一种重组毕赤酵母表达的新型乳糖酶的食用安全研究
发布时间：2019-02-14 15:09
【摘要】：乳糖酶全称为β-半乳糖苷酶,在食品、药品、保健品行业的应用潜力很大。乳糖酶在医药领域的应用方面可以作为酶类药物,治疗乳糖消化不良症状,工业领域通过乳糖酶制备低乳糖或无乳糖乳制品,应用于发酵乳制品、干乳清的处理和排放、生产低聚半乳糖。基因工程技术的飞速发展特别是真核表达系统的发展,使得具有优良酶学性质的不同来源的乳糖酶可以利用基因工程技术进行异源表达以及对乳糖酶在分子水平上进行改造加工,能够使乳糖酶制剂得到规模性的生产和应用。但是由于人们对转基因食品的食用安全性存在疑虑,通过生物技术获得的乳糖酶要推广应用就必须提供食用安全的科学依据,因此,对微生物表达的乳糖酶食用安全性研究对于保障我国转基因食品安全,促进重组乳糖酶的进一步推广应用具有重要意义。本课题中研究的对象是由中国农业科学院生物技术研究所研发的重组毕赤酵母表达乳糖酶。本研究通过借鉴已有的转基因植物性食品的食用安全性评价方法,对重组毕赤酵母表达乳糖酶进行了系统的全面的食用安全性研究。主要对重组毕赤酵母表达乳糖酶采用胃肠模拟消化试验进行潜在毒性研究；通过Ames试验、小鼠骨髓微核试验及小鼠精子畸形试验进行致突变研究；对SD大鼠进行致畸毒性研究；90天大鼠喂养试验进行亚慢性毒性研究。研究结果表明：1、胃肠模拟消化试验显示,重组毕赤酵母表达乳糖酶在胃液中极易消化,在肠液中极难消化,毒性风险小；2、重组毕赤酵母表达乳糖酶样品对TA97、TA98、TA100、TA102菌株的回变菌落数均在正常范围内,并无剂量反应关系,各剂量组微核率、精子畸形率与正常对照组比较,没有明显差异；3、对SD大鼠的致畸指数10,致畸危害数300,致畸可能性小：4、用添加重组毕赤酵母表达乳糖酶的日粮喂养大鼠90天后,大鼠的生长指标和毒理指标与对照组没有明显差异,没有发现亚慢性毒性作用。本研究结果表明在正常值范围内使用重组毕赤酵母表达乳糖酶潜在毒性与过敏性的风险较低。本研究旨在对重组毕赤酵母表达乳糖酶的食用安全性评价基础之上,为我国转基因食品的食用安全性评价标准体系的完善提供一些理论依据,同时为酵母表达乳糖酶的商业化生产提供科学依据,为生产者和消费者提供一定的科学指导,促进转基因食品市场的规范化。
[Abstract]:Lactase, known as 尾-galactosidase, has great potential in food, medicine and health products. The application of lactase in medicine can be used as an enzyme to treat lactose dyspepsia. In industry, lactase is used to produce low lactose or lactose free dairy products, which can be used in fermented dairy products, dry whey treatment and discharge. Production of galactooligosaccharides. The rapid development of genetic engineering technology, especially the development of eukaryotic expression system, The lactase from different sources with excellent enzymatic properties can be expressed heterogeneously by genetic engineering and modified at the molecular level, and the lactase preparation can be produced and applied on a large scale. However, because people have doubts about the food safety of genetically modified foods, the lactase obtained by biotechnology must provide scientific basis for food safety if it is to be popularized and applied. The study on the edible safety of lactase expressed by microorganism is of great significance to ensure the safety of transgenic food in China and to promote the application of recombinant lactase. In this study, recombinant Pichia pastoris expressed lactase was developed by Institute of Biotechnology, Chinese Academy of Agricultural Sciences. In this study, the food safety of recombinant Pichia pastoris expressed by lactase was studied by using the existing methods of food safety evaluation of transgenic plant food. The potential toxicity of recombinant Pichia pastoris expression lactase was studied by gastrointestinal mimic digestion test; mutagenicity was studied by Ames test, bone marrow micronucleus test and sperm malformation test of mice; teratogenicity of SD rats was studied. The 90-day feeding test was used to study the subchronic toxicity of rats. The results showed that: 1, the recombinant Pichia pastoris expressed lactase was easy to digest in gastric juice, which was very difficult to digest in intestinal fluid, and had little risk of toxicity. 2. The colony number of recombinant Pichia pastoris expressing lactase was within normal range, and there was no dose-response relationship. There was no significant difference in micronucleus rate and sperm abnormality rate between each dose group and normal control group. 3. The teratogenicity index of SD rats was 10, the number of teratogenic hazards was 300, and the possibility of teratogenicity was small. 4. After 90 days of feeding with the diet supplemented with recombinant Pichia pastoris to express lactase, there was no significant difference in growth index and toxicological index between the rats and the control group. No subchronic toxicity was found. The results showed that the risk of using recombinant Pichia pastoris to express lactase in normal range was lower. The purpose of this study was to evaluate the food safety of recombinant Pichia pastoris expressing lactase, and to provide some theoretical basis for the improvement of food safety evaluation standard system of transgenic food in China. At the same time, it provides scientific basis for the commercial production of lactase expressed by yeast, provides certain scientific guidance for producers and consumers, and promotes the standardization of genetically modified food market.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TS201.25
，
本文编号：2422323
[Abstract]:Lactase, known as 尾-galactosidase, has great potential in food, medicine and health products. The application of lactase in medicine can be used as an enzyme to treat lactose dyspepsia. In industry, lactase is used to produce low lactose or lactose free dairy products, which can be used in fermented dairy products, dry whey treatment and discharge. Production of galactooligosaccharides. The rapid development of genetic engineering technology, especially the development of eukaryotic expression system, The lactase from different sources with excellent enzymatic properties can be expressed heterogeneously by genetic engineering and modified at the molecular level, and the lactase preparation can be produced and applied on a large scale. However, because people have doubts about the food safety of genetically modified foods, the lactase obtained by biotechnology must provide scientific basis for food safety if it is to be popularized and applied. The study on the edible safety of lactase expressed by microorganism is of great significance to ensure the safety of transgenic food in China and to promote the application of recombinant lactase. In this study, recombinant Pichia pastoris expressed lactase was developed by Institute of Biotechnology, Chinese Academy of Agricultural Sciences. In this study, the food safety of recombinant Pichia pastoris expressed by lactase was studied by using the existing methods of food safety evaluation of transgenic plant food. The potential toxicity of recombinant Pichia pastoris expression lactase was studied by gastrointestinal mimic digestion test; mutagenicity was studied by Ames test, bone marrow micronucleus test and sperm malformation test of mice; teratogenicity of SD rats was studied. The 90-day feeding test was used to study the subchronic toxicity of rats. The results showed that: 1, the recombinant Pichia pastoris expressed lactase was easy to digest in gastric juice, which was very difficult to digest in intestinal fluid, and had little risk of toxicity. 2. The colony number of recombinant Pichia pastoris expressing lactase was within normal range, and there was no dose-response relationship. There was no significant difference in micronucleus rate and sperm abnormality rate between each dose group and normal control group. 3. The teratogenicity index of SD rats was 10, the number of teratogenic hazards was 300, and the possibility of teratogenicity was small. 4. After 90 days of feeding with the diet supplemented with recombinant Pichia pastoris to express lactase, there was no significant difference in growth index and toxicological index between the rats and the control group. No subchronic toxicity was found. The results showed that the risk of using recombinant Pichia pastoris to express lactase in normal range was lower. The purpose of this study was to evaluate the food safety of recombinant Pichia pastoris expressing lactase, and to provide some theoretical basis for the improvement of food safety evaluation standard system of transgenic food in China. At the same time, it provides scientific basis for the commercial production of lactase expressed by yeast, provides certain scientific guidance for producers and consumers, and promotes the standardization of genetically modified food market.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TS201.25
，
本文编号：2422323
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