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Different content, different adding amount montmorillonite adsorption effect on Aflatoxin B1 in ELISA method
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Abstract: Evaluate adsorption effect for the Aflatoxin B1 with 5 different content montmorillonite as different adding amount in ELISA method in vitro experiment, discover that montmorillonite content are positive correlation to adsorption rate a certain degree, beyond this range, although montmorillonite content is higher, the adsorption rate doesn’t improve too much . Montmorillonite adding amount with the AFB1 potency are positive correlation, the higher AFB1 potency is, the higher the adsorption rate is.
Keyword: Adsorbent, AFB1; Adsorption capacity
1 Materials and methods
1.1 experiment material
1.1.1 Instrumental analysis balance, Constant temperature oscillator, Centrifuge, AFB1 kit( Germany R-Biopharm)
1.1.2 Reagents: AFB1; methanol; Hydrochloric acid, sodium hydroxide ( all analytical grade)
1.1.3 Adsorbent: 70%, 85%, 92%,95%,98% montmorillonite
1.2 The preparation of daily diet with AFB1
Dissolve AFB 1 standard sample in methanol, uniformity spray on a small amount of piglet feed, after blending, make AFB1 experiment sample with the 50,100(ug/kg) content, after test, AFB1 level is the 48.9, 96.8(ug/kg)
1.3 In vitro test method
1.3.1 Evaluation on combination of adsorbent and AFB1 under PH 3 condition
1.3.1.1 Adsorption rate of montmorillonite on AFB1 under low concentration toxin (ug/kg) circumstance,
Respectively weigh 5g feed with toxins 48.9 (ug/kg) , feed sample 45 pieces, put it into 50ml with lid centrifuge tube, handle it according to sheet 1, each processing three repeat, take the average.
Sheet 1 In vitro experiment design
Adsorbent adding amount | 70% montmorillonite | 85% montmorillonite | 92% montmorillonite | 95% montmorillonite | 98% montmorillonite |
0.01 g | 0.01 g | 0.01 g | 0.01 g | 0.01 g | |
Feed amount 0.3% | 0.015 | 0.015 | 0.015 | 0.015 | 0.015 |
Feed amount 0.4% | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 |
37℃constant temperature oscillate and adsorb 10 min, 4000/r min centrifuge 4min, determine supernatant AFB1 content with ELISA method, set up not add the adsorbent, eliminate nonspecific adsorption of AFB1 in the experiment
According to the formula to calculate the adsorption rate Y (%)
In the formula: Y( adsorption rate); C0：AFB1 Initial concentration（μg/ml）；C1：AFB1 concentration in supernatant when balance（μg/ml）；v：constant volume（ml）。
1.3.1.2 Adsorption rate of Montmorillonite on AFB1 under high concentration of toxin (98.8μg/kg) circumstance；
Respectively weigh 5g feed with 98.8 (ug/kg) , feed sample 45 pieces, put it into 50ml with lid centrifuge tube, handle it according to sheet 2, each processing three repeat, take the average.
Sheet 2 In virto experiment design
Adsorbent adding amount | 70% montmorillonite | 85% montmorillonite | 92% montmorillonite | 95% montmorillonite | 98% montmorillonite |
Feed amount0.2% | 0.01 g | 0.01 g | 0.01 g | 0.01 g | 0.01 g |
Feed amount0.3% | 0.015 | 0.015 | 0.015 | 0.015 | 0.015 |
Feed amount0.4% | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 |
37℃constant temperature oscillate and adsorb 10 min, 4000/r min centrifuge 4min, determine supernatant AFB1 content with ELISA method, set up not add the adsorbent, eliminate nonspecific adsorption of AFB1 in the experiment
1.4 Statistical analysis
Result is shown as average±standard deviation, using SAS 6.12 to analysis of variance, P < 0.05 as the significance of difference judgment standard
2. Results and analysis:
2.1 Vitro experiment result and analysis
Sheet 3
No. | AFB1 Content (ug/kg） | Different content montmorillonite Adding amount 0.2%(as feed amount) | Different content montmorillonite Adding amount 0.3%(as feed amount)
| Different content montmorillonite Adding amount 0.4%(as feed amount) | ||||||||||||
70% | 85% | 92% | 95% | 98% | 70% | 85% | 92% | 95% | 98% | 70% | 85% | 92% | 95% | 98% | ||
1 | 48.9 | 58.4 | 66.2 | 80.1 | 79.1 | 80.3 | 70.1 | 72.3 | 90.5 | 90.2 | 92.3 | 70.8 | 76.7 | 92.5 | 93.8 | 95．2 |
2 | 98.8 | 60.1 | 67.1 | 84.3 | 86.2 | 89.1 | 68.2 | 77.3 | 93.1 | 93.3 | 95.0 | 72.1 | 76.8 | 93.2 | 94.3 | 95.5 |
Conclusion：it can be seen from sheet 3 and sheet1、sheet 2
1、Montmorillonite has better adsorption effect under acid environment
2、The higher montmorillonite content is, the higher adsorption rate for AFB1 is, only the montmorillonite content is more than 90%, it has better adsorption for AFB1
3、It has no significant adsorption effect on AFB1 when montmorillonite content is more than 95%
4、The higher toxin rate in feed is, the higher adsorption rate of montmorillonite is.
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