International Journal of Biological Macromolecules 50 (2012) 10081010

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Short communication

Dehydration of crude protein from Ginkgo biloba L. by microwave freeze drying

Liuping Fan a , Shaodong Ding a , Yuanfa Liu a, , Lianzhong Ai b
a b

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China State Key Laboratory of Dairy Biotechnology, Technology Center, Bright Dairy & Food Co. Ltd., Shanghai 200436, China

a r t i c l e

i n f o

a b s t r a c t
The paper optimized the parameters of microwave freeze drying (MFD) of crude Ginkgo biloba protein (CPG) using response surface methodology (RSM) based on the analysis of its proximate composition. The results showed that coefcients of determination, R2 values for drying time and protein solubility were greater than 0.9500. The drying time and protein solubility of CPG varied curvilinearly with increase of microwave power and pre-freeze temperature, and drying time varied linearly with material thickness. The optimum MFD condition was microwave power of 408421 W, material thickness of 15 mm and pre-freeze temperature of 20 C to 21 C, respectively. 2012 Elsevier B.V. All rights reserved.

Article history: Received 14 February 2012 Accepted 22 February 2012 Available online xxx Keywords: Ginkgo biloba L. Protein Microwave freeze drying

1. Introduction Numerous techniques can be used to dry solutions of proteins and obtain protein powders with desirable characteristics [1]. However, the most widely used techniques to dehydrate proteins are vacuum freeze drying (FD). The FD dehydration can minimize the losses of active components of foods owing to low temperature and low absolute pressure [2]. In the other hand, FD is also recognized as an expensive technology, because it requires much energy. The costs are 48 times higher compared to air drying [3]. There are lots of researches related to reducing FD costs by improving heat and mass transfer [4,5]. Among these improvements, MFD appears to be one of the most promising techniques to accelerate the rate of dehydration and enhance overall quality. When applied to the FD process, microwave energy can penetrate very well into ice and reduces the drying time by as much as 75% [6]. Recently, MFD has been used successfully for drying beef, skim milk, cabbage et al. [79]. Ginkgo biloba L. (usually called ginkgo) can date back to 200 million years ago and is considered as a living fossil. The proteins of ginkgo with special biological activity have attracted extensive attention [1012]. These researches indicated that Ginkgo biloba seed proteins exhibited favorable bioactivity, and could be applied in the food industry as functional additives. However, few of studies have been conducted on the effects of drying methods on the characteristics of Ginkgo seeds protein. The purpose of this paper is to optimize the microwave freeze drying parameters of crude protein powders from Ginkgo biloba.

2. Materials and methods 2.1. Materials The Ginkgo biloba L. cv Dafozhi seeds were purchased from Taixing of Jiangsu province (China). All reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (China). All results represent the average of duplicate determinations.

2.2. Chemical composition analysis Protein content was calculated from the nitrogen content (%N 6.25) analyzed by Kjeldahl method. Water and fat content were determined by the ofcial method of AOAC. Total sugar content was examined using phenolsulfuric acid colorimetric method. The polyphenol content was measured by the FolinCiocalteau method.

2.3. Extraction of protein from Ginkgo biloba L. seeds Crude Ginkgo seed protein (CPG) was obtained by an alkaline dissolving and acid precipitating method [13]. The defatted our of Ginkgo biloba L. was dispersed in distilled water (10%, w/v), and the pH was adjusted to 9.0 using 1 M NaOH. The suspension was stirred for 30 min at room temperature, and then centrifuged at 6000 g for 15 min. The extracts were combined and acidied to pH 4.4 with 1 M HCl, and then left to stand for 30 min to separate into two layers. The precipitates were recovered by centrifugation, then neutralised by 1.0 M NaOH to pH 7.0 and dialysed in distilled water for 24 h. The neutralised precipitate was dried using MFD.

Corresponding author. Tel.: +86 0510 85876799. E-mail address: fanliuping@jiangnan.edu.cn (Y. Liu). 0141-8130/$ see front matter 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2012.02.027

L. Fan et al. / International Journal of Biological Macromolecules 50 (2012) 10081010 Table 1 The coded values of the independent variables. Variable Codes 1 X1 (W) X2 (mm) X3 ( C) 350 10 25 0 400 15 20 1 450 20 15

1009

Table 2 The results of response surface analysis of the variation of drying time (Y1 ) and solubility (Y2 ) with microwave power (X1 ), material thickness (X2 ) and pre-freeze temperature (X3 ). Run Coded variables X1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 1 1 1 0 0 0 0 1 1 1 1 0 0 0 X2 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 X3 0 0 0 0 1 1 1 1 1 1 1 1 0 0 0 8.8 11.2 4.4 5.2 4.9 8.3 7.3 10.1 10.5 4.5 11.9 10.1 5.4 5.9 5.3 48 50 56 55 48 60 59 57 53 52 50 59 67 66 66 Drying time (h) Solubility (%)

2.4. Drying experiments of CPG Experiments were carried out in a lab-scale microwave freeze dryer (YT2S-01, Nanjing Yatai Microwave Power Technology Research Institute, China). Before microwave freeze drying (MFD), CPG was rst frozen for 24 h. During MFD, the pressure was maintained at 100 Pa by a vacuum pump, and the cold trap temperature (40 to 45 C) was low enough to condense vapor. The microwave frequency was 2450 MHz and the power could be regulated continually from 0 to 2000 W.

3.2. Process optimization 2.5. MFD optimization of CPG A RSM was used to optimize the MFD whilst taking short drying time and retaining high solubility of CPG. The three independent variables in this experiment were microwave power (X1 ), material thickness (X2 ) and pre-freeze temperature (X3 ). The two dependent variables were the drying time (Y1 ) and protein solubility (Y2 ). The coded values of the three independent variables are summarized in Table 1. The coded values of the three independent variables and the results are summarized in Table 2. Results revealed that the drying time of CPG varied from 4.4 h to 11.9 h, solubility of CPG varied from 48% to 67%. Data were analyzed by the SAS multivariate regression program and could be tted to the following equation.
2 2 2 Y = b0 + b1 x1 + b2 x2 + b3 x3 + b11 x1 + b22 x2 + b33 x3 + b12 x1 x2

+ b13 x1 x3 + b23 x2 x3

(1)

2.6. Properties analysis of CPG by MFD Drying time was determined according to the moisture content of the samples which dropped to 5% on the wet basis. For the protein solubility test, CPG of 1% was dispersed in phosphate buffer of pH 7.0 at room temperature. The protein suspension was stirred using a magnetic stirrer for 60 min at a speed of 500 rpm and were centrifuged at 6000 g for 30 min. The soluble protein content in the supernatant was determined by Kjeldahl method.

The regression coefcients and analysis of variance of CPG are listed in Table 3. The high coefcients of determination R2 (0.9875 and 0.9751) indicated that the variables were adequately tted to the regression equation. The probability (p) values of all regression models were less than 0.01. The coefcient of variation (CV) is the ratio of the standard error of estimate to the mean value of observed response expressed as a percentage. It is a measure of reproducibility of the models. The CV of the model was calculated as 6.6% and 3.01%, respectively. As a general rule, a model can be considered reasonably reproducible if its CV is not greater than 10% [14]. 3.3. Effects of MFD on the drying time The drying time (Y1 ) of CPG was signicantly (P < 0.05) affected by the linear terms of X1 , X2 and X3 , interact terms of X1 X3 and
Table 3 Regression coeffcients, R2 and p values for the response function (Eq. (1)). Coefcient b0 b1 b2 b3 b11 b22 b33 b12 b13 b23 R2 CV P
a b c

2.7. Statistical analysis Data were analysed by using the Statistical Analysis System (SAS, version8.0, SAS Institute Inc., Cary, NC, USA). Analyses of variance were performed by the anova procedure. Mean values were considered signicantly different when p < 0.05.

3. Results and discussion 3.1. Proximate composition The proximate composition results of the de-hulled Ginkgo biloba L. cv Dafozhi showed that water was the dominant component (48.36 1.89% in wet basis), followed by starch and protein with 71.38 1.27% and 11.57 0.16% in dry basis, respectively. The protein content of sample was in agreement with the reports by Deng et al. and they found that there is 913% crude protein (dry basis) in Ginkgo biloba seed [10]. The protein content is high on a dry matter basis, which makes Ginkgo biloba a good supplement to protein resource. Low contents of lipid (5.45 0.43%, d.b.), total sugar (3.17 0.18%, d.b.) and total phenolic (3.21 0.15, d.b.) were found in de-hulled Ginkgo biloba L. sample.

Y1 (drying time) 5.53 2.28c 0.93b 1.65c 1.73b 0.13 1.98c 0.4 1.05b 0.15 0.9875 6.60 0.0003

Y2 (solubility) 66.33 2.63b 1.13 1.75a 8.29c 5.79b 4.54b 0.75 2.5a 3.5b 0.9751 3.01 0.0017

Signicant at p < 0.05. Signicant at p < 0.01 Signicant at p < 0.001.

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L. Fan et al. / International Journal of Biological Macromolecules 50 (2012) 10081010

indicated that the medium concentration in microwave power and pre-freeze temperature extracted higher solubility of CPG. The solubility could be above 66% when the material thickness was xed at 15 mm, microwave power varied from 393 W to 422 W and prefreeze temperature from 21 C to 17 C. The solubility increased with increasing material thickness and pre-freeze temperature and then decreased when material thickness was above 16 mm and pre-freeze temperature was below 20 C. 3.5. Optimum drying conditions for the higher solubility and shorter drying time By analyzing the effects of MFD conditions on the drying time and solubility of CPG, the drying time and solubility changed signicantly with microwave power (X1 ) and pre-freeze temperature (X3 ). Contour plot of both drying time and solubility as a function of microwave power and pre-freeze temperature is shown in Fig. 1. It suggested that optimum drying condition was microwave power of 408421 W and pre-freeze temperature of 20 C to 21 C, respectively suggested by the cross hatched area. The drying time and solubility of CPG were expected to be about 5 h and 60%, respectively. Acknowledgements quadratic terms of X1 2 and X3 2 . The drying time linearly increased with increasing material thickness and varied curvilinearly with pre-freeze temperature, and this may be related to the penetration depth of microwave. The shorter drying time was observed at the pre-freeze temperature from 22 C to 20 C. Both microwave power and pre-freeze temperature exerted a quadratic effect on drying time. The drying time dramatically declined with increasing microwave power. This result was in agreement with that of Abbasi [5] and the drying process accelerated owing to higher mass transfer rate and vapour pressure difference between the central and the external parts of the products. When the CPG was dried at the microwave power more than 400 W and material thickness lower than 15 mm, drying time will be shortened to less than 5 h. 3.4. Effects of MFD on the solubility of CPG The protein solubility (Y2 ) of CPG was signicantly (P < 0.05) affected by the linear terms of X1 , X3 , interact terms of X1 X3 , X2 X3 and quadratic terms of X1 2 , X2 2 and X3 2 . Material thickness did not seem to affect protein solubility in the selected range whereas microwave power exerted a signicant effect. CPG has the higher solubility at the microwave power of 394421 W. The loss of solubility of CPG in high-power microwaves can be attributed to the protein denaturation under higher temperature. Deng et al. found the total contents albumin and globulin were 90.8% in Ginkgo seed protein isolate [10]. However, the albumins and globulin appeared as the most readily denatured proteins and their solubility index dropped signicantly as the drying temperature increased [15,16]. The loss of solubility in low-power microwaves can be attributed to the long drying time and this result was in agreement with that of Joshi et al. [15]. Both microwave power and pre-freeze temperature exerted a quadratic effect on protein solubility of CPG. The results This work was supported by the National Natural Science Foundation of China (31101361), the National High Technology Research and Development Program of China (2011AA100806-3) and the Fundamental Research Funds for the Central Universities (JUSRP211A30). References
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Fig. 1. The contour plots of the drying time and solubility of CPG as affected by microwave power and pre-freeze temperature (material thickness 15 mm).