Intestinal Mucus Alters the Ability of Probiotic Bacteria To Bind Aflatoxin B₁ In Vitro

Abstract

Several probiotics are known to bind aflatoxin B₁ (AFB₁) to their surfaces and to adhere to intestinal mucus. In this study, preincubation of two probiotic preparations with either AFB₁ or mucus reduced the subsequent surface binding of mucus and AFB₁, respectively, in a strain-dependent manner.

Aflatoxin B₁ (AFB₁) is an unavoidable food contaminant: current decontamination methods have failed to eliminate aflatoxins from crops. Novel approaches focus on preventing its absorption in the gastrointestinal tracts of animals (15) and humans (4).

Several probiotic bacteria, commonly used in food products, have been shown to bind AFB₁ efficiently in vitro (2, 6, 9), but ex vivo results are controversial [3; S. Gratz, H. El-Nezami, and H. Mykkännen, Abstr. 5th Congr. Toxicol. Developing Countries, Guilin, China, abstr. C26, Toxicology 191(Suppl.):50, 2003]. These differences may be caused by intestinal mucus, since several intestinal and probiotic bacteria have been shown to adhere to mucus (8). Additionally, in vitro studies have focused on bacterial binding sites involved in mucus adhesion (8, 12, 13, 14, 16, 19) and AFB₁ adsorption to the bacterial surface (2, 3, 5, 6, 9). Therefore, the present study investigated whether AFB₁ and intestinal mucus influence each other's binding by surface structures of two probiotic preparations.

Lactobacillus rhamnosus GG (hereafter referred to as GG) is a potent binder for AFB₁ in vitro (1, 5) and ex vivo (3) and for mucus (8, 12). A mixture of L. rhamnosus LC-705 and Propionibacterium freudenreichii subsp. shermanii JS (LC-705 plus JS), a biopreservative (Bioprofit), showed moderate AFB₁ binding in chicks [Gratz et al., Toxicology 191(Suppl.):50, 2003] and was also used in a human study (4). Both were obtained from Valio, Ltd. (Helsinki, Finland), as lyophilized powder and stored at −80°C. Bacterial counts were determined with a flow cytometer as described previously (1). Lyophilized bacteria (1, 2.5, or 5 × 10¹⁰ CFU) were suspended in 1 ml of phosphate-buffered saline (PBS), incubated for 30 min at 37°C, and centrifuged (7 min, 3,000 × g, and 4°C) prior to use. Crystalline AFB₁ (product no. A 6636; Sigma, St. Louis, Mo.) was dissolved in methanol and stored at −20°C. Aqueous AFB₁ solutions were prepared in 10 mM PBS (pH 7.4) (2). Intestinal mucus was collected from three porcine colon samples kindly provided by the University of Kuopio Animal Centre. Mucus was removed and prepared as described previously (12), and protein content was determined with a protein assay kit (Bio-Rad, Espoo, Finland).

Interference in surface binding was assessed in two assays, either by allowing bacteria to bind AFB₁ first and subsequently incubating them with mucus or by allowing bacteria to bind mucus first and then incubating them with AFB₁. In the first assay, the bacterial pellet was suspended in aqueous AFB₁ solutions of various concentrations (0.32 to 64.1 μM) and incubated (30 min and 37°C) to allow AFB₁ binding to the bacterial surface. After centrifugation, the supernatant was analyzed for free AFB₁ by reverse-phase high-performance liquid chromatography as described previously (1). The pelleted complex of bacteria and AFB₁ was resuspended in intestinal mucus with different protein concentrations (0.1 to 10 mg of protein per milliliter) and incubated at 37°C for 1 h. Following centrifugation, the supernatant was analyzed for free mucus proteins. As controls, bacteria without AFB₁ preincubation were included in the mucus binding assay. The stability of this bacterium-mucus complex was tested by washing the solution with 0.5 ml of Milli-Q water, centrifuging it, and measuring mucus proteins released in the supernatant. To estimate the impact of heat treatment on bacterial mucus binding ability, boiled (1 h in PBS) bacteria were included in the assay. In the second assay, bacteria were incubated with mucus and centrifuged, and the bacterium-mucus complex was resuspended in AFB₁ solutions as described above. In order to rule out the possibility that AFB₁ binds to the mucus glycoproteins instead of binding to the bacterial surface, AFB₁ and mucus were mixed, incubated (1 h at 37°C), and centrifuged, and the amounts of free mucus and free AFB₁ were determined. All data are expressed as means of results of four replicates and were analyzed by Student's t test. P values of <0.05 were considered significant.

Both GG and LC-705 plus JS were able to remove mucus from the incubation mixture (Fig. 1). This could be caused by bacterial degradation of mucus, since killing the bacteria with heat disabled mucus binding (data not shown). However, although lactobacilli (10) and propionibacteria (18) possess proteolytic activity, they do not degrade intestinal mucus (17). Therefore, surface binding of intestinal mucus must be at least partly involved in mucus removal, since aqueous washing released 30.7% ± 3.9% of bound mucus from LC-705 plus JS and 67.1% ± 1.1% from GG. This finding suggests that intestinal mucus is reversibly bound to the bacterial surface and that the unrecoverable part of mucus may be bound more tightly, hindering the release by aqueous washing. Perhaps more-rigorous washing or sonication could result in greater release of bound mucus. The lower level of mucus binding by LC-705 plus JS is not surprising, since both LC-705 and JS have poor adhesion ability to intestinal mucus and do not influence each other's adhesion capacity in vitro (13, 19), whereas GG was found to adhere well (8, 11, 12, 14, 19). Furthermore, the mixture LC-705 plus JS also binds AFB₁ less efficiently in vitro than GG does (S. Gratz et al., unpublished data). After preincubation with AFB₁, subsequent mucus binding by both probiotics was reduced (Fig. 1), confirming results of Kankaanpää and coworkers (7), who found that AFB₁ binding by GG reduced its subsequent adhesion to Caco-2 cells.

FIG. 1.

Effect of AFB₁ preincubation (3.2 μM) on mucus binding at different mucus concentrations (1 to 10 mg/ml) by L. rhamnosus GG (GG) and a mixture of L. rhamnosus LC-705 and P. freudenreichii subsp. shermanii JS (LC705+JS). R² values were calculated from original replicates as follows: 0.8399 for GG, 0.8447 for GG-AFB₁, 0.9013 for LC-705 plus JS, and 0.8184 for LC-705 plus JS-AFB₁.

Binding capacities of both probiotics for mucus and AFB₁ were not saturated, as indicated by linear slopes in Fig. 1 and 2. AFB₁ binding by the bacterium-mucus complex must occur to the bacteria rather than to mucus structures, since incubating AFB₁ and mucus alone did not remove any of the free AFB₁ from the incubation mixture (data not shown). At the highest AFB₁ concentration used (Fig. 2), mucus preincubation significantly (P < 0.01) reduced AFB₁ binding by 23.8% for GG and 61.2% for LC-705 plus JS, although the mixture is less capable of binding mucus initially. These strain differences may be explained by the fact that LC-705 plus JS binds mucus more tightly (smaller recovery by washing), possibly interfering more with AFB₁ binding than was the case with GG. After preincubation with mucus with different protein concentrations, AFB₁ binding by LC-705 plus JS was significantly reduced by low mucus concentrations (1 mg/ml), while for GG, significant reduction was achieved only at mucus concentrations above 8 mg/ml (Fig. 3). The reductive effect of mucus and AFB₁ on each other's binding was strongest at low bacterial counts but decreased with increasing numbers of CFU (Fig. 4). Especially at the low numbers of CFU, AFB₁ binding by LC-705 plus JS was strongly impaired. This result suggests that the probiotic mixture is less capable of binding AFB₁ in the presence of mucus and is more susceptible to interfering factors in the intestinal tract, which may explain poorer results in AFB₁ binding in animals [Gratz et al., Toxicology 191(Suppl.):50, 2003].

FIG. 2.

Effect of preincubation of mucus (10 mg/ml) on AFB₁ binding at different AFB₁ concentrations (0.32 to 64.1 μM) by L. rhamnosus GG (LGG) and the mixture of LC-705 and P. freudenreichii subsp. shermanii JS (PJS). R² values were calculated from original replicates as follows: 0.9965 for GG, 0.9892 for GG-mucus, 0.9386 for LC-705 plus JS, and 0.9599 for LC-705 plus JS-mucus.

FIG. 3.

Effect of mucus preincubation at different concentrations (0 to 10 mg/ml) on AFB₁ binding (percentage of the level obtained with the 3.2 μM concentration) by GG and the mixture of LC-705 plus JS. * indicates a significant (P < 0.05) difference compared to values for controls without mucus preincubation (0 mg/ml).

FIG. 4.

Changes in levels of binding of AFB₁ (3.2 μM) by L. rhamnosus GG (GG) (A) and the mixture of LC-705 and P. freudenreichii subsp. shermanii JS (LC705+JS) (B) due to preincubation of mucus (10 mg/ml) at different numbers of CFU (1, 2.5 and 5 × 10¹⁰ CFU) and changes in levels of mucus binding by GG (C) and the LC-705-JS mixture (D) due to AFB₁ preincubation at different bacterial concentrations.

Despite the influence of AFB₁ and mucus on each other's binding, similar binding sites are unlikely to be involved, since heat-treated bacteria lost their ability to bind intestinal mucus, whereas AFB₁ binding was previously found to be enhanced by heat treatment (2, 9). Tuomola and coworkers (19) also showed that heat treatment disturbs mucus adhesion by GG and LC-705 and concluded that proteins must be involved in the binding of mucus, whereas carbohydrates are known to bind AFB₁ (5). Other mechanisms, such as steric hindrance, may cause interference in AFB₁ and mucus binding by bacteria.

Nevertheless, these findings are important, since probiotics adhering to the intestinal wall are less likely to bind and consequently accumulate AFB₁ in the host. On the other hand, probiotics with AFB₁ bound to their surfaces are less likely to adhere to the intestinal wall and prolong exposure to dietary AFB₁. Hence, specific probiotics may be potent and safe means to reduce absorption and increase excretion of dietary AFB₁ from the body.