| 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.
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