【作者】 朱影恬；

【导师】 王远兴；

【作者基本信息】 南昌大学 ， 营养与食品卫生学， 2010， 硕士

【摘要】 断奶应激是目前困扰养猪业发展的主要问题之一。而现在已有的研究又证明了精氨酸在仔猪应激状态下的重要作用。因此解决仔猪断奶后生长过程精氨酸的不足是有效应对断奶应激的手段。N-氨基甲酰-L-谷氨酸(N-Carbamyl-L-glutamic acid, NCG)是N-乙酰-L-谷氨酸(N-Aceyl-L-glutamic acid, NAG)的结构类似物。它能代替NAG成为氨基甲酰磷酸合成酶-Ⅰ(CPS-Ⅰ)的变构催化剂,从而促进脯氨酸和谷氨酰胺向瓜氨酸和精氨酸转化,增加精氨酸的内源合成,且保持体内精氨酸环境的平衡稳定。由此可见,NCG对于应对仔猪断奶期间因精氨酸不足引起的应激状况具有很好的效果,它是一种潜在的有效新型饲料添加剂。目前关于NCG的研究并不多,现有的合成方法耗时较长,不适合工业生产；其生物活性也缺乏系统深入的研究。本文对NCG及其锌盐的合成方法、NCG分析检测方法以及生物活性进行研究,以期为NCG产品的开发和其他衍生物的研究提供一定的理论基础,从而为养猪业的发展提供一种高效的饲料添加剂,促进养猪业的发展。这具有较高的经济价值和现实意义。现将主要研究概括如下：1.本文以味精和尿素为原料,分别采用微波加热和传统加热两种方法合成NCG,结果表明微波加热法优于传统加热法,可行有效。单因素实验结果表明,微波功率对产物得率的影响最大,其次是溶剂水的用量,原料味精与尿素的配比的影响效果最小；正交实验优化所得最佳工艺为：N味精：N尿素为1：2,水用量0.3ml/mmol,微波功率700W,反应时间15min。在该反应条件下合成NCG,得率可达到57.6%,以丙酮与水复合溶剂重结晶法对粗产品进行纯化,纯的NCG为白色粉状固体。热失重分析测定结果表明,NCG的热稳定性不是很好,其熔点约为170℃。合成产物经紫外光谱、红外光谱、质谱、核磁共振氢谱和元素分析等进行结构表征确定为NCG；合成过程中的跟踪检测以及产物含量的测定采用高效液相色谱法(HPLC),确定色谱条件为：色谱柱：Kromasil C18柱；流动相：甲醇：水=5：95(pH2.90)；检测器：光电二极管阵列检测器；检测波长：220nm；流速：0.6ml/min。方法学考察结果表明,该方法进样精密度、重现性、稳定性好,加样回收率高。以此方法测得合成产物中NCG含量为95.3%；然后进一步以NCG和氧化锌合成出N-氨基甲酰-L-谷氨酸锌,分别通过元素分析、EDTA络合滴定法以及HPLC测定锌等成分的含量,测定结果为：C：27.83%,H：3.85%,N：9.97%,Zn：23.14%,NCG：73.83%(按C6H9ZnN2O5计算,C：28.2%,H：3.9%,N：10.9%,Zn：25.59%,NCG：74.41%)。2.考察了NCG及其锌盐的抗氧化活性和抑菌活性。研究表明,NCG及其锌盐均表现出一定的抗氧化能力和抑菌性。抗氧化实验研究了NCG及其锌盐对超氧阴离子、羟基自由基、DPPH的清除能力和对铁离子的还原能力。结果表明,NCG及其锌盐对超氧阴离子自由基和羟基自由基的清除能力相对要强于对DPPH的清除力。而NCG及其锌盐对铁离子几乎没有还原能力。在所选浓度范围内,NCG及其锌盐的抗氧化能力均随浓度的增大而增强。在样品浓度为3mg/ml时,NCG和NCG-Zn对超氧阴离子的清除能力分别为55.32%和79.27%,对羟基自由基的清除率分别为38.09%和54.03%；而对DPPH的清除试验结果表明,NCG的清除率要高于NCG-Zn,分别为23.13%和7.87%。抑菌试验中分别考察了NCG以及NCG-Zn对大肠杆菌、枯草芽孢杆菌和金黄色葡萄球菌的抑菌效果,结果表明,NCG-Zn的抑菌效果要优于NCG。其中NCG-Zn对大肠杆菌的抑制效果又要比对其他两种菌的抑制效果强,当浓度为2mg/ml时,大肠杆菌、枯草芽孢杆菌和金黄色葡萄球菌的抑菌圈直径分别为27mm、13mm和14mm。而10mg/ml的NCG对这三种菌的抑菌圈直径仅分别为11mm、9mm和10mm。3.研究了不同添加水平的NCG对断奶仔猪生长性能和腹泻情况的影响。与对照组相比,日粮中中添加0.08%NCG,可显著提高仔猪的ADFI和ADG,分别比对照组提高了42.36%和89.43%(P<0.05)。同时,料肉比显著降低(P<0.05)；腹泻率和腹泻指数也明显低于对照组,腹泻率从15.83%降低到2.96%。添加0.16%的NCG仔猪与对照组的相比,ADFI和ADG有增加的趋势,F/G也有降低的趋势,但是差异均不显著(P>0.05),但与0.08%NCG相比,ADFI和ADG反而更低,腹泻程度也更为严重。由此可知,日粮中添加0.08%NCG可以有效缓解仔猪断奶应激,而高水平的NCG(0.16%)则具有一定的副作用。更多还原

【Abstract】 Early weaning stress is one of the main difficulties which hinder the development of pig industry. Now, it has been approved that the arginine is very important for weaning stress of piglets. So it is an effective method to resolve the deficiency of arginine for the weaning stress of piglets.N-carbamoyl-L-glutamate (NCG) is a metabolically stable analogue of N-acetyl-L-glutamate (NAG). It can replace NAG to be the metabolically stable activator of CPS-I to promote the conversion of proline and glutaminate to arginine and increase endogenous synthesis of arginine. So NCG can play a crucial role in maintaining arginine homeostasis in piglets. It can be seen that NCG can effectively solve the weaning-stress problem originated by the deficiency of arginine. Now there is not much studies about NCG and it is always need too long time to synthesis NCG to be used in commercial manufacture. In addition, the systematic deep research on the bioactivities of NCG was still lacked. This review reseached the synthesis of NCG and the zinic salt of NCG、analysis and detection by HPLC、bioactivities in the purpose of providing reliable scientific foundation for the exploitation of NCG and its derivatives. Then it can provide a high performance animal feeds to promote the development of swine breeding. The main results in the paper are mentioned as follows:1. In this paper two kind of heating methods which are microwave and conventional heating were used to synthesis NCG with monosodium glutamate and urea as material. The results showed that the microwave method is better than the conventional heating method. From the single factor experiments, we can know that the power of microwave has the most important effect on the yield of NCG, then is the volum of solvent water, the effect of raw material ratio is smallest. The L9(34) orthogonal test was used to evaluate the factors optimum conditions based on single factor tests. The optium technology were established as below:the molar ratio of monosodium glutamate and urea 1:2, microwave power 700W, microwave irradiation time 15min, solvent water 0.25ml/mmol. The yield percent is 57.6% in this condition and the product was purified by recrystallization with acetone and water. The purified NCG is white powder crystal. The thermogravimetric analysis shows that the thermostability of NCG is not good and the melting point of NCG is 170℃. The product was detected by ultraviolet spectrum, infrared spectrum, mass spectrum, H-NMR and elemental analysis to characterize the structure. The HPLC was used for tracking detection and the content determine of NCG. The chromatographic conditions were Kromasil C18 column (4.6 mm×150 mm,5μm) using isocratic elution with a mobile phase of pH 2.90 water-methanol(95:5); the analytes were monitored by the diode array detector at 220 nm. The flow rate is 0.6ml/min. The developed method has good precision, high recovery and reproducibility. The content of NCG determined with this method was 95.3%.The zinc salt of NCG was synthensised with NCG and ZnO as the raw material. The contents of zinc, NCG and every element which were detected respectively by EDTA complexing titration, HPLC and element analysis were Zn 23.14%, NCG 73.83%, C 27.83%, H 3.85%, N 9.97%, and in agreement with the theoretical value(C:28.2%, H:3.9%, N:10.9%, Zn: 25.59%, NCG:74.41%).2. The antioxidant activity and biocidal property were researched. The results showed that NCG and the zinc salt have antioxidant activity and biocidal property. The antioxidation of NCG and zinc salt of NCG were determined using superoxide anion free radicals, hydroxyl free radicals, DPPH free radicals and reducing capacity system. The scavening effects on hydroxyl radicals and superoxide anion free radicals were stronger than that of DPPH free radicals. NCG and its zinc salt almost had no effect on ferric ion. And the antioxidation of NCG and its zinc salt increases as the concentration. When the sample concentration is 3mg/ml, the scavening effects on superoxide anion free radicals of NCG and NCG zinc salt were 55.32% and 79.27% respectively and the effects on hydroxyl radicals respectively were 38.09% and 54.03%. And the results of the DPPH system showed that the scavening effects of NCG is stronger than NCG-zinc, respectively is 23.13% and 7.87%. In the bacteriostatic experiment, Escherichia coli, Bacillus subtillis and Staphylococcus aureus were used. The results showed that the bactericidal capacity of NCG-zinc were strongger than that of NCG. And the effects of NCG zinc salt on Escherichia coli is strongest. When the sample concentration is 2mg/ml, the inhibition zone diameters against Escherichia coli, Bacillus subtillis and Staphylococcus aureus were 27mm, 13mm,14mm respectively. The inhibition zone diameters of 10mg/ml NCG on the strain were 11mm,9mm and 10mm.3. The effects of NCG on the growth performance and diarrhea of early-weaned piglet were studied. Compared with the piglets in control, supplementation of 0.08% NCG improved ADFI and ADG, respectively increased 42.36% and 89.43%. And F/G was reduced(P＜0.05). Diarrhea rate and diarrhea index of the weaned piglets also significantly reduced, diarrhea rate reduced from 15.83% to 2.96%. Compared with the piglets in control, supplementation of 0.16% NCG also improved ADFI and ADG, and reduced F/G, but had no obvious difference. When compared with 0.08% NCG, the ADFI and ADG of the piglets were lower, and the diarrhea degree is more serious. The results showed that dietary supplementation of 0.08% NCG may help overcome weaning stress of piglets, while higher level of NCG (0.16%) may have side effect.更多还原