708 Modern Food Microbiology 87 Rosset, J.S., K.D McClatchey, G.I Higashi, and A.S Knisely 1982 Anisakis larval type I in fresh salmon Am J Clin Pathol 78:54–57 88 Roy, S.L., A.S Lopez, and P.M Schantz 2003 Trichinellosis surveillance—United States, 1997–2001 Morb Mort Wkly Rep 52(SS-6):1–7 89 Sacks, J.J., R.R Roberto, and N.F Brooks 1982 Toxoplasmosis infection associated with raw goat’s milk J Am Med Assoc 248:1728–1732 90 Salata, R.A., A Martinez-Palomo, L Canales, H.W Murray, N Revino, and J.I Ravdin 1990 Suppression of Tlymphocyte responses to Entamoeba histolytica antigen by immune sera Infect Immun 58:3941–3946 91 Schantz, P.M 1983 Trichinosis in the United States—1947–1981 Food Technol 37(3):83–86 92 Sherwood, D., K.W Angus, D.R Snodgrass, and S Tzipori 1982 Experimental cryptosporidiosis in laboratory mice Infect Immun 38:471–475 93 Shlim, D.R., M.T Cohen, M Eaton, R Rajah, E.G Long, and B.L.P Unger 1991 An alga-like organism associated with an outbreak of prolonged diarrhea among foreigners in Nepal Am J Trop Med Hyg 45:383–389 94 Smith, H.V., C.A Paton, M.M.A Mtambo, and R.W.A Girdwood 1997 Sporulation of Cyclospora sp oocysts Appl Environ Microbiol 63:1631–1632 95 Smith, J.L 1993 Documented outbreaks of toxoplasmosis: Transmission of Toxoplasma gondii to humans J Food Protect 56:630–639 96 Smith, P.D 1989 Giardia lamblia In Parasitic Infections in the Compromised Host, ed P.D Walzer and R.M Genta, 343–384 New York: Marcel Dekker 97 Smith, H.V., R.W.A Girdwood, W.J Patterson, R Hardie, L.A Green, C Benton, W Tulloch, J.C.M Sharp, and G.J Forbes 1988 Waterborne outbreak of cryptosporidiosis Lancet 2:1484 98 Soave, R 1996 Cyclospora: An overview Clin Infect Dis 23:429–437 99 Sturbaum, G.D., Y.R Ortega, R.H Gilman, C.R Sterling, L Cabrera, and D.A Klein 1998 Detection of Cyclospora cayetanensis in wastewater Appl Environ Microbiol 64:2284–2286 100 Sole, T.D., and N.A Croll 1980 Intestinal parasites in man in Labrador, Canada Am J Trop Med Hyg 29(3):364–368 101 Sterling, C.R., and M.J Arrowood 1986 Detection of Cryptosporidium sp infections using a direct immunofluorescent assay Pediatr Infect Dis 5:139–142 102 Sterling, C.R., K Seegar, and N.A Sinclair 1986 Cryptosporidium as a causative agent of traveler’s diarrhea J Infect Dis 153:380–381 103 Todd, E.C.D 1989 Foodborne and waterborne disease in Canada—1983 annual summary J Food Protect 52:436–442 104 Tzipori, S 1988 Cryptosporidiosis in perspective Adv Parasitol 27:63–129 105 U.S Department of Agriculture 1982 USDA advises cooking pork to 170 degrees Fahrenheit throughout News release, USDA, Washington, DC 106 Zimmermann, W.J 1983 An approach to safe microwave cooking of pork roasts containing Trichinella spiralis J Food Sci 48:1715–1718, 1722 107 Zimmermann, W.J., and P.J Beach 1982 Efficacy of microwave cooking for devitalizing trichinae in pork roasts and chops J Food Protect 45:405–409 108 Zimmermann, W.J., D.G Olson, A Sandoval, and R.E Rust 1985 Efficacy of freezing in eliminating infectivity of Trichinella spiralis in boxed pork products J Food Protect 48:196–199 Chapter 30 Mycotoxins A very large number of molds produce toxic substances designated mycotoxins Some are mutagenic and carcinogenic, some display specific organ toxicity, and some are toxic by other mechanisms Although the clear-cut toxicity of many mycotoxins for humans has not been demonstrated, the effect of these compounds on experimental animals and their effect in in vitro assay systems leaves little doubt about their real and potential toxicity for humans At least 14 mycotoxins are known to be carcinogens, with the aflatoxins being the most potent.84 It is generally accepted that about 93% of mutagenic compounds are carcinogens With mycotoxins, microbial assay systems reveal an 85% level of correlation between carcinogenicity and mutagenesis.84 Mycotoxins are produced as secondary metabolites The primary metabolites of fungi as well as for other organisms are those compounds that are essential for growth Secondary metabolites are formed during the end of the exponential growth phase and have no apparent significance to the producing organism relative to growth or metabolism In general, it appears that they are formed when large pools of primary metabolic precursors such as amino acids, acetate, pyruvate, and so on, accumulate The synthesis of mycotoxins represents one way the fungus has of reducing the pool of metabolic precursors that it no longer requires for metabolism Some methods for the detection of mycotoxins in foods can be found in Chapter 11 For an extensive review of these substances in fruits, fruit juices, and dried fruits, see reference 27 AFLATOXINS Aflatoxins are clearly the most widely studied of all mycotoxins Knowledge of their existence dates from 1960, when more than 100,000 turkey poults died in England after eating peanut meal imported from Africa and South America From the poisonous feed were isolated Aspergillus flavus, and a toxin produced by this organism that was designated aflatoxin (Aspergillus flavus toxin—A-fla-toxin) Studies on the nature of the toxic substances revealed the following four components: 709 710 Modern Food Microbiology A flavus produces AFB1 and AFB2 ; and A parasiticus produces all four of the major aflatoxins (B1 , G1 , B2 , and G2 ) AFB1 is produced by all aflatoxin-positive strains, and it is the most potent of all Other known aflatoxin producers include A nominus,47 A bombycis, A pseudotamartii, and A ochraceoroseus among the aspergilli; and Emericella venezuelensis These compounds are highly substituted coumarins, and at least 18 closely related toxins are known AFM1 is a hydroxylated product of AFB1 , and it appears in milk, urine, and feces as a metabolic product.30 AFL, AFLH1 , AFQ1 , and AFP1 are all derived from AFB1 , AFB2 is the 2,3-dihydro form of AFB1 , and AFG2 is the 2,3-dihydro form of AFG1 The toxicity of the six most potent aflatoxins decreases in the following order: B1 > M1 > G1 > B2 > M2 = G2 When viewed under ultraviolet (UV) light, six of the toxins fluoresce as noted: B1 and B2 —blue G1 —green G2 —green–blue M1 —blue–violet M2 —violet They are polyketide secondary metabolites whose carbon skeleton comes from acetate and malonate The proposed partial pathway for AFB1 synthesis is as follows: acetate > norsolorinic acid > averantin > averufanin > averufin > versiconal hemiacetal acetate > versicolorin A > sterigmatocystin > O-methylsterigmatocystin > AFB1 Versicolorin A is the first in the pathway to contain the essential C2 C3 double bond Requirements for Growth and Toxin Production No aflatoxins were produced by 25 isolates of A flavus/parasiticus on wort agar at 2, 7◦ , 41◦ , or 46◦ C within days, and none was produced below 7.5◦ C or over 40◦ C even under otherwise favorable conditions.75 In another study employing Sabouraud’s agar, maximal growth of A flavus and A parasiticus occurred at 33◦ C when pH was 5.0 and water activity (aw ) was 0.99.41 At 15◦ C, growth occurred at aw 0.95 but not at 0.90, while at 27◦ and 33◦ C, slight growth was observed at an aw of 0.85 The optimum temperature for toxin production has been found by many to be between 24◦ and 28◦ C In one study, maximal growth of A parasiticus was 35◦ C, but the highest level of toxin was produced at 25◦ C.78 Mycotoxins 711 Figure 30–1 Growth and aflatoxin B1 production on malt extract-glycerine agar at various water activity values and temperatures White columns: rate of growth; black columns: average AFB1 production Source: From Northolt et al.,63 copyright c 1976 by International Association of Milk, Food and Environmental Sanitarians The limiting moisture content for AFB1 and AFB2 on corn was 17.5% at a temperature of 24◦ C or higher, with up to 50 ng/g being produced.96 No toxin was produced at 13◦ C Overall, toxin production has been observed over the aw range of 0.93–0.98, with limiting values variously reported as being 0.71–0.94.58 In another study, no detectable quantities of AFB1 were formed by A parasiticus at aw values of 0.83 at 10◦ C.63 The optimum temperature at aw 0.94 was 24◦ C (Figure 30–1) Growth without demonstrable toxin appeared possible at aw 0.83 on malt agar containing sucrose It has been observed by several investigators that rice supports the production of high levels of aflatoxins at favorable temperatures but none is produced at 5◦ C on either rice or cheddar cheese.68 Overall, the minimal and maximal parameters that control growth and toxin production by these eukaryotic organisms are not easy to define, in part because of their diverse habitats in nature and in part because of their eukaryotic status It seems clear that growth can occur without toxin production AFG1 is produced at lower growth temperatures than AFB1 , and while some investigators have found more AFB1 than AFG1 at around 30◦ C, others have found equal production With regard to A flavus and A parasiticus, the former generally produces only AFB1 and AFG1 22 Aeration favors aflatoxin production, and amounts of mg/g can be produced on natural substrates such as rice, corn, soybeans, and the like.22 Up to 200–300 mg/l can be produced in broth containing appropriate levels of Zn2+ The release of AFB1 by A flavus appears to involve an energy-dependent transport system Production and Occurrence in Foods With respect to production in foods, aflatoxin has been demonstrated on fresh beef, ham, and bacon inoculated with toxigenic cultures and stored at 15◦ , 20◦ , and 30◦ C,9 and on country-cured hams 712 Modern Food Microbiology during aging when temperatures approached 30◦ C, but not at temperatures below 15◦ C or relative humidity (RH) above 75%.10 They have been found in a wide variety of foods, including milk, beer, cocoa, raisins, soybean meal, and so on (see below) In fermented sausage at 25◦ C, 160 and 426 ppm of AFG1 were produced in 10 and 18 days, respectively, and 10 times more AFG1 was found than B1 51 Aflatoxins have been produced in whole-rye and wholewheat breads, in tilsit cheese, and in apple juice at 22◦ C They have been demonstrated in the upper layer of 3-month-old cheddar cheese held at room temperature52 and on brick cheese at 12.8◦ C by A parasiticus after week but not for A flavus.78 AFB1 was found in of 63 commercial samples of peanut butter at levels less than ppb.69 From a 5-year survey of around 500 samples of Virginia corn and wheat, aflatoxins were detected in about 25% of corn samples for every crop year, with 18–61% of samples containing 20 ng/g or more and 5–29% containing more than 100 ng/g.80 The average quantity detected over the 5-year period was 21–137 ng/g Neither aflatoxins nor zearalenone and ochratoxin A were detected in any of the wheat samples The 1988 drought led to an increase in the amount of aflatoxin produced in corn in some midwestern U.S states that received less than inches of rain in June and July About 30% of samples contained more than 20 ppb compared to 2–3 ppb levels during normal rainfall.83 In a study of AFB1 in foods and feedstuffs in Cuba for the years 1990–1996, 17% of 4529 samples were positive with 83% of sorghum and 40% of peanuts being the most contaminated.32 The incidence in corn was 23 and 25% in wheat In Botswana, 120 peanut samples were tested for aflatoxin and 78% were positive at levels from 12 to 329 µg/kg, and 49% contained >20 µg/kg.59 Twenty-one percent of these samples contained cyclopiazonic acid at levels from to 10 µg/kg Cyclopiazonic acid is produced by some A flavus strains and it is thought to contribute to the toxicity of aflatoxin In an examination of seven truckloads of corn, it was found in four at levels of 25–250 ng/g.49 Also found in four of five loads was deoxynivalenol (DON, vomitoxin) at levels of 46–676 ng/g The effect of temperature cycling between 5◦ and 25◦ C on production in rice and cheese has been investigated A parasiticus produced more toxin under cycling temperatures than at 15◦ , 18◦ , or 25◦ C, while A flavus produced less under these conditions.68 On cheddar cheese, however, less aflatoxin was produced than at 25◦ C, and these investigators noted that cheese is not a good substrate for aflatoxin production if it is held much below the optimum temperature for toxin production Aflatoxin production has been demonstrated to occur on an endless number of food products in addition to those noted Under optimal conditions of growth, some toxin can be detected within 24 hours—otherwise within 4–10 days.18 On peanuts, Hesseltine40 has made the following observations: Growth and formation of aflatoxin occur mostly during the curing of peanuts after removal from soil In a toxic lot of peanuts, only comparatively few kernels contain toxin, and success in detecting the toxin depends on collecting a relatively large sample, such as kg, for assay The toxin will vary greatly in amount even within a single kernel The two most important factors affecting aflatoxin formation are moisture and temperature The U.S Food and Drug Administration (FDA) has established allowable action levels of aflatoxins in foods as follows: 20 ppb for food, feeds, Brazil nuts, peanuts, peanut products, and pistachio nuts; and 0.5 ppb for milk.48 A committee of the Codex Alimentarius Commission has recommended the following maximum levels of mycotoxins in specific foods: 15 µg/kg of aflatoxins in peanuts for further processing; 0.05 µg/kg of aflatoxin M1 in milk; 50 µg/kg of patulin in apple juice and apple juice ingredients in other beverages; and µg/kg of ochratoxin A in cereals and cereal products.61 ... Production and Occurrence in Foods With respect to production in foods, aflatoxin has been demonstrated on fresh beef, ham, and bacon inoculated with toxigenic cultures and stored at 15◦ , 20◦ , and... cultures and stored at 15◦ , 20◦ , and 30◦ C,9 and on country-cured hams 712 Modern Food Microbiology during aging when temperatures approached 30◦ C, but not at temperatures below 15◦ C or relative... moisture and temperature The U.S Food and Drug Administration (FDA) has established allowable action levels of aflatoxins in foods as follows: 20 ppb for food, feeds, Brazil nuts, peanuts, peanut