Abstract: The experiment was to evaluate the adsorption effect of montmorillonite on AFB1 in vitro, and to explore the effects of AFB1 concentration, effective content and addition amount of montmorillonite, pH value, adsorption time and adsorption temperature on adsorption of AFB1 by montmorillonite. Two kinds of AFB1 contaminated feeds with high (98.8μg/kg) and low (48.9μg/kg) concentrations were prepared. Five kinds of montmorillonite with different effective contents were added into contaminated feed with three kinds of supplemental amounts to adsorb AFB1,  and the supernatant was centrifugally extracted after adding the extract. The residual AFB1 content in supernatant was determined by ELISA, and the adsorption rate was calculated. After determining the effective content and addition amount of montmorillonite with better adsorption effect, the adsorption rate of montmorillonite on AFB1 was determined under different pH value (2~7), adsorption time (5~115min) and adsorption temperature (20~40℃). The results showed that the adsorption effect of montmorillonite at high toxin concentration was higher than that at low toxin concentration. When the effective content of montmorillonite was 70% ~ 92%, the adsorption rate of AFB1 was positively correlated with the effective content of montmorillonite, Only when the effective content of montmorillonite was higher than 90%, the good adsorption effect of AFB1 can reach more than 80%, When the effective content of montmorillonite was higher than 95%, the adsorption effect of montmorillonite on AFB1 was relatively stable. The adsorption effect of different adding amount of montmorillonite was 0.4% adding amount > 0.3% adding amount > 0.2% adding amount. The effect of pH value on detoxification effect of montmorillonite was not obvious. The adsorption rate of AFB1 was above 90% in the range of pH 2 ~ 7, The adsorption of montmorillonite on AFB1 was close to equilibrium at 55 min, and the adsorption rate reached 93.2%. After 55 min, the adsorption curve was nearly stable, and the adsorption rate reached the highest (94.8%) at 95 min. In the range of 20~40℃, the higher the temperature, the better the adsorption effect. At 35℃, the adsorption rate reached the highest (93.2%).

Key words: montmorillonite; aflatoxin B1; adsorption; influence factors

CLC number :S 816

Document id :A

Article code :1002-2813 (2021) 15-0088-04

Doi: 10.13557 / j.carol carroll nki issn1002-2813.2021.15.020

Aflatoxin (AFs) is a secondary metabolite produced by a variety of strains such as Aspergillus aspergillus parasiticus and Aspergillus opticus. Natural AFs include AFB1, AFB2, AFG1, AFG2, which were found and isolated in milk. AFM1, AFM2 and AFs are the most toxic AFB1 [1] Carcinogenic and immunosuppressive effects [2] can cause a series of damage to liver, kidney, spleen, mouth, digestive tract, brain and nervous system [3] Among all kinds of animals, ducklings are most sensitive to AFs, pigs, turkeys, broilers are sensitive to AFs, ruminants are not sensitive to AFs [1] Studies show that AFB1 positive detection rate in poultry feed is high, and AFB1 level and mold content in about 14.29% of poultry feed exceed the specified limit [4] poultry feed intake, tibia strength and immune performance will decrease after feeding AFB1 contaminated feed [5,6] Pig AFB1 poisoning of feed intake reduced, growth performance, immune function of liver function and antioxidant function is impaired, serious when killed [7-10], the sow poisoning serious when still birth deformities of AFs for ruminant production has a certain tolerance ability, but also can appear poisoning phenomenon, mainly for feed conversion rate decreased, growth performance Immune capacity and reproductive capacity are affected, and milk contains AFM1. Although AFM1 is less toxic than AFB1, AFM1 can also cause DNA damage, gene mutation and chromosome aberration [7]

Mycotoxin binder added to feed is one of the most commonly used methods to remove mycotoxin from compound feed. [11-13] Montmorillonite is a silicate clay ore with 2:1 structure composed of 2 layers of silica tetrahedron and 1 layer of alumina octahedron [14] Montmorillonite can use a large specific surface area of chemical and mechanical stability of the lamellar structure High cation exchange capacity adsorbs and immobilized mycotoxin heavy metal pathogens and bacterial toxins [15-17]. In mycotoxins such as AFs zealenone (ZEA) emetic toxin (DON), the adsorption effect of AFs is the best [18] liang Xiaowei et al. [19] added montmorillonite containing AFB1 In artificial gastric juice or artificial intestinal juice of ZEA and T-2 toxin, it was found that the adsorption reached equilibrium after 1h. The adsorption rate of sodium montmorillonite to AFB1 and T-2 toxin was 96.8% 38.0%, respectively, and the adsorption effect of ZEA was poor, the adsorption rate was less than 10% Studies have shown that adding calcium montmorillonite to dairy feed can effectively reduce the concentration of AFs in milk, without affecting the quality composition and yield of milk [20]. The adsorption rate of AFB1 by montmorillonite is affected by the adsorption conditions of AFB1 concentration (temperature, pH value, adsorption time, etc.) in the feed with montmorillonite content At present, the research on the adsorption effect of montmorillonite on AFB1 under different conditions is still not comprehensive and systematic. Therefore, this experiment selected 5 montmorillonite with different effective content as the research object, and studied the adsorption effect of montmorillonite on AFB1 under different conditions through in vitro adsorption test, so as to provide reference for its application in feed demildew

1. Materials and methods

1.1 Test Materials

5 Five montmorillonite products with different contents (effective contents 70% 85% 92% 95% 98%) were purchased from Inner Mongolia Hezhengmei Biology Science and Technology Co., Ltd. Suckling pig feed was purchased from Chifeng Dabeinong Agriculture and Animal Husbandry Technology Co., Ltd.

1.2 Test reagents

Methanol (analytical pure) AFB1 Standard Hydrochloric acid (analytical pure) Sodium hydroxide (analytical pure), AFB1 ELISA Kit (Bayfa, Germany)

1.3 Instruments and Equipment

METTLER TOLEDO AB204E Electronic analytical balance (METTLER TOLEDO, Shanghai Co., LTD.); Wd-9405b Horizontal shaker (Beijing Liuyi Biotechnology Co., LTD.); 5810R Large capacity high speed refrigerated centrifuge (Eppendorf, Germany); Bio-rad Full wavelength Microplate tester (Bole); Ghp-9270 Water-proof constant temperature incubator (Shanghai Yiheng Technology); Fw-100 Multi-functional mill (Tianjin Tester Instrument Co., LTD.)

1.4 Preparation of AFB1 contaminated feed

AFB1 standard was dissolved in methanol and evenly sprayed on a small amount of Suckling pig feed. After being mixed, test samples with AFB1 content of 50， 100 μg/kg were prepared. The AFB1 level was 48.9， 98.8 μg/kg

1.5 Determination of AFB1 content in feed

Weigh the 5.00g sample and put it into a 50 mL centrifuge tube with a cover. Add the extraction agent provided by the kit (25 mL in 70% methanol solution) to the centrifuge tube according to the proportion required by the kit instruction. After constant temperature oscillation for 3 min, take the supernatant and dilute it with deionized water before using AFB1 made by Bayfa Germany The content of AFB1 in supernatant was determined by ELISA

1.6 AFB1 concentration, montmorillonite addition amount, montmorillonite content on the adsorption of AFB1 in vitro

A total of 45 samples with 5.00 g toxin content of 48.9, 98.8 μg/kg were taken and placed in a 50 mL centrifuge tube with a cover. The montmorillonite with 5 different contents was divided into 0.2% 0.3% of the feed amount 0.4% was added into the centrifuge tube, and 25ml of 70% methanol extraction solution was added according to the instructions of the kit, followed by constant temperature oscillation extraction for 1h, and centrifugation at 4000r/min for 4min. The supernatant was absorbed, and the pH value was adjusted to 3. AFB1 content in the supernatant was determined by ELISA A total of 90 samples were tested, which were divided into 30 treatments with 3 replicates for each treatment. The average value of the test data was taken to calculate the adsorption rate Y according to the formula.

Where, C0 is the initial concentration of AFB1 (mg/L); C is the concentration of AFB1 in supernatant at equilibrium (mg/L)

1.7 Influence of PH value on AFB1 adsorption of montmorillonite in vitro

After studying the effect of AFB1 concentration and montmorillonite addition amount on AFB1 adsorption in vitro, and obtaining the best and most appropriate montmorillonite content and addition amount (the best combination of montmorillonite addition), sodium hydroxide and hydrochloric acid solution were used to adjust the pH value, and the pH value was 2, 3, 4, 5,6 respectively The adsorption rate of AFB1 for montmorillonite under the optimum combination was determined

1.8 Effect of adsorption time on AFB1 adsorption by montmorillonite in vitro

Montmorillonite was added to the toxic feed according to the optimal combination, and the extraction solution was added according to the kit instruction. The extraction solution was extracted with 37 constant temperature oscillation for 5 15 25 35 55 75 95 115 min, respectively, and centrifugation was carried out at high speed. The content of AFB1 in the supernatant was determined by ELISA, and the adsorption rate was calculated

1.9 Effect of temperature on AFB1 adsorption by montmorillonite in vitro

After montmorillonite was added in the toxic feed according to the optimal combination, the extraction solution was added according to the kit instruction, and at 20, 25, 30, 35, 40, The supernatant was extracted with constant temperature oscillation for 1h and centrifuged at high speed. The content of AFB1 in the supernatant was determined by ELISA, and the adsorption rate was calculated. The treatment group without adsorbent was set in 1.6, 1.7, 1.8 and 1.9 to eliminate the non-specific adsorption of AFB1 in the test process

2. Results and analysis

2.1 Effects of AFB1 concentration montmorillonite effective content in feed and addition amount on adsorption of AFB1 by montmorillonite (Table 1)

According to Table 1, the adsorption effect of montmorillonite on AFB1 in high-concentration toxin feed is better than that in low-concentration toxin feed. AFB1 adsorption rate increased with the increase of montmorillonite effective content in the range of 70-92%, but only when montmorillonite effective content is higher than 90%, the good adsorption effect of more than 80% can be achieved. When the effective montmorillonite content was higher than 95%, the AFB1 adsorption rate was not significantly increased by increasing the effective montmorillonite content. The adsorption effect of different montmorillonite content was 0.4% ＞ 0.3% ＞0.2%, that is, with the increase of montmorillonite, the adsorption rate of AFB1 by montmorillonite also increases. Therefore, the effective montmorillonite content of 92% and the addition amount of 0.4% were selected as the combination of montmorillonite addition, and the high-concentration toxin feed was used for subsequent experiments

2.2 Influence of pH value on AFB1 adsorption by montmorillonite (See Table 2)

Table 2 shows that montmorillonite has a good adsorption effect on AFB1 under acidic conditions, and the adsorption rate is 93.2% when pH value is 2. When pH value increased from 2 to 7,AFB1 adsorption rate only decreased by 2.1%, and both of them were above 90%. The results showed that the detoxification effect of montmorillonite was better under acidic conditions, and pH value had no obvious effect on the detoxification effect of montmorillonite

2.3 Influence of adsorption time on AFB1 adsorption by montmorillonite (See Table 3)

Table 3 shows that the adsorption of Montmorillonite to AFB1 approached equilibrium at 55 min, when the adsorption rate of montmorillonite to AFB1 reached 93.2%. Before this, the adsorption rate of montmorillonite to AFB1 increased significantly with the increase of adsorption time. The adsorption rate increased by 71% from 5 min to 55 min. After 55 min, the adsorption curve tends to be stable, and the adsorption rate reaches the highest at 95 min, which is 94.8%. At 115 min, the adsorption rate was slightly lower than that at 95 min. The results showed that the optimal adsorption time of montmorillonite was 95 min

2.4 Influence of adsorption temperature on AFB1 adsorption by montmorillonite (See Table 4)

As can be seen from Table 4, the adsorption rate of AFB1 by montmorillonite is greatly affected by temperature in the range of 20-40℃. The adsorption rate reaches the highest at 35℃, which is 93.2%. From 20 to 35℃, the adsorption rate increased significantly, up to 47.3%. At 40℃, the adsorption rate is 93.1%, which is not much different from that at 35℃. The results show that the optimal adsorption temperature of montmorillonite is 35-40℃

3. Discussion

3.1 Effects of AFB1 concentration montmorillonite effective content and supplemental amount on adsorption of AFB1 by montmorillonite in feed

The results showed that the higher the concentration of AFB1 in the feed, the better the adsorption effect of MONTmorillonite on AFB1. The results are slightly different from those of Xu Rui et al. [3] Xu Ruietc research found, With the increase of AFB1 concentration in the feed, the adsorption rate of montmorillonite to AFB1 gradually decreased, and the reduction of adsorption rate increased with the increase of toxin concentration in the feed Reason may be that montmorillonite relative to the amount of mycotoxin, montmorillonite existence saturated adsorption capacity, and the saturated adsorption amount of small [21], montmorillonite in this test the amount of total adsorption capacity relative AFB1 slants big, at the end of the adsorption of saturation state, and [3] experiments such as Mr Montmorillonite total small relative to the amount of AFB1 adsorption capacity, adsorption in not fully It has reached the saturation state, so the adsorption rate decreases with the increase of toxin concentration. If the concentration of AFB1 is increased and the amount of montmorillonite is increased simultaneously, better adsorption effect can be achieved.

The effective content of montmorillonite in the product and the increase of the amount of montmorillonite in the feed will increase the adsorption rate of AFB1. In this test, when the effective content of montmorillonite is higher than 90%, the effect of toxin adsorption is not significantly affected by the effective content of montmorillonite, and the effect is the best under the concentration of both toxins is 0.4%, so it is determined that the content of montmorillonite is 92% and the dosage of 0.4% It is a good combination of AFB1 in adsorbed feed. However, in actual production, it is impossible to determine the amount of montmorillonite only from the concentration of AFB1 in feed, and it is also necessary to consider the influence of feed supplemented with montmorillonite on the performance of livestock and poultry as well as economic factors Excessive montmorillonite supplementation has a negative impact on the health and production performance of livestock and poultry. Studies have shown that montmorillonite can absorb minerals and nutrients from the feed, leading to the deficiency of micronutrients such as iron, potassium and vitamins in animals fed with the feed [22] Qi Zhiguo et al. [14] study showed that 0.3% was added to the diet of Beijing youji and laying hens 0.4% montmorillonite significantly decreased serum total antioxidant capacity and performance of laying hens, while 0.2% montmorillonite supplementation significantly increased average daily egg weight of laying rate and total superoxide dismutase content. In the case of mycotoxin don't overweight, 0.2% amount of Beijing chicken fodder recommend adding dose As a result, not a single from the AFB1 content in the feed to determine the content of montmorillonite, need from forage, tolerance ability of mycotoxins in feed concentration, additive in the effective content of montmorillonite and economical aspects of considering montmorillonite appropriate adding amount.

3.2 Influence of pH value, adsorption time and temperature on adsorption of AFB1 by montmorillonite

After animals eat, the food is digested in the stomach, and then enters the small intestine for further digestion and absorption. The stomach secretes gastric acid and the digestive system is acidic with a pH value of about 2~3. Small intestine digestive system is weak alkaline,pH value is about 7~8 This experiment adjusted pH value is 2~7, explore the influence of pH value on the adsorption of AFB1 test found that the influence of pH value on the adsorption rate is not obvious, in the range of pH value is 2~7, the adsorption rate of AFB1 montmorillonite reached more than 90%, the adsorption effect is good. The isoelectric point of AFB1 is 4.17, and the montmorillonite surface has a large negative charge [21]. From the perspective of electrostatic adsorption,AFB1 has a positive charge at pH value 2-4.17, which is conducive to the adsorption of montmorillonite. Under the condition of pH 4.17-7,AFB1 has negative charge, which is not conducive to the adsorption of Montmorillonite. However, due to the low content of AFB1 and the adsorption of AFB1 by montmorillonite is mainly chemical adsorption, the influence of pH value on the adsorption of AFB1 by montmorillonite is not obviously consistent with the results of previous studies [23]

In this experiment, the adsorption of montmorillonite to AFB1 approached adsorption equilibrium at 55 min, which was similar to the adsorption equilibrium time obtained by Liang Xiaowei et al. [19]. However, some experiments found that the adsorption equilibrium time was inconsistent at 20 min [23], which might be due to the test environment, montmorillonite type, In the initial stage of experimental adsorption, the adsorption rate of montmorillonite increases rapidly until it reaches equilibrium. After equilibrium, the effect of prolonged adsorption time on adsorption is no longer obvious, because in the initial stage of adsorption, the adsorption sites on the surface of montmorillonite are sufficient, and the concentration of AFB1 is relatively high. AFB1 can be quickly fixed by montmorillonite and combined with it to form a complex. With the increase of adsorption time, the content of AFB1 and the free sites of montmorillonite decreased, and the adsorption rate slowed down until the adsorption equilibrium

The common body temperature of animals is generally between 37 and 40℃. In this experiment, 20 to 40℃was selected as the temperature range combined with air temperature to test the adsorption effect of Montmorillonite on AFB1. It was found that montmorillonite had a better adsorption effect on AFB1 between 35 and 40℃, indicating that montmorillonite can play a good detoxification effect in animals.

4. Conclusion

The adsorption rate of AFB1 by montmorillonite was positively correlated with the effective content of montmorillonite. When montmorillonite content is higher than 95%, the adsorption effect of Montmorillonite on AFB1 is stable. The higher the level of AFB1 and the amount of montmorillonite added in the feed, the higher the adsorption rate of AFB1 by montmorillonite. However, the pH value of montmorillonite added in the feed should not exceed the tolerance capacity of animals, which had no obvious effect on the adsorption effect of AFB1 by montmorillonite, but it was favorable for the adsorption of AFB1 by montmorillonite under acidic conditions. The adsorption equilibrium was reached about 1h, and the adsorption effect was positively correlated with the adsorption time within 1h. After 1h, the adsorption effect of montmorillonite on AFB1 was stable. AFB1 adsorption rate of montmorillonite was positively correlated with the adsorption temperature.