Perspectives on aflatoxin control for human food and animal feed

40 Dickinson,E., Elverson,D.I.and Murray,B.S,(1989)Food Hydrocolloids3, 101-114 41 Reichman,D, and Garti,N. (1991)in FoodPolymers,Gelsand Colloids(Dickinson.E.,ed.),pp. 549-556,RoyalSocietyof Chemistry 42 Garti,N. and Reichman,D. FoodStructure(in press) 43 Dickinson,E.and Woskelt,C.M.(1989)in FoodColloids(Bee,R.D, Richmond,P.and Mingins,I., eds),pp. 74-96, RoyalSocietyofChemistry 44 Dickinson,E.(1993)in Interactionsof Surfactantswith Polymersand Proteins(Goddard,E.D.andAnanthapadmanabhan,K.P.,eds), pp. 295-317,CRCPress

45 Dickimon,F. and Galazka,V.B.(1991)FoodHydrocolloids5. 281-296 46 Dickinson,E.(1993)in FoodColloidsandPolymers:Stabifityaod MechanicalProperties(Dickinson,E.and Walslra,P., eds),pp. 77-93, RoyalSocietyofChemistry 47 Nakamura,R., Mizutani,R., Yano,M.and Hayakawa,S. 119881 I. Agric.FoodChem. 36, 729-732 48 Kalo,A.,Mifuru,R.,Matsudomi,N. and Kobayashi,K.(1992)Biosci. Biotech. Biochem.56, 567-571 49 Bj6rkling,F., Godlfredsen,S.E.and Kirk,O. (1991)TrendsBiotechnol. 9, 360-363

i

Perspectives on aflatoxin

Review iLL i~

1, i

improper storage of agricultural commodities t. The major agricultural commodities affected with aflatoxins are corn (maize), peanuts, figs, tree nuts, rice, dried fruit, pumpkin seeds and cottonseeds. Aflatoxin residues can also occur in the milk of lactating dairy animals following the ingestion of aflatoxin-contaminated feed 2. Human exposure to aflatoxins can result from the direct consumption of contaminated commodities, or from the consumption of foods from animals previously exposed to aflatoxin in feeds (milk and egg products). The significance of the risk due to aflatoxin contamination is dependent on the toxicological properties of the particular compound (acute, subacute, reproductive or longAflatoxins are potent carcinogenic, mutagenic and terato- term toxicity; mutagenicity; teratogenicity) as well as on genic metabolites produced by molds that grow on food and the extent of the exposure (occurrence, incidence, level feed. Their toxicity has caused severe health and economic of contamination). Unquestionably, prevention is the best method for problems worldwide. Human exposure to aflatoxins can arise controlling mycotoxin contamination. However, prefrom the direct consumption of contaminated commodities harvest invasion of the mold and its subsequent prosuch as corn (maize), peanuts or figs, or from the consump- duction of aflatoxin are currently unavoidable. Hazards associated with the toxins must be removed tion of foods from animals previously exposed to aflatoxin in feeds (milk and egg products). Food safety monitoring pro- if the products are to be used for food or feed purposes. Mycotoxin control programs include the establishment grams for aflatoxins have been established for raw and finof regulatory limits or guidelines, monitoring programs ished products; these include establishment of regulatory for mycotoxin levels in susceptible products, and deconlimits or guidelines, monitoring programs for aflatoxin levels tamination procedures and/or strategies for the diversion in susceptible products, and decontamination procedures of contaminated products to lower-risk uses. Research and/or the diversion of contaminated producls to lower-risk into the development of decontamination procedures has been underway for over 20 yea~. The decontamination uses. procedures currently used are based on physical, chemical or biological removal, or on physical or chemical inactivation. The ammoniation of corn, peanuts, cottonseed and meals to alter the toxic and carcinogenic Aflatoxins, potent carcinogenic, mutagenic and terato- effects of contaminating aflatoxins appears to have pracgenie metabolites produced by the fungal species tical applications and has been the subject of intense AspergiUus flavus and AspergiUus parasiticus, can con- research efforts by scientists in various government taminate human foods and animal feeds. Such contami- agencies and universities worldwide. nation is the result of (currently unavoidable) invasion This review describes the acute and chronic by the molds betbre and during harvest, or because of toxicity/carcinogenic potential of aflatoxins, the rationale for the control of human exposure, and monitoring Douglas t. Park and Bailin tiang are at the Department ot Nutritional and decontamination programs that have been put into

control for human food and animal feed

Douglas L. Park and Bailin Liang

Sciences, Universily of Arizona, Tucson, AZ 85721, USA.

334

~'~ ~.El,~,,,,.rst,,.,,~,P~hl,,h~,r~U,i.~Ut
use.

Trends in Food Science & TechnologyOctober 1993 IVol. 41

Table 1. Currentaflatoxinat!ion levelsestablishedby the US Foodand Drug Adminislrationa Health hazards to humans from aflatoxins Acute toxicity potential of aflatoxins in humans There is ample epidemiological evidence that humans are not immune to acute aflatoxicosis, and the US Food and Drug Administration (FDA) has established 'actian levels' for aflatoxins in various foods and feeds (Table I). The first report of acute intoxication wi~h aflatoxin came from Taiwan in 1967 (Ref. 4). A few years later, in Uganda, a 15-year-old boy died two days after hospitalization. P o s t m o r t e m examination found organ and cellular damage that was similar to that described for aflatoxicosis in the African monkey-~; the examination of foodstuffs in the child's home revealed moldy cassava containing 1700 pg/kg aflatoxin. In 1974, in India, there was an outbreak of hepatitis affecting 397 patients in 200 villages and resulting in 106 deaths 6. The outbreak lasted for two months and was confined to tribal population groups whose staple food was corn. Adverse rains had drenched the standing crop an~l the outbreak began a few weeks after harvesting. Aflatoxin B~ was detected in seven serum samples. Daily aflatoxin B~ intakes from the corn were estimated to have been at least 55 p.g per kilogram of body weight for an undetermined number of days 7. An additional outbreak of aflatoxicosis occurred in 1982 in the Machakos area of Kenya s, where 20 patients developed hepatitis; 12 died. Aflatoxin B~ was detected in the livers of two patients at levels of 391~g/kg and 89 p.g/kg. Levels of aflatoxin B0 found in corn samples from affected households were high (3200-12 000 llg/kg); unaffected homes had levels not exceeding 500 ~g/kg. Chronic toxic/carcinogenic potential Aflatoxins, although highly carcinogenic in all experimental animal species studied, and tumorigenic in many organs, act predominantly in the liver and biliary tract, alkr oral, subcutaneous or intraperitoneai administration '~. Although a direct cause-effect relationship has not been demonstrated, there is a general correlation between the incidence of liver cancer in humans in specific areas of Africa and Asia and dietary exposure to aflatoxins m. Several epidemiological studies have shown that there is a positive correlation between aflatoxin levels in the diet and the incidence of primary hepatocellular carcinoma (PHC) in specific regions of the world. These include certain regions of the People's Republic of China, Kenya, Mozambique, The Philippines, Swaziland, Thailand and Transkei in South Africa. However, the prevalence of hepatitis B virus (HBV) infection was also found to be correlated to liver cancer incidence in these regions". In contrast, low PHC incidence was observed in certain regions such as the southeastern states of the USA, where the estimated dietary exposure to aflatoxin was high ~-'. Although it is difficult to differentiate the roles of aflatoxins and HBV on the pathogenesis of PHC in humans, recent studies have indicated that aflatoxin exposure is more important than the prevalence of HBV. In Swaziland, where there is little geographic difference in HBV exposure throughTrends in Food Science & Technolog,/October 1993 [Vol. 4]

Food/feed

Action level (~g/kg)

Human foods (except milk! Milk Animal feeds (except as listed below) Cottonseed meal (for mature beef, swine and poultly) Corn (for breeding beef cattle, swine or mature poultry) Corn (for finishing swine) Corn (for feedlot beef cattle)

2o o.5 20 3o0 loo 200 3o0

a Datatakenfrom Ref. 3

out the area, PHC incidence was strongly associated with the level of aflatoxin exposure in the subregions ~3. Studies in Fusui of the People's Republic of China, where the incidence of PHC is among the highest in the world (120 cases/IO~), also revealed the existence of a sub-population in which there was a positive and almost perfectly linear relation between dietar/ aflatoxin B~ level and PHC death rate ~~. Several epidemiological studies have been published on the correspondence between aflatoxin exposure and liver cancer in Africa. In one of these reports, BulataoJayme et al.~4 compared the mean dietary intake of 90 patients with confirmed primary liver cancer with that of 90 age-sex-matched controls. By using dietary recall, the frequency and amounts of food items consumed were calculated into units of aflatoxin load per day using a standardized Philippine table of the aflatoxin levels in these items. Of the subjects' total aflatoxin load, 51.2% came from cassava, 20.3% from corn, 6.8% from peanuts and 5.8% from sweet potato. The mean aflatoxin load per day for the liver cancer patients was found to be 440% that of the controls. Furthermore, dietary aflatoxin loads and alcohol intakes were subjectively allocated into 'heavy' and 'light' exposure groups; comparison of the numbers of patients versus controls in each group generated a measure of the relative risks of developing primary liver cancer from the ingestion of contaminated foods. The following foods were found to be statistically significant in the following order of rank: cassava > peanuts > sweet potato > corn > alcohol. Boiled rice, which had negligible aflatoxin content, gave no difference in risk. Indirect human exposure to aflatoxins Aflatoxins can also cause significant health problems in animals. The premise 'until demonstrated other wise, any animal carcinogen should be considered a human carcinogen, and that, as an unavoidable carcinogenic contaminant, aflatoxin should be controlled at the lowest practical level' has been applied to the regulation of aflatoxin in animal feeds and requires that levels in feeds result in toxicologically insignificant levels of aflatoxin and/or its metabolites in edible tissues and dairy products 7. Extensive studies on feed-to-tissue transfer rates have been conducted. Knowledge of these ratios played a major role in the FDA's decision to 335

increase the allowable levels of aflatoxins in cottonseed meal ~5. The highest feed-to-tissue transfer rates (i.e. the ratio of the level of aflatoxin B~ in the feed to the level of aflatoxin B~ and its metabolites in the edible tissue) are 75 : 1, 800 : 1 and 2200 : I for milk (dairy cows), liver (swine). and eggs (poultry), respectively (Table 2). Aflatoxin M~ is the primary metabolite found in milk; aflatoxico[ has also been identified in milk. Aflatoxicol has been shown to be almost as carcinogenic as aflatoxin B~ in the rat and in rainbow trout; however, the feed-to-tissue transfer rate is high (195 000: 1)~5. Thus, aflatoxin metabolites in milk, particularly aflatoxin M~, are of potential concern for human health. Because of the presence of aflatoxins and metabolites in foods and feeds and strong evidence of their association with human aflatoxicosis and carcinogenesis, aflatoxins are still a serious threat to human health even after almost 30 years of research.

concentrations were highly variable, the estimated average concentration of ~140~g/kg aflatoxin B~ in prepared mixed cattle feed yielded a daily intake for workers via the respiratory route of -170 ng. The calculated risk was established on the basis of the number of cancer cases among male workers whose employment with one o f the companies was the single job that they had held for the longest time since 1964. A two- to threefold increase in risk for liver cancer and for cancers of the biliary tract was observed. Exposure to aflatoxins in the imported crops was judged to be the most probable explanation for these findings, although the influence of lifestyle factors (e.g. alcohol consumption) on the results cannot be fully disregarded. Increased risks for salivary gland tumors and multiple myeloma were also detected ~.

Rationale of aflatoxin control Mode of contamination Aflatoxins occur worldwide. AspergiUus species are Occupational exposure to aflatoxins capable of growing on a variety of substrates and under The possible role of aflatoxins as an occupational risk a variety of environmental conditions. Therefore, most factor has been considered in only a few studies. In a foods and feeds are susceptible to invasion by aflatoxiregistry-based analysis of occupational risks for primary genie Aspergillus species at some stage during proliver cancer in Sweden, significantly increased risk was duction, preharvesting, harvesting, processing, transporobserved in both male and female workers in grain tation and storage ~9. The presence of aflatoxigenic mills; this finding was associated with potential expo- molds on a substrate does not automatically result in the sure to aflatoxins tr. In a follow-up study of 71 Dutch presence of aflatoxin. Conversely, the absence of ariaoil-press workers exposed to aflatoxins, mortality from toxigenic molds does not guarantee the absence of ariaall cancers and from respiratory cancer was higher than toxins, since the toxins may have diffused into the subexpected. Although no cases of primary liver cancer strate. Aflatoxin formation in agricultural commodities is were recorded, two of the 30 deaths were due to an likely to occur in warm and moist weather conditions, and unspecified liver disease, in addition, case reports have in faulty or inadequate storage facilities. The most importbeen published that provide circumstantial evidence for ant factors influencing growth of, and aflatoxin production an association between human cancer and the inhalation by, A. flavus and A. parasiticus are the relative humidity of aflatoxin-contaminated dust tT. surrounding the substmte, which in most cases is 88-95%, A study of cancer risk among male employees at 241 and a storage temperature of 25-30°C ( Ref. 20). livestock feed-processing facilities in Denmark was The life cycle of an aflatoxinogenic fungus consists of conducted utilizing the data linkage system established entry into the plant, seed infection, and finally aflatoxin at the Danish Cancer Registry tbr the detailed investi- tbrmation in the seed (the inoeulum may be from prigation of occupational cancer, providing employment mary or secondary sources)2L The potential sources of histories back until 1964. Crops imported tbr t~ed pro- primary inoculum in the form of spores of A. flavus in duction were often contaminated. Although aflatoxin the spring appear to be propagules in the soil, mycelium that overwinters in plant debris and litter on the soil, insects, or sclemtia of A. flavus in the soil. The proTable 2. Relationship belween aflatoxin levels in feed and duction of conidia on floral parts and leaves may be an aflaloxin residue levels in edible tissuesa important source of secondary inoculum, while insects as vectors can cause infestation of the plant. The infecFeed : tissue tion via flower, stem bract, peduncle, developing fruit, Animal Tissue Aflatoxin ratioh kernels and seed is reviewed well in Ref. 21. Bee(cattle Liver Bi 14 000 Meteorological factors, potential insect vectors and the plant interact to allow for the formation of aflatoxins Dairy cattle Milk Mt 75 prior to harvest. Attempts to develop aflatoxin-resistant Atlatoxicol 195 0t')0 hybrid corn crops have been disappointing due to the Swine Liver BI 800 difficulty of selecting for drought-stress resistance Layers Egg B~ 2 200 applicable to various different geographical locations 2-~. Biological control agents provide a promising approach to Broilers Liver BI 1 200 preventing the preharvest formation of aflatoxin -'3. Certain bacteria, and a highly competitive, non-aflatoxin-producing '~Datalakenfrom Ref, 15 I t, Levelof al'laloxinin feeddividedb)~levelin the specifiedtissue strain of A. parasiticus have been shown to replace or inhibit wild toxic strains in peanut test plots. Integrated ]]6

Trends in Food Science & Technology October 1993 IVol. 41

pest management programs, which through the use of pesticides and other agronomic procedures effectively control insect populations, have also been effective in reducing preharvest formation of aflatoxin. None of these programs, however, completely eliminate the possibility of aflatoxin formation in selected field crops. Risk assessment Unquestionably, prevention is the best method for controlling mycotoxin contamination. Should contamination occur, however, the hazard associated with the toxin must be removed if the product is to be used for food or feed purposes. As described by Park and Stoloff7, the ideal process is usually divided into four components: hazard evaluation, exposure determination, risk determination and management of the risk. A hazard evaluation for a toxin provides a description of the expected toxic effect and of the dose-response relationship. Ideally, the test animal used should be the target animal of interest. The most important "target animals" from a public health agency's viewpoint are humans, but domestic animal welfare also comes within the regulatory jurisdiction of public health agencies. Since experimentation with humans is not a viable option, and unless the target animal is some species of livestock, laboratory animals must be used. It must be determined, usually by comparative toxicology, whether the test animal is an appropriate surrogate for the target animal, and a scaling factor must be established for translation of the dose-response data from test species to target species. When the toxic agent is a recently recognized component of the environment, the laboratory data must be examined in relation to epidemiological evidence of past exposure to the agent. After the development of suitable analytical and sampling procedures, a determination must be made of the geographic and temporal distribution of the toxic agent in each major commodity subject to contamination. Combined with commodity consumption data, this information can lead to an estimate of exposure to the toxic agent by various human and animal populations. Method development and the surveys by which such data are accumulated are :~really more costly in both time and resources than is ~ ~erally acknowledged. With dose-response, s~,:amg and exposure data in hand, an estimate of risk to the exposed population can be made. For a newly recognized natural toxicant, regardless of the theoretical basis for calculation, the risk based on laboratory data must be compared with the observed risk from epidemiological data. When the two approaches produce divergent results, comparative toxicology observations may shed light on the reasons for the difference and lead to a regional conclusion. Risk management decisions are usually made with an inadequate knowledge of all the foregoing factors. Decisions can be forced by societal or political pressures, economic necessity, or legal mandates, and can be molded by the same factors. The management plan can take the form of maximum tolerated levels that are legally enforced to limit human exposure to these toxins Trends in Fcod Science& TechnologyOctober 1993 [Vol. 4l

(Table I), educational programs leading to voluntary compliance by the industry, and research programs leading to a better understanding of how and where the toxicant occurs, with prevention of the occurrence or removal of the toxicant as the ultimate goals. Risk analysis and management should be an evolving process as more and better knowledge is acquired. For example, yearly data from the peanut certification program in the USA, supported by routine regulatory surveillance data accumulated by the US FDA and Canadian Health and Welfare, and by a comprehensive survey of consumer peanut product manufacturers by the FDA in 1973, provided considerable information on which to base an estimate of the expected incidence and level of aflatoxin exposure from peanuts as consumed. Data on the consumption of peanut products were available from decennial surveys carried out by the US Department of Agriculture (USDA) Agricultural Research Service on food consumption patterns of US households. However, this exposure information was not used in developing proposed tolerance levels, because there was no toxicological information available with which to determine a safe level on which to base a tolerance. Instead the Commissioner of the FDA 'sought to bring four factors into balance: the need to minimize human exposure to aflatoxins; the capabilities of sampling procedures and analytical methods to detect, measure and confirm aflatoxins; the capability of agricultural and manufacturing technology to prevent and remove contaminated peanuts; and the need for continued availability of a low cost protein source (i.e. peanuts)'. In the end, taking into account sampling uncertainties and analytical capabilities at the margin below the required tolerance, to assure compliance with regulations, dictated a minimum practical tolerance level of 15 lag/kg. In response to comments received on the tolerance proposal, the FDA undertook an "assessment estimated risk resulting from aflatoxin in consumer peanut products and other food commodities'. This assessment was presented for comment by way of a Federal Register notice -'4. At this time it was abundantly clear that the major exposure to aflatoxin in the US population was in the Southeastern states, as a result of three coincidental factors: dry-milled corn products (meal and grits) are consumed in those states at a much higher rate than in any of the other states; most of the dry-milled corn products consumed in the Southeast are made from local corn, locally milled2S; and corn from this Southeastern region suffers by far the highest incidence and level of aflatoxin contamination in the USA2~. The analysis focused on this region, and the lifetime cancer risk was calculated based on aflatoxin exposure levels es6mated from the consumption of corn and peanut products and aflatoxin contamination levels derived from commodity survey data. Calculations of low-dose response were made using averaged dose-response data from five published lifetime feeding studies with rats and both Mantel-Bryan-~7and 'one hit '~-~models, and also from a dose-response relationship derived from African and

337

Asian epidemiological studies -'9. Additional calculations of lifetime cancer risk, using the relationship derived from epidemiological studies, were made, with the average contamination assumed under various possible tolerance levels for peanut products. The ultimate conclusion could have been reached without all the laborious calculations; the expoSUre of the S0u~east population to aflatoxin from corn products was so much greater than exposure from peanut products that even a zero tolerance in peanut products could have little effect on the calculated risk. This conclusion, together with all the uncertainties expressed in the risk assessment, supported the basic reasoning in the original regulation proposal that a reduction of the regulatory action level from 20 lag/kg to 15 ~tg/kg would not significantly reduce the risks posed by exposure to aflatoxins at these levels. Monitoring programs In addition to the establishment of regulatory limits, monitoring programs to identify high-risk products and decontamination procedures to reduce the risks associated with aflatoxin contamination are integral parts of an aflatoxin control program. For example, the Arizona Department of Health has had an extensive monitoring program for aflatoxin in milk for almost two decades (Collier, R., Arizona Dairy Commissioner, pets. commum), However, during the year 1978, Arizona dumped almost 910 000 lb (409 500 kg) of milk with contamination levels as high as lO~tg/1 aflatoxin M v As a result of this contamination event, the State instituted a comprehensive program to monitor aflatoxin levels in whole cottonseed and cottonseed products, the primary source of aflatoxin contamination within the State. All cottonseed produced in the state is tested tbr aflatoxin content. The maximum size of each lot tested is 100t and the testing is conducted in state-certified laboratories. The end use of the product is dictated by the aflatoxin levels |bund, Cottonseed lots testing over 20p.glkg aflatoxin are usually treated with ammonia to reduce aflatoxin levels, Cottonseed products containing less than 20 I,tg/kg aflatoxin, either with or without ammonia decontamination treatment, can be used tbr dairy rations. Since 1980, the Arizona aflatoxin feed monitoring program has resulted in over 21 000 analyses. A summary of the results of the monitoring program showed 75.8%, 23.7% and 0.7% of the lots of cottonseed tested contained aflatoxin levels of <20, 21-300 and >300 I.tg/kg, respectively.k Of the lots analysed between 1983 and 1989, 5% were subjected to ammonia decontamination procedures. An average of 2731 lots were tested, and 143 lots treated with ammonia per year. Since the passage of statutes allowing the use of ammonia to reduce aflatoxin levels (Arizona Revised Statutes 36-904.01: Regulation R 9-17-318, 13 May 1981), the destruction of milk due to violative levels of afiatoxin Ms (>0.5 lag&g) was ordered only once; it was later determined that the event had resulted from a feeding error, in which a pile of cottonseed that had been designated for ammonia treatment was fed before it could be treated. A summary of the results of testing for 338

aflatoxin residues in Arizona milk show that from 1979 to 1982 and in 1989, no samples contained aflatoxin M~ at a level ab')ve 0.5 lag/kg; however, detectable levels of aflatoxin Mt were found in 11-28% of the samples. The use of ammonia to treat cottonseed feed for lactating cows has been a significant benefit, keeping Arizona's milk supply free from aflatoxin contamination. The key aspect of the aflatoxin decontamination program is that the product is tested for aflatoxin levels both before and after the ammonia treatment, as appropriate. Many organizations worldwide continually monitor aflatoxin-susceptible commodities for the incidence and levels of aflatoxin contamination. Although, as indicated earlier, many different commodities are susceptible to aflatoxin contamination, here we concentrate on corn, corn-based products, peanuts, and peanut products subject to international trade. Significant aflatoxin contamination levels in corn and corn-based commodities have been reported in Latin America and the Caribbean. Mexico, Guatemala and Costa Rica have the three highest frequencies (87.8%, 54.0% and 50.0%, respectively); the range of aflatoxin levels was 0-1650 Iag/kg, with most of the samples containing >201ttg/kg aflatoxin (Table 3; Ref. 30). Other reports from Canada, France, Senegal, Japan, the UK and Italy (Table 4) have shown that Senegal, Italy and France are the three countries with the highest frequencies (60%, 45% and 23%, respectively)~°. High incidence levels are evident in tropical regions worldwide. Relatively lower levels of aflatoxin contamination occur in temperate regions. The results of a FAO/WHO/UNEP (Food and Agriculture Organization/World Health Organization/ United Nations Environmental Program) monitoring program tbr the years 1976-1983 indicated that Kenya had the highest contamination level; 90% of the samples contained 30-19201ag/kg aflatoxin, compared to <4% in Canada. Again higher levels of aflatoxin were evident in tropical regions-~t. US FDA compliance data for milled corn products (meal, grits, hominy and hominy grits) obtained from retail outlets show that aflatoxin contamination levels in these products in the Southeastern states is significantly higher than in other states. From 1980 to 1983, 72%, 21%, 8% and 41% of the samples from the Southeast contained aflatoxins, whereas for other states no more than 19% of the total samples were contaminated. The average contamination levels in the Southeast were 27, 21, 34 and 16 lag/kg, respectively for these years, much higher than that in other states (<15 lag/kg)3". In 1983, 45% and 43% of samples of shelled corn from the Southeast and South contained aflatoxins. FDA compliance data for 1986 for shelled corn, milled corn products and manufacturcd corn-based products revealed that 32%, 28% and 47% of the samples, respectively, contained afiatoxins. Again this indicates that the Southern and Southeastern regions of the USA have the highest contamination levels". Although aflatoxin contamination can occur at significant levels in the raw product, processing procedures Trends in Food Science & l-~chnology October 1993 [Vol. 41

Table 3. Aflatoxin conlamination in corn and corn-based commodities from Latin America and the Caribbeana

such as screening and milling can Contamination Afiatoxin Mean Total number frequency level/range aflatoxin reduce these levels. This is evident Country Year Product of samples (%) (pg/kg)b level (p~kg) c from the results of USDA aflatoxin tests of processed corn products for 1989-1991, which revealed negative Argentina 1983-1984 Corn 87 33.4 0-150 ND screening results (<20pg/kg aria1986-1989 Corn 644 21.6 2-230 1.1 toxins) for 14 Ol 3 analyses~°. However, it is not clear whether levels are Brazil South ND Corn 165 18.0 5--900 14.3 reduced to a similar extent by proSoutheast ND Corn 163 8.5 5-148 3.0 cessing in lesser-developed countries. Significant aflatoxin contamination Costa Rica 1985-1987 Corn 3000 50.0 >20 156.1 1979-1990 Corn 3000 17.0 20-200 50.0 levels in raw peanuts are evident in Latin America, Caribbean regions Cuba ND Corn 443 25.3 10-95 ND and West Africa. Canada, France and Guatemala 1987 Corn on cob 103 0 0 0 Japan, which import peanuts, show 1987 Corn drink 100 1.0 4 0.04 comparatively low levels of aflatoxins 1987d Tortilla 50 2.1 >20 I1 in finished products. Hydar et aL "~4 1987d Tortilla 50 16.0 >20 6 reported aflatoxin B~ and aflatoxin B., 1987d Tortilla 52 26.7 >20 I0 levels of <3 pg/kg for raw and roasted 19874 Tortilla 57 54.0 >20 51 peanut products from Syria. The Dry season 1976 Corn 18 16.7 21-30 ND results of 188 analyses for aflatoxin Wet season 1976 Corn 42 23.8 21-I 650 ND in edible peanuts imported into the Mexico NO Corn, kernel 41 87.8 5-465 (B0 ND UK during 1982-1984 show that 2-57 (G0 ND 74% had aflatoxin aflatoxin B, levels of <5 lig/kg (Ref. 35). The incidence a DatatakenfromResnik,S. and FerroFontan,C., pets.commun. of measurable aflatoxius in consumer h Rangeof detectablelevels;detectionlimitcan be as lowas 0.1 pg/kg peanut products in the USA for the c Forall samplesInotonlythosetestingpositive! d Fourdifferentsamplestakenat randomintervalsthroughoutthe calendaryear time periods 1980-1984 and 1986 ND, Notgiven varied from year to year, but the overall incidence of products containing violative levels (total aflatoxins A recent review of the different approaches to re>20 pg/kg) was low. Other commodities, including figs, almonds, pecans, ducing aflatoxin levels in agricultural commodities 3' is walnuts, raisins and spices are considered to have a summarized below. lower risk of aflatoxin contamination. Soybeans, beans, cassava, grain sorghum, millet, wheat, oats, barley and Food and feed processing The stability of aflatoxins in food products from rice are moderately susceptible to aflatoxin contamipeanuts, corn and milk is the major concern. Since corn nation in the field *t. However, it should be reiterated and peanuts are staple foods for many countries and are that resistance to aflatoxin contamination in the field does not guarantee that the commodities remain free of usually grown in climates that are favorable to fungai aflatoxin contamination during storage. Thus, the analy- growth and aflatoxin production, they are probably the sis of stored susceptible commodities should also be commodities of greatest worldwide concern 37. The thermal inactivation of aflatoxins has been included in monitoring programs. attempted. However, aflatoxins are resistant to thermal inactivation, and therefore procedures based on this Decontamination strategies Decontamination procedures have focused on re- phenomenon can only result i n modest reductions in moval (physical, chemical or biological) or inactivation aflatoxin levels following treatment with boiling water (physical or chemical) ~. Specific criteria have been and autoclaving 38, roasting 3'J and baking 4°. Afiatoxin M~ is apparently stable in milk exposed to pasteurization established for evaluating the acceptance of a given and processing4L decontamination procedure. The process must: Irradiation has been shown to result in a marked • inactivate, destroy or remove the toxin; reduction in the concentration of aflatoxins in contami• not produce or leave toxic residues in foods from ani- nated peanut oil exposed to short-wave and long-wave ultraviolet (UV) light 42. A 14-hour exposure to sunlight mals led the decontaminated product; destroyed -77-90% of aflatoxin B~ added to peanut • retain the nutritive value and feed acceptability of the flakes, whereas only 50% of the toxin was destroyed in product; naturally contaminated product 43. Gamma-irradiation • not alter significantly the technological properties of could not degrade aflatoxin in contaminated peanut meal, and UV treatment produced no observable change the product; in the fluorescence or toxicity of the samples 44. • if possible, destroy fungal spores. 339 Trends in Food Science& TechnologyOctober 1993 lVol. 41

Table 4. Aflatoxin contamination in corn and corn products in Canada, France, Senegal, lapan, the UK and Italy a Contamination Number

frequency

Aflatoxin level/range

Mean aflatoxin

tCountry

Year

Commodity

Canada

1992

Corn,all products

39

0

<0.5-1.3

<0.5

France

1992

Corn gluten, raw Corn gluten,feed

78 78

0 23.0

>200 >20

ND ND

Senegal

1988

Yellow corn White corn

58 60

60.0 40.0

ND ND

90 (B~) 119 (B0

371

0.5

0.7-52

6.4 (B0

29 13 2

3.4 0 0

>10 >10 >10

ND ND ND

111

45.0

0.02-1.2

ND

Japan

1972-1989

UK

ND

Italy

1982-1984

Corn Corn Corn fleur Flakes Corn

of samples

(%)

(p~1(g)b

level (pg/Eg) c

'1 Data taken from Ref, 30 I~Range of detectable levels; detection limit can be as low as 0.1 pg/kg ~ For all samples (not only those testing positive) ND, Not given

Ariatoxin M~ levels in contaminated milk containing 0.05% peroxides after exposure to UV light at 25°C for 20 minutes was decreased by 89.1%, compared with a reduction of 60.7% in peroxide-free milk 45. Various suitable solvent systems can be used for the extraction and mechanical separation of aflatoxins from different commodities, with minimal effects on protein content or nutritional quality 46. These include 95% ethanol, 90% aqueous acetone, 80% isopropanol, hexane-ethanol, hexane-methanoi, hexane-acetonewater and hexane-ethanol-water. However, current extraction technology using these compounds for the detoxification of aflatoxin-eontaminated oilseed meals is still impractical and cost-prohibitive~7. An effective method has been reported Ibr reducing aflatoxin levels in corn~ and peanuts4' by flotation and density segregation of toxic kernels. Electronic and hand sorting of contaminated peanuts have been used extensively by the peanut industry to reduce aflateein levels in peanut products destined for human consumption (Table 5; Cole, R,, USDA, pets. commun.; Ref, 50). Various processing procedures such as the dry and wet milling of corn can result in lower aflatoxin levels in the final product-~L However, where there are significant contamination levels in the raw products, the final products will usually have a low level of contamination. Adsorbent materials, including activated carbon-~-~, clay and zeolitic minerals .~,~,can bind and remove ariatoxins from aqueous solutions such as water, Sorensen buffer, Czapek's medium, Pilzer beer, sorghum beer, whole milk and skimmed milk, Clay, which is incorporated to remove pigments from crude oils, significantly reduces contamination levels in refined peanut and corn oils by adsorbing the aflatoxins. Machen et aL ~4 reported that a phyllosilicate clay (HSCAS, or 'NovaSil') effectively removed aflatoxins from contaminated peanut oil and prevented its mutagenicity and toxicity ht vitro. Finally, microbial inactivation and fermentation have also been studied as means of degrading and removing ariatoxins~''. Fhn'obacterittm atttzttlliacttm and selected J40

acid-producing molds have been successfully used to remove aflatoxins from liquid media. It was postulated that the reduction in aflatoxin levels was a result of acid production and subsequent transformation of aflatoxin B~ to aflatoxin B:~. These processes have not yet been put into production applications. Structural degradation Many chemicals have been tested for their ability to bring about the structural degradation and/or inactivation of aflatoxins. These include numerous acids, bases, aldehydes, bisulfite, oxidizing agents and gases. A number of these chemicals can effectively destroy (or degrade) aflatoxins, but most are impractical and/or potentially unsafe because they tbrm toxic residues or damage the nutrient content, flavor, odor, color, texture or functional properties of the product. Ammoniation and reaction with sodium bisulfite are the major procedures that have received industrial attention. The treatment of grain with ammonia appears to be a viable approach to the detoxification of ariatoxins. Ammoniation (under appropriate conditions) results in a significant reduction in the level of aflatoxins in contaminated peanut and cottonseed meals5-~and com~% Due to the severity of ariatoxin contamination in selected agricultural commodities in various locales, specific decontamination procedures have been approved and put into use~,55.In the USA, Arizona, California and Texas permit the ammoniation of cottonseed products, and Texas, North Carolina, Georgia and Alabama have approved use of the ammoniation procedure for the treatment of aflatoxin-contaminated corn. Mexico and South Africa have approved the procedure for use on corn. Peanut meal is widely used in animal leeds in Europe and elsewhere; consequently, ammoniation is routinely used in France, Senegal, Sudan and Brazil. Several member countries of the European Community import ammonia-treated peanut meal on a regular basis, The ammoniation process, using either ammonium hydroxide or gaseous ammonia, has been shown to Trends in Food Science& TechnologyOctober 1993 [Vol. 4]

Table 5. Effectivenessof poslharvestaflatoxin management strategiesat the processingleveP reduce aflatoxin levels in corn, peanut meal-cakes and whole cottonseed and cottonseed products by more than 99%. If the reaction is allowed to proceed sufficiently, the process is irreversible. Primarily, two procedures are used: a high-pressure, high-temperature process used at feed mills; or an atmospheric-pressure, ambienttemperature procedure that can be used on the farm (Table 6). Other ammonia-related processes (e.g. treatment with monomethylamine-lime and urea-urease) have also been shown to be effective. Sodium bisulfite can react with aflatoxins B,, G~, M, and aflatoxicol, at various temperatures and concentrations and for various times, to form water-soluble products sT. The addition of 0.04g potassium bisulfite per 10ml milk resulted in a 45% reduction in the level of aflatoxin M~ after five minutes 58. The afla~oxin B,-sodium bisulfite reaction products s9 and sulfonat~~,derivatives of aflatoxins G,, M, and aflatoxicol6° have been tentatively identified. Dietary chemicals that alter the normal responses of mammalian biological systems may significantly influence the toxicity and carcinogenicity of aflatoxins. Dietary components that result in modification of the effects of aflatoxins include nutritional components (e.g. dietary proteins and fats, lipotropic agents, vitamins and trace metals)6', numerous food and feed additives (e.g. phenolic antioxidants and ethoxyquin) 62 and endogenous chemical constituents of foods and feeds (e.g. constituents of cruciferous vegetables, naturally occurring steroids, bark extract, derivatives of chlorophyll, organosulfur compounds of garlic, and water-soluble chemicals from licorice). Currently available as an anticaking agent for animal feeds, HSCAS, or 'NovaSil' (a phyilosilicate clay) has been used to reduce levels of bioavailable aflatoxins by selective chemisorption. HSCAS has been reported to:

Aflatoxin level Reduction Cumulative Technology

(pg/kg)

(%)

reduclion (%)

Farmers' stock Belt separator Shelling plantt'

217 140 100

35 29

35 54

Color sortingb

30

70

86

Gravity tableb Blanching/ color sorting Re-color sortingb

25

16

88

2.2 1.6

91 27

99.0 99.3

a Dalataken[rom Ref.7. Resultsfrom the processingof a 40 000 kg segregationI lot of contaminatedpeanuts Databasedon medium-categorypeanutsonly

Table 6. Parametersand applicationsof aflatoxin decontaminationproceduresinvolvingammoniationa High pressure, high temperature procedure

Ambient pressure, atmospherictemperature procedure

Ammonia level 0.2-2%

1-5°1o

Pressure

35-50 psi

Atmospheric

Temperature

80-120°C

Ambient

Duration

20-60 minutes

14-21 days 12-16%

Moisture (%)

12-16%

Commodities

Whole cottonseed, Whole cottonseed, cottonseed meal, corn peanut meal, corn

Application

Feed mill

Farm

a Datatakenfrom Re[.55

• remove aflatoxins from aqueous suspensions; limits based on use, the monitoring of commodities susceptible to aflatoxin contamination, and analysis of these results to dictate end use as well as whether the • prevent aflatoxicosis in farm animals, including product should be subjected to a decontamination chickens, turkey poults, goats, pigs and mink; processes significantly reduces the risks posed by alia• reduce aflatoxin M, residues in milk from lactating toxin contamination. The elements of this program can also be applied to other commodities. dairy cattle exposed to aflatoxin-contaminated feeds. For a number of years, the Codex Committee on Food HSCAS had been reported to be effective in the removal Additives and Contaminants (CCFAC) and the Codex Committee on Cereals, Pulses and Legumes (CCCPL) of aflatoxins from contaminated skim milk63. Various other dietary supplements and chemisorbents have discussed the establishment of aflatoxin levels have been evaluated as possible feed decontamination acceptable for international trade. In these discussions, methods6L Although some show great potential, data levels have largely reflected importer requirements for supporting the efficacy and safety of their use are lack- internal market purposes. In recommending levels for ing. Other than ammoniation, most of the techniques so raw peanuts as well as for consumer-ready products, far studied are impractical, ineffective, or unproven with Codex recognized the ability of processors to improve the quality of finished goods through manufacturing respect to their safety for long-term use. techniques (blanching, additional sorting, roasting, etc.) and the need to remove unnecessary barriers to interConclusions In summary, using the Arizona program for control- national trade. For example, >95% of processed peanut product ling aflatoxin levels in cottonseed as an example, a program combining the application of different regulatory samples evaluated in the USA during the late 1980s by • significantly reduce the uptake and distribution of aflatoxin in biological systems;

Trends in Food Science & Technology October 1993 [Vol. 4]

341

manufacturers showed levels below 5 ~glkg (Ref. 30). Levels were similar for corn and for the situation in Europe, The establishment of uniform regulatory limits for raw products would be the most appropriate point of international harmonization - provided that the limit does not penalize the exporting country. This is of particular significance for countries in the process of economic development, where procedures to reduce aflatoxin levels i n t h e exported product may result in the redirection of contaminated product to local consumers. The International Agency for Research on Cancer (IARC) has classified aflatoxin B~ as a probable human carcinogen*4; however, in its evaluation of acceptable aflatoxin intake levels, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) did not identify an acceptable daily intake value. They did recommend, however, that chronic exposure to aflatoxin contamination be reduced to the lowest practical level.

26 Stoloff,L. (1976)Prec. Am. PhytopathnL Soc. 3, 156-172 27 28

29 30 31

32

(Krogh,P., ed.),pp. 35-64,AcademicPress 33 Wood,G.E.(1989)1. Assoc. Off. Anal. Chem. 72, 543-548 34

Busby,W.F., Jr and Wogan, G.N. 11984)in Aflaloxins in Chemical Carcinogens (Searle,C.E,, ed.), pp. 945-1136, American Chemical Society 2 Price,R.L.,Lough, O.G. andBrown, W,H.(1982)l. FoodProtect. 45, 341-344 3 Park,D.L (199311,FoodAddit. Contain. 10, 49-60 4 Ling,K.H., Wang, 1,1.,Wu, R., Tung, T.C., Lin, CK., Lin, S.S.and Lin, T.M. (19671]. FormosanMed. Assoc. 66, 517-525 S Serck-Hanssen,A. (19701Arch. Environ. Health 20, 729-731 6 Krishnamachari,K.A.V.R.,Bhat, R.V., Nagerajan,V. and Tilak, T.B.G. (1975) Lancet i, 1061-1063 7 Park,D.L. and Stoloff, L. (19891Regular. Toxicol. Pharmacol. 9,

36

37 38

39 40

41 42

43 44 45

46 47

109-130

8 9

10 11 12 13 14 15 16

17 18

Ngindu, A., Johnson,B.K. and Kenya, P.R. (1982) Lan.ceti, 1346-1348 IARC119871IARCMonographs on the Evaluation of the Carcinogenk" Risk of Chentir'als to Humans, Stq)pl. 7, pp 8:1-87, Internalional Agency for Researchon Cant ~r, Lyon, France Stoloff, I.. 11986)in Mycotoxins and Phycotoxins (Sleyn, P,S.and Vleggaar, R., eds), pp, 457-471, Elsevier Chu, F,S,(19(tl) Murat, Res.58, 291-306 Stoloff, L, (19831 Nutr. CancerS, 165-186 Van Rensberg,S.J.,Cook-Moazfbri, P., Schalkwyle, D.J., Van Der Watt, LI., Vincenl, T,I, and Puff:hase,J.F,(19851 Br, l, Cancer 51, 713-726 Bulalao-Jayme,l., Almero, A.M., Castro,C,A., lardeleza, T.R. and Salamat, L. (1982l Int. I. Epidemiol. 11(21,112 Park, D.L. and Pohland, A.E. (19861in Mycotoxins and Phycotoxins (Steyn. P.S. and Vleggaar,R., eds),pp. 473-482, Elsevier Mclaughlin, I.K. etal. (19871Cancer Res.47, 287 Deger, G,E,119761Ann. Intern. Mc~l. 85, 254 Olsen, I.H., Dragsle\'l,L. anti Autrup, H. (19881 Br. I. Cancer 58,

392-396 19

Ellis, W.O., Smith, I.P. and Simpson. B.K. (1991) Crit. Roy. FoodSci. Nutr. 30(.~,),403-439 20 Bullerman,L.B.(1979) I. Food Protect.42, 65 21 Urhan. L. et al. (I 987) Ann. Rev. Phytol, Pathol. 25, 249-270 22 Wicklov,,. D3. (I 990~ in A Perspectiveon Aflatoxin in Field Crops and Animal Food Products in the United States tARS-83), pp. 53-61, NationalTechnical Information Services.Springfield, VA,USA 23 Cole, R.I. and CottV. P.I. I19901 in Perspectiveson Aflatoxin m Fiekl Crt)psand,,tnimal Food Prodoctsin the United St,liestARS-83Lpp,62-66, National Technical Information Sea,ices, Springfield, VA, USA 24 Fed. R~:qist.I1978) 43.8808 25 5toloff, t. and Dalrvmple, B. t l977~ I. Assoc. Off. Anal. Chem.60.

579-582

342

Hydar, M., Benelli, L. and Brera, C. (1990) Food Chem. 37, 261-268

35 Kershaw,S.I,(1985)J. Food Technol. 20, 647-649

References 1

Mantel, N. and Bryan,W.R, (1961) J. Natl Cancer Inst. 27, 455-470 Heel, D.C. (197511. Toxicol. Environ. Health 1, 133-151 Peers,F.G. and Linsell, C.A. (19771Ann. Nutr. Aliment. 31, 1005-1018 FAO (1993) Technical Consultation on Sampling Plans for Aflatoxin Analysis, Food and Agriculture Organization, Rome, Italy Jelinek,C.F., Pohland, A.F. and Wood, G.E. (198911.Agric. Food Chem. 29, 555-558 Pohland,A.E.and Wood,G.E.(1987) in Myeotoxins in Food

48

49 50

51 52

33 54 55

56

$7 58

59

60 61

Phillips, T.D., Clement, B.A. and Park, D.L. (1993) in The Toxicology of Aflatoxins: Human Health, Veterinaryand AgriculturalSignificance (Eaton, D.L and Groopman, J.D., eds), pp. 287-291, Academic Press CAST(!989)Mycotoxins: Economic and Health Risks (CAST Task Force Report No. f 16), Council for Agricultural Scienceand Technology, Ames, IA, USA Christensen.C.M., Mirocha, CJ. and Meronuch, R.A. (1977) Mold, Mycotoxins, and Mycoloxicoses fAgricultural ExperimentSection Miscellaneous Report 1421,University of Minnesota, St Paul, MN, USA Marth, E. and Hadpoyle, M.P. (1979) Food Technol. 33, 81-87 Reiss,J. (1978) Cereal Chem. 55, 421-423 Stoloff,L. (198011. Food Protect. 43, 226-230 Shantha,T. and Sreenivasa,M. (19771Ind. I. Technol. 15, 453 Shantha,T., Sreenivasa,M,, Raft, E.R.and Prema,V. (1986) 1. Food Safety 7, 225-232 Feuell,A.J, (1966) Trop. Sci. 8, 61-70 Yousef,A.E. and Marlh, E.H. (1986) J. Dairy Sci. 69, 2243-2247 Rayner,E.T., Koltun, S.P. and Dollear, F.G. (1977) ]. Am, Oil Chem. Soc. 54, 242A-244A Shanlha,T.(1987) in Summaryand Recommendationsof the International Workshop on Aflatoxin Contamination of Groundnut, p, 16, Icrisal Center, India Huff, W.E. and Hagler, W.M., Jr 11985l I. Food Protect. 48, 416-420 Cole, R.J, 11989)in Mycotoxins and Phycotoxins 1988 (Natori, S,, Hashimoto, K, and Ueno, Y., ecls),pp. 177-184, Elsevier Dickens,I.W. and Whitaker, T.B. (1975) PeanutSci. 2, 45-50 Brekke,O.L. et al. (1975) Cereal Chem. 52,205-211 Decker,W.J,(19801Vet. HumanToxicol. 22, 388-389 Masimanco,N., Remacle,J. and Ramaut,I. 119731Ann. Nutr. Alimen. 23, 137-147 Machen,M.D., Mayura, K,, Clemenl, B.A. and Phillips, T.D. (1991) Toxicologist 11,280 JAbstract] Park,D.L., Lee, L.S,, Price, R.L. and Pohlanfl, A.E. (1988)). Assoc. Off. Anal. Chem. 71,685-703 Brekke,O.L,, Peplinski, A.I., Nofsinger, G.W., Conway, H.F., Stringfellow, A.C., Montgomery, R.R., Silman, R,W., Sohns, V.E, and Bagley, E.8. (19791 Trans.Am. Soc. Agric. Eng. 22, 425-432 Hagler, W.M, Ir, Hulchins, J.E.and Hamilton, P.B. (1982)J. Food Protect. 45, 1287-1291 Doyle, M.P., Applebaum, R.S., BrackelL R.E.and Marlh, E.H. (19821 I. Food Protect. 45,964-971 Yagen,B., Hulchins, I.E., Cox, R.H., Hagler, W.M,, Ir and Hamilton, P.B, (1989) l, Food Protect. 52, 574-577 Lee,LS, (1989) l. Am. OiI. Chem. Soc. 66,1398-1413 Newberne,P.M.(1987)in Mycotoxins in Food IKrogh,P., ed.),

pp, 177-216,AcademicPress 62 63 64

Kensler,T.W., Egner, P.A., Davidson, N.E., Roebuck, B.D., Pikul, A, and Groopman, I.D. 11986) Cancer R~. 46, 3924-3931 Ellis,I.A., Bailey, R.H., Clement, B.A. and Phillips, T.DI (1991) Toxicologist 11,96 (Abslractl IARC(1993) IARCMonograph on the Evaluation of Carcinogenic Risk to Humans. 56, Inlernalional AgenQ, for Researchon Cancer, Lyon, France

Trends in Food Science & Technology October 1993 IVol. 4i