Safety of Fungal Fermented Foods

There are two main reasons why the safety of Asian, fungal-fermented foods has been questioned. First, the Aspergillus sp. used in the production of soy sauce, miso, sake, and related products are taxonomically similar to the my-cotoxigenic aspergilli that produce aflatoxins, ochratoxin, and other toxins. Despite these similarities, however, surveys in which these foods have been analyzed for the presence of myco-toxins indicate that mycotoxins are not present. Recent studies have shown that very subtle genetic differences exist between industrial strains and mycotoxin-producing strains that would account for these findings (Box 12-5).

A second reason for the concern is due to the suggested epidemiological relationship between consumption of indigenous foods from the Far East and the development of certain

Box 12—5. Mycotoxins in Fungal Fermented Products: Cause for Concern?

In nature, growth of fungi is often accompanied by production of mycotoxins, produced as secondary metabolites. Some of these mycotoxins—and aflatoxins, in particular—are extremely carcinogenic and mutagenic. In fact, aflatoxins are considered to be among the most toxic of all naturally occurring compounds found on the planet.

There are as many as sixteen different types of aflatoxins, of which B1,B2,G1, and G2 are most commonly found in human and animal foods.They are produced mainly by Aspergillus flavus and Aspergillus parasiticus, members of the Aspergillus section Flavi (or the A. flavus group). This taxonomic group also includes Aspergillus sojae and Aspergillus oryzae, species that are used in the manufacture of koji, soy sauce, miso, sake, and other Oriental fermented products.

Given the morphological and genetic similarities between these four different species, questions have been raised regarding the potential ability of A. sojae and A. oryzae to produce my-cotoxins in foods in which these fungi are used. Indeed, several recent studies have revealed that some of the genes (or homologs) that encode for proteins involved in the aflatoxin biosynthesis pathway may be present in these food production strains (see below). Despite the apparent metabolic potential of A. sojae and A. oryzae to produce aflatoxins, however, there are no reports of aflatoxins being produced by these fungi or for the presence of aflatoxins in fermented soy products (Watson et al., 1999).These observations have led investigators in Japan, the United Kingdom, and the United States to explore, in detail, why A. sojae and A. oryzae do not synthesize aflatoxins.

The biochemical pathway for aflatoxin biosynthesis consists of at least twenty-three enzymatic reactions and more than fifteen intermediates (Yu et al, 2004A and 2004B). In 2004, the complete gene cluster encoding for aflatoxin biosynthesis in A. flavus and A. parasiticus was identified and sequenced (Yu et al., 2004A; Figures 1 and 2).There are twenty-five genes within this 70 Kb cluster (four additional sugar utilization genes are located immediately downstream).

Because enzymatic activities associated with the aflatoxin pathway are generally not detectable in A. sojae, it would appear that the relevant genes must also be absent. However, according to one

Box 12—5. Mycotoxins in Fungal Fermented Products: Cause for Concern? (Continued)

report (Matsushima et al., 2001B), three aflatoxin genes (aflR, nor1, andpksA~) were present in a commercial soy sauce strain (strain 477), based on Southern hybridization analysis.Another study showed that omtA, nor1, and ver1 were also present in A. sojae and A. oryzae and that these genes had high sequence similarity to those from aflatoxin-producing strains of A. parasiticus (Watson et al., 1999). However, none of these genes were transcribed, even when cells were grown in alfatoxin-conducive media.Two other genes, avfA and omtB, were also reported to be present in A. sojae and were 99% identical to those found in A.parasiticus (Yu et al., 2000). Furthermore, complementation assays showed that the A. sojae avfA gene was fully functional.Thus, if aflatoxin genes are present in A. sojae and A. oryzae, and they appear to be functional, why don't these fungi produce aflatoxin in foods?

The answer to this question can now be revealed.The aflatoxin pathway in A. parasiticus is regulated mainly by the aflR gene product AflR.An adjacent gene, aflS, appears to modulate aflR expression, but its exact role is not known.AflR is a 47 kDa protein that acts as a positive regulator by binding to the promoter region of the afl gene cluster, activating transcription of the structural genes. Mutant strains of A.flavus, defective in aflR, do not express alfatoxin genes, indicating that AflR is required for aflatoxin biosynthesis.Although a homolog of the aflR gene is present in A. sojae and A. oryzae, afl genes are still not transcribed in these strains.The non-aflatoxin-producing phenotype is not due to the absence of a promoter-binding region, since that region has been shown to be functional (Takahashi et al., 2002). Rather, it is AflR that is nonfunctional, not only in the native A. sojae and A. oryzae backgrounds, but also in an A. parasiti-cus aflR deletion mutant transformed with aflR from A. sojae (Takahashi et al., 2002). Also, whereas introduction of an additional copy of the A. parasiticus aflR gene into A. parasiticus increased aflatoxin production, no such effect was observed when the aflR from A. sojae was introduced into A.parasiticus (Matsushima et al., 2001A).

Sequence analysis has shown that there are two mutations located within the A. sojae aflA gene (Watson et al., 1999).One mutation consists of a duplication of histidine (H) and alanine (A) residues at amino acid positions 111 to 114, resulting in a HAHA motif (no joke).The second mutation consists of a C- T transition, and subsequent conversion of an arginine codon at position 385 to a premature stop codon.This results in a truncated protein that is short sixty-two amino acid residues at the carboxy end, which is where the transcription-activating domain is located. The HAHA mutation, it turns out, however, does not appear to affect AflR activity or synthesis of aflatoxin, because cells harboring a hybrid gene containing the intact carboxy-terminal region— but with the HAHA motif—have a normal aflatoxin phenotype (Matsushima et al., 2001A).Thus, the defect in aflR must be due to the missing sixty-two residue region.The truncated AflR is then unable to activate transcription of alfgenes, resulting in the lack of aflatoxin biosynthesis.

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