防龋食品功能学评价程序的建立与应用研究
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摘要
龋病是人类最普遍的疾病之一,世界卫生组织已将其与癌症、心血管疾病列为人类三大重点防治疾病。龋齿的防治成为备受关注的公共卫生问题。要从根本上解决龋病问题,必须立足于预防。近二十年来,防龋功能性食品应运而生并快速发展。我国防龋功能性食品生产与销售规模日趋壮大,然而与之配套的功能性评价及规范化管理却相对滞后,对其进行全面科学评价在国内外尚无统一的标准。在我国卫生部公布的二十四项保健功能中并没有包含防龋功能,对防龋功能性食品的功能学评价尚属空白。为了建立防龋功能性食品的功能学评价方法,本研究以FDA、ADA等权威机构的文献资料为基础,在对防龋功能性食品进行安全性评价的基础上,根据《保健食品检验与评价技术规范》(2003年版),拟定从离体实验、动物实验二个方面对该功能进行评价。本研究的创新之处在于:在国内首次建立一套针对防龋功能性食品的功能评价程序和检测方法,并以特异性鸡蛋黄抗体为原料开发的一种防龋食品为主要研究对象,对其安全性和防龋功能进行检测,从而验证防龋功能评价程序的科学性、全面性和可操作性。本研究的主要内容及结论如下:
主要研究内容:
(1)参照《食品安全性毒理学评价程序和方法》GB15193.1-15193.19—94程序进行特异性鸡蛋黄抗体制剂安全性实验。对特异性鸡蛋黄抗体制剂进行急性毒性实验、小鼠骨髓微核实验、小鼠精子畸形实验、Ames实验和30d喂养实验。结果表明:在急性毒性实验中,动物无明显中毒反应,无死亡,雌雄小鼠经口LD50均大于20ml/kg.Bw,根据急性毒性分级标准,判定其属无毒级;微核实验、精子畸形实验和Ames实验结果均为阴性。在大鼠30d喂养实验中,按急性毒性实验灌胃量的1/16、1/8、1/4,设计低、中、高剂量组(即0.625、1.25、2.50ml/kg.BW),相当于人群推荐日摄入量0.0167ml/kg.Bw的37.5、75、150倍;另设正常对照组,连续经口给予30d。结果表明:各剂量组与正常对照组比较,对SPF级Wistar大鼠的临床检查、体重、食物利用率无不良影响。血液学检查、血生化检查均在正常范围内,且对脏器重量和系数以及病理组织学等指标无明显影响,未发现该受试物有明显的毒性作用。以上实验证实特异性鸡蛋黄抗体制剂安全无毒,可用于食品。
(2)观察特异性鸡蛋黄抗体制剂对变形链球菌生长、粘附、产酸的影响。将一定浓度的样品加入变形链球菌培养管中,培养一定时间,经革兰氏阳性染色和分光光度计测量观察变形链球菌的生长情况,测定其百分粘附率,并测定细菌产酸后pH值。结果表明特异性鸡蛋黄抗体制剂对变形链球菌生长无明显影响,但易促使其形成长链;变形链球菌粘附率降低;pH值升高至pH6.0以上。
(3)评价特异性鸡蛋黄抗体制剂对大鼠龋齿的预防作用。采用变形链球菌建立大鼠
Dental caries is a kind of wide spread human diseases, and has been ranked in the three diseases emphasized to be prevented and treated. Prevention and remedy of dental caries, as a public health problem, have been received more and more attention. To solve it radically, prevention must be grounded on. In recent years, many functional foods to prevent it have springed up and developed rapidly. Up to now, the manufacture scale and sale volume of anticaries food are enlarged day after day, while the function assessment and normalized administration lags behind. Unitary standards to assess such foods all-sidedly and scientifically still need to complete. In the 24 items of health foods assessments issured by health ministry, the ones evaluating the function of caries protecting food were not involved in. Based on the the authority literatures of FDA and ADA and "technical standards for testing and assessment of health food"(2003), a series of function assessment procedures and testing methods of caries protection foods were established, and a kind of caries protecting food, developed from a specific antibody of fowl yolk was used to verify the objectivity, all-sidedness and operability of our methods. Methods: The security of the sample was evaluated according to "technical standards for testing and assessment of health food"(2003) GB 15193.1-15193.19—94. Streptococcus mutans (S. mutans) were cultured with the sample of olitin tooth protecting spray. Then S. mutans were viewed through spectrophotometer and the percentages of the adherent S. mutans were calculated in vitro. 40 wistar rats were divided into 4 groups: group A as control group; group B、 C、 D were dealed with our sample in low, middle, high dose respectively. They were all fed with caries-inducing diet 2000*. The sums of the caries scores were assessed by the procedure of Keyes'.Results: The LD50 of the sample was more than 20ml/ kg. BW, and there were no significant difference between the numbers of micronuclens of born marrow cell and sperm shape abnormality of test groups and control. There were no distinctive effects of the sample on blood test, body weight and biochemical index. All these indicated that the sample has no toxicity to the body.It was showed that the growth of S. mutans was not inhibited, but the long chain was occurred. The adherence of S. mutans cells on the glass decreased, the pH value increased to above 6.0. There was a significant lower mean of caries score of test group than those
of the control group (p<0.05).Conclusion: It was demonstrated that IgY preparation was nontoxic and may be added infood. The sample could prevented efficiently the occurrence and progress of caries in rats,the mechnism of the pretection lied in the enhancement aggregating, inhibition adhesionand decreasing sour products of S. muntans.It was also proved that the function evaluation procedure and testing methods could givea scientific, all-sideness, and operative assessment to foods function, so they providedtheoretic references and application experiences to assess caries protecting foods andenhanced supervision and administration of anticaries food.
主要研究内容:
(1)参照《食品安全性毒理学评价程序和方法》GB15193.1-15193.19—94程序进行特异性鸡蛋黄抗体制剂安全性实验。对特异性鸡蛋黄抗体制剂进行急性毒性实验、小鼠骨髓微核实验、小鼠精子畸形实验、Ames实验和30d喂养实验。结果表明:在急性毒性实验中,动物无明显中毒反应,无死亡,雌雄小鼠经口LD50均大于20ml/kg.Bw,根据急性毒性分级标准,判定其属无毒级;微核实验、精子畸形实验和Ames实验结果均为阴性。在大鼠30d喂养实验中,按急性毒性实验灌胃量的1/16、1/8、1/4,设计低、中、高剂量组(即0.625、1.25、2.50ml/kg.BW),相当于人群推荐日摄入量0.0167ml/kg.Bw的37.5、75、150倍;另设正常对照组,连续经口给予30d。结果表明:各剂量组与正常对照组比较,对SPF级Wistar大鼠的临床检查、体重、食物利用率无不良影响。血液学检查、血生化检查均在正常范围内,且对脏器重量和系数以及病理组织学等指标无明显影响,未发现该受试物有明显的毒性作用。以上实验证实特异性鸡蛋黄抗体制剂安全无毒,可用于食品。
(2)观察特异性鸡蛋黄抗体制剂对变形链球菌生长、粘附、产酸的影响。将一定浓度的样品加入变形链球菌培养管中,培养一定时间,经革兰氏阳性染色和分光光度计测量观察变形链球菌的生长情况,测定其百分粘附率,并测定细菌产酸后pH值。结果表明特异性鸡蛋黄抗体制剂对变形链球菌生长无明显影响,但易促使其形成长链;变形链球菌粘附率降低;pH值升高至pH6.0以上。
(3)评价特异性鸡蛋黄抗体制剂对大鼠龋齿的预防作用。采用变形链球菌建立大鼠
Dental caries is a kind of wide spread human diseases, and has been ranked in the three diseases emphasized to be prevented and treated. Prevention and remedy of dental caries, as a public health problem, have been received more and more attention. To solve it radically, prevention must be grounded on. In recent years, many functional foods to prevent it have springed up and developed rapidly. Up to now, the manufacture scale and sale volume of anticaries food are enlarged day after day, while the function assessment and normalized administration lags behind. Unitary standards to assess such foods all-sidedly and scientifically still need to complete. In the 24 items of health foods assessments issured by health ministry, the ones evaluating the function of caries protecting food were not involved in. Based on the the authority literatures of FDA and ADA and "technical standards for testing and assessment of health food"(2003), a series of function assessment procedures and testing methods of caries protection foods were established, and a kind of caries protecting food, developed from a specific antibody of fowl yolk was used to verify the objectivity, all-sidedness and operability of our methods. Methods: The security of the sample was evaluated according to "technical standards for testing and assessment of health food"(2003) GB 15193.1-15193.19—94. Streptococcus mutans (S. mutans) were cultured with the sample of olitin tooth protecting spray. Then S. mutans were viewed through spectrophotometer and the percentages of the adherent S. mutans were calculated in vitro. 40 wistar rats were divided into 4 groups: group A as control group; group B、 C、 D were dealed with our sample in low, middle, high dose respectively. They were all fed with caries-inducing diet 2000*. The sums of the caries scores were assessed by the procedure of Keyes'.Results: The LD50 of the sample was more than 20ml/ kg. BW, and there were no significant difference between the numbers of micronuclens of born marrow cell and sperm shape abnormality of test groups and control. There were no distinctive effects of the sample on blood test, body weight and biochemical index. All these indicated that the sample has no toxicity to the body.It was showed that the growth of S. mutans was not inhibited, but the long chain was occurred. The adherence of S. mutans cells on the glass decreased, the pH value increased to above 6.0. There was a significant lower mean of caries score of test group than those
of the control group (p<0.05).Conclusion: It was demonstrated that IgY preparation was nontoxic and may be added infood. The sample could prevented efficiently the occurrence and progress of caries in rats,the mechnism of the pretection lied in the enhancement aggregating, inhibition adhesionand decreasing sour products of S. muntans.It was also proved that the function evaluation procedure and testing methods could givea scientific, all-sideness, and operative assessment to foods function, so they providedtheoretic references and application experiences to assess caries protecting foods andenhanced supervision and administration of anticaries food.
引文
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