Optimization of reaction conditions for resistant Canna edulis Ker starch phosphorylation and its structural characterization

Industrial Crops and Products 30 (2009) 105–113

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Optimization of reaction conditions for resistant Canna edulis Ker starch phosphorylation and its structural characterization Juan Zhang, Zheng-Wu Wang ∗ Department of Food Science and Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China

a r t i c l e

i n f o

Article history: Received 13 November 2008 Received in revised form 18 January 2009 Accepted 16 February 2009 Keywords: Canna edulis Ker starch Phosphorylation Resistant starch Structural characterization

a b s t r a c t Response surface methodology was employed to study phosphorous (P) and resistant starch (RS) content of resistant Canna edulis Ker starch phosphate monoester by starch phosphorylation with sodium mono- and dihydrogen orthophosphate. A five-level-four-factor central composite design was used for maximizing the P and RS content. The optimal reaction conditions obtained were NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio of 1:0.44, phosphate sodium to starch weight ratio of 1.76:1, reaction temperature of 131.6 ◦ C and reaction time of 2.43 h. The theoretical P and RS content were attained to about 0.58 and 73.17%, respectively. P and RS content experimentally obtained were 0.55 and 71.93%, in agreement with the predicted values. In addition, the structural characteristic of the purified starch phosphate monoester was investigated by 31 P-nuclear magnetic resonance (31 P-NMR), fourier transform-infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD) and scanning electron microscopy (SEM). Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.

1. Introduction As reported, resistant starch (RS) played a beneficial role on human health, such as altering lipid metabolism, improving cholesterol metabolism, affecting on colon microflora, reducing the risk of ulcerative colitis and the glycemic index of foods so on (Kim et al., 2008). RS is classified into four categories based on its resistance to digestion, and RS4 is chemically modified starch (Englyst et al., 1992). Now, the studies on RS4 focus on citrate, glutarate and phosphorylated starches (Xie et al., 2006). Phosphorylated starch was produced using a mixture of sodium trimetaphosphate and sodium tripolyphosphate (Sang and Seib, 2006). It is well known that RS content was positively related to P content of phosphorylated starch based on the results obtained in the previous references (Sang et al., 2007). Whereas, there was no studies carried out on RS from starch phosphate monoester, the dispersive solution of which has been reported to give clear pastes of high consistency, with good freeze–thaw stability and emulsifying properties. And these properties could allow interesting applications of starch phosphate monoester in food. Canna edulis, which contains abundant phenolic compounds with a higher nutritional quality than that of other rhizomes, is largely cultivated in South America, Taiwan, Vietnam, Thailand and China. The dry rhizome of C. edulis contains 70–80% starches which are reported for one of the most potential raw materials for

∗ Corresponding author. Tel.: +86 21 34205748; fax: +86 21 34205748. E-mail address: [email protected] (Z.-W. Wang).

industrial production. Some studies have been conducted on physiochemical properties and modification of C. edulis starch (Hung and Morita, 2005; Pérez and Lares, 2005; Saartrat et al., 2005; Lares and Pérez, 2006; Chuenkamol et al., 2007; Puncha-arnon et al., 2007). However, there has been no report on modification of C. edulis starch for RS production in food industry. Therefore, it is greatly significant to exploit resistant C. edulis starch phosphate monoester. As proved, statistical experimental design has become a powerful tool to determine the effects of experimental factors and their interactions (Wen et al., 2008). In this study, we reported optimization strategy for resistant C. edulis starch phosphate monoester production through vacuum-oven reaction. Central composite design (CCD) was used to describe the nature of the response surface in the experimental region, to search optimal reaction conditions for maximizing the resistant phosphate monoester content. Moreover, the structure of the phosphate monoester was characterized by FT-IR, 31 P-NMR, XRD and SEM. 2. Materials and methods 2.1. Samples and reagents C. edulis rhizomes were obtained from Guizhou Ziyun Jiahe Chemical Co. Ltd (Guizhou, China). The starches were prepared using the method in our previous study (Zhang et al., 2009). In brief, the slurry of fresh C. edulis rhizomes was prepared through crushing cleaned rhizomes in ascorbic acid (0.1%) aqueous solution by food processing machine (JYL-390, Joyoung Co. Ltd, Beijing, China). The pulp in slurry was removed carefully by screening through gauze

0926-6690/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2009.02.006

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Table 1 Factors and their levels for central composite design. Variables

Symbol

NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio Orthophosphate to starch weight ratio Reaction temperature (◦ C) Reaction time (h)

X1 X2 X3 X4

and the suspension obtained was filtered through a 200 ␮m sieve. The filtrated solution was allowed to deposit for starch layer. Then starch cake obtained was rewashed three times, and dried in the air for 48 h at 30 ± 2 ◦ C for further analysis. And all the chemicals used in the experiment were of analytical grade and water was doubly distilled. 2.2. Preparation of starch phosphate monoester The starch was phosphorylated using the method described by Sitohy et al. (2000) with slight modification. In brief, the mixture of sodium dihydrogen phosphate dihydrate (NaH2 PO4 ·2H2 O) and disodium hydrogen phosphate twehydrate (Na2 HPO4 ·12H2 O) with different molar ratios was dissolved in the distilled water at 40 ◦ C to prepare the salt solution for phosphorylation. Then starch with variable quality was slurred in the salt solution and the mixture was stirred for 10 min. The slurry was filtered with suction on a fritted glass funnel. Then the filter cake was lyophilized and heated in a vacuum oven at differently scheduled temperature for varied heating time. The product was recovered on a Büchner funnel and then dehydrated by washing with absolute ethanol. A 10% (w/v) paste in water was dialyzed 24 h against distilled water. The starch phosphate was recovered by flocculation with acetone. The latter precipitate was recovered by vacuum filtration, and freed from water and acetone by washing with anhydrous ethanol. At last, the dialyzed product was lyophilized.

Coded factor levels −2

−1

0

1

2

1:1 1:1 120 1

1:0.8 1.25:1 130 1.5

1:0.6 1.5:1 140 2

1:0.4 1.75:1 150 2.5

1:0.2 2:1 160 3

perature, and centrifuged at 3000 × g for 12 min. Amyloglucosidase (0.05 ml) and 0.2 ml deionized water was added to the supernatant solution (2.7 ml). This enzyme solution was freshly prepared for each digestion. Aliquots (0.1 ml) were taken at intervals and mixed with 1 ml of 80% ethanol. The hydrolyzed glucose content was measured by 3,5-dinitrosalicylic acid reagent. The rapid digestible starch (RDS) was defined as the total starch digested within the first 20 min, and the slowly digestible starch (SDS) was the starch digested between 20 and 120 min (Englyst et al., 1992). The resistant starch content was calculated as follows: RS (%) =

(total starch − RDS − SDS) × 100 % total starch

(1)

2.5. Experimental design In order to optimize the reaction conditions, a five-level fourfactor CCD was adopted in this study, requiring 30 experiments, which included sixteen factorial points, eight axial points, and six central points to provide information regarding the interior of the experiment region, allowing for the evaluation of the curvature. The parameters and their respective levels were shown as follows: NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio (1:1 to 1:0.2), orthophosphate to starch weight ratio (1:1 to 2:1), reaction temperature (120–160 ◦ C) and reaction time (1–3 h). Table 1 shows the coded independent factors and their levels. 2.6. Statistical analysis

2.3. Determination of P content Phosphorus was colorimetrically determined by the reaction with ammonium molybdate according to the method described by Murphy and Riley (1962). In brief, the starch sample was digested with the mixture of sulfuric acid and nitric acid (1:1) (v/v) in the digestion vessel in aid of electric oven. The digestion liquid was neutralized with sodium hydroxide and diluted to predetermined volume with distilled water. The a aliquot of the liquid was added into 8 ml of the mixed reagent which was prepared through mixing thoroughly 125 ml of 5N sulfuric acid and 37.5 ml of ammonium molybdate and then adding 75 ml of ascorbic acid solution and 12.5 ml of potassium antimonyl tartrate solution. Then the mixed liquid was diluted to the volume and mixed well. The optical density was determined at 825 nm using UV–visible spectrophotometry (Beckman Coulter DU800, USA), with fresh distilled water as blank. And the calibration curve was made using sodium dihydrogen phosphate.

Experimental data (Table 2) were analyzed via response surface methodology, in order to fit the following second-order polynomial equation generated by Design-Expert Version 7 software for Windows (Stat-ease Inc., Minneapolis, MN, U.S.A.). Second-order coefficients were generated via regression. The response was initially fitted to the factors via multiple regressions. The quality of the fit of the model was evaluated using the coefficients of determination and analysis of variance. The quadratic response surface model was fitted to the following equation: Y = ˇ0 +

4 

ˇi Xi +

i=1

4  i=1

ˇi Xi2 +

4 

ˇij Xi Xj

(2)

i
in which Y is the response factor, Xi is the independent factor, ˇ0 is the intercept, ˇi is the first-order model coefficient, ˇii is the quadratic coefficient for the factor i, and ˇij is the linear model coefficient for the interaction between factors i and j. 31 P-nuclear

magnetic resonance

2.4. Determination of resistant starch

2.7.

Resistant starch was determined according to the method described by Englyst et al. (1992). Briefly, Starch (100 mg) in 2 ml of sodium acetate buffer (0.1 M, pH 5.2) was cooked in a boiling water-bath for 30 min. The starch dispersion was cooled down to 37 ◦ C, mixed with an enzyme solution (0.5 ml) consisting of pancreatin extract and amyloglucosidase, and incubated in a water-bath at 37 ◦ C. The pancreatin extract was prepared as follows; pancreatin (0.6 g) was dispersed in water (4 ml), stirred for 10 min at room tem-

31 P-NMR was used to ascertain the binding site of P group into C. edulis starch. The sample prepared under the optimized conditions obtained was treated according to the method described by Sang et al. (2007). The proton-decoupled 31 P-NMR data were acquired on Mercury plus 400 spectrometer (Varian, Inc., USA), operating at 400 MHz for 1 H and 202.34 MHz for 31 P respectively, with a 3 mm NMR probe. Chemical shifts were reported in ı (ppm) from the reference signal of external 85% phosphoric acid.

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107

Table 2 Central composite design, experimental data for five-level-four-factor response surface analysis. Std

Run

Molar ratioa

Weight ratiob

Reaction T (◦ C)

Reaction time (h)

P content (%)

RSc content (%)

18 1 20 28 24 19 8 17 16 11 22 26 21 10 4 25 27 3 7 6 30 15 13 9 5 2 12 29 23 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

2 −1 0 0 0 0 1 −2 1 −1 0 0 0 1 1 0 0 −1 −1 1 0 −1 −1 −1 −1 1 1 0 0 1

0 −1 2 0 0 −2 1 0 1 1 0 0 0 −1 1 0 0 1 1 −1 0 1 −1 −1 −1 −1 1 0 0 −1

0 −1 0 0 0 0 1 0 1 −1 2 0 −2 −1 −1 0 0 −1 1 1 0 1 1 −1 1 −1 −1 0 0 1

0 −1 0 0 2 0 −1 0 1 1 0 0 0 1 −1 0 0 −1 −1 −1 0 1 1 1 −1 −1 1 0 −2 1

0.342 0.045 0.133 0.498 0.261 0.086 0.506 0.092 0.521 0.072 0.573 0.461 0.579 0.547 0.317 0.422 0.484 0.069 0.334 0.112 0.405 0.067 0.158 0.401 0.241 0.054 0.557 0.451 0.080 0.301

53 17 26 53 45 13 48 24 56 35 53 63 69 62 49 65 63 13 52 21 63 12 24 62 37 13 68 63 16 46

a b c

NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio. Orthophosphate to starch weight ratio. RS: resistant starch.

2.8. Fourier transform-infrared spectroscopy

3. Result and discussion

In order to further determine the structure of phosphate C. edulis starch monoester produced in the optimized experimental conditions, FT-IR spectra was obtained using FT-IR (Paragon 1000, PerkinElmer Inc. USA). The spectra were recorded in transmission mode from 4000 to 400 cm (mid-infrared region) at a resolution of 0.44 cm−1 . Samples were diluted with KBr (1:100, v/v) before acquisition and the background value from pure KBr was acquired before the sample was scanned.

3.1. Optimization of reaction conditions by response surface methodology

2.9. X-ray powder diffraction To determine the change of crystalline type and crystallinity of C. edulis starch phosphate monoester prepared in the optimized conditions, X-ray diffractograms were obtained with a Rigaku D/max2200/PC X-ray diffractometer with a chart speed of 10 mm/min. The starch powder was scanned through the 2 range of 5–40◦ . Traces were obtained using a Cu-K␣ radiation detector with a nickel filter and scintillation counter operating under the following conditions: 40 kV, 20 mA, 1/2◦ /1/2◦ divergence slit/scattering slit, 0.30 mm receiving slit, 1 s time constant and scanning rate of 3◦ /min. The degree of crystallinity of samples was quantitatively estimated following the method of Nara and Komiy (1983). 2.10. Scanning electron microscopy In order to determine the physical form of C. edulis starch phosphate monoester obtained under the optimized conditions, scanning electron microscopy (SEM) was conducted on an S-2150 microscope (Hitachi Ltd, Tokyo, Japan) at an accelerating voltage of 15 kV. The dried starch materials were deposited on copper stubs using double-adhesive tape and coated with gold.

In order to optimize the reaction conditions of resistant C. edulis starch phosphorylation, CCD was selected with five-level-fourfactors. Table 2 listed the experimental factor settings and results on the basis of experimental design. All 30 of the designed experiments were conducted, and the results were analyzed via multiple regression. The coefficients of a full model were evaluated via regression analysis and tested for significance. Finally, the best fitting model was determined via regression. This showed that quadratic mode was significant for two response values (p < 0.0001). The ANOVA for the response surface quadratic model was provided in Tables 3a and 3b. And the coefficients of the response surface model as provided by Eq. (2) were also evaluated. For all response values, according to the ANOVA analysis of factors (Tables 3a and 3b), a low lack of fit was noted, indicating that the model indeed represented the actual relationships of reaction parameters, which were well within the selected ranges (Table 1). And the model coefficients and probability values (code value) were shown in Tables 4a and 4b. The model proved suitable for the adequate representation of the real relationship among the selected factors. Fig. 1 represented the effects of NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio and orthophosphate to starch weight ratio on P and RS content at a constant reaction temperature of 140 ◦ C and reaction time of 2 h. At the designed ratio of orthophosphate and starch from 1:1 to 1.75:1, the augmentation of both P and RS content resulted in a linear increase in NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio. An increased value of P content was observed with orthophosphate to starch weight ratio varied from

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Table 3a Results of analysis of variance (ANOVA) for quadratic model using P content as response. Source

SSa

dfb

MSc

F-value

Probability (p) > F

Model X1 X2 X3 X4 X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4 X1 2 X2 2 X3 2 X4 2 Residual (error) Lack of fit Pure error Total

1.017414 0.171366 0.019154 0.001148 0.071286 0.088506 0.003844 0.053824 0.026244 0.058081 0.09579 0.095546 0.202371 0.0259 0.136891 0.024475 0.018158 0.006318 1.017414

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 10 5 14

0.072672 0.171366 0.019154 0.001148 0.071286 0.088506 0.003844 0.053824 0.026244 0.058081 0.09579 0.095546 0.202371 0.0259 0.136891 0.001632 0.001816 0.001264 0.072672

44.53846 105.0244 11.73853 0.703672 43.68877 54.24248 2.355857 32.98691 16.08406 35.59588 58.7066 58.55709 124.0261 15.87341 83.89612

<0.0001 <0.0001 0.0038 0.4147 <0.0001 <0.0001 0.1456 <0.0001 0.0011 <0.0001 <0.0001 <0.0001 <0.0001 0.0012 <0.0001

1.437093

0.3614

2

2

2

44.53846

<0.0001

2

R = 0.9381; CV = 16.02; Adj R = 0.8803. R = 0.9765; CV = 13.22; Adj R = 0.9546. a SS: sum of squares. b df: degrees of freedom. c MS: mean square. Table 3b Results of analysis of variance (ANOVA) for quadratic model using RS content as response. Source

SSa

dfb

MSc

F-value

Probability (p) > F

Model X1 X2 X3 X4 X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4 X1 2 X2 2 X3 2 X4 2 Residual (error) Lack of fit Pure error Total

10685.55 1190.042 247.0417 126.0417 1247.042 715.5625 22.5625 473.0625 52.5625 588.0625 1501.563 864.6458 2946.503 0.002976 1590.36 705.25 611.9167 93.33333 11390.8

14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 15 10 5 29

763.2536 1190.042 247.0417 126.0417 1247.042 715.5625 22.5625 473.0625 52.5625 588.0625 1501.563 864.6458 2946.503 0.002976 1590.36 47.01667 61.19167 18.66667

16.23368 25.31106 5.254342 2.680787 26.5234 15.21934 0.479883 10.06159 1.117955 12.50753 31.93681 18.3902 62.66933 6.33E-05 33.82545

<0.0001 0.0001 0.0368 0.1224 0.0001 0.0014 0.4991 0.0063 0.3071 0.0030 <0.0001 0.0006 <0.0001 0.9938 <0.0001

3.278125

0.1011

R2 = 0.9381; CV = 16.02; Adj R2 = 0.8803. R2 = 0.9765; CV = 13.22; Adj R2 = 0.9546. a SS: sum of squares. b df: degrees of freedom. c MS: mean square. Table 4a Regression coefficients and significance of response surface quadratic model for P content. Factor

Coefficient Estimatea

df

Standard Error

95% CI Low

95% CI High

VIF

Intercept X1 X2 X3 X4 X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4 X1 2 X2 2 X3 2 X4 2

0.4535 0.0845 0.02825 0.006917 0.0545 0.074375 −0.0155 0.058 0.0405 −0.06025 −0.07738 −0.05902 −0.0859 0.030729 −0.07065

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.017175 0.008588 0.008588 0.008588 0.008588 0.010518 0.010099 0.010518 0.010518 0.010518 0.010518 0.008033 0.008033 0.008033 0.008033

0.41709 0.066295 0.010045 −0.01129 0.036295 0.052078 −0.03702 0.035703 0.018203 −0.08255 −0.09967 −0.07605 −0.10293 0.0137 −0.08768

0.48991 0.102705 0.046455 0.025122 0.072705 0.096672 0.006024 0.080297 0.062797 −0.03795 −0.05508 −0.04199 −0.06887 0.047758 −0.05362

1 1 1 1 1 1 1 1 1 1 1.05 1.05 1.05 1.05

a

Coefficient estimate values in terms of coded factors.

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Table 4b Regression coefficients and significance of response surface quadratic model for RS content. Factor Intercept X1 X2 X3 X4 X1 X2 X1 X3 X1 X4 X2 X3 X2 X4 X3 X4 X1 2 X2 2 X3 2 X4 2 a

Coefficient Estimatea

df

Standard Error

95% CI Low

95% CI High

VIF

61.66667 7.041667 3.208333 −2.29167 7.208333 6.6875 −1.1875 −1.1875 5.4375 1.8125 −6.0625 −9.6875 −5.61458 −10.3646 0.010417

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

2.799305 1.399653 1.399653 1.399653 1.399653 1.714218 1.714218 1.714218 1.714218 1.714218 1.714218 1.714218 1.309255 1.309255 1.309255

55.70009 4.058377 0.225044 −5.27496 4.225044 3.033732 −4.84127 −4.84127 1.783732 −1.84127 −9.71627 −13.3413 −8.40519 −13.1552 −2.78019

67.63325 10.02496 6.191623 0.691623 10.19162 10.34127 2.466268 2.466268 9.091268 5.466268 −2.40873 −6.03373 −2.82397 −7.57397 2.801028

1 1 1 1 1 1 1 1 1 1 1 1.05 1.05 1.05

Coefficient estimate values in terms of coded factors.

1:1 to 1.582:1 with code values from −2 to 0.33 at a constant ratio of NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O. However, for RS content, orthophosphate to starch weight ratio was changed from 1:1 to 1.577:1 with code values from -2 to 0.31. The maximal P content of 0.48 and RS content of 63.70%, predicted, were determined at NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio of 1:0.53 and 1:0.54, and orthophosphate to starch weight ratio of 1.54:1 and 1.52:1, with code value of 0.16 and 0.09, respectively.

Fig. 2 showed the effects of NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio and reaction time on P and RS content at a constant orthophosphate to starch weight ratio of 1.5:1 and reaction temperature of 140 ◦ C. A complicated interaction was observed between NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio and reaction time. At a constant reaction time, an increase in the molar ratio of NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O increased P and RS content. With any NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio, reaction

Fig. 1. Response surface plots representing the effect of NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio, and orthophosphate to starch weight ratio as well as their reciprocal interaction on (a) P content and (b) RS content. Other factor is constant at zero levels.

Fig. 2. Response surface plots representing the effect of NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio, and reaction time as well as their reciprocal interaction on (a) P content and (b) RS content. Other factor is constant at zero levels.

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Fig. 3. Response surface plots representing the effect of orthophosphate to starch weight ratio and reaction temperature as well as their reciprocal interaction on (a) P content and (b) RS content. Other factor is constant at zero levels.

time had a little impact on P and RS content. In the comparison to the results exhibited in Fig. 1, the high molar ratio of NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O was beneficial to phosphate monoester reaction and RS formation, which can be ascribed to the increase of NaH2 PO4 ·2H2 O, with high acidity, which reduced to the amount of hydrogen ions and accelerate the dehydrate rate of orthophosphate in order to high reaction efficiency. The effect of orthophosphate to starch weight ratio and reaction temperature on P and RS content at a constant NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio of 1:0.6 and reaction time of 2 h was provided in Fig. 3. The reaction temperature seemly had no obvious effect on P content with different ratio of orthophosphate and starch. In contrast, P content was influenced complicatedly by the ratio of orthophosphate and starch at any reaction temperature. Similarly, for RS content, at any weight ratio of orthophosphate and starch, RS content was slightly affected by reaction temperature, whereas, with the increase of orthophosphate to starch weight ratio, RS content changed obviously and maximized when the weight ratio was between 1.25:1 and 1.75:1. As shown in Fig. 4, an increase of orthophosphate to starch weight ratio increased the P and RS contents within the ratio from 1:1 to 1.525:1 and from 1:1 to 1.55:1, respectively, at a constant reaction time. And reaction time had a linear effect on P and RS content in the range from 1 to 2.14 h, and from 1 to 2.25 h, respectively, at a constant ratio of orthophosphate and starch. The maximized P and

Fig. 4. Response surface plots representing the effect of orthophosphate to starch weight ratio and reaction time as well as their reciprocal interaction on (a) P content and (b) RS content. Other factor is constant at zero levels.

RS content were determined at orthophosphate to starch weight ratio of 1.525:1 and 1.55:1, and reaction time of 2.14 and 2.25 h, respectively. Compared to the results shown in Figs. 1, 3 and 4, the appropriated ratio of orthophosphate and starch (1.5:1 ∼ 1.55:1) was necessary to the acquisition of high P and RS content, as a result of common effect of other three factors. The effect of reaction temperature and reaction time as well as their reciprocal interaction at a constant molar ratio of NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O of 1:0.6 and weight ratio of orthophosphate and starch of 1.5:1 was exhibited in Fig. 5. For both P and RS content, the reciprocal interaction between reaction temperature and time was complex. The prolonged reaction time was advantageous to starch phosphate monoester reaction and RS formation at low reaction temperature, in accordance with the results revealed in Figs. 2 and 4. And the proper reaction temperature was affected intricately by other factors, in Figs. 3 and 5. As compared to the results shown in Figs. 1–4, the optimal condition for the maximal acquisition of P and RS contents was achieved with high NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio, orthophosphate to starch weight ratio (1.5:1 ∼ 1.55:1), low reaction temperature and prolonged reaction time. The optimal values of the variables were obtained using DesignExpert software. As exhibited in Fig. 6, the best conditions for resistant C. edulis starch phosphate monoester reaction estimated were NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio of 1:0.44, orthophosphate to starch weight ratio of 1.76:1, reaction tempera-

J. Zhang, Z.-W. Wang / Industrial Crops and Products 30 (2009) 105–113

Fig. 5. Response surface plots representing the effect of reaction temperature and reaction time as well as their reciprocal interaction on (a) P content and (b) RS content. Other factor is constant at zero levels.

ture of 131.6 ◦ C, and reaction time of 2.43 h. The theoretical P and RS content predicted under the above conditions was 0.58 and 73.17%, respectively. In order to verify the prediction of the model, the experiment was carried out under the optimized conditions, and P and RS content obtained were 0.55 and 71.93%, respectively, demonstrating that the application of response surface methodology for the optimization of factors in the chemical reaction. This would provide useful information regarding the development of efficient and economic production of resistant starch phosphate monoester. 3.2.

31 P-nuclear

111

Fig. 6. Response surface plot representing the effect of NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio, and orthophosphate to starch weight ratio of the optimized (a) starch phosphate monoester reaction and (b) RS formation condition at the stationary point (reaction temperature of 131.6 ◦ C and reaction time of 2.43 h).

the presence of C6 P–OH. These well approved by the results in the measure of 31 P-NMR. 3.2.2. X-ray powder diffraction As shown in Fig. 9 and Table 5, X-ray spectrum of native starch was B-type representative of tuber starches with two main peaks at around 2 of 16.97◦ 4 and 22.15◦ . However, the peaks 2 at 5.57◦ and 24.05◦ , which were typical for potato starch (B-type), were

magnetic resonance

As exhibited in Fig. 7, the 31 P-NMR spectrum of the ␣, ␤-dextrin of C. edulis starch phosphate monoester showed a strong set of two signals for starch phosphate monoester at ␦ 4.076 and 4.564 ppm, indicating the phosphorylation of hydroxyl groups at C-2 or C3, and C-6, respectively. (Lim and Seib, 1993; Tarelli et al., 1997; Sang et al., 2007). In addition, the signal at ␦3.454 was attributed to be orthophosphate, as a result of residual NaH2 PO4 ·2H2 O and Na2 HPO4 ·12H2 O in the reaction mixture (Sang et al., 2007). 3.2.1. Fourier transform-infrared spectroscopy As shown in Fig. 8, the adsorption spectrum was presented at near 1400 cm−1 , belonging to P O stretching vibration. In addition, very strong adsorption spectrum at about 1000 cm−1 verified

Fig. 7. 31 P-NMR spectrum of the ␣,␤-dextrin prepared from resistant Canna edulis Ker starch phosphate monoester.

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Fig. 8. Fourier transform-infrared spectrum of resistant Canna edulis Ker starch phosphate monoester.

not present (Imberty et al., 1991; Franco et al., 1995). However, the starch phosphate monoester exhibited C-type characterization, with the presentation of two peaks 2 at 15.58◦ and 19.63◦ , signifying that the structure of the starch changed after the reaction. Moreover, compared to that of native starch, the crystallinity of starch phosphate monoester almost remained, suggesting that the layer structure did not been destroyed in the process of reaction.

Fig. 9. XRD spectra of (a) native Canna edulis Ker starch and (b) resistant Canna edulis Ker starch phosphate monoester.

3.2.3. Scanning electron microscopy After the treatment, the physical shape of starch remained (Fig. 10), in accordance with the result in the measurement of XRD which exhibited that the degree of crystallinity did not nearly changed for C. edulis starch phosphate monoester. And it well also suggested that reaction did not disrupt the starch structure.

Table 5 X-Ray diffraction intensities of the major peak and crystallinity of native Canna edulis Ker starch and resistant Canna edulis Ker starch phosphate monoester. Starch source

Native starch Starch phosphate monoester a

2 (◦ ) with intensities (CPS)a

Crystallinity (%)

5◦

15◦

17◦

18◦

22◦

5.69◦ (68)

15.57◦ (252)

16.95◦ (345) 17.12◦ (323)

19.53◦ (232)

22.95◦ (255) 22.75◦ (242)

16.78 16.32

counts per second.

Fig. 10. Scanning electron microscopy of native Canna edulis Ker starch (A and B) and its resistant phosphate monoester (C and D). And the second column corresponds to higher magnification of the same samples in the first column.

J. Zhang, Z.-W. Wang / Industrial Crops and Products 30 (2009) 105–113

4. Conclusion In summary, the conditions of resistant C edulis starch phosphorylation were optimized through signal factor experiment, and further through central composite design using Design Expert software. And the optimal values of the variables were shown as follows: NaH2 PO4 ·2H2 O to Na2 HPO4 ·12H2 O molar ratio of 1:0.44, orthophosphate to starch weight ratio of 1.76:1, reaction temperature of 131.6 ◦ C, and reaction time of 2.43 h. At this predicted optimum condition, the theoretical P and RS content were attained to about 0.58 and 73.17%, respectively. And the experimental values corresponded well with the estimated values of the model. Moreover, the structure of the starch phosphate monoester was identified by FT-IR, 31 P-NMR, XRD and SEM. This study has a great significance to the production of resistant C. edulis starch phosphate monoester. Furthermore, high RS content undoubtedly increased value to the application of C. edulis starch phosphate monoester. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No: 20676051), the Major Project Regarding Scientific and Technological Development of Science and Technology Commission of Shanghai Municipality (07DZ195080, the Technology Standard Project of Science and Technology Commission of Shanghai Municipality (07DZ05019) and The Innovation Fund for Graduate Student of Shanghai Jiao Tong University. References Chuenkamol, B., Puttanlek, C., Rungsardthong, V., Uttapap, D., 2007. Characterization of low-substituted hydroxypropylated canna starch. Food Hydrocolloids 21, 1123–1132. Englyst, H.N., Kingman, S.M., Cummings, J.H., 1992. Classification and measurement of nutritionally important starch fractions. Eur. J. Clin. Nutr. 46, S30–S50. Franco, M.L., Preto, S.J.R., Ciacco, F.C., Tavares, D.Q., 1995. Effect of the heat moisture treatment on the enzymatic susceptibility of corn starch granules. Starch/Stärke 47, 228–233.

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