OPTIMIZATION OF THE ENZYME ASSISTED EXTRACTION OF BETALAIN FROM DRAGON FRUIT (HYLOCEREUS UNDATUS)

PEEL BY PECTINASE PREPARATION

Nguyen Thanh Sang1, Hoang Kim Anh2, Le Van Viet Man1

1Department of Food Technology, Ho Chi Minh City University of Technology

2Department of Food Technology, Saigon Technology University

*E-mail: lvvman@hcmut.edu.vn

Received: XX September 2014, Accepted: XX October 2014

ABSTRACT

Pectinex Ultra SP-L with polygalactorunase activity was used for betalain extraction from dragon fruit (Hylocereus undatus) peel. Firstly, Plackett-Burman model was used to select the technological factors for extraction optimization. Material : solvent ratio and extraction time were the factors that hightly affected the betalain yield. Response surface methodology (RSM) was then applied for maximizing the betalain yield. The optimal material : solvent ratio and biocatalytic time were 1:28 and 66 min, respectively. Under these conditions, the maximal betalain yield achieved 1,46 mg/g material dry weight and this value was 2,1 times higher than that in the control without enzymatic treatment.

Keywords: betalains, dragon fruit peel, pectinase, Plackett-Burman, response surfacemethodology.

1. INTRODUCTION

In recent years, natural colorants have attracted great interest mainly because of the apparent lack of toxicity and eco-friendliness. In addition, natural colorants are found to be nutritional antioxidants and their presence in the diet can reduce the risk of cardiovascular diseases, cancer and diseases associated with ageing [1].

Betalains are water-soluble nitrogen-containing pigments, which consist of the red-violet betacyanins and the yellow-orange betaxanthins. Dragon fruit (Hylocereus undatus) peel is rich in betalains [2]. However, pectin in dragon fruit peel increased solvent viscosity during the extraction and reduced the betalain yield [3].The application of pectinase preparation to betalain extraction from dragon fruit peel is therefore essential. Our preliminary study showed that material : solvent ratio, enzyme concentration, pH, temperature and biocatalytic time were important factors affecting extraction efficiency. There has been no report on optimization of enzyme-assisted extraction of betalains from dragon fruit peel.

The objective of this study was to optimize the extraction variables for maximizing the betalain yield from dragon fruit peel.

2. MATERIALS AND METHODS

2.1. Materials

Peel of dragon fruit (Hylocereus undatus) was obtained from Rong Xanh company (Phan thiet, Vietnam).

Pectinex Ultra SP-L produced by Aspergillus aculeatus was obtained from Novozymes (Switzerland). The preparation contains polygalacturonase and other activities such as pectinlyase, cellulase, polyphenol oxidase, β-glucosidase. The catalytic activity of Pectinex Ultra SP-L is 4188 polygalacturonase unit (PGU) per mL. Theoptimal temperature and pH of this preparation are 50

oC and 4,5; respectively.

Distilled water - an eco-friendly solvent - was used for betalain extraction.

2.2. Experimentation

Peel of dragon fruit was firstly washed and the blanched in hot water at 90-95oC for 3 min.

The peel was then ground in a blender (Panasonic, Malaysia). The peel mash was then sieved through a 0,5 mm-mesh screen, kept at -18

oC and used for the experimentation. Before use, the

peel mash of dragon fruit was mixed with distilled water at the appropriate ratio and adjusted to pH 4,5 for further enzyme-assisted extraction of betalains. For each assay, samples of 30g peel mash were taken and placed into 250 mL beakers, which were covered with aluminium-foil paper to prevent the oxidative change from light.

2.2.1. Selection of technological factors of the enzyme-assisted extraction by Plackett–Burman design

Different technological factors of the enzyme-assisted extraction including material : solvent ratio (Z1) (g material dry weight per mL solvent), pectinase concentration (Z2) (PGU/g material dry weight), extraction temperature (Z3) (

oC), pH of solvent (Z4) and biocatalytic time

(Z5) (min) were tested using Plackett–Burman design [5]. Each technological factor was examined at two levels: low (-) and high (+) values. The values of two levels were selected according to our preliminary experimental results (Data not shown).The Plackett-Burman design is shown in Table 1.

2.2.2. Optimization of the enzyme-assisted extraction by RSM

Based on the results of section 2.2.1, two technological factors which had the highest impact on the betalain yield were selected for optimization of the enzyme-assisted extraction. RSM with central composite circumscribed (CCC) design was used. In this experiment, a two-

factor, five-level ( , –1, 0, +1, ) CCC design with 13 runs were employed. The two

technological factors were denoted as X1 and X2. The dependant variable was betalain yield (mg/g material dry weight).

2.3.Analytical methods

Betalain content in the extract was determined by spectrophotometric method [3]. The results were expressed as mg of betalain per 1 g of dry weight of dragon fruit peel.

2.4.Statistical analysis

Modde version 5.0 was used for experimentation design as well as for regression and graphical analysis of the experimental data obtained.

3. RESULTS AND DISCUSSION

3.1.Selection of technological factors of the enzyme -assisted extraction by Plackett–Burman design

The experimental results of Plackett-Burman design are visualized in Table 1. Analysis of the regression coefficients and Pvalues of 5 factors is shown in Table 2. The factor with confidence level above 95% is considered as significant factor. It was clear that variables Z1, Z2, Z4, and Z5

were the significant factors, while factor Z2 was considered insignificant. According to the affecting coefficient, Z1, Z4, Z5 had positive effects on betalain yield whereas Z2 had negative effects. The absolute value of Z1 and Z5 were the highest. Therefore, the material : solvent ratio (Z1) and biocatalytic time (Z5) were selected for further optimization. In a previous study, material : solvent ratio and extraction time were also considered as important variables and used to optimize oil extraction from sunflower seeds [4]. Although three factors Z2, Z3, Z4 were not selected for optimization but their impacts on the extraction yield must be confirmed.

Table 1.Results of Plackett-Burman model for the effect of variables on the betalain yield (mg/100g of material dry weight)

Run Technological factors Betalain

yield Run

Technological factors Betalain yield Z1 Z2 Z3 Z4 Z5 Z1 Z2 Z3 Z4 Z5

1 1:30 5,2 25 6 30 1,19 9 1:30 5,2 25 6 30 1,17

2 1:30 15,6 25 4 90 1,11 10 1:30 15,6 25 4 90 1,10

3 1:30 15,6 35 4 30 1,06 11 1:30 15,6 35 4 30 1,05

4 1:20 15,6 35 6 30 1,02 12 1:20 15,6 35 6 30 0,94

5 1:30 5,2 35 6 90 1,28 13 1:30 5,2 35 6 90 1,26

6 1:20 15,6 25 6 90 1,08 14 1:20 15,6 25 6 90 1,10

7 1:20 5,2 35 4 90 1,12 15 1:20 5,2 35 4 90 1,07

8 1:20 5,2 25 4 30 0,99 16 1:20 5,2 25 4 30 0,92

Table 2.Statistical analysis of Plackett-Burman model for the effectsof variables

onbetalain yield

Tech. factors Affecting coefficient PValue at 5% significant level

Z1 0,529 0,0000159*

Z2 -0,288 0,0018* Z3 0,072 0,3188

Z4 0,337 0,0006* Z5 0,412 0,0001*

3.2.Optimization of the enzyme-assisted extraction by RSM

Table3.Experimental design and results

Run X1 X2 Betalain yield

(mg/100g dry weight) Run X1 X2

Betalain yield

(mg/100g dry weight)

1 1:20 30 0,48 8 1:25 102 1,25

2 1:30 30 1,17 9 1:25 60 1,35

3 1:20 90 0,74 10 1:25 60 1,34

4 1:30 90 1,11 11 1:25 60 1,34

5 1:17 60 0,51 12 1:25 60 1,40

6 1:32 60 1,37 13 1:25 60 1,39

7 1:25 18 0,66

The experimental design and the results are presented in Table 3. Multiple regression analysis was used to analyze the data and thus a second-order polynomial equation was derived as follow:

Table 4 shows that the coefficient of determination (R2) was calculated as 0,956 indicating

good agreement between the experimental and the predicted values [5]. Material : solvent ratio (X1) and biocatalytic time (X2) had significantly positive effects on betalain yield, while their quadratic effects were also observed, but negative. Material : solvent ratio (X1) had higher effect on betalain yield than biocatalytic time (X2).

Table 4.Coefficient list for response surface

quadratic model

Betalain content

Coefficient Standard

Error Pvalue

Constant 1,36401 0.0432 8,31E-009

X1 0,28153 0.0341 7,57E-005

X2 0,13055 0.0341 0,00655

X12 -0,23266 0.0366 0,00039

X22 -0,22266 0.0366 0,00051

X1X2 -0,07750 0.0483 0,15293

N = 13 DF = 7 Q2

= 0,702 R2 = 0,956

A B

Figure 1. Effect of material:solvent ratio and extraction time onbetalainyield (A: two-

dimensional contour plots, B: three-dimensional

response surface plots)

Figure 1 shows the response surface plots and their corresponding contour plots. An important increase in Y could be achieved when the value of material : solvent ratio decreased from 1:17 to 1:28 g material dry weight/mL solvent, and the biocatalytic time increased 18 to 66 min.The predicted maximal betalain yield was 1,45 mg/g material dry weight at the material : solvent ratio of 1:28 and biocatalytic time of 66 min. In order to verify the accuracy of the model, three independent replicates were conducted under the optimal conditions. The betalain yield was 1,46±0,004 mg/g material dry weight. The experimental values were therefore similar to the predicted value from equation (1). The betalain yield obtained was 2,1 times higher than that in the control without enzymatic treatment. Recently, Maran et al. (2013) optimized the conditions of thebetalain extraction from prickly pear fruits and showed that the maximal betalain yield was 0,42 mg/g material [1]. Our results showed higher extraction yield; that was

probably due to different distributions of the solutes in different plant materials and enzyme application to betalain extraction.

4. CONCLUSION

In the enzyme-assisted extraction of betalains from dragon fruit peel, material : solvent ratio and biocatalytic time were the most important technological factors. Maximal betalain yield achieved 1,46 mg/g material dry weight when the material : solvent ratio and biocatalytic time were 1:28 and 66 min, respectively. The betalain yield obtained from RSM was 2,1 times higher than that in the control without enzymatic treatment.

REFERENCES

1. Maran, J.P., ManikandanS., Mekala V. - Modeling and optimization of betalain extraction from Opuntiaficus-indica using Box–Behnken design with desirability function,Industrial Crops and Products 49 (2013): 304-311.

2. Wybraniec S., Nowak-Wydra B., Mitka K., Kowalski P., MizrahiY. - Minor betalains in fruits of Hylocereusspecies, Phytochemistry 68 (2007): 251-259.

3. SchweiggertR.M., Villalobos-Gutierrez M.G., Esquivel P., Carle R. - Development and optimization of low temperature enzyme-assisted liquefaction for the production of colouring foodstuff from purple pitaya (Hylocereus sp.[Weber] Britton & Rose), European Food Research and Technology 230 (2009): 269-280.

4. Sineiro J., o gue H., Núñez M.J., Lema J.M. - Optimization of the enzymatic treatment during aqueous oil extraction from sunflower seeds, Food Chemistry 61 (1998): 467-474.

5. ZhouJ.,Yu X., Ding C., Wang Z., Zhou Q., Pao H., Cai W.- Optimization of phenol degradation by Candida tropicalis Z-04 using Plackett-Burman design and response surface methodology, Journal of Environmental Sciences 23 (2011): 22-30.

TÓM TẮT

TỐI ƯU HÓA QUÁ TRÌNH XỬ LÝ ENZYME ĐỂ TRÍCH LY BETALAIN TỪ VỎ QUẢ THANH LONG (HYLOCEREUS UNDATUS)VỚI CHẾ PHẨM PECTINASE

Nguyễn Thanh Sang1, Hoàng Kim Anh2, Lê Văn Việt Mẫn1

1Bộ môn Công nghệ thực phẩm, Khoa Kỹ thuật hóa học, Trường Đại học Bách khoa thành phố

Hồ Chí Minh 2Khoa Công nghệ thực phẩm, Trường Đại học Công nghệ Sài Gòn

*E-mail: lvvman@hcmut.edu.vn

Đến tòa soạn: XX September 2014, Chấp nhận: XX October 2014

Pectinex Ultra SP-L với hoạt tí h polygalactoru ase được sử dụ g để trích ly betalain từ vỏ quả thanh long (Hylocereus undatus). Đầu tiên, mô hình Plackett-Bur a được sử dụng để chọn

các yếu tố công nghệ cho thí nghiệm tối ưu hóa quá trì h trích ly. Tỉ lệ nguyên liệu:dung môi và thời gian trích ly là những yếu tố ả h hưởng nhiều đến hiệu suất trích ly betalain. Tiếptheo, phươ g pháp đáp ứng bề mặt được áp dụ g để hiệu suất trích ly betalain đạt giá trị cực đại. Tỉ lệ nguyên liệu:dung môi và thời gian xúc tác tối ưu của enzyme lần lượt là 1:28 và 66 phút. Khiđó, hà lượng betalain tối đa thu được là 1,46 mg/g chất khô nguyên liệu và giá trị ày cao hơ gấp 2,1 lần so với mẫu đối chứng không xử lý enzyme.

Từ khóa: betalains, vỏ thanh long, pectinase, Plackett-Bur a , phươ g pháp đáp ứng bề mặt