玉米主要霉菌毒素体外评定与吸附剂研发

发布时间：2018-11-08 14:03

【摘要】：霉菌毒素是霉菌在基质(饲料、饲料原料等)上生长的过程中所产生的有毒的次级代谢产物,包括霉菌使基质(饲料)的成分转变而形成的有毒物质。霉菌毒素能够干扰许多靶器官和系统,尤其是肝、肾、神经系统、内分泌系统和免疫系统,引发人类和动物疾病。研究表明当植物受到霉菌毒素的侵害时,会进行自我保护,将非极性的毒素与糖、氨基酸或硫酸盐等结合,转化为极性更强的代谢产物,储存在液泡中或结合在器官、组织、细胞器等的生物大分子上,从而产生霉菌毒素的结合态。常规的霉菌毒素检测方法只能检测到游离态的霉菌毒素,而检测不到结合态的霉菌毒素,因而这部分结合态的毒素也被称为隐蔽性毒素。然而研究表明结合态的霉菌毒素经动物消化后会被部分或全部释放出来,危害畜禽的健康。因此,建立一种快速真实地评定饲料及饲料原料中霉菌毒素(游离态和结合态)的含量显得尤为重要。本研究通过3个试验分别研究了霉菌毒素的体内外释放测定,在此基础上分析了现有的霉菌毒素吸附剂对黄曲霉毒素B1、玉米赤霉烯酮和呕吐毒素的吸附效率,并开展了新型吸附剂的开发研究。为了能够真实地评定结合态和游离态的霉菌毒素,本试验建立了体外消化评定法。通过体外消化法模拟动物体内的消化过程对发霉玉米进行消化。玉米样品在60目的筛下物样品,先后用胃蛋白酶和胰蛋白酶进行体外消化,然后再用ELISA的方法对消化前后的霉菌毒素进行检测。试验结果表明玉米中的霉菌毒素AFB1 (P0.001)、 DON (P0.01)、ZEN (P0.05)经过体外消化后毒素含量会明显升高。采用了高效液相色谱法对ELISA检测的结果进行了验证,试验结果表明表明玉米中的霉菌毒素AFB1 (P0.05)、DON (P0.001)、ZEN (P0.001)经过体外消化后毒素含量会明显升高,高效液相色谱法进一步验证了玉米中的霉菌毒素AFB1、DON、ZEN经过体外消化后毒素含量会明显升高。为了验证体外消化试验结果的真实性,还进行了肉鸡体内消化试验来验证体外消化试验的结果。试验挑选10只体重基本一致的健康AA肉仔鸡,单笼饲养,禁食12h,自由饮水,自由采食含1%三氧化二铬的发霉玉米4h,采集十二指肠、空肠、回肠内容物。分别检测饲料、十二指肠内容物、空肠内容物、回肠内容物中的三氧化二铬(湿法消化法)及霉菌毒素AFB1、DON、ZEN含量(ELISA)。试验结果表明发霉玉米经肉鸡体内消化后AFB1 (P0.05)、DON (P0.05)、ZEN (P0.01)的含量显著升高。上述结果表明：霉菌毒素在玉米体内存在结合态和游离态两种形式,结合态霉菌毒素经消化后会被充分的释放出来,转变为游离态的霉菌毒素。为了真实地评价霉菌毒素吸附剂的吸附效果,本试验还通过体外消化评定法对市场上收集的10种霉菌毒素吸附剂的吸附效果进行了评定,试验结果表明3#、9#吸附剂对三种毒素均有较好的吸附效果,其它吸附剂对各个毒素的吸附效果各有侧重,3#、9#是酵母细胞产品,这说明酵母细胞产品对霉菌毒素的吸附效果较好。为了有效地去除饲料中的霉菌毒素,本课题研发了一种新型经济有效的霉菌毒素吸附剂,并用体外消化评定法对该吸附剂的吸附效果进行评价。试验结果表明：该吸附剂对黄曲霉毒素B1(AFB1)的吸附率达49.17%,对呕吐毒素(DON)的吸附率达67.27%。
[Abstract]:The mycotoxin is a toxic secondary metabolite produced during the growth of a mold on a substrate (feed, feed, etc.), including a toxic substance formed by the transformation of the components of the substrate (feed) by mold. The mycotoxin can interfere with many of the target organs and systems, in particular the liver, the kidney, the nervous system, the endocrine system and the immune system, triggering human and animal diseases. The research shows that when the plant is attacked by the mycotoxin, self-protection is carried out, the non-polar toxin is combined with sugar, amino acid or sulfate and the like, converted into a more polar metabolite, stored in the vacuole or combined with the biological macromolecules such as organs, tissues, organelles, and the like, so as to produce the bound state of the mycotoxin. The conventional mycotoxin detection method can only detect the mycotoxin of the free state, but the mycotoxin of the bound state can not be detected, and thus the toxin of the bound state is also referred to as the hidden toxin. However, the study shows that the bound mycotoxin can be partially or completely released after the animal is digested, which is harmful to the health of the livestock and poultry. Therefore, it is very important to establish a fast and realistic evaluation of the content of mycotoxins (free and bound) in feed and feed materials. In this study, the in vitro release of mycotoxin was studied by three experiments, and the adsorption efficiency of the existing mycotoxin adsorbent to the aflatoxin B1, the zearalenone and the Vomiting toxin was analyzed, and the development and research of the new type of adsorbent were also carried out. In order to be able to truly assess the bound and free mycotoxins, an in vitro digestion and evaluation method was established. the musty corn was digested by an in vitro digestion process to simulate the digestion process in the animal. The corn sample was digested with pepsin and trypsin in vitro at 60 mesh, and then the mycotoxin before and after digestion was detected by ELISA. The results showed that the content of mycotoxin AFB1 (P0.01), DON (P0.01) and ZEN (P0.05) in maize increased significantly after in vitro digestion. The results of ELISA were validated by high performance liquid chromatography. The results showed that the content of mycotoxin AFB1 (P0.05), DON (P0.01) and ZEN (P0.001) in maize increased significantly after in vitro digestion. The content of mycotoxin AFB1, DON and ZEN in maize was increased significantly after in vitro digestion with high performance liquid chromatography. In order to verify the authenticity of the in vitro digestion test results, the in vivo digestion test was conducted to verify the results of the in vitro digestion test. 10 healthy AA broilers with almost the same body weight were selected for feeding, fasting for 12h, free drinking water, and free feed of musty corn with 1% chromium oxide for 4h, and the contents of the duodenum, the jejunum and the ileum were collected. The contents of the feed, the contents of the duodenum, the contents of the jejunum, the chromium trioxide in the contents of the ileum (wet digestion method) and the mycotoxin AFB1, DON and ZEN (ELISA) were respectively detected. The results showed that the content of AFB1 (P0.05), DON (P0.05) and ZEN (P0.01) increased significantly after the body was digested with musty corn. The above results show that the mycotoxin is in the form of binding and free state in the corn, and the bound mycotoxin can be fully released after being digested and transformed into a free-form mycotoxin. In order to evaluate the adsorption effect of the mycotoxin adsorbent, the adsorption effect of 10 kinds of mycotoxin adsorbent collected on the market was evaluated by in vitro digestion and evaluation. The results showed that 3 # and 9 # adsorbent had good adsorption effect on the three kinds of toxins. The effect of other adsorbents on the adsorption of each toxin is different, and the 3 # and 9 # are the product of the yeast cell, which indicates that the yeast cell product has good adsorption effect on the mycotoxin. In order to effectively remove the mycotoxin from the feed, a new type of economical and effective mycotoxin adsorbent was developed and the adsorption effect of the adsorbent was evaluated by in vitro digestion and evaluation. The results showed that the adsorption rate of Aflatoxin B1 (AFB1) was 49. 17%, and the adsorption rate of VON was 67. 27%.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816;S859.8

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