Document Sample
					International Journal of Food Sciences and Nutrition,
February 2011; 62(1): 91–96

Optimization conditions for anthocyanin and phenolic content extraction
form purple sweet potato using response surface methodology

 Department of Food Science and Technology and Institute of Biotechnology, Chonnam National University, Gwangju,
South Korea, and 2Department of Food Processing and Preservation, Hajee Mohammad Danesh Science and Technology
University, Dinajpur, Bangladesh

Purple sweet potato flour could be used to enhance the bioactive components such as phenolic compounds and anthocyanin
content that might be used as nutraceutical ingredients for formulated foods. Optimization of anthocyanin and phenolic
contents of purple sweet potato were investigated using response surface methodology. A face-centered cube design was used to
investigate the effects of three independent variables: namely, drying temperature 55 – 658C, citric acid concentration 1– 3% w/v
and soaking time 1– 3 min. The optimal conditions for anthocyanin and phenolic contents were 62.918C, 1.38%, 2.53 min
and 60.948C, 1.04% and 2.24 min, respectively. However, optimal conditions of anthocyanin content were not apparent.
The experimental value of anthocyanin content was 19.78 mg/100 g and total phenolic content was 61.55 mg/g. These data
showed that the experimental responses were reasonably close to the predicted responses. Therefore, the results showed that
treated flours could be used to enhance the antioxidant activities of functional foods.

Keywords: Purple sweet potato, response surface methodology, phenolic compounds, anthocyanin content

Purple-fleshed sweet potatoes have an intense purple                  ethanol, acetone, water or mixture (Pathirana and
color in the storage roots due to the accumulation of                Shahidi 2005, Huang et al. 2006). The stability of
anthocyanins (Terahara et al. 2004).The anthocyanins                 anthocyanin and phenolic content were influenced by
in purple sweet potato are mono-acylated or di-acylated              several factors (Jiang 2000). Among them, polyphenol
forms of cyanidin and peonidin (Yang and Gadi 2008).                 oxidase plays an important role in the degradation of
Sweet potatoes had intermediate antioxidant activity                 anthocyanin and phenolic content. Citric acid has been
among 43 vegetables (Huang et al. 2006). Recently                    used extensively for the inhibitory activity on poly-
natural antioxidants have attracted considerable atten-              phenol oxidase and the anti-browning activity in
tion due to their positive health benefit (Huang et al.               minimally processed fruits and vegetables. Citric acid
2006). Rumbaboa et al. (2009) reported that antho-                   extracts have a double inhibitory effect by chelating
cyanin from purple sweet potato has better radical                   copper at lower pH (Altunkaya and Gokmen 2009).
scavenging activity than that of red cabbage, grape skin,               Sweet potatoes can be processed into flour, which
elderberry and purple corn. Anthocyanins from purple                 are less bulky and more stable than the highly
sweet potatoes have many biological functions, such as               perishable fresh root. Flour can be used as a thickener
scavenging free radicals, anti-mutagenicity, anti-carci-             in soup, gravy, fabricated snacks and bakery products.
nogen activity and antihypertensive effect (Oki et al.               It could be used to enhance food products through
2002). Several extraction methods have been used to                  color, flavor, natural sweetness and nutrients. Sing et al.
obtain extracts rich in anthocyanin and phenolic                     (2003) used potassium metabisulphite, citric acid and
content based on different solvents such as methanol,                sodium chloride to improve the quality of chips from

Correspondence: Jong-Bang Eun, Department of Food Science & Technology, Chonnam National University, 77 Yongbong-ro Buk-gu,
Gwangju 500-757, South Korea. Tel: 82 62 530 0255. Fax: 82 62 530 2149. E-mail: jbeun@jnu.ac.kr

ISSN 0963-7486 print/ISSN 1465-3478 online q 2011 Informa UK, Ltd.
DOI: 10.3109/09637486.2010.511167
92    M. Ahmed et al.

sweet potatoes. Response surface methodology (RSM)                     6 – 7%) was obtained by milling the dried slices using a
has been successfully used to optimize biochemical                     blender (FM-681C; Hanil, Gwangju, Korea), and
and biotechnological process related to food systems                   sieved through an 80-mesh (Chung gye sang gongsa,
(Cacace and Maza 2003, Pathirana and Shahidi                           Seoul, South Korea) screen.
2005). Therefore, the goal of the present study was to
optimize different pretreatments such as drying                        Experimental design for RSM analysis
temperature, citric acid concentration and soaking
                                                                       A three-factor (X1, X2 and X3) and three-level (– 1, 0
time for production of sweet potato flour with
                                                                       and 1) face-centered cube design were employed in
high retention of anthocyanin and phenolic content
                                                                       this study, and 15 individual run points were taken for
using RSM.
                                                                       analysis (Wanasundara and Shahidi 1999). The actual
                                                                       and corresponding values are presented in Table I.
Materials and methods                                                  The multiple regression equation was used to fit the
Raw materials                                                          second-order polynomial equation based on the
                                                                       experimental data as follows:
Sweet potato (Ipomoea batatas L. Lam variety,
Sinjami) was purchased from a local farm. Roots
                                                                        Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X3 þ b11 X1 X1
were washed with tap water to remove dirt and soil
and allowed to dry at ambient temperature (, 208C).                              þ b22 X2 X2 þ b33 X3 X3 þ b12 X1 X2 þ b13 X1 X3
The washed sweet potatoes were stored at 148C for                                þ b23 X2 X3
15 days without curing.
                                                                       where Y is the response variable, b0 is the intercept, b1,
Sample preparation and treatment                                       b2, b3, b11, b22, b33 and b12, b13, b23 are linear,
Sweet potatoes were peeled with a hand peeler (Han                     quadratic and interaction coefficients respectively, and
Sung 27 stainless; Namdong Industry Park, Incheon,                     X1, X2 and X3 are the coded independent variables.
South Korea). Then peeled samples were cut into slices
(1 mm thickness) using a slicing machine (HFS 350G;                    Verification of model
Hankook fujee Industries Co. Ltd. Suwon-si,                            RSM was used to optimize anthocyanin and phenolic
Gyeonggi-do, Fujee, South Korea). Various levels of                    contents from purple sweet potato. The experimental
citric acid concentration (1 – 3% w/v) were solubilized                and predicted values were compared to confirm the
in deionized water at room temperature (20 ^ 18C).                     validity of the model.
After that, peeled slices were dipped in aqueous citric
acid solutions (1 –3% w/v) for different soaking times                 Analysis of anthocyanin contents
(1 – 3 min) at room temperature.
                                                                       The content of anthocyanin was determined following
                                                                       the procedures of Proctor (1974) and Huang et al.
Preparation of sweet potato flour
                                                                       (2006) The sweet potato flour (1 g) was treated with
The slices were dried using a convection drying oven                   15 ml HCl – methanol (0.15% HCl:methanol ¼
(Dasol Scientific Co. Ltd, Seoul, South Korea) at                       15:85) for 4 h. The extract was filtered and its
different temperatures 558C, 608C, and 658C for                        absorbance was determined at 530 nm. The antho-
7 – 8 h. The sweet potato flour (moisture content                       cyanin content was calculated on the basis of the
                                 Table I. Three-factor, three-level, face-centered cube design for RSM.

                      Factor X1                 Factor X2             Factor X3                               Variable

Assay number    Drying temperature (8C)    Concentration (%)     Soaking time (min)     Anthocyanin (mg/100 g)      Total phenolics (mg/g)

 1                     55 (21)                    2(0)                  3(þ 1)                 40.32 ^ 1.32              56.85 ^ 8.28
 2                     55(21)                     1(21)                 2(0)                   39.56 ^ 1.03              47.77 ^ 1.53
 3                     55(21)                     2(0)                  1(21)                  40.79 ^ 0.60              47.18 ^ 5.73
 4                     55(21)                     3(þ 1)                2(0)                   37.40 ^ 2.08              47.52 ^ 4.02
 5                     60(0)                      3(þ 1)                3(þ 1)                 34.17 ^ 4.72              44.64 ^ 0.34
 6                     60(0)                      1(21)                 1(21)                  32.53 ^ 2.73              51.69 ^ 0.19
 7                     60(0)                      3(þ 1)                1(21)                  24.45 ^ 4.96              56.38 ^ 0.39
 8                     60(0)                      1(21)                 3(þ 1)                 24.51 ^ 0.37              63.38 ^ 1.02
 9                     65(þ 1)                    2(0)                  3(þ 1)                 29.16 ^ 3.64              46.46 ^ 1.07
10                     65(21)                     1(21)                 2(0)                   26.49 ^ 1.83              51.65 ^ 2.37
11                     65(þ 1)                    2(0)                  1(21)                  24.55 ^ 0.29              51.65 ^ 2.37
12                     65(þ 1)                    3(þ 1)                2(0)                   22.45 ^ 0.28              52.03 ^ 2.43
13                     60(0)                      2(0)                  2(0)                   23.94 ^ 0.57              45.70 ^ 0.41
14                     60(0)                      2(0)                  2(0)                   20.02 ^ 1.18              49.39 ^ 0.81
15                     60(0)                      2(0)                  2(0)                   21.63 ^ 0.17              58.91 ^ 1.18
                                              Optimizing anthocyanin and phenolic content of purple sweet potato flour                                              93

following equation:                                                    (a)

Anthocyanin content ¼ A £ MW £ DF £ 100=ðe £ WÞ

where A is the absorbance, MW is the molecular
weight of cyanidin-3-glucoside (MW ¼ 449.2), DF is

                                                                          Anthocyanins (mg/100 g)
the dilution factor, 1 is the molar absorptivity
(34,300), and W is the sample weight (g).                                                           39.39

Analysis of total phenolic contents
The total phenolic content was determined using                                                     29.55
Folin – Ciocalteau reagent according to a slightly                                                                                                           3.00

modified method (Swain and Hills 1959). The sample                                                                                                      2.33

(0.1 g) was extracted three times with 20 ml of 75%


methanol and was filtered through Whatman No. 2                                                                                                      1.67

filter paper. Extracts were combined and concentrated


in a rotary vacuum evaporator (Rikakikai Co. Ltd,                                                               Tempe           58.33        1.00
                                                                                                                      ra  ture (°C       55.00
Tokyo, Japan) at 408C; the volume was adjusted to                                                                                  )
20 ml with 75% methanol. One milliliter of extract,
5 ml distilled water and 2 ml of 10% Folin – Ciocalteau                (b)
reagent were added into a Falcon tube. After 3 min at
room temperature, 2 ml of 7.5% Na2CO3 solution was
added and the sample was diluted to 20 ml with
distilled water. Each sample was allowed to stand for                                               49.22
                                                                        Anthocyanins (mg/100 g)

Table II. Regression coefficients of predicted quadratic polynomial                                  39.39
for the response anthocyanin and phenolic contents.

Coefficient           Anthocyanin content          Phenolic content
  b0                      1240.40***                2176.82                                                                                                3.00

  b1                      236.84***                      6.19                                                                                         2.33

  b2                      224.72                        21.65

  b3                      247.49***                     37.69                                       19.72

Quadratic                                                                                              65.00

  b11                        0.28***                    20.04                                                    61.67

                                                                                                                             58.33           1.00
  b22                        3.12***                    24.76                                                  Tempe                    55.00
                                                                                                                     rature (°C
  b33                        3.92***                    21.95                                                                   )
  b12                        0.04                       20.16          Figure 1. Response surface plots of the anthocyanin content of
  b13                        0.39                       20.30          purple sweet potato flour as affected by temperature, citric acid
  b23                        4.43***                    25.97**        concentration and soaking time. (a) Temperature and concentration.
R2                           0.97                        0.80          (b) Temperature and soaking time.

R 2, coefficient of multiple determination. ***Significant at
P # 0.01. **Significant at P # 0.05.
                                                                       1 h at room temperature and absorbance was
                                                                       measured at 760 nm (UV-1201; Shimadzu, Kyoto,
Table III. Analysis of variance for the response surface quadratic     Japan). The total phenolic content was calculated on
model for anthocyanin and phenolic contents.
                                                                       the basis of standard curves of gallic acid, and
                  Degree          Sum                                  expressed as milligrams of gallic acid equivalents per
Source          of freedom     of squares   Mean square     F value    gram of sample on a wet weight basis.
For anthocyanin
  Lack of fit          3           7.50           2.50                  Statistical analysis
  Pure error          2           7.76           3.88           0.64
  Total error         5          15.26           3.05                  All determinations were carried out in triplicate and
For total phenolic                                                     the experimental results are expressed as means ^
  Lack of fit          3          18.75          6.25
  Pure error          2          92.91         46.45            0.93
                                                                       standard deviation. Statistical analysis of the verifica-
  Total error         5         111.67         22.33                   tion results was carried out by analysis of variance and
                                                                       Duncan’s multiple-range tests using SAS (version
94                   M. Ahmed et al.

  (a) 65.0                                                                      (a)

  Temperature (°C)


                                                                                Phenolic content (mg/g)
                     57.5                                                                                 56.82

                         1.0        1.5         2.0         2.5          3.0                              49.17
                                          Concentration (%)                                                                                                       3.00

                                           21.20    25.62    30.05   34.47                                                                                 2.33

             Anthocyanins (mg/100 g)
                                           38.90    43.32    47.75



  (b) 65.0                                                                                                               61.67

                                                                                                                                     58.33       1.00

                                                                                                                      Tempera                55.00
                                                                                                                              tu re (°C)
  Temperature (°C)




                                                                                Phenolic content (mg/g)

                         1.0        1.5          2.0         2.5        3.0
                                          Soaking time (min)                                              56.82
                                          21.20     25.62    30.05   34.47
          Anthocyanins (mg/100 g)
                                          38.90     43.32    47.75

Figure 2. Contour plots showing the effects of temperature, citric                                        49.17
acid concentration and soaking time on anthocyanin content of

purple sweet potato flour. (a) Temperature and citric acid


concentration. (b) Temperature and soaking time.



9.1). The optimal conditions were estimated through                                                                                 58.33        1.00

                                                                                                                      Tempera                55.00
three-dimensional response surface analyses of the                                                                            tu re (°C)
three independent variables and each dependent
variable.                                                                      Figure 3. Response surface plots of the phenolic contents of purple
                                                                               sweet potato flour as affected by temperature, citric acid
                                                                               concentration and soaking time. (a) Temperature and
                                                                               concentration. (b) Temperature and soaking time.
Results and discussion
Fitting the models
Analysis of variance using SAS was performed to                                Effect of pretreatment on anthocyanin and phenolic
determine the significance of the linear, quadratic,                            contents
cross-product (Table II) and the lack of fit (Table III)                        The anthocyanin content of sweet potato flours ranged
of the independent variables on the anthocyanin and                            from 20.02 to 40.79 mg/100 g wet weight basis
phenolic contents. The lack-of-fit test is a measure of                         (Table I). The contents of anthocyanin were much
the failure of a model to represent data in the
                                                                               higher than those of sweet potato puree (Steed and
experimental domain at the points that were not
                                                                               Truong 2008) and of steamed or kneaded flours
included in the regression (Montgomery 1984).
However, the R 2 value of the dependent variables                              (Huang et al. 2006). The results of multiple regression
was approximately 0.80, indicating that a high                                 analysis showed that the anthocyanin contents were
proportion of variability was explained by the data                            significantly (P # 0.001) affected by the linear term of
(Varnalis et al. 2004). Therefore, the results showed                          temperature and soaking time, the quadratic of all
that the experimental model was adequate due to no                             terms and the interaction term of concentration and
significant lack of fit and satisfactory levels of R 2.                          soaking time (Table II). The predicted model obtained
                                                                       Optimizing anthocyanin and phenolic content of purple sweet potato flour                    95

                                                                                                     the anthocyanin structure could be varying with pH
(a) 65.0
                                                                                                     (Cevallos-Casala and Cisneros-Zevallos 2004).
                                                                                                        The total phenolic content of sweet potato
 Temperature (°C)

                                                                                                     flours ranged from 44.64 to 64.32 mg/g wet weight
                                                                                                     basis (Table I). These results were much higher
                     60.0                                                                            than those reported in literature for fresh and
                                                                                                     steamed sweet potato flours (Yang and Gadi 2008).
                     57.5                                                                            The results of multiple regression showed that the
                                                                                                     total phenolic content was significantly affected by the
                     55.0                                                                            interaction term of concentration and soaking time
                         1.0                  1.5           2.0            2.5             3.0       (X2, X3, P # 0.05). The final predictive model for
                                                     Concentration (%)                               phenolic content is given below:
                                                    42.68     46.12       49.56    53.00
                     Phenolic content (mg/g)
                                                    56.43     59.87       63.31                                      Y ¼ 2176:82 2 5:97X2 X3

                                                                                                     The response surface plots in Figures 3 and 4 show the
(b) 65.0
                                                                                                     relationship between the phenolic content and drying
                                                                                                     temperatures, citric acid concentrations and soaking
 Temperature (°C)

                                                                                                     times. The total phenolic content increased with
                                                                                                     increasing drying temperatures (Figure 3a,b). This
                     60.0                                                                            might release more bound phenolic compounds from
                                                                                                     the breakdown of cellular constituents. Huang et al.
                     57.5                                                                            (2006) found that steaming treatment increased the
                                                                                                     total phenolic content of purple sweet potato flour.
                     55.0                                                                            Dewanto et al. (2002a) also found that the free
                         1.0                  1.5           2.0            2.5             3.0       phenolic content of sweet corn increased with
                                                     Soaking time (min)                              increasing heating temperature and time. However,
                    Phenolic content (mg/g)
                                                    42.68    46.12       49.56     53.00             thermal processing had no effect on the phenolic
                                                    56.43    59.87       63.31                       content of tomato (Dewanto et al. 2002b). On the other
                                                                                                     hand, total phenolic contents decreased with increas-
Figure 4. Contour plots showing the effects of temperature, citric                                   ing the concentration and soaking time (Figure 4a,b).
acid concentration and soaking time on phenolic contents of purple
                                                                                                     This was probably because some phenolic compounds
sweet potato flour. (a) Temperature and citric acid concentration.
(b) Temperature and soaking time.                                                                    were more hydrolyzed or oxidized because dispersions
                                                                                                     were prepared in the presence of ambient oxygen.
for Y is given below:
                    Y ¼ 1240:40 2 36:84X1 2 47:49X3 þ 0:28X2
                                                           1                                         Optimization of pretreatments and verification of models

                            þ 3:12X2 þ 3:92X2 þ 4:43X2 X3                                            The predicted and experimental results are presented
                                   2        3
                                                                                                     in Table IV. For the phenolic content, the predicted
Figures 1 and 2 show the response surface plots of the                                               response surface of the stationary point was a saddle
relationship between anthocyanin content and drying                                                  point. Thus the estimated surface did not have a
temperatures, citric acid concentrations and soaking                                                 unique optimum. However, for the anthocyanin
times. Anthocyanin contents decreased with increas-                                                  content, the predicted response surface of the
ing drying temperatures (Figure 1a,b). This was as                                                   stationary point was a minimum. Therefore, different
expected because heating opened the structure of                                                     optimum conditions were obtained for both responses.
anthocyanin to form chalcones, which was degraded                                                    The optimal conditions for anthocyanin and phenolic
further to form brown products (Delgado-Vargas et al.                                                contents were 62.918C, 1.38%, 2.53 min, whereas for
2000). However, the anthocyanin content increased                                                    total phenolic contents they were drying temperature
with increasing soaking time and concentration                                                       60.948C, citric acid concentration 1.04% and soaking
(Figure 2a,b). This might be due to interaction                                                      time 2.24 min. However, optimal conditions of the
between citric acid and anthocyanin. In acidic media,                                                anthocyanin content were not apparent. This is due to

                             Table IV. Comparison of predicted and experimental values for the response of anthocyanin and phenolic contents.

                                                                                  Optimum conditions                                                Values

Response variable                        Stationary point         Temperature (8C)         Soaking time (min)   Soaking concentration (%)   Experimental     Predicted

Anthocyanin                              Minimum                       62.91                     2.53                     1.38              19.78 ^ 0.97      19.71
Total phenolics                          Saddle                        60.64                     1.04                     2.24              61.55 ^ 2.9       52.89
96     M. Ahmed et al.

the fact that the optimization point was a minimum.                    Fan G, Han Y, Gu Z, Chen D. 2008. Optimization conditions for
The optimal value of anthocyanin content was                               anthocyanins extraction from purple sweet potato using response
                                                                           surface methodology (RSM). LWT Food Sci Technol 41:
lower than expected values. This might be related to
                                                                           155 –160.
the anthocyanin extraction conditions by second-
                                                                       Huang YC, Chang YH, Shao YY. 2006. Effects of genotype and
order polynomials (Fan et al. 2008). The corres-                           treatment on the antioxidant activity of sweet potato in Taiwan.
ponding experimental responses of anthocyanin and                          Food Chem 98:529–538.
total phenolic contents were 19.78 mg/100 g and                        Jiang Y. 2000. Role of anthocyanins, polyphenoloxidase and phenols
61.55 mg/g, respectively. These data showed that the                       in lychee pericarp browning. J Sci Food Agric 80:305–310.
experimental responses were reasonably close to the                    Montgomery DC. 1984. Design and analysis of experiments. 2nd ed.
predicted responses.                                                       New York: John Wiley and Sons.
                                                                       Oki T, Masuda M, Furuta S, Nishiba Y, Terahara N, Suda I. 2002.
                                                                           Involvement of anthocyanins and other phenolic compounds in
                                                                           radical-scavenging activity of purple-fleshed sweet potato
The results of anthocyanin and phenolic contents were                      cultivars. J Food Sci 67:1752–1756.
higher than the previous reported values for raw, steam                Pathirana LC, Shahidi F. 2005. Optimization of extraction of
                                                                           phenolic compounds from wheat using response surface
and kneaded sweet potato flours. Therefore, treated
                                                                           methodology. Food Chem 93:47– 56.
flours could be used to make the higher quality products                Proctor JTA. 1974. Colour stimulation in attached apples with
that would be more attractive to product developers and                    supplementary light. Can J Plant Sci 54:499–503.
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Declaration of interest: The authors report no                             (Ipomoea batatas) varieties. Food Chem 113:1133–1138.
conflicts of interest. The authors alone are responsible                Sing S, Raina CS, Bawa AS, Saxena DC. 2003. Optimization of
for the content and writing of the paper.                                  processing variables in the preparation of sweet potato chips
                                                                           using response surface methodology. Euro Food Res Technol
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