【作者】 孙莹；

【导师】 夏雨；

【作者基本信息】 江南大学 ， 食品科学与工程， 2020， 硕士

【摘要】 黄曲霉毒素B₁（Aflatoxin B₁,AFB₁）是一种天然的高毒性次级代谢产物,广泛存在于自然界,易污染花生、玉米、大米等谷物,被世界卫生组织的癌症研究机构（IARC）划定为Ⅰ类致癌物。AFB₁对人和家畜的健康安全都产生了极大的威胁,因此研究AFB₁的降解脱毒对食品和饲料加工工业具有重要意义。锰过氧化物酶（Manganese peroxidase,MnP）能够裂解AFB₁呋喃环上的8,9位双键,将AFB₁氧化生成AFB₁-8,9环氧化物,经Mn P氧化产生的环氧化物会自发水解为AFB₁-8,9-二氢二醇,从而达到对AFB₁降解脱毒的目的。本文在食品级乳酸克鲁维酵母（Kluyveromyces lactis）中实现了三种不同来源的锰过氧化物酶的分泌表达,并对降解AFB₁效果最优的重组菌的诱导条件和反应条件进行优化,从而实现直接利用其发酵液高效降解AFB₁。具体研究如下:1、本研究实现了将三种不同来源的Mn P在食品级乳酸克鲁维酵母中的分泌表达,成功构建了重组酵母GG799（p KLAC1-Phsmnp）、GG799（p KLAC1-Plomnp）和GG799（p KLAC1-Phcmnp）,发现构建成功的三个重组菌发酵液对AFB₁降解率分别为35.55±3.30%,40.02±1.77%和50.52±3.69%,其中重组菌GG799（p KLAC1-Phcmnp）发酵液对AFB₁的降解效果最好,和另外两个重组菌对AFB₁的降解存在显著差异。2、本研究对重组菌GG799（p KLAC1-Phcmnp）诱导表达条件进行了优化,最优的诱导表达条件为:诱导温度为30°C,氯高铁血红素（Hemin）浓度为1.0 mmol/L,诱导时间为96 h,Mn SO₄浓度为1.0 mmol/L,转速为200 rpm,初始培养基p H为6.5,半乳糖浓度为50.0 g/L。在该条件下诱导表达,重组菌GG799（p KLAC1-Phcmnp）发酵液对AFB₁的降解率为67.40±0.74%,与优化前相比,降解率提高了16.88%。3、本研究对重组菌GG799（p KLAC1-Phcmnp）发酵液降解AFB₁反应条件进行了优化,最优的反应条件为:反应体系中pH为4.5,反应温度为40°C,Mn SO₄浓度为1.2mmol/L,葡萄糖添加量为2.5 mmol/L,反应时间为40 h,发酵液中PhcMnP含量为3.0g/L,葡萄糖氧化酶添加量为1.5 U/mL。在该条件下进行降解反应,发现重组菌GG799（pKLAC1-Phcmnp）发酵液对AFB₁的降解率为75.71±1.21%,与优化前相比,降解率提高了8.31%。4、本研究还对重组菌GG799（p KLAC1-Phcmnp）发酵液降解AFB₁后生成的产物进行分析。发现大量m/z 346.681的[M+H]⁺离子,推测认为是分子式为C₁₇H₁₄O₈的化合物,即AFB₁-8,9二氢二醇。5、本文还研究了重组菌GG799（p KLAC1-Phcmnp）发酵液降解花生样品中AFB₁的降解效果,发现其对不同程度AFB₁污染的花生样品降解率均达到74.5%以上。6、本文构建的重组菌GG799（pKLAC1-Phcmnp）连续传代6次,前5代菌株发酵液对AFB₁的降解效果与原始菌株发酵液对AFB₁的降解效果无显著性差异（P>0.05）,且降解率均达到72.0%以上,因此,本文构建的基因工程菌稳定性较好。更多还原

【Abstract】 Aflatoxin B₁（Aflatoxin B₁,AFB₁）was a natural high-toxic secondary metabolite,widely presented in nature,could easily contaminate peanuts,corn,rice and other grains,and it was classified as a ClassⅠcarcinogen by the World Health Organization’s Cancer Research Agency（IARC）.AFB₁ posed a great threat to the health and safety of humans and livestock.Therefore,studying the degradation and detoxification of AFB₁ was of great significance to the food and feed processing industry.Manganese peroxidase（MnP）could cleave the double bond at the 8,9 position of the AFB₁ furan ring,oxidize AFB₁ to form AFB₁-8,9 epoxide,and the epoxide produced by MnP oxidation will spontaneously hydrolyze to AFB₁-8,9-dihydrodiol,so as to achieve the purpose of detoxifying AFB₁.In this paper,the secretory expression of manganese peroxidase from three different sources was achieved in food-grade Kluyveromyces lactis,and the induction conditions and reaction conditions of the recombinant bacteria with the best AFB₁degradation effect were optimized,so as to realize the direct use of its fermentation broth to efficiently degrade AFB₁,the specific research is as follows:1.This study realized the secretion and expression of MnP from three different sources in K.lactis,and successfully constructed recombinant yeasts GG799（p KLAC1-Phsmnp）,GG799（p KLAC1-Plomnp）and GG799（pKLAC1-Phcmnp）.It was found that the AFB₁ degradation rates of the three crude recombinant fermentation enzymes that have been successful constructed were 35.55±3.30%,40.02±1.77%,and 50.52±3.69%.Among them,the fermentation broth of recombinant GG799（pKLAC1-Phcmnp）had the best degradation effect on AFB₁,and was significantly different from the other two recombinant bacteria.2.The recombinant GG799（p KLAC1-Phcmnp）induction expression conditions were optimized.The optimal induction expression conditions were:induction temperature 30°C,Hemin concentration 1.0 mmol/L,induction time 96 h,MnSO₄ concentration 1.0 mmol/L,speed 200 rpm,initial medium pH 6.5,galactose concentration 50.0 g/L.Expression was induced under this condition,and the degradation rate of recombinant enzyme GG799（p KLAC1-Phcmnp）fermentation broth towards AFB₁ was 67.40±0.74%.The degradation rate increased by 16.88%compared with that before optimization,.3.The reaction conditions of AFB₁ degradation by recombinant enzyme GG799（p KLAC1-Phcmnp）crude fermentation broth were optimized.The optimal reaction conditions were:pH in the reaction system was 4.5,reaction temperature was 40°C,and MnSO₄concentration was 1.2 mmol/L The glucose content was 2.5 mmol/L,the reaction time was 40h,the protein content in the fermentation broth was 3.0 g/L,and the glucose oxidase content was 1.5 U/mL.The degradation reaction was carried out under this condition,and it was found that the degradation rate of recombinant enzyme GG799（pKLAC1-Phcmnp）fermentation broth towards AFB₁ was 75.71±1.21%.The degradation rate increased by 8.31%compared with that before optimization,.4.The products produced after the degradation of AFB₁ by recombinant GG799（p KLAC1-Phcmnp）fermentation broth were analyzed.A large number of[M+H]⁺ions of m/z346.681 were found.It was speculated that Mn³⁺,peroxy free radicals,formate free radicals and superoxide anion free radicals generated during the self-circulation reaction of Mn P could oxidize the 8,9 vinyl group of AFB₁,and the resulting epoxide would spontaneously hydrolyze to produce AFB₁-8,9 dihydrodiol.5.The degradation effect of recombinant enzyme GG799（p KLAC1-Phcmnp）fermentation broth on the degradation of AFB₁ in contaminated peanut samples was studied,and it was found that the degradation rate of peanut samples contaminated by AFB₁ in different degrees reached more than 74.5%.Therefore,it was considered that the fermentation broth of recombinant bacteria GG799（p KLAC1-Phcmnp）fermentation had potential value for the degradation of AFB₁ in actual products.6.The recombinant strain GG799（pKLAC1-Phcmnp）constructed in this paper was passaged 6 times in succession.There was no significant difference in the degradation effect on AFB₁ between the first 5 generations strain fermentation fermentation broth and the original strain fermentation fermentation broth（p>0.05）,and the degradation rates were all more than72.0%.Therefore,the stability of the genetically engineered bacteria constructed in this paper was good.更多还原