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134-136 137,138

terants in meat (129,130). This method was also used to test insect blood meats and identify the species of animals that these hematophagous insects had fed on (131).

Rias have not been extensively used for meat protein detection due to the short shelf life of the reagents, expensive equipment required for analysis, potential health hazards of exposure to radioisotopes, and governmental regulations pertaining to radioisotope use. A RIA was developed that could distinguish cattle meat from sheep, donkey, pig, horse, and kangaroo meats. Contamination as low as 5% could be determined (132).

A number of different eias have been developed for fresh meat speciation. Current slaughter methods leave residual blood, as much as 1.5-2.0% in the muscle tissue (140). Some of the blood proteins, namely the albumins and immunoglobulins, have served as immunogens for producing antibody-based meat species testing kits (133,141). An indirect competitive ELISA for meat speciation that enabled detection of 0-40% horse meat in beef has been developed (133).

Competitive ELISAs have been employed for quantitative determinations, whereas qualitative species identification of fresh meats have been obtained with indirect noncompetitive ELISAs (133-136,142,143). Horse meat was differentiated from beef and mutton with an indirect ELISA in which an antibody raised against horse serum albumin was used. Adulteration of beef with goat and kangaroo was also elucidated with an indirect ELISA.

The ELISA variants are not applied for quantification because they have relied on the presence of serum antigens that can vary greatly in meat and are not indicative of the meat content (24). Stunning and bleeding methods greatly affect the content of serum proteins in the muscles (142). The stability of the antigen with time is also a factor (143). Therefore, antigens that are genuine muscle components that are indicative of the meat content must be developed (124).

Sandwich ELISAs also permit speciation of meat (137,138). Adulteration of raw meat mixtures with pig meat was identified (129), and improved speciation of raw meat was achieved using this ELISA format. Levels as low as 0.5% (137) and 17% adulteration have been detected (138).

The task of identifying the species of origin of cooked or heat-processed meats becomes more difficult than speciation of fresh meat. Heating denatures or changes the species-specific epitopes that are used for identifying the species of fresh meat (24). When meat is heated at 45°C denaturation of myofibrillar and sarcoplasmic proteins occurs (144), and at 60°C collagen shortens and changes into a more soluble form (145). As the temperature is raised from 60 to 90°C, the percentage of collagen solubilized increases gradually (146). Therefore, to develop an immunoassay that permits identification of heat-treated meat, antigens must be isolated that will retain their species specificity following the heating process. They must not be lost in cooking juices and must be antigenic and immunogenic.

The first EIA developed (147) enabled detection of origin of heat-treated meats. Kangaroo meat was detected in frankfurter-type sausages and dry sausages. Antisera produced against thermostable muscle antigens (TMAs) were used to develop an indirect ELISA that enabled the detection of origin of heat-treated meats from closely related species (148). Immunodiffusion tests have demonstrated that TMAs are species, organ, and tissue specific (149151).

Monoclonal antibodies were used to achieve speciation of heat-treated meats because cross-reactions could be eliminated. Monoclonal antibodies were produced against heat-treated kangaroo and cattle meats, which differentiated the two kinds of meat in an elisa (152). Use of monoclonal antibodies has the advantage of being specific without requiring absorptions needed when polyclonal antisera are used; therefore, the future trend would be to make use of monoclonal antibodies for speciation of meats from closely related species.

NATURAL CONTAMINANTS Natural Toxicants

There are natural components of food that can potentially be toxic to consumers; however, attention has been largely focused on detecting food additives and contaminants and not these natural toxicants. It is true that many of the plant and animal portions that might cause toxic effects have over time been avoided by humans or are deemed safe for consumption when they are prepared in a certain manner. When normal practices are followed the naturally occurring toxins do not present acute problems to the consumer. It is when these normal practices are changed that problems can arise (153).

Until recently a dearth of information existed concerning toxin occurrence, toxicology, and metabolism following ingestion of foodstuffs containing natural toxins. One of the biggest problems has been the dilemma presented by analysis. Chemical methods (HPLC and mass spectroscopy) have helped improve toxin analysis. Immunoassays have greatly furthered this effort. They can be highly spe cific, which is desirable, because compounds with very similar structures will exhibit widely different toxicity potential. The high specificity will eliminate chances of mis-classification of compounds. Immunoassays can also be very sensitive, which would permit detection of toxin metabolism following ingestion. The ability to quickly test large numbers of samples is also desirable. It is common for natural toxicants to exhibit nonhomogeneous patterns of distributions so that multiple subsamples must be tested to obtain data about overall concentrations. The multiple samples can also be tested at low cost (153).

A microtiter-well elisa for quinine detection in soft drinks has been developed (154). It is, of course, added as a bittering agent. Although the risk from consumption of soft drinks containing it is low, quality-control procedures for its analysis are important to the soft drink industry. The ability to test easily for the compound permits the industry to provide greater control on a potentially dangerous substance, which will boost consumer confidence in the product containing the substance.

A number of immunoassays have been described for caffeine which is found in a variety of food products, namely those containing teas, coffee, and chocolate. A radioimmunoassay and a substrate-labeled fluoroimmunoassay for caffeine detection have been described (155,156). Assays have been developed for analysis of naringin and li-monin, which are bitter substances isolated from citrus fruits. The ria for naringin (157) and the elisa for limonin (158) permit the analysis of the distribution and metabolism of the compounds in the fruit. Assays of this nature may also be of use to citrus growers for quality-control testing of both raw materials and finished products.

Many toxic compounds are found in the potato, Solarium tuberosum. One group is collectively known as the potato glycoalkaloids. Ninety-five percent or more of the tuber is normally composed of a-chaconine, a-solanine, and derivatives of solanidine (159).

A ria, the first immunoassay described for glycoalkaloid analysis (160), employed an antiserum that did not recognize a-chaconine and a-solanine. A more useful elisa was developed (161) that enabled detection of all three compounds. It has been shown to have good correlation with conventional chemical methods (162,163). Immunoassays have been developed for testing glycoalkaloid concentrations in body fluids. Solanidine can be detected in serum (164,165) and saliva using a RIA.

Immunoassays that permit lycoalkaloid detection can be used by plant breeders to screen tubers of all potential new varieties. Testing can be performed on the retail level as well to prevent glycoalkaloid concentrations from exceeding 20 mg/1.0 g (166).

Mycotoxins

Mycotoxins are a chemically diverse group of secondary fungal metabolites that are toxic following ingestion or environmental exposure (167). Mycotoxins are haptens with molecular weights of300-400. Many of these molecules are nonpolar and exhibit low solubility in water. The fungi that produce the mycotoxins are ubiquitous. They can grow and elaborate the toxins in numerous agriculture commodities either in the field or following harvest during storage and processing. The factors that influence toxin elaboration are temperature, relative humidity, and moisture content of the substrate. As a result of their toxicity, mycotoxins can cause severe economic losses to farmers and livestock producers as well as pose a threat to humans consuming contaminated foods. Affected agricultural commodities include corn, wheat, peanuts, cottonseed, rice, sorghum, almonds, walnuts, and milk (168).

The target tissues that can be affected by mycotoxins are the liver, kidney, spleen, gastrointestinal tract, lymphoid tissue, reproductive organs, skin, and nervous system. In the United States the most important toxins are the aflatoxins, zearalenone, the trichothecenes and to a lesser extent ochratoxin. These toxins are produced by species of Aspergillus and Fusarium and cause a variety of health problems to livestock and humans (Table 4).

Classical methods of analysis of mycotoxins include TLC, HPLC, GC, and mass spectroscopy (MS). TLC methods have found wide use because they are easy to perform, are inexpensive, and are relatively sensitive. Many of the mycotoxins naturally, fluoresce and absorb in the UV region, so that TLC methods (169-171) while TLC determination of mycotoxins are frequently only semiquantitative and require elaborate sample cleanup. GC and HPLC methods offer sensitive quantitative determinations of mycotoxins (172-174). Sample cleanup is also extensive and labor intensive prior to analysis by these methods. Due to these drawbacks, development of immunochemical methods for mycotoxin detection have become popular.

The Aflatoxins. The majority of work on mycotoxins has focused on the aflatoxins, the family of difurancoumarins, which act as hepatotoxins and hepatocarcinogens (175). Af-latoxin (AFBj) is reported to be the most prevalent and most potent carcinogen. Results from the modified salmonella mutagenesis test show that AFBX is about 30 times more mutagenic than AFGj or AFM1( which are more mutagenic than AFB2, AFG2, or AFM2 (Fig. 1) (176). Similar patterns exist for hepatotoxicity and carcinogenicity. The biological effects of the toxin are attributed to the difuran ring with 8:9 vinyl ether, while the coumarin ring absorbs at 360 nm and fluoresces. The Food and Drug Administration (FDA) has set an action level of 20 ppb total aflatoxins in foods or feeds.

AFBj can be metabolized by hepatic enzymes {in vivo or in vitro) to different metabolite (173,174). The carcinogenicity results from the reaction of the 8,9 epoxide with cellular nucleophiles (RNA, DNA, and proteins). Other detoxification pathways involve reduction or hydroxylation, which form more polar compounds that are then removed from the animal. One hydroxylated metabolite, AFMj, is also hepatocarcinogenic and will be found in milk of lac-tating animals that have ingested AFBj contaminated feed (177). The action set by the FDA for AFMX in dairy products is 0.5 ppb.

The Trichothecenes. Many of the mycotoxins produced by Fusarium species and other fungi (Cephalosporium, Verticimonosporium, Myrothecium, and Stachybotrys) are called trichothecenes (178). These mycotoxins are tetracyclic sesquiterpenoid molecules, all possessing an epoxy group. T-2 toxin and deoxynivalenol (DOH), also known as vomitoxin (Fig. 2), are frequently identified in Fusarium culture filtrates. The toxins cause a strong dermatitis reaction when applied to the skin of shaved guinea pigs, characterized by severe local irritation, inflammation, desquamation, and necrosis (175).

There have been more than 148 trichothecenes identified, but only four have been unambiguously identified in naturally contaminated food samples. Trichothecenes are difficult to analyze especially because, unlike the aflatoxins, they do not possess any useful spectroscopic characteristics. Analysis of the trichothecenes have been accomplished using TLC (179), HPLC (180,181), GC (182), and MS (183,184).

The trichothecenes are believed to function as protein synthesis inhibitors. The initiation or elongation termination steps of protein synthesis are believed to be affected.

The Zearalenones. Fusarium species, primarily F. Sra-minearium, produce zearalenones which are resorcylic lactones (Fig. 3). The parent compound is zearalenone, which does not exhibit acute toxicity. However, high levels of zearalenone can interrupt normal estrus in sows and cause vulvar prolapse and enlargement of the uterus. In young males, the toxin causes testicular atrophy and mammary gland hyperplasia. Physiological response in swine occur when the zearalenone level in corn used for feeds exceeds about 1 ppm (185). Rats, mice, sheep, monkeys, and humans can be affected by zearalenone (168).

The Ochratoxins. Ochratoxin contamination of grains and food products is less of a concern in the United States than contamination of products with aflatoxins, zearalenones, and trichothecenes. It is frequently detected in Scandinavian and Balkan countries and occasionally in the United States in barley, corn, wheat, oats, rye, peanuts, hay, and green coffee beans (186). Ochratoxins are a group of structurally related metabolites produced by Aspergillus ochraceus and related species as well as Pénicillium viri-

Table 4. Abbreviated List of Major Mycotoxins and the Potential Health Hazards Following Exposure

Mycotoxin

Fungi

Potential Health Risk

Aflatoxins Trichothecenes

Zearalenones

Ochratoxins

Aspergillus flavus, A. parasiticus Fusarium sp.

Fusarium roseum and other Fusarium sp. Aspergillus sp. and Pénicillium sp.

Mutagenic, teratogenic, hepatotoxic, and carcinogenic Feed refusal, emesis, immunotoxicity, gastrointestinal tract hemorrhaging, alimentary toxic aleukia Estrogenic, infertility, and reproductive problems in cattle, swine, and poultry

Nephrotoxic, teratogenic, and immunotoxic

Aflatoxin Bx

Aflatoxin B2

Aflatoxin Beans

Aflatoxin Gi

Aflatoxin G2

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Homemade Pet Food Secrets

Homemade Pet Food Secrets

It is a well known fact that homemade food is always a healthier option for pets when compared to the market packed food. The increasing hazards to the health of the pets have made pet owners stick to containment of commercial pet food. The basic fundamentals of health for human beings are applicable for pets also.

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