Growth and Enterotoxin Production by Staphylococci in Genoa Salami 1

325

I. C. LEE2, L. G. HARMON, andJ. F. PRICE Department ofFood Science and Human Nutrition Michigan State University, East Lansing, Michigan 48824 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/5/325/1649781/0362-028x-40_5_325.pdf by guest on 30 September 2021 (Received for publication August 30, 1976)

ABSTRACT staphylococcal strains tested grew and produced Staphylococcus aureus strains 265 and 243 which produce enterotoxin aerobically at pH 5.t in broth media. Control enterotoxins A and B, respectively, were inoculated into meat beinJ of S. aureus in sausage by lactic cultures and chemical made into Genoa salami in the amount of lOl. lOS, and 107. cells/g. No acidulation was also reported by Daly et al. (4). Partial lactic starter culture was added. Samples were taken at different stages of processing to determine the microbial populations, percenta,e inhibition of staphylococcal cells was observed by using 8 moisture, tntal acidity, pH, and enterotoxin content. Staphylococcal the starter culture at t0 cells/g, or by chemical populations varying from about HY to 5 x 10'/g were detected during acidulation with 0.75% GDL and O.t% citric acid. They tempering of the salami. Enterotoxin A was detected in surfKe but not also suggested using a combination of chemical in core samples of salami inoculated with 105 and 107 S. aureus 265 acidulation and lactic culture. The effect of water activity cells/g. However, no enterotoxin B was detected in the salami inoculated with S. aureus 243, which requires a relatively high 9.w for (3w) on enterotoxin production and growth of S. aureus enterotoxin production. Staphylococcal counts were higher in surface was reported by Troller (JO, 11) who demonstrated the samples than in core samples, attributable to the difference in oxysen, production of enterotoxin B by S. aureus C-243 was but there was no significant difference in microaerophilic lactic Kid strongly inhibited by a reduction in aw from 0.99 to 0.98 bacteria in different portions of the salami. in broth despite the attainment of populations of 101 cells/mi. However, S. aureus t96E produced enterotoxin A at an 3w of0.90 and fmal cell counts were tO'/ml. Meat products such as ham, bacon, and fermented The purpose of this investigation was to evaluate sausage have been incriminated in staphylococcal food growth of staphylococci and production of enterotoxins poisoning, caused by careless manufacturing techniques in Genoa salami. which render these products vulnerable to staphvlococcal development. Use of cultures of lactic acid bacteria has YIATERIALS A:"pork was thawed, cut into strips and ground through a still used to some extent in industry (4). In 1971, several 1.2-cm plate. Spices and curing agents (fable 1) were miXJ:d in by outbreaks of gastroenteritis were traced to Genoa salami TABLE 1. Genoa salami ingredients containing up to to• coagulase positive staphylococci/a Quantity and type A enterotoxin was detected in some of these samples (12. 13). Niskanen and Nurmi (6) found Pork Sodium chloride J06.20g measurable amounts of enterotoxin A in a 200-g sample 11.34 g 1 White pepper of dry sausage containing per g 10 cells of Staphylococ· Whole pepper 2.84g cus aureus which produce type A toxin but enterotoxin B Sodium nitrite 0.71 g was not detected in corresponding samples containing Sodium nitrate 5.68g Garlic 1.89 g lQB cells per g of S. aureus which produce type B toxin. 68.10 g Barber and Deibel (2) studied the effect of pH and oxygen tension on staphylococcal growth and enterotoxin stirring. The meat was then inoculated with washed cells rA S. aureu.r formation in fermented sausage. They indicated growth 265 or 243 in the amount of about 10l, 10', and 107 cells/g. After could be controlled with l.So/o glucono-delta-lactone inoculation, meat was spread in layers and for 2days at4 C. (GDL), whereas a hig.h inoculum of Pediococcus It was re-ground and stuffed into pre-soaked, tied collaiFO CISings (9 x 56 em; Brechteen Co.). The casings of salami were refriaeratecl 1t cerevisiae failed to suppress aerobic growth. Most 4 C for 4 davs and then put in a tempering room at 20 to 25 C ano !!Oo/o relative- humidity (RH) for 2 days. FoUowinJ temperlna, the 1 MichiganAgriculturalExperiment Station Journal Article No. 7372. casings were heated in air at 38 C for 20 h, 43 C for 2 b, 49 C for 4 11: 1 Present address: Central Research Dept., Anheuser-Busch. Inc. 1101 and S4 C for J hat 80 to 90% RH. The salami was then dr1ed at 12 C Wyoming Street, St. Louis, Mo 63118 and 72%RH torabout60days. 326 LEE, HARMON, AND PRICE

Sampling methods Samples from the outer 1 em of surface and sample~~ of the core were 7 taken from salami inoculated with S. aureus 265. Samples of the emire cross section were taken from salami inoculated with S. aureus 243. 6 The salami were examined at aifferent stages of processing as follows: (a) after inoculation, (b) before tempering, (c) after tempering, (d) after 5 heating, and (e) at various intervals during drying. 4 Enumeration ofmicrobial populations --- ..... -.... _~ Staphylococcal counts were made using spread plate technique 3 01 on Mannitol Salt Agar (MSA; Difco). Following incubation at 37 C for 2 48 h. coagulase tests were made on a representative number of typical '1/) S. aurous colonies. The aerobic counts were made in Plate Count Agar Q) () 8~ (PCA; Difco). Lactic acid bacteria were enumerated in plates of Lactobacillus Selective Agar (LBS: BBL). c 7~ 0 L------~~ Lactic acid and pH determination +- 6 /~ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/5/325/1649781/0362-028x-40_5_325.pdf by guest on 30 September 2021 c I I Twenty grams of salami and 180 mi of de-ionized water were mixed ::1 in a Waring belnder for 2 min. The pH of the homogenate was a. 5 h.. __ ------..c... measured on a Beckman pH meter. The homogenate was then filtered a..0 through Whatman #1 filter paper and portions of filtrate corresponding 01 to 5 g of sample were titrated with 0.1 N NaOH to pH 8.3. The total 0 ;[ _J titratable acid was calculated as percent lactic acid. 9 Moisture determination A 5-g sample of salami was spread in an aluminum moisture dish 5.5 em in diameter (Sargent and Co.} and dried in a convection oven at

100 C for 16 to 18 h and cooled. The weight loss was expressed as I percent moisture. I 6 I Determination ofwater activity --I ,I.. 5 b------.t>.- ---c. A moisture sensing element (No. 547535, Hygrodynamics. Inc.) was mounted in a rubber stopper on a 170-ml jar containing 20 g of salami 4L------~------~------~~~ and attached to a hygrometer indicator. Wate~ activity measurements 0 10 20 30 63 were carried out after the samples were equilibrated for 24 hat 22 C. Days Extraction and detection of enterotoxin Enterotoxin was enracted from 100-g samples of salami and determined by the serological procedure described by Casman and Figure 1. Populations of S. aureus strain '?65 determined on MSA Bennett (])with modifications described by Barber and Deibel (2). plates and enterotoxin A produced in salami inoculated with 10' (top), 105 (middle), and 107 (bottom) cells/fl. Legend: -o- surface sample;-/::,. core sample. Solid symbols indicate enterotoxin A was detected. RESULTS AND DISCUSSION Genoa salami inoculated with S. au reus 265 of the microbial populations was mainly due to the Samples of salami were obtained for analyses at 0 day difference in the oxygen tension. Total population trends (after inoculation), 6 days (after curing in the cooler), 8 for organisms enumerated by aerobic plate counts (Fig.2) days (after tempering), 9 days (after heating), 29 and 63 were similar to those of the staphylococci, except that days (during drying). Data in Fig. 1 illustrate the growth during the drying period the total populations decreased patterns of S. aureus 265 in the inoculated salami. The less than the staphylococcal populations. Data in Fig. 3 staphylococcal population remained the same or illustrate the population changes of the lactic acid decreased slightly during 6 days of curing in the cooler. bacteria in salami inoculated with S. aureus 265. The After tempering, counts of 1.5 x 107, 2.8 x 101, and original population of these organisms in the pork was 4.9 x 108 cells/g were obtained from the surfaces of the less than 150/g of meat, but the count was more than salami inoculated with 103 , 105 , and 107 staphylococci/ 105/g in samples taken after the salami was heated. The g, respectively. In the core samples, however, increases of anaerobic condition in core samples caused a decrease in 300-fold and 15-fold occurred in the salami inoculated the lactic acid bacteria, since they are microaerophilic. with 10 3 and 10 5 cells/g, respectively, while only a slight Samples taken from different locations ofthe salami did increase occurred in the salami inoculated with 10 7 not show any significant difference in the populations of cells/g. Heating caused a reduction of 10- to 100-fold in lactic acid bacteria. Also, there was no significant populations in both surface and core samples. During decrease in the population of lactic organisms during the drying period the populations gradually decreased. drying. After drying, counts were more than 101 cells/g on the The pH of salami made in this experiment was surface of each salami and less than 1.0 x 103, 1.7 x 103 relatively high, ranging from 6.06 to 6.33 and probably and 1.2 x 105 cells/gin the core of salami inoculated with would have been lower if the meat had been inoculated 103, 105, and 107 cells/g, respectively. The staphylococcal with lactic organisms. However, low pH values may not counts were always higher in surface samples than in inhibit growth and enterotoxin production by staphylo core samples. These results verify the finding of Barber cocci. Enterotoxin B can be produced in cured meat at and Deibel (2) who reported that the uneven distribution an initial pH of 5.0 (7), and an initial pH of 4.5 in STAPHYLOCOCCI IN SALAMI 327

reconstituted nonfat milk solids permitted enterotoxin A 8 production according to Tatini et at. (9). The lowest pH 7 e------~--~~ value reported to permit formation of staphylococcal ~ enterotoxin was 4.0 in broth medium inoculated with J\------cz..-~-{;.. 6 1 Dt------a 1 x 108/ml (5). Total acidity developed during the curing I 5 period varied from 0.64 to0.79o/oexpressed as lactic acid. More lactic acid was present in the surface samples of 4 salami inoculated with 103 staphylococci than in those 3 inoculated with 105 and 107/g. The lactic fermentation of 01 the salami was dependent on the indigenous bacterial ...... 9 Ill flora of the meat and the lactic organisms reached .a 5 Q) 8 population of only about 10 /g. Acton et al. (J) 0 ~------~~~~ 1

reported more than 10 /g of lactic acid bacteria in Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/5/325/1649781/0362-028x-40_5_325.pdf by guest on 30 September 2021 c 7 0 sausage after 48 h in a controlled fermentation when 6 ~~---- lactic culture was inoculated at 2 x 108/g. .Q- / ------<"----lr---..<:1 :::3 ,<1!----tf In the samples taken after 8 or more days, a. 5 0 approximately 0.2 ~ of enterotoxin A was detected in !-----'-----...... ______.__-1 f-----J a... 4 100-g portions of surface samples from salami 01 0 inoculated with 105 and 107 S. aureus 265/g of meat, but _J 10 no enterotoxin was detected in any of the core samples or 9 in the surface samples of salami inoculated with 10-1/g. 8 Absence of enterotoxin in core samples can be explained by restricted growth of the organisms due to the reduced 7 I l)xygen tension in the center ofthe salami. 6 b------6- - --II- -- -6 Genoa salami inoculated with S. aureus 243 5 L------~-----~-----L-~~ Samples were taken from cross sections of this salami 0 10 20 30 63 instead of from the surlace and core as was done with the Days previous group. Figure 4 illustrates the data obtained on Figure 2. A••rob1c ])[ate counts of salami inoculated with 103 (top), 105 (middle), and 101 (bottom) S. aureus strain 265 celts/g. Legend: ·o· swface sample; ·.6. core sample. 8

7 6 7

5 I 'fP------er----1~-~/..-___,.. --...... 01 ··········.J 01 Ill ...... Ill 3

~------~------~------~~~ 10 20 30 63 10 20 30 60 Days Days Figure 3. Populatioll8 of lactic acid bacteria in salami inoculated with 1()3 (top). Ufl (middle), and 101 fbottom) S. aureus strain 265 Figure 4. Populations (}(S. aureus 243 as determined on MSA plates 3 1 cells/g. Legend: ·o· sur(ace sample; ·Ll • core samples. from salami inoculated with 10 (D). J(J6 (.)and /0 (o) cells/g. 328 LEE, HARMON, AND PRICE the staphylococcal populations in the inoculated salami. competitive effect was observed between the lactic acid The counts decreased slightly during curing for 6 days in bacteria and the staphylococcal inocula at the various the cooler. Determinations after tempering indicated that concentrations. the populations increased to 1.0 x 106, 9.0 x 1Q6, and Data. showing the moisture content and aw of the 8 1.3 x 10 /g in the salami inoculated with 103, 105, and salami are recorded in Table 3. The moisture content 107/g, respectively. Heating on the 9th day reduced the populations 5000-to 59,000-fold in different samples of TABLE 3. Moisture content and aw ofa representive blend qf' salami salami but a slight increase in population at the next inoculated with S, au reus 265 sampling time on the 24th day occurred in two of the Days three samples. Thermally injured cells are inhibited on a N.D. highly selective medium and the staphylococcal popula 6 58.2 0.99 tions in samples taken immediately after heating 8 58.4 0.98 9 59.9 N.D. probably did not include the heat injured cells. On the 23 55.3 0.97 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/5/325/1649781/0362-028x-40_5_325.pdf by guest on 30 September 2021 59th day, a population of 2.3 x 104/g remained in the 59 43.4 0.84 salami inoculated with 10 7/g. The data in Fig. 5 show the fluctuated within the range of sampling error during heating and decreased 16.5% during the drying period. Substantial decreases occurred in both moisture and aw 8 between the 23rd and 59th day. Enterotoxin B was not detected in any of the samples taken from the inoculated salami, although the population of S. aureus 243 reached 1.3 x 108 /g. The variations in individual staphylococ cal strains and environmental conditions have a great influence on the production of enterotoxin. Although the production of enterotoxin is always dependent upon attainment of high cell populations, good growth of -c staphylococci is not necessarily an indication of the :::::l presence of enterotoxin (2, 7, 8). In this investigation, the a. 0 aw dropped from 0.99 to 0.98 during tempering of the a.. 5 salami and is a limiting factor in the production of Cl 0 enterotoxin A (J 1). Although enterotoxin B was not ....l detected, the high population ofthe staphylococci in the 4 product could be a potential health hazard since measurable toxin production sometimes occurs in foods containing only a few million coagulase positive staphylococci/g, and some strains of S. aureus produce multiple types of toxins detectable only by testing for each toxin individually. Days ACK!'>O\HEDG.\1E;o.;'I Figure S. Aerobic plate counts of non-inoculated salami (.6.) and of' salami inoculated with S. aureus 243 at 103 ( []), IO' (.), and J07 (o) This project was partially supported by Public Health urant No. FD cells/g. 00163 of the Food and Drug Administration. aerobic plate counts of the salami. Again, the REFERENCES populations increased during tempering and decreased 1. Acton, J. C .. J. G. Williams, and M. G. Johnson. 1972. Effect ~ during heating, whereas little change in the population fermentation temperature on changes in meat properties and Davor: ocLurred during dtying. Data on the population ot-lactic of summer sausage. J. Milk Food Techno!. 35:264-268. 2. Barber, L. E., and R. H. Deibel. 1972. Effect of pH and oxygas acid bacteria are summarized in Table 2. No significant tension on staphylococcal growth and enterotoxin tormatiou in TABLE 2. Population of lactic acid bacteria in non-inoculated fermented sausage. Appl. Microbiol. 24:1191-898. salami and in salami inoculated with S. aureus 3. Cas man, E. P ., and R. W. Bennett. 1965. Detection of staphyloeoc· cal enterotoxin in food. Appl. Microbiol. 13:1111-189. 4. Daly, C., M. Chance, W. E. Sandine, and P. R. Elllker. 11173. Control of St .. phylococcus aureus m sausage oy starter culture ana chemical acidulation J. Food Sci. 38:426-432. <150 5. Genigeorgis, C., M.S. Foda, A. Mantis, and W. W. Sadler. 1971. 6

7. Reimann, H .. W. H. Lee, and C. Genigeorgis. 1972. Control of 11. Troller. J. A. 1972. Effect of water activity on enterotoxin A Clo.stridium botulinum and Staphylococcus aurew in semi l-":oduction and growth of Staphylococcu aurew. Appl. Microbiol preserved meat products. J. Milk Food Techno!. 35:514-523. 24:440-443. 8. Scheusner, D. L., and L. G. Harmon. 1973. Growth and entero 12. United States Department of Health, Education aDd Welfare. toxin production by various strains of Staphylococeu~ aurew in 1971. Staphylococcal gastroenteritis associated with salami - selected foods.J. Food Sci. 38:474-476. United States, Morbidity and Mortality 20:253, 261. Center for 9. Tatini, S. R .. J. J. Jezeski, J. C. Olson, Jr. and E. P. Casman. 1971. Disease Control. Atlanta, Georgia. Factors influencing the production of staphylococcal enterotoldn 13. United States Department of Health, Education and Welfare. A in milk. J. Dairy Sci. 51:312-320. 1971. Gastroenteritis attributed to Hormel San Remo stick Genoa 10. Troller, J. A. 1971. Effect of water activity on enterotoxin B salami Maryland. Morbidity and Mortality 20:370. Center for production and growth of Staphylococcus aureus. Appl. Microbiol Disease Control. Atlanta, Georgia. 21:435-439. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/40/5/325/1649781/0362-028x-40_5_325.pdf by guest on 30 September 2021

Heat-Resistant Psychrotrophic Bacteria Isolated from Pasteurized Milk

C. J. WASHAM, H. C. OLSON, and E. R. VEDAMUTHU

Department ofDairy Science Oklahoma State University. Stillwater, Oklahoma 74074

Table 10 was inadvertently omitted from this paper. It should have been provided as follows:

TABLE 10. Tentative identification ofnon-spore·producing types Type Tentative identification Variations from Bergey's A Microbacterium flavum Acid from maltose and lactose B Arthrobacter aurescens c Microbacterium lacticum Starch not hydroloyzed D Corynebacterium equi E Arthrobactersp. Did not agree with any specie described in Bergey's Manual F Arthrobacter sp. G Streptococcus faecalis var. liquefociens H Streptococcus faecalis