Effects Of Different Treatment Condition On The Properties Of Walnut Protein Isolates

Walnut protein is a high-quality resource of plant protein,which contains18 kinds of amino acids,has a well-balanced amino acids,and is rich in essential amino acids.The high proportion of gluten in walnut protein results in its low solubility.As a result,walnut protein is less widely used in food production process.At present,the product type of walnut protein is relatively simple,and it is mainly applied to produce walnut powder,walnut milk,walnut fermented milk etc,Therefore,in the present work,the walnut protein was modified by means of ultrasound,glycation and enzymatic hydrolysis in this work,and the functional properties and structural characteristics of the modified products were further studied,to expand the application of walnut protein in food processing and other fields.The main results were as follows:Firstly,walnut protein isolates were prepared by alkali extraction and acid precipitation.The effects of different factors?pH,ionic strength,gum arabic?on the solubility,emulsification activity and emulsification stability of WNPI were studied.The results showed that the solubility,emulsification activity and emulsification stability were the lowest around isoelectric point?PI 5.0?.At p H3.0 and pH 8.0,the addition of NaCl,CaCl₂ and gum arabic decreased the solubility of the proteins.Arabia gum had greater effect on the solubility of the proteins in acidic condition,whereas,NaCl and CaCl₂ had greater effect on the solubility of proteins in alkali condition.In addition,the solubility of proteins was more greatly affected by CaCl₂ than by NaCl.At different pH,NaCl decreased the emulsifying activity and emulsifying stability of the proteins.Moreover,gum arabic increased the proteins'emulsifying activity.However,the emulsifying stability of the proteins first drop and then increased with increasing gum arabic.Secondly,the impact of high-intensity ultrasound treatment on the molecular,physicochemical,and functional WNPI were investigated.Aqueous walnut protein suspensions were sonicated at varying power levels?200,400 or600 W?and times?15 or 30 min?,and then any alterations in protein properties were determined.SDS-PAGE indicated that there were no changes in protein electrophoretic patterns,indicating that sonication did not break covalent bonds.However,circular dichroism spectroscopy showed a change in protein secondary structure,with a decrease in?-helix and increase in?-sheet,?-turn and random coil after sonciation.There was an increase in surface free sulfhydryl?SH?groups and a decrease in fluorescence intensity after sonication,indicating that appreciable changes in tertiary structure occurred.Differential scanning calorimetry?DSC?confirmed that sonication caused appreciable denaturation of the protein,with a decrease in both the temperature and enthalpy of thermal denaturation.The size of the particles in aqueous walnut protein dispersions decreased with increasing sonication power and time as confirmed by static light scattering and scanning electron microscopy,suggesting that sonication dissociated protein aggregates.The SEM images showed that the intact lamellar structure of WNPI was destroyed after sonication.Moroever,the water-solubility?+22%?,emulsifying activity index?+26%?,and emulsifying stability index?+41%?all increased after sonication.These results suggest that sonication is a valuable tool for improving the functional attributes of walnut proteins.Thirdly,the effects of glycation on the function and structure of WNPI were studied.WNPI-MD conjugates were obtained by classical heating?12,24 h?and ultrasound?200,400,600 W,for 40 min?at 80?.Compared with classical heating,ultrasound could accelerate the graft reaction between WNPI and polysaccharides.Both treatments could improve the solubility,emulsifying activity and emulsifying stability of WNPI.However,the isoelectrical point of WNPI-MD shifted to lower pH values after classical heating,and its emulsifying activity and emulsifying stability were higher than sonicated conjugates.Ultrasonic treatment was able to produce WNPI-MD conjugates with higher color lightness.There was an decrease in surface free sulfhydryl?SH?groups after glycating with MD,and tertiary structure tended to be much looser.SDS-PAGE showed the formation of macromolecular polymers after glycation,accompanied by the disappearance of some low molecular weight bands.SEM images displayed that the WNPI-MD conjugates had thinner and more complete lamellar structure.WNPI-MD conjugates had less?-helix,?-turn and more?-sheet and disordered structure.In addtion,the content of?-helix and?-turn slightly increased with increasing ultrasonic power?400 W,600 W?.Finally,WNPI was hydrolyzed by trypsin and alcalase?30,60 or 90 min?and the effect of the two enzymes on the physico-chemical properties of WNPI were compared.The results showed that alcalase had better hydrolysis ability than trypsin.With the increasing of hydrolysis degrees,the solubility of hydrolyzate increased.Also,the emulsifying activity and emulsifying stability of trypsin hydrolyzates were significantly improved.The change in?potential of trypsin hydrolyzate was opposite to that of alcalase.with its absolute value decreasing.The content of surface free sulfhydryl?SH?groups of hydrolyzates increased with the increase of hydrolysis degree.The intrinsic fluorescence spectrum demonstrated that alcalase treatment had larger impact on tertiary structure of WNPI,and the maximum fluorescence emission wavelength redshift about 8¹0 nm.Secondary structure of hydrolyzates obviously changed,and the content of?-helix and?-turn decreased significantly,?-sheet and disordered structure increased sharply.However,the secondary structures of trypsin and alcalase hydrolyzates were not obviously different.The SDS-PAGE profile showed that as the degree of hydrolysis increased,some of the high molecular weight bands disappeared,and low molecular weight bands were formed.The microstructure of trypsin hydrolyzates were flaky and porous,and the size of alcalase hydrolyzates were much smaller.