Potential Mycotoxin Problems in Mould-Fermented Sausage H..J
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Potential Mycotoxin Problems in Mould-fermented Sausage H..J. 1IlliTZL.AFF, A. CIEGLER, and L. LEISTNER :Mitteilung aus der Bundcsanstalt fUr Fleischforschung, Kulmbach, (BRD) und Northern Regional Research Laboratory, Peoria, (USA) Received August 3, 1972 Summary. A considerable proportion of the sausage consumed in many European countries is produeed by a mould-fermentation process that does not involve pure culture technics. 422 Penicillium cultures isolated from mould-ripened sausages produced in 11 European countries were analyzed for their ability to produce the following nine mycotoxins: aflatoxin B, and Gl , ochratoxin A, penicillic acid, patulin, citrinin, tremortin A, zearalenone, rubratoxin B; 88 isolates were found capable of toxin synthesis (20.9 %). 44 of these cultures produced penicillic aeid, 17 ochratoxin A, 11 tremortin A, 10 citrinin, and 6 patulin. 3 cultures produced both patulin and citrinin. Sausages were ripened with 6 moulds producing penicillic acid, 17 producing ochratoxin, 5 producing citrinin and 3 each producing patulin or tremortin. No mycotoxins were detected up t{) 70 days of ripening. Direct addition of penicillic acid to raw sausage resulted in disappearance of the mycotoxin. Amino acids normally occuring in meat were found capable of rapidly reacting with penicillic acid to produce adducts that were nontoxic to laboratory animals. Although our data indicate that consumption of mould ripened sausage is not a health hazard with respect to the 9 mycotoxins analyzed for, it is recommended that manufacturers of these products adopt pure culture technics using moulds known to be toxicologically safe. Zusammenjassung. In zahlreichen Liindern Europas werden seit altersher mit Schimmel pilzen gereifte Rohwiirste hergcstellt. Es handelt sich hierbei um traditionelle Produktionsver fahren, bei denen jedoch keine SchimmelpiIz-Reinkulturen verwendet werden. 'Vir untersuchten das Toxinbildungsvermogen von 422 Penicillium-Stammen, die von schim melpilzgereiften Rohwiirsten isoliert worden waren, die aus 11 verschiedenen Landen;' stammten. Insbesondere interessierten uns die :\Iykotoxine Aflatoxin B, und Gl , Ochratoxin A, Penicillin saure, Patulin, Citrinin, Tremortin A, Zearalenon und Rubratoxin B. Bei SS Stammen (20,9%) 134 war eine Toxinbildung naehweisbar. 44 dieser Kulturen bildeten Penieillinsiiure, 17 Ochratoxin A, 11 Tremortin A, 10 Citrinin und 6 Patulin. Von drei Stii=en wurde sowohl Patulin als aueh Citrinin gebildet. Wurden frisch hergestellte Roh'll'iirste mit Schimmelpilzen, die die geriannten Toxine bilden, beimpft, so konnten wiihrend der 70 Tage dauerriden Reifung keine Mykotoxine in der Wurst nachgewiesen werden. Den Rohwiirsten direkt zugesetzte Penicillinsaure wurde inaktiviert, da dieses lIykotoxin sehr rasch mit Aminosiiuren reagiert, die iIll Fleisch vorhanden sind. Obwohl diese Untersuchungen darauf hinwciscn, daB schimmelpilzgcreifte Rohwiirste, zumin dest beziiglieh der 9 genannten Mykotoxine, keine potcntieUe Gefahr iiir den Verbraucher dar steUen, soUte man dennoch dazu iibergehen als Starterkultur nur technologisch und toxikologisch gepriifte Schimmelpilz-Stamme in Reinkultur einzusetzen. Introduction The occurrence of moulds on meat is not lmcommon and in some speciality-type items, such as Italian- or Hungarian-style sausages, so-called salami, are actually a part of the production process. Throughout much of Southern Europe including some of the Balkan countries, these sausages rcpresent a major item of food commerce. The moulds involvedin the ripeningprocess although confinedto the outer casing affect the appearance, taste, odor and keeping qualities of the finall}roducts. The fermen tation is generally completed in five days but the sausages are often permitted to .ripen for up to 70 days for finishing. 'With but few exceptions, pure culture technic is not practiced in commercial production. The moulds that develop on the casing depend for the most part on the fungal flora of the ripening chamber. Similar non pure culture technics are practiced in the production of other mould-fermented meats such as country cured hams in the Southeastern United States. This reliance on nature makes quality control haphazard and, at best, a matter of chance. Conse quently, production problems are not uncommon. ,,'lith the recognition that moulds are capable of producing highly toxic substances, another clement of uncertainty is introduced into the production process. The flmgal flora of salamis has been determined by several investigations and, although there is some variation, most of the data indicate that members of the genus Penicillium predominate [1]. In particular, the following species have bcen reported: Penicillium expansum, P. commune, P. simplicissimum, P. miczynskii and P. janthi nellum. Recognizing the need for controlled inocula in the production process, Raco vita et al. [2] recommended the use or P. expan-mm since in their experiments it proved to be nontoxigenic, grew rapidly on sausage with a desirable grey white color and imparted the proper organo-leptic properties. E::\.-pcrimcntal In our own investigation we isolated 25 strains of P. e;cpansum from salamis originating in 11 European cOlmtries. 'When these were i,rrown on sausage they pro duced an undesirable greenish appeal'Ul1ce but, more important, filtrates of these strains when grown onpotato-dextrose broth proved toxic for chick embryos, chicken, japanese quails and mice whether administered i. p. or per os. The organs, liver in particular, of chicken dosed with these cultme filtrates showed typical toxic lesions. The histopathological finclings could be designed as a "hepatitis interstitialis lympho c:y-taria" ·with pcribiliar and l}erivascular lymphocytic infiltrations [3, 4]. In subse quent studies many of these cultures were found to produce patulin as well as other mycotoxins. Under these circumstances it would appear undesirable to recommend the use of P. expanswn as a starter cultuTe for fermented sausage production. vI,!e enlarged the scope of ourulvestigation to include a survey of the toxinogenic capacity of the other Penicinium isolates we had obtained from commercial salamis. Using YES-meclium [5] we assayed for the production of the following toxins: aflatoxins Bl and Gl , citrinin, ochratoxin A, patulin, penicillic acid, rnbratoxin B, 135 trcmortin A, and zearalcnonc. Other media provedlcss suitablc for toxin production. A summary of the data obtained from 422 cultures is sho1\"11 in Table 1. Table 1. ilIycotoxins produced by 422 strains of the Penicillium species 1Iycotoxins No. produc- Percentage of Toxin production ing strains the total strains in YES broth . analyzed mg/100 ml Penicillic acid 44 10.4 0.1- 50 Ochratoxin A 17 4.0 0.4- 64 Tremortin A 11 2.6 0.1-15 Citrinin 10 2.4 0.1-40 Patulin 6 1.4 2.0 -48 Total 88 20.9 This table shows that five of the nine toxins analyzed for were detected and that on this basis 20.9 % of the cultures were toxinogenic. Of course we recognize certain inherent linlitations of our assay procedure. The media and other physical and physiological conclitions were limited, other knO\\"ll toxins were not included in the assay, and we did not attempt to determine if unkno,vn toxins were sY"11thesized.Thus it is easily conceivable that an even larger percentage could prove toxinogenic. The 88 toxinogenic Penicillium strains represent 13 identified species. Table 2 shows those toxinogenic isolates that we identified to species and the toxins they produced. ~qjl demonstrated mycotoxins were produced by several Penicillium species. Table 2. lIIycotoxins produced by identified Penicillhlm species lIIycotoxins Penicillium species No. toxigenicl No. surveyed Penicillic acid P. 'O'iridicatum 14/32 P. expan.sum 9141 P. janthinellllm 3/13 P. cyclopium 2/3 P. simplici.ssimum 2/11 P. commune 1/5 P. marten.sii 1/3 not identified 12/275 Ochratoxin A P. viridicatllm 12/32 P. mriabile 212 P. commune 1/5 P. cyclopium 1/3 P. purpurescens III Tremortin A P. palitans 4/7 P. commune 3/5 P. /requentans 1/4 not identified 3/275 Citrinin P. citrinum 1/4 P. clavi/orme III P. expansum 1/41 not identified 7/275 Patulin P. expansum 3/41 not identified 3/275 Patulin and P. expansum 1/41 Citrinin not identified 2/275 The most striking data are those sho,vn for P. expansurn and P. viridicaturn. These two species represent over half thc cultures identified and between them constitute the largest total number of toxin producers. P. viridicatum, in particular, 136 is noteworthy for over 80 % of the isolates wcre toxigenic. This species appears to be assuming increasing importance for it has been reported by Krogh [6J to occur in Danish fcedstuffs, by van \Valbeek et al. [7J and other investigators in Canada and in the United States it has been isolated from grain and corn. However, the data obtainedon the ability of moulds to produc3 toxins onlaboratory media should be interpreted carefully and ummrranted assumptions not made. Thus, from our standpoint, a more important question is: Can these moulds isolatedfrom sausage produce toxins when grown on sausage under commercial production condi tions? We inoculated raw sausage \\ith representative producers of the five toxins detectcd including 16 of the 17 ochratoxin produccrs. The sausages were fermented under the regimen of changing temperature and relative humdity used in industrial production. None of the five toxins were detected after periodic analyses for up to 70 days [8]. However, the interpretation of our data was tempered