Characterization and In Vitro digestib ility o

Characterization and In Vitro digestibility of rice protein prepared by
enzyme-assisted microfluidization:Comparison to alkaline extraction
Ning Xia a,b,1,Jin-Mei Wang a,1,Qian Gong c,Xiao-Quan Yang a,*,Shou-Wei Yin a,Jun-Ru Qi a
a College of Light Industry and Food Sciences,South China University of Technology,Guangzhou510640,PR China
b Department of Light Industry and Food Engineering,Guangxi University,Nanning530004,PR China
c Nanchang Institute for Foo
d and Drug Control,Nanchang330038,PR China
a r t i c l e i n f o
Article history:
Received27February2012
Received in revised form
11June2012
Accepted19June2012
Keywords:
Rice protein
Microfluidization
Structural properties
In vitro digestibility
a b s t r a c t
Microfluidization followed by density-based separation was employed to extract protein from broken
rice by disrupting protein-starch agglomerates.Follow-up enzyme treatments(amylase and glucoamy-
lase)were performed to further improve the purity of the protein-rich fraction.High protein recovery
(81.87%)and purity(87.89%)were obtained.The protein composition,solubility,structural properties,
and in vitro digestibility of rice proteins prepared by enzyme-assisted microfluidization(EM-RP)and
alkaline extraction(AE-RP)were compared.EM-RP was mainly composed of glutelin,which had low
solubility and native structure.By contrast,large quantities of prolamin and globulin appeared in the AE-
RP except glutelin,leading to the richness of glutamic acid/glutamine,leucine,aromatic and charged
amino acids in the pared to AE-RP,EM-RP showed higher digestibility due to the richness of
glutelin(an easy-to-digest protein),as evidenced by higher nitrogen release during pepsin-trypsin
digestion.The presence of prolamin(an indigestible protein)in AE-RP decreased protein digestibility
although alkaline extraction improved its hydrolysis.These results suggest that enzyme-assisted
microfluidization could be an effective technique to non-destructively and selectively extract rice
glutelin.
Ó2012Elsevier Ltd.All rights reserved.
1.Introduction
Rice protein has attracted considerable attention owing to its
high nutritional and health properties,such as well-balanced
amino acid,hypoallergenicity,hypocholesterolemic and hypolipi-
demic effects,as well as high anti-cancer activity(Juliano,1985;
Helm and Burks,1996;Morita et al.,1996;Ni et al.,2003).Broken
rice,an unavoidable by-product during milling operations,contains
about80%starch and8%protein and can be used to extract starch
and protein because of much lower commercial value compared to
whole rice.However,the separation of these two ingredients is
greatly limited due to the presence of compact protein-starch
agglomerates in the rice endosperm cell(Guraya and James,
2002).It is well known that rice protein has complex composi-
tion,including albumin,globulin,prolamin,and glutelin.These
proteins exist in the form of protein bodies(protein body I,PB-I;
protein body II,PB-II),which closely wrap around starch granules
(Juliano,1985;Hamada,1996).Glutelin is the major storage protein
of rice(about80%of total proteins)and mainly accumulates in the
PB-II.This protein has limited solubility in rge macromo-
lecular complexes linked by extensive disulfide bond and hydro-
phobic interactions are believed to appear in glutelin,which may
partly account for the difficulty in protein extraction(Cagampang
et al.,1966;Utsumi,1992).
Several methods such as alkaline extraction(Kumaga et al.,
2006;Paraman et al.,2008;Cao et al.,2009)and enzymatic treat-
ments(Morita and Kiriyama,1993;Shih and Daigle,2000;Paraman
et al.,2006)have been extensively attempted to prepare rice
proteins.Alkaline extraction,a most common method,obtains high
protein recovery(>80%)by dissolving the insoluble rice proteins in
dilute alkaline solution and subsequently precipitating protein
under acidic conditions(the isoelectric point of protein).Alkali
treatment is considered to break some of the hydrogen,amide,and
disulfide bonds in rice glutelin,leading to the reduction in molec-
ular size of protein and the improvement in protein extraction
(Hamada,1997).Recently,it has been reported that this method
improves in vitro and in vivo digestibility of rice protein,which is Abbreviations:EM-RP,rice proteins prepared by enzyme-assisted micro-
fluidization;AE-RP,rice proteins prepared by alkaline extraction.
*Corresponding author.Tel.:þ862087114262;fax:þ862087114263.
E-mail address:fexqyang@(X.-Q.Yang).
1These two authors contributed equally to this study and sharefirst authorship.
Contents lists available at SciVerse ScienceDirect
Journal of Cereal Science
journal ho mep age:/lo cate/jcs
0733-5210/$e see front matterÓ2012Elsevier Ltd.All rights reserved.
/10.1016/j.jcs.2012.06.008
Journal of Cereal Science56(2012)482e489
微流化
氨基酸丆低过敏性丆降胆固
醇丆降脂丆抗癌
associated with the modification of protein body structure and amino acid composition(Kumaga et al.,2006;Kubota et al.,2010; Yang et al.,2012).Unfortunately,exposing protein to severe alkaline conditions may cause some undesirable side reactions,degradation of protein,and potential toxicity,such as lysinoalanine,resulting in the loss of nutritional value(Shewry and Miflin,1985).
Protease treatment has been widely applied to extract rice protein by improving the solubility of protein hydrolysates and to modify its physicochemical and functional properties(Anderson et al.,2001;Paraman et al.,2007).This method could cause the generation of bitter hydrolysates.In addition,various carbohydrate-hydrolyzing enzymes have also been used to prepare rice protein with high protein purity(>80%)by removing carbo-hydrate components(mainly starch)(Morita and Kiriyama,1993; Shih and Daigle,2000;Paraman et al.,2006).However,this protein separation process is accompanied by severe damage of rice starch. Rice protein and starch are therefore not simultaneously obtained. To overcome this disadvantage,in previous studies,high pressure homogenization followed by density-based separation,efficient natural-type processes,has been described to separate rice starch from protein by breaking rice protein-starch agglomerates(Guraya and James,2002).Different to the above mentioned chemical and enzyme methods,these mechanical processes can non-destructively separate rice starch with high recovery and purity. However,detailed information on rice protein obtained by homogenization treatment has not been provided.
The objective of this research was to evaluate the effects of microfluidization and subsequent density-based separation on protein extraction from broken rice.Enzyme treatments(amylase and glucoamylase)were used to further improve protein purity of protein-rich fraction by hydrolyzing residual starch.The protein composition,solubility,structural properties,as well as in vitro digestion of rice protein prepared by enzyme-assisted micro-fluidization(EM-RP)were compared with those of rice protein prepared by alkaline extraction(AE-RP).
2.Materials and methods
2.1.Materials
Broken rice was obtained from Zhong-Dao Industrial Co.Ltd. (Shandong,China),and its protein content was8.20Æ0.87%, determined by the Kjeldahl method(NÂ5.95,wet basis).Food-grade amylase(35,000units/g)and glucoamylase(12,0000units/ g)were purchased from Su-Hong Biological Co.Ltd.(Suzhou, China).Fluorescein isothiocyanate(FITC)and Rhodamine B(RhB) were purchased from Sigma e Aldrich Co.(St.Louis,MO,USA). Bovine serum albumin(BSA),pepsin,trypsin,and low molecular weight protein markers were obtained from Ding-Guo Biotech. Co.,Ltd.(Shanghai,China).All other chemicals were of analytical grade.
2.2.Preparation of rice proteins by enzyme-assisted
microfluidization and alkaline extraction
The broken rice was suspended in distilled water at a solid/ solvent ratio of1:20(w/v),and a preliminary wet mill,called colloid milling,was performed for30min using Labor-pilot2000/4 colloid mill(IKA Co.,Germany).Microfluidization was then done twice by forcing milled slurry through a narrow orifice at a pressure of100MPa using a microfluidizer(Microfluidics Co.,USA).The treated slurry was centrifuged at8000g for10min in a CR22G centrifuge(Hitachi Co.,Japan),and the supernatant was decanted. Due to density differences between protein and starch,two different layers in the precipitates were observed,corresponding to protein-rich and carbohydrate-rich fractions,respectively.The supernatant,protein-rich fractions,was carefully scraped with a spatula and redispersed with10times distilled water(w/v).
To further improve the purity of the protein-rich fraction, follow-up enzymatic treatments(amylase and glucoamylase)were carried out according to the method of Shih and Daigle(2000). Amylase treatment was performed by adding amylase to the dispersion(0.5%,w/v)and incubating at pH6.0and90 C for1h, and glucoamylase treatment was then done with same additive amount of enzyme(0.5%,w/v)by incubating at pH4.6and60 C for 1h.Rice protein prepared by enzyme-assisted microfluidization (EM-RP)was recovered by neutralization to pH7.0,centrifugation, and freeze-drying.Protein content determined by the Kjeldahl method(NÂ5.95)was used to calculate the recovery yield and purity of protein.Protein recovery was expressed as grams of protein in protein-rich fraction/total protein.
Another common method for preparing rice protein was alka-line extraction.Broken rice was mixed with0.1%NaOH solution, stirred at room temperature for1h,and then left overnight.The mixture was centrifuged at8000g for10min.The supernatant was adjusted to pH4.8to precipitate rice protein and centrifuged at 8000g for20min.Subsequently,rice protein prepared by alkaline extraction(AE-RP)was recovered by dispersing the protein precipitate in distilled water(1:10,w/v),neutralizing to pH7.0,and freeze-drying.
2.3.Deagglomeration of starch-protein aggregates during
microfluidization
The microstructure and particle size of the carbohydrate-rich fraction were studied to indirectly monitor the deagglomeration of rice starch-protein aggregates during colloid milling pretreat-ment and microfluidization.The microstructure of samples was observed using confocal laser scanning microscopy(CLSM,Leica Microsystems Inc.,Heidelberg,Germany)with a100mm oil immersion objective lens according to the method of Funamia et al. (2008).The carbohydrate-rich fraction was dispersed in distilled water(5.0%,w/v)and heated at90 C for20min to gelatinize the rice starch.The gelatinized samples(500m L)were immediately mixed with staining solution(20m L)containing2mg/mL FITC and 2mg/mL RhB(FITC for starch and RhB for protein).All images of the stained sample were collected using an argon krypton laser (488nm)and a helium neon laser(543nm).Furthermore,the partial samples were dispersed in distilled water(2%,w/v),and the particle size was determined using a Malvern Mastersizer2000 (Malvern Instruments Ltd.,Worcestershire,UK)at25 C.The refractive index of water was1.33,and the volume distribution was calculated as an indication of particle size.
2.4.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE(12%separating gel and4%stacking gel)was per-formed on a discontinuous buffered system according to the method of Laemmli(1970).The protein samples were dissolved in suitable volumes of sample buffer(0.125M Tris e HCl buffer,1%SDS (w/v),20%glycerol(v/v),2%2-mercaptoethanol(2-ME,v/v),pH 6.8),and heated at95 C for5min.The protein samples for SDS-PAGE under non-reducing conditions were prepared by the same process,just without the presence of2-ME.Gels were stained with Coomassie Brilliant Blue R-250(0.05%)in methanol/acetic acid/ water(25:10:65,v/v/v)and destained in methanol-water solution containing10%acetic acid(methanol/acetic acid/water¼1:1:8, v/v/v).
N.Xia et al./Journal of Cereal Science56(2012)482e489483赖丙氨
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小孔
磨刀
2.5.Amino acid analysis
The amino acid analysis was determined by monitoring the absorption at254nm using an automatic amino acid analyzer (Waters M510,USA)equipped with a PicoTag column.The samples were put in hydrolysis tubes,and suitable volumes of6M HCl were added.The tubes were evacuated,sealed,and hydrolyzed at110 C for24h.The determination was carried out at38 C and aflow rate of1mL/min.Amino acid composition was reported as g/100g protein.Tryptophan was not determined because of amino acid destruction during acid hydrolysis.
2.6.Protein solubility
Protein solubility as a function of pH was determined according to the method of Wang et al.(1999),with minor modification. Protein samples were dispersed in distilled water(1%,w/v)and the pH of dispersions was adjusted to2.0e10.0.Samples were then centrifuged at10,000g for10min after stirring for30min.After appropriate dilution,the protein content of the supernatant was measured by Lowry’s method,with BSA as the standard.Protein solubility was expressed as grams of soluble protein/100g of sample protein.
2.7.Differential scanning calorimetry(DSC)
The thermal analysis of rice proteins was performed using a TA Q100-DSC thermal analyzer(TA Instruments,New Castle,DE, 19720,USA).Proteins(2mg)were weighed into aluminum pans, and10m L of50mM phosphate buffer(pH7.0)was added.The pans containing protein samples and buffers were hermetically sealed and placed at room temperature for more than4h to achieve protein hydration.The samples were heated in the calorimeter from40to100 C at a rate of10 C/min.A sealed empty pan was used as reference.Denaturation temperature(T d)and denaturation enthalpy(D H)were calculated by Universal Analysis Software, version4.1D(TA Instruments-Waters LLC).
2.8.Raman spectroscopy
Raman spectra of rice proteins were measured using a LABRAM-Aramis spectrometer(Horiba Jobin Yvon,Villeneuve d’Ascq, France)at room temperature.The spectrometer was operated under the following conditions:exciting source,He e Ne laser (632.8nm);laser power,10mW;number of scans,32;exposure time,2s;spectral resolution,2cmÀ1.The spectral data were baseline-corrected and normalized to the intensity of the phenyl-alanine band at1004cmÀ1.
2.9.Sequential in vitro digestion
Sequential in vitro digestion experiments(pepsin e trypsin digestion)were carried out according to the method of Wang et al.(2010),with some modifications.Protein dispersions(pH 1.5,1%,w/v)were mixed with pepsin(enzyme:protein¼1:100,w/ w),and the mixture was gently stirred at37 C for120min.The mixture was then neutralized with1.0M NaOH to stop the diges-tion reaction.The trypsin digestion was performed by adding trypsin(enzyme:protein¼1:100,w/w)into the neutralized pepsin-digested mixture.After incubating at37 C for120min, protein dispersions were heated at95 C for10min to stop the trypsin digestion.
To evaluate pepsin e trypsin digestibility of protein,protein content of the precipitate obtained by adding an equal volume of trichloroacetic acid(TCA,10%,w/v)was determined using the micro-kjeldahl method(NÂ5.95).Protein digestibility(%)was calculated as(N0ÀN t)Â100/N tot,where N0and N t represent TCA-precipitated nitrogen content at0and t min,respectively,N tot is total nitrogen content of protein samples.In addition,SDS-PAGE was also used to monitor the polypeptide hydrolysis of rice proteins.Unless specified otherwise,all above-mentioned experi-ments were conducted in duplicate or triplicate,and the results were given as meansÆstandard deviations.
2.10.Statistical analysis
Unless specified otherwise,three independent trials were carried out.All of the tests were executed in triplicate and the results are given as meansÆstandard deviations.An analysis of variance(ANOVA)of data was carried out using SPSS13.0statistical analysis system to compare significant differences(p<0.05) between results.
3.Results and discussion
3.1.Preparation of rice protein by enzyme-assisted
microfluidization(EM-RP)
During the processes of enzyme-assisted microfluidization fol-lowed by density-based separation,protein recovery yield and purity of the protein-rich fraction are determined,as shown in Fig.1.Without physical treatment,distilled water extraction of broken rice only recovered rice protein of5.03Æ0.15%,and protein purity was only8.86Æ0.28%.Colloid milling significantly(p<0.05) increased protein recovery yield to63.79Æ1.04%,accompanied by an evident increase in the purity of the protein-rich fraction. Follow-up microfluidization further distinctly improved rice protein extraction yield to81.87Æ 4.97%,with a significant (p<0.05)increase of protein purity,which was associated with the disruption of protein-starch interactions and consequential liber-ation of starch-bound protein.It has been reported that colloid milling and microfluidization can break protein-starch agglomer-ates under high shear force in the wet process,leading to better density-based separation of two ingredients(Guraya and James, 2002).In fact,the protein-rich fraction has relatively low purity (44.50%Æ 1.24)although high protein recovery yield
was
Fig.1.Protein recovery yield and protein purity of protein-rich fraction during the processes of microfluidization followed by density based separation and enzyme treatments(amylase and glucoamylase).Water,water extraction;C:colloid milling pretreatment;CþM,colloid millingþmicrofluidization;CþMþE,colloid millingþmicrofluidizationþfollow-up enzyme treatments;AE,alkaline extraction. Different letters(a e e)indicate significant difference at the p<0.05level.
N.Xia et al./Journal of Cereal Science56(2012)482e489 484
successfully obtained (Fig.1).Thus,residual starch in the protein-rich fraction was removed using carbohydrate-hydrolyzing enzymes to further signi ficantly (p <0.05)improve protein purity.The purity of EM-RP reached to 87.89Æ3.22%.
To aid in clarifying the disruption of starch-protein agglomer-ates,the microstructure and particle size distribution of the carbohydrate-rich fraction were determined using CLSM and light scattering technologies,as presented in Fig.2.Confocal micro-graphs of untreated samples (broken rice)indicated that protein bodies (red)were distributed around the surface of starch granules (green)and compactly combined with starch granules (Fig.2A).In this case,a monomodal droplet size distribution with a mean pore size of about 100m m was also observed,suggesting the existence of particles with large size.Overall,it appeared that the red area in the CLSM pro file gradually decreased after colloid milling and subse-quent micro fluidization,indicating the reduction of rice protein in the carbohydrate-rich fraction.These phenomena are consistent with the increase in recovery yield of the protein-rich fraction (Fig.1).
It seemed that colloid milling of broken rice resulted in some microstructural changes of the starch granule,as evidenced by the disappearance of regular starch-protein agglomerates.A particle size peak at 10m m was found for this milled sample (Fig.2B).Micro fluidization of the milled slurry further decreased protein content,accompanied by the decrease of particle size,although
small quantities of rice protein remained in the carbohydrate-rich fraction (Fig.2C).The existence of protein-starch agglomerates and/or the structural properties of two protein bodies (PB-I and PB-II)in rice endosperm,such as particle size,may be responsible for the incomplete extraction of rice protein.PB-I is a spherical and lamellar protein body with a diameter of 1e 2m m,while PB-II is a crystalline one,which has uniform structure and an irregular shape with a diameter of 2e 4m m (Tanaka et al.,1980;Krishnan and White,1995;Ashida et al.,2011).In brief,these results not only clearly support the viewpoint of deagglomerates of starch-protein aggregates but also demonstrate that micro fluidization followed by density-based separation is an effective method for separating rice protein from starch.3.2.Protein composition
Alkaline extraction,another usual method for extracting rice protein,was performed to compare with enzyme-assisted micro-fluidization.It should be pointed out that,after alkaline extraction,the recovery yield and purity of AE-RP were 77.30Æ0.24%and 81.12Æ0.19%,respectively (Fig.1).SDS-PAGE under non-reducing and reducing conditions could give information on polypeptide composition of rice proteins prepared by these two methods (EM-RP and AE-RP),as shown in Fig.3.Major polypeptides of prolamin (13and 16kDa),globulin (26kDa),glutelin (22e 23and 37e 39kDa for basic and acidic subunits,respectively),and proglutelin (57kDa)were identi fied (Kumaga et al.,2006).Under non-reducing condi-tions,large macromolecule complexes (>43kDa)were observed for EM-RP (lane 1).By contrast,in the case of AE-RP (lane 2),bands with small molecular weight (about 14kDa)appeared,and pro-glutelin was transformed into acidic and basic subunits.These results may be attributed to the degradation of some amino acids and/or the disruption of hydrogen,amide,and disul fide bonds under high alkaline condition (Shewry and Mi flin,1985;Hamada,1997).
Under reducing conditions,EM-RP was mainly composed of glutelin subunits,with the presence of small quantities of prolamin (Fig.3,lane 1),suggesting that enzyme-assisted micro fluidization selectively extracts rice glutelin.However,AE-RP (lane 2)had complex polypeptide composition,including large quantities of
Fig.2.CLSM pro files of carbohydrate-rich fraction obtained by micro fluidization and subsequent density-based separation.FITC and RhB were used to stain starch (green)and protein (red),respectively.Particle size distribution of protein samples was superimposed on the micrographs.(For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)
N.Xia et al./Journal of Cereal Science 56(2012)482e 489485
prolamin and globulin except glutelin,consistent with previous studies (Kumaga et al.,2006).Rice proteins with different protein composition could be obtained by two extraction methods.It has been recognized that various rice storage proteins are deposited into separate protein bodies.Prolamin is accumulated in the spherical PB-I,whereas glutelin and globulin are localized in the irregular-shaped PB-II (Krishnan and White,1995).Based on these structural properties of protein bodies,it could be speculated that micro fluidization at 100MPa may preferentially disrupt the binding between starch and PB-II.This is in accordance with the deduction that the structure of protein bodies may in fluence deagglomerates of starch-protein aggregates,as proposed by CLSM images (Fig.2).
Table 1shows amino acid composition of rice proteins prepared by two extraction methods.Both the two protein samples were rich
in total essential amino acids (34.98and 36.4g/100g protein for EM-RP and AE-RP,respectively)(Shih and Daigle,2000).Lower glutamic acid/glutamine,leucine,as well as aromatic and charged amino acids (acidic and basic amino acids)were found for EM-RP.However,the lysine content of this protein was higher than that of AE-RP.The difference in the protein composition as supported by SDS-PAGE (Fig.3)may be a main reason for the above-mentioned differences in the amino acid composition.Hibino et al.(1989)re-ported that rice prolamin polypeptides were rich in glutamic acid/glutamine and leucine but poor in lysine.This fact further supports the result that prolamin appears in the AE-RP (Fig.3).On the other hand,we could not exclude the possibility of acid amino compo-sition modi fication during alkaline extraction.It was noteworthy that EM-RP exhibited higher arginine content (Table 1).Dietary arginine,as one of the important factors to affect cholesterol metabolism,is believed to be involved in the regulation of digest-ibility even if the mechanism of action was not clear (Yang et al.,2012).The richness of arginine in this protein may be favorable to improve the digestibility of rice protein.
3.3.Protein solubility and structural properties
Protein solubility,thermal property,and secondary structure of rice proteins prepared by enzyme-assisted micro fluidization (EM-RP)and alkaline extraction (AE-RP)were investigated,as shown in Fig.4.Low solubility (<25%)in the whole pH range from 2.0to 10.0was observed for EM-RP (Fig.4A).In view of the fact that this protein mainly consists of glutelin (Fig.3),it could be deduced that rice protein keeps its native structure during micro fluidization.In contrast,AE-RP showed the minimum solubility at pH 5.0,and the solubility gradually increased below and/or above pH 5.0,which was similar with the solubility pro file of globulin.Protein solu-bility reached 97.25Æ2.07%when the pH was increased to 10.0.High solubility of AE-RP at alkaline pH is in accordance with previous studies (Paraman et al.,2006,2008),which may be related to high content of charged amino acids of protein (Table 1).These distinct differences in protein solubility may be attributed to the differences in their protein composition and structural changes of protein occurred under severe alkaline condition.Hence,more details in structural properties of rice proteins prepared by the two methods are desired to support this speculation.
Fig.4B shows the DSC thermograms of rice proteins prepared by the two extraction methods.A major endothermic peak with denaturation temperature (T d )of 66.46 C and enthalpy value (D H )of 6.67J/g was observed for EM-RP.However,in the case of AE-RP,there was no detectable thermal transition signal,probably sug-gesting the disruption of ordered protein structure due to alkali-induced protein denaturation.This data may further support the viewpoint that enzyme-assisted micro fluidization protects the native structure of rice protein (mainly glutelin),as proposed by protein solubility (Fig.4A).
Raman spectroscopy,as a tool to characterize secondary struc-ture of protein,can be used for liquid and solid samples,particularly suitable for studying proteins with low solubility.This technology can give information on different backbone conformations of protein and the environment around the side chains.Fig.4C pres-ents Raman spectra of rice proteins obtained by two extraction methods.The major bands attributed to amide I (1654and 1663cm À1),III (1233cm À1)and C e H bending vibration (1442and 1445cm À1)peaks were observed for rice proteins (Ellepola et al.,2006).In the Raman spectrum of EM-RP,the presence of a band at 1663cm À1with high intensity indicated that this protein (mainly rice glutelin)was a predominantly disordered structure (Ellepola
Table 1
Amino acid compositions of rice proteins prepared by enzyme-assisted micro-fluidization (EM-RP)and alkaline extraction (AE-RP)(g/100g protein).Amino acids Proteins Amino acids Proteins EM-RP AE-RP EM-RP AE-RP Asp 9.08Æ0.458.23Æ0.20Val a
5.81Æ0.15 5.72Æ0.14Glu 17.35Æ0.6020.84Æ0.22Met a 2.51Æ0.16 2.76Æ0.23Ser 5.15Æ0.52 5.24Æ0.18Cys 0.61Æ0.010.59Æ0.01Gly 5.83Æ0.24 4.19Æ0.13Ile a 3.61Æ0.08 4.09Æ0.10His a 2.96Æ0.14 2.52Æ0.08Leu a 7.54Æ0.108.04Æ0.24Arg 10.15Æ0.357.98Æ0.32Phe a 4.58Æ0.11 5.89Æ0.13Thr a 3.89Æ0.11 4.13Æ0.17Lys a
4.08Æ0.10 3.25Æ0.23Ala
5.94Æ0.23 5.42Æ0.15Acidic b 2
6.43Æ0.1929.07Æ0.14Pro 6.03Æ0.25 5.59Æ0.13Basic c
8.19Æ0.2313.75Æ0.30Tyr
4.88
Æ0.20
5.52
Æ0.10
Aromatic d
9.46
Æ0.17
11.41
Æ0.33
a
Essential amino acids.[Requirement by FAO/WHO for weaned (2e 5years)child or adult].Trp was not determined.b
Acidic amino acids,Asp and Glu.c
Basic amino acids,Lys,Arg,and His.d
Aromatic amino acids,Phe and Tyr.
Fig. 3.SDS-PAGE pro files of rice proteins prepared by enzyme-assisted micro-fluidization (EM-RP)and alkaline extraction (AE-RP)under non-reducing and reducing ne 1:EM-RP;Lane 2:AE-RP;AS:acid subunits;BS:basic subunits.
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486。