【作者】 李敏；

【导师】 杨晓光； 朴建华；

【作者基本信息】 中国疾病预防控制中心 ， 营养与食品卫生学， 2009， 博士

【摘要】 目的抗性淀粉(Resistant Starch,RS)是一种“不被健康人体小肠所吸收的淀粉及其分解物的总称”。国内外研究发现,抗性淀粉通过在结肠内发酵产生有益的发酵产物促进肠道健康,抗性淀粉还能改善脂代谢以及控制餐后血糖和胰岛素升高,因此人们认为抗性淀粉是一种功能性的食物成分。转双反义SBE基因水稻是一种富含抗性淀粉的水稻,本研究将通过动物实验和人体实验对该转基因水稻的食用安全性及功效进行研究。材料与方法1.转双反义SBE基因水稻亚慢性毒性实验(90天喂养实验)初断乳Wistar大鼠100只,雌雄比例1:1,体重80g～90g,适应7天后按体重随机分为5组,每组20只,雌雄各半。分别喂饲亲本大米高剂量掺入饲料(No-GM)、转基因大米高剂量掺入饲料(GM)、转基因大米中剂量掺入饲料(Half-GM)、转基因大米低剂量掺入饲料(Quarter-GM)和AIN-93G正常对照饲料(ND)。所有动物自由进食饮水。每周称量2次进食量,1次体重。实验中期和实验结束时各检测血常规和血生化1次。90天后处死大鼠,取右股骨测量骨密度;取脑、心、肝、脾、肾、睾丸(或子宫)、胸腺称重,计算脏器系数;上述脏器及胃、十二指肠、空肠、回肠、盲肠、结肠和直肠等要进行常规病理学检查。2.转双反义SBE基因水稻对大鼠肠道健康的影响健康成年雄性SD大鼠48只,体重210g～230g,适应7天后按体重随机分为4组,每组12只。分别喂饲亲本大米高剂量掺入饲料(No-GM)、转基因大米高剂量掺入饲料(GM)、转基因大米中剂量掺入饲料(Half-GM)和AIN-93M正常对照饲料(ND)。喂养5周后连续收集4天新鲜粪便,测定粪便重量、水分、干重、pH值和短链脂肪酸含量,6周后处死大鼠并收集盲肠、结肠内容物,测定内容物和肠壁重量、内容物pH值和短链脂肪酸含量。3.转双反义SBE基因水稻预防高脂饲料诱导的大鼠血脂异常健康成年雄性SD大鼠50只,体重210g～230g,适应7天后按体重随机分为5组,每组10只。分别喂饲亲本大米高剂量掺入高脂饲料(NoGM-HF)、转基因大米高剂量掺入高脂饲料(GM-HF)、转基因大米中剂量掺入高脂饲料(HGM-HF)、高脂饲料(HF)和AIN-93M正常对照饲料(ND)。所有动物每天定量给予各组饲料,分别于实验开始后第4周、第8周、第13周取血,分离血清测定甘油三酯、胆固醇、高密度脂蛋白胆固醇含量。13周后处死大鼠并收集肝脏,测定肝脏甘油三酯和胆固醇含量。4.转双反义SBE基因水稻重要营养素消化率的体内实验研究五指山小型去势公猪8只,体重30kg～35kg。术前单个放入代谢笼内适应喂养7天,期间用通灭(多拉菌素注射液)行肠道驱虫处理。7天后,动物禁食36小时,禁水12小时,进行回肠造瘘手术。手术后2周,选择7只恢复良好的小型猪作为实验对象,将小型猪分为两组,采用交叉法分别喂饲转基因大米饲料和亲本大米饲料,饲料中加入三氧化二铬作为指示剂。每种饲料适应喂养4天后连续收集3天的食糜,然后换另一种饲料,重复上述步骤。最后统一喂饲5%酪蛋白饲料以测定内源性氨基酸的排出量。食糜收集后用均浆机混匀,冷冻干燥后再次混匀过60目筛子,分析两种大米食糜中主要营养素含量并计算消化率。采用氨基酸评分和经蛋白质消化率校正的氨基酸评分评价两种大米的蛋白质质量。实验结束后处死小型猪,并取瘘管处肠段及瘘管处上下肠段进行病理检测。5.转双反义SBE基因水稻餐后血糖和胰岛素效应以及在人体大肠中发酵情况的研究男女各10名受试者参加本实验,经过常规体检和葡萄糖耐量实验筛选后,合格受试者16名(9名男受试者平均年龄24.3±1.0,7名女受试者平均年龄24.6±1.0),将男女各分为3组采用交叉设计分别食用40g葡萄糖、相当于40g碳水化物的转基因大米和亲本大米蒸煮后的米饭,洗脱期为1周,3周内交叉食用受试物。受试者于实验当天上午6点到达实验室,静坐半小时后,于前臂静脉埋入静脉留置针收集空腹血样,分别于餐后15min、30min、45min、60min、90min、120min、180min和240min采取静脉血2mL(血糖专用的血液收集管),分离血浆-20℃保存备检。食用转基因大米和亲本大米米饭前先测定空腹呼气中氢气含量,餐后3.5h～14h每隔半小时测定1次,14h～16h每隔1小时测定1次,至16小时结束。由于要监测食用两种米饭后16小时内呼气中氢气含量,因此受试者实验前1天晚餐和实验当天的午餐及晚餐的种类和数量要统一安排,避免产氢食物,如乳制品、麦面制品和豆类制品。结果1.转双反义SBE基因水稻亚慢性毒性实验(90天喂养实验)各组大鼠平均每日摄食量之间的差异和体重之间的差异均没有显著性(P＞0.05)。实验中期,雌性大鼠转基因大米GM组平均红细胞容积显著高于亲本大米No-GM组(P＜0.05),单核细胞百分比显著低于ND对照组(P＜0.05),谷草转氨酶活性显著高于ND对照组(P＜0.05)。雌性大鼠转基因大米Half-GM组谷丙转氨酶活性显著高于亲本大米No-GM组和ND对照组。雄性大鼠转基因大米GM组平均红细胞容积显著高于ND对照组(P＜0.05)。实验末期,雌性大鼠转基因大米GM组平均红细胞血红蛋白量显著高于亲本大米No-GM组(P＜0.05),谷丙转氨酶和谷草转氨酶活性高于ND对照组。雄性大鼠转基因大米GM组红细胞压积和尿素氮显著低于亲本大米No-GM组(P＜0.05),单核细胞百分比显著高于ND对照组(P＜0.05)。转基因大米GM组雌性大鼠大脑的脏器系数显著大于亲本大米No-GM组(P＜0.05),转基因大米GM、Half-GM、Quarter-GM组雌性大鼠肾脏脏器系数显著小于ND对照组(P＜0.05)。实验大鼠各组之间血脂、血钙和骨密度均无统计学差异,内脏病理检查未发现明显异常。2.转双反义SBE基因水稻对大鼠肠道健康的影响转基因大米GM组大鼠体重增长趋势接近ND对照组大鼠,显著低于No-GM组大鼠体重的增长(P＜0.05)。与No-GM组和ND对照组大鼠相比,转基因大米GM组和Half-GM组粪便量、粪便水分、盲肠壁以及内容物含量显著增加(P＜0.05),并且上述指标在GM组和Half-GM组之间的差异也存在显著性(P＜0.05)。GM组结肠壁重量显著高于No-GM组和ND对照组(P＜0.05)。各组大鼠盲肠、结肠和粪便中短链脂肪酸的含量逐渐降低,除了Half-GM组结肠中丁酸含量与其他各组的差异没有显著性外,GM组和Half-GM组盲肠、结肠中短链脂肪酸的含量与No-GM组和ND对照组相比都有显著增加(P＜0.05),各组大鼠粪便中丁酸含量的差异消失,但是乙酸和丙酸含量的差异仍然存在(P＜0.05)。GM组和Half-GM组大鼠粪便和盲肠的pH值显著低于No-GM组和ND对照组(P＜0.05)。3.转双反义SBE基因水稻预防高脂饲料诱导的大鼠血脂异常实验前各组大鼠体重的差异和每日进食量的差异均没有显著性(P＞0.05),实验4周后,NoGM-HF组、GM-HF组、HGM-HF组和HF组大鼠体重均显著高于ND对照组,这种显著性差异一直持续至实验结束。NoGM-HF、GM-HF、HGM-HF和HF组大鼠血清甘油三酯含量在实验进行至13周时与ND对照组相比才出现显著性升高(P＜0.05),但NoGM-HF、GM-HF、HGM-HF和HF组血清甘油三酯之间的差异没有显著性(P＞0.05)。实验4周后,NoGM-HF、GM-HF、HGM-HF和HF组大鼠血清胆固醇之间的差异没有显著性(P＞0.05),但是与ND对照组相比,均有显著性升高(P＜0.05),并且这种显著性差异一直持续至实验结束。大鼠血清HDL-C从实验开始至结束,各组之间的差异均没有显著性(P＞0.05)。实验结束时各组大鼠肝脏胆固醇含量之间以及甘油三酯含量之间的差异也没有显著性(P＞0.05)。4.转双反义SBE基因水稻重要营养素消化率的体内实验研究转基因大米中18种氨基酸、蛋白质的表观消化率和真消化率与亲本大米的差异没有显著性,但是转基因大米中碳水化合物和能量的消化率显著低于亲本大米(P＜0.05)。转基因大米和亲本大米的AAS评分分别为75%和62%,PDCAAS评分分别为65%和56%。5.转双反义SBE基因水稻餐后血糖和胰岛素效应以及在人体大肠中发酵情况的研究食用转基因米饭和亲本米饭前空腹血糖浓度分别为4.7±0.3mmol/L、4.6±0.4mmol/L,差异没有显著性(P＞0.05);餐后血糖最大值分别为6.8±0.4mmol/L、7.2±0.6mmol/L,存在显著性差异(P＜0.05)。食用转基因米饭后30min、45min、60min、90min和120min的血糖值均显著低于食用亲本米饭后的血糖(P＜0.05)。以葡萄糖GI值为100作为参照,转基因大米的GI值为48.4±21.8,亲本大米GI值为77.4±34.9,两者之间存在显著性差异(P＜0.05)。食用转基因米饭和亲本米饭前空腹血浆胰岛素浓度分别为6.7±2.3μIU/mL、6.8±2.4μIU/mL,差异没有显著性(P＞0.05)。食用转基因米饭后45min、60min、90min和120min的血浆胰岛素值均显著低于食用亲本米饭后的血浆胰岛素(P＜0.05)。以葡萄糖Ⅱ值为100作为参照,转基因大米的Ⅱ值为34.2±18.9,亲本大米Ⅱ值为54.4±22.4,两者之间存在显著性差异(P＜0.05)。受试者食用转基因米饭后5h时呼气氢明显升高并维持在较高水平,最高值(38.9±10.5ppm)显著高于食用亲本米饭后呼气氢的最高值(17.6±3.7ppm)(P＜0.05)。食用转基因大米米饭后5h～16h之间各监测点上呼气氢含量均显著高于亲本大米米饭(P＜0.05)。结论1.转双反义SBE基因水稻亚慢性毒性实验(90天喂养实验)虽然个别指标在各组之间存在差异,但是大多数存在差异的指标并没有同时与两个对照组都存在差异;即使在统计学上存在差异显著性的指标,数值相差也不大,大部分在文献报道的范围内。研究认为这些改变应与转基因操作无关,并且病理检查也未发现显著异常,所以现有实验结果不能证实该转基因大米对大鼠有亚慢毒性作用。2.转双反义SBE基因水稻对大鼠肠道健康的影响转双反义SBE基因水稻能促进大鼠肠道健康,包括增加粪便体积和水分,增加大肠和粪便中SCFA含量,降低粪便和盲肠pH等。3.转双反义SBE基因水稻预防高脂饲料诱导的大鼠血脂异常虽然构建了高脂饲料诱导的大鼠血脂异常模型,但是没有发现转基因大米缓解大鼠血清胆固醇和甘油三酯升高的作用,也未见其改善肝脏脂质的效果。4.转双反义SBE基因水稻重要营养素消化率的体内实验研究转基因大米中抗性淀粉含量的增加并没有显著影响到大米中蛋白质、氨基酸的表观消化率和真消化率。由于转基因大米中抗性淀粉保持了在小肠不被吸收的特性,因此转基因大米中碳水化合物和能量消化率显著低于亲本大米。转基因大米AAS评分和PDCAAS评分均略高于亲本大米,因此该转基因大米氨基酸的营养价值和食用价值与亲本大米具有“实质等同性”。5.转双反义SBE基因水稻餐后血糖和胰岛素效应以及在人体大肠中发酵情况的研究转双反义SBE基因大米能有效控制餐后血糖和胰岛素升高,并促进其在人体中与发酵相关的产物氢气含量的显著增加。更多还原

【Abstract】 ObjectiveResistant starch (RS) is the sum of starch and products of starch hydrolysis that are not absorbed in the small intestine of healthy individuals. Consumption of RS-enriched foods have shown beneficial effects on the health of large bowel where the RS is fermented by anaerobic bacteria. Resistant starch also can modify lipid metabolism and reduce postprandial glycemic and insulinemic responses. The genetically modified rice with double antisense SBE gene is enriched with resistant starch. This study aimed to evaluate the feeding safety and functional properties of the genetically modified rice through animal and human trials.Methods1. Subchronic toxicity test of the genetically modified rice with double antisense SBE gene100 male and female healthy weanling Wistar rats with an initial weight of 80-90g were randomly sorted into five groups, each consisting of 10 males and 10 females, as follows: No-GM (nongenetically modified rice) group, GM (genetically modified rice) group, Half-GM (half genetically modified rice) group, Quarter-GM (quarter genetically modified rice) group and ND (AIN-93G normal diet) group. During the experiment, food consumption was recorded two times and body weight was measured once in a week. At the middle and end of the experiment, the hematological and biochemical parameters were monitored. At termination, all animals were anaesthetized and killed by exsanguination for gross and histopathological examinations. The main organs were weighed: brain, heart, liver, spleen, kidneys, testicle, uterus, thymus. The organ coefficients were measured and the right legs were isolated for bone density testing.2. Effects of the genetically modified rice with double antisense SBE gene on the large bowel health in ratsForty-eight healthy and adult male SD rats with an initial weight of 210-230g were randomly assigned into four groups as follows: No-GM (nongenetically modified rice) group, GM (genetically modified rice) group, Half-GM (half genetically modified rice) group, and ND (AIN-93M normal diet) group. After five weeks, 4-day faecal samples were collected. After six weeks, all animals were anaesthetized and killed by exsanguination. Contents of cecum and colon were collected. Large bowel function was evaluated by determining many indexes related with large bowel health, such as the weight of cecum, colon and their contents, pH and short-chain fatty acid concentration of the contents and feces.3. The preventive effects of the genetically modified rice with double antisense SBE gene on high fat diet induced blood lipids abnormalities in ratsFifty healthy and adult male SD rats with an initial weight of 210-230g were randomly divided into five groups as follows: NoGM-HF (nongenetically modified rice with high fat) group, GM-HF (genetically modified rice with high fat) group, HGM-HF( half genetically modified rice with high fat) group, HF (high fat diet) group and ND (AIN-93M normal diet) group. All rats were given equal amount of individual diets every day and at 4w, 8w, 13w after the experiment, serum TG、TC and HDL-C were measured. At 13w, all animals were anaesthetized and killed by exsanguination. Liver lipids including TG and TC were also measured.4. Study on the digestibility of important nutrients in the genetically modified rice with double antisense SBE gene in vivoEight Wuzhishan healthy adult barrows with an initial weight of 30-35kg were housed in adjustable metabolism cages. Pigs were injected with Doramectin injection which is indicated for the treatment and control of the following endoparasites and ectoparasites in cattle during the 7-day adaptation period. After adaptation, pigs were surgically fitted with a simple T-cannula at the terminal ileum. After surgery, seven pigs were chosen as experimental animals. Three diets were prepared. Diet 1 and diet 2 mainly contained nongenetically modified rice and genetically modified rice, respectively. A low-protein (5% casein) diet (diet 3) was fed to determine endogenous amino acid losses. Chromic oxide (0.3%) was includes in all diets as an inert marker. The whole experiment contained three periods. In the first period, four pigs were fed diet 1, the other three pigs were fed diet 2. In second period, diets 1 and diet 2 were exchanged to feed the seven pigs. At last period, all pigs were fed diet 3. Each experimental period lasted seven days. The initial 4-day of each period were considered an adaptation period to the diet. Ileal digesta were collected for 12 h on the last 3-day of the each period. Digesta was immediately frozen at -20℃to prevent microbial degradation of the amino acid in the digesta. At the end of the experiment, ileal digesta were thawed, freeze-dried and ground through a 0.2 mm screen before analysis. At termination, all animals were anaesthetized and killed by exsanguination for determining whether cannulation had caused intestinal abnormalities.5. Postprandial glycemic and insulinemic responses to genetically modified rice with double antisense SBE gene and its fermentation in the large bowel of healthy peopleTwenty health adult people were recruited for this study. All subjects were firstly subjected to routine medical examination and oral glucose tolerance test. After screen, nine voluntary men at 23-26 years of age (24.3±1.0) and seven women in 24-26 years of age (24.6±1.0) took the study. They were randomized into three groups (three men and two-three women per group) and tested simultaneously. They consumed one of the 40g glucose, 40g carbohydrate of RS rice (genetically modified rice) and WT rice (nongenetically modified rice) meal in 300 mL water with a washout period of 7-day. The WT and RS rice were cooked for rice meal. One week later, they were administrated with the second type of food and after another week, they were provided with the third type of food. Individual subjects arrived at the study site at 6 am. After resting for 30 min, individual was inserted with a catheter into the antecubital vein by a registered nurse. Their blood samples were collected and hydrogen breath was tested as the baseline values. At 7am, those subjects consumed individual food within 10 min. Their blood samples (2 mL) were collected at 0, 15, 30, 45, 60, 90, 120, 180, and 240 min post food intake and simultaneously subjected to hydrogen breath tests for indicated time points. The collected blood samples in grey-top BD Vacutainer blood tubes (special for blood glucose test) were centrifuged at 3000 g for 15 min at room temperature. The plasma was collected and stored at -20℃for less than 3 days for analysis, which did not significantly change the value of plasma glucose in our preliminary studies. Hydrogen breath testes for individual subjects were performed at 0 and 3.5-16h post food consumption with a half-hour interval between 3.5-14h and one-hour interval between 14-16h on a portable breath hydrogen analyzer. Subjects were provided special diner on the day before testing, lunch and dinner after the last blood collection (5h and 11h after the beginning of experiments) with little hydrogen-producing foods. The amount and kind of foods individuals consumed were recorded. The subjects were requested to consume equal amount of the same kind of foods at lunch and dinner when they participated in testing for the second and third type of foods.Results1. Subchronic toxicity test of the genetically modified rice with double antisense SBE geneThe weigh of rats and daily intake were not different among all the groups (P>0.05). At the middle of the experiment, MCV in female rats of GM group was higher than in those of No-GM group (P<0.05), Mo less than that in ND group (P<0.05), AST activity higher than that in ND group (P<0.05). ALT activity in female rats of Half-GM was higher than in those of ND and No-GM groups (P<0.05). Male rats of GM group had higher MCV than that in ND group (P<0.05). At the end of the experiment, MCH in female rats of GM group was higher than in those of No-GM group (P<0.05), AST and ALT activity higher than that in ND group (P<0.05). HCT and BUN level in male rats of GM group were less than in those of No-GM group (P<0.05), Mo level higher than that in ND group (P<0.05).To female rats, brain index of GM group was higher than that in No-GM group and kidney index of ND group was higher than that in other groups(P<0.05). To male rats, all index had no significant difference among all the groups (P>0.05). Blood lipids, calcium and bone mineral density were also no significant difference(P>0.05). Among all the groups, no notable abnormity was found in the pathological examination on the main purtenances (P>0.05).2. Effects of the genetically modified rice with double antisense SBE gene on the large bowel health in ratsRats of GM group had similar body weight with ND group and significantly less than that of No-GM group (P<0.05). In comparison with No-GM and ND groups, fecal bulk and moisture, cecum weight and contents weigh in rats of GM and Half-GM groups had enhanced significantly(P<0.05). Colon weight in rats of GM group also were higher than in those of No-GM and ND groups (P<0.05). The concentration of short-chain fatty acid (SCFA) in the cecum, colon and fecal dropped gradually among all the groups. Compared with No-GM and ND groups, SCFA level of cecum and colon enhanced significantly in rats of GM and Half-GM groups (P<0.05) except colon butyric acid in rat of Half-GM group. In all groups, there were differences of acetic acid and propionic level in feces (P<0.05) but no difference of butyric acid. Cecal and fecal pH were lower in rats of GM and Half-GM groups than in those of other groups. (P<0.05).3. The preventive effects of the genetically modified rice with double antisense SBE gene on high fat diet induced blood lipids abnormalities in ratsThe initial weigh of rats and daily intake were not different among all the groups (P>0.05). At 4w, rats of NoGM-HF、GM-HF、HGM-HF and HF groups had higher weight than that of ND group (P<0.05), and the significant difference kept in the end. Compared with ND group, serum TG concentration in rats of other groups had no difference until termination. Serum TC contents in rats of NoGM-HF、GM-HF、HGM-HF and HF groups were significantly higher than in those of ND group from 4w to 13w, but there were not different among these groups (NoGM-HF、GM-HF、HGM-HF and HF) (P>0.05). Serum HDL-C contents were not different among all the groups (P>0.05), so as liver TG and TC concentration.4. Study on the digestibility of important nutrients in the genetically modified rice with double antisense SBE gene in vivoThe apparent and true digestibility of all amino acids and crude protein had no significant difference in the two rices (P>0.05). The digestibility of carbohydrate and energy in genetically modified rice was significantly lower than that in nongenetically modified rice (P<0.05). The AAS value of genetically modified rice and nongenetically modified rice were 75% and 62%, corresponding PDCAAS value were 65% and 56%, respectively.5. Postprandial glycemic and insulinemic responses to genetically modified rice with double antisense SBE gene and its fermentation in the large bowel of healthy peopleThe mean baseline blood glucose levels before the RS, WT rice, or glucose intake were similar (4.7±0.3 mmol/L vs 4.6±0.4 mmol/L vs 4.7±0.3 mmol/L, P>0.05), respectively. The value of plasma glucose for the RS rice meal was significantly smaller than that for the WT rice meal (P<0.05), particularly at 30, 45, 60, 90 and 120min post intake of meals. The highest levels of blood glucose after consuming RS rice (6.8±0.4 mmol/L) were significantly lower than that with WT rice (7.2±0.6 mmol/L, P<0.05). Importantly, the GI for the RS rice meal (48.4±21.8) was lower than of the WT rice meal (77.4±34.9, P<0.05).The mean baseline insulin levels before the RS, WT rice, or glucose intake were similar (6.7±2.3μIU/mL vs 6.8±2.7μIU/mL vs 6.1±1.5μIU/mL, P>0.05), respectively. The levels of plasma insulin in subjects with the RS rice were significantly lower than that with WT rice at 45, 60, 90 and 120 min after food intake. After adjusting to the reference glucose (100%), the mean value of II in subjects with the RS rice meal (34.2±18.9) was significantly lower than that with the WT rice meal (54.4±22.4, P<0.05).There was no significant difference in the baseline levels of fasting breath hydrogen before intake of RS and WT rice meal. In contrast, the levels of breath hydrogen after the RS rice were remarkably higher, as compared with that after the WT rice (P<0.05). The levels of hydrogen significantly increased 5 h after the RS rice, reached the highest level near 7 h and flatted until 14 h, followed by declining slightly. The peak levels of breath hydrogen after the RS rice meal (38.9±10.5ppm) were significantly higher than after the WT rice (17.6±3.7ppm, P<0.05).Conclusions1. Based on the results of the 90-day safety study in Wistar rats fed genetically modified rice with double antisense SBE gene, there were no enough evidences to confirm that the genetically modified rice had adverse effects on the rats.2. Consumption of the genetically modified rice can improve large bowel health-related indexes and have active healthy effects on rat’s large bowel.3. Blood lipids abnormalities were successfully induced in the rats after feeding them high fat die. But consumption of the genetically modified rice had no preventive effect on the development of blood lipids abnormalities in rats.4. The apparent and true digestibility of all amino acids and crude protein were not greatly changed by the increase of resistant starch content in the genetically modified rice. The digestibility of carbohydrate and energy in genetically modified rice was significantly lower than that in nongenetically modified rice owing to its resistant starch, which kept its character that are not absorbed in the small intestine. The AAS and PDCAAS value of genetically modified rice were higher than that of the nongenetically modified rice, so the two rices have substantial equivalence in the nutrition and feeding value of amino acid.5. Consumption of the genetically modified rice meal decreased the postprandial glycemic and insulinemic responses and promoted resistant starch fermentation -related production of hydrogen in the large bowel of young and healthy Chinese adults.更多还原