黄曲霉毒素解毒酶项目介绍 Aflatoxin-detoxifizyme Application Development

1. 国家863高科技项目 National "863" Key Project 黄曲霉毒素解毒酶项目介绍Aflatoxin-detoxifizyme Application Development 广州科仁生物工程有限公司 Guangzhou Co-win Bioengineering Co., Ltd

2. 黄曲霉毒素（Aflatoxin，AFT）What is AFT? • 是一类由黄曲霉和寄生曲霉产生的真菌毒素； • A kind of mycotoxin produced by Aspergillus flavus and Aspergillus parasiticus Speare • 已发现B1、B2、G1、G2、M1、M2等十几种，其中以黄曲霉毒素B1（AFB1）的毒性最强. • Including Aflatoxin B1,B2,G1,G2,M1and M2 etc, among which AFB1 is the most toxic. 黄曲霉菌 (Aspergillus flavus) AFB1的化学结构 Structure of AFB1

3. AFT的污染广泛AFTcontamination is wide-spread • 黄曲霉和寄生曲霉适宜在温暖潮湿的环境下生长并产生毒素，广泛污染饲料、粮食和食品，例如花生、玉米、大米、小麦、牛奶等。 • Aspergillus flavus prefer warm and moist conditions to grow and produce toxins; Most feed and food could be contaminated, such as peanut, corn, rice and milk. 发霉的棉籽(a) 发霉的玉米(b) 发霉的大米(c) Contaminated cottonseed (a), corn (b) and rice (c)

4. AFT的毒性巨大 AFT is very toxic • 被国际癌症研究机构（IARC）列为天然存在的1类（Group-A）致癌物，人类摄入被污染食品可诱发原发性肝癌、胃癌、肺癌等。 • AFT had been classified as carcinogen of Group A by IARC, could cause human gastric carcinoma, Iiver cancer and lung cancer etc. • 毒性(toxicity)是氢氰酸的10倍，砒霜的68倍。 • Toxicity of AFT is 10 times of hydrocyanic acid and 68 times of arsenic. • 0.294毫克/公斤剂量的AFB1就能引起敏感动物的急性中毒死亡。 • 0.294mg/kg AFB1 could cause susceptive animals die of acute toxicity

5. AFT毒性的危害Ways for AFT to harm human beings 污染饲料 feed contamination 污染食品 food contamination AFT 人类 human 禽、畜 Birds and beasts 食物链food chain 轻度中毒 毒素蓄积 Light poisoning 严重中毒 Serious poisoning 毒素蓄积 Toxin accumulation 体重下降、生病lose weight, get sick 死亡 death 诱发肿瘤 tumor

6. 各国食品中AFT含量的标准Standards of AFT in Food

7. 我国的AFT污染情况AFT contamination in China • 污染广泛，形势严峻 • Wide-spread and Serous • 2004年，我国31个储藏稻谷样品检测真菌毒素含量，结果显示AFT阳性率达90.32%。 • In 2004, AFT contamination ratio of 31 examined stored paddy samples is 90.32% • 1999年，韶关钢铁集团花生油调查,AFT大于20 g/kg的检出率为20.7%。 • In 1999, among examined peanut oil, percentage of samples with AFT concentration above 20 μg/kg is 20.7% • 2000年，某地区市售部分有品牌酱油抽样检查,结果：样品30个，AFB1呈阳性10个。 • In 2000, results of a spot check of 30 samples of soy sauce for certain brand showed that 10 of 30 was AFT positive. GRAIN STORAGE。2004 Vol.33 No.4 P.49-51 Journal of Jinan University（Natural Science & Medicine Edition）2002，23（5）：95-100

8. 过往常用的AFT 去毒方法Common Methods to detoxify AFT • 一、物理方法（physical） • 1.筛选法(pick out) 2.漂洗法(rinse)3.热处理法(heat) • 局限性： 去毒不彻底；经过处理的食物保存时间短 ，易产生二次污染 • Shortage: not thoroughly; shorter reserve time, easy to secondary contamination • 二、化学方法（chemical） • 1.氨化处理(amination) 2.粘土或沸石处理(clay or zeolite) • 3.生石灰处理(calces) • 局限性：成本高；去毒不彻底 • Shortage: high cost; not thoroughly • 三、生物学方法（biological） • 1.乳酸菌发酵法(lactobacillus fermentation) • 局限性：仅适用于轻度发霉的饲料；处理后的饲料禁止作为主要饲料喂用 • Shortage: only for lightly contaminated feed; treated feed can only be used as supplement

9. 黄曲霉毒素解毒酶（Aflatoxin-detoxifizyme,ADTZ）A new fungi-found enzyme: ADTZ • 从食用真菌中提取出来的一种蛋白质。 • Protein extracted from a kind of edible fungi • 在温和的作用条件下，可以与AFB1作用，破坏毒性结构双呋喃环，使其开环而起到解毒作用。 • under mild conditions, ADTZ could interact with AFB1 to break the toxic structure bifuran and function as antidote against AFT • 是国际上首先发现并分离到的针对黄曲霉毒素的解毒酶,是一种新蛋白质酶； • First discovered and isolated in international wide, was proved to be a new enzyme。 • 为我国源头创新性的发现； • An innovative, China-source discovery • 具有完全独立的自主知识产权。 • Independent Intellectual Property Rights

10. 重组黄曲霉毒素解毒酶（rADTZ）Our recombinant enzyme：rADTZ • 利用分子生物学技术获得了真菌ADTZ的全长基因序列，并将该基因转入酵母中表达，构建了酵母基因工程菌． • full-length ADTZ gene had been cloned from the fungi and expressed in the gene engineer yeast reconstructed. 真菌培养,蛋白提纯 基因克隆技术 Fungi cultivation protein purification Gene cloning 产酶起始真菌 Original fungi 新蛋白质酶ADTZ New enzyme ADTZ ADTZ基因 ADTZ gene 基因重组技术 工程菌发酵 Gene recombination fermentation 重组蛋白质酶rADTZ Recombinant enzyme rADTZ 产ADTZ工程菌 Gene engineer yeast

11. rADTZ的研究背景Research background of rADTZ • 本项目研究历时10年，先后获得过国家“863”计划、国家自然科学基金、广东省自然科学重点项目和科技攻关计划项目等资助 • Research lasted 10 years; obtained financial supports from “863”project, national science foundation, key science projects of Guangdong province etc. • 申请了国内发明专利6项，国际发明专利1项，部分已获授权 • Applied 6 national patents and 1 international patent, part of which have been licensed. • 已经成功构建rADTZ的重组酵母基因工程菌 • Successfully constructed rADTZ-producing gene engineering yeast • 目前已经完成20升小试规模的发酵工艺 • Small-scale of 20L fermentation technology of gene engineering yeast has been achieved.

12. rADTZ的应用领域What can rADTZ do? • 饲料脱毒添加剂 detoxifying additive for feed • 食品脱毒添加剂 detoxifying additive for food • 酶生物传感器 enzyme biosensor • 药用酶制剂 pharmaceuticals enzyme • 农作物转基因改造 Transgenic reconstruction of special crops rADTZ的应用开发技术平台 Platform of rADTZ application development

13. rADTZ的应用前景Promising prospects of rADTZ • 一、饲料行业(Feed Industry) • 全世界每年平均至少约有2%的粮食因污染霉菌发生霉变而不能食用和饲用。 • Every year about 2% of foodstuffcannot be fed or eaten because of mustinessin the world. • 全世界供应的谷物中有25％受到霉菌毒素污染。 • 25% of the world’s grain supply was polluted by mycotoxin. • 目前市场上还没有一种有效的办法对霉菌毒素的污染进行安全而廉价的脱毒处理 ; • Currently no method can detoxify mycotoxin effectively, safely and economically. • 除本项目拟研发的重组黄曲霉毒素解毒酶产品，是特异性的针对毒性最强的霉菌毒素---黄曲霉毒素之外，目前没有同类产品. • No product except ours has been developed to specifically detoxify AFB1, the most toxic mycotoxin. • 本项目拟开发的产品具有巨大的市场前景。 • Our products have great market prospect.

14. rADTZ的应用前景Promising prospects of rADTZ • 一、饲料行业(Feed Industry) • 2005年全国饲料总产量1.07亿吨，工业总产值为人民币2742亿元。用于脱毒的工业酶成本占饲料生产成本的1%，本项目产品在国内饲料市场的总价值为人民币27亿元/年。 • Feed output of China was 107 million in 2005, with the industry value of 274.2 billion RMB. The cost of industrial enzyme was 1% of the total cost of feed, thus our enzyme will have the total value of 2.7 billion per year in the domestic feed market. • 目前饲料用酶已成为世界工业酶产业中增长速度最快、势头最强劲的一部分，2000年饲料用酶的市场值达到了2.5亿美元，近5年的年增长率达到11％，远远高于别的酶种，预计到2010年，饲料用酶的市场值将达到8亿美元。 • Enzymes used for feed has been the fastest increasing one in the world industrial enzyme industry, whose market value was 250 million dollar in 2000, with a increasing speed of 11% in the recent 5 years. It was said that in 2010, its market value will be 800 million dollar. • 由于本项目产品已获国际专利，故本项目产品在国际工业酶市场上发展潜力无可限量，盈利前景极为可观。 • Owing to the patents we had applied, our product has infinite developing potential and promising prospect the international industrial enzyme market.

15. rADTZ的应用前景promising prospects of rADTZ • 二、食品行业(food industry) • 我国食物和花生油的黄曲霉污染水平达23～500μg/kg • The AFB1 contamination level of food and peanut oil in China was 23～500μg/kg. • 牛奶目前尚没有现成可供使用的脱毒方法。 • Currently no method can be used to detoxify AFT-polluted milk. • 常用脱毒方法如碱处理法和紫外线照射法等对花生油的品质和保质都有一定的影响。 • Methods used to detoxify peanut oil such as alkali treatment and UV radiation may affect the quality. • 黄曲霉毒素解毒酶对花生油的脱毒条件温和，是和AFB1发生特异性反应，对花生油的品质和保质无影响。具有巨大的开发潜力和广阔的市场前景。 • ADTZ could detoxify AFB1 specifically under mild conditions and has no effect on the peanut oil quality and storage, thus has great developing potential and promising prospects.

16. rADTZ的应用前景Promising prospects of rADTZ • 二、食品行业(Food Industry) • 2005年全国花生油总产量230万吨，工业总产值为人民币225亿元。本项目产品在国内花生油市场的市场总价值为人民币2.2亿元/年。 • In 2005, annual output of peanut oil was 2 million 300 thousand ton, total industrial value at 22.5 billion RMB in China. Market value of our product will be 220 million RMB per year in the domestic peanut oil market. • 2005年中国调味品、发酵制品的累计工业总产值为540亿元，增长速度高达35％，酱油行业正以每年30％—50％的速度递增，市场潜力很大。本项目产品在国内酱油等调味品市场的市场总价值为人民币5.4亿元/年。 • Total industrial value of spice and fermented food was 54 billion RMB in China in 2005, a growth of 35%. Soy sauce market in China is rapidly growing at 30-50% annually. Our product will have a total value of 540 million per year in domestic spice market. • 2005年全国乳制品总产量2300万吨，工业总产值为人民币2300亿元。本项目产品在国内乳制品市场的市场总价值为人民币23亿元/年。 • Total output of milk product was 23 million ton in China in 2005, with the total value of 230 billion RMB. Total market value of our product is 2. 3 billion RMB per year.

17. rADTZ的应用前景Promising prospects of rADTZ • 三、健康行业(Health Industry) • 研制药用酶制剂，用于原发性肝癌 、胃癌、肺癌的防治 • For prevention and cure of gastric carcinoma, primary hepatocarcinoma (PHC) and lung cancer • 原发性肝癌高危人群：HBV携带者、肝炎患者比率在全球至少有10%以上，在中国的某些地区达到了15-20%，全球高危人群人口约6亿人，中国高危人群人口约1.3亿。 • Percentage of PHC extremely dangerous population, such as HBV carriers and hepatitis patients, is more than 10% in the world and 15%-20% in some chinese regions. PHC extremely dangerous population is 130 million in china, 600 million in the world. • 本项目的药用酶制剂产品在国内用于原发性肝癌高危人群的市场总价值将超过人民币100亿元/年。 • Our enzyme product will have a value of more than 10 billion RMB per year in domestic market.

18. rADTZ的应用前景Promising prospects of rADTZ • 四、检测行业(Detection) • 研制AFT酶生物传感器，用于食品、饲料加工生产、海关、卫生监测等部门的质量安全检测。 • To develop enzyme biosensor for quality and safety detection of AFT in food and feed process, custom and sanitation inspection. • 由于目前使用的检测方法均存在玷污危险，检测人员在操作时需要冒接触剧毒的危险，而酶生物传感器最大的优点是不用接触毒性标准品，具有很好的推广价值，市场潜力很大。 • AFT enzyme biosensor can operate without physical contact with toxic standard samples. It can be used for a variety of applications, thus huge market potential. • 本项目产品在国内酶制剂及AFT生物传感器市场的市场总价值为人民币1亿元/年。 • Our product has a market value of 100 million RMB per year in the enzyme and AFT biosensor market.

19. rADTZ的应用前景Promising prospects of rADTZ • 五、对特殊的农作物进行转基因改造 Transgenic reconstruction of special crops • 例如，将ADTZ基因转入花生的基因组中，使其表达，生产抗AFT的转基因花生。 • Transfer of ADTZ gene to the genome of peanut and expression of ADTZ gene will confer the peanut the property of AFT-resistant.

20. 广州科仁生物工程有限公司Guangzhou Co-win Bioengineering Co., Ltd • Founded in June 2006 • Guangzhou Konzern Medicine Co., Ltd is the wholly owned subsidiary of China Medicine Corporation which is a US public company. Konzern distributes approximately 1,100 medicine products in China, including traditional pharmaceutical medicines, traditional Chinese medicines (finished medicines made of Chinese herbs), Chinese herbs and nutritional supplements. These products include both prescription drugs and over-the-counter drugs. Its network reaches 28 provinces in China, over 300 hospitals, 500 medicine companies and 1700 drug stores.

21. 广州科仁生物工程有限公司Guangzhou Co-win Bioengineering Co., Ltd • The company is seeking the co-developer to transmit the lab results to the final products, and will apply these products in different industries. • The company will produce the ADTZ products which will be used to remove the AFT toxin from feed, dairy products, flavoring , peanut oil and other foods . • The company will make ADTZ products targeting the health industry sectors dealing with liver, stomach and other cancers • The company will make the ADTZ biological sensor products for the border customs, hygiene units, food and feed factories.

22. 广州科仁生物工程有限公司Guangzhou Co-win Bioengineering Co., Ltd We are looking forward to developing the ADTZ project together with you!