Chlorophyll-Amended Organoclays for the Detoxification of Ochratoxin A

Abstract

Climate change has been associated with outbreaks of mycotoxicosis following periods of drought, enhanced fungal growth, and increased exposure to mycotoxins. For detoxification, the inclusion of clay-based materials in food and drinking water has resulted in a very promising strategy to reduce mycotoxin exposure. In this strategy, mycotoxins are tightly sorbed to high-affinity clay particles in the gastrointestinal tract, thus decreasing bioavailability, uptake to blood, and potential toxicity. This study investigated the ability of chlorophyll and chlorophyllin-amended montmorillonite clays to decrease the toxicity of ochratoxin A (OTA). The sorption mechanisms of OTA binding to surfaces of sorbents, as well as binding parameters such as capacity, affinity, enthalpy, and free energy, were examined. Chlorophyll-amended organoclay (CMCH) demonstrated the highest binding (72%) and was better than the chlorophyllin-amended hydrophilic clay (59%), possibly due to the hydrophobicity of OTA (LogP 4.7). In silico studies using molecular dynamics simulations showed that CMCH improves OTA binding in comparison to parent clay in line with experiments. Simulations depicted that chlorophyll amendments on clay facilitated OTA molecules binding both directly, through enhancing OTA binding on the clay, or predominantly indirectly, through OTA molecules interacting with bound chlorophyll amendments. Simulations uncovered the key role of calcium ions in OTA binding, particularly in neutral conditions, and demonstrated that CMCH binding to OTA is enhanced under both neutral and acidic conditions. Furthermore, the protection of various sorbents against OTA-induced toxicity was carried out using two living organisms (Hydra vulgaris and Caenorhabditis elegans) which are susceptible to OTA toxicity. This study showed the significant detoxification of OTA (33% to 100%) by inclusion of sorbents. Organoclay (CMCH) at 0.5% offered complete protection. These findings suggest that the chlorophyll-amended organoclays described in this study could be included in food and feed as OTA binders and as potential filter materials for water and beverages to protect against OTA contaminants during outbreaks and emergencies.

Keywords: enterosorption; food contamination; isotherms; kinetics; molecular dynamics; mycotoxin; ochratoxin; thermodynamics.

Figure 1

Sorbent screening with reduction percentage of ochratoxin by the sorbents. * p < 0.01 when compared to CM or SM. CM: calcium montmorillonite; CMCH: chlorophyll-amended calcium montmorillonite; CMCHin: chlorophyllin-amended calcium montmorillonite; SM: sodium montmorillonite; SMCH: chlorophyll-amended sodium montmorillonite; SMCHin: chlorophyllin-amended sodium montmorillonite.

Figure 2

Adsorption isotherms of OTA onto binding surfaces of (A) CM-amended clays and (B) SM-amended clays at pH 2. CM: calcium montmorillonite; CMCH: chlorophyll-amended calcium montmorillonite; CMCHin: chlorophyllin-amended calcium montmorillonite; SM: sodium montmorillonite; SMCH: chlorophyll-amended sodium montmorillonite; SMCHin: chlorophyllin-amended sodium montmorillonite.

Figure 3

Desorption isotherms of OTA onto binding surfaces of (A) CM-amended clays and (B) SM-amended clays at pH 6. CM: calcium montmorillonite; CMCH: chlorophyll-amended calcium montmorillonite; CMCHin: chlorophyllin-amended calcium montmorillonite; SM: sodium montmorillonite; SMCH: chlorophyll-amended sodium montmorillonite; SMCHin: chlorophyllin-amended sodium montmorillonite.

Figure 4

Effect of contact time on the adsorption of OTA. CM: calcium montmorillonite; CMCH: chlorophyll-amended calcium montmorillonite; CMCHin: chlorophyllin-amended calcium montmorillonite; SMCH: chlorophyll-amended sodium montmorillonite.

Figure 5

Toxicity effects of OTA exposure to hydra (A), protection with SM-derived sorbents (B), and CM-derived sorbents (C). CM: calcium montmorillonite; CMCH: chlorophyll-amended calcium montmorillonite; CMCHin: chlorophyllin-amended calcium montmorillonite; SM: sodium montmorillonite; SMCH: chlorophyll-amended sodium montmorillonite; SMCHin: chlorophyllin-amended sodium montmorillonite.

Figure 5

Toxicity effects of OTA exposure to hydra (A), protection with SM-derived sorbents (B), and CM-derived sorbents (C). CM: calcium montmorillonite; CMCH: chlorophyll-amended calcium montmorillonite; CMCHin: chlorophyllin-amended calcium montmorillonite; SM: sodium montmorillonite; SMCH: chlorophyll-amended sodium montmorillonite; SMCHin: chlorophyllin-amended sodium montmorillonite.

Figure 6

Effect of toxicity of OTA on the body length (A), nose touch response (B), and survival rate of Caenorhabditis elegans after 24 h and 48 h of exposure (C). Data represent the average value from triplicate analysis  ±  the standard deviation. * indicates a significant difference (p  ≤  0.05) compared to the vehicle control group.

Figure 7

Protective effect of parent clays and amended clays against OTA toxicity on the body length (A,D), nose touch response (B,E), and survival rate of Caenorhabditis elegans (C,F). Data represent the average value from triplicate analysis  ±  the standard deviation. * indicates a significant difference (p  ≤  0.05) compared to the vehicle control group. # indicates a significant difference (p  ≤  0.05) compared to the OTA-alone group. 1: 0.2% and 2: 0.5% clay inclusions; CM: calcium montmorillonite; CMCH: chlorophyll-amended calcium montmorillonite; CMCHin: chlorophyllin-amended calcium montmorillonite; SM: sodium montmorillonite; SMCH: chlorophyll-amended sodium montmorillonite; SMCHin: chlorophyllin-amended sodium montmorillonite.

Figure 7

Protective effect of parent clays and amended clays against OTA toxicity on the body length (A,D), nose touch response (B,E), and survival rate of Caenorhabditis elegans (C,F). Data represent the average value from triplicate analysis  ±  the standard deviation. * indicates a significant difference (p  ≤  0.05) compared to the vehicle control group. # indicates a significant difference (p  ≤  0.05) compared to the OTA-alone group. 1: 0.2% and 2: 0.5% clay inclusions; CM: calcium montmorillonite; CMCH: chlorophyll-amended calcium montmorillonite; CMCHin: chlorophyllin-amended calcium montmorillonite; SM: sodium montmorillonite; SMCH: chlorophyll-amended sodium montmorillonite; SMCHin: chlorophyllin-amended sodium montmorillonite.

Figure 8

Average (%) probability of interaction between OTA molecules in the presence of CMCH, CMPHO, and CM. Blue corresponds to acidic conditions; red and yellow correspond to neutral conditions, simulating monoanionic and dianionic OTA, respectively. Average values are calculated from triplicate runs. Error bars denote standard deviation values calculated from the triplicate runs.

Figure 9

Average (%) probability of OTA molecules interacting with CMCH, CMPHO, and CM in acidic condition (A) and neutral condition (B,C), simulating monoanionic and dianionic OTA, respectively. Direct, direct-assisted, and indirect-assisted interactions are shown in blue, red, and yellow, respectively. Average values are calculated from triplicate runs. Error bars denote standard deviation values calculated from the triplicate runs.

Figure 9

Average (%) probability of OTA molecules interacting with CMCH, CMPHO, and CM in acidic condition (A) and neutral condition (B,C), simulating monoanionic and dianionic OTA, respectively. Direct, direct-assisted, and indirect-assisted interactions are shown in blue, red, and yellow, respectively. Average values are calculated from triplicate runs. Error bars denote standard deviation values calculated from the triplicate runs.

Figure 10

Average (%) probability of CM participating in interactions with different OTA groups (defined in Figure S2). Group 1 corresponds to blue, group 2 corresponds to red, group 3 corresponds to yellow, group 4 corresponds to green, and group 5 corresponds to orange. Additionally, the average (%) probability of CM-bound OTA molecules interacting with calcium is shown in cyan. The results were normalized, i.e., they were calculated given an interaction between OTA and CM. Values correspond to parent (CM) and amended clays (CMCH and CMPHO) in acidic conditions (left), as well as neutral conditions (middle) and (right), of simulations including monoanionic and dianionic OTA, respectively. Average values are calculated from triplicate runs. Error bars denote standard deviation values calculated from the triplicate runs.

Figure 11

Average (%) probability of CMCH (A) and CMPHO (B), respectively, participating in interactions with different OTA groups (defined in Figure S2). Group 1 corresponds to blue, group 2 corresponds to red, group 3 corresponds to yellow, group 4 corresponds to green, and group 5 corresponds to orange. Additionally, the average (%) probability of CMCH-bound (A) or CMPHO-bound (B) OTA molecules interacting with calcium is shown in cyan. The results were normalized, i.e., they were calculated given an interaction between OTA and CMCH and CMPHO. Values correspond to systems in acidic conditions (left), as well as neutral conditions (middle) and (right), of simulations including monoanionic and dianionic OTA, respectively. Average values are calculated from triplicate runs. Error bars denote standard deviation values calculated from the triplicate runs. All values shown above are normalized over the total number of interactions per system.

Figure 12

Simulation snapshots of CMCH in complex with OTA in (A) acidic conditions, as well as (B,C) neutral conditions, investigating OTA in monoanionic and dianionic states, respectively. Panels (D–F) show zoomed-in representation of particular interactions, marked as (i) and (ii), that occur within panels (A–C), respectively. CM, chlorophyll, and calcium are shown in vdW representation, while OTA is shown in licorice representation. Atoms are colored by atom type, except for carbon atoms of chlorophyll in green and calcium in tan. Calcium ions that are at a greater distance than 3.5 Å from all OTA molecules were omitted. Hydrogen atoms are also omitted for clarity.