【作者】 王媛；

【导师】 葛毅强； 陈颖；

【作者基本信息】 中国农业大学 ， 食品科学与工程， 2005， 硕士

【摘要】 为揭示转基因大豆食品从原料到成品的加工过程中内、外源基因的降解变化规律,为我国的转基因标识条例的实施及转基因食品安全性的评价提供科学依据,本论文以转基因和非转基因大豆为试材,采用定性PCR、多重PCR、实时荧光定量PCR等方法,分析了豆腐、豆奶、豆粉、酱油等四种大豆加工食品中磨浆、点浆、调配、均质、喷雾干燥、发酵、杀菌等关键加工工艺对基因组DNA、调控元件、目的基因的片段大小及含量的影响。结果如下: 1.建立了大豆加工食品中转基因成分的定性PCR技术、多重PCR方法及实时荧光定量PCR检测方法,这三种方法分别能有效、准确地检测出大豆加工食品中转基因成分及其含量。 2.通过内源、筛选及目的基因片断大小的变化,研究了不同加工工艺中基因组DNA、调控元件及外源基因的降解变化规律。 磨浆、点浆、高温杀菌、喷雾干燥、制曲及发酵各工艺使大豆基因组DNA发生严重地降解;针对外源目的基因,磨浆、挤压成型、调配、均质、喷雾干燥、制曲及发酵等加工过程对其产生了较大的影响,高温煮浆过程对内、外源基因降解产生的影响较小。 针对CaMV35S启动子,引物35S₁/35S₁’、35S₂/35S₂’和35S₄/35S₄’的扩增片段基本不受加工工艺的影响,但35S₃/35S₃’的扩增片段受加工工艺的影响较大。对于终止子nos基因,所设计的两种引物的扩增产物在各个工艺过程中均能检测到,nos基因降解程度变化很小。 3.应用实时荧光定量PCR技术,研究了大豆加工食品中不同加工工艺对加工食品中转基因含量的变化规律。 磨浆、煮浆过程对外源基因破坏得很严重,转基因含量迅速下降至0.435%;点浆是一个生物化学变化,对基因组DNA的破坏较为严重,随着内源基因片断的降解,转基因含量有所回升;外源基因在受到挤压成型过程后,比内源基因更易受到破坏,终产品中转基因含量下降到0.797%。 豆粉加工中喷雾干燥对内源基因的破坏较为严重,终产品豆粉中转基因成分较前一过程有所上升;豆奶加工过程中调配和均质过程使外源基因遭到严重破坏,转基因含量下降,但杀菌工艺又对内源基因的降解程度更加严重,转基因含量有所上升;酱油各个加工过程对外源基因的破坏都比内源基因更加严重,转基因含量不断下降。更多还原

【Abstract】 In order to reveal the rule of the degradation of DNA, exogenous gene and non-target genes during different manufacturing processes from the soybean raw material to products, to provide the scientifically basement for the estimation of GMO food, to put the GMO food management in practice, the degradation of genome DNA, epsps gene, promoter CaMV35S and terminator nos genes in the steps such as milling, cooking, blending, homogenizing, sterilizing, spray drying, yeast-refining, and fermenting in the processes of Tofu, soymilk, soybean flour and sauce were investigated using GMO soybean and non-GMO soybean as the experimental materials by sensitive polymerase chain reaction system, multiplex polymerase chain reaction and real-time quantitative polymerase chain reaction detection method.The results were summarized as follows:1. Methods for qualitative and quantitative detection of transgenic component in genetically modified soybean products were established respectively, which provide sensitive, specific, simple and useful tools for soybean food products.2. The degradation of genome DNA, non-target genes and exogenous gene in various processing methods were studied according to the changes of the fragments’ sizes of the three genes mentioned above.The lectin gene was affected during milling, sterilizing, spray drying, yeast-refining and fermenting manufacturing processes; the fragment of exogenous gene epsps had been degraded in milling, blending, homogenizing, spray drying, yeast-refining and fermenting manufacturing processes. Cooking had no effect on the DNA and epsps gene.CaMV35S promoter considered, the amplication fragment of primer 35S₁/35S₁, 35S₂/35S₂, and 35S₄/35S₄,had no change in the different manufacturing processes except primer 35S₃/35S₃’. The terminator nos gene was nearly not degraded in each step of manufacturing process of RR soybean products.3. The quantitative change of transgenic component of the four soybean products during different manufacturing process was investigated using real-time quantitative PCR detection method.The epsps gene degraded so that the content of GMO dropped to 0.435%; depositing is a biochemical reaction, this process degraded genome DNA and the content of GMO raised. The exogenous gene epsps was affected worse than genome DNA after shaping; the content of GMO in Tofu was 0.797%.The endogenous gene was seriously damaged during the process of the soybean flour by spray drying; therefore the content of transgenic component in the terminal product was higher than before. The content of transgenic component decreased because the exogenous gene was degraded when blending and homogenizing in the manufacturing of soymilk. However, sterilizing could aggravate the degree of the degradation of the lectin gene, so the component increased. The damage of epsps gene was more serious than that of lectin gene in all the steps of sauce process, meanwhile the content of GMO dropped continuously.更多还原