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Effect of heat treatment on efficacy of Thai bentonite for adsorption of aflatoxin B1 in vitro

S Wongtangtintan, P Saipan, U Tengjaroenkul*, S Suthimun** and B Tengjaroenkul

Khon Kaen University, Khon Kaen 40002, Thailand
* Chiang Mai University, Chiang Mai 50200, Thailand
** Rajamangala University of Technology Isan, Sakon Nakhon 30000, Thailand


This research evaluated the effects of heat treatments on the efficacy of Thai bentonite (TBN) for adsorption of aflatoxin B1 (AfB1) in vitro. The TBN clays were ground to sizes less than 40, 60, 100 and 200 m and heated for 1 hour at eleven heating temperatures at 25C and from 100C to 1000C, at increments of 100C. Clays were mixed separately with 5 ppm AfB1 solution and shaken at 1,000 rpm for 24 hours. Adsorption capacities for AfB1 by the TBN clays were determined using a UV-spectrophotometer.  

Results revealed that adsorption capacities were significantly increased, when temperatures reached 400C and 500C as compared to lower temperatures (p<0.05). The highest adsorption capacity was observed in clay heated at 600C, which was significantly greater than at other temperatures (p<0.05). Significantly lower adsorption capacities were observed in the clays heated at 800C, 900C, and 1000C. Furthermore, sizes of the TBN were not significantly related to the adsorption capacities among the clay samples (p>0.05). The results support the conclusions that TBN can efficiently adsorb AfB1 in vitro and heating the clay at optimal temperatures (600C) can increase the adsorption capacity for AfB1. 

Keywords: binder, clay, heat, mycotoxin, size, temperature


Aflatoxin B1 (AfB1) is widely known as the most acute hepatotoxin of the natural occurring aflatoxins that can cause human and animal health problems (Williams et al 2004, WHO 2005). One encouraging approach to detoxify aflatoxin in animal feed is to add toxin binders to the feed, which reduces the bioavailability of the toxin, absorbed by the intestinal tract (Phillips et al 1988). This strategy is considered practical and cost-effective. Several in vitro studies have shown that binders, such as phyllosilicate, zeolite and bentonite are capable of binding aflatoxin (Kubena et al 1991; Phillips 1999; Pimpukdee et al 2004). Our preliminary study found that Thai bentonite (TBN) from Lopburi province was capable of sequestering AfB1 and was not significantly different using a modified Freundlich model analysis (p>0.05) from several commercial available toxin binders (Tengjaroenkul et al 2011). Phillip et al (1995) reported that heat at 650C modified the structure of phyllosilicate clay and resulted in an increase in efficacy for the adsorption of AfB1. Similarly, Pimpukdee et al (2000) demonstrated that heated hectorite clay at 700C had the greatest adsorption of AfB1. At present, the effect of heat treatments on the adsorption capacity of the Thai clay on AfB1 has not been reported. Thus, the primary objective of this study was to investigate effect of different heating temperatures on efficacy of TBN clay for adsorbing AfB1 in vitro.

Materials and Methods

Standard methods were used as reported by Phillips et al (1995) and Pimpukdee et al (2000). The TBN was ground using jaw crusher (Retsch, Germany) to obtain clay particles with sizes less than 40, 60, 100 and 200 m. Clay samples were subsequently heated for 1 hr, at eleven specific temperatures: 25C (non-heated parent clay), 100C, 200C, 300C, 400C, 500C, 600C, 700C, 800C, 900C and 1,000C. To determine the adsorption capacity, a stock 5 μg/ml solution of AfB1 (Sigma-Aldrich, USA) was prepared by dissolving pure crystals in acetonitrile and 10 mg of TBN was added to purified water to make 2 mg/ml suspension. Then, a 50 μl clay suspension was mixed with 5 ml of the AfB1 solution. Three controls were made for the study: a purified water control, a toxin control containing 5 μg/ml of AfB1 solution without clay, and a clay control containing 5 ml purified water and 100 μg of BN. Controls and the TBN solutions were shaken at 1000 rpm for 24 hr at 25C. After shaking, all samples were centrifuged at 2000 rpm for 30 minutes to separate clays from their supernatants. Approximately 2 ml of supernatant of each sample was analyzed for absorbance using a UV-visible spectrophotometer at 362 nm in order to determine the AfB1 concentration remaining in the solution. The AfB1 adsorption data were represented as an arithmetic mean of the three replications per sample. Means having significant differences in analysis of variance (ANOVA) were compared using Duncan’s multiple range test.

Results and Discussion

The results indicated that different heating temperature affected the AfB1 adsorption capacity of the TBN. No significant difference on adsorption of AfB1 was observed when the clay was heated at 100C, 200C and 300C, as compared to the parent clay (non-heated). Significantly increasing binding capacities were observed in clay heated at 400C and 500C and the highest toxin adsorption was observed at 600C (96.04%), which was not significantly difference from 700C (Figure 1).

 Figure 1: Average percent adsorption capacity for AfB1 by non-heated TBN (25C)
and heated TBN from 100C to 1000C, with increments of 100C.

It has been shown that heat treatment causes the evaporation of moisture and interlayer water and the higher the temperature, the larger the amount of water that is released (Hansen et al 2012, Bergaya and Lagaly 2013). Thus, the heated clays offer more AfB1 binding sites by increasing interlayer spaces and/or suitable pore sites for AfB1 adsorption (Onal and Sarikaya 2008, Sakizci 2009). Phillip et al (1995) showed that heating clays at 650C increasing their adsorption capacity by modifying the structure of phyllosilicate clay, and increasing its efficacy for adsorbing AFB1. Bara (2008) reported on sepiolite, where aflatoxin quantities adsorbed at a ratio of 9 to 1 when comparing the heated to non-heated clays. Pimpukdee et al (2000) reported that hectorite heated at 700C for 1 hr had the highest AfB1 adsorption, while Bayram et al (2010) concluded that heated clays offered more AfB1 binding sites, by increasing the number of pores and/or spaces for the toxin to be adsorbed. 

In this study, significant lower adsorption was found in TBN heated at 800C, 900C and 1000C, suggesting that at these temperatures, the interlayer spaces in clay could be collapsed, which impaired clay binding sites and/or their function. This hypothesis is supported by previous studies (Merkel et al 2006, Hansen et al 2012, Bergaya and Lagaly 2013). In addition, this study demonstrated that the particle size of TBN was not significantly related to the adsorption capacity of the toxin (p>0.05)(Table 1), suggesting that smaller particle sizes, with greater surface areas do not significantly affect the efficacy of TBN to adsorb AfB1.

Table 1:  Average percent of adsorption capacity for AfB1 by parent TBN (25C) and heated TBN from 100C to 1000C, with different particle sizes (m).

  Temperature, C

     Particle Size of Thai Bentonite (m)




























































*Average percent of adsorption capacity for AfB1



Financial and technical supports were provided by the Higher Education Research Promotion and National Research University Project of Thailand, the Research Group on Toxic Substances in Livestock and Aquatic Animals, Khon Kaen University, and the Faculty of Veterinary Medicine, Khon Kaen University.


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Received 1 November 2013; Accepted 7 December 2013; Published 1 January 2014

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