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Functional properties and in vitro trypsin digestibility of red kidney bean

(Phaseolus vulgaris L.) protein isolate: Effect of high-pressure treatment
Shou-Wei Yin, Chuan-He Tang *, Qi-Biao Wen, Xiao-Quan Yang, Lin Li
Department of Food Science and Technology, South China University of Technology, Guangzhou 510640, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: The effects of high-pressure (HP) treatment at 200–600 MPa, prior to freeze-drying, on some functional
Received 18 September 2007 properties and in vitro trypsin digestibility of vicilin-rich red kidney bean (Phaseolus vulgaris L.) protein
Received in revised form 9 February 2008 isolate (KPI) were investigated. Surface hydrophobicity and free sulfhydryl (SH) and disulfide bond (SS)
Accepted 27 February 2008
contents were also evaluated. HP treatment resulted in gradual unfolding of protein structure, as evi-
denced by gradual increases in fluorescence strength and SS formation from SH groups, and decrease
in denaturation enthalpy change. The protein solubility of KPI was significantly improved at pressures
Keywords:
of 400 MPa or higher, possibly due to formation of soluble aggregate from insoluble precipitate. HP treat-
Red kidney bean (Phaseolus vulgaris L.)
Protein isolate
ment at 200 and 400 MPa significantly increased emulsifying activity index (EAI) and emulsion stability
High pressure index (ESI); however, EAI was significantly decreased at 600 MPa (relative to untreated KPI). The thermal
Functional property stability of the vicilin component was not affected by HP treatment. Additionally, in vitro trypsin digest-
Emulsifying property ibility of KPI was decreased only at a pressure above 200 MPa and for long incubation time (e.g., 120 min).
In vitro digestibility The data suggest that some physiochemical and functional properties of vicilin-rich kidney proteins can
be improved by means of high-pressure treatment.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction (Tang, 2007). This seems to be closely related with its unique struc-
tural peculiarity, e.g. least susceptible to trypsin digestion, as com-
Kidney bean (Phaseolus vulgaris L.) is the most widely produced pared to other vicilin components (Di Lollo, Alli, Biliarderis, &
and consumed food legume in Africa, India, Latin America and Barthakuri, 1993; Jivotovskaya, Vitalyi, Vitalyi, Horstmann, & Vain-
Mexico (FAO, 1993). This bean usually contains 20–30% protein traub, 1996).
on a dry basis, and the protein has a good amino acid composition High static pressure (HP), a promising technology, has been
but is low in sulfur-containing amino acids (notably methionine) widely applied to modify food proteins, since this treatment has
and tryptophan (Gueguen & Cerletti, 1994; Sathe, 2002). Vicilin, little influence on the nutritional compositions or flavor of related
sometimes also named as phaseolin (or G1 globulin), is the major foods. Pressure-induced modification of protein is a complex phe-
storage protein of this bean. It is an oligomeric protein consisting nomenon, concerning the disruption of interactive forces (espe-
of three polypeptide subunits a-, b- and c-phaseolin with molecu- cially hydrophobic bonds and electrostatic interactions) and
lar weight distribution from 43 to 53 kDa (Bollini & Vitale, 1981; changes in structural conformation (Huppertz, Fox, De Kruif, &
Hall, McLeester, & Bliss, 1977; Romero, Sun, McLeester, Bliss, & Kelly, 2006; Messens, van Camp, & Huyghebaert, 1997; Trujillo,
Hall, 1975). This kind of protein shows pH-dependent associa- Capellas, Saldo, Gervilla, & Guamis, 2002). The degree of modifica-
tion–dissociation behavior between tetrameric, protomeric, and tion of the target properties (e.g. emulsifying activities) of proteins
polypeptide forms of the molecule (Romero et al., 1975; Sun, McLe- greatly depend on the protein system (e.g., type of protein, pH and
ester, Bliss, & Hall, 1974), and its subunits also display molecular ionic strength), applied pressure level and duration time of pres-
heterogeneity, attributed to differential degrees of glycosylation sure treatment. When compared to milk proteins and food emul-
(Paaren, Slightom, Hall, Inglis, & Blagrove, 1987). In our recent sions, information on the application of HP treatment to modify
study, it was shown that the protein isolate (rich in vicilin compo- seed storage proteins is limited.
nent) from kidney bean had much higher subunit homogeneity and To date, HP-induced changes in physicochemical and/or func-
gelation ability, relative to those from other Phaseolus legumes tional properties have been reported for soybean protein or its
purified protein components (Molina, Papadopoulou, & Ledward,
* Corresponding author. Tel.: +86 20 87114262; fax: +86 20 87114263. 2001; Puppo et al., 2004, 2005; Wang et al., 2008) and lupin pro-
E-mail address: [email protected] (C.-H. Tang). tein (Chapleau & De Lamballerie-Anton, 2003). On the whole, the

0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.02.090
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S.-W. Yin et al. / Food Chemistry 110 (2008) 938–945 939

emulsifying activity is the functional property mostly influenced sure transmitting medium in the vessel, and its temperature was
by HP treatment. In some cases, the protein solubility is also neg- kept at 25 ± 2 °C during processing. Both untreated (0.1 MPa) and
atively influenced by HP treatment, due to HP-induced protein HP-treated KPI solutions were freeze-dried in the same process.
aggregation. The literature about the application of HP technique All the protein samples were obtained from duplicate experiments.
to modify other seed storage proteins is very limited.
The main objective of this study was to investigate the effects of 2.4. Protein solubility (PS)
additional HP treatment prior to freeze-drying on some physico-
chemical and functional properties of KPI at neutral pH. Addition- PS was determined according to the method of Petruccelli and
ally, the influence on in vitro trypsin digestibility was evaluated. Añón (1994), with minor modification. One hundred milligrams
The protein isolate from kidney bean was chosen for the experi- of untreated and HP-treated KPIs (freeze-dried) were dispersed
ments, because of the two following reasons: (1) this protein is rich and stirred in 10 mL of de-ionized water for 1 h at room tempera-
in vicilin (a representative 7S globulin), and its nutritional charac- ture. Then, the dispersions were centrifuged at 12,000g for 20 min
teristics have been well recognized and (2) this protein is impor- at 20 °C in a CR22G centrifuge (Hitachi Co., Japan). Protein content
tant for the diet in many countries, and thus the related of the supernatants was determined by micro-kjeldahl method
conclusions would be of importance for the commercial utilization (N  6.25). The PS was expressed as gram of soluble protein per
of this bean. 100 gram of total protein. All determinations were conducted in
triplicate.

2. Materials and methods 2.5. Emulsifying activity index (EAI) and emulsion stability index (ESI)

2.1. Materials EAI and ESI were determined according to the method of Pearce
and Kinsella (1978), with minor modifications. For emulsion for-
Red kidney bean (Phaseolus vularis L.) seeds, cultivated in mation, 15 mL of 0.1% (w/v) untreated or HP-treated dispersions
Gangshou Province of China were purchased from a local super- in de-ionized water (pH 7.0) and 5 mL of corn oil were homoge-
market (Guangzhou, China). The seeds were stored at 20 °C, be- nized in FJ-200 High-Speed Homogenizer (Shanghai Specimen
fore use. Trypsin powder (from porcine pancreas; 96 catalog no. Model Co., China) for 1 min at the maximum velocity (close to
T4799, 1000–5000 BAEE units/mg solid), 5,50 -dithio-bis 2-nitro- 10,000 rpm). Fifty micro-liters of emulsion were taken from the
benzoic acid (DTNB), Folin & Ciocalteu’s Phenol Reagent (F-9252), bottom of the homogenized emulsion, immediately (0 min) or
Guanidinium thiocyanate and 1,8-anilinonaphthalenesulfonate 10 min after homogenization, and diluted (1:100, v/v) in 0.1%
(ANS) reagents were purchased from Sigma (St. Louis, MO, USA). (w/v) SDS solution. After shaking in a vortex mixer for 5 s, the
Bovine serum albumin (BSA) was obtained from Fitzgerald Indus- absorbance of dilute emulsions was read at 500 nm using a spec-
tries International Inc. (Concord, MA, USA). All other chemicals trophotometer (Spectrumlab 22PC, Shanghai Lengguang Technol.
used were of analytical or better grade. Ltd. Co., Shanghai, China). EAI and ESI values were calculated using
the following equations:
2.2. Preparation of protein isolates
2  2:303  A0  DF
EAI ðm2 =gÞ ¼ ;
c  /  ð1  hÞ  10; 000
The seeds were soaked in de-ionized water for 12 h at 4 °C and
A0
de-hulled manually. The de-hulled seeds were freeze-dried, ESI ðminÞ ¼  10;
A0  A10
ground and defatted by Soxlet extraction with hexane. Protein iso-
lates were extracted according to the procedure of Fan and Sosulski where DF is the dilution factor (100), c the initial concentration of
(1974), with some modifications. Defatted flours were dispersed in protein (g/mL), U the optical path (0.01 m), h the fraction of oil used
de-ionized water (1:20, w/v), and the pH of the dispersions was ad- to form the emulsion (0.25), and A0 and A10 the absorbance of the
justed to 8.0 with 2 N NaOH. The resultant dispersions were gently diluted emulsions at 0 and 10 min, respectively. Measurements
stirred at room temperature for 2 h, and then centrifuged at 8000g were performed in triplicate.
at 20 °C for 30 min in a CR22G centrifuge (Hitachi Co., Japan). The
pellets were discarded, and the supernatants were adjusted to pH 2.6. Differential scanning calorimetry (DSC)
4.5 with 2 N HCl to precipitate the proteins. Last, the precipitates
obtained by centrifugation at 5000g for 20 min were re-dispersed DSC experiments were performed on a TA Q100-DSC thermal
in de-ionized water. The dispersions were homogenized and ad- analyzer (TA Instruments, New Castle, DE), according to the proce-
justed to pH 7.0 with 2 N NaOH. For HP processing, the protein dure of Meng and Ma (2001), with some modifications. Approxi-
concentration was adjusted to 30 g/L (determined by micro-Kjel- mately 2.0 mg of untreated or HP-treated KPI samples were
dahl method, using a nitrogen conversion factor of 6.25). accurately weighed into aluminum liquid pans, and 10 lL 50 mM
phosphate buffer (pH 7.0) was added. The pans were hermetically
2.3. High-pressure processing sealed and heated in the calorimeter from 20 to 120 °C at a rate of
5 °C/min. A sealed empty pan was used as a reference. Peak or
HP processing was carried out in a 5 L reactor unit (Datong Pres- denaturation temperature (Td), enthalpy change of denaturation
sure Systems, KEFA 75 Hitech Food Machine Company Co. Ltd., (DH) and width at half peak height of endothermic peak (DT1/2)
Baotou, China), equipped with temperature and pressure regula- were computed from the thermograms by the universal analyzer
tion. Prior to pressure processing, aliquots of KPI solutions were 2000, version 4.1D (TA Instrument-Waters LLC, USA). All experi-
packaged in a polyethylene bag (with a size of about ments were conducted in triplicate.
50  150 cm) under vacuum condition. Temperature during HP
treatment was controlled to avoid overheating. Protein solutions 2.7. Free sulfhydryl (SH) content
were subjected to HP treatment at 200 ± 10, 400 ± 10 and
600 ± 10 MPa for 20 min, respectively. The target pressure was Sulfhydryl groups of protein isolates were determined accord-
reached at a rate of about 250 MPa/min, and released at a rate of ing to the method of Ellman (1959), as modified by Beveridge,
about 300 MPa/min. The dioctyl sebacate oil was used as the pres- Toma, and Nakai (1974), with some modifications. Ellman’s
中国科技论文在线 http://www.paper.edu.cn
940 S.-W. Yin et al. / Food Chemistry 110 (2008) 938–945

reagent was prepared by dissolving 4 mg of DTNB reagent in 1 mL was determined, based on the Debye plot (Zhao et al., 2004). The
of Tris-glycine buffer (0.086 M Tris, 0.09 M glycine, 4 mM EDTA, pH dn/dc is reckoned to be constant (±0.185 mL/g) across the sample
8.0). Total and exposed SH contents for the proteins were obtained peak, and nearly independent of its amino acid composition, where
by suspending 15 mg of protein samples in 5.0 mL of reaction buf- n and c present the refractive index and sample concentration for
fer, Tris-glycine buffer with (total SH) or without 8 M urea (ex- each data slice. Bovine serum albumin monomer (Sigma, St. Louis,
posed SH), respectively. Then, 50 lL of the Ellman’s reagent were MO) was used for normalizing various detectors’ signals relative to
added. The resultant suspensions were incubated for 1 h at room the 90° laser light detector signal.
temperature (25 ± 1 °C), with occasional vibrating, and then centri-
fuged at 12,000g for 10 min. The absorbance of the supernatant 2.10. Intrinsic fluorescence emission spectra
was determined at 412 nm with the reagent buffer as the blank.
The protein contents of isolates were determined by micro-Kjel- Intrinsic emission fluorescence spectra of protein samples were
dahl method (N  6.25). The SH contents were calculated by use determined in a RF-5301 PC spectrofluorometer (Shimadzu Corp.,
of the extinction coefficient of 2-nitro-5-thiobenzoate (NTB) at Kyoto, Japan). Protein solutions (0.15 mg/mL) were prepared in
412 nm, 13,600 M1 cm1 (Ellman, 1959), and expressed as lmol/g 10 mM phosphate buffer (pH 7.0). To minimize the contribution
of protein. of tyrosyl residues to the emission spectra, protein solutions were
excited at 290 nm, and emission spectra were recorded from 300 to
2.8. Determination of disulfide bond (SS) content 400 nm at a constant slit of 5 nm for both excitation and emission.

Synthesis of 2-nitro-5-thiosulfobenzoate (NTSB) was performed 2.11. Measurement of surface hydrophobicity (H0)
according to the method of Thannhauser, Konishi, and Scheraga
(1984), as modified by Petruccelli and Añón (1995). First, DTNB H0 was determined using ANS, according to the method of Kato
(0.1 g) was dissolved in 10 mL of 1 M Na2SO3. Then, the pH of the and Nakai (1980), as modified by Haskard and Li-Chan (1998). In
reaction mixture was adjusted to 7.5, and 50 lL of a 0.1 M ammo- brief, stock solutions of 8  103 M ANS, and 1.5% (w/v) protein
niacal solution of CuSO4 was added. The reaction was magnetically were prepared in 10 mM phosphate buffer (pH 7.0). To successive
stirred at 38 °C; it was ended when more than 99% of DTNB was samples containing 4 mL of buffer and 20 lL of ANS stock solution
transformed into NTSB (the reaction was followed by measuring were added 10, 20, 30, 40, and 50 lL of 1.5% protein solution. The
the concentration of NTB by its absorbance at 412 nm). The mixtures were shaken in a vortex mixer for 5 s. Fluorescence inten-
remaining NTB was determined by measuring absorbance at sity (FI) was measured at wavelengths of 390 nm (excitation) and
412 nm. The stock solution was stored at 20 °C. The test NTSB 470 nm (emission) using the same spectrofluorometer at
solution was prepared by diluting the stock solution (1:100) with 20 ± 0.5 °C, with a constant excitation and emission slit of 5 nm.
a fresh 0.2 M Tris-base buffer containing 0.1 M Na2SO3, 10 mM The FI for each sample with probe was then computed by subtract-
EDTA, and 3 M guanidinium thiocyanate. This solution was ad- ing the FI attributed to protein in buffer. The initial slope of the FI
justed to pH 9.5 with l N HC1. versus protein concentration plot was calculated by linear regres-
Determination of disulfide bonds was carried out according to sion analysis and used as an index of H0.
the method of Thannhauser et al. (1984). Protein isolates (25 mg/
mL) were previously dispersed in Tris-base buffer. Then, 100 lL 2.12. In vitro trypsin digestibility
of the protein solution was mixed with 3 mL of the NTSB test solu-
tion prepared just prior to use. Absorbance at 412 nm was deter- The in vitro trypsin digestibility of protein samples was deter-
mined using the test NTSB solution as the reference. The mined as follows: 5 mL of protein dispersions (1%, w/v) in 10 mM
extinction coefficient of NTB used to transform absorbance values phosphate buffer (pH 8.0) was mixed with 1 mg of trypsin powder,
into concentration values was 13,600 M1 cm1. and the mixtures were incubated at 37 °C for 30 or 120 min. The
reactions were stopped by adding an equal volume of 20% (w/v)
2.9. High-performance size-exclusion chromatography (HPSEC) trichloroacetic acid, and the protein precipitates were removed
combined with multi-angle laser light scattering (MALLS) by centrifugation at 10,000g for 20 min. The TCA-soluble nitrogen
in the supernatants was determined by micro-Kjeldahl method
The HPSEC and the MALLS systems were the same as those de- (N  6.25). The %N release during the digestion was calculated as
scribed by Zhao, Mine, and Ma (2004). Two TSK columns (G4000 % N release = (NS  N0)  100/Ntot, where NS is TCA-soluble nitro-
PWXL + TSK G6000 PWXL) were connected in series (TOSOH Corp., gen in supernatant phase, N0 (mg) TCA-soluble nitrogen at 0 min,
Montgomeryville, PA). The fractionation range of these two col- and Ntot (mg) total nitrogen of protein.
umns was 2000–8,000,000 for proteins. The mobile phase
(50 mM phosphate buffer, pH 7.4, containing 0.05 M NaCl) was fil- 2.13. Statistics
tered through 0.2 lm (Whatman International Ltd., Maidstone,
England) and then 0.02-lm filters (Millipore Corp., Bedford, MA). An analysis of variance (ANOVA) of the data was performed, and
The flow rate was 0.8 mL/min. a least significant difference (LSD) or Tamhane’s with a confidence
A Dawn EOS photometer (Wyatt Technology Corp., Santa Bar- interval of 95 or 99% was used to compare the means.
bara, CA) was used. Two auxiliary analogue inputs enabled inter-
facing to external detectors such as RI and UV detectors. The
instrument was placed directly before the RI detector and after 3. Results and discussion
the SEC columns and UV detector to avoid backpressure on the
RI cell. Dynamic light scattering measurement was performed 3.1. SEC–MALLS–RI analysis
on-line in the flow cell using a QELS meter (Wyatt Technology
Corp., Santa Barbara, CA). An optical fiber receiver was mounted Typical SEC elution profiles of untreated (0.1 MPa) KPI, com-
in the read head of one of the MALLS detectors (detector 13 in bined with MALLS (at 90°) and RI detection are shown in Fig. 1.
our works). Chromatographic data were collected and processed In the RI profile, one major peak (peak 3) eluting at 20.6 mL and
by the ASTRA software (Wyatt Technology Corp.). The Mw of pro- several minor peaks eluting before or after the major peak were
tein eluting in small and individual slices of the SEC chromatogram observed. In the corresponding LS profile, another observable peak
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S.-W. Yin et al. / Food Chemistry 110 (2008) 938–945 941

Total area fraction I

A 2.5 1
fraction II fraction III
0.35

2.0 0.30

Integrated area (A.U.)

LS signal (A. U.)

0.25
1.5
0.20
3
1.0
2 0.15

600 MPa 0.10

0.5
400 MPa
200 MPa 0.05
0.0 0.1 MPa
0.00
10 15 20 25 30
Control 200 MPa 400 MPa 600 MPa
Elution volume (mL)
Fig. 2. Total or individual integrated area(s) of RI peaks for SEC profiles of untreated
and HP (200–600 MPa)-treated KPI samples. Total area: total integrated RI peak
B 0.30 3
area; Fraction I: <17.5 mL (peak 1); Fraction II: 17.5–23.0 mL (peaks 2 and 3); Fr-
action III: >23.0 mL (peak 4 and others).
0.25
RI signal (A. U.)

0.20 ered to be due to the increase in integrated area of peak 1 (aggre-

gate). This is an indication that in the present case, HP at pressures
0.15 of 400 MPa or higher led to transformation of insoluble precipitate
4 to soluble aggregate.
0.10 1 2
600 MPa
3.2. Emission fluorescence spectroscopy analysis and surface
0.05 400 MPa
hydrophobicity (H0)
200 MPa
0.00 0.1 MPa
The emission fluorescence spectroscopic technique was used to
10 15 20 25 30 characterize the conformational changes of KPI, induced by HP treat-
Elution volume (mL) ment, as shown in Fig. 3. The fluorescence spectrum is determined
chiefly by the polarity of the environment of the tryptophan and
Fig. 1. Superimposed SEC–MALLS–RI elution profiles of untreated and HP-treated
tyrosine residues and by their specific interactions and provides a
(200–600 MPa) KPI samples: (A) MALLS detection (at 90°) and (B) RI detection.
sensitive means of characterizing proteins and their conformation,
since the fluorescence emission maximum suffers a red shift when
at about 15.0 mL (peak 1) and a shoulder peak (peak 2) appeared, chromophores become more exposed to solvent, and the quantum
indicating the presence of large molecular weight (Mw) aggregates, yield of fluorescence decreases when the chromophores interact
but the amount was very low, since the RI signal of peak 2 was with quenching agents either in a solvent or in the protein itself (Pal-
nearly invisible. The Mw of peak 3, calculated from the combined larès, Vendrell, Avilès, & Ventura, 2004). The intrinsic fluorescence
LS and RI signals, was about 161 kDa, in agreement with the Mw spectrum of untreated KPI shows an emission maximum at around
of vicilin in the trimeric form. The Mw of the shoulder peak was cal- 332 nm (Fig. 3A). This is a characteristic fluorescence profile of tryp-
culated to be about 6.2  105 Da (with a standard deviation of 4%), tophan residues in a relatively hydrophobic environment, such as
suggesting that it was mainly composed of tetramers of vicilin. the interior of the globulin (Dufour, Hoa, & Haertlé, 1994). This max-
From the RI profile, the vicilin (peak 3 plus peak 2) constituted imum wavelength was nearly unaffected by HP treatment at a pres-
about 78% of total protein in KPI. The data suggest that this protein sure of 200 and 400 MPa, while it red-shifted to about 335 nm when
isolate was rich in vicilin component. Furthermore, peak 4 or other HP treatment at 600 MPa was applied. On the other hand, the fluo-
peaks eluting at similar volumes may correspond to albumin or rescence intensity of untreated KPI was increased by HP treatment
other components with small Mw in KPI. at pressures of 400 MPa or higher. The increase in fluorescence
The effects of HP (200–600 MPa) treatment prior to freeze-dry- intensity after HP treatment was also observed for b-lactoglobulin
ing on SEC elution profiles (LS and RI signals) are also presented in (Yang, Powers, Clark, Dunker, & Swanson, 2002). These observations
Fig. 1. In the RI profile, HP-induced changes in total or individual indicated that HP treatment at higher pressure levels (especially at
fraction integrated area(s) are shown in Fig. 2. HP treatment re- 600 MPa) resulted in an increased exposure of the tryptophan resi-
sulted in a slight but gradual increase of total integrated area, sug- dues of the protein to the solvent.
gesting that the HP treatment might improve protein solubility of The fluorescence emission spectra of ANS (a polarity-sensitive
KPI in the applied buffer (pH 7.4). However, the changes of relative fluorescent probe) upon binding to untreated and HP-treated KPI
integrated area for various elution fractions were different. HP samples were also analyzed. At a constant pH (e.g. pH 7.0), ANS
treatment at 200 MPa resulted in dissociation of aggregate (peak binds to exposed hydrophobic surfaces in partially unfolded pro-
1) into peaks 2 or 3 (Figs. 1B and 2). Concomitantly, LS signal of teins with much higher affinity than to native or completely un-
peak 1 of untreated KPI was considerately higher than that of HP folded proteins, resulting in an increase in fluorescence emission
(200 MPa)-treated KPI (Fig. 1A). However, HP at a pressure above compared with the emission of free ANS in aqueous solution (Pal-
200 MPa markedly increased the LS and RI signals of the aggregate larès et al., 2004). As expected, HP treatment at pressures of 200
peak (peak 1) (Figs. 1 and 2). Interestingly, from Fig. 2, it was ob- and 400 MPa led to similar increases in fluorescence emission
served that the integrated areas for peaks 2, 3 or 4 were nearly intensity, while further increase in intensity was observed at a
not affected by HP treatment. Thus, the increase in total integrated pressure of 600 MPa (Fig. 3B), indicating the gradual exposure of
area by HP treatment at a pressure above 200 MPa can be consid- hydrophobic clusters initially buried in the untreated proteins.
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942 S.-W. Yin et al. / Food Chemistry 110 (2008) 938–945

400 MPa. These results are consistent with the data of maximum
A 350
Relative influorecence intensity (A.U)

emission fluorescence intensity (Fig. 3B), indicating gradual expo-

300 0.1MPa sure of hydrophobic groups initially buried in the interior of the
200MPa molecules. Molina et al. (2001) used a different method based on
250 400MPa SDS binding and Puppo et al. (2004) used the same method as in
600MPa
the present to determine H0, and also observed a pressure-induced
200
increase in H0 of SPI and/ or its 7S and 11S fractions in the range of
150 200–600 MPa (though Puppo et al. performed the study at pHs 3
and 8).
100
3.3. SH and SS contents
50
Fig. 5 shows free SH (including total SH and exposed SH) and SS
0
300 320 340 360 380 400 contents of untreated and HP-treated KPI samples. With pressure
Wavelength (nm) increasing from 0.1 to 600 MPa, total free SH content of SPI signif-
icantly and gradually decreased with a concomitant increase in SS
content (Fig. 5). The data clearly indicate that HP treatment re-
B
Relative Fluorescence intensity (A.U)

500
sulted in formation of SS bonds among protein components, in a
pressure level dependent manner. Similar results of exposed SH
0.1 MPa
400 content change have been observed in egg white proteins (Van
200MPa
der Plancken, Van Loey, & Hendrickx, 2005), and soy protein (Pup-
400MPa
300 600MPa po et al., 2004). It is reasonably supposed that the formation of new
SS bonds is involved in the formation of soluble aggregate by HP
treatment.
200 The decreases in total SH content clearly corresponded with the
increases observed in SS content (Fig. 5). This observation suggests
100 that the total SH and SS content changes are primarily due to oxy-
gen-catalyzed oxidation of the free SH groups. In the present study,
although the protein solutions were packaged in a polyethylene
0
400 450 500 550 600
Wavelength (nm)
A Total SH Exposed SH
SH content (μmol/g protein)

Fig. 3. Conformational changes in KPI monitored by intrinsic and ANS fluorescence 12 a

emission at various pressure levels. Panel A: Intrinsic fluorescence spectra of unt- ab
b
reated and HP-treated (200–600 MPa) KPI samples, in 10 mM phosphate buffer (pH
7.0). Panel B: Fluorescence emission spectra of ANS upon binding to untreated and
c
10
HP-treated KPI samples (0.15 mg/mL). a
a
b
b
8
Fig. 4 shows surface hydrophobicity (H0) values of untreated
and HP-treated KPIs, as determined from ANS emission fluores-
cence spectra. HP treatment at pressures of 200 and 400 MPa sig- 6
nificantly increased the H0, however, there was no significant
difference of H0 between 200 and 400 MPa. The HP treatment at
600 MPa further significantly increased H0, relative to that at 4
0 200 400 600
Pressure levels (MPa)
2500
a B
Surface hydrophobicity (H0)

b b
5 a
SS content (μmol/g protein)

2000
bc b
c
c
1500 4

1000
3
500

0 2
0 200 400 600 0 200 400 600
Pressure levels (MPa) Pressure levels (MPa)

Fig. 4. Surface hydrophobicity (H0) of untreated (0.1 MPa) and HP-treated (200– Fig. 5. SH (A) and SS (B) contents of untreated (0.1 MPa) and HP-treated (200–
600 MPa) KPI samples in 10 mM phosphate buffer (pH 7.0). Different characters 600 MPa) KPI samples. Different characters (a–c) on the columns represent signif-
(a–d) on the top of columns indicate significant (P < 0.05) difference between dif- icant difference at P < 0.05 level among various pressure levels. Within panel A, two
ferent pressure levels. kinds of SH are included: total and exposed.
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S.-W. Yin et al. / Food Chemistry 110 (2008) 938–945 943

bag under vacuum condition, prior to HP treatment, residual oxy- 40 EAI ESI 400
gen was still present. Cheftel, Hayashi, Heremans, and Masson
35 a a 350
(1992) had pointed out that, free sulfhydryl groups of protein could
b
be oxidized under pressure above 300 MPa, when oxygen was 30 300
present. On the other hand, only HP treatment at pressures of

ESI (min)
EAI (m /g)
400 and 600 MPa led to a significant (P < 0.05) increase in exposed 25 250
c

2
free SH content, relative to the control (untreated) (Fig. 5A). This e
20 200
was a result of HP-induced protein unfolding and denaturation at
high-pressure levels. 15 f 150

10 100
3.4. Functional properties g g
5 50
3.4.1. Protein solubility 0 0
As shown in Fig. 6, protein solubility (PS) of untreated KPI was 0 200 400 600
about 77%, which is higher than that of amorphous protein isolates Pressure levels (MPa)
from white kidney bean (about 65.7%), and lower than that of the
crystalline (85–90%) (Di Lollo et al., 1993). The PS was significantly Fig. 7. Emulsifying activity index and emulsion stability index of untreated (0.1 -
MPa) and HP-treated (200–600 MPa) KPI samples in de-ionized water (pH 7.0).
improved by HP treatment at a pressure of 400 MPa or higher
Different characters (a–g) on the top of columns represent significant difference
(P < 0.05). This result was consistent with SEC analysis using RI (P < 0.05) in EAI or ESI among different pressure levels.
detection as shown in Figs. 1 and 3, indicating that the PS improve-
ment at pressures above 200 MPa was due to the transformation of
insoluble aggregates to soluble ones with lower molecular weight. 3.4.3. DSC characteristics
The formation of stable soluble aggregate seems to be closely re- The thermal properties of untreated and HP-treated SPI samples
lated to the newly formed SS bonds after HP treatment at higher in 50 mM phosphate buffer (pH 7.0) were evaluated by DSC, and
pressure levels (above 200 MPa). A contrary result has been ob- related DSC characteristics are summarized in Table 1. In the DSC
served in lupin proteins, wherein PS decreased after HP treatment profiles of all tested KPI samples, there was a prominent endother-
at a pressure above 200 MPa (Chapleau & De Lamballerie-Anton, mic peak, clearly attributed to thermal denaturation of vicilin com-
2003). The difference may be attributed to differences of protein ponent (7S) (data not shown). The on-set temperature of
type (11S or 7S globulins and/or albumin), nature and conforma- denaturation (To) and thermal denaturation temperature (Td) of
tional stability between these two proteins. the vicilin component were unaffected by HP treatment at 200–
600 MPa (Table 1), suggesting that the structure of this protein
3.4.2. Emulsifying properties component was very compact and not easily susceptible to HP
Fig. 7 shows emulsifying activity index (EAI) and emulsion sta- treatment. Jivotovskaya et al. (1996) also pointed out that the vic-
bility index (ESI) values of untreated and HP-treated KPIs at pH 7.0. ilin (also called phaseolin) in KPI has unique structural peculiarities
HP treatment at 200 and 400 MPa resulted in a similar and signif- relative to other vicilins, since it was least susceptible to protease
icant increase in EAI, while a significant decrease was observed at digestion.
600 MPa, relative to untreated KPI. As for EAI, highest ESI was also However, HP treatment led to a gradual decrease in enthalpy
obtained at 400 MPa. With pressure level increasing from 0.1 to change (DH) of the endothermic peak of vicilin (Table 1), indicating
400 MPa, ESI gradually and significantly increased, while there HP-induced unfolding of undenatured protein in KPI (Arntfield &
was no significant difference of ESI between 600 MPa and the con- Murray, 1981). On the other hand, the width at half peak height
trol (Fig. 7). The results suggest that moderate protein unfolding by of the endotherm, related to cooperativity of transition from native
HP treatment could improve emulsifying activities of KPI, and se- to denatured state (Privalov, 1982), also slightly decreased with in-
vere HP treatment on the contrary impaired its emulsifying activ- crease in pressure (Table 1). This situation is very different from
ities. In the case of HP-treated soy 7S globulin, highest EAI was also the influence of HP on other 7S globulins. For example, Molina
obtained at 400 MPa, and the increase in EAI was also attributed to et al. (2001) indicated that HP treatment at a pressure of
protein unfolding of its subunits (Molina et al., 2001). 400 MPa and higher led to remarkable decrease in DH, and the Td
was also significantly decreased by HP treatment at 600 MPa. In
this work, the HP influence was attributed to the dissociation of
100
7S fraction (b-conglycinin) of SPI into partially or completely dena-
tured monomers. The differences of structural characteristic and
90
Protein solubility (%)

a a
b b
80
Table 1
DSC characteristics of untreated (0.1 MPa) and HP (200–600 MPa)-treated KPI
70 samples

To (°C) Td (°C) DH (J/g) DT1/2 (°C)

60
Control 88.3 ± 0.08a 93.9 ± 0.16a 16.2 ± 0.50a 5.47 ± 0.09a
200 MPa 88.1 ± 0.40a 93.8 ± 0.27a 13.8 ± 0.10b 5.37 ± 0.08ab
50 400 MPa 88.7 ± 0.26a 93.9 ± 0.22a 12.4 ± 0.18c 5.32 ± 0.05bc
600 MPa 88.5 ± 0.05a 93.7 ± 0.06a 11.2 ± 0.84c 5.19 ± 0.08c
40
The scan rate was 5 °C per min. The KPI samples were dispersed at about 20% (w/v)
0 200 400 600
in 50 mM phosphate buffer (pH 7.0).
Pressure levels (MPa) Values were expressed as mean and standard deviations of triplicate measure-
ments. To, on-set temperature of denaturation; Td, thermal denaturation tempera-
Fig. 6. Protein solubility of untreated (0.1 MPa) and HP-treated (200–600 MPa) KPI ture; DH, enthalpy changes of the endotherm and DT1/2, width at half peak height of
samples in de-ionized water (pH 7.0). Different characters (a–b) on the top of col- endothermic peak. Superscript letters (a–c) indicate significant (P < 0.05) difference
umns indicate significant difference (P < 0.05) among different pressure levels. within the same column.
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944 S.-W. Yin et al. / Food Chemistry 110 (2008) 938–945

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