| Aflatoxin B1(AFB1) is a toxic metabolite mainly produced by the fungal species Aspergillus flavus and Aspergillus parasiticus. It is a potent teratogen, mutagen, and hepatocarcinogen. Peanut and its products are often contaminated by AFB1, which seriously threat the health of human and animals. It leads to enormous economic loss each year for the peanut producers and processors due to the contamination of AFB1. The prevention or control of the contamination of aflatoxins in peanuts is a difficult problem in the world. In the 60’s of the last century, ozone had been used to degrade AFB1 in cottonseed and peanut meals. Ozone has high reactivity, penetrability, and spontaneous decomposition into a non-toxic product(oxygen) without forming any hazardous residues on treated products. So it has been widely applied in the researches in detoxifying aflatoxins in agricultural products. However, the ozonolysis mechanism of AFB1 has not been detaily studied and reported by scholars up to now, and the oxidation products of AFB1 have not been identified. And most researches in the ozonolysis of AFB1 were restricted in the experiment room, and the corresponding equipments in detoxifying AFB1 have not been developed and applied in practical production. Firstly, the ozonolysis mechanism of AFB1 was studied in the mode system in this research, and identified its oxidation products. Secondly, two sets of equipments in degrading AFB1 in agricultural products developed by ourselves had been used to decomposed AFB1 in peanuts. The safety of peanuts contaminated by AFB1 before and after being detoxified were evaluated. It provides an important theory evidence and technical support for applying these results in the food industry. The main results are as follows:1. AFB1 was rapidly degraded by ozone in the mode system with the increase of exposure time. AFB1 in acetonitrile solution was degraded completely within 25 seconds, and produced many intermediate products. In these products, thirteen products were identified, i.e. C17H14O10(m/z 379.0667), C17H12O9(m/z 361.0554), C16H12O8(m/z 333.0611), C16H10O8(m/z 331.0452), C18H16O10(m/z 393.0822), C15H10O5(m/z 271.0599), C15H10O6(m/z 287.0551), C16H10O7(m/z 315.0499), C16H10O6(m/z 299.0556), C17H12O8(m/z 345.0605), C19H15NO9(m/z 402.0820), C17H12O9(m/z 361.0555), C17H12O9(m/z 361.0555), and C17H12O6(m/z 313.0707). Among the thirteen products, six products were the main ones, i.e. C17H14O10(m/z 379.0660), C18H16O10(m/z 393.0816), C16H10O6(m/z 299.0556), C19H15NO9(m/z 402.0820), C17H12O9(m/z 361.0555), and C17H12O9(m/z 361.0555).Based on the intermediate products, two ozonolysis pathways of AFB1 were deduced. The first pathway pass through the Criegee reaction, which is the main oxidation pathway. AFB1 was oxidized to C16H10O8(m/z 331.0452), C17H14O10(m/z 379.0667) and C18H16O10(m/z 393.0822) by Criegee reaction in this pathway. The second pathway is methoxyl reaction on benzene ring, i.e. AFB1 is oxidized to C16H10O7(m/z 315.0502) and C15H10O6(m/z 287.0551).2. According to the relation between the structure of AFB1 and its toxicity, the C8-C9 double bond of the furan ring on the left and the lactone ring on the right of AFB1 are essential for its toxic and carcinogenic activity. Seen from the structures of thirteen oxidation products, the C8-C9 double bonds on the furan ring of nine oxidation products were destroyed by ozone, and the lactone rings of two oxidation products were broke. The methoxyls on the benzene ring of the remained two oxidation products were replaced by hydroxyl. Among the six main oxidation products, the C8-C9 double bonds on the furan ring of five products were destroyed by ozone except the oxidation product C16H10O6(m/z 299.0556). Compared with the toxicity of AFB1, the toxicity of most oxidation products of AFB1 were reduced, and even lost their toxicity except the oxidation product C16H10O6(m/z 299.0556), which has still some toxicity because the C8-C9 double bonds on the furan ring had not been destroyed.3. Both dry and humid gaseous ozone can degrade AFB1 in peanuts effectively. After being treated for 30 h with dry ozone, AFB1 in peanuts was significantly reduced from 85.83±3.36 μg/kg to 14.70±1.92 μg/kg with a degradation rate of 82.88%(P<0.05). Under the same conditions, the humid ozone could decompose AFB1 in peanuts from 84.25±3.23 μg/kg to 8.61±0.74 μg/kg with a degradation rate of 89.78%(P<0.05). The degradation efficiency of humid ozone is better than that of dry ozone.4. The detoxifying equipment of mycotoxin in grain and oil developed by ourselves was used to degrade AFB1 in peanuts. AFB1 in peanuts were decreased from 189.53±3.23 μg/kg to 20.08±0.89 μg/kg by dry ozone with a content of 50 mg/L at the flow rate of 5.0 L/min for 60 h, and to 14.60±1.69 μg/kg for 120 h(P<0.01). After being treated for 60 h with this equipment, the concentration of AFB1 in peanuts was closed to the limited requirement of the GB 2761-2011(20 μg/kg).5. The detoxifying cooker of aflatoxins with ozone developed by ourselves was used to decompose AFB1 in peanuts. The detoxifying technology is as follows: the initial moisture content of peanuts is 9~10%; 4~5% of ozonated water(based on the weight of peanuts) is added; the concentration of gaseous ozone is 50 mg/L(the concentration of ozonated water is about 5.0 mg/L); the final moisture content of peanuts is about 14%; the temperature of the wetting layer is at 90℃ and the time is 20 min; the temperature of cooking layer is at 160℃ and the time is 40 min; the temperature of roasting layer is at 160℃ and the time is 30 min; the final temperature of peanuts with a moisture content of 2~3% reach to 110~120℃. The peanuts were pressed immediately after being cooked, and produced the pressed oil. AFB1 in peanut oil was decreased from 41.29±0.68 μg/kg for the control to 6.09±0.28 μg/kg for the treated one by ozone(P<0.01), which reduced by 85.25%. It reaches the limited requirement for AFB1 in peanut oil in GB 2761-2011(20 μg/kg).6. It had insignificant changed in the protein, fat, and unsaturated fatty acid contents in peanuts after being treated by ozone within 40 h(P>0.05). However, the content of protein in peanuts was reduced obviously after being treated for more than 40 h(P<0.05). The content of fat had a tendency of decrease with the increase of ozone treating time(P>0.05). There were insignificant changes in the contents of oleic acid, linolic acid, linolenic acid, and eicosenoic acid in peanut oils. Dry ozone treatment reduced moisture contents of peanuts, and humid ozone treatment increased their moisture contents with the increase of treating time. Dry gaseous ozone made the red of peanut skins more deep, and even changed to black, while humid gaseous ozone made the red skins faded with the increase of treating time. The polyphenols in peanut skins were destroyed during the process of ozone treatment.The ozone treatment of exceeding 40 h could increase the acid values(AV) and peroxide values(POV) of peanuts significantly. The AV of peanuts increased from 0.40±0.05 mg/g for the control to 0.91±0.20 mg/g(dry ozone) and 1.72±0.21 mg/g(humid ozone) after being treated for 40 h(P<0.05). The POV of peanuts increased from 0.25±0.05 mmol/kg for the control to 0.48±0.02 mg/g(dry ozone) and 0.52±0.07 mg/g(humid ozone)(P<0.05). The AV and POV of peanuts treated with ozone for 40 h were far less than the limited requirements of GB 16326-2005(AV ≤ 4 mg/g, POV ≤ 6.3 mmol/kg).The peanut oils obtained from ozonated peanuts were more clear and transparent than the control. The AV and POV of peanut oils obtained from ozonated peanuts were significant increased(P<0.05), but they were far less than the limited requirements of GB 1534-2003(AV ≤ 1.0 mg/g, POV ≤ 6.0 mmol/kg).7. The storage stability was good for ozonated peanuts. Storing for 28 days at 40℃, the moisture contents of ozonated peanuts were decreased with the increase of storage time, whether ordinary or vacuum packing. The AV and POV of ozonated peanuts were increased with the increase of storage time, while they were far less than the limited requirements of the Chinese national standard, and vacuum packing was better than ordinary one.8. Ozone treatment can significantly reduce the toxicity of AFB1 in contaminated peanuts, and improve their edible safety. Peanuts contaminated AFB1(189.53±2.38 μg/kg) were treated with ozone for 60 and 120 h, and AFB1 reduced to 20.08±0.89 μg/kg and 14.60±1.69 μg/kg, respectively. The ozonated peanuts were ground into paste, and used the paste to feed Wistar rats by intragastric administration. The rats did not have unusual changes in behavior, and no signs of intoxication were observed. Compared with the positive control group, the body weight gain, food intake, and feed conversion efficiency of the rats fed with the peanuts ozonated for 60 h had significantly increased(P<0.05). And the weights of the livers and the ratios of liver to body weight were significant reduced(P<0.05). In addition, the male and female rats fed with peanuts ozonated for 60 h showed significant increase in total protein(TP) and albumin(Alb), but significantly reduce in glucose(GLU), Urea nitrogen(Urea), total cholesterol(TC), triglycerides(TG), and maleic dialdehyde(MDA)(P<0.05). The activity of alanine aminotransferase(ALT) was significantly decreased, but the activity of total superoxide dismutase(T-SOD) was significant increased. No histopathological changes were observed in the livers and kidneys of rats except for minor fatty changes and vacuolar degeneration. Compared with the negative control group, all the indexes above mentioned for the rats fed with detoxified peanuts had insignificant differences(P>0.05). Ozone itself did not show any toxic effects on animals in this processing. Ames tests and Hep G2 cytotoxicity further verified that ozone treatment can significantly reduce the toxicity of AFB1 in contaminated peanuts, and improve the edible safety of peanuts. |
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