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Structural and functional properties of hydrolyzed/glycosylated ovalbumin under spray drying and microwave freeze drying

2020-05-28LiliLiuXiaoningDaiHuaibinKangYunfengXuWeimingHao

食品科学与人类健康(英文) 2020年1期

Lili Liu,Xiaoning Dai,Huaibin Kang,Yunfeng Xu,Weiming Hao

Food and Biology Engineering College,Henan University of Science&Technology,471003,Luoyang,China

Keywords:

ABSTRACT

1.Introduction

As a functional ingredient,egg white protein(EWP)is widely used in the food industry because it has high nutritional quality and a wide range of functional properties[1].The functional properties of EWP must be improved to expand its industrial utilization.Many methods,including chemical modification and enzymatic approaches,have been utilized to improve EWP’s properties,including emulsification,foaming,gelation,and water-bonding capability.In terms of food safety,enzymatic phosphorylation is the most desirable among all these methods[2].In enzymatic modification,selecting the proper proteolytic enzyme,conditions,and degree of hydrolysis is important for the improvement of the functional properties of proteins[3].Meanwhile,the protein side chain reaction in chemical modification can preserve or alter the electrostatic charge and enhance the functional properties of proteins[4-6].However,chemically phosphorylated food proteins are unacceptable to consumers because of the intense reaction and difficulty of removing remaining chemicals.Glycosylation,which is also known as the Maillard reaction,improves the functional properties of food proteins under mild and safe conditions,and this method does not require extraneous chemicals,making it a promising approach for protein modification in the food industry[5,6].The protein-sugar products obtained via the Maillard reaction are usually formed by the conjugation of protein-sugar mixtures in certain conditions[7,8].

Ovalbumin(OVA)is the main ingredient in EWP,and it accounts for about 54% of the total protein content;its behavior plays a main role in the functional properties of EWP[9].OVA considerably affects the application of EWP.Thus,improving OVA’s functional properties is beneficial for the further application of EWP in the food industry.Several effective methods have been developed to enhance the functionalities of OVA,and these include dry-heating phosphorylation[10],molten globule state caused by pH alternation[11],and the Maillard reaction[12].For OVA improved by the Maillard reaction,investigating the physicochemical and structural changes can help clarify the relationship between the structure of dried EWP and its properties after the Maillard reaction.On the basis of these findings,we hypothesized that synergistic modification of enzymatic hydrolysis and glycation could improve the functional characteristics of EWP.

At present,dried EWP has numerous applications in food preparation,such as baking and confectionary and meat products,because of its microbiological safety and reduced volume compared with unshelled or liquid eggs.However,food processing and storage conditions affect the physicochemical and functional properties of EWP[13].

OVA is sensitive to heat and has a critical denaturation temperature of 70°C[14].In high-temperature drying,EWP denatures,and its functional properties are reduced[15].Typically,through the modification of protein structures,protein drying can induce several stresses that can denature proteins[16].Spray drying(SD)is one of the most frequently used operations for the drying of heatsensitive powder in the food industry.SD,which produces dry,stable,and small-volume food materials,has a low operation cost and short processing time.However,due to the high-temperature drying strategy adopted in the SD process,this method could decrease the quality of EWP.Freeze drying can remove water by sublimation under vacuum conditions to prepare high-quality dried products because the proteins experience reduced thermal and water evaporation-related stresses[17].However,freeze drying is uneconomical for food processing because of its low efficiency and high energy consumption,which limit its industrial-scale application.Therefore,new drying methods should be developed to provide alternative options for producing high-quality products in the egg protein industry.

Compared with the conventional hot-air drying method,microwave drying is more rapid,uniform,and energy efficient.When used as the heat source in freeze drying,microwaves can heat a material in a vacuum environment and may considerably improve the freeze drying rate;this method is called microwave freeze drying(MFD).In recent years,MFD has been investigated as a potential method of obtaining high-quality dried food products with low energy consumption.To date,only a few reports on MFD have been published.Wang et al.[18]used MFD to dry a liquid material,and Duan et al.[19]utilized MFD to dry type I collagen from a bovine bone.These studies showed that the drying time can be considerably reduced by MFD,and the method can be used to dry liquid materials because of its high efficiency.Therefore,MFD has a potential to dry high-quality EWP,but the effects of microwave heating on the native structure and functional properties of modified OVA are unclear.

To the best of our knowledge,no studies have been conducted on the combined effect of hydrolysis and glycation treatments.Given that SD is an important drying method for preparing various protein powders in the food industry,the objectives of the present work are to(i)study the effect of hydrolysis followed by glycation with D-lactose of OVA(HGOVA)and(ii)investigate and compare the influences of two drying methods(SD and MFD)on the functional and structural properties of HGOVA.This study is expected to provide useful information on the potential commercial applications of egg protein powders.

2.Materials and methods

2.1.Preparation of native ovalbumin

Native ovalbumin(N-OVA)was prepared.First,OVA was purified from fresh infertile egg white(less than 10 days after being laid;purchased from Dennis Supermarket,Luoyang,China)by a crystallization method in Na2SO4(60% m/m)solution and recrystallized three times[20].Second,the final protein was brought into the solution and dialyzed in deionized water for 24 h at room temperature.Lastly,the dialysate was lyophilized for further use.Three replicates of the lyophilized material(OVA)were analyzed with the Bradford method to determine protein contents above 91.0%.

2.2.Preparation of HGOVA

OVA was hydrolyzed to prepare polypeptides and eventually obtain HGOVA systems.The optimal conditions for enzymatic hydrolysis were pH 8.5(1 M NaOH was added to adjust the pH),alcalase(E/S=5500 U/g),5%(m/V)of the substrate concentration(OVA),and temperature of 55°C for 5 h.At the end of hydrolysis,the samples were heated at 100°C for 20 min to inactivate the enzyme.The protein hydrolysates were centrifuged at 6000×g for 20 min,and the soluble fractions were freeze dried(-50°C).Then,glycation was performed with D-lactose(Sigma-Aldrich,St.Louis,USA).OVA polypeptides were dissolved in 20 mM carbonate buffer(pH 9.0)at a fixed protein concentration(8%,m/V)with D-lactose(12%of the protein dry weight).The OVA-lactose solutions were lyophilized,and the dried samples were incubated at pH 9.0,temperature of 65°C,and relative humidity of 60%by using a saturated KBr solution for 24 h.The glycated proteins were dialyzed overnight at 4°C to remove free sugar.The dialysis retentates were collected and stored at 4°C for subsequent testing.

2.3.SD

To obtain spray-dried glycosylated OVA(SD-HGOVA),the prepared HGOVA was homogenized before spray drying on a rotary atomizer(185°C at the inlet and 90°C at the outlet)using a with a 0.5 mm nozzle(GEA Niro Process Engineering,Denmark).The resulting powder was stored at 4°C.

2.4.MFD

A multifunctional microwave dryer developed by Duan et al.[19]was used in the drying process.HGOVA was dried in the MFD chamber after being frozen at-25°C for 12 h to reach the final moisture content of 3.0% w.b.MFD was performed at 80 Pa cavity pressure,-40°C cold trap temperature,and 2 W/g microwave power.The dried sample was obtained and referred to as MFDHGOVA.

All the experiments were repeated three times,and the dehydrated samples were stored in polyethylene bags immediately after drying for further use.

2.5.Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)analysis of SD-HGOVA and MFD-HGOVA was performed in accordance with the discontinuous buffer system of Laemmli[21]at 5% stacking gel and 10% separating gel by using a Mini Protean II Slab cell equipment(Bio-Rad,Richmond,California,USA).The molecular weight markers(Fermentas,St.Leon-Rot,Germany)were run simultaneously to determine the molecular weight.The gels were stained with Coomassie brilliant blue R-250(40%methanol-10%acetic acid solution,v/v).

2.6.Fourier transform infrared spectroscopy

The secondary structures of SD-HGOVA and MFD-HGOVA were characterized with a Fourier transform infrared(FTIR)instrument(Nicoler-SX-170,Thermo Nicolet Corporation,USA).The dried protein samples were mixed with solid KBr,ground,and pressed into pellets(1-2 mm thick).The scanning range and wave number were set to 4000-400 cm-1and 128 scans,respectively.The medium(KBr)with no protein was recorded as the reference spectrum.OMNIC software was used to analyze the FTIR spectral data.

2.7.Differential scanning calorimetry

Samples of SD-HGOVA and MFD-HGOVA were analyz ed using a Pyris Diamond differential scanning calorimeter(DSC)(Perkin Elmer,USA).The samples(5-8 mg)were weighed in hermetic aluminum pans,sealed,and placed in the heating cell of the calorimeter.The samples were heated at temperatures ranging from 20°C to 200°C at a rate of 10°C/min.The thermal transition temperature(onset and maximum temperature peak,Td)was calculated with an empty aluminum pan as a reference.All experiments were performed in triplicate to ensure good reproducibility.

2.8.Scanning electron microscopy

To examine the effect of MFD and SD on HGOVA,the gel structure morphologies of SD-HGOVA and MFD-HGOVA were determined by scanning electron microscopy(SEM)(S-3000 N,Hitachi Limited,Japan).Before imaging,all samples were mounted on carboncoated SEM stubs and sputter-coated with a thin layer of gold.An acceleration voltage of 20 kV was used during imaging.The microappearances of the gel with a magnification of 5000 were easily observed.

2.9.Particle size measurement

The particle sizes of SD-HGOVA and MFD-HGOVA were measured using a NICOMP 380/ZLS(PSS Nicomp,Santa Barbara,CA,USA).The results were displayed as volume-averaged sizes.Before measurements,the prepared 1% HGOVA solutions were diluted with 10-fold deionized water,and all experiments were performed at 25°C with a 90°scattering angle.

2.10.Color detection

The color properties(L*,lightless;a*,redness;b*,yellowness)of the SD-HGOVA and MFD-HGOVA powders were obtained using a Konica Minolta chroma meter(model CR-400,Osaka,Japan)with a CIELAB scale.

2.11.Determination of functional properties

2.11.1.Solubility

Protein samples(1 g/L)were dissolved in 50 mmol/L Tris-HCl buffer(pH 7.0)and centrifuged at 4500×g for 15 min.The concentration of protein in the supernatant was determined by referring to the method of Lowry et al.[22].

2.11.2.Emulsifying properties

The emulsifying activity and stability of the protein samples were determined as described by Pearce and Kinsella[23].The emulsifying activity index(EAI)and the emulsifying stability index(ESI)were calculated according to Eqs.(1)and(2),respectively.

where A500represents the absorbance at 500 nm and 0.25 is the oil volume fraction.

whereΔA=A0-A10,Δt=10 min,and A10and A0represent the absorbance values at 10 min and at the start of the experiment(zero time)at 500 nm wavelength,respectively.

2.11.3.Foaming properties

Foaming ability(FA)and foam stability(FS)were measured in accordance with the procedure of Miller and Groninger[24].FA was calculated with Eq.(3).

where A represents the volume after homogenizing(mL)and B represents the volume before homogenizing(mL).The homogenized sample was left to stand at 20°C for 3 min,and the volume was recorded.FS was calculated with Eq.(4).

where N is the volume of the foam after standing(mL)and M is the volume of the foam before homogenizing(mL).

2.11.4.Water-and oil-absorption capacity

The capacity of the protein samples to bind water and oil was evaluated using the method adopted in a previous study[25].

Exactly 0.5 g of samples(W0)of SD-HGOVA and MFD-HGOVA were dispersed in 6 mL of soybean oil or deionized water in a pre-weighed centrifuge tube.The volume(V1)of the samples was recorded after standing at 30°C for 30 min.The dispersion was centrifuged at 3000×g for 20 min.The supernatant was carefully poured into a 10 mL graduated cylinder,and the volume(V2)was recorded.WAC or OAC(mL of water or oil per gram of protein)was calculated with Eq.(5).

2.11.5.Gel properties

The sample protein powders(SD-HGOVA and MFD-HGOVA)were added to distilled water and blended gently at 20°C to prepare an 11% protein solution.About 20 mL of the protein solution was transferred to a 25 mL beaker,heated in a water bath at 80°C for 1 h,and subsequently cooled at room temperature for at least 4 h.The gelled samples were then cut into cylinders(diameter 10 mm,height 20 mm).The texture of the gels was detected with a TA-XT2 texture analyzer(Stable Micro System Ltd.,Leicestershire,UK).A 20 mm diameter plate probe was used,and double compression tests(penetrate to 50%depth)at a speed of 2 mm/s were performed.The tests were conducted in triplicate,and the average firmness values were used as the gel strength of the samples.

2.12.Statistical analysis

The statistical significance of the data was analyzed using SPSS version 11.5.1.The analyses of variance were performed via the ANOVA procedure.The significance of data was defined as P<0.05.

3.Results and discussion

3.1.Structural characteristics

3.1.1.Molecular weight

The changes in the molecular weight of the different samples were examined through SDS-PAGE.As shown in Fig.1,N-OVA(lane 1,control)exhibited a main band with a molecular weight of 45 kDa,which is in accordance with the report of Mine et al.[26].The molecular weight of hydrolyzed OVA(HOVA)was reduced by enzymatic hydrolysis(lane 2),indicating that the protein molecules were degraded into small molecule peptides,and their molecular weight distribution ranged from 14.3 kDa to 29 kDa.After OVA hydrolyzation/glycosylation with D-lactose(lanes 3 and 4),the molecular weights of SD-HGOVA and MFD-HGOVA changed considerably compared with that of HOVA(lane 2).However,the patterns of SD-HGOVA and MFD-HGOVA were almost identical,indicating that SD and MFD processing did not lead to the dissociation of the HGOVA subunits(lanes 3 and 4).

Fig.1.SDS-PAGE profiles of the different samples(5% stacking gel and 10% separating gel).Lane 1:N-OVA(control);Lane 2:hydrolyzed OVA(HOVA);Lane 3:SD-HGOVA;Lane 4.MFD-HGOVA;Lane Mark,standard protein markers:aprotinin(6.5 KDa);lysozyme(14.3 Kda),trypsin inhibitor(20.1 kDa),Carbonic Anhydrase(29.0 kDa),ovalbumin(44.3 kDa),bovine serum albumin(BSA)(66.4 kDa),Phosphatase b(97.2 kDa),β galactosidase(116.0 kDa),myosin(200.0 kDa).

Fig.2.The DSC curves for different samples.a.N-OVA;b.hydrolyzed OVA(HOVA);c.SD-HGOVA;d.MFD-HGOVA.

3.1.2.DSC analysis

Fig.2 shows the DSC profiles of the different samples,including HGOVA dried by the two different methods.The thermal properties of OVA were affected by enzymatic hydrolysis and the drying methods.The denaturation peak temperature(Tp)can be used to reflect the thermal stability of proteins.DSC thermograms of N-OVA and HOVA show that their Tpwas 77.55°C and 85.04°C,respectively.The Tpof MFD-HGOVA(95.05°C)was much higher than those of SD-HGOVA(89.45°C),N-OVA,and HOVA.These results indicate that denaturation temperature may increase significantly if proteins are conjugated with lactose.Lechevalier et al.[27]reported that EWP denaturation occurs easily in SD due to the use of hightemperature hot air.Therefore,the denaturation extent of EWP in the SD process,wherein hot oscillating gas of 185°C is used,must be determined.In this study,MFD was assumed to be 0% protein denatured because MFD-HGOVA was obtained by drying the initial frozen HGOVA to a dry solid with a final moisture of 3.0%in an MFD oven using a low temperature,which was considerably lower than the denaturation temperature of HGOVA(about 95.05°C).By calculating and comparing the total denaturation enthalpies using the DSC curves shown in Fig.2,the protein non-denaturation degree of SD-HGOVA relative to MFD-HGOVA was determined to be 94.2%,indicating that some protein denaturation occurred in the SD process.The reason may be the short residence time of the egg white in the SD dryer due to its high drying rate.Given that the residence time was less than 1 s and the gas temperature was only 65°C at the drying chamber outlet,the temperature of SD-HGOVA did not even increase to its denaturation temperature at the end of drying.EWP did not have sufficient time to denature completely in such a short residence time[13].

Fig.3.FTIR spectra for different samples.

3.1.3.FTIR spectroscopic analysis

The information on the secondary structures of N-OVA,HOVA,SD-HGOVA,and MFD-HGOVA was investigated via FTIR(Fig.3).The changes in the peak position of amides I(1700-1600 cm-1),II(1530-1550 cm-1),and III(1260-1300 cm-1)indicate the transformation of protein structures[28].Our results showed that several peak positions of four samples changed remarkably,suggesting that the transformation of the secondary structure of OVA occurred.For different samples,the changes in the wavelengths of bands at 3500-3000 cm-1and 1700-1100 cm-1(COO-region,related to sugar presence)were associated with their hydrogen-bond sensibility in relation to sugar conjugation.Meanwhile,the enzymatic hydrolysis treatment resulted in N-H stretching vibration and the formation of a hydrogen bonding complex,which enhanced absorption.The peak position of amide I shifted from 1645.63 cm-1(N-OVA)and 1647.21 cm-1(HOVA)to 1651.10 cm-1(SD-HGOVA)and 1656.10 cm-1(MFD-HGOVA).An increase in wave number revealed that part of the β-sheet in OVA turned into a α-helix or random coil.Amide I was caused by either a stretching vibration of C=O or a hydrogen bond.This may be due to the cleavage of the OVA peptide chain by enzymatic hydrolysis and glycation,which affected the stretching vibration of C=O.The peaks of SD-HGOVA and MFD-HGOVA were different,indicating that the protein structures were changed by the drying methods.In general,structural transformation of MFD-HGOVA and SD-HGOVA was the cause of the differences in the functional properties of proteins.Thus,the significant difference in the structural compositions observed in this study confirmed the structural-functional relationship.

Fig.4.SEM images of N-OVA(control),SD-HGVOA and MFD-HGVOA(Magnification:5000×).

Fig.5.Pictures of N-OVA(control),SD-HGVOA and MFD-HGVOA.

3.1.4.SEM analysis of the gel structure

The SEM images revealed the differences in gel shape among N-OVA,SD-HGOVA,and MFD-HGOVA(Fig.4).The gel of the unmodified protein(N-OVA,control)showed a porous,loose,irregular net-like structure.The microstructures of the synergistic modified proteins(SD-HGOVA and MFD-HGOVA)exhibited a dense and smooth net-like structure.No obvious difference in terms of gel microstructure was observed between SD-HGOVA and MFDHGOVA.The gel microstructure of SD-HGOVA was tighter and denser than that of MFD-HGOVA.The differences in size,texture,and color were apparent even to the naked eye(Fig.5).

3.1.5.Particle size and color

Table 1 The particle size and color(L*,a*and b*)of HGOVA powders dried by SD and MFD.

Table 1 shows the size distributions of the N-OVA(control),SD-HGOVA and MFD-HGOVA powders.Dynamic light-scattering measurements within the diameter range of 3-5000 nm revealed that N-OVA,SD-HGOVA and MFD-HGOVA had wide size distributions in the aqueous state.When measured with the DC-P3 colorimeter,The SD powders had average L*,a*,and b*values of 89.74±0.05,-3.26±0.01,and 8.17±0.06,respectively.The MFD powders had mean L*,a*,and b*values of 98.73±0.07,-3.32±0.02,and 5.71±0.05,respectively(Table 1),which agrees with the findings of Zhou et al.[29].N-OVA had the highest L*value,and the lowest a*and b*values among the three samples.This might be due to the browning reaction caused by glycosylation,which made the colors of SD-HGOVA and MFD-HGOVA darker.MFD-HGOVA had a higher lightness value but almost the same redness value compared with SD-HGOVA.During MFD,due to the efficiency of microwave drying under vacuum conditions,the temperature of the sample surface was low,and most of the ice in the sample sublimated into vapor directly.This condition might have helped maintain the good color properties of the MFD-HGOVA powder.However,the color of SD-HGOVA was much yellower than that of MFD-HGOVA possibly because SD used a longer drying time compared with MFD.The mechanism of color change was complex,which could be due to the oxidization reaction that occurred during drying.Although the powder had a short residence time in the SD dryer,the oxygen concentration in the hot air used in traditional spray dryers is higher than that in MFD.In this environment,the oxidization reaction of protein is a major influencing factor in the SD dryer,and it causes a change in the color of HGOVA.

Fig.6.The solubility of N-VOA,SD-HGVOA and MFD-HGVOA at different pHs.

3.2.Functional properties

3.2.1.Solubility

Solubility is another important functional property of proteins that affects emulsifying and foaming properties[30].The solubility of SD-HGOVA and MFD-HGOVA in the pH range of 2-9 is shown in Fig.6.The solubility values of both proteins subjected to different drying methods were significantly higher(P<0.05)than that of N-OVA(control).SD-HGOVA had higher(P<0.05)solubility than MFD-HGOVA.However,SD-HGOVA and MFD-HGOVA exhibited similar pH dependence.Solubility increased with the increase in pH value when pH was above its isoelectric point at 4.0(lower than N-OVA with an isoelectric point at 4.5).The difference in solubility may be related to the difference in protein surface charges because of the exposure/formation of charged residues,which followed the protein structural transitions due to de-protonation[31].Moreover,the differences in particle size distribution between SDHGOVA and MFD-HGOVA may result in different solubility values.In terms of solubility properties,SD HGOVA has great potential for application in the food industry.

Table 2 Functional properties of N-OVA,SD-HG OVA,and MFD-HG OVA at pH 7 and 25°C.

3.2.2.Emulsifying activity and emulsion stability

EAI and ESI are two important functional characteristics of proteins that influence the behavior of various products[32].Table 2 describes the effects of drying on the EAI and ESI of native and modified OVA.The high emulsifying capacities in three protein samples indicated that they are suitable for application in food and pharmaceutical products as emulsifiers[33].Generally,the emulsifying properties of HGOVA(SD and MFD)were better than those of NOVA(P<0.05),indicating that synergistic modification can improve the emulsifying properties of OVA.The EAI values of the SD and MFD samples were similar,but the EAI of SD-HGOVA was higher than that of MFD-HGOVA at pH 7(P>0.05).The difference may be due to the smaller particle size and higher solubility of the protein powder obtained by SD.

The ESI value of MFD-HGOVA was higher than that of SD-HGOVA(P<0.05).A high ESI implies high stability at the interface and sufficient flexibility to form strong interfacial membranes,which enhance the stability of the emulsion[34].The reason might be due to the microwave treatment.After the microwave process,the hydrophobic and hydrophilic portions of the proteins were stretched out;exposure of these polar and apolar moieties at the oil-water interface enhanced their emulsifying stability[35].In addition,emulsifying properties depend not only on the solubility of proteins but also on certain factors,such as pH,droplet size,net charge,interfacial tension,viscosity,and protein conformation[30].

3.2.3.FA and FS

In addition to protein glycation,enzymatic hydrolysis is another suitable method for altering the foaming properties of proteins as a function of the molecular weight distribution and composition of different components,which in turn depend on the degree of hydrolysis[36].

FA and FS are important parameters for characterizing the functional properties of proteins.The FA and FS of SD-HGOVA and MFD-HGOVA are presented in Table 2.The foam properties of SD-HGOVA and MFD-HGOVA were higher than those of N-OVA(P<0.05),indicating that synergistic modification improved the foam properties of OVA.Although the FA value of MFD-HGOVA was less than that of SD-HGOVA(P>0.05),the FS value of MFDHGOVA was significantly higher than that of SD-HGOVA(P<0.05).This finding suggests that the drying method influenced FA and FS values.The reason was that SD might cause more unfolding of the proteins than MFD could[37],thus improving the surface activities of these proteins.Given that SD-HGOVA has a smaller particle size and higher protein solubility than MFD-HGOVA,SD-HGOVA is adsorbed faster during whipping or bubbling and therefore exhibits higher foaming capacity than MFD-HGOVA.In addition,the electrical conductivity of the MFD protein samples was likely increased;thus,the distributions of charged,polar,and nonpolar residues of the protein molecules were altered,thereby decreasing the FA of MFD-HGOVA compared with that of SD-HGOVA.

3.2.4.WAC/OAC and gel properties

As shown in Table 2,SD-HGOVA and MFD-HGOVA showed significant changes in WAC and OAC in comparison with N-OVA(P<0.05).The proteins obtained from both drying methods exhibited good WAC,which can be attributed to the exposure of polar groups that exert a substantial effect on the amount of water absorbed[35].WAC of HGOVA prepared by MFD was significantly higher than that prepared by SD at pH 7(P<0.05)because of the less compact/condensed and more porous particle morphology.Although the solubility of SD-HGOVA was higher than that of MFD-HGOVA at pH 7(P<0.05;Fig.5),no direct correlation existed between WAC and solubility[38].reported that protein structures might be responsible for the differences in the WAC of proteins obtained by diverse drying methods.OAC is a vital functional characteristic of proteins utilized in the manufacture of meat and confectionery products.The OAC of HGOVA obtained by MFD in this study was much higher than that obtained by SD at pH 7(P<0.05;Table 2).This result can be attributed to the good porous structure of MFD powders that contributes to satisfactory and rapid oil absorption.The MFD process forms a porous structure that generally promotes the preferential migration/diffusion of proteins[19].Therefore,SD-HGOVA has a lower OAC than MFDHGOVA because of the more compact and less porous structure of the former compared with the latter.

Gel property is another important functional characteristic of OVA.When OVA is used as an ingredient,it can affect the adhesive and hydration properties of meat products[28].As indicated in Table 2,SD-HGOVA and MFD-HGOVA had higher values of gel hardness than N-OVA(P<0.05).The MFD-HGOVA sample had a gel hardness of(883.65±35.7)g,which was lower than that of SD-HGOVA.These results suggest that a relatively lower extent of denaturation occurred in MFD-HGOVA because of its higher denaturation temperature compared with SD-HGOVA,and this condition decreased the hardness value of MFD-HGOVA.

4.Conclusions

The effects of SD and MFD methods on the structural and functional properties of HGOVA were studied.The results showed that synergistic modification altered the structure of the protein and improved its functional properties.SD-HGOVA had higher protein solubility,emulsifying activity,foaming capacity,and gel hardness than MFD-HGOVA.However,MFD-HGOVA had higher emulsion stability,foam stability,water/oil absorption capacities,and thermal stability than SD-HGOVA.These findings indicate that the two drying methods lead to different changes in conformations associated with their physicochemical and functional properties.Generally,structural transformation of MFD-HGOVA by unfolding into a higher-ordered conformation with stronger intra-molecular hydrogen bonds could be responsible for the higher thermal stability compared with that in SD-HGOVA and/or cause differences in other functional properties.However,further study is necessary to understand further the relationship between structure and functionality.This research revealed that the two drying methods for HGOVA preparation can significantly affect structural and functional properties.Selecting an appropriate drying method can enhance the potential applications of HGOVA in the food industry.Therefore,although SD is an excellent choice in egg manufacturing,MFD can be an alternative because it substantially reduces the denaturation of proteins at high temperatures while maintaining good product quality.

Declaration of Competing Interest

The authors declare that they do not have any conflict of interest.

Acknowledgements

This study was supported by Natural Science Foundation of China(No.U1704114),and Key Scientific Research Program of Henan Province(No.182102110346,161100110900).