Degradation of Aflatoxin B1 by a Sustainable Enzymatic Extract from Spent Mushroom Substrate of Pleurotus eryngii

Ligninolytic enzymes from white-rot fungi, such as laccase (Lac) and Mn-peroxidase (MnP), are able to degrade aflatoxin B1 (AFB1), the most harmful among the known mycotoxins. The high cost of purification of these enzymes has limited their implementation into practical technologies. Every year, tons of spent mushroom substrate (SMS) are produced as a by-product of edible mushroom cultivation, such as Pleurotus spp., and disposed at a cost for farmers. SMS may still bea source of ligninolytic enzymes useful for AFB1 degradation. The in vitro AFB1-degradative activity of an SMS crude extract (SMSE) was investigated. Results show that: (1) in SMSE, high Lac activity (4 U g−1 dry matter) and low MnP activity (0.4 U g−1 dry matter) were present; (2) after 1 d of incubation at 25 °C, the SMSE was able to degrade more than 50% of AFB1, whereas after 3 and 7 d of incubation, the percentage of degradation reached the values of 75% and 90%, respectively; (3) with increasing pH values, the degradation percentage increased, reaching 90% after 3 d at pH 8. Based on these results, SMS proved to be a suitable source of AFB1 degrading enzymes and the use of SMSE to detoxify AFB1 contaminated commodities appears conceivable.


Introduction
Aflatoxins (AFs) are mycotoxins produced mainly by fungi of the genus Aspergillus. Aflatoxin B 1 (AFB1) is the most harmful among the Afs and exhibits extremely high hepatotoxic, mutagenic, and carcinogenic effects on humans and animals [1]. AFB1 has been classified by the International Agency for Research on Cancer [2] as a Group 1 substance, that is of proven carcinogenicity to humans. Many agricultural commodities such as groundnuts (peanuts), corn, sorghum, rice, spices, nuts, and several other cereals are subjected to infestation by aflatoxigenic molds and thereby contamination with AFB1 [3,4]. Human exposure to AFB1 can result directly from the ingestion of contaminated food or indirectly from the consumption of products from animals fed with contaminated feed [5]. According to the Rapid Alert System for Food and Feed (EU RASFF) reports [6], in the last ten years,

SMSE Enzymatic Activities
In SMSE, the activity of two important ligninolytic enzymes, Lac and MnP, was determined by spectrophotometric assays. Among ten different SMS production batches, some variability in the enzymatic activity was observed. High Lac activity (4 U ± 1 g −1 of dry matter, DM) and low MnP activity (0.4 U ± 0.09 g −1 DM) were found in SMSE. The total protein content in SMSE was about 600 mg g −1 DM.

Degradation of AFB1 by SMSE
AFB1 remained stable for 7 days when incubated in the extraction buffer and no significant variation in concentration (p < 0.05) was observed during this period of time (data not shown). The SMSE supplemented to AFB1 was able to determine the degradation of the mycotoxin in solution, as shown in Figure 1 by the reduction of the AFB1 UPLC/FLD (Ultra High Performance Liquid Chromatography equipped with a Fluorescence Detector) peak area of the SMSE treatment at the retention time of 3.7 min, corresponding to the AFB1 standard. The chromatographic peak at the retention time of 3 min in the positive control ( Figure 1, black line) was aflatoxin B 2 (AFB2), which was present as an impurity and was completely degraded by the treatment with SMSE ( Figure 1, red line). No other peak is highlighted in the obtained chromatograms. No significant variation in the proportion of AFB1 degradation by SMSE was observed at increasing concentration of AFB1, in the range 154 ± 3-885 ± 22 ng mL −1 (Table 1); therefore, subsequent experiments were performed using the single dose of 885 ± 22 ng mL −1 of AFB1.  The time course of AFB1 degradation by SMSE is shown in Figure 2. More than 50% AFB1 degradation occurred within 1 day of incubation, whereas after 3 days of incubation, the degradation percentage raised to 80% and did not change significantly from there on, till the 7th day of incubation. Based on these results, the maximum incubation time for the following experiments was 3 days. Data are expressed as mean ± SD (n = 3) of the percent AFB1 degradation with respect the control (AFB1 without SMSE). Statistically significant differences with the control by ANOVA are indicated by asterisks: * for p < 0.05 and *** for p < 0.001. A statistically significant difference with the previous value by Tukey-Kramer Multiple Comparison Test is indicated by daggers: † for p < 0.05 and † † † for p < 0.001.
The effect of temperature and pH on SMSE ability to degrade AFB1 is shown in Figure 3A,B, respectively. The results show that at temperatures between 25 and 37 • C, about half of AFB1 degradation occurred after 1 day of incubation, while the maximum degradation (80%) was reached after 3 days. When the temperature was lowered to 15 • C, the degradation was inhibited, reaching a maximum value of 25% after 3 days, and no degradation was observed after 1 day. The pH greatly influenced the degradative capability of SMSE. In particular, AFB1 degradation increased with the rise of pH from pH 4.5 to pH 8, exceeding at pH 8 the percentage of 70% and 90%, respectively after 1 and 3 days of incubation ( Figure 3). The degradative ability of SMSE was positively correlated with Lac activity ( Figure 4); however, it was not possible to evaluate its correlation with MnP, due to the very low enzyme activity. The correlation analysis of data showed a strong relationship between Lac activity and AFB1 degradation, with R Squared values higher than 0.90 both at 1and 3 days of incubation ( Figure 4). In particular, it was observed that in the presence of 5 U mL −1 of Lac, the percentages of AFB1 degradation were 50% and 70% after 1 and 3 days, respectively. Conversely, SMSE containing 2.5 and 1.25 U mL −1 of Lac degraded AFB1, respectively, by 50% and less than 40% after 3 days of incubation.

Storability of SMS
Considerable variation in DM of different batches of newly produced SMS was found, with average values of about 50% and minus or plus variations of about 10%, depending on the place, period, and method of production. SMS stored at room temperature showed a progressive loss of water with a consequent increase of DM by about 25% in 15 days of storage. Subsequently, no significant variations of DM% were observed from there on, even after 30 days of storage ( Figure 5). The loss of Lac activity of SMS stored at room temperature showed the same trend of the water loss, with a reduction of Lac activity by 30% (from 2.65 to 1.7 U g −1 DM) within 15 days and no further loss up to 30 days of storage.  SMS stored in refrigeration at +4 • C did not show significant loss of Lac activity over 6 months of storage (data not shown).

Storability of SMSE
The effect of storage temperature on Lac activity of SMSE over a 70-day long period is shown in Figure 6. When stored at +30 • C, the Lac activity of SMSE decreased by 49% after 30 days and further decreased by 90% after 70 days of storage. At +25 • C, the loss of activity was less severe, but still high, with a decline of 34% and 47% of initial activity after 30 and 70 days, respectively. Furthermore, both at +25 • C and +30 • C storage temperatures, the development of molds in the extract was observed. On the contrary, storage under refrigerated (+4 • C) or freezing (−20 • C) conditions preserved 68% or 62%, respectively, of Lac activity over 70 days of storage. After lyophilization, Lac activity of SMSE decreased by 57%.

Discussion
As previously reported, Pleurotus spp. are efficient producers of ligninolytic enzymes, mainly laccase (Lac) and manganese peroxidase (MnP), whose application for aflatoxin degradation has been proposed [23,24,40]. The species P. eryngii (king oyster mushroom) has been shown to produce and release AFB1-degrading enzymes in the substrate used for mushroom cultivation [19,35]. The spent mushroom substrate (SMS) that remains as a waste after harvest, at the end of production cycle of the king oyster mushroom, is therefore a potential low-cost source of enzymes for biodegradation of aflatoxin and bioremediation of AFB1-contaminated commodities. With the aim to develop a new "green" method for the detoxification of aflatoxin-contaminated feed, we investigated the AFB1-degrading activity of an extract of SMS (SMSE) obtained from king oyster mushroom cultivation.
In SMS of P. eryngii obtained from a mushroom farm, we found high levels of Lac activity and low levels of MnP activity, consistent with the findings of Li et al. [35]. The correlation that we found between Lac activity and AFB1 degradation by SMSE suggests that Lac is a major factor in AFB1 degradation, even if the presence of other catalysts or catalysis mediators is conceivable; in fact, Loi et al. [25] reported that a pure Lac from P. pulmonarius oxidized AFB1 with low efficiency, whereas it was able to degrade up to 73% of AFB1 when combined with mediator systems. Consistently, Li et al. [35] found that biodegradation of polycyclic aromatic hydrocarbons was higher with the use of crude extracts from P. eryngii than that with pure Lac.
The AFB1-degrading capability of P. eryngii SMSE, which in the optimal conditions of pH 8 and temperatures between 25 and 37 • C reached 70% and 90% after 1 and 3 day of incubation, appears noteworthy, when compared to the performances reported for other fungal or plant extracts or for purified laccases from other sources. Filtered cultures of T. versicolor containing 1.8 U mL −1 of Lac were able to degrade approx. 30% and 50% of AFB1 in solution at 30 • C, at the same incubation times of 24 and 72 h used in the present study [41]. An aqueous extract from the aromatic seed spice ajowan (Trachyspermum ammi) was reported to inactivate approximately 50% and 75% at 26 • C, and 75% and 90% at 37 • C of a mixture of aflatoxins B 1 , B 2 , G 1 , and G 2 in 24 and 72 h, respectively, by an unidentified mechanism [42]. Alberts et al. [24] obtained 87% degradation of AFB1 in 72 h at 30 • C by incubating with 1 U mL −1 of a pure Lac from T. versicolor. A pure laccase enzyme (Lac2) from P. pulmonarius was tested by Loi et al. [40] for AFB1 degradation. However, the degradation achieved by incubating for 72 h at 30 • C with 2.5 U of Lac2 without redox mediators (namely 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), acetosyringone, and syringaldehyde) was low, accounting for just 23% reduction of AFB1 content. With respect to the above-proposed applications, the technology that we experimented appears advantageous under various aspects. It does not require the dedicated cultivation of ligninolytic fungi in culture media to obtain active filtrates or extracts but, on the contrary, it relies on the recycling of disposable agricultural waste that, in an environment-friendly fashion, is thus transformed into a valuable by-product of mushroom cultivation. The production of SMSE is also a low-cost process and more economically sustainable than the use of purified enzymes, especially for developing countries and in marginal economies, where consumption of AFB1-contaminated foods and feeds has the highest social impact. Moreover, the possibility to store for long periods SMS (one month at room temperature) and SMSE (two months in refrigerated conditions) makes this waste and its raw extract particularly suitable for technological applications.
While the identification of the by-products and their toxicological characterization are gaps in the knowledge of chemical and microbial degradation of AFB1 that need to be bridged to implement these technologies into practical control measures, in the large majority of studies in this field, the degradation products still have to be identified. Branà et al. [19] analyzed the SMS of a laboratory-scale P. eryngii cultivation where 86% of the AFB1 had been degraded and did not find detectable levels of aflatoxicol, the main toxic metabolite of AFB1. An approach based on the cytotoxicity or mutagenicity testing of enzyme-treated materials has been used to claim the cleavage of AFB1 into non-toxic, or at least less toxic, fragments by laccases and other enzymes from various fungal or bacterial sources [17,24,41,43,44]. Alberts et al. [24] reported no significant differences in the mutagenic response of AFB1-containing samples treated with 1 U mL −1 of a pure Lac from T. versicolor with respect to negative control samples, in the Salmonella typhimurium mutagenicity assay. The treatment of 150 ng of AFB1 with a culture filtrate having laccase activity of 3.5 U mL −1 for 72 h at 25 • C also resulted in the complete loss of cytotoxicity on two human lymphoma cancer cell lines [41].
Several reports on the detoxification of feed-or foodstuff by treatments with water solutions of detoxifying agents can be found in the literature. Aqueous treatments with citric acid were used to reduce AFB1 in contaminated maize [45] as well as in extruded sorghum [46]. Scarpari et al. [41] sprayed AFB1-contaminated maize with culture filtrates of T. versicolor containing Lac and obtained 50% degradation with 3.5 U and 70% degradation with 7.0 U of the enzyme in 48 h. Recently, a similar approach has been used for the enzymatic detoxifying of maize from fumonisin mycotoxins [47] and the technology has been proposed to be utilized as a safer alternative to water washing that is practiced in subsistence farming communities in Africa for the treatment of whole maize intended for human consumption. A major advantage of the proposed technology was that the bulk solution of the residual enzyme and the less toxic degradation products could be easily separated from the treated maize kernels. A similar application is conceivable also for the degradation of AFB1 with SMSE, that qualifies as a simple, eco-friendly, low-cost, and efficient technology for remediation mainly of contaminated feedstuff, particularly in marginal areas. As previously noted by van der Westhuizen et al. [48]: "In many Sub-Saharan countries, where both maize contamination and maize consumption are high, regulatory mechanisms to control mycotoxin levels ( . . . ) are either lacking or are not enforced. Therefore, reducing exposure levels by intervention, specifically those based on simple low-cost measures acceptable to these communities, becomes critical to protect the population at greatest risk".

Conclusions
Based on these preliminary results, SMS from P. eryngii proved to be a suitable source of ligninolytic enzymes and in particular of Lac, a "green catalyst" able to degrade AFB1. The crude extract from SMS is easy to be obtained, not toxic, inexpensive, eco-friendly, and is able to remove up to 90% of AFB1. Further research is needed to confirm the absence of by-products with any residual toxicity. More studies are also needed to identify and fine-tune practical applications of SMSE to reduce AFB1 levels in animal feeds. In particular, "in vivo" tests on the major commodities affected by AFB1 are needed to set the most suitable soaking times and conditions and to experiment with different methods of SMSE application, such as spraying and washing techniques.

Enzymes Extraction
For extraction of the enzymes, a portion of SMS was taken in order to obtain a representative sample that included both the internal and external parts of SMS balls. Preliminarily, several extraction buffers were compared for efficient recovery of ligninolytic enzymes from SMS in an eco-friendly manner and at a low cost (data not shown). Finally, SMS crude extracts (SMSE) were obtained by blending 50 g of wet SMS with 150 mL of 0.1 M sodium phosphate (pH 7.3) at high speed for 3 min with a Sorvall Omnimixer. The extract was filtered with cheesecloth and centrifuged at 14,000 rpm for 15 min and then filtered through 0.22 µm pore size cellulose acetate filters (Sartorius AG, Muggiò, Italy). The enzyme activities (Lac and MnP) and total protein content of the resultant supernatants were estimated as described below.

SMSE Enzymatic Activities
Lac (EC 1.10.3.2) activity in SMSE was spectrophotometrically determined by oxidation of 2,2 -azino-bis 3-ethylbenzothiazoline-6-sulphonate (ABTS) at 37 • C. The reaction was performed in 100 mM of sodium malonate buffer (pH 4.5), 2 mM ABTS, and an appropriate amount of enzyme solution in a final volume of 1.5 mL. The increase in absorbance at 420 nm was evaluated with a spectrophotometer (Varian Cary 50, Agilent Technologies, Santa Clara, CA, United States. The enzymatic activity was determined by the molar extinction coefficient of ABTS (ε = 3.6 × 10 4 M −1 cm −1 ). One unit (U) of Lac was defined as the amount of enzyme able to oxidize 1 µmole ABTS min −1 [49].
MnP (EC 1.11.1.13) activity was assayed by the oxidation of 1.0 mM MnSO 4 in 50 mM of sodium malonate buffer, pH 4.5, in the presence of 0.05 mM H 2 O 2 . Manganese ions (Mn 3+ ) form a complex with malonate, which absorbs at 270 nm (ε = 7.8 × 10 3 M −1 cm −1 ). One unit of MnP is defined as the amount of enzyme producing 1 µmole of product per minute under the assay condition [49]. To increase Lac activity, SMSE was concentrated by ultrafiltration (AMICON YM1, cutoff size 10 kDa). Protein concentration was determined by the Bradford method, using bovine serum albumin as standard [50].

AFB1 Determination
The standard solution of AFB1 was prepared by dissolving the commercial toxin (Sigma-Aldrich, Milan, Italy) in toluene/acetonitrile (9:1, v/v) into amber silanized vials to obtain a 1 mg mL −1 solution. The exact concentration of aflatoxin solution was spectrophotometrically determined according to AOAC Official Method 971.22 [51]. The residue was dissolved with water/methanol (60:40, v/v) to obtain calibrant standard solutions with 0.2, 0.4, 1.2, 2.0, 4.0, 5.0, and 10.0 ng mL −1 of AFB1. Standard solutions were stored at −20 • C and warmed to room temperature before use.
The fluorometric detector was set at wavelengths of 365 nm (excitation) and 435 nm (emission). The mobile phase was a mixture of water/acetonitrile/methanol (64:18:18, v/v/v) at a flow rate of 0.4 mL min −1 . The temperature of the column was maintained at 40 • C. AFB1 was quantified by measuring the peak areas at the retention time of AFB1 standard and comparing these areas with the calibration curve of AFB1 in the range from 0.2 to 10.0 ng mL −1 . With this mobile phase, the retention time of AFB1 was about 3.7 min. The limit of quantification (LOQ) of the method was 0.2 ng mL −1 for AFB1, based on a signal-to-noise ratio of 10:1.
The experiments were performed in 1.5 mL Eppendorf-tubes in a final volume of 1 mL. The assay mixture contained 995 µL of SMSE and 5 µL of AFB1 solution. In control samples, the SMSE was replaced by an equal volume of extraction buffer. The degradation was conducted in the dark at 25 • C under continuous shaking at 120 rpm for 3, 6, 12, 24, 72, and 168 h. The effect of temperature (15,25,37 • C) and pH (4.5, 5.5, 6.5, 8) on SMSE degradative activity was also studied. The pH range was obtained by adding suitable amounts of concentrated HCl or NaOH solutions to reach the required pH values. To determine the AFB1 degradation obtained with varying Lac activities (5, 2.5, 1.2 U mL −1 ), the SMSE was diluted with sterile extraction buffer and incubated with AFB1 in the same conditions described above. All the experiments were performed in triplicate.
After the enzymatic digestion, 500 µL of each sample were diluted with 500 µL of ultrapure water produced by a Milli-Q system, filtered through 0.2-µm-pore-size regenerated cellulose (RC) filters and 10 µL of the filtrate were injected directly into the UPLC apparatus through a full loop injection system. The percent degradation (D) was calculated by the formula D (%) = (C f /C i ) × 100, where C f was the concentration of AFB1 in the treatment with SMSE and C i was the concentration of AFB1 in the control.

Storability of SMS
The persistence of Lac activity in stored SMS was assessed over a 30-day long period of time as a function of desiccation and temperature of storage (room temperature or +4 • C). Desiccation of SMS at room temperature was assessed by the percentage of dry matter over the total fresh weight (DM%). About 50 g of fresh SMS were precisely weighted to the nearest 0.01 g and then dried in a forced ventilation oven at 65 • C until a constant weight to determine the dry weight. The dry matter percentage was calculated as: DM% = dry weight/fresh weight × 100. SMS was stored on a shelf at room temperature or under refrigeration in a cool room at +4 • C for 30 days and then extracted and assayed for Lac activity as previously described. The experiments were carried out with six replicates per treatment.

Storability of SMSE
SMSE was obtained as previously described and stored at +30, +25, +4, and −20 • C for 70 days. SMSE was sampled at days 0, 3,9,24,54, and 70 to determine the Lac activity as previously described. Moreover, the possibility to lyophilize the SMSE was also evaluated. The experiment was carried out with three replicates per treatment.

Statistical Analysis
Data were analyzed by one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison test. The statistical analyses were performed using the GraphPad Instat 3.0 software (GraphPad Software, San Diego, CA, USA).