A comparative study of physicochemical characteristics and functionalities of pinto bean protein isolate (PBPI) against the soybean protein isolate (SPI) after the extraction optimisation

Food Chemistry 152 (2014) 447–455

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

A comparative study of physicochemical characteristics and functionalities of pinto bean protein isolate (PBPI) against the soybean protein isolate (SPI) after the extraction optimisation Ee-San Tan a,b, Ying-Yuan Ngoh b, Chee-Yuen Gan b,⇑ a b

Bioprocess Division, School of Industrial Technology, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia Centre for Advanced Analytical Toxicology Services, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia

a r t i c l e

i n f o

Article history: Received 24 June 2013 Received in revised form 25 November 2013 Accepted 3 December 2013 Available online 10 December 2013 Keywords: Functionalities Optimisation Physicochemical Pinto bean Protein isolate

a b s t r a c t Optimisation of protein extraction yield from pinto bean was investigated using response surface methodology. The maximum protein yield of 54.8 mg/g was obtained with the optimal conditions of: temperature = 25 °C, time = 1 h and buffer-to-sample ratio = 20 ml/g. PBPI was found to obtain high amount of essential amino acids such as leucine, lysine, and phenylalanine compared to SPI. The predominant proteins of PBPI were vicilin and phytohemagglutinins whereas the predominant proteins of SPI were glycinin and conglycinins. Significantly higher emulsifying capacity was found in PBPI (84.8%) compared to SPI (61.9%). Different isoelectric points were found in both PBPI (4.0–5.5) and SPI (4.0–5.0). Also, it was found that PBPI obtained a much higher denaturation temperature of 110.2 °C compared to SPI (92.5 °C). Other properties such as structural information, gelling capacity, water- and oil-holding capacities, emulsion stability as well as digestibility were also reported. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Pinto bean (Phaseolus vulgaris cv. Pinto) is an underutilised legume containing high amount of protein as well as numerous other phyto nutrients. It has been a stable source of traditional foods in several countries given its high nutritional value. The word ‘‘pinto’’ in Spanish means ‘‘painted’’, referring to its beige background strewn with reddish brown speckles of colour. These beans are often consumed in United States and northwestern Mexico. The top five producers of pinto bean are the United States, China, Brazil, India and Indonesia. Seyam, Banasik, and Breen (1983) had studied the use of this protein source in producing macaroni. Result showed that it improved the cooking quality of the macaroni and increased its lysine content. Also, Ye and Ng (2001) reported that the bean peptides exhibit antifungal activity towards a few fungal species, mitogenic activity toward mouse splenocytes and HIV-1 reverse transcriptase-inhibitory activities. It was also reported that phenolic acids and flavonols extracted from pinto beans, possess significant antioxidant activity (Xu & Chang, 2009), however there is no study on the protein isolation from pinto bean. In this study, an extraction optimisation of this new protein isolate (i.e. pinto bean protein isolate) will be conducted followed by the comparison of the physicochemical characteristics, and

⇑ Corresponding author. Tel.: +604 6534261; fax: +604 6534688. E-mail address: [email protected] (C.-Y. Gan). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.12.008

functionalities of the extracted protein isolate against the commercially available soybean protein isolate. Due to the presence of genetic modified soybean, public is concerned about the health issue and its long-term side effects. The reason to compare between PBPI and SPI was to find the possibility of PBPI to be an alternative to SPI. For a successful utilisation of plant proteins in food application or research, it is important to investigate the intrinsic physicochemical characteristics and functionalities such as water and oil holding capacities, protein solubility, gelling properties and foaming properties. These properties manipulate the behaviour of proteins and therefore it is crucial for future protein research.

2. Material and methods 2.1. Materials Pinto beans (15 kg) were purchased from three different local markets (i.e. Lip Sin Market, Jelutong Market and Bayan Baru Market) located in Penang, Malaysia. These three different sources of pinto bean were used as replicates for the experiment. The beans were separated from the pods and rinsed with distilled water and lyophilised. The dried beans were subsequently milled into powder form, sieved (60 mesh) and stored at 4 °C prior to extraction. All chemicals and reagents used in the experiment were of analytical grade purchased from Sigma–Aldrich (Malaysia) company or otherwise mentioned.

448

E.-S. Tan et al. / Food Chemistry 152 (2014) 447–455

2.2. Extraction of protein Extraction of pinto bean protein isolate (PBPI) was carried out according to Adebowale, Adeyemi, Oshodi, and Niranjan (2007) with slight modification. Briefly, pinto bean flour was added to phosphate buffer solution (pH 8 ± 0.1) at different buffer-to-sample ratio. Subsequently, the sample was incubated at designated temperatures and extraction periods (Table 1) with constant agitation at 250 rpm. The resulting slurry was then centrifuged at 4000g for 30 min after incubation. The supernatant was collected and the extracted protein content of the sample was determined using Bradford assay. The extraction yield was expressed as total extracted protein per gram of pinto bean flour. 2.3. Experiment design The extraction parameters (i.e. incubator temperature (X1), extraction time (X2) and buffer-to-sample ratio (X3)) were optimised using RSM. A Box–Behnken design (BBD) was employed in this regard. The range and centre point values of these three independent variables displayed in Table 1 were based on the results of preliminary experiments. The experimental design consists of twelve factorial points and five replicates of the central point (Table 1). Extraction yield (mg extracted protein/g sample) was used as the response for the combination of the independent variables given in Table 1. Three experimental replicates of each condition were performed and the mean values were stated as experimental responses. Experimental runs were randomised to minimise the effects of unexpected variability in the observed responses.The variables were coded according to the equation:

x ¼ ðX i  X o Þ=DX

ð1Þ

where x is the coded value, Xi was the corresponding actual value, Xo was the actual value in the centre of the domain, and DX is the increment of Xi corresponding to a variation of 1 unit of x.The mathematical model corresponding to the design is:

Y ¼ bo þ

3 3 2 X 3 X X X bi X i þ bii X 2i þ bij X i X j i¼1

i¼1

ð2Þ

i¼1 j¼1þ1

Table 1 Experimental domain of Box–Behnken Design (BBD) with the observed responses and predicted values for yield of protein (mg/g). Xj

Uncoded

Temperature (°C) Time (h) Buffer to solid ratio (ml/g)

X1 X2 X3

Coded

x1 x2 x3

Std

Run

X1

X2

X3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

15 12 17 16 5 11 1 4 10 9 7 6 13 3 8 14 2

25 50 25 50 25 50 25 50 37.5 37.5 37.5 37.5 37.5 37.5 37.5 37.5 37.5

1 1 3 3 2 2 2 2 1 3 1 3 2 2 2 2 2

35 35 35 35 20 20 50 50 20 20 50 50 35 35 35 35 35

Factor levels 1

0

1

25 1 20

37.5 2 35

50 3 50

Yield (experimental) 54.8 47.0 52.4 33.8 57.9 34.8 56.0 37.4 42.7 37.2 42.2 38.6 46.7 40.3 43.7 40.3 43.7

Yield (predicted) 55.9 44.3 55.2 32.7 55.9 36.7 54.1 39.3 43.5 36.4 43.0 37.7 42.9 42.9 42.9 42.9 42.9

where Y was the dependent variables (i.e. extraction yield), bo was the model constant, and bi, bii and bij were the model coefficients. They represent the linear, quadratic and interaction effects of the variables. Analysis of the experimental design data and calculation of predicted responses were carried out using Design Expert software (version 6.0, USA). Additional confirmation experiments were subsequently conducted to verify the validity of the statistical experimental design. 2.4. Characterisation of pinto bean protein isolate (PBPI) PBPI was prepared according to Section 2.2 at the optimised condition and the protein was precipitated by adjusting the pH to 4.5. Subsequently, the precipitate was collected after centrifugation at 4000g for 15 min, lyophilised and ground into powder prior to analyses. Commercial soy protein isolate (SPI) was used as protein reference in all the analyses. 2.4.1. Amino acid composition determination PBPI (0.1 g) was hydrolysed with 6 N HCl (5 ml) at 110 °C for 24 h in a sealed tube under a vacuum condition. Subsequently, a 400 ll of 50 lmole/ml of L-a-amino-n-butyric acid (AABA) (used as internal standard) was add in the resulting hydrolysates and made up to 100 ml with distilled deionised water. Before conducted the amino acids analysis, the samples were filtered through Whatman No 1 filter paper followed by passing through a 0.22 lm Milipore filters. Following, derivation of the PBPI hydrolysate amino acids was performed by incubating 10 ll of the hydrolysed samples with 20 ll of AccQFluor™ reagent (AQC: 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) for 1 min at room temperature. Samples were then placed on the deactivated 150 ll glass insert with poly spring which was equipped in the screw-capped vial and the vial was heated for 10 min at 55 °C before subjected to analysis. Injection of 10 ll of the sample into the column was carried out and elution was conducted with a flow rate of 1 ml/min. Determination of amino acid was carried out according to the AccQTag™ method using a Waters-HPLC-System (U.S.A) with Waters 1525 Binary Pump, Waters 717 plus Autosampler, Waters 2475 Mutli k Fluorescence Detector and a Waters AccQTag™ Amino Acid Analysis Column (3.9 mm  150 mm; packing material: silica based bonded with C18). The column was fixed at temperature of 37 °C with fluorescence detection of 250 nm for excitation and 395 nm for emission. AccQTag™ Eluent A and Acetonitrile/ Water (60%/40%) was used as eluents and calibration of the HPLC-system was performed using the amino acids standard H (PIERCE, U.S.) as reference. Methionine and cysteine were analysed separately by using performic acid procedure. Breeze Workstation version 3.20 was used data analysis. The amino acid composition of the PBPI sample was presented as per 1000 residue and the analysis was carried out in three replicates. 2.4.2. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) Isolated protein in PBPI was identified using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Resolving gel of 15% and stacking gel of 4% were used in the experiment. The sample was treated under two different conditions (i.e. reducing and non-reducing conditions). In reducing condition, the sample was added with 20 ll of Laemmli buffer and 1 ll of b-mercaptoethanol. On the other hand, the sample was only added with 20 ll of Laemmli buffer in non-reducing condition. Subsequently, the mixture was heated at 95 °C for 5 min prior loading. The electrophoresis experiments were ran at 80 V for 10 mins and subsequently ran at 120 V until the dye reached the bottom of the gel. The gel was stained with 0.1% Commassie-Blue G-250

E.-S. Tan et al. / Food Chemistry 152 (2014) 447–455

containing 40% of methanol and 10% of acetic acid solution for 2 h. Subsequently, the get was destained in a solution containing 40% of methanol and 10% of acetic acid for 2 h. The image of the gel was captured using Fujifilm Luminescent Image Analyser LAS-3000 version 1.1 and the protein bands were then analysed using Fujifilm MultiGauge V3.0. Prestained SDS-PAGE standard (Broad Range, Biorad Catalogue 161-0318) was used as protein standard marker. 2.4.3. Fourier transform infrared (FTIR) spectroscopy PBPI lyophilized powder was dessicated prior to FTIR analysis. FTIR spectra of the samples were recorded using Cary 600 Series FTIR spectrometer with attenuated total reflectance (ATR) system (Agilent Technologies, U.S.) from 4000 to 650 cm1. The measuring resolution was 4 cm1 and iterations were performed for 32 times. The scan speed was set at 5 kHz and sensitivity of 1. The resultant spectra were analysed using Resolution Pro version 5.2.0 software. 2.4.4. Thermal property determination using differential scanning calorimetry (DSC) Differential scanning calorimetry experiment was performed as described by Sousa, Mitchell, Ledward, Hill, and Beirão da Costa (1995). PBPI sample was equilibrated at 5 ± 1 °C for 16 h and heated from 27 °C to 157 °C at a rate of 5 °C/min. Thermal stability is expressed as denaturation peak temperature (Td, °C). At least three runs average of the calorimetric results for PBPI was determined. 2.4.5. Protein solubility The 0.2% (w/v) suspensions of the PBPI were dissolved in 0.1 M NaOH solution. The pH of the suspension was then adjusted to the desired pH values (pH 2–11) using 0.1 M HCl or 0.1 M NaOH. The precipitate was separated via centrifugation at 4500g for 15 min. The resulting precipitates were then resolubilised with 10 ml of 0.1 M NaOH. The soluble protein was then measured using Bradford assay. The percentage of protein solubility was calculated as follows:

%Solubility ¼ 100 



P  100% T



449

For the emulsion stability analysis, the sample was prepared as mentioned above followed by incubation at 80 °C for 30 min. Sample was then centrifuged at 1200g for 5 min and the emulsion layer was measured. The emulsion stability was determined as:

 %ES ¼ 100 

 R  100 Ri

ð5Þ

where R was the emulsified layer volume that heated at 80 °C for 30 min and Ri was the initial emulsified layer volume. 2.4.8. Gelling capacity Sample suspensions of 6–16% (w/v) were prepared in phosphate buffer with the pH 7. Each of the prepared dispersions (6 ml) was heated to 95 °C in a water bath for 1 h and cooled at 4 °C. Gel formed was visually observed and least gelling concentration (LGC) was determined. 2.4.9. In-vitro protein enzyme digestibility PBPI was subjected to enzymatic digestion as described by Hsu, Vavak, Satterlee, and Miller (1977). Fifty millilitres of aqueous PBPI suspension (6.25 mg sample/ml) were prepared by dissolving the PBPI with distilled water and the suspensions were adjusted to pH 8 with 0.1 N HCl or 0.1 N NaOH at 37 °C in a water bath. A multi-enzyme mixture containing 1.6 mg trypsin, 3.1 mg chymotrypsin and 1.3 mg peptidase was prepared in an ice bath and adjusted to pH 8 with 0.1 N HCl or 0.1 N NaOH. Subsequently, 5 ml of the multi-enzyme solution was added to the PBPI suspension with constant stirring at 37 °C. The pH value was recorded every 1 min interval for total digestion period of 10 min. Percentage of in vitro digestibility was calculated by using the following equation:

%Digestibility ¼ 210:46  18:10X

ð6Þ

where X is expressed as the pH of the sample after 10 min in-vitro digestion with multi-enzyme. 2.5. Statistical analysis

ð3Þ

where P was defined as the protein content of the precipitate at different pH levels whereas T was defined as the total protein content of the sample.

Experimental data were statistically evaluated using Tukey’s test and reported as mean values with standard deviation. Mean values were considered significantly different at p < 0.05. 3. Results and discussion

2.4.6. Water- and oil-holding capacity The PBPI (0.1 g) was mixed with 1.5 ml of distilled water or soybean oil (density of 0.912 g/ml) in a pre-weighed microcentrifuge tube and vortexed for 1 min. Sample was incubated at room temperature for 30 min. The sample was then centrifuged at 5000g for 30 min. The resulting supernatant was carefully decanted and sample was weighed. The WHC and OHC were expressed as gram of water or oil bound per gram sample. 2.4.7. Emulsifying activity and stability (EA & ES) Briefly, a 10 ml of 1% (w/v) and 5% (w/v) PBPI suspensions were prepared in phosphate buffer (pH 7 ± 0.05) prior to homogenisation using Omni TH Tissue homogenizer (Omni International, United States) for 2 min. The slurries was then added with 10 ml of palm oil and homogenised for 2 min. The emulsions were centrifuged at 1200g for 5 min. The emulsion layer was then measured. The percentage of emulsifying activity was determined as:

 %EA ¼

VE VT



 100

ð4Þ

where VE was the emulsified layer volume and VT was the total volume.

The effects of three process variables (i.e. temperature (X1), time (X2) and buffer-to-sample ratio (X3)) were studied during experimentation. The response of interest was extraction yield. The results of 17 runs using BBD design are presented in Table 1 that includes the design, experimental responses and the predicted values. Results showed that there was a close agreement between experimental and predicted values. The extraction yield seemed to be varied depending on the conditions given. It could be observed that the yield ranged from 33.8 to 57.9 mg/g. The maximum yield (57.9 mg/g) was found under the experimental conditions of X1 = 25 °C, X2 = 2 h and X3 = 20 ml/g (Table 1). 3.1. Model fitting Table 2 presents the results of fitting quadratic models to the data. The results of analysis of variance (ANOVA) indicate that the contribution of quadratic model was significant. The fitted quadratic model for extraction yield in coded variables is given in Eq. (6). The significance of each coefficient was determined using the F-test and p-value. The corresponding variables would be more significant if the absolute F-value becomes greater and the p-value

450

E.-S. Tan et al. / Food Chemistry 152 (2014) 447–455

Table 2 ANOVA for quadratic model: estimated regression model of relationship between response variable (yield) and independent variables (X1, X2, X3). Source

Sum of squares

Model X1 X2 X3

817.21 579.22 76.39 0.30 114.71

9 1 1 1 1

X 21 X 22 X 23 X1X2 X1X3 X2X3 Residual Lack of Fit Pure error Cor total R-Squared Adj R-squared C.V.

DF

Mean square

F Value

Prob > F

90.80 579.22 76.39 0.30 114.71

9.86 62.88 8.29 0.03 12.45

0.0032 <0.0001 0.0237 0.8616 0.0096

5.36

1

5.36

0.58

0.4706

11.10

1

11.10

1.20

0.3087

29.56 4.86 0.84 64.48 34.58 29.90 881.69

1 1 1 7 3 4 16

29.56 4.86 0.84 9.21 11.53 7.47

3.21 0.53 0.09

0.1164 0.4910 0.7710

1.54

0.3341

0.9269 0.8328 6.88

becomes smaller. It can be seen that the variable with the largest effect on extraction yield was linear term of extraction temperature (X1) followed by quadratic term of extraction temperature X 21 and linear term of extraction time (X2). However, the linear term of buffer-to-sample ratio (X3), quadratic terms of extraction time and buffer-to-sample ratio (X 22 and X 23 ) as well as all the interaction terms (X1X2, X1X3 and X2X3) were found insignificant (p > 0.05). These results shown in Table 2 suggested that the change of extraction temperature and time had significant effects (p < 0.05) on the extraction yield of protein however there is no significant (p > 0.05) effect in buffer-to-sample ratio.

Yield ¼ 42:93014  8:50896x1  3:09003x2 þ 0:194096x3 þ 5:219453x21  1:12797x22  1:6235x23  2:71838x1 x2 þ 1:102749x1 x3 þ 0:459202x2 x3

ð7Þ

The lack of fit test measured the failure of the model to represent the data in the experimental domain at points which were not included in the regression. The result showed that the lack of fit p value of 0.3341 (p > 0.05) indicating the experimental data fitted well to the model and adequate for predicting the extraction yield. The value of the determination coefficient R2 was 0.9269 while the value of adjusted determination coefficient R2adj was 0.8328, indicating a high degree of correlation between the experimental and predicted values. Coefficient of variation (CV) was a standard deviation expressed as a percentage of the mean. The lower the CV, the smaller residuals relative to the predicted value was. A low CV of 6.88% suggesting a good precision and high reliability of the experiment performed. 3.2. Interpretation of response surface model Three-dimensional (3-D) plots for extraction yield as a function of buffer-to-sample ratio and extraction time at different temperature conditions are given in Fig. 1a–c. It can be seen that increasing the extraction temperature would decrease the extraction yield. At 25 °C (Fig 1a), the extraction yield was ranging from 52.9 to 56.8 mg/g whereas the response decreased to 36.4–44.9 mg/g and 29.3–44.4 mg/g at higher extraction temperature of 37.5 and 50 °C, respectively. This occurrence might due to the presence of higher phytate content in the resulting slurries at these temperatures (i.e. 37.5 and 50 °C). The interaction between phytate and proteins caused the decreasing in protein solubility and consequently led to lower protein extraction yield. Phytate or phytic

acids are natural constituents found in leguminous seeds, cereal grains and oil seeds which act as storage form of phosphorus. Deak and Johnson (2007) reported that very low phytate content was obtained when protein extraction was performed at room temperature compared to higher extraction temperature. Therefore, higher extraction yield was obtained in lower extraction temperature of 25 °C. Also, it could be observed that the extraction yield decreased with time at extraction temperature of 37.5 and 50 °C. Whereas, the yield increased when the extraction period extended up to 2 h at temperature of 25 °C. Further extraction to 3 h had a slight decrease in the yield. Eromosele, Arogundade, Eromosele, and Ademuyiwa (2008) reported that prolonged extraction time was not conducive to increase protein extraction yield. This might be due to the formation of protein–phytic acid complexes which make the protein less soluble. In addition, pH value of the solution above the protein isoelectric point(pH 4.5–5 to 9) would also lead to formation of ternary complexes via interaction between phytic acid, divalent cations (Ca2+, Mg2+) and proteins (Ali, Ippersiel, Lamarche, & Mondor, 2010). Therefore, it is suggested that extraction at room temperature with shorter extraction period (<2 h) would produce higher yield of protein. No significant changes in yield with the change of buffer-tosample ratio at higher temperature (37.5 and 50 °C) whereas a slight quadratic effect was found at temperature of 25 °C. Hence, it is suggested that buffer-to-sample ratio of 20 ml/g is sufficient for PBPI extraction reduce wastage of buffer solution and reduce the cost of production. 3.3. Verification of predictive models Based on the above findings, an optimisation study was performed to evaluate the optimal operating conditions for the extraction with high extraction yield of protein. One combination was found to maximise these response taking into consideration of the efficiency, the energy conservation and the feasibility of the experiment. The optimal conditions for desired extraction yield corresponded to extraction temperature of 25 °C, extraction time of 1 h, and buffer-to-sample ratio of 20 ml/g. These optimum conditions yielded protein of 54.8 mg/g. Only small deviations were found between the experimental value and predicted value (55.7 mg/g) with desirability of 0.976. Thus, the model can be used to optimise the process of protein extraction from pinto bean. 3.4. Amino acid content of PBPI The amino acid composition of PBPI is shown in Table 3. PBPI has a low content of sulphur-containing amino acids such as methionine and cysteine, which could be due to their destruction during the preparation of protein isolates using aqueous-alkaline extraction approach (Adebowale et al., 2007). It was also reported that methionine and cysteine were the limiting amino acids present in legumes (Wu et al., 1996). On the other hand, PBPI is rich in aspartic acid and glutamic acid which would suggest that this protein isolate possesses acidic characteristic. The lysine content for PBPI (78 per 1000 residues) was found higher than that of SPI (60 per 1000 residues). It is important to note that lysine is nutritionally important as cases of growth retardation in children increase as a consequence of deficiency in dietary lysine. In comparison with the reported SPI, the ratio of essential to total amino acid (E/TN) in PBPI was higher, which had met the minimum E/TN ratio (0.36) suggested by FAO/WHO/UNU. It could also be observed that PBPI could fulfill the requirements of amino acid recommended by FAO/WHO/UNU. Hence, the result suggested that PBPI could be used as an alternative source for cereal-based foods which are low in lysine. The amino acid characteristics profile (e.g.,

451

E.-S. Tan et al. / Food Chemistry 152 (2014) 447–455

(i)

(ii) lot

yield

50.00

53.6

54.2

53.6

54.9

5 6 .8

(a)

42.50

55.5

5 5 .9

0

C: ratio

5 4 .9

yield

5 3 .9

5 2 .9

35.00

56.2

27.50

5 0 .0 0 3 .0 0 4 2 .5 0

54.9 2 .5 0 3 5 .0 0 2 .0 0

C: ratio

2 7 .5 0

20.00 1.00

1 .5 0

1.50

2.00

2.50

3.00

B: time 2 0 .0 0

1 .0 0

B: time 50.00

4 4 .9

(b)

42.50

4 2 .8

4 0 .7

C: ratio

yield

3 8 .6

3 6 .4

42.7 43.9

35.00

41.5

5

40.3

27.50

5 0 .0 0 3 .0 0 4 2 .5 0 2 .5 0 3 5 .0 0 2 .0 0 20.00

C: ratio

2 7 .5 0

1 .5 0

1.00

B: time 2 0 .0 0

1.50

2.00

2.50

3.00

1 .0 0

B: time 50.00

4 4 .4

(c)

42.50

4 0 .7

3 6 .9

C: ratio

yield

3 3 .1

2 9 .3

42.7 43.9

35.00

5

41.5

40.3

27.50

5 0 .0 0 3 .0 0 4 2 .5 0 2 .5 0 3 5 .0 0 2 .0 0

C: ratio

20.00

2 7 .5 0

1 .5 0

B: time 2 0 .0 0

1.00

1.50

2.00

2.50

3.00

1 .0 0

B: time Fig. 1. Response surfaces: (i) three-dimensional plot and (ii) contour plot for extraction yield as a function of buffer-to-sample ratio and extraction time at (a) 25 °C, (b) 37.5 °C and (c) 50 °C.

452

E.-S. Tan et al. / Food Chemistry 152 (2014) 447–455

Table 3 Amino acid composition of PBPI and SPI (per 1000 residue) as well as FAO recommended amino acid requirement for adult.

a b c d e

Amino acid

PBPI

SPI

FAO/WHO/UNU requiremente (mg/g protein)

Nonessential amino acid Asp Ser Glu Gly Arg Ala Pro Tyr Cys

111 63 153 41 66 47 48 53 0

117 55 207 40 80 40 52 37 10

– – – – – – – – –

Essential amino acid Lys Ile Leu Phe Met Thr Val His

78 48 89 60 7 46 58 20

60 49 77 54 9 37 46 24

18 15 21 (Phe + Tyr) 21 (Met + Cys) 20 11 15 15

Percentage of amino acid with different characteristics Acidica Basicb Hydrophobicc Uncharged polard

264 164 357 203

324 164 331 179

E/TN ratio Total essential amino acids Total nonessential amino acids

0.41 406 582

0.36 356 638

Acidic: aspartic acid, glutamic acid. Basic: lysine, arginine, histidine. Hydrophobic: alanine, isoleucine, leucine, methionine, phenylalanine, proline, valine. Uncharged polar: glycine, serine, threonine, tyrosine, cysteine. Adapted from WHO (1985).

acidic, basic, hydrophobic and uncharged polar) of PBPI was also summarised in Table 3. PBPI was found to be significantly higher (P < 0.05) in hydrophobic and uncharged polar amino acid residues than the SPI. Therefore, it could be expected that the physicochemical/functional properties of PBPI would be different from SPI. 3.5. SDS–PAGE profiles Fig. 2a shows the SDS–PAGE profile of PBPI and SPI under both non-reducing (lane 1 and 2, respectively) and reducing (lane 3 and 4, respectively) conditions. The profiles of PBPI under both nonreducing and reducing conditions were similar. Proteins with their subunits found in these profiles were 7S vicilin (41 kDa) and phytohemagglutinins (30, 29, 27, 26, 23 and 16 kDa). The former is a trimer devoid of disulphide bonds which has been reported as a major fraction protein that mainly contained in the Phaseolus bean (Rui, Boye, Ribereau, Simpson, & Prasher, 2011). The latter, which is also known as lectins, posses sugar binding properties and hemagglutinating ability (Nasi, Picariello, & Ferranti, 2009). This component has shown to be toxic if it is injected into animals in high doses. Also, it was reported that it could induce growth inhibition when incorporated into diet. However, it can be reduced to safe levels by proper cooking (Liener, 1974). In addition, some minor bands with high molecular weight (55, 76 and 98 kDa) were also observed under both conditions. Disappearance of bands with molecular weight of 195 and 9 kDa were observed under reducing condition, which might be attributed to the presence of 11S legumin subunit (Rui et al., 2011). For SPI, the presence of a’, a and b subunits of b-conglycinin (84, 76 and 46 kDa) were found in both reducing and non-reducing conditions. Moreover, four intense bands at around 33, 29, 26

and 14 kDa were also observed in SPI and might belong to the acidic and basic polypeptides which were dissociated from glycinin. It was also revealed that the SDS–PAGE profiles for SPI under non-reducing (lane 2) and reducing (lane 4) conditions were different as shown in Fig. 2a. Bands with molecular mass above 121 kDa were not found in the lane 4 whereas three additional bands (37, 21 and 18 kDa) were observed in this profile. Therefore, these new bands might represent the subunits of acidic and basic polypeptides of glycinin which are linked by disulphide bonds. 3.6. FTIR spectra The IR spectra of PBPI and SPI are shown in Fig. 2b. A broad peak was found in a region of 3500–3200 cm1 (amide A) indicating the –NH stretching which corresponding to the flexural vibration frequencies of the intra- and inter-molecular hydrogen bonds. The peak at 3283 cm1 is reported to be similar with a helical formation that formed by the hydrogen bond between functional groups of C@O and N–H (Gupta et al., 2006). A minor peak of 3075 cm1 was also found in the spectra. According to literature, this peak is due to the @CAH stretching vibration mode of the unsaturated hydrocarbons (HC@CH groups) or aromatic C–H vibrations of lipid molecules (Zeier & Schreiber 1999). It is therefore suggesting a trace amount of the lipid could be presence in the protein isolate. The bands appears from 2990 to 2850 cm1 represented the CH antisymmetric and symmetric stretching modes of methyl (CH3) and methylene (CH2) that normally found in aliphatic side chain of proteins (Chen et al., 2013). Therefore, the peaks (2959, 2927 and 2867 cm1) found in the spectra were corresponding to the stretching aforementioned.

453

E.-S. Tan et al. / Food Chemistry 152 (2014) 447–455

(a)

(b)

(i)

(ii)

(c) % solubility

PBPI

SPI

100.0 80.0 60.0 40.0 20.0 0.0 1

2

3

4

5

6

7

8

9

10

11

12

pH

Fig. 2. (a) SDS–PAGE profile of PBPI and SPI at non-reducing and reducing conditions. Lane M: standard protein marker; lane 1: non-reduced PBPI; lane 2: non-reduced SPI; lane 3: reduced PBPI and lane 4: reduced SPI. AS: acidic subunit; BS: basic subunits; (b) IR spectra of (i) PBPI and (ii) SPI; (c) Protein solubility profile of pinto bean protein isolate (PBPI) and soybean protein isolate (SPI) as a function of pH. Data points are mean ± standard deviation. (n = 3).

Three major peaks at 1643, 1536, 1402 cm1 which were known to be assigned as amide l, amide ll and amide lll, respectively, were also found in the samples. They were corresponding to C@O stretching, N–H bending or C–N stretching and C–N stretching, respectively. Other researchers showed that the IR peak located between 1640–1648 cm1 in amide l region could be due to the unordered structure (random coil) (Zhao, Chen, Xue, & Lee, 2008). This observation implying that the secondary structure of both samples might be partly destroyed (Zhao et al., 2008). It is believed that destroy of ordered secondary structure might attributed to the effect of the condition used in protein extraction (Zhao, Li, Wang, & Ji, 2006). Apart from that, peaks at around 1449 and 1236 cm1 was attributed to CH2 bending (scissors) vibration and C–N stretching modes, respectively (Chen et al., 2013) whereas peaks around 1152 and 1069 cm1 could be assigned to antisymmetric stretching variation of C–O–C.

It is worth noting that four distinct peaks (1742, 990, 942, and 859 cm1) were only found in PBPI. These peaks were corresponding to C@O stretching modes,@CH out-of-plane deformation, CH2 out-of-plane wag and O–OH stretching modes, respectively (Chen et al., 2013). Conversely, a distinct peak of 1308 cm1 which was due to in-plane hydroxyl deformation vibration (Carpenter & Crowe, 1989) was observed only in SPI. Therefore, it is suggested that both protein isolates could have differences in terms of structure and conformation due to differences in amino acid content reported in Section 3.4. 3.7. Thermal properties The DSC thermogram of PBPI showed a single endothermic peak with the denaturation temperature of 110.2 °C (Table 4). This peak most probably represents the denaturation of 7S vicilin. The result indicated that three subunits of the vicilin were denatured

454

E.-S. Tan et al. / Food Chemistry 152 (2014) 447–455

Table 4 Comparisons of physico-chemical/functional properties between PBPI and SPI. No.

Properties

1 2 3 4

Denaturation temperature, Td (°C) Water holding capacity (g/g) Oil holding capacity (g/g) Emulsifying activity (EA) (%)

5

Emulsion stability (ES) (%)

6 7

Least gelling concentration (%, w/v) Digestibility (%)

PBPI

SPI b

At At At At

concentration concentration concentration concentration

of of of of

1% 5% 1% 5%

110.2 ± 4.2 1.65 ± 0.02a 1.43 ± 0.05a 63.6 ± 2.50b 84.8 ± 1.25c 91.9 ± 6.60a 90.1 ± 3.17a 16b 71.3 ± 0.0a

92.5 ± 2.1a 6.13 ± 0.18b 1.51 ± 0.04a 47.4 ± 2.30a 61.9 ± 3.31b 100.0 ± 5.55a 100.0 ± 6.51a 12a 85.2 ± 0.0b

Note: comparison within the column in the table with the data written as mean. (n = 5). Means within the same column not followed by the same letter are significantly different at p < 0.05 level of significance, according to Tukey’s test.

co-dependently since only one peak was observed in thermogram. In comparison to other bean species, including soy protein isolate (92.0 °C) (Sousa et al., 1995), dark red kidney bean protein isolate (91.1 °C) (Rui et al., 2011), and white bean (90.6 °C) (Rui et al., 2011), the Td value of PBPI in the present investigation was higher. Assessment of the thermal stability of globular proteins is determined by the Td value implying that the protein has high value of Td exhibit a superior thermal stability. Therefore, the result suggests that PBPI has better thermal stability than the SPI. 3.8. pH-dependent protein solubility The solubility profiles of PBPI and SPI (Fig. 2c) showed typical U-shaped curves. The minimum solubility of PBPI fell into the pH range from 4.0 to 5.5, whereas its maximum solubility was obtained at pH values lower than 3 and higher than 6. On the other hand, the lowest solubility of SPI protein was at pH 4.0–5.0. The minimum solubility of PBPI and SPI found in the present study was due to their isoelectric pH of the samples and the net charge of the proteins will be little or zero. Electrostatic repulsive forces among the proteins were reduced and led to protein aggregation (i.e. formation of protein precipitation) thus causing the decrease in solubility (Singh, Kaur, & Sandhu, 2005). When the pH values above or below the isoelectric pH of a protein, there is a net charge on the protein surface in which induced the protein-solvent interaction and consequently led to a higher solubility. As shown in the Fig. 2c, PBPI exhibited excellent solubility at acidic and alkaline pH which is an important prerequisite for food formulation and thus it is possible for PBPI employed in various food applications. 3.9. Water- and oil holding capacity Table 4 shows that the water holding capacity (WHC) of PBPI was significantly (p < 0.05) lower than that of SPI. A possible reason could be due to lack of polar amino acid groups on the surface of the protein molecules because these polar groups were responsible for protein–water interaction. This explanation is supported by the amino acid result shown in Section 3.4. Conversely, results showed that no significant (p > 0.05) difference in oil holding capacity (OHC) between PBPI (1.43 g/g) and SPI (1.51 g/g) (Table 4). 3.10. Emulsifying activity (EA) and emulsion stability (ES) EA was defined as the ability of a protein to form an emulsion by adsorbing oil at the water-oil interface. On the other hand, ES was defined as the ability to stabilize emulsion without forming coalescence and flocculation over a period of time (Can Karaca, Low, & Nickerson, 2011). Emulsifying activity (EA) and emulsion stability (ES) of PBPI was performed in order to evaluate their ability to act as emulsifiers in various foods such as soup, sauce, confectionary product, and dairy product. In general, PBPI exhibited

higher emulsifying activity compared to SPI regardless of concentration. A high emulsion stability (>90%) was observed in all samples, as shown in Table 4. Sikorski (2001) reported that the emulsifying properties of legume proteins were greatly affected by molecular size, surface hydrophobicity, net charge, steric hindrance, and molecular fleaxibility, however, among these physicochemical characteristics, surface hydrophobicity and net surface charge were found to be the most significant factors in these functional properties. Apart from this, it has been observed that the presence of hydrophobic patches on the surface of the protein molecule was important for protein adsorption at the water-oil interface during the formation of the emulsion (Can Karaca et al., 2011). A high value of EA for PBPI might be due to present of globulins (i.e. protein fractions of PBPI) which have been reported to possess higher surface hydrophobicity than albumins and the result is supported by the amino acid data shown in Table 3. Overall, the result exhibited that PBPI could be an effective emulsifiers in food application. 3.11. Gelling capacity The least gelling concentration (LGC) is an indicative of the gelling capacity of a food protein. For PBPI, no gels were formed at concentration from 6% to 14%. However, at 16% (LGC) concentration, the PBPI formed a weak gel. On the other hand, SPI started to form a strong gel at concentration of 12%. The differences in LGC of PBPI and SPI could be due to the differences in their thermal stability. As shown in Section 3.7, PBPI has a high resistance toward thermal denaturation due to its higher thermal stability. Therefore, the heating temperature (100 °C) might not be sufficient to completely denature and unfold the PBPI. It has been reported by Matsumura and Mori (1996) that heating temperature is one of the factors which influence the formation of gel network of globular protein. Apart from this, it was observed that at 16% of LGC, PBPI formed a soft fragile gel whereas SPI formed firm, tough, and resilient gel at 12% of LGC. It could be due to the differences of their protein components, i.e., 11S and 7S globulins. Previous studies found that 11S globulin formed firmer with more resilient gel than those formed from 7S globulin (Kinsella, 1979). The formation of soft fragile gel of PBPI could be explained by their predominant protein (7S globulin) which is supported by the SDS-PAGE result (Section 3.5). 3.12. In vitro digestibility Table 4 shows that PBPI showed lower digestibility than that of SPI. This could be attributed to the majority storage protein of PBPI was vicilin. Previous studies reported that vicilin had resistance toward enzymatic hydrolysis due to their unique compact structure and the presence of the carbohydrate moiety which led to enhance their stability of three dimensional structural by impeding the

E.-S. Tan et al. / Food Chemistry 152 (2014) 447–455

proteolyitc enzyme access to some peptide bonds on the protein surface (Deshpande & Damodaran, 1989). Therefore, it was not readily attacked by these proteolytic enzymes as a consequent low digestibility. Similar result was reported in vicilin-rich protein isolate from Phaseolus legumes (Tang, Xiao, Chen, Yang, & Yin, 2009). 4. Conclusion The extraction yield of PBPI was successfully optimised under the condition of: extraction temperature of 25 °C, extraction time of 1 h, and buffer-to-sample ratio of 20 ml/g. This protein isolate was significantly different from soybean protein isolate in terms of physicochemical characteristics as well as functionalities. It was suggested that PBPI could have a great potential in food research as it showed high qualities in its characteristics. Acknowledgement This work is financially supported by ERGS Grant (203/CAATS/ 6730103). References Adebowale, Y. A., Adeyemi, I. A., Oshodi, A. A., & Niranjan, K. (2007). Isolation, fractionation and characterization of proteins from Mucuna bean. Food Chemistry, 104, 287–299. Ali, F., Ippersiel, D., Lamarche, F., & Mondor, M. (2010). Characterization of lowphytate soy protein isolates produced by membrane technologies. Innovative Food Science and Emerging Technologies, 11, 162–168. Can Karaca, A., Low, N., & Nickerson, M. (2011). Emulsifying properties of chickpea, faba bean, lentil and pea proteins produced by isoelectric precipitation and salt extraction. Food Research International, 44, 2742–2750. Carpenter, J. F., & Crowe, J. H. (1989). An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry, 28, 3916–3922. Chen, X., Ru, Y., Chen, F., Wang, X., Zhao, X., & Ao, Q. (2013). FTIR spectroscopic characterization of soy proteins obtained through AOT reverse Micelles. Food Hydrocolloids, 31, 435–437. Deak, N. A., & Johnson, L. A. (2007). Fate of phytic acid in producing soy protein ingredients. Journal of the American Oil Chemists’ Society, 84, 369–376. Deshpande, S. S., & Damodaran, S. (1989). Structure–digestibility relationship of legume 7S proteins. Journal of Food Science, 54, 108–113. Eromosele, C. O., Arogundade, L. A., Eromosele, I. C., & Ademuyiwa, O. (2008). Extractability of African yam bean (Sphenostylis stenocarpa) protein in acid, salt and alkaline aqueous media. Food Hydrocolloids, 22, 1622–1628. Gupta, A., Mehrotra, R., Klimov, E., Siesler, H. W., Joshi, R. M., & Chauhan, V. S. (2006). Thermal stability of dehydrophenylalanine-containing model peptides as probed by infrared spectroscopy: A case study of a-Helical and a 310-Helical peptide. Chemistry and Biodiversity, 3, 284–295.

455

Hsu, H. W., Vavak, D. L., Satterlee, L. D., & Miller, G. A. (1977). A multienzyme technique for estimating protein digestibility. Journal of Food Science, 42, 1269–1273. Kinsella, J. E. (1979). Functional properties of soy proteins. Journal of the American Oil Chemists’ Society, 56, 242–258. Liener, I. E. (1974). Phytohemagglutinins. Their nutritional significance. Journal of Agricultural and Food Chemistry, 22, 17–22. Matsumura, Y., & Mori, T. (1996). Gelation. In G. M. Hall (Ed.), Methods of testing protein functionality (pp. 76–106). London: Blackie Academic & Professional. Nasi, A., Picariello, G., & Ferranti, P. (2009). Proteomic approaches to study structure, functions and toxicity of legume seeds lectins. Perspectives for the assessment of food quality and safety. Journal of Proteomics, 72, 527–538. Rui, X., Boye, J. I., Ribereau, S., Simpson, B. K., & Prasher, S. O. (2011). Comparative study of the composition and thermal properties of protein isolates prepared from nine Phaseolus vulgaris legume varieties. Food Research International, 44, 2497–2504. Seyam, A. A., Banasik, O. J., & Breen, M. D. (1983). Protein isolates from navy and pinto beans: Their uses in macaroni products. Journal of Agricultural and Food Chemistry, 31, 499–502. Sikorski, Z. E. (2001). Functional properties of proteins in food systems. In Z. E. Sikorski (Ed.), Chemical and functional properties of food proteins (pp. 113–135). Boca Raton: CRC Press. Singh, N., Kaur, M., & Sandhu, K. S. (2005). Physicochemical and functional properties of freeze-dried and oven dried corn gluten meals. Drying Technology, 23, 1–14. Sousa, I. M. N., Mitchell, J. R., Ledward, D. A., Hill, S. E., & Beirão da Costa, M. L. (1995). Differential scanning calorimetry of lupin and soy proteins. Lebensmittel Untersuchung and Forschung, 201, 566–569. Tang, C. H., Xiao, M. L., Chen, Z., Yang, X. Q., & Yin, S. W. (2009). Properties of cast films of vicilin-rich protein isolates from Phaseolus legumes: Influence of heat curing. LWT-Food Science and Technology, 42, 1659–1666. WHO. Energy and protein requirements. Report of a Joint FAO/WHO/UNU Expert Consultation. Geneva, World Health Organization, 1985 (WHO Technical Report Series, No. 724). Wu, W., Williams, W. P., Kunkel, M. E., Acton, J. C., Huang, Y., Wardlaw, F. B., & Grimes, L. W. (1996). Amino acid availability and availability-corrected amino acid score of red kidney beans (Phaseolus vulgaris L.). Journal of Agricultural and Food Chemistry, 44, 1296–1301. Xu, B., & Chang, S. K. C. (2009). Total phenolic, phenolic acid, anthocyanin, flavan-3ol, and flavonol profiles and antioxidant properties of pinto and black bean (Phaseolus vulgaris L.) as affected by thermal processing. Journal of Agricultural and Food Chemistry, 57, 4754–4764. Ye, X. Y., & Ng, T. B. (2001). Peptides from pinto bean and red bean with sequence homology to cowpea 10-kDa protein precursor exhibit antifungal, mitogenic, and HIV-1 reverse transcriptase-inhibitory activities. Biochemical and Biophysical Research Communications, 285, 424–429. Zeier, J., & Schreiber, L. (1999). Fourier transform infrared-spectroscopic characterisation of isolated endodermal cell walls from plant roots: Chemical nature in relation to anatomical development. Planta, 209, 537–542. Zhao, X. Y., Chen, F. S., Xue, W. T., & Lee, L. T. (2008). FTIR spectra studies on the secondary structures of soybean 7S and 11S globulins using AOT reverse micellar extraction. Food Hydrocolloids, 22, 568–575. Zhao, C., Li, L. Z., Wang, L., & Ji, H. W. (2006). Circular dichroism spectral studies on the recombinant human neuroglobin. Chinese Science Bulletin, 51, 2581–2585.