| Food safety and health are topic in nowadays, sythentic additives used in food are increasingly queried by customers because of their toxic side effect. Hence, the natural, efficient, safety antioxidants and preservative are the urgent need for food enterprise. Phellodendron amurense Rupr. is pharmacal plant of Traditional Chinese Medicine cortex phellodendri. Its bia-activities including antimicrobial, antioxidant, antiphlogistic activity and so on were all further sduied. As by-product, Phellodendron amurense Rupr. fruits contain essential oils which have seldom been investigated detaily in application. As far as the efficacy of homology is concerned, the EOs must have potential bio-activity. So it is remarkable significance to implement the investigation. In this study, Phellodendron amurense fruits as materials are to conduct following researches, which include essential oils extraction, separation, bioactivity and microcapsules. The research can provide a reference for further study and exploit the natural antioxidant and preservative. The main results are as follows:(1) The optimum extraction conditions of EOs from the fruits of Phellodendron amurense Rupr. through steam distillation (SD) and super critical CO2fluid extraction (SCFE-CO2) were obtained by single factor and response surface experiments.The optimum condition of SD method:extraction time 6.7 h, liquid to material 8.5:1, pierce degrees 16, extraction rate 1.39%. The optimum condition of SCFE-CO2:extraction pressure 17 Mpa, extraction temperature 40℃, extraction time 3 h, material particle size 55, the extraction rate 1.43%.(2) The composition of Phellodendron amurense Rupr. fruits EOs extracted by SD and SCFE-CO2 method were analyzed by GC-MS. Their constituents existed in some difference. For EOs extracted by SD,74 components were identified, which accounted for 91.42%. While 59 components were identified, which accounted for 82.75% of EOs extracted by SCFE-CO2. The dominant constituents were myrcene, a-pinene, gimaene-D, y-limonene, m-camphorene, p-Camphorene and so on. Besides cis-Verbenol was found accidentally in EOs extracted by SCFE-CO2.Effect of picking time on the composition of EOs is significant. Altogether, about 63 constituents were identified. Among of these, there were 35 mutual compounds existed in four various stages. The EOs composition were dominated by a-Myrcene (20.27%). In the third stage the prominent constituent were a-Myrcene(22.76%), a-pinene(8.96%), a-phellandrene (1.85%),δ-elemene (1.05%), farnesyl alcohol (3.19%), which were higher than those of the other three stages. At the same time, α-Ylangene,8-Elemene and β-cyclocitral were special constituent in third stage EOs. And Fenretinide was found in the EOs of the fourth stage by chance.EOs distilled by molecular distillation, four new components were identified by GC-MS, including Santolina triene in distilled fraction and Nimesulide, Ambrosin and Cryptomeridiol in residue fraction, which were firstly found in Phellodendron amurense Rupr. fruits EOs.There were 38 mutual compounds existed in distilled fraction. These components were concentrated by molecular distillation. In distilled fraction, the content of (+)-Citronellal is 2.29 times that of EOs control, the following of 1-Terpinenol 2.81 times, myrcene 1.25 times, cis-β-Terpineol 1.7 times, a-Terpineol 1.45 times. While 49 mutual compounds in residue fraction and 41 out of them were concentrated. Among of them, the concent of m-camphorene, p-Camphorene, (-)-a-Cadinene,4,7-Methano-lH-indenol,hexahydro-, Citronellyl acetate, Eugenol were all increased over two times than untreated EOs, which were 2 times,2.17 times,2.5 times,2.45 times,2.13 times and 2.16 times, respectively.(3) The comprehensive evaluation on antioxidant activity of Phellodendron amurense Rupr. fruits EOs at different picking time was conducted by means of scavenging free radical and inhibit lipid peroxidation. And found by analysis of GC-MS that the difference of EOs composition is related with their activity. Then the optimal harvesting time was to determined the third stage. The IC50 value of the third stage EOs was 1.613 mg/mL,2.601 mg/mL and 2.601 mg/mL using in scavenging DPPH assay,·OH and inhibiting β-carotene peroxidation, respectively.(4)Research on antimicrobial activity by Oxford cup method and gradient plate method, which the former is suitable for bacteria and the latter for mould. At the same time, determination of minimum inhibitory concentration(MIC) and minimum bactericidal concentration (MBC) were obtained according to the method of two fold serial dilutions. The results showed that gram-negative bacteria is more sensitive to the Phellodendron amurense Rupr. fruits EOs than that of gram-positive. And the ability of EOs inhibiting Geotrichum candidum is stronger than Aspergillus niger.The MIC of Salmonella and Escherichia coli was 0.25 mg/mL, while Staphylococcus aureus and Bacillus cereus was 0.5mg/mL, repectively. The MIC of mold on Aspergillus niger and Geotrichum candidum was 0.125 mg/mL and 0.0625 mg/mL, repectively. The MBC of tested four bacterias was 2 mg/mL while the minimum fungicidal concentration (MFC) of tested two moulds was 1 mg/mL.Effect of pH on antimicrobial activity of EOs is significant. When the pH of envioromental condition is 4-5, it showed much stronger antimicrobial activity. The Phellodendron amurense. Rupr. fruits EOs has much better thermal stability and it’s antimicrobial activity is still stronger at 121℃.(5) The antibacterial mechanism of Salmonella was conducted by Reagent Kit method. The results indicated that Phellodendron amurense Rupr. fruits EOs can change permeability and integrity of Salmonella cell membrane, which can lead to improving relative electrical conductivity, increasing the content of protein, reducing sugar and malondialdehyde (MDA) in medium. Besides it can decrease ATP content of bacteria. Some related enzymes activity, including superoxide dismutase (SOD), catalase (CAT) and ATP enzyme were all evaluated and their activity were lower. The structure of Salmonella treated by Phellodendron amurense Rupr. fruits EOs was observed by scanning electron microscopy, the result showed that the cell wall was destructively affected and presented some defect, even disappeared. These results showed that Phellodendron amurense Rupr. fruits EOs can destroy structure of Salmonella and disturb the activity of some protective or metabolic enzymes, then enventurally led to death.(6)Study on four factors which affect on the embedding rate of microcapules, including wall material and core material ratio, concentration of Calcium chloride, concentration of Carboxymethyl chitosan, the concentration of span 80 as emulsifier. The final optimized conditions:ratio of wall/core 3:1, concentration of CaCl2 2%, concentration of Carboxymethyl chitosan, concentration of span 800.1%. Using the optimized process, Phellodendron amurense Rupr. fruits EOs microcapsules entrapping rate was 90.86%.Study on microcapules stability of Phellodendron amurense Rupr. fruits EOs affected by temperature, oxygen, light. The results showed that the environmental sensitivity of the microcapules was weak and the residue rate of EOs was high.The experimental results of light inocubation showed that the stability of EOs embedded was obviously improved. |
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