Study On The Synthesis And Biological Activity Of N-Carbamyl-L-glutamic Acid

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.