Detoxification of the Mycotoxin Citrinin by a Manganese Peroxidase from Moniliophthora roreri

Abstract

Citrinin (CIT) is a mycotoxin found in foods and feeds and most commonly discovered in red yeast rice, a food additive made from ordinary rice by fermentation with Monascus. Currently, no enzyme is known to be able to degrade CIT effectively. In this study, it was discovered that manganese peroxidase (MrMnP) from Moniliophthora roreri could degrade CIT. The degradation appeared to be fulfilled by a combination of direct and indirect actions of the MrMnP with the CIT. Pure CIT, at a final concentration of 10 mg/L, was completely degraded by MrMnP within 72 h. One degradation product was identified to be dihydrocitrinone. The toxicity of the CIT-degradation product decreased, as monitored by the increased survival rate of the Caco-2 cells incubated with MrMnP-treated CIT. In addition, MrMnP could degrade CIT (with a starting concentration of up to 4.6 mg/L) completely contaminated in red yeast rice. MrMnP serves as an excellent candidate enzyme for CIT detoxification.

Keywords: citrinin; detoxification; manganese peroxidase; mycotoxin; red yeast rice.

Figure 1

Effects of the buffer components on CIT degradation. (a) Degradation of CIT by MrMnP in presence of various buffers. (b) Degradation of CIT when no MrMnP was added. The reactions were carried out by incubating 10 mg/L of CIT and 500 U/L with or without MrMnP in one of the buffers containing malonate, acetate, oxalate, citrate, lactate, phosphate, MES, and HEPES at 30 °C for 72 h. The data shown is the average ± standard deviation.

Figure 2

Mn²⁺ played an important but not exclusive role in degrading CIT. The reactions were carried out by incubating 500 U/L of MrMnP with 10 mg/L of CIT in the acetate or malonate buffer either in presence or in absence of Mn²⁺ at 30 °C for 72 h. Then, the reaction products were analyzed by HPLC. The data demonstrated in the figure were prepared by integrating the HPLC traces from analyses of multiple reactions. The bar on the left represents a signal of 100 mV.

Figure 3

MrMnP treatment of CIT led to detoxification as determined by using Caco-2 cell as a model. The cells were sub-cultured in 96-well plates with an inoculation density of 1 × 10⁵ cells per well. Before mycotoxin treatment, the cells were left for adhesion to the wall for 24 h. CIT was added to cells at a concentration of 10 mg/L. Degradation of CIT was achieved by treating with MrMnP for 72 h. Then, the culture was continued for 48 h. Each value in the panel represents the mean ± SD (n = 6).

Figure 4

Identification of dihydrocitrinone as one degradation product of CIT. CIT was incubated with 500 U/L of MrMnP in 50 mM malonate buffer (pH 5.0) supplemented with 1 mM MnSO₄ and 0.1 mM H₂O_2. The reaction was carried out at 30 °C for 72 h. The degradation products were analyzed by UHPLC–MS/MS and the secondary mass spectrum of the identified degradation product dihydrocitrinone was shown. In [M+H]⁻, the M was referring to dihydrocitrinone. Its two daughter ions were with m/z values of 223.1 and 179.2, respectively, reflecting continuous loss of the carbon dioxide from dihydrocitrinone.

Figure 5

MrMnP could degrade CIT in presence of red yeast rice. Two kinds of commercially available red yeast rice containing different concentrations of CIT (4.6 and 2.1 mg/L of CIT in 12.5 and 6.25 g/L of Brand A, respectively; and 2.4 and 1.2 mg/L of CIT in 12.5 and 6.25 g/L of Brand B, respectively) were used. MrMnP (500 U/L) was incubated with each of the two kinds of red yeast rice and the samples taken out at 72 h were analyzed by HPLC. The data demonstrated in the figure were prepared by integrating the HPLC traces from analyses of multiple reactions. The bar on the left represents a signal of 100 mV.