Inhibition of Aflatoxin Production by Citrinin and Non-Enzymatic Formation of a Novel Citrinin-Kojic Acid Adduct

Abstract

Screening for microorganisms that inhibit aflatoxin production from environments showed that Penicillium citrinum inhibited aflatoxin production by Aspergillus parasiticus. The inhibitory substance in the culture medium of P. citrinum was confirmed to be citrinin (CTN). RT-PCR analyses showed that CTN did not inhibit expressions of aflatoxin biosynthetic genes (aflR, pksL1, and fas-1) of A. parasiticus, whereas feeding experiments using A. parasiticus showed that CTN inhibited the in vivo conversion of dihydrosterigmatocystin to AFB2·AFG2. These results suggest that CTN inhibits a certain post-transcriptional step in aflatoxin biosynthesis. CTN in the culture medium of A. parasiticus was found to be decreased or lost with time, suggesting that a certain metabolite produced by A. parasiticus is the cause of the CTN decrease; we then purified, characterized, and then analyzed the substance. Physico-chemical analyses confirmed that the metabolite causing a decrease in CTN fluorescence was kojic acid (KA) and the resulting product was identified as a novel substance: (1R,3S,4R)-3,4-dihydro-6,8-dihydroxy-1-(3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)-3,4,5-trimethyl-1H-isochromene-7-carboxylic acid, which was named “CTN-KA adduct”. Our examination of the metabolites’ toxicities revealed that unlike CTN, the CTN-KA adduct did not inhibit aflatoxin production by A. parasiticus. These results indicate that CTN’s toxicity was alleviated with KA by converting CTN to the CTN-KA adduct.

Detoxication of Citrinin with Kojic Acid by the Formation of the Citrinin-Kojic Acid Adduct, and the Enhancement of Kojic Acid Production by Citrinin via Oxidative Stress in Aspergillus parasiticus

“…After SYS-4 and the resulting mutants were, respectively cultured in the medium without or with 0.1 mg mL −1 CTN for 4 days, metabolites in each culture medium were analyzed by TLC ( Figure 3 ). The AF productivity of SYS-4 had already recovered after 4-day incubation, whereas the CTN first added completely disappeared ( Figure 3 a, upper panel); this is the same result obtained in our earlier study [ 12 ]. The KA productivity of SYS-4 was remarkably enhanced by CTN, suggesting that the production of KA was induced by CTN ( Figure 3 a, lower panel).…”

“…In the cases of supplementation, the regulation value of CTN is relatively high (European Food Safety Authority [EFSA] value: 2000 μg/kg). Although our previous work showed that 1–1.3% of the CTN-KA adduct was decomposed to CTN after incubation at either 37 °C or 50 °C for 48 h. in aqueous condition [ 12 ], the resulting concentration seems to be very low. The consideration of the effects of KA on the health of animals and humans thus does not seem necessary.…”

“…The formation of the CTN-KA adduct does not require high (>100 °C) temperatures. Incubation at 28 °C, 37 °C, or 50 °C was sufficient for the conversion of CTN to the CTN-KA adduct, and the CTN-KA adduct was a sole product of CTN and KA [ 12 ]. This method also does not require any special equipment, unlike the solvent extraction method.…”

“…Aspergillus and Penicillium species are common in moderate-temperature regions, and fungi belonging to both of these genera are often detected in the same environments (e.g., soils and plants) [ 12 ]. We observed that AFs (Yabe, personal data) and KA ( Figure 8 ) did not affect the growth of P. citrinum , whereas the addition of KA slightly changed the color of the conidiospores of P. citrinum , suggesting that P. citrinum might also be affected by Aspergillus species via the formation of KA.…”

“…Regarding the inhibition of AF contamination with microorganisms, in our recent work we observed that Penicillium citrinum strains isolated from the environment inhibited the production of AF by A. parasiticus and the fungal growth of this strain; moreover, the inhibitory substance produced by P. citrinum was confirmed to be CTN [ 12 ]. We also observed that CTN in the culture medium of A. parasiticus was converted to a novel compound that we named ‘CTN-KA adduct’: (1 R ,3 S ,4 R )-3,4-dihydro-6,8-dihydroxy-1- (3-hydroxy-6-(hydroxymethyl)-4-oxo-4 H -pyran-2-yl)-3,4,5-trimethyl-1 H -isochromene-7-carboxylic acid ( Figure 1 ).…”