ; IOI ..I l .l g. . h to i .r u i \ r t- .l l ." o ' u ' o o o 3O N.Y.), and 0.1% sodium metabisulfite. The basal medium was autoclaved at 121 C for 15 minutes. Seromycin (Eli Lilly & Co., Indianapolis, Ind.), a brand of D-cycloserine, was used as the antibiotic in pflace of polymyxin and sulfadiazine (Harmon 2E.Elfir 1971). The seromycin powder was removed from the cap- sules which contained a filler (1.678 g seromycin powder = 1.00 g of D-cycloserine). The D—cycloserine was mixed with distilled water (1.00 g/12.5 m1) and filtered using a Millipore 3 um pore-size filter (Millipore Corp., Bedford, Mass.). Five ml of filtrate were placed into tubes, steamed for 20 minutes for sterilization, and frozen at -73 C until used. D-cycloserine was used at a final concentration of 400 ug/ml. The pouch method developed by Bladel and Greenberg (1965) was used for the isolation and enumeration of S5- perfringens. A polyester film was used for the Pouch material (Kapak Pouches B-1215-2; Scotchpack-BM Ck3., Kapak Ind. Inc., Minneapolis, Minn.). The Pouches did not need to be sterilized when properly handled . The pouches were placed into a holder with walls Spaced 0.5 cm apart and 23 ml of agar were added to each Pouch. It was not necessary to seal the pouches since the agar, which formed in the neck of the pouch, pro- fiuced an oxygen barrier. .I' 126.1 r- c .o 31 The plastic pouches with agar were held in a 55 C incubator until inoculated with a sausage sample. After solidification of the agar the pouches were then removed from the holder and placed in an incubator at 37 C for 24 hours. During growth, _C_. perfringens reduces sulfites to sulfides producing black colonies when the ferric citrate is converted to ferrous sulfide. A11 black colonies from one of the middle dilution pouches suspected of being 9. perfringens were tested in trypticase nitrate-motility agar and iron milk tubes; both media were prepared in the laboratory. Colonies Were considered to be C. perfringens if they contained nonmotile, nitrate-reducing bacteria which produced a Stormy fermentation in iron milk. Enteropathogenic Escherichia coli. A three-tube MPN technique was employed for the enumeration of EEC. The turkey sausage dilutions were placed into lauryl tryptose broth (Difco) tubes which contained Durham Vials. hours . The tubes were incubated at 35 C for 24-48 A loopful from each tube exhibiting gas was Streaked onto EMB agar and incubated at 35 C for 24 hours. Typical E. coli colonies, showing a metallic sheen and dark centers, were transferred to EC medium and incubated in a water bath at 45.5 C for 24-48 hours. The samples showing gas production were considered to 32 contain EEC organisms. The organisms were identified using the biochemical tests contained in the API system for the identification of Enterobacteriaceae (Analytab Products Inc., N.Y.). The slide agglutination test was used for serological confirmation which employed the use of antisera OB poly A and B, and OK poly C (Difco). Coagulase-Positive Staphylococcus aureus. E Dilutions were made using phosphate buffered dilution j blanks (APHA, 1966). Samples were spread in 0.1 ml volumes over the surfaces of Vogel-Johnson agar plates using a bent glass rod. The inoculum was allowed to atmorb into the agar and the plates were incubated at 137 C for 48 hours. Black colonies larger than 1 mm in (iiameter were picked, transferred into BHI broth, and :incubated for 24 hours at 37 C. One drop of BHI culture ‘vas added to 0.5 ml of citrated rabbit plasma (Difco) Jin.a 13 X 100 mm test tube. The tubes were incubated eat 37 C and coagulation of the plasma within 6 hours .indicated the presence of coagulase-positive staphylococci. Detection of_Staphylococcus Aureus Enterotoxin The micro-slide gel double diffusion method developed by Casman and Bennett (1965) was used, with slight modifications, for the extraction, purification, and detection of enterotoxin in fermented turkey sausage. After centrifugation at 32,800 X g_for 20 minutes, "I .n 33 the supernatant fluid was heated in a 56 C water bath followed by chilling in ice (Barber and Deibel, 1972) in order to eliminate substances which.might interfere with serological test for enterotoxin. The fluid was again centrifuged at 32,800 X g_for 15 minutes. The supernatant fluid was adjusted to a pH of 4.5 and cen- trifuged at 32,800 X g for 15 minutes. The supernatant I was saved and the pH was adjusted to 5.7 before being applied to a carboxymethyl cellulose (CMC) column. The enterotoxin was absorbed onto the CMC column and eluted E 1 , E] from the column with 0.2 M sodium phosphate buffer con- L~¥+“ taining 0.2 M NaCl, pH 7.4. Enumeration of Nonpathogenic Microorganisms Aerobic plate counts. For aerobic plate counts, 50—g portions of sausage mixture were added to 450 m1 of 0.1% peptone and blended for 2 minutes. Serial dilutions of the homogenate were prepared and l-ml portions were used as inocula for pour plates of plate count agar (Difco). The plates were incubated at 30 C for 72 hours (APHA, 1966). Lactic acid bacteria. LBS agar (BBL) was used for pour plates for the enumeration of lactic acid bac- teria (Rogosa e_t_: 91., 1951). These plates were incubated atzl30 C for 72 hours. Three percent hydrogen peroxide 34 was poured over the surface of the agar plates to deter- mine the presence of the enzyme catalase. Most organisms growing on aerobically incubated plates possess the enzyme catalase which will release oxygen from the hydrogen peroxide causing an effervescence. However, the lactic acid bacteria, including the Lactobacillus and Pediococcus genera, do not normally produce a detectable catalase (Harrigan and McCance, 1969). Therefore, a negative catalase test indicated the presence of lactic acid bacteria. Coliform. Coliforms were determined using violet red bile agar (Difco) as described in Recommended Methods for the Microbiological Examination of Foods (APHA, 1966). The plates were incubated for 48 hours at 35 C. Dark red colonies, at least 0.5 mm in diameter, were con- sidered to be coliforms. Yeast and molds. Yeast and molds were enumerated using potato dextrose agar (Difco) acidified as described in Microbiological Examination of Foods (APHA, 1966). The plates were incubated at 25 C for 48 hours. pH and Total Acidity Determinations The pH and total acidity of the fermented Sausage products were determined with the aid of an automatic titrimeter model No. 36 (Fisher Scientific, Pittsburgh, Pa.) . The standard AOAC method for 35 determining total acidity in cheese was used to determine total acidity in sausage products (AOAC, 1970). Ten 9 of meat were placed in a blender containing 100 ml of distilled water at 4.4 C. After blending for two minutes, the homogenate was filtered through a Seitz filter (Hercules Filter Corp., Hawthorne, N.J.). Portions of filtrate corresponding to 2.5 g of sample were titrated against 0.1N sodium hydroxide. An end- point of pH 8.7 was used for the sausage sample titration by plotting pH vs ml of titrant which was determined using the automatic titrimeter. The point on the titration curve at which the pH changes most rapidly with addition of titrant was chosen as the endpoint. This endpoint was used throughout the eXperiments. Percent total acidity, expressed as lactic acid, was determined by the formula: (ml N/lO NaOH) (.009) X 100 = % total acidity of 2.5 g sample the sausage product RESULTS AND DISCUSS ION Spice Contamination Table II indicates the aerobic plate counts of each spice and ingredient used in the formulation of the turkey sausage. The results show sorbitol and glucose supplied some microorganisms to the product. However, the spices contributed more organisms than the other ingredients. An average of 9 X 103 organisms/g meat was contributed to the sausage mixture by the spices. No lactic acid bacteria or coliforms were recovered from the spices and ingredients. Some molds and yeasts were found in allspice (5.5 X 102 /g meat) and red pepper (2.5 X 104 /g meat). The microorganisms intro- duced to the product by the spices could be reduced by using gas-sterilized spices. Krishnaswamy E£,El° (1971) enumerated the micro- organisms present in spices and spice mixtures. Their results for numbers of total organisms were similar to those in Table II. Processing Parameters Table III shows the pH, total acidity, and per- cent 1083 of moisture obtained in the sausage when 36 37 TABLE II. Microbial counts of spices and ingredients added to the turkey meat mixture. Aerobic Plate Count/ Ingredient g/5 lbs Meat Gram of Spice Allspice 1.41 3.7 x 106 Black pepper 8.49 3.5 X 105 Red pepper 5.66 8.5 X 105 Paprika 5.66 1.3 X 106 Garlic powder 0.80 1.4 X 104 Sorbitol 15.0 1.5 x lo1 Glucose 16.95 1.0 x 101 Salt 45.40 nc Sodium nitrate 0.177 nc Sodium nitrite 0.177 nc 1 nc represents < 1.0 X 10 38 TABLE III. Processing results for a fermented turkey sausage product inoculated with four dif- ferent pathogenic organisms. % Loss of % Total Acidity Organism . pH With Respect to Moisture Lactic Acid Control 35.2 5.3 .31 Salmonella spp. 35.4 After heating 5.8 .10 After processing 5.9 .14 Clostridium s22. 37.5 After heating 5.6 .12 After processing 5.7 .17 Enteropathogenic E. coli 46.6 After heating 5.5 .09 After processing 5.2 .21 Staphylococcus aureus 38.2 After heating 5.4 .15 After processing 5.6 .23 | 1 “'14-- I l 39 various pathogenic inocula were used. The indicated parameters were examined before processing (after inocu- lation of pathogens, addition of spices, and incubation at 50 F for 24 hours) and after processing (addition of E. cerevisiae, heat processing, and storage at 50 C and 72% relative humidity for 8 days). There was a difference in percent loss of moisture between the control sausages and the inoculated sausages. This was the result of fluctuation in the control chamber due to mechanical problems. A pH of 5.3 provided an acceptable organoleptic product. The sausages inoculated with Clostridium gpp., Salmonella spp., and Staphylo- coccus gpp, had a slight pH increase during processing. The pH did not drop as in the control sausage which.may be the result of competition of the inoculated organisms with the E. cerevisiae. The sausages inoculated with E. coli showed a decrease in pH after processing. This may be due to acid products produced by the E, coli strains or the inability of E. coli to compete with P. cerevisiae. The percent total acidity, expressed as lactic acid, increased during the processing indicating that lactic acid was being produced. The sausages inoculated with the Salmonella Egg. and Clostridium gpp. had a lower total acidity than the control sausage. .\ P4 40 Microbial Contamination During Processing Data in Table IV represent the microbial develop- ment at various processing stages. Initially the fresh processed turkey meat had a low count because the turkeys were processed in our own facilities under sanitary con- ditions which may not be obtained with continuous pro- If] cessing. The total initial aerobic count of 2.7 X 104 ' 1 organisms/g meat was an acceptable figure and no off-odor or discoloration was noted in the product. The final aerobic plate count was measured after the heating and drying process. In general a lO-fold increase in aerobic plate counts occurred during pro- cessing. One experiment with a sausage product which did not contain the starter culture, E. cerevisiae, showed a similar increase in aerobic plate counts. This indicates that other organisms besides the starter culture are able to grow during the processing. Yeast and molds were completely destroyed by processing in all runs. For each sausage batch inoculated with a selected pathogen, lactic acid bacteria, coliform, and yeast and mold counts were obtained. The lactic acid bacteria counts of the sausage mixture prior to inoculation with the starter culture were high. Even without the addition of the lactic starter culture, E. cerevisiae, a lO-fold increase in numbers of lactic acid bacteria was noted. 41 TABLE IV. Aerobic plate counts of turkey sausage at various processing steps. Aerobic Plate Count Sample Org/ Gram Fresh processed turkey meat 1.3 X 104 Added spices 9.0 x 103 Pathogenic inoculation 6.0 X 103 Initial sausage mixture 2.7 X 104 Sausage mixture after inoculation of pathogens, addition of spices, and incubation at 7 50 F for 24 hours 1.3 X 10 Sausage in casings, after the addition of Pediococcus 8 cerevisiae and processing 5.0 X 10 Sausage in casings, after processing without addition of Pediococcus cerevisiae 4.1 X 10 42 Coliforms were found at low levels in the initial product, and they were completely destroyed by the pro- cess when E. perfringens were used as the pathogenic inoculum. However, there were some coliform type colonies found after processing when Salmonella Egg. and E. coli were used as the pathogenic inocula. These coliforms could have been part of the salmonellae inoculum or E. coli inoculum that had survived the processing. Studies of Selected Pathogens Salmonellae. As shown in Figure 1, during pro- cessing, Salmonella pullorum was reduced in numbers by 2 4.1 to 2 5.1 log cycles when the initial inoculum was 99,000 to 380,000 organisms/g meat. E. pullorum.wou1d be expected on poultry products although not in as high a number as used in the inoculation studies. Figure 2 indicates that an initial inoculum of > 2,200 to 61,000 organisms/g meat of Salmonella senften- berg 775 W was reduced by 1.3 to 1.7 log cycles during processing. These results indicate E. senftenberg has a higher tolerance than E. pullorum to the processing conditions used. The higher processing tolerance may be due to factors other than heat since the heating process was very mild. Possibly tolerance to pH, dry- ing, and greater ability to compete with other organisms, or a combination of these factors were responsible for the greater survival of E. senftenberg. fif. Emir ass! in: Ira-r .osveccomu 2m: when women on» an omucHoEdcmstm mcowumuucmocoo usmuwmmep mourn um omumasoocw Esuoaasm maamcoEme mo Hm>w>usm ecu so mswmmoooum wmmmscm horns» coucmEhmm mo nowmmm .H mesmem 43 , E 053009.“. .22 E 05330:. _IL .L . otoeom nu N — wow bud sues onqom lo 001 r «a: '0 U) o' 44 .mswwsnomu zmz menu owns» may he pouchEscm can mcowumuucmocoo ucmummmwv mono» um ooumasoosw 3 men mumncmumcwm caamcosaom mo Hm>w>usm on» so mcfimwmooum mummscm hmxusu pmucmfiumw mo vacuum ' .N madman mEuuoooi .22 g octuoooi 9.30m U l7/7// W/////4, [///////// A A ' V v 1 '3‘ to N ~ loan 0 and snag «(pm )0 001 I U) c5 45 A comparison of the results obtained using E. pullorum and E. senftenberg 775 W as inocula indicates that a difference in tolerance to processing exists between salmonellae species. This result emphasizes the importance of determining the species, and presumably the strains, in solving salmonellae problems. Matches and Liston (1972a) indicated that three serotypes of salmonellae were capable of growth.when incubated at 12 C in a medium containing 0 to 4% sodium chloride. This indicates that the salt concentration used in the sausage mixture may not be sufficient to inhibit the growth of Salmonella Epp. In another study Matches and Liston (1972b) suggested that some salmonellae grew at low temperatures in a narrow pH range. Growth was reported at pH 6.0 with a temperature of 5 C. There- fore, with the temperature used in these studies, 10 C or higher, the possibility of salmonellae growth exists. Takacs and Simonffy (1970) studied the fate of Salmonella E22. during maturation and storage of inocu- lated dry sausages. Declines in the concentration of Salmonella E22. were apparent after 7-9 days of storage and were related to pH, salt concentration, and water content. However, when initial counts were > 20,000 organisms/g meat, the sausages contained viable salmon- ellae up to the time of consumption. élflf‘! g j {P_ L. In 46 Goepfert and Chung (1972) reported growth of salmonellae occurred in a low-acid sausage product heated to 46 C during processing. Reduction of the number of viable salmonellae occurred during refrigerated storage. The results of the present study indicated that even when salmonellae were inoculated in high numbers into turkey sausage, the salmonellae were greatly reduced in numbers, but complete destruction of the organisms did not occur. Clostridium perfriggens. Figure 3 demonstrates the survival of the heat-sensitive strain of E. perfringens ATCC 3624. The numbers inoculated varied from 14 to 250,000 organisms/g meat. The number of log cycle reductions obtained after processing declined as the inoculum declined. The range of reduction was 0.55 to 3.4 log cycles, depending on the inoculum. This result may be due to a protection phenomenon at the lower con- centrations. However, it should be noted that the plate count technique has a relatively high degree of error at the lower concentrations measured in this investi- gation. Figure 4 shows the survival of the heat-resistant E. perfringens strain NCTC 8238. The range of inoculation was 21.5 to 27,300 organisms/g meat. The numbers of E. perfringens were reduced by 1.0 to 2.1 log cycles during processing. The samples were not heat shocked to deter- mine if spores were present. The results indicate that 47 .oocume cocoa mmm on» an pwumumescmchm msomumuucwocoo usenmumap usom um omumHsoocw «Non Quad mammcwumumm EdwowuumoHU mo Hm>fl>nsm may no mcfimmmooum mommsmm mmxusu ooucmeumm mo uommmm b' b ’ .m musmwm @ 05332.. 9.200 U .i a a _. wow 0 19d snao olqom lo 601 3 I) 1 T 19 . NI 7 _7 .- ..o I‘" l s [- .oosumE nosom mmm can he pwumuwescw mam mcofiucuucmocoo ucmumm umwo owns» an pmumasoocw mmmm uaoz mammswumuwm Edaowuumoao mo Ho>w>usw can so mswmmmooum mmcmscm hmxusu nonsmEumm mo pommmm .v musmwm 48 33:09:. .2: E 05339:. :2... _U E 2 <- u; a} .1 wow 6 19d moo alqom #0 601 MD 0 49 E. perfringens can survive the process, even when present at low concentrations, and, if mishandling of the fer- mented product occurred, the surviving organisms could multiply and cause a foodborne disease outbreak. Solberg and Elkind (1970) conducted a study with "frankfurters" inoculated with E. perfringens strain 1362 and S-80. After heating the "frankfurters" to an internal temperature of 68-69 C in 30-48 min., they reported growth of these strains of E. perfringens at 12 C during storage, but not at 10 C. E. perfringens strain 1362, inoculated at 4.5 X 103 to 7.0 X 103 organisms/g meat, was reduced in numbers by .16 to .62 log cycles. The results presented here vary from those reported by Solberg and Elkind (1970). This may be due to the addition of a starter culture which.may compete with E. perfringens, the 8-day drying process, the higher levels of organisms used for inoculation, the use of different strains of E. perfringens, or the addition of both nitrate and nitrite to the fermented sausage mixture. Enteropathggenic Escherichia coli. Three enteropathogenic strains of Escherichia coli were used in these studies. Figure 5 shows the results using EEC strain 026. The initial inoculum of < 1,000 to 10,000 organisms/g meat was reduced by 2.5 to 3.0 log cycles during processing. 50 .msowcoomu zmz was» noun» on» an consumescm use msomucuucmo Icon econommwo omen» um omumasoosw one swcuum wHoo mwcowumnomm mo Hc>w>usm man so mcwmmmooum mommsmm moxusu omucmeuow mo uommmm .m musmam i! E - W 3.8300...“ .2: E 9530065 22... Pli— L _A A N. J 70' wow 610d suoo olqom JO 001 ‘f' 7 ‘5 A V 51 In contrast to the results reported above, EEC strain 0128 was relatively resistant to the fermented turkey sausage process (Figure 6). The initial numbers of organisms, 10,000 to 130,000 organisms/g meat, were reduced by only .83 to 1.8 log cycles. As shown in Figure 7, EEC strain 0125 was inoculated at concentrations of 5,500 to 55,000 organisms/g meat which were reduced by 2.4 to 2.7 log cycles. The resistance of EEC strain 0125 was comparable to strain 026, and less than that of strain 0128. A comparison of Figures 5, 6, and 7 shows a variation between EEC strains. The EEC strain 0128 was very resistant to the process considering it does not produce spores. Staphylococcus aureus. Staphylococcus aureus strain 243 was used to inoculate sausages with 25,000 to 1,700,000 organisms/g meat. With an inoculum of 1.0 X 106 organisms/g meat, growth occurred during the process, i.e., the final count was larger than the inoculum (Figure 8). In sausages inoculated with lower cell concentrations, no growth occurred. In contrast to that result, in a second experiment using washed cells as the inocula, an increase in the number of cells occurred even when the inoculum was only 5,500 organisms/g meat (Figure 9). 52 .msowsnomu zmz ohsu mmuau ecu an pmumumessm use mcowuonu Ismocoo usmumwuwo mounu um pmumasoocw mNHo samuun «H00 canowumnonm mo HM>H>HSm msu so mswmmmooum mommsmm moxusu oousmEHmm mo uommmm .m museum 05:30.... 32.43 2:339... ouoeom n..— W///// / W///////////4 [///////////////2 '0. wow 6 19d suoo snow )0. 00-1 53 .mnvuceomu 2m: menu mmHeu meu he pmumumencm use mcomumuucmo Isoo ucmummwwo mmueu um omumanoosfl mmao cwmuum waoo cweowumeumu mo Hm>u>unm meu so ocummmooum mommnmm mmxunu vmusmEHmu mo uomumm .5 munmuh ’ } 0530005 .334 a 05838.. 9.83 U AA / A V — v; #1 r C) 00 I0 wow 0 106 snag omom JO 001 e I) 54 .ooeume unnoo mumao conce0b nummo> meu he omumumencm can mnemwmuucmocoo usmummmuo mmueu um pmumanoocw new cwmuum mnmunm mnOOOOOANemmum mo mHHmo pmemmznn mo Hm>u>unm meu so ocummmoouo mmmwnmm mmxunu omucmEHmm mo uommmm ' .m munmwm A ///// 4 W///////7////) /////////// L mcfiuooota . 05330.... 333 D 7* L /////////////// W 3 Cd 4' a; wow 6 19d moo anqom lo 001 I) (O .ooeume ucnoo mundm somceOH nammo> meu we omumnmennm mam msoaumuunmocoo ucmummmao mmneu um omumanoocw mvN cumuum unmunm mnoooooawemmum mo mHHmo omens; mo Hm>w>unm meu so mcummmuoum mommnmm hmxunu omucmeumm mo uommum 55 A I — CU fl) * @5330; .22 E 053395 9.33 U : ID wow 0 led snao apqom )0 601 V/////////////// 2 W//////////////// 2 W///////////////// / o f~ (0 IO 9. 56 Using the micro-slide double gel diffusion method no enterotoxin was detected in any of the three sausage samples or in the spent medium. A sausage mixture in which 1 ug enterotoxin per g of meat was incorporated into the blender before mixing of the sample, gave a positive result for enterotoxin indicating that the system used for detection of enterotoxin was satis- factory. Aerobic plate counts and counts of lactic acid bacteria indicated the starter culture grew during pro- cessing and a 20-fold increase in lactic acid bacteria occurred. However, Barber and Deibel (1972) showed that even with a 6:1 ratio of E. cerevisiae to staphylococci, 3 4 the staphylococci grew to papulations of 10 to 10 cells/g of surface sausage. The final pH (5.64) of the finished sausage product inoculated with E. aureus was not sufficiently acid to eliminate growth and subsequent production of enterotoxin. Morse SE EE. (1969) reported little or no production of enterotoxin occurred at pH values less than 5.0. However, Genigeorgis gg_3£. (1969) reported enterotoxin B was formed in cured hams at pH 5.3 or above and at sodium chloride concentrations up to 9.2%. Enterotoxin was detected after at least 2 weeks of incubation at 10 C and most samples contained enterotoxin after 8 weeks when the pH was > 5.6. Barber and Deibel 57 (1972) reported the majority of strains tested initiated growth and produced detectable amounts of enterotoxin aerobically in buffered BHI broth at pH 5.1. However, under anaerobic conditions most strains failed to produce detectable amounts of enterotoxin in media with pH values below 5.7. Kao and Frazer (1966) reported that type B toxin production could occur at pH values as low as 5.0 to 5.1. The number of cells required for production of detectable amounts of enterotoxin varies from strain to strain. Barber and Deibel (1972) reported that strain S-6 required 9 X 108 cells/g before enterotoxin B was formed, and that strains 272 and 334 did not pro- duce enterotoxin. Genigeorgis 2E.2lé (1969) also reported that toxic hams contained 4 X 106 cells/g. The highest cell concentration obtained in this study was 2.3 x 107 organisms/g meat. Scott (1953) was the first to report that growth of E. aureus was related to water activity (aw). He observed growth at water activities between .999 and 0.86, with a reduction in growth when the aw was less than 0.94. Troller (1971) reported that toxin pro- duction in broth cultures was greatly reduced if there was a slight decrease in aw. He indicated that the rapid growth and presence of high numbers of staphylo- cocci did not necessarily indicate the presence of enterotoxin. Therefore, although the conditions were 58 present for growth of the staphylococci in the turkey sausage, a reduction in aw during processing could have prevented the production of enterotoxin B. The starter culture did grow, and acid was produced, but not in sufficient quantities to lower the pH and thereby reduce the growth of staphylococci. 1. CONCLUSIONS A small (14 mm) diameter fermented turkey sausage was prepared using a processing time of 8 days. This sausage was produced using only turkey meat and turkey fat, i.e., no beef or pork. Binding was improved with the use of sorbitol as a humectant. The aerobic plate count and lactic acid bacteria count increased 10- to 20-fold during the pro- cessing. Salmonella pullorum and Salmonella senftenbe£g_ 775 W survived the process at the concentrations used in this investigation. However, the concen- trations used would seldom be found in poultry meat. The concentrations of Clostridium perfringens cells in the sausage were significantly reduced during processing. However, even when inoculated with low concentrations of cells, some cells survived the process. There was a surprisingly small difference in the results obtained with the heat-sensitive and heat-resistant strains. 59 60 Enteropathogenic strains of Escherichia coli varied in rate of survival although none of the strains grew during processing. E. coli strain 0128 was relatively resistant to the processing. Washed cells of Staphylococcus aureus strain 243 grew during the processing even when inoculated at low concentrations (5,500 organisms/g meat). However, even though growth occurred, no entero- toxin was produced. BIBLIOGRAPHY 1. :‘g.’ .‘tu BIBLIOGRAPHY Abrahamsson, K., and H. Riemann. 1971. Prevalence of Clostridium botulinum in semi-preserved meat products. Applied MiCrobiology 21: 543-544. Adajian, A., and B. W. Gardner. 1946. Processed meats, part II. Quartermaster food and container insti- tute for the armed forces. lst ed. American Public Health Association. 1966. Recommended methods for the microbiological examination of foods. 2nd ed. New York, N.Y. Angelotti, R., H. E. 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Batch Number 1 2 3 salmonellae before processing >1.1 X 10 >1.1 X 10 3.8 X 10 after processing 3 4.6 8.6 <:3 pH after heating 5.6 6.3 5.8 after processing 5.7 6.7 5.8 % total acidity after heating .08 .08 .10 after processing .13 .10 .13 % weight loss (moisture) 38% 70% 34% 71 72 TABLE IIA. Determination of total acidity, pH, salmonellae counts, and percent weight loss in three exper- imental batches of fermented turkey sausage inoculated with Salmonella senftenberg 775 W. Batch Number 1 2 3 salmonellae (org/g) 4 4 3 before processing 6.1 X 10 1.6 X 10 >2.2 X 10 after processing >1.1 X 103 2.4 X 102 1.2 X 102 pH after heating 6.1 5.9 5.9 after processing 5.9 6.2 6.2 % total acidity after heating .11 .12 .12 after processing .16 .12 .15 % weight loss (moisture) 40% 30.2% 28% TABLE I I IA . 73 Determination of total acidity, pH, counts of Clostridium perfringens, and percent weight loss in four expefimental batches of fer- mented turkey sausage inoculated with Clostridium perfringens ATCC 3624. Batch Number Clostridium perfringens (org/9) before pro- cessing after pro- cessing pH after heat- ing after pro- cessing % total acidity after heat- ing after pro- cessing % weight loss (moisture) 2.5 x 105 1.0 x lo2 .12 .19 47.4% 1.1 X 10 2.4 X 10 .15 .21 40.8% 2.6 x 103 1.8 x lo1 .12 .18 40.1% 1.4 x lo1 4 .12 26.8% 74 TABLE IVA. Determination of total acidity, pH, counts of Clostridium perfripgens, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Clostridium perfringens NCTC 8238. Batch Number 1 Clostridium perfringens (GIG/9) 4 before processing 2.7 X 10 after processing 2.1 X 102 pH after heating 5.3 after processing 5.0 % total acidity after heating .10 after processing .22 % weight loss 49.6% (moisture) 30.1% 75 TABLE VA. Determination of total acidity, pH, counts of Escherichia coli, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Escherichia coli 026. Batch Number 1 2 3 E. coli (org/9) 4 3 3 before processing 1.0 X 101 1.8 X 10 ‘<1.0 X 10 after processing 3.3 X 10 1.6 < 3 pH after heating 5.8 5.6 5.3 after processing 5.1 5.2 4.9 % total acidity after heating .08 .08 .08 after processing .21 .21 .25 % weight loss (moisture) 51.3% 50.9% 53.9% 76 TABLE VIA. Determination of total acidity, pH, counts of Escherichia coli, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Escherichia coli 0128. Batch Number 1 2 3 E. coli (org/g) 5 4 3 before processing 1.3 X 104 4.5 X 10 9.8 X 10 after processing 31.1 x 10 6.7 x 103 _7.9 x 102 pH after heating 5.3 5.1 5.8 after processing 5.1 5.3 5.5 % total acidity after heating .10 .10 .09 after processing .21 .19 .21 % weight loss (moisture) 51.3% 50.4% 52.4% 77 TABLE VIIA. Determination of total acidity, pH, counts of Escherichia coli, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Escherichia coli 0125. Batch Number 1 2 3 E. coli (org/9) 3* 4 4** before processing 11.1 X 10 1.1 X 10 11.1 X 10 after processing 1.2 x 101 < 3.0 x 101 2.2 x 102 pH after heating 5.7 5.8 5.5 after processing 5.7 5.5 5.0 % total acidity after heating .12 .11 .10 after processing .19 .18 .21 % weight loss (moisture) 35.2% 35.9% 37.5% * 1.1 X 103 approximately 5500 org/g ** 1.1 x 104 approximately 55,000 org/g 78 TABLE VIIIA. Determination of total acidity, pH, counts of Staphylococcus aureus, and percent weight loss in three experimental batches of fer- mented turkey sausage inoculated with Staphylococcus aureus strain 243 (unwashed cells). Batch Number 1 2 3 E. aureus (org/g) 4 5 6 before processing 2.5 X 10 1.4 X 10 1.7 X 106 after processing 6.9 X 103 1.5 X 105 4.7 X 10 pH after heating* 5.5 5.2 5.5 after processing 5.8 5.6 5.6 % total acidity * after heating .16 .14 .15 after processing .27 .20 .22 % weight loss (moisture) 39.8% 36.3% 38.5% * Samples taken after 12 hours drying. 79 TABLE IXA. Determination of total acidity, pH, counts of Stgphylococcus aureus, and percent weight loss in three experimental batches of fermented turkey sausage inoculated with Staphylococcus aureus strain 243 (washed cells). Batch Number 1 E. aureus (org/g) 3 before processing 5.6 X 10 after processing 1.8 X 106 pH after heating* 5.5 after processing 5.4 % total acidity * after heating .11 after processing .21 % weight loss (moisture) 45.9% 2 3 5.3 x 104 2.2 x 106 4.2 x 106 2.3 x 107 L 5.2 5.2 5.3 5.3 .12 .12 .22 .22 45.7% 46.9% * Samples taken after 12 hours drying TABLE XA. 80 Various nonpathogenic microorganisms enumerated in processed sausages inoculated with Salmonella pullorum and Salmonella senftenbepg 775*W. Salmonella pullorum (BatEh number 3) aerobic plate count lactic bacteria coliform yeast and mold Salmonella senftenberg 775 W (Batch number 3) aerobic plate count lactic bacteria coliform yeast and mold After Processing org/g > 1000 w > 1000 . 76 e ,- nc E‘I_E After Processing org/g 4.3 x 102 1.4 x 10 6.0 x 102 no nc = < 10 organisms detected 81 TABLE XIA. Various nonpathogenic microorganisms enumerated in processed sausages inoculated with Clostri- dium perfripgens ATCC 3624. . . . Before After Clostridium erfrin ens . . 3:31. ATCC $624 9 Proce551ng Proce881ng . '1 B t h b 3 ( a C hum er ) org/g org/9 aerobic plate count 1.5 X 10; 4.6 X 103 L lactic bacteria 5.0 X 103 1.7 X 10 7 coliform 3.2 X 103 no yeast and mold 1.0 X 10 no L nc = < 10 organisms detected 82 TABLE XIIA. Various nonpathogenic microorganisms enumerated in processed sausages inoculated with Escherichia coli strain 026. Before Processing Batch Number l 2 3 org/g org/g org/g aerobic plate 6 6 7 count 3.6 X 106 4.7 X 106 1.7 X 107 lactic bacteria 3.3 X 10 3.9 X 10 1.5 X 10 coliform 1.0 x 102 1.0 x lo2 <:l.o x 103 yeast and mold 3.4 x 103 5.0 x 104 1.1 x 103 After Processing Batch Number 1 2 3 org/g org/g org/g aerobic plate 8 8 9 count 2.0 X 108 2.5 X 108 1.1 X 10 lactic bacteria 1.6 X 104 1.5 X 10 - coliform 1.2 x 10 4.6 x 103 - yeast and mold no no nc nc = <.10 organisms detected 83 TABLE XIIIA. Various nonpathogenic microorganisms enumerated in processed sausages inoculated with Stgphylococcus aureus strain 243 (washed cells). Batch Number Before processing aerobic plate count lactic bacteria After processing aerobic plate count lactic bacteria mm U'ICD ><>< PIP 00 mm o a cow >< raw OD O\\l ubU'l o~4 x>< FJH c>o qco “fl 1.; .4