【摘要】：乳糖酶全稱為β-半乳糖苷酶,在食品、藥品、保健品行業(yè)的應(yīng)用潛力很大。乳糖酶在醫(yī)藥領(lǐng)域的應(yīng)用方面可以作為酶類藥物,治療乳糖消化不良癥狀,工業(yè)領(lǐng)域通過乳糖酶制備低乳糖或無乳糖乳制品,應(yīng)用于發(fā)酵乳制品、干乳清的處理和排放、生產(chǎn)低聚半乳糖。基因工程技術(shù)的飛速發(fā)展特別是真核表達(dá)系統(tǒng)的發(fā)展,使得具有優(yōu)良酶學(xué)性質(zhì)的不同來源的乳糖酶可以利用基因工程技術(shù)進(jìn)行異源表達(dá)以及對乳糖酶在分子水平上進(jìn)行改造加工,能夠使乳糖酶制劑得到規(guī)模性的生產(chǎn)和應(yīng)用。但是由于人們對轉(zhuǎn)基因食品的食用安全性存在疑慮,通過生物技術(shù)獲得的乳糖酶要推廣應(yīng)用就必須提供食用安全的科學(xué)依據(jù),因此,對微生物表達(dá)的乳糖酶食用安全性研究對于保障我國轉(zhuǎn)基因食品安全,促進(jìn)重組乳糖酶的進(jìn)一步推廣應(yīng)用具有重要意義。本課題中研究的對象是由中國農(nóng)業(yè)科學(xué)院生物技術(shù)研究所研發(fā)的重組畢赤酵母表達(dá)乳糖酶。本研究通過借鑒已有的轉(zhuǎn)基因植物性食品的食用安全性評價方法,對重組畢赤酵母表達(dá)乳糖酶進(jìn)行了系統(tǒng)的全面的食用安全性研究。主要對重組畢赤酵母表達(dá)乳糖酶采用胃腸模擬消化試驗進(jìn)行潛在毒性研究；通過Ames試驗、小鼠骨髓微核試驗及小鼠精子畸形試驗進(jìn)行致突變研究；對SD大鼠進(jìn)行致畸毒性研究；90天大鼠喂養(yǎng)試驗進(jìn)行亞慢性毒性研究。研究結(jié)果表明：1、胃腸模擬消化試驗顯示,重組畢赤酵母表達(dá)乳糖酶在胃液中極易消化,在腸液中極難消化,毒性風(fēng)險��；2、重組畢赤酵母表達(dá)乳糖酶樣品對TA97、TA98、TA100、TA102菌株的回變菌落數(shù)均在正常范圍內(nèi),并無劑量反應(yīng)關(guān)系,各劑量組微核率、精子畸形率與正常對照組比較,沒有明顯差異；3、對SD大鼠的致畸指數(shù)10,致畸危害數(shù)300,致畸可能性�。�4、用添加重組畢赤酵母表達(dá)乳糖酶的日糧喂養(yǎng)大鼠90天后,大鼠的生長指標(biāo)和毒理指標(biāo)與對照組沒有明顯差異,沒有發(fā)現(xiàn)亞慢性毒性作用。本研究結(jié)果表明在正常值范圍內(nèi)使用重組畢赤酵母表達(dá)乳糖酶潛在毒性與過敏性的風(fēng)險較低。本研究旨在對重組畢赤酵母表達(dá)乳糖酶的食用安全性評價基礎(chǔ)之上,為我國轉(zhuǎn)基因食品的食用安全性評價標(biāo)準(zhǔn)體系的完善提供一些理論依據(jù),同時為酵母表達(dá)乳糖酶的商業(yè)化生產(chǎn)提供科學(xué)依據(jù),為生產(chǎn)者和消費者提供一定的科學(xué)指導(dǎo),促進(jìn)轉(zhuǎn)基因食品市場的規(guī)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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：TS201.25
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本文編號：2422323