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The Microbiology of Cereals and Cereal Products
Variety of media and methods available for detection and enumeration of molds in cereal products
by Lloyd B. Bullerman and Andreia Bianchini
Editor’s Note: This article is excerpted from a longer article that appeared in “Microbiologically Safe Foods,” edited by Norma Heredia, Irene Wesley, and Santos Garcia. The book was published in 2009 by John Wiley & Sons, which also publishes Food Quality. To access a PDF of the full article, go to our website at www.foodquality.com. A PDF of another chapter from the book, “Foodborne Pathogens and Toxins: An Overview,” is also available on our website.
Cereals and cereal products are significant and important human food resources and livestock feeds worldwide. Cereal grains and legumes are food staples in many, if not most, countries and cultures and are the raw materials of many of our foods and certain beverages. The main cereal grains used for foods include corn (maize), wheat, barley, rice, oats, rye, millet, and sorghum. Soybeans are not a cereal product, but rather, are legumes or a pulse, but are often considered with cereals because of their importance as a food source.
Examples of cereal products derived from cereal grains include wheat, rye, and oat flours and semolina, cornmeal, corn grits, doughs, breads, breakfast cereals, pasta, snack foods, dry mixes, cakes, pastries, and tortillas. In addition, cereal products are used as ingredients in numerous products, such as batters and coatings, thickeners and sweeteners, processed meats, infant foods, confectionary products, and beverages such as beer.
Because of their extensive use as human foods and livestock feeds, the microbiology and safety of cereal grains and cereal products is a very important area. The sources of microbial contamination of cereals are many, but all are traceable to the environment in which grains are grown, handled, and processed. Microorganisms that contaminate cereal grains may come from air, dust, soil, water, insects, rodents, birds, animals, humans, storage and shipping containers, and handling and processing equipment. Many factors that are a part of the environment influence microbial contamination of cereals, including rainfall, drought, humidity, temperature, sunlight, frost, soil conditions, wind, insect, bird and rodent activity, harvesting equipment, use of chemicals in production versus organic production, storage and handling, and moisture control.
The microflora of cereals and cereal products is varied and includes molds, yeasts, bacteria (psychrotrophic, mesophilic, and thermophilic/thermoduric), lactic acid bacteria, rope-forming bacteria (Bacillus spp.), bacterial pathogens, coliforms, and Enterococci. Bacterial pathogens that contaminate cereal grains and cereal products and cause problems include Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Escherichia coli, Salmonella, and Staphylococcus aureus. Coliforms and enterococci also occur as indicators of unsanitary handling and processing conditions and possible fecal contamination.
Bacteria are frequent surface contaminants of cereal grains. For bacteria to grow in cereal grains, they require high moisture or water activity (aw) in equilibrium, with high relative humidity. Generally, bacteria are not significantly involved in the spoilage of dry grain and become a spoilage factor only after extensive deterioration of the grain has occurred and high moisture conditions exist. However, bacterial pathogens and spoilage bacteria, such as spore-forming bacteria that cause ropiness in bread, may survive and carry through to processed products and become problems. Lactic acid bacteria may also be present in the raw grain and carry over into flour and cornmeal and spoil doughs prepared with them. Yeasts present on cereal grains may also carry through into processed products. The main spoilage organisms in cereal grains, however, are molds.
There are more than 150 species of filamentous fungi and yeasts on cereal grains. But again, the most important of these are the filamentous fungi or molds. The filamentous fungi that occur on cereal grains are divided into two groups, depending on when they predominate in grain in relation to available moisture in the grain. These groups have been referred to as field fungi and storage fungi. Field fungi invade grain in the field when the grain is high in moisture (18 to 30%, i.e., at high aw) and at high relative humidities (90 to 100%). Field fungi include species of Alternaria, Cladosporium, Fusarium, and Helminthosporium. Storage fungi invade grain in storage at lower moisture contents (14 to 16%), lower aw and lower relative humidities (65 to 90%). These main storage fungi are species of Eurotium, Aspergillus, and Penicillium. To prevent spoilage by storage fungi, the moisture content of starchy cereal grains should be below 14.0%, soybeans 12.0%, and other oilseeds, such as peanuts, and sunflower seeds, 8.5%. Certain molds, such as Eurotium glaucus, may initiate growth at low aw and moisture contents (i.e., 15 to 16% moisture) and through their respiration increase aw and raise the moisture content, facilitating molds to grow, thus ultimately leading to spoilage. More information on storage fungi and moisture contents in various commodities is given in Table 1 (see p. 26).
The major effects of fungal deterioration of grains include decreased germination, discoloration, development of visible mold growth, musty or sour odors, dry matter loss and nutritional heating, caking, and the potential for production of mycotoxins in the grain. Decreased germination of the grain occurs when storage fungi invade the germs or embryos of the grain kernel. The embryos are weakened and die as the storage fungi attack and parasitize the embryo to utilize its oils and other nutrients. Decreased germination caused by storage fungi usually precedes discoloration. However, discoloration can be caused by both field and storage fungi and can result in brown to black germs in wheat and corn and “blue eye” in corn, due to the presence of blue Aspergillus and Penicillium species. Musty odors may become apparent before mold growth becomes visible and is an early warning of mold activity, as is heating.
Heating often starts in the fine materials or dust associated with the grain and is due to the growth of storage fungi. If sufficient heating occurs, the grain becomes dark and blackened. Further growth of storage fungi may result in surface growth and binding of the grain kernels together by mold hyphae, which is manifested as caking of the grain (i.e., large masses of the kernels bound together). By the time caking occurs, mold growth has become extensive and the grain is in advanced stages of decay. At this point the moisture content of the grain is increasing due to the respiration of the molds, and growth of yeasts and bacteria may also occur.
The word mycotoxin is derived from the Greek word mykes, meaning fungus or mold, and the Latin word toxicum, poison or toxin. Thus, mycotoxin is a general term meaning fungus poison or mold toxin. Mycotoxins are toxic secondary metabolites produced by filamentous microfungi or molds. These secondary metabolites are distinguished from primary metabolites because they are not required for the growth of the fungus and have no apparent purpose in the metabolism of the organism. It has been speculated that mycotoxins are waste products or defense mechanisms. Mycotoxins are toxic and harmful in varying degrees to humans and animals, and may contaminate cereal grains in the field and in storage. Mycotoxins are stable compounds that resist destruction by food-processing methods and may carry through and contaminate finished processed foods.
There are numerous specific mycotoxins that may contaminate cereal grains, such as aflatoxins, ochratoxin, fumonisins, moniliformin, deoxynivalenol, T-2 toxin, and zearalenone. Mycotoxin research began in 1960 with the outbreak of Turkey “X” disease in England, where thousands of turkey poults and other young farm animals were lost due to poisoning by a fungal metabolite produced by Aspergillus flavus in peanut meal. The toxic substance was called aflatoxin (A. flavus toxin). Since 1960, many other toxic mold metabolites have been described. Those mycotoxins currently thought to be most important in cereal grains are listed in Table 2 (see p. 28) along with the molds that produce them.
Mycotoxins exhibit a range of toxicological properties, including acute toxicity or poisoning, which often results in death, and subacute or chronic toxicity, which may not result in death directly but which gradually weakens and lowers the general health of an animal or human due to effects on the immune system. Chronic toxicity may result in greater susceptibility to secondary bacterial infections. Some mycotoxins are carcinogenic and may cause cancers; some are mutagenic and are capable of causing mutations; they may also be teratogenic and embryo toxic, causing deformities and death in developing embryos.
Media, Methods for Molds and Mycotoxins
There are a variety of media and methods for detection and enumeration of molds in cereal products. Many media are recommended by the International Commission on Food Mycology (ICFM) as well as given in the Compendium of Methods for the Microbiological Examination of Foods.
Dichloran rose bengal chloramphenicol (DRBC) agar is recommended as a general-purpose medium for direct plating of grain kernels and for plate counts of flours, meals, and processed products for total counts. Dichloran with 18% glycerol (DG18) is also recommended for these uses, especially for direct plating kernels for xerophilic molds, which prefer low aw and dry conditions. For plating dry grains and cereal products, DG18 may actually be better than DRBC and the medium of choice.
Two differential media may also be used for detection and enumeration of specific molds in cereals. Aspergillus flavus–parasiticus agar (AFPA) can be used as a differential medium for direct plating of kernels and plate counts of A. flavus and A. parasiticus. Czapek agar with iprodione and dichloran added (CZID) is widely used as a differential medium for detecting and enumerating Fusarium species from cereals. The simplest way to evaluate the internal microflora of seeds and kernels is the direct plating method, which involves surface sanitizing seeds or kernels in full strength or 50% household bleach for one minute to kill surface microflora. The kernels or seeds are then rinsed in sterile distilled water and dried on sterile paper towels.
The seeds or kernels are then placed directly on an agar surface in a Petri dish and incubated at 25 to 30°C to allow molds located in the interior of the seed or kernel to grow out. The number of kernels with internal mold is counted and the results are expressed as a percentage of infected kernels. The amount of internal infection of the grain is an indicator of quality and storability of the grain. The technique can also give some information about the safety of the grain if AFPA or CZID have been used, indicating whether or not potentially toxic A. flavus, A. parasiticus, or Fusarium species are present.
Methods for detecting mycotoxins are summarized in Table 3 (above). Chromatographic methods have been used from the beginning of mycotoxin research and are still used for detecting, quantifying, and confirming the presence of mycotoxins. These methods have evolved, been improved, and have become more sophisticated. Chromatographic methods in use include thin-layer chromatography, high-performance liquid chromatography, liquid chromatography combined with mass spectrometry, gas chromatography, and gas chromatography combined with mass spectrometry.
Various detection methods, such as fluorescence, ultraviolet absorption, and others have been combined with chromatographic methods. New methods based on the production of antibodies specific for individual mycotoxins have also been developed and include enzyme-linked immunosorbent assays and immunoaffinity columns. These methods allow for specific and precise detection and quantification of specific mycotoxins. This has lead to test kits for mycotoxins which are rapid and simple to use and can be used in the field, country elevators, grain-buying stations, feed mills, and processing plants.