| The walnut in China has a high yield,but its low degree of deep processing hinders the development of walnut industry.Previous studies in our laboratory have shown that walnut aqueous extract can be separated into oil bodies cream,skim and precipitate by centrifugation,of which the skim can be used for beverage preparation and the precipitate can be used to prepare protein products(protein content,more than 85%,dry basis).This study focuses on the demulsification and application of oil bodies cream.The demulsification of walnut oil body was examined by two physical methods(the combined thin film drying-vacuum filtration method and freeze-thaw method),and the walnut oil and phospholipid-membrane protein concentrate were prepared.The emulsifying property of phospholipid-membrane protein concentrate was examined,and further,whether the purified phospholipid-membrane protein could be used for preparation of nanoemulsion(nano-scale reconstituted oil body)was investigated.This study can provide a novel strategy for the demulsification of oil body,which is meaningful for the comprehensive and value-added utilizations of walnut.Firstly,the walnut variety,which was used for oil body preparation,was selected from four main walnut varieties in China based on the activity of endogenous proteases and the degree of lipid oxidation.At the same time,amino acid,fatty acid,and metabolite composition of the four varieties were systematically compared.The results showed that there were significant differences in the protein content(13.73%～17.54%,Xinxin 2 highest,Qingxiang lowest),lipid content(69.10%～72.77%,Qingxiang highest,Xinxin 2 lowest),linolenic acid content(8.77%～13.23%,Wen 185 highest,Qingxiang lowest),bioactive substances(e.g.,sphingosine),and the endogenous protease activity(Wen 185 highest,Qingxiang lowest)of the four varieties(p < 0.05).With increasing roasting temperature,the acid value and peroxide value of walnut oil pressed from the four peeled walnut kernels(prepared by wet method well used in industry)showed a rising trend,but none of them exceeded the national standard.Amongst them,the oxidation degree of the Wen 185 and Qingxiang was relatively low.Comprehensively considering,Qingxiang was selected as the variety for oil body preparation.Secondly,the lipid distribution and their properties in the three centrifugal fractions separated from walnut aqueous extract were investigated,and LC-MS/MS was used to analyze the protein component of walnut oil body.The results showed that the lipids in the three centrifugal fractions were all in the existing states of oil body,but their particle sizes were different: OBs cream(average particle size: 2750 nm)> skim(average particle size: 905 nm)>precipitate(average particle size: 417 nm).There were nine membrane proteins in walnut oil body,including seven oleosin isoforms,one caleosin and one steroleosin.The membrane protein of the OBs cream was dominated by oleosin 1,while the membrane protein of oil body in the precipitate was dominated by oleosin 5.In addition,the proteins adsorbed by oil body were also different,the oil body in OBs cream mainly adsorbed sucrose-binding protein,while the oil body in the precipitate mainly adsorbed 11 S globulins.Thirdly,the combined thin film drying-vacuum filtration method was designed to demulsify walnut oil body,and its demulsification mechanism was explored,and compared its efficiency with the optimized freeze-thaw method.The results showed that the OBs cream could be separated into walnut oil(demulsification rate: 95.45%)and phospholipid-membrane protein concentrate,and the concentrate was composed of 67.23% neutral lipid,19.41% protein(membrane proteins contributed more than 50% of protein),6.61% phospholipid and 6.75%other components(e.g.sphingosine)by this method.The demulsification mechanism of this method: the distance among oil body shortened during water evaporation,and gradually coalesced,resulting in the reduction of the specific surface area of coalesced oil body.As a result,the phospholipid-membrane protein was squeezed out from the coalesced oil body.With further coalescence,the coalesced oil body became larger and larger until demulsification.The optimized conditions of freeze-thaw method were as follows: the solid content of OBs cream was 75%,the p H was adjusted to 5.0 with lactic acid,and the maximum demulsification rate was 93.94% when frozen at-20 ℃ for 48 h and thawed at 30 ℃ for 2 h.In contrast,the combined thin film drying-vacuum filtration method showed a slightly higher breakage rate than the freeze-thaw method.Comparison of walnut oil quality: the quality of walnut oil obtained by the freeze-thaw method is superior to that of the combined thin film drying-vacuum filtration method.The fatty acid composition of walnut oil obtained by the two methods was not significantly different,and the unsaturated fatty acid content was above 90%,which met the standard of extra-grade walnut oil and the national standard of first-grade walnut oil.Finally,the emulsifying property of phospholipid-membrane protein concentrate,which was the by-product from freeze-thaw method,were investigated.The results showed that the phospholipid-membrane protein concentrate had good emulsifying property.After emulsification at 70 MPa,the emulsion with average particle size of 212 nm could be obtained,which was close to nanoemulsion(< 200 nm).Further,the phospholipid-membrane protein was prepared from purified oil body(prepared from OBs cream by p H 11 washing),and its emulsifying property and stability were examined.The results showed that the phospholipidmembrane protein could be used as a natural emulsifier for the preparation of nanoemulsion.When phospholipid-membrane protein/oil = 1:1.5,the nanoemulsion with average particle size of 129 nm could be prepared by ultrasonic emulsification(180 W,8 min).The stability of nanoemulsion was mainly maintained by electrostatic repulsion,and it remained stable at p H <4.0 or p H > 6.0.In addition,the nanoemulsion had good thermal stability and salt stability. |
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