Extraction And Characterization Of Active Ingredients In Rana Chensinensis Ovum And Preparation Of Microcapsule

In the Rana chensinensis industry,Rana chensinensis ovum is the main by-product of Rana chensinensis production,which is rich in unsaturated fatty acids and protein.However,due to insufficient basic research to support development and application,Rana chensinensis ovum is usually treated as waste materials cheaply,resulting in a serious waste of Rana chensinensis resources.In order to improve the overall utilization rate of Rana chensinensis resources and promote the sustainable development of the Rana chensinensis industry,this research is devoted to the extraction and characterization of the active ingredients in Rana chensinensis ovum and the preparation of microcapsules.Firstly,this work focused on the development of CO₂ supercritical fluid extraction（CO₂-SFE）process for Rana chensinensis ovum oil（RCOO）.The Box-Behnken design（BBD）model was used to investigate the influence of four independent factors（pressure,flow,temperature and time）on the yield of RCOO in the CO₂-SFE process,and UPLC-ESI-Q-TOP-MS and HPLC was used to identify and analyze the main unsaturated fatty acids in RCOO.According to the analysis results of the BBD response surface model,the most suitable CO₂-SFE conditions for the production of RCOO are pressure 29 MPa,flow rate 82 L/h,temperature 50°C and time 132 min.The corresponding predicted optimal yield is 13.61%,the actual optimal yield obtained through model verification is 13.29±0.37%,and the average error between them is0.38±0.27%.Six major unsaturated fatty acids were identified from the RCOO extracted by CO₂-SFE,including eicosapentaenoic acid（EPA）,α-linolenic acid（ALA）,docosahexaenoic acid（DHA）,arachidonic acid（ARA）,linoleic acid（LA）and oleic acid（OA）,which are important bioactive components in RCOO.Pearson correlation analysis showed that the yield of these unsaturated fatty acids is closely related to the yield of RCOO（the correlation coefficient is greater than 0.9）.Therefore,under the most suitable CO₂-SFE extraction conditions,the yield of RCOO and its main unsaturated fatty acids always reached the optimum value at the same time.Based on the above results,this work realized the development of CO₂-SFE green extraction process of RCOO and the determination of the main bioactive components in the extract,which laid the foundation for the green production of RCOO.Secondly,to explore the most ideal scheme of Rana chensinensis ovum protein isolate（RCOPI）as the outer wall material of microcapsules,this work was devoted to analyzing the physicochemical and functional properties of RCOPI recovered from by-products and investigating the effects of three different drying techniques including freeze drying（FD-RCOPI）,spray drying（SD-RCOPI）and vacuum drying（VD-RCOPI）.Scanning electron microscopy（SEM）was used to characterize the differences in appearance and microstructure of RCOPI prepared by different drying techniques,meanwhile,surface hydrophobicity,Fourier transform infrared spectroscopy（FTIR）and autofluorescence spectrum confirmed that drying techniques greatly affected the structure and conformation of RCOPI,leading to different physicochemical and functional properties.RCOPI had four main subunit bands distributed around 110,90,35 and 32 k Da in SDS-PAGE,and seven essential amino acids were detected,accounting for 43.27–43.65%of total amino acids,of which FD-RCOPI had a relatively large protein efficiency ratio,reflecting superior nutritional value.Besides,FD-RCOPI showed significant superiority in terms of solubility,water holding capacity（3.90±0.05g/g）,oil holding capacity（2.91±0.02 g/g）,stabilization of Pickering emulsion and inhibition of linoleic acid autoxidation.Interestingly,SD-RCOPI and VD-RCOPI had the optimal foaming capacity（FC）and foam stability（FS）respectively,while the FC and FS of FD-RCOPI were in the middle value.Overall,RCOPI had exceptional nutritional properties,especially FD-RCOPI exhibited relatively superior functional properties.FD-RCOPI is not only suitable for the production of viscous foods,bakery products and Pickering emulsions,but also could be used as an antioxidant to effectively delay or inhibit lipid oxidation,so it is the most suitable choice for the outer wall materials of microcapsule.Finally,to realize the comprehensive three-dimensional development of Rana chensinensis ovum,the two most important active parts（RCOO and RCOPI）were used to prepare microcapsules.RCOO was used as the core of the microcapsule,and RCOPI was used as the outer wall of the microcapsule.The microcapsules were prepared according to the mass ratios of 4:1,2:1,1:1,1:2 and 1:4.The results of encapsulation efficiency（EE）showed that the EE（67.09±9.37%）had reached the transition point when RCOO and RCOPI were mixed in a ratio of 1:2.At the same time,considering the loading amount of RCOO in the microcapsules,the mass ratio of oil to protein of1:2 was the most suitable choice for the preparation of microcapsules.There was no significant difference in the particle size of microcapsules prepared with different ratios of oil and protein,but the difference of surface morphology in their SEM images was consistent with the measurement results of EE.The co-localization of RCOO and RCOPI in the fluorescently labeled microcapsules indicated that the microcapsules successfully integrated the oil and protein wall materials together.Through the microencapsulation technology,the liquid RCOO（not easy to preserve）could be converted into a solid powder（convenient for storage）.And as the outer wall material,RCOPI had a great lipid autoxidation inhibitory effect,which provided a potential protective effect for the easily oxidized unsaturated fatty acid components in the core of the microcapsule.