| Objective:This research is mainly focused on the preparation of fresh pilose antler, traditional fried process, directly freeze drying process, homogenate process, freeze dring process with protective agent, freeze dring process without protective agent, freeze dring process with protective agent for 10 days at 40℃. According to medical research, pilose antler have the function of anti-fatigue. And the main component in it is protein. So this research first determine the content of soluble proteins in different pilose antler samples mentioned above. Then using iTRAQ technology to investigate the regularity of activity by freeze dring process, stablishinng chemical and molecular biological effective information of protein fingerprint database. Through data management and analysis, combining with the anti-fatigue experiment, we attend to find the regularity of activity by fresh, fried, homogenate and freeze dring process, Qualitative description the importance of freeze dring process. Providing scientific basis for improving the efficiency of fresh animal medicine bioactive protection. Theoretical basis for optimization the process technology of pilose antler.Methods:Preparation the different processions of pilose antler. Using coomassie brilliant blue G-250 method to determine the content of soluble proteins in different pilose antler samples mentioned above, and use the Kruskal-Wallis test to analyzed the data. Using iTRAQ technology coupled with 2D LC-MS to study the comparative proteomics of different processing technology for pilose antler. The main types of annotations are obtained from the gene ontology consortium website. The GO information is searched from UniProKB and NCBI, data is analyzed according to WEGO and Hierarchical clustering, discussing the differential proteomics through cellular component, molecule function, biological process. At last, making anti-fatigue experiment of pilose antler, through behavioral indexes:weight, time for swimming, and quantitative indexes:the content of blood lactic acid, urea nitrogen, MDA.Results:(1)The content of pilose antler proteins from large to small order are fresh pilose antler, directly freeze drying process, homogenate process, freeze drying process without protective agent, freeze drying process with protective agent for 10 days at 40℃, freeze drying process with protective agent, fresh pilose antler with no blood, fried pilose antler. According to Kruskal-Wallis test, comparing the fresh pilose antler, the content of proteins in pilose antler by other processed methods are lower (P≤0.05). To compare the fried pilose antler with other methods, the content of proteins in traditional fried process are lower (P<0.05). Meanwhile, the content of proteins in homogenate process are lower than that in directly freeze drying process (P<0.05). The content of proteins in freeze dring process pilose antle are lower than that in directly freeze drying process and in homogenate process(P<0.05).(2)1015 proteins are found according to MOLDI TOF/TOF 5800 mass spectrometer. A total of 87 proteins are observed to be differentially expressed (P<0.05, the ratio of>1.50 or <0.60 as the threshold selection of difference proteins). And there are 24 up regulated and 33 down regulated proteins of traditional fried process (TFP) different from fresh pilose antler(P≤0.05). Among the 87 proteins, their functions of calcium ion binding, oxidoreductase, ATP binding and hydrolase have been influenced a lot. Pilose antler proteins by traditional fried process are influenced most.19 down regulated proteins are more than other processes. And 7 proteins are significantly down regulated(P<0.001) by traditional fried process. Most of them are found to be involved in calcium ion binding, ATP binding, which are important to human. Other processions of pilose antler are little influenced compared with fresh pilose antler. Freeze dring process with protective agent (FDP) can improve the content of significantly different proteins(P<0.001). Optimizing functions of anti-fatigue and collagen, etc. GO analysis showed that bioinformatics (cellular component, molecule function and biological process) are involved in large change compared with fresh pilose antler. Among them, the main molecule function are binding(52.7%), catalytic(25.3%), structural molecule and transporter(6.6%).(3)According to weight, time for swimming and other three quantitative indexes, freeze drying process with protective agent can significantly prolong the time for swimming(P≤ 0.01), significantly increasing the body weight of mices(P≤0.01). Meanwhile, it can decrease the content of LA, BUN and MDA. Showing that freeze drying process with protective agent can significantly have anti-fatigue activity. While freeze drying process without protective agent have no influence on MDA. But it can significantly increase the weight of mices and decrease the content of LA and BUN (P≤0.01).While traditional fried process can’t prolong the time for swimming and decrease the content of MDA, but it can increase the weight of mices at beginning of the experiments(P≤ 0.01). Also, it can decrease the content of LA and BUN (P≤0.05).Directly freeze drying process and homogenate pilose antler, compared with the control group, can increase the weight of mice at beginning of the experiment(P≤0.01), but have no influence on the middle and last period of experiment. Homogenate process can decrease the content of MDA (P≤0.05), instead of directly freeze drying process.Conclusion:Through proteomics and anti-fatigue experiments, freeze dring process can save the activity of proteins and improve the anti-fatigue activity. While traditional fried process need high temperature and heat treatment, which may lead to the functional proteins significantly affected compared with other processes. While according to research, directly freeze dring process makes 15 proteins which have anti-fatigue activity destroyed. And differentially down regulate proteins make 50% of structural molecule and binding founctions destroyed. Down regulate proteins of homogenate process can influence the binding(50%), catalytic(16.7%) and structural molecule(16.7%) founctions, which have big change compared with fresh pilose antler. According to anti-fatigue research, directly freeze drying process can’t decrease the content of MDA, while homogenate process can decrease it (P≤ 0.05) but not significant. Both of them can increase the weight of mice at beginning of the experiment(P≤0.01), but have no influence on the middle and last period of experiment. Instead, after a certain amount of freeze dring parameter changes, as well as protective agent processing, freeze drying process with protective agent can improve the anti-fatigue activity of pilose antler, preservation and functional optimization of protein components. Freeze dring process with protective agent for 10 days at 40℃ damage the beneficial proteins, freeze dring process without protective agent and freeze dring process with protective agent for 10 days at 40℃ reduce anti-fatigue activity for can’t decrease the content of MDA. These indicating that with temperature increased and long storage, a certain impact on the anti-fatigue activity of pilose antler is made. |
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