Report on aflatoxin-binding activity of galactan exopolysaccharide produced by Weissella confusa KR780676

Abstract

Galactan exopolysaccharide (EPS) produced by Weissella confusa KR780676 isolated from an Indian traditional fermented food has been reported earlier. In this manuscript, we have studied aflatoxin-binding ability of this galactan EPS. Aflatoxin B₁ (AFB₁) binding ability of galactan EPS was observed in an increasing trend with increasing EPS concentration (20–100 mg/mL). At lower concentrations (< 20 mg/mL) of EPS, the binding activity was undetectable, while notable binding was seen from 30 mg/mL. Enhanced AFB₁ binding (32.40%) was recorded at 50 mg/mL of EPS and it increased gradually up to 34.79% at 100 mg/mL concentrations of EPS. The intensity of bands in high-performance thin-layer chromatography (HPTLC) analysis confirms the AFB₁ binding efficiency of galactan EPS, which shows its potential application for removal of toxins in food and feed industry. Galactan EPS binding activity to AFB₁ is further studied with particle size analysis (PSA). This is the first study reporting the aflatoxin-binding activity of any kind of EPS from lactic acid bacteria.

Introduction

Aflatoxins (AF) are naturally occurring mycotoxin that is known for its detrimental effects on the health of humans and animals, including carcinogenic, mutagenic, teratogenic, and immunosuppressive effects (Lewis et al. 2005). The various food products contaminated with aflatoxins include cereals such as maize, sorghum, pearl millet, rice and wheat; oilseeds such as groundnut, soybean, sunflower and cotton; spices such as chillies, black pepper, coriander, turmeric and ginger; tree nuts such as almonds, pistachio, walnuts and coconut; and milk and milk products (Lopez et al. 2002). There are many aflatoxins in the category, but aflatoxin B₁ (AFB₁) is predominant and more toxic, even International Agency for Research on Cancer and the US Environmental Protection Agency have classified AFB1 as a class 1 carcinogen (International Agency for Research on Cancer 2012; Marroquin-Cardona et al. 2009). Several approaches have been established for AF management in food and feed till date. Most of the strategies are aimed at the prevention of AFs production; however, removal of already produced AFs is considered to be intricate (Yiannikouris et al. 2003). The limitation of physical and chemical processes for elimination and inactivation or reducing the bioavailability of AFs have serious drawbacks such as loss of nutritional and sensory qualities of the product as well as requirement of expensive equipment, leading to the general endorsement of biological methods (Teniola et al. 2005). The use of microorganisms as a toxin binder offers an attractive management tool for the control or elimination of aflatoxins in feeds (Corassin et al. 2013). Some lactic acid bacteria (LAB) and yeast isolates have been reported to bind AFB₁ on their cell wall (Shetty and Jespersen 2006). Some probiotic LAB strains are very effective in binding AFB₁ (Huskard et al. 1998); however the most suitable candidate for aflatoxin binding is Saccharomyces cerevisiae (Shetty and Jespersen 2006; Shetty et al. 2007). However, different polysaccharides such as β-glucan and mannan from the yeast cell wall have been reported for their aflatoxin-binding activity (Devegowda et al. 1996; Yiannikouris et al. 2006; Gonçalves et al. 2014). To date,, there is no report of the EPS which shows binding activity toward AFB₁.

Galactan EPS produced by Weissella confusa KR780676 has been characterized and also its physicochemical properties were studied earlier (Devi et al. 2016; Kavitake et al. 2016). In this study, the biotechnological property of galactan EPS has been tested for the AFB₁ binding and the same has been proved with HPTLC and particle size analyzer. With this property, galactan EPS can be a potential option for detoxification or removal of the AFB from food and feed resources.

Materials and methods

Bacterial strain and chemicals

EPS producing probiotic strain Weissella confusa KR780676 was isolated from an Indian acidic fermented food (Kavitake et al. 2016) and was used for the present study. All reagents used were of analytical grade.

Extraction, isolation and purification of galactan

The EPS extraction was done according to the method reported by Kavitake et al. (2016). Briefly, W. confusa was grown in MRS broth supplemented with 2% sucrose at 30 °C for 48 h under static conditions. The cell suspension was heated at 100 °C for 10 min to inactivate enzymes and subsequently centrifuged at 12,000×g for 15 min, for removal of biomass. To the supernatant, 2% trichloroacetic acid (TCA) solution (w/v) was added and precipitated proteins were removed by centrifugation (12,000 ×g for 30 min at 4 °C). EPS was precipitated from the deproteinated solution using thrice the volume of cold ethanol and left overnight at 4 °C. After centrifugation (19,200 ×g for 15 min), the precipitate was dissolved in Milli-Q water. The crude EPS was dialyzed (12–14 kDa) at 4 °C for 48 h and the dialyzed EPS was then lyophilized and used in this binding study.

Aflatoxin-binding assay

To study the ability of galactan EPS to bind AFB₁, different concentrations (1, 5, 10, 20, 30, 40, 50, 75 and 100 mg/mL) of galactan EPS were added to PBS medium (pH 7.0) containing AFB₁ (2 µg/mL). The solutions were briefly vortexed and incubated at 30 °C for 10 h. Due to high viscosity, 1 ml PBS was added to the solution before dialysis (10–250 kDa). After dialysis, the EPS-AFB₁ residues were washed out from the dialysis membrane with PBS (1 mL), and AFB₁ was dissolved using chloroform (1 mL). The AFB₁ extraction was done thrice with chloroform and the leachate was collected, pooled and incubated in a water bath at 60 °C to evaporate the chloroform and to get the dry extract. This dried extract was further used for quantification by HPTLC (CAMAG Switzerland-ATS4).

Quantification of EPS-bound aflatoxin by HPTLC

Chromatographic separations were performed on HPTLC plate (20 × 10 cm) coated with silica gel 60 F254 (Merck Germany) as described by Liang et al. (1996) with slight modifications. To reduce the fluorescence background from any adsorbed organic impurities on the plate, the silica plate was chromatographically washed with methanol and allowed to dry at room temperature prior to sample application. The dried extract was resuspended in 1 ml of chloroform and used for HPTLC analysis. Samples (2 µL) and AFB₁ aflatoxin standard (1 and 2 µg/mL) were spotted on HPTLC plate (silica gel 60 from) and chloroform: acetone (9:1) was used as a mobile phase. After development, the plate was air dried and observed under UV light. The fluorescent intensities and Rf values of the standard band were compared with those sample bands and quantification of AFB₁ was accomplished.

Particle size analysis (PSA) for aflatoxin-binding activity

Galactan EPS (30 mg/mL), 2 µg/mL AFB₁ and combination of both (EPS-AFB₁) after 10 h incubation at 30 °C were subjected for particle size distribution by using a nanoparticle size analyzer (Zetasizer, UK) at 25 °C.

Statistical analysis

Data were assessed by analysis of variance and the significant differences were found, and the mean was separated by Duncan’s multiple range tests with a probability of p ≤ 0.05 (Duncan 1955). This analysis was done using the SPSS 18.0 for Windows (2007) computer software.

Results and discussion

Isolation and purification of galactan EPS

The strain Weissella confusa KR780676 produced a galactan EPS yield of 17.2 g/L (dry weight) in MRS broth supplemented with 2% sucrose, incubated at 30 °C for 48 h under static conditions. Van Geel-Schutten et al. (1998) reported that sucrose serves as an excellent substrate for abundant EPS synthesis; however, the EPS production was shown only 4.3 g/L under shaking condition (100 rpm), which is much lesser than the galactan EPS production. The purified EPS was light reddish and yellowish colour and in the form of odourless powder. The EPS solution was a homogeneous clear liquid with light yellow colour and without precipitation after centrifugation. It is 100% water soluble, but insoluble in ethanol and other organic solvents (Kavitake et al. 2016). This galactan EPS also possesses high oil absorption capacity (5.09 mL/g), flocculating activity (65.3–81.7%) and emulsifying activity with oils and flavours which makes it a good candidate for use as thickening and gelling agent and emulsifier to form long-term emulsions in food, pharmaceutical and cosmetic products, as well as for wastewater treatment under polluted environments (Devi et al. 2016; Kavitake et al. 2019, 2020).

Afalatoxin binding assay

The AFB₁ binding ability of galactan EPS (Fig. 1) was observed in an increasing trend with increasing concentration (20–100 mg/mL). Improved AFB1 binding (32.40%) was recorded at 50 mg/mL of EPS and it increased gradually up to 34.79% at 100 mg/mL. There is no prior report on any kind of EPS showing aflatoxin-binding activity. Peltonen et al. (2001) assessed the binding of AFB₁ with 20 strains of lactic acid bacteria and bifidobacteria and observed 50% AFB₁ binding ability with Lactobacillus amylovorus and Lactobacillus rhamnosus at 72 h. In in vitro studies on AFB₁ (50 and 100 ppb), the binding capacity of Enterococcus faecium GJ40 (24–27% and 17–24%) and E. faecium MF4 (36–42% and 27–32%) was observed at 48 h incubation (Fernandez et al. 2015). Similar to this study, increasing concentrations above 50 mg/mL did not significantly improve the binding ability of the galactan EPS. Comparing these previous studies, the binding ability of EPS seems to have more potential application towards removal of aflatoxin contaminants from food industries at a lesser time extent.

Fig. 1.

Aflatoxin-binding activity with respect to EPS concentration

Quantification of EPS bound AFB1 by HPTLC

HPTLC analysis of AFB₁ binding at different galactan–EPS concentrations was performed propitiously. In Fig. 2, wells 1 and 2 (1 and 2 µg/mL) are AFB₁ standards, and 3–11 represents AFB₁ which was bound to different concentrations of galactan EPS (1,5, 10, 20, 30, 40, 50, 75 and 100 mg/mL). The dark band in Fig. 1b indicates the AFB₁ presence, so here AFB₁ bound with EPS was released and detected in this HPTLC to prove its binding activity to EPS. Binding activity was imperceptible at lower concentrations (< 20 mg/mL), which increased further as the concentration of EPS was increased from 30 mg/mL onwards. This is the first study to show the aflatoxin-binding activity with an EPS produced by lactic acid bacteria.

Fig. 2.

HPTLC analysis of AFB₁ binding at various EPS concentrations. Wells 1, 2—AFB1 standards (1 and 2 µg/mL), 3–11 represent the band of AFB (with 2 µg of AFB) which was bound to the respective EPS concentration (1, 5, 10, 20, 30, 40, 50, 75 and 100 mg/mL)

PSA for EPS–AFB binding

PSA is one of the methods to evaluate an average particle size of a molecule individually or in combination. This analysis evidently showed the increase in the size of AFB₁ molecules after binding to the galactan EPS. As shown in Fig. 3, particle size of EPS–AFB₁ (442.25 nm) combination increased compared to AFB₁ (296.77 nm) and decreased compared to galactan EPS (486.53 nm). As explained by Yiannikouris et al. (2006), this may be due to the structural change caused by hydrogen and Van der Waals bonding between galactan EPS and AFB₁ during binding to each other.

Fig. 3.

Particle size analysis of EPS, AFB₁ and EPS–AFB₁ binding complex

Conclusion

The efficiency of galactan EPS to bind AFB₁ has been proved for the first time in this study. Galactan EPS extracted from Weissella confusa KR780676 has shown the highest binding activity of 34.79% at 100 mg/mL concentration. HPTLC analysis proved the EPS–AFLB₁ binding ability at different concentrations of EPS. PSA showed notable change in the particle size of galactan EPS after the binding with AFB₁. Therefore, the methods of aflatoxin removal employing galactan EPS have a potential application for reducing the levels of AFB₁ . Further applications can be studied to investigate the binding/removal process of AFB₁ due EPS, aiming the commercial application in food and environmental sectors.

Compliance with ethical standards

Conflict of interest

All the authors do not have any conflict of interest.