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DOI: 10.1111/jfs.12422
ORIGINAL ARTICLE
1
Center for Food Safety and Applied
Nutrition - FDA, College Park, Maryland
Abstract
2
Georgetown University Medical School, A total of 85 grain and grain product samples (including corn meal, corn muffin mix, popcorn, vari-
Georgetown University, Washington, DC ous types of rice, and self-rising, all-purpose unbleached and whole wheat flour) from U.S. retail
were tested for fungal contamination levels and profiles using conventional plating as well as
Correspondence
molecular methods. The results of this study showed that over 90% of wheat flour and corn prod-
Vasiliki Tournas, Center for Food safety
and Applied Nutrition, HFS-711 Food and uct samples and 73% of rice samples tested carried live fungi. Popcorn carried the highest fungal
Drug Administration 5001 Campus Drive levels reaching 5.45 log10 colony forming units (cfu) per gram followed by corn meal (reaching 5.38
College Park, MD, USA. log10 cfu/g). Mold and yeast counts in rice and wheat flour reached 3.30 log10 and 3.28 log10 cfu/
Email: valerie.tournas@fda.hhs.gov
g, respectively. The predominant molds in wheat flour were aspergilli and fusaria found in 50 and
46% of samples, respectively; Fusarium spp. were the most frequent contaminants of corn-based
products found in 74% of the samples followed by penicillia (present in 44% of tested samples).
Rice, conversely, contained mainly Aspergillus, Fusarium, and yeasts (each found in 21% of the
samples).
Practical applications
Toxigenic molds are often contaminating stored grains and grain products and under improper
storage conditions could cause spoilage of these commodities accompanied with production of
toxic secondary metabolites, mycotoxins. Mycotoxins are known to cause illnesses in humans and
animals. Therefore, monitoring the presence and inhibiting the growth of these organisms is critical
for achieving and maintaining high quality products, suitable for human and animal consumption,
and free of health hazards. Establishing toxigenic mold profiles in stored grains and their deriva-
tives can point to correct storage management and thus reduction/elimination of spoilage and
mycotoxin production in these products. In this study we tested several corn, rice, and wheat flour
commodities for live potentially toxigenic fungal species. Our findings can help select proper
storage management techniques for these commodities.
1 | INTRODUCTION Past studies have reported that the most common molds causing
spoilage of cereals in storage are eurotia. Wallemia and a number of
Cereal grains usually carry a variety of fungi from the field (e.g., various aspergilli are also found in these commodities. Additionally, penicillia
Fusarium and Alternaria species) and also contain some xerophilic (such as Penicillium citrinum, P. aurantiogriseum, P. brevicompactum,
storage molds. The latter are capable of growing at very low water P. chrysogenum, and P. crustosum) are present sometimes at high levels,
activity (aw) and could cause spoilage during storage, if the moisture of but it’s more likely that these organisms develop growth during drying
the commodity reaches levels above 12%. Determination of the myco- of the grains (Pitt & Hocking, 2009). Grain invasion and spoilage by the
logical quality of cereals and derived cereal products is a very important storage fungi Aspergillus and Penicillium spp., and field molds such as
issue since these commodities are used extensively as foods and animal Alternaria and Fusarium was reported in past studies (Hernandez Nopsa
feeds and because some of the organisms encountered in these prod- et al., 2015; Mislivec, 1981; Mislivec & Bruce, 1988; Talbot & Koehler,
ucts are capable of producing toxic secondary metabolites, mycotoxins. 2002). Mycotoxins, such as aflatoxins (AFs), deoxynivalenol (DON),
ochratoxin A (OTA), and B fumonisins (FBs), have also been found in 2.4 | Identification of isolated fungal strains
stored cereal grains (Birck, Lorini, & Scussel, 2006; Dhivya, 2013;
Recovered isolates were purified by reculturing on PDA agar plates and
Magan, Aldred, Mylona, & Lambert, 2010; Mylona, Sulyok, & Magan,
identified to genus or species level using the conventional methods
2012; Talbot & Koehler, 2002). Food grade preservatives such as propi-
and keys described in “Identification of Common Aspergillus Species”
onate, sorbate, and benzoate salts as well as chemical fungicides have
(Klich, 2002), “A Laboratory Guide to Common Penicillium Species”
been used to control spoilage of stored grains (Birck et al., 2006;
(Pitt, 1985), “Fusarium Species: An Illustrated Manual for Identification”
Magan et al., 2010; NASS-USDA, 2010; Vandegraft, Hesseltine, &
(Nelson, Toussoun, & Marasas, 1983), and “Fungi and Food Spoilage”
Shotwell, 1973). In recent years, however, there is a trend toward
reducing the use of certain pesticides or substituting them with other, (Pitt & Hocking, 2009). Isolates that could not be identified by conven-
less toxic chemicals. Because disease management practices evolve tional plating methods were speciated using molecular techniques as
and different fungicides are used over time while utilization of some described below.
TA BL E 1 Yeast and mold (YM) counts in various grains and grain those found in corn, not exceeding 3.30 log10 cfu/g in any of the sam-
products from U.S. retail ples. Aspergilli and fusaria were among the most common molds iso-
Number of a
YM counts range lated, occasionally reaching or exceeding 3.00 log10 cfu/g. A. flavus was
Product samples tested (log10 cfub/g) Frequencyc isolated from all commodities except Basmati rice. Black aspergilli were
Corn products found only in Basmati rice and whole wheat flour, while A. ochraceus
Cornmeal 10 3.69–5.38 100 (3.15 log10 cfu/g) was present in one Basmati rice sample. Low Alterna-
Corn muffin mix 18 <2.00–4.32 94
ria counts were found in all types of flour and in Basmati rice. Penicillia
Popcorn 11 <2.00–5.45 91
were present in whole wheat and unbleached flour and in Basmati rice,
Rice but their levels were lower than 3.00 log10 cfu/g. Low levels of eurotia
Basmati 16 <2.00–3.30 69
were found in self-rising and whole wheat flour and in some rice sam-
Otherc 8 <2.00–3.28 75
ples while yeasts were isolated from unbleached wheat flour and rice
Wheat flour
(Table 3). Yeast presence is mostly evident in products undergone
Self-rising 5 2.48–3.00 100
Unbleached 12 <2.00–3.11 83 extended processing and is usually associated with inadequate hygienic
Whole wheat 5 2.65–3.28 100 practices.
a
cfu 5 colony forming units.
b
Percent contaminated samples. 4 | DISCUSSION
c
This group included medium grain, parboiled and brown rice (two sam-
ples each), and white long grain and jasmine rice (one sample each).
Field fungi like Alternaria and Fusarium species were found in all prod-
ucts tested in this study. Fusarium spp. were found more frequently
4.00 log10 cfu/g. Low levels of Eurotium spp. were found only in one and at higher levels (sometimes exceeding 5.0 log10 cfu/g) in corn-
popcorn sample. Small numbers of yeasts were also present in all types based products such as cornmeal and popcorn. These findings are in
of corn products, but only one cornmeal sample reached 3.00 log10 agreement with results of past studies which demonstrated that corn
cfu/g (Table 2). contamination with fusaria was an on-going problem (Mislivec, Bruce,
Although live fungi were present in a high percentage of the wheat & Andrews, 1979; Vincelli & Parker, 2002). Lower levels of fusaria in
and rice samples, the levels of these contaminants were lower than corn muffin mix were probably due to destruction of these organisms
Alternaria spp. ND c
<2.00–2.60 (11) d
ND
TA BL E 3 Fungal species and levels recovered from rice and wheat flour
Contamination levels (log10 cfua/g—range)
Wheat flour Rice
Organism Self-rising (n 5 5)b Unbleached (n 5 12) Whole wheat (n 5 5) Basmati (n 5 16) Otherc (n 5 8)
Alternaria spp. <2.00–2.30 (20)d <2.00–2.00 (8) <2.00–2.00 (20) <2.00–2.00 (12) ND
Aspergillus spp. <2.00–2.48 (20) <2.00–3.00 (8) <2.00–3.00 (40) <2.00–2.30 (6) <2.00–2.30 (25)
Aspergillus flavus <2.00–2.60 (40) <2.00–2.00 (8) <2.00–2.48 (20) ND <2.00–2.48 (12)
Aspergillus niger ND e
ND ND <2.00–2.30 (6) ND
Fusarium spp <2.00–2.00 (60) <2.00–3.00 (42) <2.00–2.90 (40) <2.00–3.00 (12) <2.00–3.20 (38)
during processing. The frequent presence and high levels of Fusarium group) were found in grains and grain products during a study by Misli-
spp. in some of the products are worrisome because these organisms vec et al. (1979), whereas AF-tainted grain products were reported by
are capable of producing a wide spectrum of toxic secondary metabo- Cassel, Campbell, Draper, and Epperson (2001) and Trombete et al.
lites such as FBs, DON, T-2 toxin, nivalenol (NIV), enniatins (ENNs), (2014).
and zearalenone (ZEA) with fumonisins being the most common Fusar- Fusaria, aspergilli, eurotia, and Alternaria spp. were also recovered
ium toxins found in corn (Vincelli & Parker, 2002). Alternaria spp., from rice and wheat flour, but at much lower levels than the ones in
although potentially toxigenic, were present in very low numbers and corn products. Mislivec et al. (1979) had repeatedly isolated Alternaria
probably are not of any significance. Low levels of aspergilli, penicillia, spp. from wheat flour and brown rice while Birck et al. (2006) had
and eurotia perhaps indicated that the water activities of the commod- reported the presence of Aspergillus, Penicillium, and Fusarium species in
ities were low enough not to allow substantial growth of these organ- stored wheat grain. The most common molds isolated during the latter
isms during storage. Among the aspergilli recovered, A. flavus was study were aspergilli followed by fusaria. These investigators examined
found at relatively high levels in cornmeal and popcorn; this is a cause the same wheat samples for mycotoxin contamination and found that
of concern since many strains of this organism are capable of producing 31% of the tested samples contained FB1. Several Fusarium species
mycotoxins (B aflatoxins and/or cyclopiazonic acid) (Frisvad & Thrane, (F. graminearum, F. avenaceum, F. poae, and F. culmorum) and their
2002). A. flavus as well as members of the genus Eurotium (A. glaucus toxins (DON, ZEA, ENNs, NIV, etc.) were also reported in a study on
TOURNAS AND NIAZI | 5 of 6
stored wheat grain by Lindblad et al. (2013). According to this study, can occur when the spoilage and toxin production are spread to large
F. avenaceum, F. poae, and F. graminearum were the most common grain amounts. Richard et al. (2003) reported an estimated annual
fusaria detected while ENNs were the most prevalent mycotoxins fol- stored-grain loss of about 1 billion dollars due to mycotoxin contamina-
lowed by DON. Flour and other grain products made from mycotoxin- tion in the United States. Adherence to Good agricultural practices
containing grain can also carry active toxin, since most mycotoxins are (GAPs) and Good manufacturing practices (GMPs) during cultivation,
not completely destroyed by processing. Contamination of grain harvest, transport, and storage will help keep mold propagules at a min-
products (e.g., wheat flour, bran, and processed maize) with Fusarium imum, thus resulting in negligible contamination of the stored product.
mycotoxins such as DON, NIV, ZEA, HT-2, and T-2 toxins has been Another important factor affecting mold spoilage during storage is the
reported in the literature (Schollenberger, Terry-Jara, Suchy, Drochner, presence of various pests which can serve as vectors, transferring
& Muller, 2002, 2005). Low levels of eurotia were found in wheat flour spores, and other mold entities within and between storage units;
and some rice samples. These organisms are deemed as the most com- therefore, an effective pest control is crucial for the avoidance of mold
mon spoilers of grains in storage, but in our study they seemed to be spreading and spoilage (Stack, 2000). Even when the best practices and
of minimal significance since their levels were limited. controls are employed, some mold contaminants will remain on the
Several Penicillium species were isolated during our study from var- harvested kernels and could grow under the right conditions. Since
ious commodities tested; P. verruculosum, P. purpurogenum, P. aurantiog- mold growth and subsequent mycotoxin production are a function of
riseum (P. cyclopium), P. oxalicum, and P. polonicum were recovered from
moisture and temperature, controlling these two parameters, at levels
corn products while P. polonicum was found in whole wheat flour and
unfavorable for fungal germination and proliferation, would result in
P. brevissimum in Basmati rice. Among the isolated penicillia, only
maintaining high quality product. Quick drying of grains and grain prod-
P. verruculosum and P. purpurogenum were found at relatively high lev-
ucts to a moisture level below 12% (Aw < 0.60) and storage under cool
els. P. verruculosum is of certain significance since some members of
and dry conditions with sufficient aeration are essential for the produc-
the species have the ability to produce the potent tremorgenic myco-
tion and preservation of high quality grains, safe for human and animal
toxin verruculogen (VERR). VERR has been associated with tremor and
consumption.
blockage of the Ca21-activated K1 in animals ingesting products conta-
minated with the toxin. P. purpurogenum is a rubratoxin (RT) producer.
RT is known to cause hepatotoxicity in animals (Lockard, Watson, Siraj, 5 | CONCLUSIONS
Hayes, & O’Neal, 1981). Therefore, the presence of these organisms in
grains at elevated levels is troublesome and should be avoided. Past All types of grains and grain-derived commodities tested during the
studies have also shown the frequent incidence of Penicillium species in course of this study contained potentially toxigenic molds. Some corn
grains and grain products (Mislivec, 1981; Mislivec et al., 1979). These meal and popcorn samples fostered high levels (over 4.0 log10 cfu/g) of
researchers reported the presence of P. cyclopium, P. viridicatum, P. bre- live fusaria and A. flavus, while a high percentage of wheat flour sam-
vicompactum, and P. oxalicum in stored corn and P. urticae, P. cyclopium, ples contained low levels of Aspergillus and Fusarium species. Some of
P. viridicatum, P. islandicum, and P. citrinum in small grains and/or wheat the rice samples were also contaminated with low levels of Aspergillus,
flour. The Penicillium profiles reported by Mislivec and his co-workers Penicillium, and/or Fusarium spp. The fungal levels recovered during this
differ from ours perhaps due to different microbial control procedures study may not reflect the actual MYC contaminating these commod-
and other processes employed at that time and due to possibly ities before refining, milling, and other postharvest treatments since
different product origination. these types of processing may be detrimental to some fungi. Mycotox-
Specifications for fungal contamination of cereal grains and grain- ins, which are not likely to be removed or inactivated by the above
derived products acceptable by different manufacturers vary widely. processes, could still pose a health hazard. Therefore, testing for such
Berghofer, Hocking, Miskelly, and Jansson (2003), after analyzing over metabolites is advisable.
600 samples from various wheat milling companies, concluded that
acceptable quality limits for mold and yeast in wheat flour would be ACK NOWLE DGME NT S
103 cfu/g. The International Commission on Microbiological Specifica-
We are indebted to Jeffrey Kohn (Office of Regulatory Affairs/FDA)
tions for Foods (ICMSF) has set fungal tolerance for flour and cereal
for his assistance during the preparation of the manuscript.
products at 102–104 cfu/g (ICMSF, 1986). The fungal levels recovered
from some products during our study surpassed those recommended
by ICMSF. R EFE R ENC E S
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