| Aflatoxins are kind of carcinogenic, mutagenic and teratogenic mycotoxins, and aflatoxin B1 which is classified as first-degree carcinogen by WHO is the most toxic. A big concern is about its extensive exitance in many agricultural products, especially in peanut and maize. Aflatoxins not only have great harm to human and animal health, but also have caused enormous loss to China's export trade.The aim of this study is to screen efficient and safe probiotics which could bind aflatoxin B1.In this study, recovery of AFB1 extracted from liquid samples by silica gel column chromatography, multi-purification column and improved solvent extraction method were compared. Results showed that the AFB1 recovery by improved solvent extraction method was highest under different added levels of AFB1 with maximum recovery of 99.14%. Therefore, improved solvent extraction method was chosen to be pre-treatment method in the detection of AFB1 concentration in liquid samples.Among total of 22 examined probiotics, 14 strains were able to remove AFB1 from media. The elimination was strain specific with the AFB1 binding ranging from 0 to 35.66% for viable cells, while ranging from 8.57% to 49.53% for heat-treated cells. In addition, binding abilities of all strains were enhanced after being heated. The most effcient removal was achieved by Y1, with a maximum removal of 49.53% when being heated at 100℃for 30 min.Therefore, Y1, the most effective binding strain, was selected for further study.Mechanism was also sudied by complex stability experiment, scanning and transmission electron microscopy. Complex stability experiments showed that AFB1 was adsorped by Y1 through physical way.The combination of them was reversible, and methanol, acetonitrile as well as other solvents could extract AFB1 fromY1-AFB1 complex. Scanning and transmission electron microscope observed that Y1 cells'surface which was smooth became wrinkling, and the cell wall thickness was increased from 90.70±0.18nm to 225.58±1.06nm, which was thicker 148.7% after being heated. The above changes both could increase contact area of Y1 and AFB1, therefore, binding ability of Y1 was greatly enhanced.Effect of different factors on Y1's ability to bind AFB1 in medium was studied by single-factor experiment after the concentration of AFB1 (25ng/ml) and cell concentration(2.5×109cells/ml) was finalized. Optimized binding conditions were as follows: the cell treatment was autoclaving cells at 121℃for 30min, binding time was 60min, binding temperature was 23℃, shaking speed was 200rpm. Under the above conditions, Y1 could bind 84.17% of the AFB1 in medium.Under the optimized conditions, AFB1 concentration in medium was 4.38ng/ml after first binding, then Y1 cells were again added to the above medium with 2.0×109 cells/ml of cell concentration to implement second binding at the same conditions as the first binding. Results showed that AFB1 could not be deceted in medium by high performance liquid chromatography after the second absorption.This suggested that second binding approach could be used to completely remove the residual AFB1 when first binding could not meet requirements.Effect of two cell storge methods: first heating(autoclaving cells at 121℃for 30min) and then chilling, first chilling and then heating(autoclaving cells at 121℃for 30min) on Y1's ability to bind AFB1. Results showed that Y1's ability to bind AFB1 after being storaged at 4℃refrigeratorin for 24 days was with no significant difference compared to 0 day if cells was first storaged at 4℃refrigeratorin and then heated before use.The ability of Y1 binding AFB1 in peanut milk was also studied. Y1 could bind 88.32% of AFB1 in peanut milk when the binding conditions was as follows: 25ng/ml of AFB1 concentration, 2.5×109 cells/ml of cell concentration, autoclaving cells at 121℃for 30min, 60min of binding time, 23℃of binding temperature, 200rpm of shaking speed.
|
PDF Full Text Request