Effects Of Ozone On Aflatoxin B₁Degradation In Corn And Safety Evaluation Of The Degraded Products

Aflatoxins (AFs) are extremely hazardous secondary metabolites mainly produced byAspergillus flavus and Aspergillus parasiticus, of which AFB1, AFB2, AFG1and AFG2are mostcommon. Particularly, AFB1, which is most widely distributed and most toxic, is a class1human carcinogen. As an important material for feed and foodstuffs, corn is prone to beingcontaminated by AFB1during harvest, processing and storage. Although several physical,chemical and biological protocols have been established to degrade and to remove AFB1,efficient, safe, economical and environmentally friendly methods remain scarce. Therefore, itis of great significance to propose novel methods for degradation and removal of AFB1. AFB1has been degraded by ozone since the1960s, but most studies only referred the degradationrates of it in various raw materials. In contrast, the changes of material quality and toxicity afterozonation have seldom been accessed, which thus limits the application of this protocol inpractice. In this dissertation, by using ultra performance liquid chromatography quadrupole-time of flight mass spectrometry (UPLC Q-TOF MS), the structures of different ozonizedproducts of AFB1were analyzed, and the formation pathways and toxicity of such productswere presumed. Besides, the kinetics for AFs degradation in corn samples was studied toevaluate the physicochemical properties and toxicity after ozonation of AFB1, and to providetheoretical and practical evidences for possible application of this method. The main results aredetailed below.Study on structures of ozonized AFB1products. Ten degradation products were detectedfor the first time by reacting AFB1with gaseous ozone and ozonated water, the molecular andstructural formulas of which were postulated based on fragment ion information and accuratemasses. According to the structures of the degradation products, AFB1may be degraded in thissystem by being subjected to ozone addition. As suggested by the structure–activity relationship,the toxicity of degradation products significantly decreased.Study on kinetics for AFs degradation by ozone. With increasing ozone concentration andtreatment time, the degradation rates of AFs in corn flour and corn kernels were evidentlyelevated. After being treated with75mg/L ozone for60min, the AFB1content in corn flourwith the moisture content of17.40%reduced from53.60μg/kg to11.38μg/kg, i.e. thedegradation rate was78.76%. The AFB1contents in corn kernels with the moisture contents of13.47%and20.37%plummeted from83.0μg/kg and77.6μg/kg to4.90μg/kg and21.42μg/kgrespectively after40min of treatment with90mg/L ozone, viz., the degradation rates were94.1%and72.4%respectively. AFs in different raw materials were degraded by ozone all followingfirst order kinetics. The degradation rates of AFs in corn flour followed the descending ordersof k75mg/L>k45mg/L>k30mg/L>k15mg/Land kAFB1>kAFs>kAFG1>kAFB2, while those in corn kernelsfollowed the descending sequences of k90mg/L>k65mg/L>k40mg/Land k13.47%moisture content>k20.37%moisture content. Kinetics parameters, such as kinetic equations, reaction rate constants, correlationcoefficients and half-life periods, provided valuable evidence for controlling ozonation underoptimum conditions and for clarifying the influences of ozonation on corn quality changes andsafety. Hence, the findings allow feasible application of ozonation in degrading AFB1- contaminated corn.Study on effects of ozonation on the physicochemical properties of raw materials. After60min of75mg/L ozone treatment, the moisture content in corn flour decreased from17.40%to11.45%, while that in corn kernels dropped from13.47%to9.90%after being treated with90mg/L ozone for40min, which were both significantly lower than the safety limit for long-term storage (15.0%). In other words, ozonation was conducive to long-term storage bylowering the moisture content. The fatty acid value of corn flour remarkably exceeded thatstipulated bythe Chinese national standard of“severelyunsuitable for further storage”(>78mgKOH/100g dry basis), whereas that of corn kernels slowly increased and met the standard of“suitable for storage”(<50mg KOH/100g dry basis). Posterior to ozone treatment, the color,viscosity and gelatinization of corn flour and corn kernels, especially those of the former,changed obviously, which were adverse to the maintenance of corn quality. Being easilyoperable, economical, safe, environmentally friendly and efficient, ozone treatment coulddegrade AFB1in corn kernels satisfactorily without significantly affecting the quality.Evaluation on the safety of AFB1degradation by ozone.In vitro cytotoxicity tests showedthat AFB1in AFB1-contaminated corn kernels significantly inhibited the growth of HepG2cellsin human hepatic cancer, in addition to apparent changes of cell morphology, remarkableincrease of apoptotic cells, and significant decrease of p53protein expression. The resultsrevealed strong toxicity of AFB1(p<0.05). After ozone treatment in a degradation test onHepG2cells in human hepatic cancer, however, the survival rate of HepG2cells almostresembled that of the negative control group represented by the corn kernels that were notcontaminated by AFB1. Meanwhile, cell morphology and p53protein expression were notconsiderably affected (p>0.05). The apparent difference was that apoptotic cells weresignificantly decreased. Therefore, ozone treatment dramatically reduced the cytotoxicitycaused by AFB1contamination in corn kernels. Compared with the basic feed group, Kunmingmice fed with AFB1-contaminated corns underwent significantly decreased average weight gainand feed consumption, and significantly augmented liver-and kidney-body weight ratios(p<0.05). In the meantime, the activities of serum alanine aminotransferase, aspartateaminotransferase and alkaline phosphatase in the experimental group were significantlyenhanced, and the contents of total protein, albumin and globulin were significantly lowered(p<0.05). Moreover, their livers and kidneys suffered from obvious histological changes.Nevertheless, the average weight gain, feed consumption, as well as liver-and kidney-bodyweight ratios of the Kunming mice fed with ozone-treated AFB1-contaminated corns weresimilar to those of the control group (p>0.05). Meanwhile, the serum alanine aminotransferaseactivity, as well as the contents of total protein, albumin and globulin of the two groups werealso not significantly different (p>0.05). There were no evident histological changes in kidneytissues. The activities of aspartate aminotransferase and alkaline phosphatase were significantlyenhanced (p<0.05), which were concomitant with mild histological changes in liver tissues.Hence, AFB1-contaminated corn exerted severe deleterious effects on Kunming mice, whichcould be effectively relieved by ozone treatment.