Study on Adsorption Efficiency of Different Adsorbents for Aflatoxin B1 (AfB1)

Editor's Note: Mycotoxin contamination in feeds (materials) is a global problem encountered in animal breeding today. Common major mycotoxins are aflatoxin, trichothecenes (sinus toxin), fumagillin, zearalenone, ochratoxin A, ergot and the like. Aflatoxins are more common in cereals in tropical and subtropical regions, while aflatoxin B1 (AfB1) is an important metabolite produced by Aspergillus parasiticus. AfB1 can cause a variety of acute toxic diseases and chronic immunosuppressive diseases. The most common way to avoid animal mycotoxin poisoning during animal breeding is to use a mycotoxin adsorbent, which is mixed into the diet (feed) to absorb mycotoxins, to prevent the animals from ingesting toxins during the digestion of the diet. Currently reported as a mycotoxin adsorbent: activated carbon, hydrated sodium calcium aluminosilicates (HSCAS), bentonite and other clay materials. Although activated carbon can adsorb a variety of mycotoxins, it also adsorbs some essential nutrients in the diet because of its non-specific adsorption. In recent years, extensive research has been carried out on commercialized HSCAS, and its adsorption for AfB1 is considered to be very effective. There are also reports that another good adsorption effect on AfB1 is bentonite. This article is an experimental study conducted by Elisavet Vekiru at the Christian Doppler Mycotoxin Research Institute of the Olily Institute of Agricultural Technology. The results of this study demonstrate the importance of multi-angle evaluation of the adsorption efficiency of adsorbents in vitro, and the use of related animals for in vivo testing to verify in vitro. The need for test results.

Translator's Note: Adsorbent evaluation of mycotoxin adsorption capacity is carried out in buffer mode or gastrointestinal fluid mode, which is not used with the mycotoxin content in the test sample. The organic solvent (such as acetonitrile) used in the test sample can extract all the mycotoxin toxin in the sample and measure it. Even if the adsorbent is added to the sample, the measurement result of the sample mycotoxin will not be affected. The role of the adsorbent is to adsorb toxins from entering the digestive system and to carry the toxins out of the body with the feces, thereby reducing the damage of the toxins to the body.

Body part

The most common method for antagonizing aflatoxin B1 (AfB1) is to use an adsorbent because the adsorbent added to the feed adsorbs mycotoxins during digestion and digestion of the animal diet, reducing the bioavailability of the toxin. This study tested the adsorption capacity of different adsorbents for AfB1 at different pH values ​​and different media conditions, and also calculated the chemisorption index of different adsorbents.

Among all cereal products, economic losses and various diseases caused by aflatoxin contamination have been a worldwide problem. The most common method of antagonizing mycotoxins is to use an adsorbent (or binder) to reduce the bioactivity of mycotoxins. Among them, the widely used adsorbent is bentonite (containing a large amount of smectic silicate montmorillonite), and it is reported that bentonite can effectively reduce the content of AfB1 in the diet (material).

The adsorption of mycotoxins can be achieved by physical adsorption and chemical adsorption. An important criterion for evaluating the application effect of mycotoxin adsorbents (Huwing et al, 2001) includes: (1) strong chemisorption; (2) strong adsorption capacity (g toxin/kg adsorbent); (3) no or small amount of repeated adsorption Toxins that have been adsorbed and inactivated; (4) do not or a small amount of essential nutrients in the diet (material); (5) the adsorbent itself is non-toxic (requires that the adsorbent material does not contain heavy metals, dioxins, etc.). Based on the above requirements, under the supervision of elisavet vekiru, the research on mycotoxin adsorbents in this project was carried out at the Christian Doppler Mycotoxin Research Institute and BIOMIN GmbH.

1 Experimental purpose

The adsorption efficiency, adsorption characteristics and adsorption principle of bentonite to mycotoxin were evaluated by in vitro digestion experiments, and the effects of adsorption of mycotoxin on hydrated sodium aluminosilicate (HSCAS) products and activated carbon were compared.

2 experimental design

60 different types of bentonite treated with natural calcium, sodium or acid (labeled S3, S4...S61), from Europe, Asia, South America, etc., through this experiment, the adsorption efficiency and adsorption characteristics of the adsorbent were obtained. Information on the principle of adsorption. In the experiment, a widely acclaimed product, HSCAS, was used as a reference (labeled as R1 in Chinese), and activated carbon and bentonite with moderate adsorption were used as another reference (labeled as R2 in the text). In vitro experiments were carried out under acidic conditions (pH 1-2), focusing on the interaction between the adsorbent and AfB1 and AfB2a. The concentrations of toxins used in each experiment are 4 ml/L (ie 4 ppm) unless otherwise stated.

2.1 Adsorption test

The experiment was carried out in a buffer to evaluate the adsorption capacity of the adsorbent for AfB1 at pH 2.0, 5.0 and 7.0. In the experiment, the adsorbent content was 0.02% (w/v), that is, 200 g of adsorbent was mixed per ton of feed. The experimental method was to co-incubate the adsorbent with the buffer containing AfB1 (shake at 37 °C for 1 h), centrifuge, and take the supernatant for HPLC test (high performance liquid chromatography test).

2.2 Chemical adsorption

This experiment was performed to determine the adsorption efficiency of a 0.5% (5 kg/t) adsorbent in acetate buffer. The cells were co-incubated (shake at 37 °C for 1 h), and then the adsorbent pellets were extracted with methanol, and the chemical adsorption index (effectiveness) was calculated based on the results of the experiment.

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2.3 Comparison of adsorption effects in pH 5 buffer, synthetic gastric juice and real gastric juice

The adsorption effects of the adsorbents were compared in synthetic simulated gastric juice, swine stomach fluid and acetic acid buffer pH 5.0 to explore the adsorption characteristics of various adsorbents. The concentration of the adsorbent used in the experiment was 0.02% (w/v). Before use, the pig gastric juice sample needs to be subjected to an additional purification step before the experiment, that is, it needs to be centrifuged, filtered, and the particles are removed and co-incubated (37 °C shaking shake). Evenly 1 h), sample for testing.

2.4 Isothermal analysis

Isothermal analysis was used to determine the maximum adsorption capacity of the adsorbent. The measurement was carried out in phosphate buffer at pH 7.0 and the adsorbent concentration was 0.002% (20 g/t). The AfB1 concentration range is from 0.4 to 8.0 mg/L (ppm). After incubation of the sample for 24 h (shake at 37 °C), centrifugation was performed and the supernatant was taken for analysis. The maximum adsorption capacity (qmax, [mol/Kg]) and distribution constant (Kd), that is, the calculation method of the affinity of the adsorbent for AfB1 were reported in the literature (Grant et al, 1998).

2.5 Vitamin adsorption test

The non-specific adsorption capacity of the adsorbent for selective vitamins is 0.2% (2 kg/t) in the buffer of pH 3.0 and 6.5, 4 mg/L of AfB1, vitamin VitB12, VitH, The VitB5 content was 4 mg/L, 4 mg/L, and 10 mg/L, respectively. After co-incubation (shake for 1 h at 37 °C), samples were taken for analysis.

2.6 Mineralogy Research

The composition and other characteristics of the clay (bentonite) adsorbent selected in this experiment, such as cation exchange capacity (CEC), were determined at the Institute of Applied Geology, University of Applied Life Sciences (BOKU), Vienna, Austria.

3 Results and discussion

3.1 Adsorption test results

In general, the experimental results of the adsorption efficiency of 25 bentonites at neutral pH are similar or higher, and the adsorption rate is 45% higher than that at pH 5.0 (see Figure 1). At pH 2.0, the adsorption test results of various adsorbents on AfB1 almost reached 100%, probably because some AfB1 was converted to AfB2a under strong acidic conditions. Because AfB2a is hardly adsorbed by any adsorbent, and the level of AfB1 has been reduced to undetectable, it results in a 100% adsorption of AfB1. The amount of AfB1 converted to AfB2a observed in the experiment was closely related to pH and duration of action.

Figure 1. Comparison of the adsorption capacity of different adsorbents for aflatoxin B1 in buffer mode (column results for pH 7.0, green star labeled pH 2.0, green box for pH 5.0)

3.2 Chemical adsorption test results

18 kinds of bentonite adsorbents with an adsorption rate higher than 63% at pH 5.0 were used to test the chemisorptions index (Cα). When Cα was 1, the adsorbent reached complete adsorption to AfB1. It can be seen from Fig. 2 that the bentonite has certain adsorption capacity for AfB1 in the test, the highest adsorption efficiency is S12 (Cα=0.98), followed by S3 (Cα=0.92). Further chromatographic analysis of sample S12 indicated that it mainly adsorbed AfB2a, which may explain why this bentonite sample is more active in an acidic environment because AfB1 is converted to AfB2a under acidic conditions, which may cause adsorption. The illusion of high rates. The chemical adsorption index of sample S37 was lower because about 11% of AfB1 in the sample was desorbed.

Figure 2, the difference in chemical adsorption index of different adsorbents for aflatoxin B1, Cα range 0.81-0.98

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3.3 Adsorption comparison results in pH 5 buffer, simulated gastric juice and real gastric juice

In the first experiment, the conditions affecting the adsorption efficiency of bentonite to AfB1 in three different media were similar to those of R1 and R2. It can be seen from Fig. 3 that the adsorption capacity of bentonite to AfB1 in synthetic gastric juice was higher than that in acetic acid buffer under the same pH condition. The adsorption capacity in the liquid is low. Surprisingly, the adsorption capacity of the bentonite adsorbent is affected by factors such as the composition and proportion of the true gastric juice, while the adsorption capacity of the bentonite adsorbent is not affected by the relevant factors in the synthetic gastric juice. As can be seen from Figure 4, further detection of 47 adsorbents in buffers of the same pH and in the normal gastric juice found that 16 of the 47 adsorbents decreased the adsorption capacity of AfB1 by more than 15% when using real gastric juice. The adsorption capacity of activated carbon for AfB1 dropped sharply by about 53%.

Figure 3. Comparison of the adsorption capacity of adsorbents R1 and R2 for aflatoxin B1 in acetate buffer and simulated gastric juice and real gastric juice (R1 for wide column and R2 for narrow column)

Figure 4. Comparison of adsorption capacities of different adsorbents for aflatoxin B1

And in the real gastric juice, (the histogram shows the simulated gastric juice, the green star is labeled as the acetate buffer)

3.4 Isothermal analysis results

The results showed that each Kg reference HSCAS (R1) was able to adsorb 122 g of AfB1, ie, 1 Kg of R1 per ton of feed (material) could adsorb 122 g of AfB1. After testing 61 kinds of bentonite adsorbents, only three kinds of adsorption capacity exceeded R1 (S3, S6, S14), and S9 had the same adsorption efficiency as R1.

3.5 Vitamin Adsorption Test Results

Under the condition of pH 3, some experimental results are consistent with the experimental results obtained by the predecessors, such as activated carbon does adsorb more nutrients (see Figure 5). The bentonite used in this experiment has no adsorption to vitamin B5 and vitamin H, but adsorbs 7% to 47% of vitamin B12. At the same time, bentonite has a significant adsorption effect on AfB1.

Figure 5. Comparison of adsorption capacities of different adsorbents for vitamin B5, B12, biotin and aflatoxin B1

3.6 Mineralogy analysis

Compared with clay minerals, there is no correlation between some commonly used physical indicators such as CEC (cation exchange capacity) and particle size and adsorption capacity.

4 Conclusion

In this study, a series of in vitro tests were carried out on the mold adsorbent-bentonite to verify the adsorption capacity of 61 bentonites for AfB1, and the results were compared with the commercial product HSCAS. The results of chemical adsorption show that bentonite can adsorb most of AfB1, but only a few of the bentonite samples can meet or exceed HSCAS (R1).

The adsorption test of AfB1 on real gastric juice and vitamin mixture by bentonite showed that the activated carbon showed non-specific adsorption, so the necessary nutrients were adsorbed; the reference substance showed low adsorption rate to the refolding matrix; Some bentonite adsorbents are superior to the reference at this point. When gastric juice is used instead of buffer, some adsorbents exhibit significant adsorption of AfB1 while having different adsorption capacities for some vitamins. Therefore, a suitable bentonite adsorbent can be found, which can not only have high adsorption to mycotoxins, but also reduce non-specific adsorption of nutrients, and further testing in vitro is needed. Of course, all mycotoxin adsorbents need to be tested for efficacy, safety, and impact on the animal and its performance before being added to the animal diet.

references:

CAST (Council for Agricultural Science and Technology) (2003) Mycotoxins: Risks in Plant,

Animal and human systems. Task Force Report No. 139:13-85. CAST, Ames, IA

Grant PG, Lemke SL, Dwyer MR, Phillips TD (1998) Isothermal adsorption of aflatoxin B1 on HSCAS clay. J.Agr. Food Chem. 46: 599-605

Huwig A, Freimund S, Kappeli O, Dutler H (2001) Mycotoxin detoxication of animal feed by different adsorbents.Toxicol. Lett. 122: 179-188

Miazzo R, Peralta MF, Magnoli C, Salvano M, Ferrero S, Chiacchiera SM, Carvalho EC, Rosa CA, Dalcero A (2005) Efficacy of sodium bentonite as a detoxifier of broiler feed con?taminated with aflatoxin and fumonisin. Poult. . 84 (1): 1-8

Vekiru E, Fruhauf S, Sahin M, Ottner F, Schatzmayr G, Krska R (2007) Investigation of various adsorbents for their ability to bind Aflatoxin B1. Publication in Myc. Res., accepted 13 Nov 2006