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Extraction, Physicochemical, andFunctional Properties of Proteins FromMilk Thistle Silybum Marianum L. GaerntSeedsFang Li a , Xiangyang Wu b , Ting Zhao a , Feng Li c , Jiangli Zhao a &Liuqing Yang ca School of Food and Biological Engineering , Jiangsu University ,Zhenjiang , Jiangsu , Chinab School of Chemistry and Chemical Engineering , JiangsuUniversity , Zhenjiang , Jiangsu , Chinac School of Pharmacy , Jiangsu University , Zhenjiang , Jiangsu ,ChinaAccepted author version posted online: 13 Feb 2013.Publishedonline: 14 Jun 2013.

To cite this article: Fang Li , Xiangyang Wu , Ting Zhao , Feng Li , Jiangli Zhao & Liuqing Yang(2013) Extraction, Physicochemical, and Functional Properties of Proteins From Milk ThistleSilybum Marianum L. Gaernt Seeds, International Journal of Food Properties, 16:8, 1750-1763, DOI:10.1080/10942912.2011.608176

To link to this article: http://dx.doi.org/10.1080/10942912.2011.608176

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International Journal of Food Properties, 16:1750–1763, 2013Copyright © Taylor & Francis Group, LLCISSN: 1094-2912 print / 1532-2386 onlineDOI: 10.1080/10942912.2011.608176

EXTRACTION, PHYSICOCHEMICAL, AND FUNCTIONALPROPERTIES OF PROTEINS FROM MILK THISTLESILYBUM MARIANUM L. GAERNT SEEDS

Fang Li1, Xiangyang Wu2, Ting Zhao1, Feng Li3,Jiangli Zhao1, and Liuqing Yang3

1School of Food and Biological Engineering, Jiangsu University, Zhenjiang,Jiangsu, China2School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang,Jiangsu, China3School of Pharmacy, Jiangsu University, Zhenjiang, Jiangsu, China

Protein fractions from Silybum marianum seeds were sequentially extracted based onsolubility and then compared with protein isolate in terms of the yields and physicochemicalproperties. Molecular weights of protein fractions were in the range of 16.3–112.1 kDa.Secondary structure of the protein fractions had a high amount of β-types. Albumin frac-tion was the dominant seed protein with a characteristic profile of tryptophan residues inmost hydrophilic environments. Solubility of protein fractions was lowest at pH 4–5. Besideshigh levels of essential amino acids favorably compared with recommended levels for pre-school children, S. marianum seed proteins could also be used as protein extenders and acidformulations.

Keywords: Silybum marianum seeds, Protein fractions, Protein isolate, Physicochemicalproperties, Amino acid compositions, Functional properties.

INTRODUCTION

Milk thistle Silybum marianum L. Gaernt (family Compositae) is an annual or bien-nial plant, native to the Mediterranean area, which has now spread to other warmer anddryer regions. Various preparations of the plant, especially the fruits, have been medici-nally used to treat liver disorders for over 2000 years.[1] Besides this cytoprotective activity,there is a growing interest in its anticancer and chemopreventive activity as well as hypoc-holesterolemic, cardioprotective, neuroactive, and neuroprotective activities.[2] Silymarinis the pharmacological active component of the fruit, which is composed of an isomericmixture of the flavonolignans silychristin, silydianin, and the diastereoisomers silybin andisosilybin.[3] Thus, S. marianum is also a crop for silymarin industrial production. Recently,the increased consumption of silymarin has finally made farmers expand the harvest areaof S. marianum. In China, the growing area in Qinghai, Shanxi, Hubei, Jiangsu, andGuangdong provinces has increased year by year.[4]

Received 7 April 2011; accepted 23 July 2011.Address correspondence to Liuqing Yang, School of Pharmacy, Jiangsu University, 301 Xuefu Road,

Zhengjiang, Jiangsu 212013, China. E-mail: yangliuqing@ujs.edu.cn

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EXTRACTION AND PROPERTIES OF SILYBUM MARIANUM SEED PROTEINS 1751

The seeds of S. marianum also contain a relatively high amount of oil and crudeproteins (approximately 19.58%, dry weights of the seeds).[5] This oil contains phospho-lipids, relatively high amounts of vitamin E,[6] and a great quantity of unsaturated fattyacids, such as linoleic acid (C18:2) and oleic acid (C18:1). For plant proteins to be usefuland successful in food application, they should ideally possess several desirable charac-teristics, referred to as functional properties, as well as providing essential amino acids.[7]

These properties are intrinsic physicochemical characteristics that affect the behavior ofproteins in food systems during processing, manufacturing, storage, and preparation.[7]

Therefore, it is necessary to investigate these properties to utilize the dietary protein fromthe seeds in the food industry. However, there is little information on the properties of pro-tein fractions of the S. marianum seeds. Thus, the present study was to evaluate some basicphysicochemical properties of protein fractions from S. marianum seeds; the protein isolateof S. marianum seeds was also used as a comparison. These properties include molecularweights of major fractions, amino acid compositions, secondary and tertiary conforma-tions, and protein solubility. It was also of interest to determine some functional propertiesof the seed proteins, as it is essential for their potential use as protein extenders and as acidformulations.

MATERIALS AND METHODS

Materials

S. marianum seeds were obtained from Zhongxing Yaoyue Co., Ltd. (Zhenjiang,Jiangsu, China), and identified by Chen Li (Chief Pharmacist, Food and DrugAdministration Bureau of Zhenjiang). Low molecular weight protein markers (Code num-ber: D530S) were purchased from DINGUO Biotech. Co., Ltd. (Shanghai, China). Allsolvents and chemicals were of analytical reagent grade or better grade and obtained fromSinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Proximate Analysis

The recommended methods of the Association of Official Analytical Chemists[8]

were adopted to determine the content of crude protein, moisture, and total ash ofS. marianum seed powders (SSP). The crude fiber content was determined by the ceramicfiber filter method. The carbohydrate contents of the samples were determined as nitrogenfree extract (NFE) by the following formula: NFE % = 100 − (moisture + crude fat + crudeprotein + crude fiber + total ash).

Preparation of Defatted Powders of S. marianum Seed (DSSP)

Seeds were removed from the S. marianum fruit, husked, and air-dried at 25◦C, andthen crashed to the average particle size of 300 μm. SSP were defatted with hexane at 25◦C,air-dried in the fume hood, and stored in bottle desiccators at room temperature.

Preparation of Protein Fractions and Protein Isolate

Protein fractions of DSSP were fractionated according to the Osborne methods[9]

with modifications as follows. For albumin fraction (SA), suspensions of DSSP/de-ionized

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water (1:10, w/v) were stirred for 1 h at 25◦C and centrifuged at 13,500 g for 15 min.After dialyzing extensively with de-ionized water for 24 h, the supernatant was centrifugedagain, and lyophilized to obtain the SA. To obtain the globulin fraction (SG), the precipitateobtained by the first centrifugation was re-suspended in a solution of 0.5 mol/L NaCl (1:5,w/v), and the resulting suspension stirred for 1 h at 25◦C. After centrifugation under thesame conditions as described earlier, the supernatant was dialyzed and centrifuged again.The corresponding precipitate was dissolved in de-ionized water and then lyophilized toobtain the SG. The solid residue after SG extraction was used for the sequential extractionof prolamin fraction (SP) and glutelin fraction (SGL) by using 75% ethanol solution and0.05 mol/L NaOH stirred for 1 h at 25◦C, respectively. Ethanol in the SP was removedby evaporate at 45◦C. For SGL, the extract was adjusted to pH 4.5 with 0.5 mol/L HCl,and then centrifuged at 13,500 g for 15 min. The precipitate was then re-suspended infive volumes of de-ionized water and adjusted to pH 7.0 with 0.5 mol/L NaOH. Aftercentrifugation under the same conditions, the supernatant was dialyzed with de-ionizedwater for 24 h then lyophilized to produce SGL. The molecular weight (MW) cut-off ofdialysis membrane was above 3500 Da.

Protein isolate from S. marianum seeds (SPI) was prepared from the DSSP accord-ing to the method described by Tang[10] with necessary modifications. Briefly, the powderswere fully dispersed in a 10-fold volume of de-ionized water for 2 h at room temperature,and then the dispersion was adjusted to pH 8.0 with with NaOH (0.1 mol/L). After cen-trifugation at 13,500 g for 15 min, the resultant supernatant was adjusted to pH 4.5 usingHCl (0.5 mol/L) to precipitate the proteins. The precipitate was re-dispersed in de-ionizedwater, then dialyzed and lyophilized to obtain the SPI.

Quantification of Total, Free, and Protein-Bound Polyphenols

The contents of total, free, and protein-bound polyphenols were determined accord-ing to the method described by Tang et al.[11] with slight modifications. The freepolyphenols content was determined by the absorbance of the supernatant after proteinprecipitation with 8% trichloroacetic acid and centrifugation. All other steps and calcula-tions were done exactly as described in the original method. Rutin was used as a standardcompound for the standard curve. The contents of the polyphenols were expressed as grutin equivalent/100 g samples.

Amino Acid Analysis

The amino acid compositions of the samples were determined by an L-8800 auto-matic amino acid analyzer (Hitachi, Tokyo, Japan). The samples were hydrolyzed by using6 mol/L HCl at 110◦C in a sealed tube for 24 h. The amino acid compositions wereexpressed in proportion of the single amino acid to total amino acids. The amino acidtryptophan was not determined.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

(SDS-PAGE)

SDS-PAGE was performed on a discontinuous buffered system, according to themethod described by Laemmli,[12] using 10% separating gel and 6% stacking gel withslight modifications. The samples were dissolved in 0.06 mol/L Tris-HCl buffer (pH 6.8),

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EXTRACTION AND PROPERTIES OF SILYBUM MARIANUM SEED PROTEINS 1753

containing 2% (w/v) SDS, 2.5% (v/v) 2-mercaptoethanol (2-ME), 25% (v/v) glycerol,and 0.1% (w/v) bromophenol blue, and then heated for 5 min in boiling water beforeelectrophoresis. An aliquot of each sample (15 μL) was applied to each lane to theelectrophoresis, after that the gel was stained in 0.1% Coomassie blue (R-250) and 10%acetic acid (methanol:acetic acid:water, 9:2:9, v/v/v) and de-stained in 10% acetic acid(methanol:acetic acid:water, 1:1:8, v/v/v). The molecular weights of protein subunits foreach sample were determined using low molecular weight protein markers.

Circular Dichroism (CD) Spectroscopy

CD spectra were recorded at 25◦C on a MOS-450 spectropolarimeter (BioLogicScience Instruments, Claix, France). The CD spectroscopy measurements were performedin quartz cuvette of 2 mm with an approximate protein concentration of about 0.1 mg/mLin 10 mmol/L phosphate buffer (pH 7.0). The samples were scanned from the wavelengthsof 190 to 250 nm. The following parameters were used: step resolution, 1 nm; acquisi-tion duration, 1 s; bandwidth, 0.5 nm. The CD spectra of the samples were corrected bysubtracting of the spectrum of the protein-free buffer.

Intrinsic Fluorescence Spectroscopy

Emission spectra of intrinsic fluorescence of the samples were determined on aCary Eclipse Fluorophotometer (Varian, Palo Alto, CA, USA). Intrinsic fluorescencespectroscopy measurements were performed in quartz cuvette of 1 cm with a protein con-centration of 0.5 mg/mL in 10 mmol/L phosphate buffer (pH 7.0). Protein solutions wereexcited at 275 nm, and emission spectra were recorded from 300 to 400 nm at a constantslit of 5 nm for both excitation and emission.

Protein Solubility (PS)

The pH of aqueous solution (1%, w/v) of the protein sample was adjusted to thedesired value (pH 2.0–10.0) with either HCl (0.4 mol/L) or NaOH (0.4 mol/L). The pH wasreadjusted if necessary after being stirred for 30 min, and then the solution was centrifugedat 8000 g for 20 min. After an appropriate dilution of the supernatant, the protein content ofthe supernatant was determined. The PS was expressed as the proportion of soluble proteinto total proteins and the protein content of the dispersion (1%, w/v) in 0.1 mol/L NaOHwas used as the total protein content.

Functionality Tests

Foaming and emulsifying properties. Foam capacity (FC) and foam stability(FS%) of DSSP were determined according to the method described by Hojilla-Evangelistaet al.[13] The foam volume was measured after 1 min and the increase in volume of the foamwas expressed as foam capacity. Foam stability was measured by the decrease of the foamat 30 min. Emulsification activity (EA) and emulsification stability (ES) of DSSP weretested following the method of Wang et al.[14] EA and ES were calculated as the ratio ofemulsion layer height to the total height.

Water holding and fat absorption capacities (WHC and FAC). WHC andFAC of DSSP were determined using the method described by Tomotake et al.[15] with

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slight modifications. For WHC, briefly, accurately each 0.25 g of the DSSP was weighedinto 10-mL pre-weighed centrifuge tubes, and then 5 mL de-ionized water was added to thetubes. For FAC, accurately 0.50 g of the DSSP was weighed into 10-mL centrifuge tubespre-weighed and mixed with 3 mL of peanut oil thoroughly. All other steps and calculationswere done exactly as described in the original method.

Determination of Protein Contents

Total proteins of the SSP and DSSP were determined using the micro-Kjeldahlmethod with nitrogen conversion factor of 6.25. Whenever necessary, the contents ofsoluble proteins were analyzed by using bovine serum albumin as the standard proteinfollowing the method described by Bradford.[16] Appropriate solvent and reagent blankswere included in each assay.

Statistical Analysis

Data are reported as mean ± SD (standard deviation) of triplicate determinations.One-way analysis of variance (ANOVA) of the data was performed, and a least significantdifference (LSD) test with confidence interval of 95% was used to compare the means.

RESULTS AND DISCUSSION

Proximate Composition

The crude fat of SSP had been determined by our previous study,[17] with the contentof 46.84%. The proximate analysis showed that of the SSP contained 5.50% moisture,6.30% ash, 6.46% crude fiber, 14.87% NFE, and crude fat. The crude protein in SSPwas similar to that obtained under similar conditions (19.58%).[5] It is possible to pro-duce value-added byproducts (dietary oil or protein) resulting from the high fat and proteincontent of the S. marianum seeds.

Protein Fractions and Protein Content

The obtained protein fractions are presented in Table 1. The yields of proteinfractions indicated that albumin was the dominant fractions, followed by globulins with asmall amount of glutelins and prolamins. Conforti and Lupano also found that about 70%of the proteins of the gametophyte of araucaria angustifolia and araucaria araucana weresoluble in distilled water (albumin proteins).[18] The protein contents of protein fractionsvaried considerably. SPI had the highest protein content compared with the proteinfractions. However, albumins play an essential role in seeds because they are responsiblefor both enzymatic and metabolic activities in the seeds.[19] In addition, albumins are ofnutritional importance because of their high contents of essential amino acids and sulphuramino acids.

Polyphenol Contents

Polyphenols contents of DSSP, proteins fractions, as well as SPI are presented inTable 2. The polyphenols contents of individual protein fractions and SPI were significantlydifferent. SGL had the highest total polyphenols content, followed by SA, SG, and SPI.

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EXTRACTION AND PROPERTIES OF SILYBUM MARIANUM SEED PROTEINS 1755

Table 1 Protein contents of protein fractions as well as protein isolate from S. marianum seeds.

Samplesa Protein content (%, dry basis) % Soluble proteins

SSP 19.95 ± 0.76b —DSSP 37.52 ± 1.02d —SPI 90.13 ± 1.68g —SA 87.27 ± 0.81f 74.58 ± 0.80e

SG 85.88 ± 1.44f 16.92 ± 0.51d

SP 34.52 ± 1.40c 1.01 ± 0.05b

SGL 67.52 ± 0.69e 7.49 ± 0.18c

Each value (based on dry basis) was the mean and standard deviation of triplicatedeterminations.

aSSP: powders of S. marianum seeds; DSSP: defatted powders; SPI: protein isolates; SA:albumin fraction; SG: globulin fraction; SP: prolamin fraction; SGL: glutelin fraction.

Values in the same column followed by a different letter are significantly different (p <

0.05) according to least significant difference (LSD) test.

Table 2 Polyphenol contents of defatted powders of S. marianum seed, protein fractions, aswell as protein isolate from S. marianum seeds.

Polyphenol contents (%)b

Samplesa Total Free Protein-bound

DSSP 2.80 ± 0.12c 1.43 ± 0.02c 1.37 ± 0.08c

SPI 3.99 ± 0.13d 1.88 ± 0.06d 2.11 ± 0.09d

SA 4.37 ± 0.11e 2.12 ± 0.09e 2.25 ± 0.10d

SG 4.26 ± 0.25e 1.54 ± 0.07c 2.72 ± 0.15d

SGL 13.00 ± 0.15f 1.67 ± 0.03c 11.33 ± 0.13e

Each value (based on dry basis) was the mean and standard deviation of triplicatedeterminations.

aDSSP: defatted powders of S. marianum seed; SPI: protein isolate from S. marianum seeds.bThe total, free, and protein-bound polyphenol contents were expressed in g rutin

equivalent/100 g of sample.Values in the same column followed by a different letter are significantly different (p <

0.05) according to least significant difference (LSD) test.

The difference of total polyphenols contents among individual protein fractions and SPImay be attributed to the different extent of interactions between the polyphenols and theproteins by different preparation. In these protein fractions, the form of the polyphenolswas also diverse. Around half of the polyphenols were present in the free form in theDSSP, SA, and SPI, whereas in SG and SGL were only 36.15 and 12.85%, respectively.The result indicated that SG and SGL more preferably interacted with the polyphenolsto form polyphenol–protein complexes, through covalent linkage bonds. The interactionform of the polyphenols in DSSP and SPI was similar with that in SA, which was thedominant fraction. Most of polyphenols in the SGL (87.15%) were in the protein-boundform. The covalent interactions between polyphenols and proteins may be favored by thealkali conditions in the presence of the polyphenols,[20] which presumably account for thisphenomenon.

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Amino Acid Compositions

The amino acid compositions of the various protein fractions are shown inTable 3. The SSP, protein fractions, and SPI were richer in polar amino acids (asparticacid/asparagine, glutamic acid/glutamine, and arginine), which were the common char-acteristics of all storage proteins.[21] The seed proteins also included all essential aminoacids (exception of tryptophan, not be determined in the present study) recommended byFAO/WHO for 2–5 year olds.[22] Infants have very critical nutritional requirements due torapid growth and immaturity of gastrointestinal function, and nine amino acids have beenidentified to be essential for infants. As shown in Table 3, the percentages of histidine,valine, isoleucine, phynylalanine + tyrosine of the SSP, protein fractions and SPI werehigher than their counterparts stated in the FAO/WHO (1990) standard. However, the over-all quality of the SSP, protein fractions, and SPI were slightly compromised by the lowlevel of lysine, which accounted for between 67.07 to 76.90% of the lysine requirement forchildren as recommended by the FAO/WHO.[22]

SDS-PAGE Analysis

SDS-PAGE band patterns of protein fractions and SPI under reducing conditionsare presented in Fig. 1. SDS-PAGE band patterns of the SPI indicated a broad range of

Table 3 Amino acid composition of SSP, protein fraction, and SPI, and the FAO/WHO indicated requirements(2–5 years old) for the essential amino acids.a

Amino acid SSPb SPI SA SG SGL FAO/WHOc

Asxd 9.71 10.45 10.01 10.39 10.14Glxe 22.56 23.77 25.21 22.87 21.61Serine (Ser) 5.35 5.56 5.48 5.86 5.67Histidine (His)f 2.52 2.38 2.37 2.66 2.69 1.9Glycine (Gly) 5.92 4.65 4.80 5.36 5.46Threonine (Thr)f 3.90 3.27 3.10 3.39 3.78 3.4Arginine (Arg) 10.07 8.42 8.90 8.49 8.28Alanine (Ala) 4.28 4.28 3.97 4.57 4.75Tyrosine (Tyr) 3.52 4.12 4.01 3.95 4.41Cystine (Cys) 1.60 1.13 1.25 0.85 0.50Valine (Val)f 5.18 5.21 4.94 5.04 5.42 3.5Methionine (Met)f 1.23 1.76 1.95 1.58 1.79Phenylalanine (Phe)f 4.32 4.90 4.55 4.83 4.98Isoleucine (Ile)f 4.15 4.35 4.03 4.38 4.64 2.8Leucine (Leu)f 6.82 7.07 6.63 7.06 7.47 6.6Lysine (Lys)f 4.46 3.95 3.93 3.89 3.95 5.8Proline (Pro) 4.23 4.74 4.84 4.84 4.46Total sulfur-containing amino acids (Met and Cys) 2.83 2.89 3.20 2.43 2.29 2.5Total aromatic amino acids (Phe and Tyr) 7.84 9.02 8.56 8.78 9.39 6.3E/T(%)g 32.58 32.89 31.50 32.83 34.72

aProportion of the single amino acid to total amino acids.bSSP: S. marianum seed powders.cRecommended pattern (FAO/WHO for 2–5 years old).dAsx = Aspartic acid + Asparagine.eGlx = Glutamic acid + Glutamine.fEssential amino acid.gRatio of total essential amino acids (not including tryptophan) to total amino acids.

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Figure 1 SDS-PAGE band patterns of protein fractions and SPI under reducing conditions. Lanes 1–6: (1) molec-ular mass markers; (2) SPI: protein isolates; (3) SA: albumin fraction; (4) SG: globulin fraction; (5) SP: prolaminfraction; (6) SGL: glutelin fraction. Protein concentration, 4 mg protein/mL; Sample load volume, 15 μL. (Colorfigure available online.)

polypeptides (16.3 to 112.1 kDa) (Fig. 1, lane 2). The most intense and widest bandsresolved between 23.0 and 36.9 kDa. SA showed 12 distinct bands, similar to SPI. Themost concentrated bands of SA resolved at 23.0 kDa, 32.9–40.9 kDa. The 13 bands of SGwere in the MW range of 23.0–40.9 kDa, and 85.7–112.1 kDa (Fig. 1, lane 4). There wasonly one lighter band with MW of 23.3kDa that resolved at SP (Fig. 1, lane 5). The bandsof SGL (Fig. 1, lane 6) were comparably minor but still detectable. The most concentratedbands resolved between 23.0 and 40.9 kDa.

Conformational Characterization

Secondary conformation. Secondary structure elements, such as α-helices andβ-types, have dichroic activity in the wavelength range from 190 to 260 nm.[23] Figure 2showed far-UV CD spectra of the protein fractions and SPI at 190–250 nm, in the far-UVregion. The spectra showed a typical shape and feature of the mainly β-types secondarystructure, i.e., negative bands at 216–218 nm and 220–230 nm (weak) and positive bandsat 195–198 nm. The secondary structure compositions (including α-helices, β-strands,β-turns, and random coils) of these samples were calculated by their far-UV CD spectraaccording to the CONTIN/LL program in CDPro software (BBSRC, Swindon, UK; usedfor analysis DICHROWEB, University of London, UK). The calculations, which gave esti-mates of secondary structural components for various proteins, were shown in Table 4.The far-UV CD spectra and the secondary structure compositions of SA were similar tothose of SPI (Fig. 2, Table 4). This phenomenon was consistent with SA (the most domi-nant fraction of S. marianum seed proteins) having similar protein constituent patterns asSPI. As shown in Fig. 2 and Table 4, the SGL fraction has a typical shape and feature ofmainly β-types secondary structure, containing the highest content of β-turns structures.In the case of SG, a slight shift (about 3 nm) in band at 195 nm towards lower wavelengthwas observed (relative to SPI in band at 198 nm), which also was an indirect indication of ahigh content of α-helix. The results on the α-helices and β-strands contents were consistentwith the conclusion that the globulins from plant seed mainly belong to the class of β-typesproteins.[24]

Fluorescence spectroscopy. The tertiary conformation of these proteins wasfurther analyzed by the intrinsic emission fluorescence spectroscopy, as given in Fig. 3.

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190 200 210 220 230 240

–12

–10

–8

–6

–4

–2

0

2

4

Elli

ptic

ity (

mde

g)

SPISASGSGL

Wavelength (nm)

Figure 2 Far-UV circular dichroism spectra of protein fractions and SPI. SA (-•-); SG (-�-); SGL (-�-); and SPI(- -). (Color figure available online.)

Table 4 Secondary structure compositions of various protein fractions and SPI calculated fromfar-UV CD spectrum using the CONTIN/LL program in CDPro software.

Secondary structure compositions (%)

Protein samples α-Helicesa β-Strandsb β-Turns Random coil

SPI 0.3 61.5 11.8 26.4SA 0.4 47.8 8.0 43.8SG 4.0 15.4 9.3 71.2SGL 0.1 14.2 34.3 51.5

aCombined regular and distorted α-helices.bCombined regular and distorted β-strands.

300 320 340 360 380 4000

100

200

300

400

500

600

700

800

357.9 nm

361.1 nm

352.0 nm

Rel

ativ

e in

flu

ores

cenc

e in

tens

ity (

A.U

)

Wavelength (nm)

SPISASGSGL

347.8 nm

Figure 3 Fluorescence spectra of protein fractions and SPI. SA (dash line); SG (dot line); SGL (dash dot line);and SPI (solid line). (Color figure available online.)

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The determination chiefly carried out by the polarity of the environment of the trypto-phan residues, which provided a sensitive means of monitoring conformational changesin proteins, and protein–protein as well as ligand–protein interactions.[25] The maximumfluorescence emission spectrum suffers a red-shift when chromophores become moreexposed to the solvent.[11] The quantum yield of fluorescence decreases when the chro-mophores interact with quenching agents either in a solvent or the protein itself. TheSG exhibited a fluorescence emission spectrum with a maximum at 347.8 nm, which isthe characteristic fluorescence profile of tryptophan residues in a relatively hydrophobicenvironment.[26] Maximum emission spectrum for SGL was at 352.0 nm (4.2 nm red-shiftrelative to SG), and the quantum yield of fluorescence was less (Fig. 3), which indi-cated some tryptophan residues exposed to the exterior of the proteins. This differencein fluorescence intensity may be attributed to the difference in quenching agents (suchas polyphenols) contents, since they would cause quenching of fluorescence intensity inthe region between 300–400 nm by interaction of the aromatic ring in the polyphenolswith those of aromatic residues, e.g., tryptophan and tyrosine residues.[27] The maxi-mum of the emission spectra of SPI and SA were at 357.9 and 361.1 nm, respectively,which were the characteristic profiles of tryptophan residues in a much more hydrophilicenvironment.[28] Quantum yields for the fluorescence of SPI and SA were also much less,but the fluorescence spectra were much broader, compared with those of SG or SGL. Theresults indicated the highly flexible tertiary conformation for SPI and SA, which were inagreement with the observation described by Tang and Wang.[11] For the albumin fromcommon buckwheat seeds, the maximum of the emission spectra suffered red-shift fromglobulin, and the quantum yield of fluorescence of albumin was much less than that ofglobulin.

Protein Solubility-pH Profiles

The pH-dependent protein solubility profiles of the DSSP, protein fractions, and SPIwere presented in Fig. 4. Isoelectric points of the proteins from the S. marianum seedswere between 4.0 and 5.0. Generally, solubility was reduced as the pH increased until itreached the isoelectric point, followed by a progressive increase in solubility with furtherincrease in pH. The similar results were reported in a previous study, which found that min-imum solubility of protein isolates from Bambara Groundnut (Voandzeia subterranean L.)occurred at pH 5, and the isolates exhibited higher solubility than the correspondingisoelectric (IEP) isolates over all pH values.[29] The different PS patterns of the DSSP, pro-tein fractions, and SPI were observed. The PS profile of SA was much less pH-dependentover the tested pH range, relative to DSSP, SG, and SGL (Fig. 4). The PS of SA was inthe range of 38–90% at pH 2–10. At pH 5, the PS of DSSP, SG, and SGL was only 16.70,11.16, and 8.80%, respectively. As shown in Fig. 4, the PS profile of SA was similar to thatof SPI. This phenomenon was consistent with SA having the similar protein constituentpatterns as SPI, the most dominant of S. marianum seed proteins. In the case of SG, itexhibited a PS-pH typical profile of a number of globulins from many plant seeds, e.g.,soy globulins and buckwheat globulins. The results suggested that the albumin fractionwould have good potential for use in acidic protein-fortified beverages, e.g., protein-richcarbonated beverages,[30] since protein solubility largely affects other functionalities, suchas emulsifying, foaming, and gelating. The high solubility of the proteins indicated theirpromising food applications.

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0 2 4 6 8 100

20

40

60

80

100

Prot

ein

Solu

bilit

y

pH

DSSPSPISASGSGL

Figure 4 Protein solubility profiles of the protein fractions and SPI as a function of pH. The PS was expressed asproportion of soluble proteins to total proteins. Each datum point was the mean and standard deviation of triplicatedeterminations. DSSP: defatted powders of S. marianum seed (- -); SA (-�-); SG (- -); SGL (-�-); and SPI (-◦-).

Foaming and Emulsifying Properties

Foam capacity and its stability for DSSP were reported, since foaming is an importantcharacteristic feature of most of seed proteins. The data were presented in Table 5. TheDSSP showed a considerable amount of foam capacity, which was higher than those of seedpowders in akee (27.05%),[31] and similar to that of milkweed seeds (101 mL),[13] but lowerthan that of soyflour soybean protein (135 mL) generated by the same concentration.[32] Thefoam stability after 30 min for the DSSP was higher than that of milkweed seeds (40%),[13]

but lower than those of soybean, which was reported to retain 95% of its foam at the endof the allotted standing time.[32] The emulsion ability of DSSP was greater than that ofakee proteins (Bilphia sapida) (25.65%).[31] For the DSSP tested, EA was lower than ES,which indicated the strengthening and stabilizing of emulsion due to heating at 80◦C priorto centrifugation. Lamsal et al.[33] also reported stabilizing of alfalfa soluble leaf proteinsemulsion by heating.

Table 5 Functional properties of defatted powders of S. marianum seed.a

Functional propertiesa

Sample FC (mL) FS (%) EA (%) ES (%)WHC (g water/

g sample)FAC (g fat/g sample)

DSSP 102.0 ± 1.1 57.1 ± 0.8 48.1 ± 0.5 62.5 ± 1.2 3.24 ± 0.10 1.73 ± 0.08

The values were mean and standard deviations of triplicate determinations.aFC: foam capacity; FS: foam stability; EA: emulsion activity; ES: emulsion stability; WHC: water-holding

capacity; FAC: fat absorption capacity.

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EXTRACTION AND PROPERTIES OF SILYBUM MARIANUM SEED PROTEINS 1761

Water Holding and Fat Absorption Capacities

WHC of a protein plays an important role in imparting characteristics, such as texture,viscosity, and adhesion.[13] As shown in Table 5, the WHC of DSSP was higher than thatof Gum karaya (Sterculia urens L.) seed meal (1.11 g water/g protein),[34] nearly equal to3.70 g water/g protein reported for milkweed seed protein.[13] In the case of FAC, the valueof DSSP was higher than that of Gum karaya (Sterculia urens L.) seed meal (1.14 g fat/gprotein),[34] nearly equal to 1.50 g fat/g protein reported for buckwheat protein.[10]

CONCLUSION

Extraction, physicochemical, and functional properties of proteins from the S.marianum seeds was investigated. These properties varied among the various protein frac-tions and SPI, with albumins fraction being the most abundant seed proteins. Essentialamino acids in the seeds favorably compared with the recommended level for pre-schoolchildren. The protein fractions were composed of the glycopeptides with an estimatedmolecular mass range of 16.3–112.1 kDa. Circular dichroism (CD) spectra indicated thatthe secondary conformations of protein fractions from the S. marianum seeds were richin β-types, though these protein fractions and SPI had some differences in their secondaryconformations. The maximum emission spectrum of SA was at 361.1 nm, which was a char-acteristic profile of tryptophan residues in the most hydrophilic environment. Functionalproperties indicated that S. marianum seed proteins could be used as a protein extender inadhesives or used in formulating acid foods, such as meat and milk analogue products andprotein-rich beverages.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Professor B. A. Wallace and Dr. L. Whitmore at theUniversity of London for providing the CDPro software. The authors also are thankful to ChiefPharmacist Chen Li of the Food and Drug Administration Bureau of Zhenjiang for positivelyidentifying S. marianum seed samples.

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