Saturday, June 15, 2013

Aspergillus oryzae

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Aspergillus oryzae is one of the ascomycetous fungus that asexual species. Its used in the production of soy sauce, miso and sake without recorded incidents for hundreds of years. There are conflicting opinions about whether A. oryzae can be isolated in nature. Although the details of the genetic relationship between A. oryzae and A. flavus remain unclear, the two species are so closely related that all strains of A. oryzae are regarded by some as natural variants of A. flavus modified through years of selection for fermenting of foods. A. oryzae is regarded as not being pathogenic for plants or animals, though there are a handful of reports of isolation of A. oryzae from patients. There are also several reports of products of A. oryzae fermentations, e.g. a-amylase, that seem to be associated with allergic responses in certain occupations with high exposure to those materials. A. oryzae can produce a variety of mycotoxins when fermentation is extended beyond the usual time needed for production of these foods. While wild A. flavus isolates readily produce aflatoxins and other mycotoxins, A. oryzae has not been shown to be capable of aflatoxin production.

Aspergillus oryzae has apparently been an essential part of oriental food production for centuries and is now used in the production of many different oriental foods such as soy sauce, sake and miso. Potential uses under TSCA include fermentations of numerous enzymes, e.g., amylase, protease, B-galactosidase, lipase, and cellulase, and organic compounds such as glutamic acid. While these products have a variety of potential commercial uses, some of them are mostly frequently used in food processing.

The experience of safe commercial use of A. oryzae is extraordinarily well established. As a "koji" mold it has been used safely in the food industry for several hundred years. A. oryzae is also used to produce livestock probiotic feed supplements. Even the commercialization of byproducts of the fermentation was established nearly a century ago. The "koji" mold enzymes were among the first to be isolated and commercialized. In 1894, Dr. J. Takamine isolated and soldTakadiastase from a commercial firm he started in Clifton, New Jersey (Bennett, 1985a).

EPA has reviewed, under TSCA, two genetically modified strains of A. oryzae used for the production of enzymes (Premanufacture Notice (PMN) numbers P89-134 and P94-1475). The candidate species is a member of the genus Aspergillus and belongs to the group of fungi that are generally considered to reproduce asexually (Fungi Imperfecti or Deuteromycetes), although perfect forms (forms that reproduce sexually) of some aspergilli have been found. The form genus Aspergillus represents a taxonomic grouping of a very large number of asexual fungi which are characterized by the production of spores on large black or brown conidia in phialides arranged on a characteristic spherical conidiophore termed the vesicle. This definition leads to inclusion of a complex assortment of organisms within the taxon. To simplify the taxonomy of such a large number of organisms, the genus Aspergillus has been divided into sections or groups based on color, size and roughness of the spore, conidiophore and vesicle as well as the arrangement of phialides and the presence of sclerotia. The separation of individual species into groups is somewhat tenuous and based on distinguishing measured characters with overlapping means. This resulted in the 132 species arranged in 18 groups by Raper and Fennell (1965) due to overlapping morphological or physiological characteristics. However, it is important to remember that taxonomy is "dealing with living variable organisms and that species and group concepts must be reasonably elastic" (Raper & Fennell, 1965).

As is the case of many fungi, the taxonomy of Aspergillus is primarily based on morphological features, rather than physiological, biochemical features and genetic characteristics often used to classify bacteria. Nomenclature problems of the genus Aspergillus arise from their pleomorphic life cycle. The newer findings show that this group of fungi has both a perfect (teleomorphic) and an imperfect (anamorphic) state.

The morphological approach to taxonomy has led to the existence of several synonyms for the genus Aspergillus. They are: Alliospora Pim; Aspergillonsis Spegazzini; Cladaspergillus Ritg; Cladosparum Yuill and Yuill; Euaspergilus Ludwig; Gutturomyces Rivolta; Raperia Subramaniam and Grove; Sceptromyces Corda; Spermatoloncha Spegazzini; Sphaeromyces Montagne; Sterigmatocystis Cramer; and Stilbothamnium Hennings (Bennett, 1985b). Aspergilli are ubiquitous in nature. They are geographically widely distributed and have been observed in a broad range of habitats, because they can colonize a wide variety of substrates.

A. oryzae can produce b-nitropropionic acid, along with other food-borne molds (Gilbert et al., 1977). Its mode of action is apparently irreversible succinate dehydrogenase inhibition which can cause a variety of symptoms often neurological in nature. These symptoms have been studied in mice (Gould and Gustine, 1982; Umezawa, 1967) and rats (Hamilton and Gould, 1987) where intravenous or subcutaneous LD50s of 20-50 mg/kg were determined. Reports of livestock poisoning via ingestion in feed (James et al., 1980; James, 1983) showed that ingestion of b-nitropropionic acid could produce significant toxic effects up to and including death. When A. oryzae (ATCC 12892) was studied for its ability to produce b-nitropropionic acid on various high protein and carbohydrate-rich foods, it flourished and produced this toxin in cooked sweet potato, potato and ripe banana (Penel and Kosikowski, 1990). Ames type assays for mutagenicity (Dunkel, 1985) showed positive responses with and without activation for two Salmonella strains, but not for three others. This assay uses multiple indicator strains in order to ensure that each potential mutation mode is detectable; the failure in three strains merely implies that the mutation modes to which each is sensitive are not the ones associated with the test substance.

The closest taxon to A. oryzae is A. flavus which Kurtzman et al. (1986) regard as conspecific. Many strains of A. flavus produce aflatoxins which are acutely toxic to mammals (oral LD50s ranging from 1 to 15 mg/kg depending on test species (Ceigler, 1975). Aflatoxins are animal carcinogens (Barnes and Butler, 1964; Dickens and Jones, 1964; Sinhuber, 1968) and also probablehuman carcinogens (Council for Agricultural Science and Technology, 1989). Developmental effects have also been found (Elis and DiPaolo, 1967, Le Breton et al., 1964).

Many studies affirm that the currently available strains confirmed to be A. oryzae are not capable of producing aflatoxins (Wei and Jong, 1986; Yokotsuka and Sasaki, 1986). In one test, no strains of A. oryzae or A. sojae (another koji mold) produced detectable levels of aflatoxins, while 33% and 85% of the strains of A. flavus and A. parasiticus, respectively, were toxigenic. As mentioned above, Kurtzman, et al. (1986) regard A. oryzae and A. sojae as domesticated varieties of their respective subspecies. Only one study (El-Hag and Morse, 1976) describes aflatoxin production by a strain reported to be Aspergillus oryzae (NRRL strain 1988). This observation is notable as an exception to the rule of no aflatoxin production by A. oryzae.

Aspergillus oryzae does not appear to be a human pathogen. Available information documents infections in humans possibly caused by A. oryzae in only three instances. The first was a case of meningitis (Gordon, et al., 1976). In the second case, A. oryzae invaded the paranasal sinuses, causing fever and right periorbital swelling (Byard, et al., 1986). The third case was apulmonary aspergilloma caused by A. oryzae (Liao, 1988). Care must be exercised in evaluating these three cases as having been caused by this organism due to its close taxonomical relationship to A. flavus and the possibility of incorrect identification. The relative rarity of such cases in light of the commonplace use of A. oryzae suggests this species has a low potential for expressing pathogenic traits.

Allergic reactions are not uncommon for aspergilli in general. There is one reported case of an allergic bronchopulmonary aspergillosis due to A. oryzae in a 19-year old female (Akiyama et al., 1987). However, the a-amylase produced by A. oryzae, that is used by bakers in bread making, was reported by Birnbaum, et al. (1988) to have caused asthma in a baker. Based on an observation of a case of baker's asthma due to monovalent sensitization to a-amylase used as an additive to flour, investigators tested 31 bakers who had occupational asthma and/or rhinitis by skin tests and serologic RAST examinations. Thirty-two percent of the bakers had RAST specific IgE to a-amylase from A. oryzae. Baker's asthma is reported to be the most frequent occupational lung disease in Switzerland and West Germany (Wuthrich and Baur, 1990). However, allergic reactions in bread bakers are quite common, both to the flours of various grains, as well as to the flour additives such as fungal amylases. Allergic reactions in bakers are not specific to A. oryzae, nor the enzymes produced by A. oryzae (O'Neil and Lehrer, 1991). In addition, the exposure scenario of a bread baker to flour and the additives contained therein is quite different from that of workers in a fermentation facility using general worker hygiene and protection practices.

Hypothetically, then, if A. oryzae has evolved to non-aflatoxigenic status after centuries in culture, the question remains whether it can revert to the "wild" type. The experience of oriental food production would seem to suggest not, or at least not frequently enough as to be detectable. Recent studies (Payne, 1994; Klich, 1994) suggest homology between parts of the A. oryzae genome and structural genes for aflatoxin production. It is conceivable that reintroduction of regulatory genes or their gene products could activate a dormant aflatoxin synthetic potential. There is no evidence to show that the required gene transfer or gene rearrangement that might provide the needed functional sequences for an aflatoxin producing A. oryzae strain occurs naturally. The question is, therefore, whether this type of genetic modification is possible in culture. Gene transfer from a toxigenic strain during fermentation is highly unlikely due to the need for maintaining axenic conditions during fermentation. The theoretical possibility of genetic rearrangement occurring in culture resulting in reversion back to the "wild-type" seems unlikely. Anecdotal evidence gathered over centuries suggests that A. oryzae commercial food strains do not produce aflatoxins, nor have there been reports of any adverse human health effects from aflatoxin.

Estimates of the number of A. oryzae organisms released during production are tabulated in Table 1 (Reilly, 1991). The uncontrolled/untreated scenario assumes no control features for the fermentor offgases, and no inactivation of the fermentation broth for the liquid and solid waste releases. The containment criteria required for the full exemption scenario assume the use of features or equipment that minimize the number of viable cells in the fermentor off-gases. They also assume inactivation procedures resulting in a validated 6log reduction of the number of viable microorganisms in the liquid and solid wastes relative to the maximum cell density of the fermentation broth.

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