Effects Of Protein Modification On The Functionality And Nutritional Value Of Hempseed Protein

The increasing demand for plant proteins,along with rising awareness of the nutritional and functional roles of dietary proteins,has prompted the nutrition and food industry to explore nontraditional protein sources.Hempseed protein with its excellent nutrition of value,low allergenicity,and superior digestibility has drawn great interest in both scientific and industrial fields.In the food industry,a wide range of products have been developed from hempseed proteins,for example,beverages,functional ingredients,nutritional supplements,and various personal-care products.The value and application of hemp protein in food products is closely related to the protein structure and functional properties.The poor solubility of hemp protein due to its compact structure greatly hinders the surface activity hence interfacial behavior and other functional properties of proteins,which limits its full application in the food industry.The objective of this study was to change the structure of hemp protein by multiple modification techniques,to clarify the effects of structural changes on the functional properties of proteins,and to improve the additional nutritional value of hemp protein products.The ultimate goal was to create a theoretical foundation for the expansion of the utility of hemp proteins as value-added functional food ingredients.Firstly,in whole-hempseed milk system,the effects of pH shift（pH7→pH12→pH7）and high pressure homogenization（HH,up to 60 MPa）treatment on the microstructure,physical and oxidative stablility of hemp milk was investigated.For hemp milk without pH shift,increasing the homogenization pressure（up to 60 MPa）resulted in a more uniform distribution of emulsion droplets（2.2–2.7μm）.When pH shift was applied prior to HH,large clusters and aggregates of oil droplets（3.5–8.2μm）were formed.Interestingly,hemp milk with such interactive structures was remarkably stable,showing negligible phase separation within 3-day storage at 4°C.Moreover,hemp milk made by combined pH shift and HH exhibited delayed hydroperoxides and malonaldehyde production,suggesting the resistance of such emulsion cluster structures to radicals.On the other hand,a significant reduction of microbial population was observed in hemp milk prepared by pH shift combined with HH.Alkaline pH₁₂ shift treatment performed at varying temperatures（20 to 80°C for 1,5,and60 min）was applied to structurally modify hemp seed protein isolate（HPI）.The solubility of HPI（²0%）was remarkably improved（P?0.05）with elevating temperature and prolonging holding time,reaching 97.5%at 80°C for 60 min.The treated HPI exhibited a strong tendency of forming soluble large aggregates.To limit lysinoalanine（LAL）production,heating was methodically controlled to 60°C and 5 min where the LAL content never exceeded 100 mg/100g protein and the loss of cysteine and lysine was also minimal.Furthermore,the effects of heat-aided pH shift treatment on the properties of HPI at oil-water interface was investigated by dependant drop analysis in combination with oscillation technology.HPI were able to absorb at the air–water interface to form elastic film,and the adsorption process was dominated by protein unfolding and rearrangement at the interface.The combination treatment led to the structural unfolding of proteins and the exposure of hydrophobic groups,which can significantly increase the surface activity of HPI with a higher surface pressure（π）;the dilatational elasticity（E_d）decreased with the increasing temperature,which may be associated with the formation of multi-layer membrane structure at the oil-water interface.The enhancement of surface activity and the formation of the multilayer film structure led to a higher emulsion activity index（EAI）and more uniform distribution of emulsion droplets.Genipin,a natural electrophilic crosslinker,was applied to modify hempseed protein isolate（HPI）.Genipin treatment up to 30 mM resulted in significant losses of sulfhydryls and free amines in HPI.Surface hydrophobicity decreased by nearly 90%with 30 mM genipin,corresponding to similar attenuations of tryptophan fluorescence.The genipin treatment converted proteins into highly crosslinked polymers.The O/W emulsion-filled hydrogels formed with such polymers exhibited substantially enhanced gelling ability:up to 3.3-fold increases in gel strength and 2.6-fold increases in gel elasticity over untreated protein.The genipin-modified composite hydrogels also exhibited a superior water-holding capacity.Microstructural analysis revealed a compact gel network filled with protein-coated oil globules that interacted intimately with the protein matrix when treated with genipin.The digestibility of the HPI hydrogels was reduced by genipin crosslinking in a dosage-dependent manner.High concentrations of genipin（≥400μM）could induce the decrease of cell proliferation and the reduction of cell appptosis,where the genipin-HPI complex showed less cytotoxicity in comparison with genipin.The uptake of the genipin-HPI complex in Caco-2 cells showed that the complex could be taken up by the cells,which is closely related to the incubation time and the complex concentration.Proteins and peptides when forming complexes with zinc can increase zinc bioavailability.Such complexation was investigated on hemp protein hydrolysates（HPHs）in the present study using Pepsin,Alcalase,Flavourzyme,Papain,Protamex,and Trypsin.Two solubility fractions of Zn²⁺–HPH complexes,i.e.,P₁（water-insoluble large peptides）and P₂（water-soluble small peptides,precipitable by ethanol）,were collected.The FTIR analysis on Pepsin-HPH suggested that P₁ and P₂ peptides had different Zn²⁺-binding sites where N–H and C=O were the primary sites in P₁ and P₂,respectively.Although the Zn²⁺-binding capacity（P₁ and P₂ combined）of HPHs was lower than that of nonhydrolyzed hemp protein,the P₂-bound Zn²⁺was more abundant in HPHs（up to 63.4%）than in nonhydrlyzed protein（29.6%）.Isothermal titration calorimetry corroborated with Zn²⁺-binding capacity for different HPH samples.Peptides produced with Flavourzyme had the highest Zn²⁺-binding activity（88.8%）while those with Pepsin exhibited the maximum solubility.Compared with ZnSO₄,Zn²⁺–HPH complex showed less cytotoxicity.Moreover,zinc transport capacity was appreciably enhanced by the formation of Zn²⁺–HPH complex,resulting in the improvement of zinc bioavailability.In summary,this study provides new ideas for the improvement of functional properties and the enhancement of additional nutritional value of hemp protein.It also provides a theoretical basis for better expanding the application of hemp protein as a functional ingredient in food products.