Contaminants of Milk and Dairy Products | Environmental Contaminants

Environmental Contaminants W J Fischer, Syngenta International AG, Basel, Switzerland B Schilter, Nestle´ Research Center, Lausanne, Switzerland A M Tritscher, World Health Organization, Geneva, Switzerland R H Stadler, Nestle´ Product Technology Center, Orbe, Switzerland ª 2011 Elsevier Ltd. All rights reserved.

Introduction Milk and dairy products are remarkably susceptible to environmental contamination. Dairy animals ingest environmental contaminants while grazing on the pasture and when fed on contaminated concentrate feeds. These contaminants may be present in the soil and subsequently in fodder • naturally plants, plant toxicants, • inherent mycotoxins plants infected by fungi, and • anthropogenicfromchemicals from industrial emissions, for • example, dioxins, polychlorinated biphenyls (PCBs), or radionuclides from fallout. Milk and dairy products are consumed in significant amounts in several regions of the world. This is even more the case for the sub-population of infants and small children, who, based on their body weight, consume greater amounts of milk than adults. For certain parts of the population, milk and dairy products may therefore represent the most important source of certain contaminants in their diet. The objective of this article is to draw attention to the major sources of environmental contaminants in milk and dairy products and to highlight their occurrence and the resulting human exposure. Data are discussed in the perspective of potential human health impacts (see Contaminants of Milk and Dairy Products: Contamination Resulting from Farm and Dairy Practices). Aflatoxins will not be covered here in detail (see Yeasts and Molds: Mycotoxins: Aflatoxins and Related Compounds).

Dioxins, Polychlorinated Biphenyls, and Furans Dioxins ‘Dioxins’ is a generic term for a series of related polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) (Figure 1). Of the 210 different congeners that can be encountered, only 17 are considered toxicologically relevant (2,3,7,8chlorinated congeners). The most investigated and toxic

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representative is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), often simply referred to as ‘dioxin’ (Figure 2). Dioxins are chemically and thermally stable and highly lipophilic. Due to their environmental persistence, they bioaccumulate via the food chain and can be found at low levels in food, mainly in animal fats. Hence, traces are found in milk and dairy products, fish, meat, eggs, and other fatty foods. Sources and occurrence

Dioxins are formed as inadvertent by-products in many chemical processes involving chlorine and in any combustion process. The main sources of PCDDs/PCDFs are waste incinerators (municipal, hospital, industrial), metal sintering/recycling plants, cement kilns, and emissions by forest fires and volcanic eruptions. Dioxins, bound to particulate matter, are deposited via the atmosphere on any surface. Elevated contamination levels can be found in milk from farmland in the vicinity of latter industries. Following surveys between the early 1990s and 2004, the mean background levels in dairy products expressed as toxic equivalents (TEQ) have decreased steadily and reached a plateau at 0.3–0.5 pg TEQ g–1 fat (10–12 g g 1 fat; ppt) in industrialized countries (with seasonal and regional variations). Less industrialized areas can reach lower plateaus of 0.1–0.3 pg TEQ g 1 fat without considering the dioxin-like PCBs (DL-PCBs; see below). Health impact

Dioxins are very potent toxicants, and TCDD is one of the most potent animal carcinogens, recently classified as a human carcinogen. Apart from carcinogenicity, various effects have been demonstrated in animal models and suspected in humans, for example, on the immune system, reproduction and development, and neurobehavioral alterations. The main sources of human exposure are foods of animal origin, and the estimated average daily intake of dioxins in industrialized countries is 1–3 pg TEQ kg 1 body weight (bw) day 1. Dairy products contribute about one-fourth to one-half to the dietary intake of total dioxins, including DL-PCBs (see below). Dioxins occur as complex mixtures. They act through a common mechanism, but

Contaminants of Milk and Dairy Products | Environmental Contaminants

Figure 1 Structural formulae of polychlorinated dibenzo-pdioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs).

Figure 2 Structural formula of 2,3,7,8-tetrachlorodibenzo-pdioxin.

vary in their toxic potency. The compounds are assessed and regulated together as a group by the sum of the potency of the congeners relative to TCDD. The result is thus expressed as TEQ. DL-PCBs, which act in a similar fashion, are included in the TEQ concept. A provisional tolerable monthly intake (PTMI) for dioxins, furans, and DL-PCBs of 70 pg TEQ kg 1 body weight month 1 was set by WHO. The PTMI is a measure used by WHO for food contaminants with cumulative properties. Its value represents permissible human monthly exposure to contaminants unavoidably associated with otherwise wholesome and nutritious foods. The European Union has established a comprehensive set of maximum levels for dioxins, furans, and DL-PCBs in food and feed. For example, the maximum levels for the sum of dioxins, furans, and DL-PCBs have been set for raw milk and dairy products, including butterfat, at 6 ng WHO TEQ kg 1 fat or product. It appears that part of the population in some industrialized countries exceed the safe level of intake (PTMI). Therefore, dioxins in food are considered to be of health concern and efforts are undertaken to further reduce human exposure. Analysis

Analysis of dioxins requires determination of extremely low levels (ppt, 10 12, or even ppq, 10 15) by gas chromatography/high-resolution mass spectrometry (GC/HRMS). The analysis is complicated and expensive and performed by only a few specialized laboratories. All relevant congeners are quantified, and the congener pattern may give indications of the source of the contamination. However, there is also progress in the development of ultratrace methods, and improved chromatographic

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separation and better performance can be achieved with the help of fast-GC and GC  GC multidimensional chromatography. The latter coupled to a time-of-flight mass spectrometer (GC  GC-TOFMS) is a good alternative to conventional GC/HRMS particularly for challenging food matrices, and represents a robust and sensitive method with excellent peak resolution. Cell-based assays have been developed to determine dioxins in food and feed. Some have been commercialized such as the CALUX (chemical-activated luciferase expression) bioassay. The CALUX assay is based on a genetically engineered cell line that contains the firefly luciferase gene under transactivational control of the aryl hydrocarbon (Ah) receptor. When these cells are exposed to dioxins, the dioxin enters the cell and binds to the Ah receptor. This complex is then translocated into the nucleus of the cell and binds to the dioxin-responsive element, inducing the expression of the luciferase gene, and subsequently the synthesis of the firefly luciferase protein, which can be measured. Several ring tests have shown that the CALUX assay is suitable for monitoring dioxin in food. However, positive results from the CALUX test are confirmed by GC/HRMS. Such alternative rapid tests allow large-scale screening due to faster total analysis time and comparatively lower costs per sample versus the confirmatory MS method. Polychlorinated Biphenyls PCBs are chlorinated hydrocarbons with the general structure shown in Figure 3. In total, 209 different PCB congeners are theoretically possible, of which 36 are considered to be of environmental relevance and only about 100–140 are likely to be found in normal commercial mixtures. On the basis of structural characteristics and the associated toxicology, PCB congeners can be classified into ‘dioxin-like’ (DL) and ‘non-dioxin-like’ (NDL) PCBs. Although the manufacture, processing, and distribution of PCBs have been prohibited in almost all industrial countries since the late 1980s, their entry into the environment may still occur, especially due to improper disposal practices or leaks in electrical equipment and hydraulic systems still in use. The physicochemical properties of PCBs resemble those of dioxins, in that this group of compounds are chemically and thermally very stable and highly

Figure 3 Structural formula of polychlorinated biphenyls.

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lipophilic. They are also characterized by very low electrical conductivity, high boiling points, and fire resistance, which have led to their widespread use in various industries. Sources and occurrence

It is estimated that more than one million metric tonnes of technical PCB mixtures were commercially produced worldwide since their first commercial use in the late 1920s. Commercial PCBs are mixtures of congeners; they always contain PCDFs as impurities in the range of 0.8–5 ppm. By the 1960s, PCBs became ubiquitous in the environment, and their toxicity came under closer scrutiny after several accidents and poisoning incidents. Although the manufacture and use of PCBs were phased out from the mid-1970s onward, low levels of persistent PCBs can still be detected in the environment and via bioaccumulation in certain fatty foods. Stricter environmental controls have led to decreased levels in foods and subsequently lower human exposure over the last decade, although to a lesser degree compared with PCDDs/ PCDFs. Following exposure of farm animals, NDL-PCB will accumulate in meat, liver, and particularly in fatty tissues. In addition, NDL-PCB will be transferred into milk and eggs, and levels in these products will reach a steady state following exposure over a period of several weeks. PCB 138 and 153, both with six chlorine atoms, show the highest carryover into milk, on the order of 50–60%. After cessation of exposure, levels in milk initially decrease rapidly to about 50%, followed by a slower elimination phase. The amounts of NDL-PCBs in milk and animal fat are usually below 100 mg kg–1 (ppb) on a fat basis. For example, the analysis of butter (138 samples from 15 nonEuropean and 9 European countries) revealed the mean sum and median levels for the 7 indicator PCBs (including the DL-PCB 118) to be 8.2 and 7.1 mg kg–1 fat, respectively. Variations in reported levels in foods as well as in human intake estimates are due to analytical differences (number of PCB congeners analyzed) and dietary habits. Health impact

PCBs are of great health concern and can cause a variety of adverse effects. Accurate scientific assessment, especially of the potency, is difficult because PCBs occur only as complex mixtures and frequently together with other potent toxins such as dioxins and chlorinated pesticides. PCBs have been classified as probable human carcinogens. In animal studies, PCBs exhibit reproductive, developmental, and immunotoxic effects. The mean daily intake has been estimated as <0.1–1.9 mg person–1 day–1. At current background exposure levels, there is no real evidence of adverse effects in

humans. Of greatest concern in this context are the DLPCB congeners (coplanar PCBs) because of their similar mode of action to dioxins. This group, consisting of 12 congeners, shows toxicological properties similar to dioxins. Furthermore, no health-based guidance values have been established for NDL-PCBs. In a 2005 evaluation by EFSA, the toxicological database was considered to be too limited to develop a guidance value for NDL-PCBs. However, DL-PCBs are included in the WHO PTMI for dioxins and dioxin-like compounds, set at 70 pg TEQ kg 1 bw month 1 (see above). Many countries have set maximum residue limits for PCBs in dairy products, based mainly on the seven most abundant congeners. These are PCB 28, 52, 101, 138, 153, and 180 (all NDL-PCBs) and the DL-PCB 118. These congeners are considered as appropriate indicators of different PCB patterns in various matrices and represent about 50% of total NDL-PCB in food. Analysis

PCBs are determined by GC. However, GC identification can be difficult since different commercial mixtures give different peak patterns. The traditional approach to identify and quantify these compounds was by ‘peak pattern comparison’ using GC with electron capture detection (ECD), and comparison to higher chlorinated technical mixtures, for example, Aroclor 1260. Because many countries have now set maximum residue limits for six or seven individual congeners, it is current practice to analyze only the seven above-mentioned ‘indicator’ congeners by capillary GC-ECD, with confirmation by GC-MS. This is an alternative to the costly and timeconsuming analysis of the DL-PCBs (see above).

Other Persistent Halogenated Hydrocarbons There are other halogenated hydrocarbons that persist in the environment and can be detected in milk, such as polybrominated flame retardants (e.g., polybrominated diphenylethers), toxaphene (mixture of chlorinated boranes), chlorinated paraffins, and polychlorinated naphthalenes. Analysis is difficult and in many cases not well developed, and only limited data are available. It is generally thought that these compounds are of less health concern compared to dioxins and PCBs. However, data on exposure and toxicity are scarce and efforts are under way to reduce environmental levels. Organochlorine (OC) pesticides, which include compounds such as 1, 1, 1- trichloro- 2, 2- bis (4- chlorophenyl) ethane (DDT), hexachlorobenzen (HCB), lindane, and aldrin, are addressed in Contaminants of Milk and Dairy Products: Contamination Resulting from Farm and Dairy Practices.

Contaminants of Milk and Dairy Products | Environmental Contaminants

Metals Sources and occurrence

Metals are present in the environment either naturally or as a consequence of industrial and/or agricultural activities. They find their way to milk through several routes. Elements such as chromium and nickel from the stainlesssteel dairy equipment or tin from soldered cans may enter milk through direct contact. Heavy metals such as cadmium, lead, mercury, and arsenic are not expected to have any direct contact with milk and milk products except in accidental cases. For these elements, the main pathway to milk is through the ingestion of contaminated feeds by milk-producing animals. However, in the cow, transfer of heavy metals from feed to milk is very low. Typical analytical data for several metals in bovine milk are provided in Table 1. Data on buffalo and goat milk indicate that levels of heavy metals are likely to be similar to those encountered in bovine milk. With respect to milk products, contamination reflects the levels found in fresh milk, taking into account concentration factors. Metals may be associated with particular milk fractions. For example, lead and cadmium bind strongly to casein. The use of specific milk fractions may thus concentrate or remove metals. Health significance

Of the metals to which humans are exposed, heavy metals have raised the highest safety concern since the margins between the actual overall exposures and the safe levels of exposure (PTWI, provisional tolerable weekly intake) are narrow. For adults, estimations of intake of heavy metals from milk and milk products indicate that, in general, this category of food is unlikely to contribute significantly to the overall exposure. The situation may be different for infants and children because of their higher milk consumption on a body weight basis. For this sector of the population, intake estimates suggest that milk is likely to Table 1 Typical metal contents in bovine milk

Element

Typical (g kg–1)

Range (g kg–1)

Lead Cadmium Mercury Arsenic Chromium Nickel

<3 <2 <1 <1 <3 <3

n.d.–20 0.05–3 0.05–2 n.d.–2 1–4 0.4–6

n.d., not detectable (limit of detection not specified). Reproduced from Tahvonen R (1996) Food Reviews International 12: 1–70; Carl M (1991) Monograph on residues and contaminants in milk and milk products. IDF Special Issue 9101. Brussels, Belgium: International Dairy Federation; Blu¨thgen A, Burt R, and Heeschen WH (1997) Monograph on residues and contaminants in milk and milk products. IDF Special Issue 9701. Brussels, Belgium: International Dairy Federation.

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significantly contribute to the overall exposure. The health significance of such exposures is difficult to assess since safety standards do not cover infants less than 12 weeks of age. It is important to note that for some heavy metals such as lead, bioavailability and susceptibility are likely to be higher in infants than in adults. In this context, it is advised to limit overall exposure, and in particular exposure through milk. At the levels reported in milk, the other metals are unlikely to raise any safety issues. Some of them are thought to be essential trace elements and may be of nutritional relevance. Analysis

Methods based on atomic absorption spectrometry (AAS) are widely used to determine metals in foods. However, more sensitive multicomponent methods are available today for trace metal determination based on inductively coupled plasma (ICP) coupled to either atomic emission spectroscopy (AES) or MS. Because of the low absolute levels involved and the presence of metals in the laboratory environment, analysis may be challenging. Systematic errors and contaminations may explain some high levels reported in the older literature. It is well documented that metals such as lead, mercury, arsenic, or chromium may occur in various chemical species, which are characterized by specific toxic potentials. Presently, only little is known about the speciation of metals in milk and occurrence data are usually expressed as total metals. Radionuclides in Dairy Products Sources and occurrence

Humans have always been exposed to radioactivity. Radionuclides and their resulting ionizing radiation may stem from natural or anthropogenic sources. Contributions to natural radiation exposure comes from outer space, from radiation of soils and rocks as well as from inhaled and ingested natural radionuclides. The four most prominent and always present natural radionuclides in dairy products are potassium-40 (40K), rubidium-87 (87Rb), carbon-14 (14C), and tritium (hydrogen-3, i.e., 3H) (Table 2). Radiocontamination in the environment and consequently in dairy products may also result from anthropogenic activities. Radionuclides from ‘fallout’, that is, from nuclear weapons testing and accidents in nuclear power plants, are by far the most predominant contributors to this environmental anthropogenic radiation. Anthropogenic radionuclides may find their way into dairy animals and milk either by inhalation during actual fallout, or after deposition directly through plants and later through the soil–roots–grass–animal– milk pathway. The most significant anthropogenic

902 Contaminants of Milk and Dairy Products | Environmental Contaminants Table 2 Commonly found radionuclides in milk

Radionuclide

Half-life (t1=2)

Major type of irradiation

Rubidium-87 Potassium-40 Carbon-14 Cesium-137 Strontium-90 Tritium Cesium-134 Strontium-89 Iodine-131

49  109 years 1.3  109 years 5730 years 30.2 years 28.6 years 12.4 years 754.2 days 50.5 days 8.1 days

 ,   ,    ,   , 

Concentration in milk (Bq l 1) 0.5–3.5 40–60 14–16 <0.1a <0.04 <6.3 <0.1a b b

Predominant origin Natural Natural Natural Anthropogenic Anthropogenic Natural Anthropogenic Anthropogenic Anthropogenic

a

Values for total cesium. Data not known. Adapted from Paakkola O and Wiechen A (1990) Radionuclides in dairy products. IDF Bulletin 247, 3–15; Radioactivity in Food and the Environment (RIFE5) (1999) UK Food Standards Agency and Scottish Environment Protection Agency.

b

radiocontaminants in dairy products are strontium-89 (89Sr) and strontium-90 (90Sr), iodine-131 (131I), as well as cesium-134 (134Cs) and cesium-137 (137Cs) (Table 2). While all radionuclides will be dispersed after atmospheric nuclear explosions, in reactor accidents it is predominantly the volatile radionuclides, in particular iodine and cesium, that are liberated. This is also reflected in the dairy contamination pattern. After the Chernobyl accident in 1986, the ratio of contamination of 137Cs to 90 Sr in milk was approximately 50:1, whereas during the period of heavy nuclear weapon testing and subsequent fallouts in the 1960s, the ratio was only about 10:1. The strontium isotopes were of concern only after the 1960s due to nuclear weapons testings, and therefore both radioisotopes were ubiquitously detected in milk. Restrictions on nuclear testing have resulted in a significant drop in the amount of strontium isotopes, particularly 89Sr, attributable also to its shorter half-life, and today the overall environmental radiostrontium activity in Western Europe is only slightly elevated compared to activities measured before the nuclear weapons testing era. The fallout of volatile 131I, which is characterized by a short half-life of 8.1 days, was very high after the Chernobyl accident. Notably, nuclear weapons testing had only little impact on 131I activity, due to the fact that the radioisotope was lifted sufficiently high in the troposphere allowing ample time for decay before deposition. Therefore, levels were 100–1000 times lower as compared to the immediate post-Chernobyl period. Radioactive disintegration can be in the form of particle emission (- and -radiation) as well as in the form of energy-rich photon emission (-radiation). The energy of the emitted -, -, and -radiation is characteristic of each specific radionuclide. The activity of any radionuclide is measured in disintegrations per second in units of becquerel (1 s 1; Bq). Exposure to radiation, also referred to as the radiation dose, is measured in sievert (Sv). It is

important in the estimation of human health effect of radiation dose, and is standardized for the sensitivity of various body tissues and the type of radiation. Milk contributes considerably to the radioactive dose from foodstuffs. Mean radioactivity concentrations in milk in the United Kingdom in 1999 were reported to be <0.038 Bq l 1 for 90Sr, <0.066 Bq l 1 for total cesium, <6.3 Bq l 1 for 3H, and 16 Bq l 1 for 14C. The levels of radioactivity are considerably reduced through the food chain compared to direct intake from plant feed. Differential metabolic patterns in species of dairy animals introduce additional variation in the level of radioactivity in milk. Comparative biological trials were conducted to investigate the differential secretion into milk of radionuclides incorporated via feed. Highest 131 I contamination was found in sheep’s milk, followed by goat, cow, and buffalo milk, which contained 21, 11, and 3%, respectively, of the radioactivity found in sheep milk. A similar trend – although less pronounced – could be observed for radiocesium. Similarly, while approximately 0.1% of the amount of 90Sr given to cows daily is secreted per liter of milk, transfer to goat’s milk may be more than 10 times greater. Radionuclides partition differentially in aqueous and fatty milk fractions. These partition properties can be exploited to reduce radiocontamination of dairy products, especially in times of high contamination. Radiocesium, for example, predominantly concentrates in the aqueous fraction during physical separation procedures. In consequence, high-fat fractions, such as butter, cream, and high-fat cheeses, and high-protein fractions, such as cottage cheese curds, caseinates, and whey protein concentrates, will have comparatively low levels of radiocesium. Health impact

Adverse health effects of radiocontaminants originate from the emission of ionizing radiation, which is a

Contaminants of Milk and Dairy Products | Environmental Contaminants

known carcinogen. An estimated 95% of human exposure from man-made sources originates from medicinal applications, such as X-rays. Exposure to natural sources of radiation contributes more than 98% of the radiation dose to the general population (excluding medical exposure). The global average human exposure from natural sources is on the order of 3 mSv yr 1. In consequence, there is only a very small contribution from nuclear power generation and nuclear weapons testing to the overall human radiation exposure. The relevance of specific radionuclides to milk and consequently to human health depends on various factors. The physical half-life (Table 2) is the chief characteristic in the relative significance of radioactive contaminants in foodstuffs. Whereas 131I has a short half-life of 8.1 days, 137Cs, for example, has a half-life of 30.2 years and, once incorporated, a much longerlived effect in the body. The distinct metabolism of specific radionuclides is another important factor that determines the relative health significance of radiocontaminants. Iodine-131 acts like stable iodine and is accumulated quickly in the thyroid, where it causes local effects, while cesium isotopes that behave like potassium in the body are consequently widely distributed in soft tissues. Radiostrontium acts like calcium and therefore accumulates in bone tissue. The International Commission on Radiological Protection (ICRP) has set a maximum allowable annual dose above that from natural and medical sources at 5 mSv. The contribution of milk to the total radiation exposure is fairly small, being 0.02–0.3 mSv in the United Kingdom after the Chernobyl accident depending on the degree of deposition. In conclusion, the amounts of hazardous radionuclides in dairy products are low today and do not present immediate health hazards.

Analysis

Standard methods have been published for determining gross - and -activity concentrations in food. However, this is useful only when quick results need to be obtained, for example, during catastrophe situations. Often, the intensity of naturally present radioactivity in milk is considerably higher than radioactivity resulting from fallout. Radioactivity determination of specific radionuclides is therefore generally preferred. The radioactivity concentration of -emitting radionuclides can be determined using spectrometric analysis of -irradiation, generally without sample destruction. Determination of the activity of -radiation is comparatively more complicated, requires sample preparation, and is generally done indirectly by -scintillation spectrometry.

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Mycotoxins in Dairy Products Mycotoxins are secondary metabolites of fungi, some of which possess high potential to cause adverse effects in animals and man.

Sources and Occurrence Dairy contamination by mycotoxins can be via two different routes: indirect contamination and direct contamination. Indirect contamination stems from fungus-infected feedstuffs consumed by dairy animals. Of highest importance and significance in this respect is aflatoxin M1 (AFM1), the major metabolite of aflatoxin B1 (AFB1). In fact, approximately 3–5% of AFB1 initially present in the animal feedstuff appears as AFM1 in milk. Its acute and chronic toxicity, including hepatocarcinogenicity in several species, is similar to that of AFB1 (see Yeasts and Molds: Mycotoxins: Aflatoxins and Related Compounds). Carryover into milk of other mycotoxins (e.g., ochratoxin A, sterigmatocystin, deoxynivalenol, T-2 toxin, and zearalenone) has been reported. Their transmission rate, however, is significantly lower than that of aflatoxin and therefore does not cause immediate health concerns. The cause of direct contamination is the colonization of dairy products, in particular of cheese, by mycotoxigenic fungi. Dairy products are susceptible to contamination by molds and once infested, mycotoxins can be formed. Their formation, apart from inadvertent fungal growth, may also be attributed to fungal starter cultures used for the production of specific dairy products. Penicillium camemberti and P. roqueforti starter cultures are used in the production of white surface mold and blue-veined cheeses, respectively. While P. camemberti is a consistent producer of cyclopiazonic acid, P. roqueforti strains produce patulin, penicillic acid, isofumigaclavine A, mycophenolic acid, and roquefortine in vitro. Only the last three toxins produced by P. roqueforti could be found in commercial blue cheeses, sometimes at concentrations in the low ppm range. Cyclopiazonic acid originating from P. camemberti starter cultures could be detected in white surface mold cheese at similar concentrations. Several mycotoxins result from unintentional fungal non-starter culture infestation of dairy products. AFB1 can be formed in milk or processed dairy products infected with Aspergillus strains, although generally at lower levels than AFM1. Sterigmatocystin originates from Aspergillus versicolor and was detected upon unintentional fungal infestation in the low ppm range, mainly in hard cheeses. It is structurally similar to AFB1 and is believed to be a precursor in the biosynthesis of AFB1. The toxic properties of sterigmatocystin are much the same as aflatoxin, although much less pronounced. The

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nephrotoxic and carcinogenic ochratoxin A has also been detected in cheese. A large selection of dairy mycotoxins and causative mycotoxigenic molds is listed in Table 3, together with their typical sources relevant to human exposure. Isolated incidences of further fungal toxins in several dairy products at low levels have been reported for penitrem A, citrinin, citreoviridin, -nitropropionic acid, and PR toxin. Although Fusarium toxins, fumonisins B1 and B2, have been detected in dairy products, mycotoxins produced by species other than Aspergillus and Penicillium are of minor concern. Health impact

The aforementioned toxins occur in milk and processed dairy products generally at low concentrations. Indirect contamination of milk and dairy products with mycotoxins secreted by dairy animals is generally negligible and milk is not a major source of human exposure to these substances. The only exception is represented by AFM1, which may be secreted into and occurring in milk in significant amounts, and which is relatively stable in milk and processed milk products such as cheese and yogurt. Like AFB1, AFM1 is of considerable health concern. Although the presence of moderately toxic metabolites, particularly of Penicillium species in cheese, cannot be excluded completely, it is unlikely that consumers are endangered by the consumption of milk and mold-ripened cheeses. Milk and dairy products may be intermittently infested with mycotoxigenic fungi producing toxins such as AFB1 or ochratoxin A. In such occasional cases, high levels of mycotoxins may be anticipated, which, owing to their high toxic potency, would represent a significant health concern. Such issues are difficult to identify and

have to be dealt with through the strict application of good manufacturing practices. Comparatively little knowledge exists on potentially harmful secondary metabolites of starter cultures. Only limited data are available on the adverse effects of a number of substances occasionally produced by certain strains used in the manufacture of dairy products. Therefore, it is relatively difficult to assess the safety of human exposure to such compounds. By complying with good manufacturing practices, toxin-producing fungal infestation can be avoided or greatly reduced. Selection of starter strains with restricted capabilities for mycotoxin production can further reduce human exposure. In conclusion, with the exception of AFM1, transfer of mycotoxins from animal feed to milk is negligible. Direct contamination of dairy products with mycotoxin due to fungal infestation can occur sporadically at high levels. By and large, dairy products do not contribute substantially to the overall mycotoxin intake in man and no overt concern exists in relation to human health.

Analysis Various analytical methods have been developed for the identification and quantification of fungal toxins in milk and dairy products, ranging from simple techniques such as thin-layer chromatography (TLC) and immunochemical assays to confirmatory methods employing GC and high-performance liquid chromatography (HPLC), coupled to various detectors. For AFM1, these methods must achieve very low levels of detection and quantitation in the sub-ppm range, which can be accomplished by HPLC with fluorescence detection, preceded by a sample cleanup step using an immunoaffinity

Table 3 Mycotoxins relevant to dairy products Mycotoxin

Main sources

Main sources of human exposure

Aflatoxin B1

Aspergillus flavus, A. parasiticus, A. nomius

Aflatoxin M1 Cyclopiazonic acid

Dairy animal metabolism of aflatoxin B1 Penicillium camemberti, P. commune, Penicillium spp., Aspergillus spp. Fusarium moniliforme, F. proliferatum, Fusarium spp. Penicillium roqueforti, Penicillium spp. Penicillium roqueforti, Penicillium spp. Penicillium verrucosum, Penicillium. spp., Aspergillus ochraceus, Aspergillus spp. Penicillium spp., Aspergillus spp. Penicillium spp., Aspergillus spp. Penicillium roqueforti Penicillium roqueforti, Penicillium spp. Aspergillus versicolor, Aspergillus spp., Emericella spp.

Corn, peanuts, nuts, spices, wheat, oats, barley, rice, cheeses, etc. Milk and dairy products Grain, legumes, meat, milk, cheese (Brie, Camembert), etc. Corn and other cereal grains Blue cheeses Blue cheeses, mainly Roquefort Corn, wheat, barley, beer, wine, oats, sorghum, coffee, etc. Apples, etc. Cheese, etc. Blue cheeses Roquefort, blue cheeses Cereals, cheese (Edam, Gouda), coffee, nuts, etc.

Fumonisin B1 Isofumigaclavine A Mycophenolic acid Ochratoxin A Patulin Penicillic acid PR toxin Roquefortine Sterigmatocystin

Adapted from Weidenbo¨rner M (2001) Encyclopedia of food mycotoxins. Berlin, Germany: Springer; van Egmond HP (1989) Mycotoxins in dairy products. London: Elsevier.

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column. Commercial rapid test methods are available for AFM1, based, for example, on radioimmunoassay or solution fluorometry.

See also: Contaminants of Milk and Dairy Products: Contamination Resulting from Farm and Dairy Practices. Yeasts and Molds: Mycotoxins: Aflatoxins and Related Compounds.

Inherent Plant Toxicants – Bracken Fern Toxin

Further Reading

Sources and occurrence

The ubiquitous bracken fern (Pteridium spp.) is one of the most abundant plants on Earth. Bracken fern, due to its several inherent toxins, is well known to cause a variety of diseases in livestock that ingest large amounts of bracken or are fed bracken-containing fodder. Bracken fern is used as an example to illustrate inherent plant toxicants that may be excreted into milk. The major toxicant found to be responsible for the detrimental effects of bracken fern has been identified as the norsesquiterpene glucoside ptaquiloside (PT). The PT content of bracken fern is subject to significant regional and seasonal variations and can occasionally be extraordinarily high (up to 13 g kg 1 dry weight). It is well established that PT is excreted into milk in a dosedependent manner. Excretion rates between 1 and 11% of ingested PT have been reported, corresponding to approximately 0.1–22 mg l 1 in milk, respectively.

Health impact

Diseases reported in livestock upon ingestion of large amounts of bracken fern include thiamine deficiency in non-ruminants, acute hemorrhage, blindness due to retinal degradation, as well as bladder and intestinal cancer. There is epidemiological evidence of elevated cancer incidence in humans who consume bracken crosiers (young non-unfolded fronds) and also in people who consume dairy products from animals exposed to bracken fern through feed. Furthermore, the milk of bracken-fed cattle has been demonstrated to be carcinogenic in rats. The level of human exposure is difficult to quantify and probably inconsistent due to the seasonal variation of the PT content in bracken fern and sporadic ingestion of fern by dairy animals, which do not normally feed on bracken fern. However, during periods of drought when the ferns remain green and on overgrazed or heavily infested pastures, they may be forced to do so. Therefore, the principal strategy to minimize human risk appears to be careful management of dairy animal pastures, that is, strict adherence to good agricultural practices.

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