effects of multi-frequency power ultrasound on the enzymolysis and structural ch.
Effects of multi-frequency power ultrasound on the enzymolysis and structural characteristics of corn gluten
meal
Jian Jin a ,Haile Ma a ,b ,?,Kai Wang a ,Abu El-Gasim A.Yagoub a ,c ,John Owusu a ,d ,Wenjuan Qu a ,b ,Ronghai He a ,b ,Cunshan Zhou a ,b ,Xiaofei Ye a ,e
a
School of Food and Biological Engineering,Jiangsu University,301Xuefu Road,Zhenjiang,Jiangsu 212013,China b
Jiangsu Provincial Key Laboratory for Physical Processing of Agricultural Products,Zhenjiang,Jiangsu 212013,China c
Faulty of Agriculture,University of Zalingei,PO Box 6,Zalingei,Sudan d
Department of Hospitality,School of Applied Science and Technology,Koforidua Polytechnic,P.O.Box 981,Koforidua,Ghana e
Department of Biosystems Engineering and Soil Science,University of Tennessee,Knoxville 37996,USA
a r t i c l e i n f o Article history:
Received 25November 2014
Received in revised form 20December 2014Accepted 21December 2014
Available online 31December 2014Keywords:
Multi-frequency power ultrasound Zein Glutelin
Circular dichroism
Scanning electron microscope Molecular conformation
a b s t r a c t
The aim of this study was to investigate the effect of multi-frequency power ultrasound (sweeping frequency and pulsed ultrasound (SFPU)and sequential dual frequency ultrasound (SDFU))on the enzy-molysis of corn gluten meal (CGM)and on the structures of the major protein fractions (zein ,glutelin )of CGM.The results showed that multi-frequency power ultrasound pretreatments improved signi?cantly (P <0.05)the degree of hydrolysis and conversion rate of CGM.The changes in UV–Vis spectra,?uores-cence emission spectra,surface hydrophobicity (H 0),and the content of SH and SS groups indicated unfold-ing of zein and glutelin by ultrasound.The circular dichroism analysis showed that both pretreatments decreased a -helix and increased b -sheet of glutelin .The SFPU pretreatment had little impact on the second-ary structure of zein ,while the SDFU increased the a -helix and decreased the b -sheet remarkably.Scanning electron microscope indicated that both pretreatments destroyed the microstructures of glutelin and CGM,reduced the particle size of zein despite that the SDFU induced aggregation was observed.In conclusion,multi-frequency power ultrasound pretreatment is an ef?cient method in protein proteolysis due to its sonochemistry effect on the molecular conformation as well as on the microstructure of protein.
ó2014Elsevier B.V.All rights reserved.
1.Introduction
Protein is an essential component for human nutrition.It is esti-mated that 25%of the world population do not have adequate sup-ply of protein [1].The CGM,a byproduct of corn wet-milling process in the production of corn starch,contains 600–710g kg à1protein.The major protein fractions of the CGM are zein and glut-elin ,representing 680g kg à1and 280g kg à1of the total protein weight,respectively.Unfortunately,the CGM is underexploited due to its special composition.For instance,zein is water insoluble,and thus has limited uses in human food industry.It is reported that the water solubility of zein can be increased by enzymatic modi?cation [2],and this can increase its utilization.Nevertheless,
the traditional enzymolysis has many disadvantages such as low utilization rate of the enzyme,low conversion rate of the substrate,long enzymolysis time,and energy-extensive consumption [3,4].This is largely due to the unsuitable conformation of protein,which makes it dif?cult for the enzyme to attack the cleavage sites.Therefore,developing a more ef?cient enzymolysis method to overcome these technical bottlenecks mentioned above is in great demand.
The ultrasound technology,as a novel non-thermal physical pro-cessing technology,has many applications in food [5]and its related ?elds [6].Acoustic cavitation,resulting from the mechanical inter-action between sound waves and bubbles in liquids,is regarded as the fundamental effect that is responsible for the initiation of most of the sonochemical reactions in liquids [7–10].The collapse of cav-itation bubbles,which are formed rapidly and exploded violently during sonication,generates violent physical forces,such as micro jets,shear forces,shock waves and turbulence [11,12].The ultra-sonic frequency is one of the factors that in?uence the yield and intensity of cavitation in liquids [13].It was found that the
https://www.wendangku.net/doc/eb4415747.html,/10.1016/j.ultsonch.2014.12.013Abbreviations:CGM,corn gluten meal;CR,conversion rate of protein;DH,degree of hydrolysis;SDFU,sequential dual frequency ultrasound;SEM,scanning electron microscope;SFPU,sweeping frequency and pulsed ultrasound.?Corresponding author.Fax:+8651188780201.E-mail address:mhl@https://www.wendangku.net/doc/eb4415747.html, (H.Ma).
ultrasonic cavitation yield can be enhanced by multi-frequency son-ication[14,15].Recently,the power ultrasound(20–100kHz)is widely employed in protein enzymolysis to produce bioactive pep-tides[16–19],improvement of the functional properties of proteins [20–23]and synthesis of lysozyme microspheres[24].It is reported that power ultrasound pretreatment can improve the reaction rate of enzymolysis,and the conversion rate of substrate proteins,and the bioactivity of target products signi?cantly[4,25,26].Moreover, ultrasound treatment can lead to changes in the secondary structure [18,27]as well as in the microstructure of protein,resulting in the exposure of more hydrolysis sites to be accessible to protease [28,29].However,systematic research on the molecular mechanism of ultrasound-accelerated enzymatic hydrolysis process of CGM is unavailable.
Therefore,the objectives of this work are to investigate the mechanism of multiple frequency power ultrasound accelerated CGM proteolysis process in terms of change in the molecular con-formation as well as the microstructure of proteins.It is also hoped that the results of the present research will be of great value to the design and development of ultrasound equipment in the proteoly-sis industry.
2.Materials and methods
2.1.Materials
Corn gluten meal(particle size of0.21mm,crude protein con-tent of586g kgà1,starch content of154g kgà1)was obtained from Fenda starch Co.(Jiangsu,China).Alcalase2.4L with an activity of 23,400U mLà1was purchased from Novozymes Co.Ltd.(Tianjin, China).All reagents used in the experiment were of analytical grade.
2.2.Multi-frequency power ultrasound pretreatment of CGM
Prior to the enzymolysis reaction,the CGM was pretreated by multi-frequency power ultrasound viz.sweeping frequency and pulsed ultrasound(SFPU)and sequential dual frequency ultrasound (SDFU).The traditional enzymolysis(Control)was conducted with a magnetic stirring apparatus instead of ultrasound under the same conditions.All experiments were carried out in triplicate.
2.2.1.Ultrasound treatment with sweeping frequency and pulsed ultrasound(SFPU)
The SFPU experiments were done in an ultrasonic bath reactor (internal dimensions:362mm?294mm?502mm,Shangjia Bio-technology Co.,Wuxi,Jiangsu,China)equipped with?ve different frequency plates(Fig.1a)and the maximum output acoustic power of each plate is600W.The bath ultrasound generators provided direct contact with the surface of the solution with a bath ultra-sound(upper plate)as a power source for acoustic cavitation. Another bath ultrasound(bottom plate)was placed at the bottom of the reactor.The SFPU generator provided with function modes of sweeping frequency operation and pulsed operation.The sweeping frequency operation(f i±d kHz)refers to the sweeping frequency cycle of increasing period from f iàd to f i+d kHz and decreasing period from f i+d to f iàd kHz with the same linear speed in the form of an isosceles triangle(Fig.1b),and the pulsed operation indicates that ultrasound is generated in a pulsed mode with an on-time and an off-time cycle.A500mL CGM suspension(net pro-tein content of50g Là1)were sealed in a high pressure resistance bag and pretreated with SFPU.The ultrasonic pretreatment param-eters were obtained from our previous studies as follows:combi-nation of ultrasonic sweeping frequency28±2kHz(upper plate)on-time10s and off-time3s,cycle time of the sweeping frequen-cies of500ms,duration of40min and power density80W/L.
2.2.2.Ultrasound treatment with sequential dual frequency ultrasound (SDFU)
The SDFU(Fig.1c)which was developed by our team and man-ufactured by Meibo Biotechnology Co.(Zhenjiang,Jiangsu,China) is a probe type ultrasound equipped with?ve different frequency probes and the maximum output acoustic power of each probe is 200W.The sequential operation refers to that two kind of ultra-sound with different frequency are generated in a successive mode without interval,i.e.one is pulsed on-time while the other is pulsed off-time(Fig.1d).Similarly,an aliquot of500mL CGM sus-pension with net protein content of50g Là1was put in the reac-tion vessel,and the probes were submerged to a depth of2.0cm in the solution.The ultrasonic parameters were optimized by sin-gle factor experiment as follows:sequential frequency20kHz and 40kHz,temperature30±2°C,20kHz pulsed on-time10s during which40kHz is pulsed off-time,40kHz pulsed on-time5s during which20kHz is pulsed off-time,duration of15min and power density of400W/L.
2.3.Enzymolysis of corn gluten meal
The enzymolysis apparatus consisted of a digital thermostat water bath(DK-S26,JingHong experimental apparatus Co.,Shang-hai,China),a pH meter(FE-20,Mettler Toledo Co.,Shanghai,China) and an impeller-agitator(JJ-1,ZhongDa instrument Co.,Jiangsu, China)at a speed of100r/min.After10min preheating at50°C, the solution was adjusted to pH9.0,and1mL of enzyme was added to initial the reaction.The pH was maintained by continuous addition of0.5M NaOH during the enzymolysis process.The enzy-molysis time was60min and the reaction was terminated by boil-ing the mixtures for10min and then centrifuged at5030?g for 15min after cooling to room temperature.
2.3.1.Determination of the degree of hydrolysis(DH)
The degree of hydrolysis(DH)was calculated according to the pH-stat method described by Adler-Nissen[30]:
DHe%T?
h
h tot
?
N b?B?100
a?M p?h tote1T
where,N b is the concentration of NaOH(mol Là1);B is the volume of NaOH consumed(mL);M p is the mass of protein to be hydrolyzed (g);h tot is the total millimoles of peptide bonds per gram of protein substrate,which is9.2mmol gà1for corn protein;a is the average degree of dissociation of the a-amino groups related with the pK of the amino groups at particular pH and temperature,which is 0.99at pH9.0and50°C.
2.3.2.Determination of the conversion rate of protein(CR)
The conversion rate of protein(CR)was calculated according to the following equation:
CRe%T?
C?V
e2T
where,C is the concentration of peptides in hydrolysates (mg mLà1),which was determined by Lowry method[31]using bovine serum albumin(BSA)as standard;V is the volume of hydrol-
56J.Jin et al./Ultrasonics Sonochemistry24(2015)55–64
2.4.Measurement of the molecular conformation of corn protein
2.4.1.Extraction of zein and glutelin
A500mL of the control and multi-frequency power ultrasound pretreated CGM suspension(50g Là1)were centrifuged at5030?g for15min,and the precipitates were used to extract zein and glut-elin according to the method described by Li et al.[32]and Cheng et al.[33],respectively.The extractives were freeze-dried with a freeze dryer(ALPHA1-2,Martin Christ Inc.,Osterode,Germany) and stored at4°C for analysis.The protein content of zein and glut-elin was determined by the Kjeldahl method using an Automatic kjeldahl apparatus(UDK149,VELP Scienti?ca Co.,Ltd,Usmate, Italy).
2.4.2.Ultraviolet–visible(UV)spectroscopy
Zein and glutelin samples prepared from ultrasound treated (SFPU,SDFU)and untreated(control)CGM were dissolved in70% (v/v)ethanol and0.01M phosphate buffer(pH7.0),respectively,(UV–Vis)spectra of the sample solutions were recorded in the wavelength range of200–400nm using a Varian Cary100UV–Vis spectrophotometer(Varian Inc.,Palo Alto,USA)at25°C with a1.0cm path length quartz cell,a60nm/min scan rate and a 2.0nm bandwidth.The spectrum of70%ethanol and0.01M phos-phate buffer(pH7.0)were used as blank for zein and glutelin, respectively.
2.4.
3.Circular dichroism(CD)spectroscopy
The CD spectra of zein(0.05mg mLà1in70%,v/v)and glutelin (0.05mg mLà1in0.01M phosphate buffer,pH7.0)samples were scanned in the wavelength range190–300nm with a Jasco J-815 CD spectropolarimeter(Jasco Corp.,Tokyo,Japan)using a quartz cuvette of10mm optical path length under nitrogen?ux at room temperature(25±1°C),a scan rate of100nm/min and a band-width of1.0nm.Three scanning acquisitions were accumulated and averaged to yield the?nal spectrum in all cases.The results
1.The multi-frequency power ultrasound.(a)The sweeping frequency and pulsed ultrasound(SFPU),(b)sweeping frequency,(c)the sequential dual frequency ultrasound(SDFU),(d)sequential mode.
cm2dmolà1,using a value of110g molà1as the mean residue molecular weight.The spectrum of blank was subtracted from the average spectra to obtain a relatively corrected average spectrum for each sample.The secondary structure(a-helix,b-sheet,b-turn, and random coil)content of the samples was calculated from the [h]mrw at wavelengths190–240nm using the software CD Pro with the CONTIN/LL method.
2.4.4.Measurement of intrinsic?uorescence spectrum
The Intrinsic?uorescence emission spectra of zein (0.05mg mLà1in70%,v/v)and glutelin(0.05mg mLà1in0.01M phosphate buffer,pH7.0)samples were measured at room temper-ature(25±1°C)using a Cary Eclipse?uorescence spectrophotom-eter(Varian Inc.,Palo Alto,USA)equipped with a1cm path length cell.The excitation wavelength of280nm(slit=5nm),emission wavelength range of290–400nm(slit=5nm)and scanning speed of120nm/min were used.The spectra were an average of ten scans and the spectrum of blank was subtracted from the average spectra.
2.4.5.Surface hydrophobicity(H0)measurements
Surface hydrophobicity(H0)of protein dispersions was deter-mined using1-anilino-8-naphthalene-sulfonate(ANS(Sigma Chemical Co.,St.Louis,MO,USA))as a?uorescence probe according to the method of Kato and Nakai[34].Lyophilized zein and glutelin samples(1mg mLà1in70%ethanol and in0.01M phosphate buffer at pH7.0,respectively)were centrifuged at12,000?g at4°C for 10min.After determining the protein concentration in the super-natants according to Lowry method,each supernatant was serially diluted with the same solvent to obtain protein concentrations ranging from1.0to0.2mg mLà1.Then20l L of ANS(8.0mM in 0.01M phosphate buffer,pH7.0)was added
tein solution,mixed,and kept in the dark for
?uorescence intensity was measured at
(25±1°C)with a Cary Eclipse
Alto,USA)at excitation wavelength of280
sion wavelength305nm(slit5.0nm)for
5.0nm)for glutelin,and120nm/min of
slope of?uorescence intensity vs.protein
was calculated by linear regression analysis
of H0.
2.4.6.Determination of sulfhydryl(SH)groups,
content
The concentration of sulfhydryl(SH)
solutions was determined using Ellman’s
(2-nitrobenzoic acid),DTNB)(Sigma
USA),according to the method developed by
[35],with some modi?cation.To a1mL of
added5mL of standard buffer(86mM Tris,
4mM EDTA,pH8.0)plus8M urea and5g Là
fate(SDS),and0.05mL of Ellman’s reagent
DTNB in standard buffer).After the
and allowed to stand at room temperature
the absorbance was read at412nm.The
of protein solutions as a reagent blank.A
in which0.05mL of the buffer replaced
The concentration of the sulfhydryl
according to the following equation[36]:
C SHel mol=gT?e73:53?
D Abs412?DT
C
where,D is the dilution factor,C is the protein mined by Lowry method(mg mLà1),D Abs412D Abs412?Abs with DTNBàAbs without DTNBe4T
The content of SS bonds was determined according to the method developed by Beveridge et al.[37]with some modi?cation. In brief,0.5mL of protein solution,5mL of10M urea in standard buffer and0.10mL of b-mercaptoethanol were incubated at25°C for1h.After precipitation and washing of protein using40mL of 120g Là1trichloroacetic acid(TCA)for three times,the precipitate was dissolved in15mL of8M urea and5g Là1SDS in the standard buffer,0.15mL of Ellman’s reagent was added for color develop-ment.Absorbance was also measured at412nm.The concentra-tion of the SS groups was calculated according to the following equation:
C SSel mol=gT?eC0SHàC SHT=2e5T
where,C0
SH
is the concentration of the sulfhydryl groups derived from reduction of SS groups and total free thiol groups(C SH).Both
C0
SH
and C SH were determined according to Eq.(3).
2.5.Scanning electron microscopy(SEM)
The morphology of samples zein,glutelin,CGM as well as enzy-matic hydrolysis residues were observed with a Hitachi S-3400N scanning electron microscope(Hitachi High Technologies,Tokyo, Japan)at an acceleration voltage of15kV.Before using the scan-ning electron microscope,the samples were coated with a conduc-tive layer.
Fig.2.Effect of sweeping frequency pulsed ultrasound(SFPU)and sequential dual frequency ultrasound(SDFU)on the degree of hydrolysis and conversion rate of corn gluten meal protein.(The same index marked with different letter means signi?cantly different(P<0.05,n=3))sonication parameters:SFPU:combination of ultrasonic sweeping frequency28±2kHz(upper plate)and68±2kHz(bottom plate),temperature of the solution30±2°C,pulsed on-time10s and off-time3s, cycle time of the sweeping frequency of500ms,duration of40min and power density of80W/L;SDFU:sequential frequency20kHz and40kHz,temperature of the solution30±2°C,20kHz pulsed on-time10s during which40kHz is pulsed
58J.Jin et al./Ultrasonics Sonochemistry24(2015)55–64
3.Results and discussion
3.1.Effect of multi-frequency power ultrasound pretreatment on the enzymolysis of corn gluten meal sequential operation was higher because the implosion of the cav-itation bubbles coming from lower frequency(20kHz)irradiation can provide new cavitation nuclei not only for itself,but also for the other ultrasound(40kHz)irradiation[14].This enhancement
3.Effect of sweeping frequency pulsed ultrasound(SFPU)and sequential dual frequency ultrasound(SDFU)on the UV spectra of zein(a)and glutelin Sonication parameters:SFPU:combination of ultrasonic sweeping frequency ±2kHz(upper plate)and68±2kHz(bottom plate),temperature of the solution ±2°C,pulsed on-time10s and off-time3s,cycle time of the sweeping frequency of500ms,duration of40min and power density of80W/L.SDFU: sequential frequency20kHz and40kHz,temperature of the solution30±2 kHz pulsed on-time10s during which40kHz is pulsed off-time,40kHz pulsed
4.Effect of sweeping frequency pulsed ultrasound(SFPU)and sequential dual frequency ultrasound(SDFU)on the Far-UV CD spectra of zein(a)and glutelin Sonication parameters:SFPU:combination of ultrasonic sweeping frequency ±2kHz(upper plate)and68±2kHz(bottom plate),temperature of the solution ±2°C,pulsed on-time10s and off-time3s,cycle time of the sweeping frequency of500ms,duration of40min and power density of80W/L.SDFU: sequential frequency20kHz and40kHz,temperature of the solution30±2 kHz pulsed on-time10s during which40kHz is pulsed off-time,40kHz pulsed
J.Jin et al./Ultrasonics Sonochemistry24(2015)55–6459
($279nm),and phenylalanine ($absorbance intensity of corn multi-frequency ultrasound was of the buried hydrophobic groups ?nding was reported by Jia et al.pretreat wheat germ albumin.It of SFPU on zein is pronounced is true in case of glutelin .As far as phobicity of which is stronger exposed hydrophobic groups the hydrophobic interaction.process from taking place erated by SFPU (a bath tion is more even than that generates high temperatures,the probe tip [44].
3.2.2.CD spectra analysis
Circular dichroism (CD)is conformational changes of cially the far-UV spectra of CD protein conformation in terms of spectra of zein and glutelin were tively.Analysis of the far-UV depicted presence of two a positive peak at 192nm A positive peak at 195nm and a characteristics of b -sheet,represents the random coil.The turn,and random coil were TIN/LL,and the results were considered,SFPU pretreatment dom coil by 2.5%while it ment increased a -helix by 24.4%by 8.6%and 3.7%,respectively.SDFU pretreatment decreased a b -sheet by 12.4%,SFPU while it decreased a -helix,b The changes in the content of the been attributed to disruptions of sequences of amino acids and ecule.An increase in b -sheet and loosening of the protein molecule,sible to enzyme.In contrast,the to protein aggregation.Ren et al.resulted in slight increases in coils,which coincides with the results of the present study.How-ever,Stathopulos et al.[45]stated that sonication induced aggre-gates had an increased b -structure with a concomitant decrease in a -helix structure.This difference in the interpretation of the protein secondary structure,suggested to be responsible for aggre-gation,is that they did not distinguish the b -turn from b -structure as we did in this study.
3.2.3.Intrinsic ?uorescence analysis
Fluorescence spectra can also provide a sensitive means of char-acterizing proteins and their conformations.Tyrosine,tryptophan and phenylalanine residues in a protein,particularly the trypto-phan residue [28],will ?uoresce in a manner critically dependent
Table 1
Effect of multi-frequency power ultrasound pretreatment on the secondary structure content (%)of zein and glutelin .
Zein
Glutelin
a -Helix
b -Sheet b -Turn Random coil a -Helix
b -Sheet b -Turn Random coil Control 16.4
33.7
21.8
28.1
11.5
29.9
23.9
34.7
SFPU 15.4(à1.0)*34.6(0.9)21.3(à0.5)28.8(0.7)8.4(à3.1)34.7(4.8)22.8(à1.1)34.1(à0.6)SDFU
20.4(4.0)
30.8(à2.9)
21.0(à0.8)
27.9(à0.2)
8.3(à3.2)
33.6(3.7)
23.3(à0.6)
35.0(0.3)
*
Data are means of three scanning acquisitions.Values in parentheses indicate the increase or decrease (with negative sign)in secondary structure (a -helix,b -sheet,turn,and random coil)content of SFPU and SDFU pretreated zein and glutelin compared with that of control (ultrasound untreated);SFPU,sweep frequency and pulsed ultrasound;5.Effect of sweeping frequency pulsed ultrasound (SFPU)and sequential dual frequency ultrasound (SDFU)on the ?uorescence emission spectra of zein (a)glutelin (b).Sonication parameters:SFPU:combination of ultrasonic sweeping frequency 28±2kHz (upper plate)and 68±2kHz (bottom plate),temperature solution 30±2°C,pulsed on-time 10s and off-time 3s,cycle time of sweeping frequency of 500ms,duration of 40min and power density of 80W/L.SDFU:sequential frequency 20kHz and 40kHz,temperature of the solution ±2°C,20kHz pulsed on-time 10s during which 40kHz is pulsed off-time,kHz pulsed on-time 5s during which 20kHz is pulsed off-time,duration min and power density of 400W/L.
60
on the folding of the protein,and hence they act as sensitive mon-itors of conformational change in tertiary structure.As presented in Fig.5(a),the emission?uorescence intensities of untreated and sonicated zein(excited at280nm)had maximum value at wavelength of305nm.Sonication of the zein protein increased the?uorescence intensity over that of the native protein.In Fig.5(b),the maximum emission?uorescence intensity of the native glutelin(excited at280nm)was found at352nm.The SFPU and SDFU pretreatments of glutelin shifted the maximum wave-length of the emission?uorescence of the control to a higher value (354nm),coupled with an increase in amounts of the?uorescence intensity.This red shift indicates an increase in the polarity of the Trp due to molecular unfolding.So far,zein had higher emission ?uorescence intensity than glutelin,this is because zein contains more hydrophobic amino acids than glutelin does.This increase in?uorescence intensity might be attributed to the fact that multi-frequency power ultrasound pretreatment destroyed hydro-phobic interactions of protein molecules,induced molecular unfolding,caused more hydrophobic groups and regions inside the molecules to be exposed to the outside,and thus increasing the?uorescence intensity[46].
3.2.
4.Surface hydrophobicity(H0)
Protein surface hydrophobicity(H0),showing the number of hydrophobic groups exposed on the surface of a protein molecule, is one of the structural characteristics to evaluate the change in protein conformation.As can be seen from Table2,zein is charac-terized by higher H0than glutelin.The multi-frequency power ultrasound pretreatment increased signi?cantly(P<0.05)the sur-face hydrophobicity of zein and glutelin over that of the control. This?nding was consistent with previous similar studies on the H0of bovine serum albumin[46],whey protein concentrate[36], black bean protein isolate[47]and wheat germ protein[29].This increase in H0indicates that the hydrophobic groups and regions which were located in the interior of the protein molecules were exposed to the surface under the cavitation phenomenon induced by ultrasound.However,unlike the UV–Vis and?uorescence spec-tra,there was little increase between SFPU and SDFU pretreated samples,this might be due to the fact that the H0values were obtained from the initial slope of?uorescence intensity vs.protein concentration.The results of Jiang et al.[47]also showed that the ?uorescence spectra and H0did not follow the same trend.
3.2.5.Sulfhydryl groups and disul?de bond content
The changes in sulfhydryl groups(SH)and disul?de bond(SS) contents of zein and glutelin due to sonication are presented in Table2.As far as zein was concerned,the SFPU pretreatment did not change the content of SH and SS signi?cantly(P>0.05),while the SDFU decreased SH content and increased SS content signi?-cantly(P<0.05).The might be due to the differences in the applied ultrasound.Having undergone ultrasound pretreatment,the SH groups of glutelin increased signi?cantly(P<0.05),especially the SDFU pretreatment,by which the SH content of glutelin was dou-bled compared with that of the control.The change in SS content was just opposite to the change in SH content.This increase in SH content might be mainly attributed to the fact that the ultra-sonic treatment broke the disul?de bonds,causing reduction in SS to form SH groups.Zhou et al.[29]reported increase in SH con-tent and reduction in SS content when using ultrasound to pretreat wheat germ protein,and they also found that these changes were in function of ultrasound frequency as well as power and duration. Some researchers reported that ultrasonic treatment can reduce the size of particle[20,28,39,40].Therefore,the potential exposure of buried sulfhydryl groups to the surface of protein molecules might have happened during the process of reducing the size of protein under the action of micro jets,shear forces,shock waves, and turbulence which are induced by the cavitation phenomenon.
3.3.Scanning electron microscopy(SEM)
The microstructures of glutelin,zein,CGM,enzymolysis residues of CGM from both untreated and samples sonicated by multi-fre-quency power ultrasound are shown in Fig.6.The native glutelin exists in the form of massive texture(Fig.6a).Having undergone the multi-frequency power ultrasound pretreatment,the structure of glutelin became incompact,and either existed in the form of lamellar structure(Fig.6b)or the surface of which was full of micro pores(Fig.6c).This?nding was in agreement with the results obtained by some researchers[48,49].Untreated zeins were aggre-gated into large protein masses because of their high hydrophobic-ity(Fig.6d),the shapes of zein which were treated by ultrasound were deformed and the size reduction occurred to different extent (Fig.6e and f).In addition,more compact aggregation was observed in zein pretreated by SDFU.Some researchers also found that ultrasonic treatment can reduce the size of the particles [20,28,39,40]and Stathopulos et al.[45]discovered that sonication of proteins caused formation of aggregates.These changes in struc-tures of glutelin and zein might result in an increase in intensity of UV and?uorescence spectra,surface hydrophobicity,SH groups content and decrease in SS bonds.Moreover,these changes in microstructures could be employed to explain why SFPU was supe-rior to SDFU when pretreating zein while SDFU was better than SFPU as far as glutelin was concerned.Fig.6(g)–(i)shows the microstructures of untreated and ultrasound pretreated CGM. The texture of the native CGM is very compact with starch granules associated with proteins[50]while those of CGM for both samples treated by SDFU or by SFPU became loose.Thereto,the surface of SDFU pretreated CGM is a curl-shaped with many holes while that of SFPU pretreated sample is similar to the surface of glutelin which was pretreated by SDFU but with some breakage.This increase in speci?c surface areas by multi-frequency power ultrasound undoubtedly resulted in enhancement of the contact areas between the enzyme and matrix,and resulted in the exposure of more cleavage bonds to alcalase,thus increasing the degree of hydrolysis and conversion rate of the protein.These changes in structure mentioned above can be attributed to the formation of localized hot spots upon collapse of bubbles,and to the formation of shear forces created by micro streaming and shock waves[48], which destroyed the cross-link between the protein molecules and caused the breakdown of the hydrogen bonds and Van der
Table2
Effect of multi-frequency power ultrasound pretreatment on the surface hydrophobicity(H0),sulfhydryl content(SH)and disul?de bond(SS)of zein and glutelin.*
H0SH content(l mol/g)SS bonds content(l mol/g) Zein Glutelin Zein Glutelin Zein Glutelin
Control555.9±10.87a182.87±8.53a36.35±0.97b11.87±1.60a63.54±1.48a74.99±0.80c SFPU622.25±7.82b278.87±6.17b37.27±1.60b20.20±0.40b63.08±0.80a70.83±1.20b SDFU619.36±8.86b286.37±6.33b32.92±0.82a24.06±0.49c66.44±1.41b68.89±1.24a
*Means±SD(n=3).Within a column,means with different superscript letters are signi?cantly different(P<0.05);SFPU,sweeping frequency and pulsed ultrasound;
J.Jin et al./Ultrasonics Sonochemistry24(2015)55–6461
Waals interactions in the polypeptide chains[51,52].The struc-tures of hydrolysis residues from untreated CGM(Fig.6j)is still very compact,and thus the enzymolysis might only have happened on the surface of CGM.Accordingly,large cavities even the hollow structures were observed in the residues which came from SDFU pretreated CGM(Fig.6k).As far as the residues from SFPU pre-treated CGM was concerned,its surface was very uniform and was covered with many small fragments,and the potential col-lapse might have taken place because obvious?ssure stack layers are visible(Fig.6l).Jia et al.[28]reported that hydrolyzed defatted pretreatment.The contradictory result might be attributed to the type of ultrasound used as well as the properties of proteins.In conclusion,the increases in DH and CR are bene?ts from the change in microstructures of zein,glutelin and CGM,and this change will undoubtedly further lead to changes in the structures of hydrolysis residues.
4.Conclusions
Multi-frequency power ultrasound(SFPU,SDFU)pretreatment
a b c
d f
e
g h i
k l
j
sweeping frequency pulsed ultrasound(SFPU)and sequential dual frequency ultrasound(SDFU)on the microstructure of glutelin,zein,CGM, glutelin,(b)SDFU treated glutelin,(c)SFPU treated glutelin,(d)Native zein,(e)SDFU treated zein,(f)SFPU treated zein,(g)Native CGM,(h)SDFU (j)enzymolysis residues from CGM,(k)enzymolysis residues from SDFU treated CGM,(l)enzymolysis residues from SFPU treated CGM.
?5000.Sonication parameters:SFPU:combination of ultrasonic sweeping frequency28±2kHz(upper plate)and68±2kHz(bottom
±2°C,pulsed on-time10s and off-time3s,cycle time of the sweeping frequencies of500ms,duration of40min and power density frequency20kHz and40kHz,temperature of the solution30±2°C,20kHz pulsed on-time10s during which40kHz is pulsed off-time,40kHz kHz is pulsed off-time,duration of15min and power density of400W/L.
62J.Jin et al./Ultrasonics Sonochemistry24(2015)55–64
molecular mechanism of multi-frequency power ultrasound which promoted enzymolysis includes the exposure of hydrophobic groups,redistribution of the secondary structure,decrease in the SS bonds content,and changes in the microstructures.In addition, it can be concluded from the results that it is better to take the hydrophobicity of a protein into consideration when choosing the type of ultrasound(bath or probe)because SDFU induced aggregation of zein was observed in the present study.Generally, the SFPU is superior to SDFU provided that the hydrophobicity of the protein is strong,otherwise the SDFU is more suitable.How-ever,from the perspectives of energy ef?ciency,the SFPU pretreat-ment is still the best method.
Acknowledgements
The authors wish to express their appreciation for the support obtained from grant(2013AA100203)of the Project of National 863Plan of China,National Natural Science Foundation of China (31471698,31301423),Research-Innovation Program of Postgradu-ate in General Universities of Jiangsu(CXZZ13-0695),Natural Science Foundation of Jiangsu Province(BK2012708),Key University Science Research Project of Jiangsu Province(12KJA550001),Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD).
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