Food Research International 50 (2013) 603609

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Stabilization of grape skin anthocyanins by copigmentation with enzymatically modied isoquercitrin (EMIQ) as a copigment
Qiuli Yan a, Linhan Zhang a, Xiaofei Zhang a, Xuan Liu a,b, Fang Yuan a, Zhanqun Hou a, Yanxiang Gao a,
a b

College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China Beijing Institute of Nutritional Resources, Beijing 100069, PR China

a r t i c l e

i n f o

a b s t r a c t
The thermal and light stability of grape skin anthocyanins with enzymatically modied isoquercitrin (EMIQ) as a copigment was investigated at different pH levels of 3, 4 and 5. The ratios of anthocyanins to EMIQ were 2:1, 1:1, and 1:2 (w/w), respectively, in the thermal experiments at 90 C, and EMIQ concentrations (0.25, 0.5, and 1%, w/w) were evaluated respectively in the light experiments. Results revealed that the degradation of anthocyanins copigmented with EMIQ followed rst-order reaction kinetics. The half life of anthocyanins extended signicantly with the increase of EMIQ concentration (p b 0.05), moreover, the color stability increased due to the addition of EMIQ as the total color difference values E* were smaller for the copigmented anthocyanins. The magnitude of the bathochromic (max) shifted to the longest wavelength absorption band with the increasing copigment concentration for all pH levels. Results demonstrated that EMIQ was an effective copigment to stabilize grape skin anthocyanins. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 11 November 2010 Accepted 5 April 2011 Keywords: Anthocyanins Enzymatically modied isoquercitrin Copigment Stability

1. Introduction Color is one of the most important characteristics of fruit and vegetable products signicantly determining customers' choice (Stintzing & Carle, 2004). To improve the visual appearance and restore initial color shades, synthetic colorants have commonly been applied in food processing. However, toxic effects have been described, resulting in a ban of some of these synthetics. A recent study demonstrated that synthetic colorants were shown to increase hyperactivity in children (attention-decit hyperactivity disorder ADHD) syndrome (McCann et al., 2007).For these reasons, synthetic food additives are increasingly rejected by consumers. So far, natural pigments, such as anthocyanins, have not been broadly used in foods and beverages since they are not as stable as synthetic colorants. Therefore, great attentions have been paid to the intrinsic and extrinsic factors affecting pigment stability and color shade, such as temperature, the presence of light, pH value (Baranac, Petranovi, & Dimitri-Markovi, 1996) and thermal processing, such as pasteurization, sterilization or concentration particularly causing the degradation and color loss (Sadilova, Stintzing, Kammerer, & Carle, 2009). Copigmentation is a natural phenomenon which plays a major role in the expression of a wide range of colors provided by anthocyanins in plants, fruits, vegetables, and food products. The study carried out by Mazza (1993) showed that the molecular complexation of anthocyanins with other phenols named copigments is the main

Corresponding author. Tel.: + 86 10 62737034; fax: + 86 10 62737986. E-mail address: gyxcau@126.com (Y. Gao). 0963-9969/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2011.04.007

color-stabilizing mechanism in plants. The color of the solution can be greatly enhanced when the copigment was added. The copigments promote the stabilization of the colored structural forms of the anthocyanins and consequently enhance the retention of their color (Liao, Cai, & Haslam, 1992).The copigments may be one of avonoids, alkaloids, amino acids, organic acids, nucleotides, polysaccharides, metals, and anthocyanins themselves (Mazza & Brouillard, 1990). The complexation of a copigment with an anthocyanin causes a bathochromic shift and hyperchromic effect which means an increase in color intensity (Mazzaracchio, Pifferi, Kindt, Munyaneza, & Barbiroli, 2004). Several studies have evaluated the stability of anthocyanins present in juices, wines and fruit products due to the interaction between pigments and phenols. The presence and amount of hydroxyl and/or glycosylated groups on the copigment structure had a great inuence on the copigmentation as well as on anthocyanins stabilization (Cavalcanti, Santos, & Meireles, 2011). Gonzlez-Manzano, Dueas, Rivas-Gonzalo, Escribano-Bailn, and Santos-Buelga (2009) testied the importance of the qualitative phenolic composition, determined in the wine by the type of grape and winemaking practices, to the production of an effective copigmentation process. Meanwhile, Pacheco-Palencia and Talcott (2010) investigated the inuence of polyphenolic copigments on the phytochemical and color stability of anthocyanins in aai fruit and the results revealed that avone-C-glycoside is a stabilizing agent. The results were in accordance with previous study carried out by Chen and Hrazdina (1981) which showed that avonoids that contained many hydroxyl groups are the best copigments. Enzymatically modied isoquercitrin(EMIQ) which was approved as generally regarded as safe (GRAS) by FDA in 2003, was the world's

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rst commercial water-soluble avonol glycoside obtained from rutin manufactured by transglycosylation with cyclodextrin glucanotransferase (CGTase) (Akiyama, Washino, Yamada, Koda, & Maitani, 2000). The glycosylated groups were important for EMIQ as a copigment and the solubility of isoquercitrin was also improved by adding glycosyl instead of rhamnose. EMIQ has been used in various beverages and foods to prevent the deterioration of avor and color caused by sunlight or uorescent light irradiations in stores. The structure of EMIQ was shown in Fig. 1. There are two main methods to stabilize anthocyanin: encapsulation and copigmentation. Although the stability of anthocyanins could be increased by encapsulation, it had some drawbacks as the requirement for sophisticated equipments and high percentage of losses obtained in the encapsulation process (Cavalcanti et al., 2011). However, as a copigment, EMIQ was available and could be used easily in food industry. For the high antioxidant ability of EMIQ, furthermore, the positive effect of EMIQ was not only related to the stability of anthocyanins, but also to the functionality and possible improvement of the antioxidant capacity of food products. The objective of the present work was to study the effect of EMIQ as a new copigment on the stability of grape skin anthocyanins during thermal process and light exposure. 2. Materials and methods 2.1. Materials Grape skin anthocyanin solution (AC 3 WS colorant) was obtained from Chr. Hansen (Hrsholm, Demark). EMIQ was provided by San-Ei Gen. (Osaka, Japan). The content of EMIQ was 20% (w/w). All the reagents (HPLC grade) were obtained from Merck (Shanghai, China). Other chemicals and solvents (analytical grade) used were purchased from Beijing Chemical Co. (Beijing, China). 2.2. Thermal stability test of anthocyanins with EMIQ An aliquot of 5 ml grape skin anthocyanins solution was diluted with McIlvaine buffers (citric acid/disodium hydrogen phosphate) to 250 ml at pH 3, 4, and 5, respectively. After 1 h equilibrium at room temperature before heating, solutions with and without copigment (8 ml) in tubes were placed in a water bath at 90 C. Samples were collected with an interval of 30 min, and then cooled in ice bath for 3 min. 1 ml of the treated solution was collected and diluted to a volume of 10 ml. The total anthocyanin content of the solution was determined by the pH-differential method. Each sample was used for one measurement, in order to minimize the effect of oxygen. 2.3. Light stability test of anthocyanins with EMIQ Light stability test was involved to evaluate the effect of EMIQ on the stability of anthocyanins exposed to light and elevated temper-

atures. An aliquot of 5 ml grape skin anthocyanins solution was diluted with McIlvaine buffers (citric acid/disodium hydrogen phosphate) to 250 ml at pH 3, 4, and 5, respectively. The concentration of EMIQ added was 0.25%, 0.5% and 1% (w/w) of anthocyanins, respectively. Each colored solution was transferred to an IWAKI ask (70 ml, Tokyo, Japan) and exposed to accelerated light in a controlled light cabinet (SUNTEST CPS+, 450 W/m2, 45 C) at 30 min interval. 2.4. Spectrophotometric measurements of anthocyanins All the spectrophotometric measurements were performed by SHIMADZU UV-1800 UVvis spectrophotometer, using 1 cm path length glass cuvettes. After 1 h of equilibration at room temperature, visible spectra from 450 to 700 nm with a 1 nm bandwidth were recorded for the co-pigmentation reactions and colorimetric calculations. 2.5. HPLC-DAD-ESI/MS/MS analysis of anthocyanins Analysis of anthocyanins by HPLCMS/MS was carried out according to the methods described by Wu and Prior (2005) with some modications. Chromatographic analyses were performed on an Agilent 1100 series HPLC equipped with diode array detector (DAD). A Zorbax Stablebond Analytical SB-C18 column (4.6 250 mm, 5 m, Agilent Technologies, Rising Sun, MD) was used for separation. Elution was performed using mobile phase A (aqueous 5% formic acid solution) and mobile phase B (methanol).The ow rate was 1 ml/min, and detection was at 520 nm. Gradient was used as follows: 01 min, 5% B; 115 min, 525% B; 1520 min, 25% B; 2022 min, 2527% B; 2230 min, 2733% B; 3036 min, 33% B; 3643 min, 3340% B; 43 45 min, 47% B; 4551 min, 4750% B; and 5160 min, 505% B. Lowresolution electrospray mass spectrometry was performed with an Esquire 3000 on trap mass spectrometer (MS) (Bruker Daltoniks, Billerica, MA). The experimental conditions were as follows: ESI interface, positive ion, nebulizer pressure, 35 psi; dry gas, nitrogen 11.0 psi; dry temperature, 350 C; collision gas, helium; and MS/MS scan from m/z 100 to 1000. 2.6. Analysis of the total anthocyanin content The total anthocyanin content (TAC) was determined by using the pH-differential method described by Lee, Durst, and Wrolstad (2005) with modication, using two buffer systems: potassium chloride buffer (0.025 M, pH 1.0), and sodium acetate buffer (0.4 M, pH 4.5). Briey, aliquots of 1 ml of the samples were mixed with 9 ml of the pH buffer solution, and the absorbance was measured at 520 and 700 nm. Total anthocyanin content was calculated using the following equation: Total anthocyanin mg = L = A MW DF 1000 1 1

where A = (A520A700)pH=1.0 (A520A700)pH=4.5, MW = molecular weight, DF = dilution factor, 1 = path length (1 cm), Pigment contents were calculated as malvidin-3-O-glucoside using an extinction coefcient of 28,000 L/mol/cm and a molecular weight of 493.2 g/mol, 1000 = conversion from g to mg. 2.7. Thermal degradation kinetics of anthocyanins with EMIQ Previous study showed that the thermal degradation of anthocyanins in aqueous solutions followed the rst order kinetics (Mourtzinos et al., 2008). The reaction rate constants (k) and half lives (t1/2, the time needed for 50% degradation of anthocyanins) were calculated by the following equations: Ln Ct C0 = kt 2 3

Fig. 1. The structure of EMIQ.

t12 = 0:693k

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605

Where C0 is the initial content of TAC, Ct is the anthocyanin content at time t (min). 2.8. Light irradiation of anthocyanins with EMIQ Colorimetric parameters L*, a*, b* (CIE 1976) were directly measured on the IWAKI asks during the treatments using a DataColor SpectraFlash SF450 spectrophotometer (color space CIELAB). The colorimetric coordinates of the colorant solutions were computed in the CIELAB scale in a CIE D65/10 illuminant/observer condition. Color change was calculated as E* = (L*2 + a*2 + b*2)1/2. The untreated samples were used as control. 2.9. Statistical analysis The whole experiment was conducted in duplicate. Data were subjected to analysis of variance (ANOVA) using the software package SPSS 12.0 for Windows (SPSS Inc., Chicago, USA). Means of treatments were separated at the 5% signicance level using the LSD method. 3. Results and discussion 3.1. Characterization and quantication of grape skin anthocyanins The anthocyanins prole of the grape skin was shown in Fig. 2 and Table 1. The 3-mono-glucoside was the main structure of the anthocyanins. Malvindin-3-O-glucoside (oenin) and delphinidin-3O-glucoside were the major components, which represented 45.4% and 16.1% of the total anthocyanin content, respectively. The total anthocyanin content of the grape skin, which calculated as that of malvidin-3-O-glucoside, was 399.84 0.20 mg/ml. 3.2. Analysis of thermal degradation kinetics Kinetic modeling may be employed to predict the inuence of thermal processing on critical quality parameters. Key parameters of degradation kinetics, including rate constant, half life, are vital to predict food quality loss during thermal process. Therefore, kinetic studies are required in order to minimize the undesired change and to optimize the quality of specic foods (Patras, Brunton, O'Donnell, & Tiwari, 2010). The degradation of anthocyanins in juices and concentrates of blackberries (Wang & Xu, 2007), strawberry (Garzon & Wrolstad, 2002) and sour cherry (Cemero lu, Velio lu, & Iik, 1994) under isothermal heating was found to follow the rst order kinetics. In the present study, the stability of grape skin anthocyanins against heating with or without the copigment was investigated at different

Table 1 LCMS data of grape skin extract anthocyanins. Peak Retention [M]+ MS/MS fragments Peak assignment no. time (min) (m/z) (m/z) 1 2 3 4 5 6 7 18.3 20.8 23.5 27.2 29.2 34.9 52.3 465 449 479 463 493 517 535 303 287 317 301 331 355 331 Delphinidin-3-O-glucoside Cyanidin-3-O-glucoside Petunidin-3-O-glucoside Peonidin-3-O-glucoside Malvindin-3-O-glucoside Malvidin-3-O-glucoside-acetal Malvidin3-O-(6-p-coumaroyl) glucoside (trans)

pH levels. Table 2 showed degradation kinetics and parameters of grape extract anthocyanins combined with EMIQ at different pH levels. The effect of copigmentation on the stability of grape extract anthocyanins varied at different ratios of anthocyanins to EMIQ. Fig. 3 illustrates the kinetic plots of each sample. As indicated by the high values of the correlation coefcient (R2 N 0.93), the degradation of the anthocyanins copigmented with EMIQ ideally followed rst order reaction kinetics. With increasing heating times, the stability of grape extract anthocyanins was enhanced in the presence of EMIQ. These ndings are in accordance with previous studies reporting degradation kinetics for anthocyanins both in model systems and food matrices (Mollov, Mihalev, Shikov, Yoncheva, & Karagyozov, 2007). As shown in Table 2, It was found that the t1/2 increased signicantly at pH 3 and 4 (p b 0.05), while there was no signicant difference between anthocyanins with and without copigment at pH 5. The t1/2 increased by 12.6%, 55.7%, and 36.1% for the EMIQ/anthocyanin ratios of 2:1, 1:1 and 1:2, respectively, while 39.3%, 53.7%, and 15.1% at pH 4. The t1/2 values increased more rapidly at pH 3 and 4 than pH 5, especially at pH 3. The anthocyanin half life increased signicantly due to the addition of EMIQ, reecting the lower rate of pigment degradation in the presence of the copigment, EMIQ may prevent the pigment degradation that took place during heating. The results were in agreement with that reported by Brenes et al. (Brenes, Del Pozo-Insfran, & Talcott, 2005). The copigmentation effect of EMIQ was strongly pH-dependent. Rapid degradation was observed when the pH was increased from 3 to 5, and the concentration of vulnerable colorless forms simultaneous elevated. At high temperature, the dissociation of the complex between avylium cation and copigment was increased (Brouillard, Wigand, Dangles, & Cheminat, 1991). The copigmentation effect was maximized in mildly acidic aqueous solutions, where anthocyanins exist essentially in the structures of hemiacetal form B and cis-chalcone CE, which were

Table 2 Degradation kinetics parameters of grape skin extract anthocyanin with EMIQ. Ratio pH 3 Control 2:1 1:1 1:2 pH 4 Control 2:1 1:1 1:2 pH 5 Control 2:1 1:1 1:2 Fig. 2. HPLC separations (520 nm) of grape skin extract anthocyanin. k(min 1) 0.0026 0.0019 0.0023 0.0017 t1/2(min) 266.6a 362.9b 300.1a 415.1c R2 0.93 0.99 0.94 0.94

0.0027 0.0019 0.0017 0.0023

260.6a 362.9b 400.7b 300.1a

0.99 0.95 0.98 0.98

0.0028 0.0025 0.0022 0.0023

249.3a 278.4a 309.4a 300.1a

0.95 0.94 0.99 0.94

Values followed by same letter within a column are not signicant (p b 0.05).

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acylated ones under same conditions (Malien-Aubert, Dangles, & Amiot, 2001). According to the HPLCMS results, there were two acylated forms of malvidin in the grape extract. Nevertheless, they were not dominant components. The main anthocyanin was malvindin-3-O-glucoside (Mv3-G), which was unstable to thermal processing and light exposure. Therefore, intermolecular copigmentation was proposed to play a major role in the stabilization of the colorant. The thermal experiment results suggested that intermolecular copigmentation between anthocyanins and EMIQ dominated the color stability during thermal treatment. The reversible conversion of avylium ions into chalcone forms was an important step in the overall mechanism of anthocyanin thermal degradation (Furtado, Figueiredo, Chaves das Neves, & Pina, 1993). 3.3. Analysis of light irradiation stability Light stability of anthocyanin is another important characteristic for food products. Anthocyanins were sensitive to light exposure especially UV, while adding the copigment could improve their stability (Bkowska, Kucharska, & Oszmiaski, 2003). Figs. 4, 5 and 6 showed the color attributes of grape skin anthocyanins with and without EMIQ as a copigment at different pH levels. For all pH levels, with the time extension of light exposure, the addition of EMIQ to anthocyanin solution caused the decrease of L*, a*, b* and E* values, compared with the control sample without EMIQ. The color stability increased due to the addition of copigment as the total difference values E* were smaller for the copigmented anthocyanins, especially upon extended heating (Shikov, Kammerer, Mihalev, Mollov, & Carle, 2008). At different pH levels, E* changed very distinctly: more than 5 units with each ratio of anthocyanins/EMIQ at pH 3, however, the value changed obviously around 2 units with EMIQ concentration of 1% at pH 4 and 5, which meant that a greater inuence of EMIQ on the anthocyanin was disappeared at pH 4 and 5 than pH 3. With the elevation of EMIQ concentration, no signicant difference was observed for E* values. All these changes were more pronounced with the increase of the concentration of EMIQ at different pH levels, which was coherent with the results about magnitude of the copigmentation when evaluated at maximum wavelength of absorption (Gonnet, 1998). These observations indicated that the presence of the copigment dominated the color change in anthocyanin solution during light exposure and the color proles were more signicantly controlled with the addition of EMIQ. E* indicates the magnitude of color difference among samples. Differences in perceivable color can be analytically classied as very distinct (E* N 3), distinct (1.5 b E* b 3) and small difference (E* b 1.5) (Tiwari, Muthukumarappan, O'Donnell, & Cullen, 2008). In general, the eye is able to discriminate two colors when E* N 1 (Gonnet, 1998), while for the effect of bottle or test tube, the color discrimination is worse and the acceptable tolerance for the human eye in distinguishing the color is 3 units (Malien-Aubert et al., 2001). By contrast, most of the solutions containing EMIQ surpassed the value of 3.0 units for E*, it revealed that EMIQ was more effective copigment to anthocyanins. Color differences between anthocyanins solutions copigmented with and without EMIQ were always easily detectable by the human eyes, since E* values changed more than 5 units especially at pH 3. Kucharska, Oszmianski, Kopacz, and Lamer-Zarawska (1998) investigated the inuence of UV irradiation on the stability of the cyanidin-copigment complex. They found that the presence of copigments in anthocyanin solutions inhibited the UV degradation, the copigmentation that lowers the ratio between the colorless forms and colored forms via selective complexation of the colored forms must efciently compete with the rst step of anthocyanin degradation (formation of the colorless forms). Moreover, copigments from the avonol subgroup, which strongly absorb damaging UV radiations, must provide additional protection against degradation of the colored forms. Such a protection has been already evidenced by Sweeny, Wilkinson,

Fig. 3. The effect of copigmentation of grape skin extract anthocyanin with EMIQ at different pH levels: (a) pH3, (b) pH4, and (c) pH5.

in fast equilibrium with each other (Brouillard, Mazza, Saad, AlbrechtGary, & Cheminat, 1989). The anthocyanins structure is the main element to determine their instability. The acylated anthocyanins were more stable than non-

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Fig. 4. Color changes (L*, a*, b*, and E*) of grape skin extract anthocyanin with and without EMIQ at pH 3: (a) L*, (b) a*, (c) b*, and (d) E*.

and Iacobucci (1981) and Santhanam, Hautala, Sweeny, and Iacobucci (1983) with avylium ions and avonoid sulfonates. Therefore, the structure of EMIQ had subgroup to absorb damaging UV radiations which was effective in stabilizing color through an intermolecular association between anthocyanins. The sandwich-type association between EMIQ and anthocyanin could stabilize the color. This stacking can prevent color loss caused by hydration reactions.

3.4. Spectral characteristics of grape skin anthocyanins with EMIQ The spectral properties of the anthocyanins solution could be modied when a copigment was added. Because the anthocyanin content was constant in each solution, it was obvious that the magnitude of spectral variations depended on the concentrations of EMIQ. The ratios of anthocyanin to EMIQ were 2:1, 1:1, and 1:2 (w/w, anthocyanin/EMIQ), respectively. As shown in Table 3, the max value increased signicantly (p b 0.05) from 525.4 nm to 535.7 nm at pH 3, from 527.5 to 535.7 at pH 4, and from 528.5 to 541.3 at pH 5. Under the same ratio of 1:2, max amounted to 10.3, 8.2, 12.8 nm at pH 3, 4 and 5, respectively, the shift of max at pH 5 changed much more than pH 3 and 4. The magnitude of the bathochromic (max) shifted to the longest wavelength absorption band with the increase of copigment concentration at different pH levels. As the concentration increased, the copigment frequently formed a complex with anthocyanins. Similar results were also reported by Dimitri Markovi, Petranovi, and Baranac (2000) who have studied the copigmentation of Mv-3-G with well-known copigments, such as rutin, quercetin and morin. Therefore, the results of bathochromic shift were in accordance with the stability enhancement of anthocyanins with EMIQ during thermal and light irradiation.

It has been demonstrated that the copigment effect on the stability occurs from low pH levels to neutrality (Williams & Hrazdina, 1979). The copigmentation is affected by pH, and the max is strongly pHdependent. To describe the intermolecular copigmentation, these changes at different pH levels can be interpreted by the mechanism proposed by Brouillard et al. (1991). At pH 3, there was a little color loss for anthocyanins and the color retention for the solution containing the anthocyanins, therefore, the copigment was signicant. At pH 4 and 5, the solutions were practically colorless, however, the solution with anthocyanin and copigment was still colored. Thus, the copigment is to inhibit the production of the colorless carbinol pseudobase. In those pH levels, quinoidal bases were formed and color retention was due to decrease in the amount of the carbinol pseudobase in the solution.

4. Conclusion The results have provided a more detailed understanding of copigment effect of EMIQ on anthocyanins. The degradation of the anthocyanins copigmented with EMIQ followed rst order reaction kinetics and the half life extended signicantly. Less difference was observed of the E* values throughout the time for each case. Color differences between anthocyanins with and without EMIQ were easily detectable by human eyes. In conclusion, EMIQ acting as a copigment, which might have effect with anthocyanins to form a new complex, remarkably improve the thermal and light stability of anthocyanins at different pH levels. Therefore, the present ndings are much valuable to the food products which were required to promote their stability for color and avor. Further research will be carried out to understand the mechanism of EMIQ-anthocyanin complexes.

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Fig. 5. Color changes (L*, a*, b*, and E*) of grape skin extract anthocyanin with and without EMIQ at pH 4: (a) L*, (b) a*, (c) b*, and (d) E*.

Fig. 6. Color changes (L*, a*, b*, and E*) of grape skin extract anthocyanin with and without EMIQ at pH 5: (a) L*, (b) a*, (c) b*, and (d) E*.

Q. Yan et al. / Food Research International 50 (2013) 603609 Table 3 Spectral characteristics of grape skin extract anthocyanins with or without EMIQ at different pH levels. Ratio max pH 3 Control 2:1 1:1 1:2 525.4 0.1 530.6 0.2b 534.7 0.2c 535.7 0.1d
a

609

pH 4 527.5 0.6 533.6 0.1b 534.9 0.2c 535.7 0.3d

a

pH 5 528.5 0.3a 535.8 0.2b 536.5 0.2b 541.3 0.1b

Values followed by same letter within a column are not signicant (p b 0.05).

Acknowledgments This work has been nanced by the National Key Technology R&D Program of the Chinese Ministry of Science and Technology (2006BAD27B04) and Inter-governmental (China and Poland) S&T cooperation project. References
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