Construction Of Lotus Seed Protein Isolate Emulsion System And Study On Its β-carotene Loading Effect

Plant-based proteins have the characteristics of natural non-toxicity,abundant resources and high nutritional value.Compared with small molecule surfactants,it is safer in food processing.The emulsions formed by them have the characteristics of convenient preparation,low cost,adjustable morphology,continuous reprocessability,load and sustained release of bioactive substances.Lotus seed protein is one of the main components of lotus seed,accounting for about 14%-20%of the dry weight of lotus seed,which has an important influence on the quality of lotus seed products and the design and development of new food.At present,the research on lotus seed protein mainly focuses on extraction,preparation,enzymatic hydrolysis,structure and functional properties,etc.,and there is no relevant application research on emulsifying functional properties of lotus seed protein.Based on this,this paper extracted the protein isolates from lotus seeds,and systematically studied the physicochemical properties and spatial molecular structure of lotus seed protein isolates.The interfacial properties and stability of low internal phase emulsion（protein concentration,p H value,ion concentration）and high internal phase emulsion were studied by using lotus seed protein isolate as emulsifier.On this basis,the formation mechanism,interface properties and stability of single lotus seed protein isolate emulsion and lotus seed protein isolate-polysaccharide composite emulsion,as well as the digestion properties and sustained release effect of loadedβ-carotene were compared and studied.The stability of lotus seed protein isolate at the oil-water interface and the load and sustained release mechanism of in vivo digestion were clarified.The purpose of this study is to provide reference for further processing of lotus seed protein isolate and emulsion products.The main conclusions are as follows:（1）Study on the structure and physicochemical properties of lotus seed protein isolate.The isoelectric point of lotus seed protein isolate（LSPI）was near 4.9,and its solubility was low,and its emulsifying property and emulsifying stability are 82.35%and 96.02%respectively.When the lotus seed protein isolate forms an aqueous solution,its Zeta potential value was-20.71 m V,and d_4,3was 77.51 nm,showing a single peak distribution,and the soluble part was easy to form a stable solution system.The composition and content of amino acids showed that the E/T and E/N ratios of lotus seed protein isolate were similar to those proposed by FAO/WHO,which indicated that that the amino acid composition of lotus seed protein isolate was balanced,which was a high-quality plant-based protein resource and rich in hydrophobic amino acids,accounting for 34.62%of the total amino acid content.Compared with soybean protein,the initial denaturation temperature of lotus seed protein isolate was higher（70.10℃）,and theα-helix content of 17.42%higher than13.05%-19.50%.Compared with wheat protein,theα-helix content of lotus seed protein isolate is lower than 30%-60%,and theβ-sheet structure is more,which reflects that the molecular chain structure of lotus seed protein isolate is both flexible and rigid.The above analysis will help to deeply understand the molecular characteristics of lotus seed protein isolate,clarify the structure-activity relationship between its physical and chemical characteristics and molecular structure,and provide theoretical basis for the application of lotus seed protein isolate in emulsion.（2）Study on emulsification properties and oil-water interface adsorption behavior of lotus seed protein isolate.In view of the good emulsifying properties of lotus seed protein isolate,it can be applied to the construction of emulsion system.Therefore,the effects of different factors and conditions on the formation and stability mechanism of emulsion were investigated with lotus seed protein isolate as emulsifier.When the protein concentration c increased from 0.5 wt.%to 3.0 wt.%,the droplet size decreased,the interfacial pressure and swelling modulus increased,the mutual flocculation between droplets was delayed,and the emulsion stability was improved.After 30 days of storage,the droplet size increased and the emulsion stability decreased.The stability of lotus seed protein isolate emulsion was affected by p H value（2.0-7.0）,and the droplet size was smaller and more uniform in the range of p H 2.0-4.0 and p H 5.0-7.0,and the emulsion has low flocculation and good emulsification stability.In addition,the aqueous solution and emulsion of lotus seed protein isolate were sensitive to Na⁺ions.With the increase of ionic strength（0-500 m M）,the solubility and particle size of aqueous solution increased,while the Zeta potential and fluorescence intensity decrease.Emulsification and droplet size of the emulsion increased significantly,the emulsification efficiency and flocculation increased,and the interfacial adsorption and viscoelasticity decreased,thus reducing the stability of the emulsion.The above experiments revealed the changing law of stability and interface characteristics of low internal phase emulsion of lotus seed protein isolate,which provided reference for constructing high internal phase emulsion system of lotus seed protein isolate.（3）Study on the mechanism of lotus seed protein isolate stabilizing high internal phase emulsionBased on the construction and stability of low internal phase emulsion system,the high internal phase emulsion system of lotus seed protein isolate was constructed,and the effects of different factors and conditions on the stability mechanism of high internal phase emulsion were investigated.When the protein concentration c increases,the droplet size decreases,the interfacial adsorption and viscoelasticity increase,forming a semi-solid state that is not easy to flow.When the oil phase volume fractionφincreases,the droplet size increases,the interfacial adsorption decreases,resulting in emulsion instability,and the stabilityφcan be as high as 88%,with high load capacity.In different environments,the emulsion remained stable during storage（30 days）and high temperature（100℃）,but the droplet size increased significantly in the freeze-thaw environment,and the emulsion system lost its stability,but it had three freeze-thaw-repreparation cycles.Recoverability provides a reference for the construction of high-load active substance delivery systems and the storage of emulsions in extreme environments.（4）Study on the stability and digestibility of lotus seed protein isolate emulsion loaded withβ-caroteneTheβ-carotene composite emulsion was constructed with lotus seed protein isolate as emulsifier.After 30 days of storage,the retention rates ofβ-carotene at 4℃,25℃ and 35℃ were 88.76%,74.67%and 45.67%,respectively.High temperature treatment at 100℃ caused the molecular structure of lotus seed protein isolate to gradually unfold and expose hydrophobic groups,forming a spatial network structure to give thermal stability to the LSPI-BC system and reduce the degradation ofβ-carotene.In the stage of simulated oral and gastric digestion in vitro,the emulsion potential changed from negative to positive,and the protein aggregates gradually dispersed.In the simulated intestinal digestion stage,the droplet size decreased significantly,the potential（absolute value）increased,and the free radical scavenging ability increased significantly after hydrolysis.At the same time,the free fatty acids were gradually released with the prolongation of digestion time,and the release amount increased significantly at the initial 30 min of digestion,reaching 61.94%at60 min,indicating that lotus seed protein isolate was suitable for embeddingβ-carotene,maintaining the stability of active substances in acidic medium,delaying the release ofβ-carotene in vitro digestion stage and improving its bioavailability（58.51%）.（5）Construction of oil-in-water emulsion stability by lotus seed protein isolate/polysaccharide covalent complex and its applicationBased on the conclusion of the previous chapter on the sustained release and digestibility of lotus seed protein isolate to construct emulsion and loadedβ-carotene,in order to further improve the sustained release and bioavailability ofβ-carotene,the protein and polysaccharide complex was selected to construct the emulsion system.Compared with the aqueous solution composed of lotus seed protein isolate and its glycated complexes（c-LSPI,p-LSPI,p H 7.0）formed with chitosan or pectin,the glycated complex has a lower potential value compared to a single lotus seed protein isolate.The content ofα-helix is reduced and transformed intoβ-sheet andβ-turn.The emulsion formed has a smaller droplet size,an increase in apparent viscosity,a decrease in creaming and coalescence between droplets and an uneasy flow state.In the simulated oral and gastric digestion stage in vitro,the release rates ofβ-carotene in c-LSPI and p-LSPI emulsions were only 3.42%,20.33%and 8.71%,27.41%,respectively.In the simulated intestinal digestion stage,β-carotene was gradually released,and the bioavailability was increased from 51.37%of lotus seed protein isolate emulsion to 80.44%（c-LSPI）and 71.22%（p-LSPI）.The above conclusions show that the glycosylated complex has better plasticity,stability and load capacity than the emulsion system constructed by lotus seed protein isolate,and can effectively delay the release ofβ-carotene in the body.