Accepted Manuscript Optimization of ultrasonic-assisted extraction of phenolic compounds, antioxidants, and anthocyanins from sugar beet molasses Mingshun Chen, Yi Zhao, Shujuan Yu PII: DOI: Reference:

S0308-8146(14)01495-2 http://dx.doi.org/10.1016/j.foodchem.2014.09.110 FOCH 16466

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

6 June 2014 10 September 2014 19 September 2014

Please cite this article as: Chen, M., Zhao, Y., Yu, S., Optimization of ultrasonic-assisted extraction of phenolic compounds, antioxidants, and anthocyanins from sugar beet molasses, Food Chemistry (2014), doi: http:// dx.doi.org/10.1016/j.foodchem.2014.09.110

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Optimization of ultrasonic-assisted extraction of phenolic compounds, antioxidants, and anthocyanins from sugar beet molasses

Mingshun Chen, Yi Zhao, Shujuan Yu∗ College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety

Please send this correspondence to: * Shujuan Yu, Dr. Professor College of Light Industry and Food Sciences South China University of Technology 381 Wushan road Guangzhou, 510640 Phone: (86) 20-87113668, Fax: (86) 20-87113668 E-mail: [email protected]

∗

Corresponding author: Shujuan Yu; e-mail:[email protected]; Tel.: +86 2087113668, Fax: +86

2087113668 1

Abstract: Response surface methodology was used to optimize experimental conditions for ultrasonic-assisted extraction (UAE) of functional components from sugar beet molasses. The central composite design (CCD) was used for the optimization of extraction parameters in terms of total phenolic contents, antioxidant activities and anthocyanins. Result suggested the optimal conditions obtained by RSM for UAE from sugar beet molasses were as follows: HCl concentration 1.55-1.72 mol/L, ethanol concentration 57-63% (v/v), extraction temperature 41-48 °C, and extraction time 66-73 min. In the optimal conditions, the experimental total phenolic contents were 17.36 mg GAE/100 mL, antioxidant activity was 16.66 mg TE/g, and total anthocyanins were 31.81 mg/100g of the sugar beet molasses extract, which were well matched the predicted values. Teen compounds, i.e. gallic acid, vanillin, hydroxybenzoic acid, syringic acid, cyanidin-3-O-rutinoside, cyanidin-3-O-glucoside, catechin, delphinidin-3-O-rutinoside, delphinidin-3-O-glucuronide and ferulic acid were determined by HPLC-DAD-MS/MS in sugar beet molasses.

Keywords: Sugar beet molasses; Ultrasonic-assisted extraction; Response surface methodology; Phenolic compounds; Antioxidants; Anthocyanins

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1. Introduction Sugar beet is the important crops for the production of sugar (Balbach, Royer, & Rogers, 1998; McDill, 1947). However, by-product in sugar industry, sugar beet molasses is always produced, and readily available at relatively low cost (Roukas, 1996). Early researchers have elaborated the recycle of molasses for livestock feed, preparing alcohol and fermentation medium (Ahmedna, Marshall, & Rao, 2000; Paturau, 1989). Sugar beet molasses contain abundant antioxidants (Filipčev, Lević, Bodroža-Solarov, Mišljenović, & Koprivica, 2010; Koprivica, Mišljenović, Lević, & Kuljanin, 2009), which means that they might be used as raw material to produce antioxidants. Methods such as cold pressing, heating reflux, soxhlet and solvent extraction have been widely used to extract bioactive components from natural products (Contini, Baccelloni, Massantini, & Anelli, 2008; Du, Xiao, & Li, 2007; Mandal, Mohan, & Hemalatha, 2007; Wang, Sun, Cao, Tian, & Li, 2008). However, disadvantages including safety hazards, high energy input, low product quality, environment risk and toxicological effects were always found during extraction (Contini, Baccelloni, Massantini, & Anelli, 2008; Mandal, Mohan, & Hemalatha, 2007). Therefore, improving the extraction methods in antioxidants production is necessary. Recently, novel extraction methods such as accelerated solvent, ultrasound-assisted, microwave-assisted, supercritical fluid and enzyme-assisted extraction have been developed for the antioxidants (Luque de Castro & Garcıa-Ayuso, 1998; Sporring, Bøwadt, Svensmark, & Björklund, 2005; Vilkhu, Mawson, Simons, & Bates, 2008). Among them, ultrasound-assisted extraction (UAE) has been confirmed as a more economic and efficient extraction means (Vilkhu, Mawson, Simons, & Bates, 2008). Enzyme-assisted extraction is also an eco-friendly extraction technology, however, it has a low range of temperatures and high cost (Puri, Sharma, & Barrow, 2012).

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Therefore, ultrasonic-assisted extraction might be a potential means to extract bio-activity compounds better than other modern extraction techniques. Response surface method (RSM) is a mathematical and statistical tool, which has been widely used to optimize various parameters in industry processing (Deepak, Kalishwaralal, Ramkumarpandian, Babu, Senthilkumar, & Sangiliyandi, 2008). RSM can evaluate the impact of different multiple parameters, and simultaneously optimize experimental conditions (Shekarchizadeh, Kadivar, Ghaziaskar, & Rezayat, 2009; Xu, Skands, Høy, Mu, Balchen, & Adler-Nissen, 1998). Different RSM methods such as box-behnken design (BBD), central composite design (CCD) and three-level full factorial designs (TFFD) have been widely used in various fields such as food, biology and chemistry. Among these means, CCD has been proved for its fitness in model and experiment design, which is a powerful and common design for RSM, less time-using and more efficient than many other designs (Aybastıer, Işık, Şahin, & Demir, 2012). Therefore, using CCD to optimize the antioxidants extraction by ultrasound-assisted extraction is necessary for further study. RSM has been recently used to determine the most influential parameters for simultaneous production of lactic acid, xanthan and ethanol from molasses (Ergun & Ferda Mutlu, 2000; Kalogiannis, Iakovidou, Liakopoulou-Kyriakides, Kyriakidis, & Skaracis, 2003; Kotzamanidis, Roukas, & Skaracis, 2002). However phenolic compounds, antioxidants, and anthocyanins were not given on the previous literature. In the present study, the UAE parameters such as HCl concentration, ethanol concentration, extraction temperature and time were optimized by RSM, in order to obtain the optimal extraction ratio of total phenolic contents, antioxidant activities and anthocyanins from sugar beet molasses. In

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addition, we identified the composition of extracts by HPLC-MS/MS, and assessed the influence of ultrasound on the extraction efficiency and chemical compositions of extracts.

2. Materials and methods 2.1. Materials and reagents Sugar beet molasses was provided by Xinjiang Green Xiang Sugar Industry Co., Ltd (Tacheng, China). Folin-ciocalteu (FC) reagent, 2,2’-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), trolox

[(±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic

acid]

were

purchased

from

Sigma-Aldrich (St. Louis, MO, USA). Analytical grade ethanol, methanol, gallic acid and sodium carbonate were purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). HPLC grade methanol and acetic acid were purchased from Tianjin Kermel Chemical Reagent Co., Ltd. (Tianjin, China).

2.2. Ultrasound-assisted extraction Ultrasound-assisted extraction was performed in an ultrasonic cleaner RK102H (BANDELIN SONOREX, Germany). The sugar beet molasses (2.0 g) was placed into a beaker (100 mL), and 60 mL acidic ethanol solvent was added, and then underwent ultrasonic treatment at 35 kHz, 450 W. The acidic ethanol solution was prepared across the range of concentrations of HCl (0.8-2.4 mol/L) and ethanol concentration (50-90%, v/v). The extract was concentrated at 45 °C in vacuum and analysed.

2.3. Experimental design CCD methods with five level and four-variable (30 experiments) was used for the optimization of extraction variables. The independent variables and their levels are as follows: HCl concentration 0.8-2.4 mol/L, ethanol concentration 50-90% (v/v), extraction temperature 20-60 °C and extraction time 30-90 min. Total phenolic contents, antioxidant activity and total anthocyanins were selected as

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the response of the design experiments (Y). Table 1 showed the coded and actual values for box-behnken design of the independent variables. The predicted response was calculated by a second-order polynomial model. The second-order polynomial model for the response surface analysis was shown as follows: 4

Y = b0 +

4

3

∑ bX + ∑ b X i

i=1

i

ii

i=1

2 i

+

4

∑ ∑b XY ij

i=1

i

j

（1）

j=i+1

Where Y is response value, b0 is the offset term, bi is the linear effect, bii is the squared effect, bij is the interaction effect and Xi and Xi are independent variables. The data were analyzed using Design Expert (7.1.3 version) program and the coefficients were analyzed by F -value. In order to optimize experimental conditions of total phenolic contents, antioxidant activity and total anthocyanins, an analysis of variance (ANOVA), regression analysis and plotting of response surface figures were performed.

2.4. Analysis for total phenolic contents Total phenolic contents of the sugar beet molasses extracts were determined using Folin-Ciocalteu method according to the literature (Şahin, Aybastıer, & Işık, 2013b) with a slight modification. 20 µL of sugar beet molasses sample, 2.0 mL FC reagent and 1.2 mL sodium carbonate solution (7.5% w/v) were mixed and allowed to place at 30 °C for 30 min. The absorbance was measured at 765 nm (Gil, Tomás-Barberán, Hess-Pierce, Holcroft, & Kader, 2000). Total phenolic contents were expressed as mg of gallic acid equivalent (GAE) per g of dried weight.

2.5. Analysis for antioxidant activity The antioxidant activity of extract was determined with ABTS method, as described in the literatures (Re, Pellegrini, Proteggente, Pannala, Yang, & Rice-Evans, 1999; Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Hawkins Byrne, 2006) with some modification. ABTS+ was produced by reacting 6

20 mM ABTS solution with 2.45 mM potassium persulphate solution and placed in the dark at 30 °C for 12-16 h before use. 0.25 mL of sugar beet molasses extract, 3.75 mL of ethanol and 1 mL of the ABTS+ radical cation solution which was diluted with ethanol at a ratio of 1:10 were mixed. The absorbance was measured at 734 nm against blank after 6 min. The results were expressed as mg trolox equivalent (TE) per g dried weight.

2.6. Determination of anthocyanins Anthocyanins were used to indicate the contents of anthocyanins extracted from sugar beet molasses. Anthocyanins were based on the method described by Fuleki and Francis (Fuleki & Francis, 1968), and expressed as mg cyanidin-3-glucoside equivalent per 100 g dried weight. Visible absorbance was measured at 520 nm (Ghafoor, Choi, Jeon, & Jo, 2009; Ku & Mun, 2008).

2.7. HPLC-DAD-MS/MS analysis HPLC-DAD-MS/MS was be used to identify compounds in sugar beet molasses extract (He, Liu, Xu, Gong, Yuan, & Gao, 2010; Romani, Vignolini, Isolani, Ieri, & Heimler, 2006). Waters HPLC system consist a Waters 600E pump and a diode array detector (DAD) was used for analysis. The chromatographic separations were carried out using an Agilent C18 column (250 mm × 4.6 mm, 5µm; Agilent Technologies Co., Ltd., Shanghai, China), analyzed at 25 °C. The mobile phase was 1% acetic acid aqueous solution (A) and methanol (B) with a gradient program of 0-5 min, linear gradient 5-10% B; 5-35 min, linear gradient 10-20% B; 35-45 min, 20% B isocratic; 45-65 min, linear gradient 20-40% B; 65-70 min, linear gradient 40-100% B and 70-90 min 100% B isocratic elution at a flow rate of 1 mL/min. 20 µL of samples or standard solutions were injected. The quantitative analysis of the major components in sugarcane molasses extracts was calculated using peak areas from calibration curves established using external standards ((+)-catechin for flavanol quantification, ferulic acid for phenolic

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acids quantification and cyanidin-3-O-glucoside for anthocyanins quantification).The PDA was set at 280 nm for flavanols, 320 nm for phenolic acids and 520 nm for anthocyanins (Ghafoor, Choi, Jeon, & Jo, 2009; Mané, Souquet, Olle, Verries, Veran, Mazerolles, et al., 2007). A Thermo Scientific LCQ Ion-Trap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) with electrospray ionization (ESI) interface was used for Triple-quadrupole tandem mass spectrometric detection. The ESI source was set in negative ionization mode. Operating conditions were: capillary voltage, 3.50 kV; nebuliser, 30 psi; dry temperature, 350 °C; dry gas flow, 35 L/min; cone gas flow, 5 L/min, dwell time, 5ms. Nitrogen was used as the dry and cone gas. Ion trap full-scan analysis was conducted from m/z 50 to 1300 with an upper fill time of 200 ms. Scan mass spectra of the phenolic compounds were measured from m/z 100 up to 1000. All data were acquired and processed using Xcalibur™ software with Virtual Instrument Partner Program (Thermo Fisher Scientific, San Jose, CA, USA.

2.8 Statistical analysis All experiments were carried out in triplicates. Means and standard deviations of the data were calculated for each treatment. Analysis of variance (ANOVA) was carried out to determine any significant differences (p < 0.05).

3. Results and discussion 3.1. Fitting the model The responses of each independent variable were listed in Table 2. Among the experiments carried out in the present study, experiment #28 (HCl concentration 1.6 mol/L, ethanol concentration 70% (v/v), temperature 40 °C and time 60 min) provided the highest total phenolic contents (17.03 mg GAE/g), and the experiment #3 (HCl concentration 1.2 mol/L, ethanol concentration 80% (v/v),

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temperature 30 °C and time 45 min) produced the least phenolic content (12.54 mg GAE/g ). The extract of experiment #29 (HCl concentration 1.6 mol/L, ethanol concentration 70% (v/v), temperature 40 °C and time 60 min) showed the highest antioxidant activity (17.34 mg TE/g), and extract of experiment #12 (HCl concentration 2.0 mol/L, ethanol concentration 80% (v/v), temperature 30 °C and time 75 min) showed the lowest antioxidant activity (12.54 mg TE/g). The extract of experiment #25 (HCl concentration 1.6 mol/L, ethanol concentration 70% (v/v), temperature 40 °C and time 60 min) showed the highest anthocyanins (31.99 mg/100g), and extract of experiment #3 (HCl concentration 1.2 mol/L, ethanol concentration 80% (v/v), temperature 30 °C and time 45 min) showed the lowest anthocyanins (26.64 mg/100g). Analysis of variance (ANOVA) showed the quadratic polynomial model was highly significant with the p-value is less than 0.0001 for total phenolic contents and anthocyanins, 0.0069 for antioxidant activity (Table 3). The F-value, i.e. 11.65, 3.87 and 9.74 for total phenolic contents, antioxidant activity and anthocyanins, respectively, suggested the significant of the model was higher than 95% confidence level. As shown in Table 3, the absence of lack of fit and the value of pure error indicated well reproducibility of the experimental data.

3.2. Response surface analysis of total phenolic contents The effects of extraction factors such as X1 (HCl concentration), X2 (ethanol concentration), X3 (extraction temperature) and X4 (extraction time) were carefully studied. The significance of each coefficient was determined by F-values and p-values. The regression equation in coded level neglecting insignificant terms was generated: Y1 (mg GAE/g) = 16.47 - 0.47X2 + 0.68X3 + 0.49X1X2 + 0.41X1X3 - 0.83X1X4 + 0.43X2X3 + 0.33X3X4 - 0.32X22 - 0.55X32 - 0.53X42

(2)

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Results indicated that the quadratic of relationship between the total phenolic contents and extraction factors had a good regression coefficient (R2=0.9158). Higher F-value with lower p-value always led to more significant corresponding among various independent variables. X2, X3, X1X2, X1X3, X1X4, X2X3, X3X4, X22, X32 and X42 were significant as p-value was less than 0.05. However, X1, X4, X2X4 and X12 were not significant due to a higher p-value (i.e. higher than 0.05). Fig. 1 showed the complex interaction between HCl concentration, ethanol concentration, extraction temperature and time. The highest total phenolic contents were observed at higher HCl concentrations and lower ethanol concentrations (Fig. 1A). However, the increase in ethanol concentrations at a fixed HCl concentration led to an increase in the total phenolic contents, and reached a maximum at the lowest ethanol concentration tested. Fig. 1B showed the interaction between HCl concentration and extraction time on total phenolic extraction. An increase in the total phenolic contents were observed as increasing extraction time up to 73 min, but decreased thereafter. Significant increases in the total phenolic contents were observed at high extraction temperature. However, the trend was reversed as the extraction temperature reached to 43 °C (Fig. 1C). High temperature might increase the antioxidants yield due to the increase of solubility and diffusion of compounds. However, over-elevated temperatures always degraded phenolic compounds, leading to reducing of the antioxidants (Dorta, Lobo, & Gonzalez, 2012; Ling, Yap, Radhakrishnan, Subramaniam, Cheng, & Palanisamy, 2009).

3.3. Response surface analysis of antioxidant activity Table 3 showed a good regression value for antioxidant activity (R2=0.8826) and the relationship between the antioxidant activity and extraction parameters of HCl concentration, ethanol concentration, temperature and time. The quadratic polynomial equations with significant terms of the antioxidant activity of sugar beet molasses which neglecting the insignificant terms are given below:

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Y2 (mg TE/g) = 16.43 + 0.54X3 + 0 .53X2X3 - 0.45X12 - 0.49X32 - 0.59X42

(3)

X3, X2X3, X12, X32 and X42 were the most significant parameters on the ultrasonic-assisted extraction of the antioxidant activity of sugar beet molasses. X1, X2, X4, X1X2, X1X3, X1X4, X2X4, X3X4, and X22 had less effect on antioxidant activity from ultrasonic-assisted extraction. Fig. 2 showed the relationship between the antioxidant activity and extraction factors. Fig. 2A represents the effect of varying HCl concentration, extraction time their mutual interaction on the antioxidant activity. Antioxidant activity increased with increasing time at the high HCl concentration. Highest antioxidant activity was observed at higher HCl concentration (1.66 mol/L) and higher time (66 min). The effects of extraction temperature, ethanol concentration and their interaction on antioxidants activity were shown in Fig. 2B. Obviously, increasing extraction temperature before 48 °C with a lower ethanol concentration increased the antioxidants extraction, while, overheated temperature always decreased the antioxidants extraction ratio. And higher antioxidant activity of extract was tested at higher extraction temperature (48 °C) and longer time (66 min) (Fig. 2C). Moreover, higher extraction temperature can produce increasingly repulsive solute-solvent interaction, leading to a reduction of antioxidants in extract. The highest antioxidant activity of extracts was tested at 48 °C for 66 min.

3.4. Response surface analysis of anthocyanins A good regression value for the anthocyanins (R2=0.9009) and the relationship between the antioxidant activity and extraction parameters were shown in Table 3. The quadratic polynomial equations with significant terms of the anthocyanins of sugar beet molasses which neglecting the insignificant terms are given below:

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Y3 (mg/100g) = 31.42 + 0.34X1 - 0.69X2 + 0.66X3 - 0.53X1X4 + 0.61X2X3 - 0.44X12 - 0.33 X22 - 0.62X32 - 0.54X42

(4)

X1, X2, X3, X1X4, X2X3, X12, X22, X32 and X42 were the most significant parameters on the ultrasonic-assisted extraction of the anthocyanins of sugar beet molasses. X4, X1X2, X1X3, X2X4, and X3X4, had less effect on anthocyanins from ultrasonic-assisted extraction. Fig. 3 showed the relationship between the anthocyanins and extraction factors. Fig. 3A shows the effect of HCl concentration, ethanol concentration and their mutual interaction on the anthocyanins. A decrease in anthocyanins was observed with increasing ethanol concentration. An increase in the anthocyanins was observed with increasing HCl concentration at first, but the trend was reserved when the HCl concentration reached to 1.72 mol/L and anthocyanins decreased thereafter. Fig. 3B represents the effect of varying HCl concentration, extraction time their mutual interaction on the anthocyanins. Anthocyanins increased with increasing time at the high HCl concentration. Highest anthocyanins were observed at higher HCl concentration (1.72 mol/L) and higher time (68 min). The effects of extraction temperature, ethanol concentration and their interaction on anthocyanins were shown in Fig. 3C. Obviously, increasing extraction temperature before 41 °C with a lower ethanol concentration increased the anthocyanins extraction, while, overheated temperature always decreased the phenolic extraction ratio. And higher anthocyanins of extract were tested at higher extraction temperature (41 °C) and longer time (68 min) (Fig. 3C).

3.5. Optimization of extraction craft parameter and the model validation The optimum ultrasonic-assisted extraction conditions for total phenolic contents, antioxidant activity and anthocyanins of sugar beet molasses extract was shown in Table 4. HCl concentration range from 1.55 to 1.72 mol/L, ethanol concentration range from 57 to 63% (v/v), extraction

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temperature from 41 to 48 °C and time from 66 to 73 min produced the optimal total phenolic contents (17.36 mg GAE/g), antioxidant activity (16.66 mg TE/g) and anthocyanins (31.83 mg/100g). The predicted results showed no significant difference was found comparing with experimental results. The parameters such as HCl concentration, ethanol concentration, extraction temperature and time have significantly impacted the extraction of phenolic compounds from sugar beet molasses (Castañeda-Ovando, Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal, 2009). According to literature (Herrera & Luque de Castro, 2005) a low HCl concentration was necessary to rupture the cell walls of the fruit and extract the phenolic compounds. Water and Ethanol are preferred as a solvent in the food industry and recommended by the US Food and Drug Administration for extraction purposes (Tabaraki, Heidarizadi, & Benvidi, 2012). It has been reported that high temperature favors extraction, increasing both the diffusion coefficient and the solubility of the solute; however, elevated temperatures, phenolic compounds can be denatured (Şahin, Aybastıer, & Işık, 2013a). Because UAE could greatly decrease the extraction time, the use of it for the extraction of total phenols, antioxidants, and anthocyanins was more effective and energy saving than any other high temperature long time extraction process (Ghafoor, Choi, Jeon, & Jo, 2009). According to literature (Toma, Vinatoru, Paniwnyk, & Mason, 2001), the explanation for the efficiency of UAE that sonication simultaneously enhanced the hydration and swelling process while facilitating the mass transfer of soluble constituents to the extraction solvent. We may conclude that UAE enable improve existing extraction processes and can provide commercially attractive advantages and outcomes (Vilkhu, Mawson, Simons, & Bates, 2008).

3.6. Chemical composition

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The chemical composition of extracts from sugar beet molasses using the predicted optimum conditions are shown in Table 5. The major components of the extracts which were identified by HPLC-DAD-MS/MS, were gallic acid (1), vanillin (2), hydroxybenzoic acid (3), syringic acid (4), cyanidin-3-O-rutinoside (5), cyanidin-3-O-glucoside (6), catechin (7). delphinidin-3-O-rutinoside (8), delphinidin-3-O-glucuronide (9), ferulic acid (10). The amounts of predominant components of extract from sugar beet molasses determined by HPLC-DAD are shown in Table 6. Similar results were obtained with three extraction procedures taking into account the total phenolic contents, antioxidant activity and anthocyanins values. Syringic acid, vanillin, ferulic acid, hydroxybenzaldehyde, hydroxybenzoic acid, luteolin/kaempferol, feruloyl-arabinose-arabinose and caffeoyltartaric acid were determined in literature (Valli, Gómez-Caravaca, Di Nunzio, Danesi, Caboni, & Bordoni, 2012). There was no detectable amount of hydroxybenzaldehyde, luteolin/kaempferol, feruloyl-arabinose-arabinose and caffeoyltartaric acid in our study. The amounts of syringic acid, vanillin, ferulic acid, and hydroxybenzoic acid determined were higher than previously reported in literature (Valli, Gómez-Caravaca, Di Nunzio, Danesi, Caboni, & Bordoni, 2012). On the other hand, there is no information in literature about gallic acid, cyanidin-3-O-rutinoside, cyanidin-3-O-glucoside, catechin delphinidin-3-O-rutinoside and delphinidin-3-O-glucuronide contents in sugar beet molasses.

4. Conclusions In the present study, the response surface methodology using CCD method was successfully employed to optimize the important functional components from sugar beet molasses. The optimum conditions of HCl concentration, ethanol concentration, extraction temperature and time were determined for maximum extraction yield of phenolic compounds with respect to the total phenolic contents, antioxidant activity and anthocyanins. We can observe that among the total phenolic contents,

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antioxidant activities, and anthocyanin levels of the extracts from sugar beet molasses using UAE have a strong correlation. Gallic acid, vanillin, hydroxybenzoic acid, syringic acid, cyanidin-3-O-rutinoside, cyanidin-3-O-glucoside, catechin, delphinidin-3-O-rutinoside, delphinidin-3-O-glucuronide and ferulic acid were determined by HPLC-DAD-MS/MS in sugar beet molasses. The study indicates that ultrasound-assisted extraction of the important functional components from sugar beet molasses is a green process for the preparation of extracts rich in natural antioxidants aimed at replacing synthetic antioxidants. There could be a clear potential for the utilization of sugar beet molasses in food industry.

Acknowledgments All authors acknowledge the ministry of science and technology in agriculture science and Technology Achievements Transformation Fund Project(No.2013GB23600669), the Science and Technology Planning Project of Guangzhou Municiple, China(No. 2011Y2-00012), and this work was supported by the basic research foundation of SCUT(2012ZM0072).

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21

Figure Legends: Figure 1 Response surface plots showing the operating parameter effects on total phenolic contents. (A) the total phenolic contents vs. HCl concentration and ethanol concentration at fixed extraction temperature of 43 ºC and time of 73 min; (B) the total phenolic contents vs. HCl concentration and extraction time at fixed ethanol concentration of 57% and extraction temperature of 43 ºC; (C) the total phenolic contents vs. ethanol concentration and extraction temperature at fixed HCl concentration of 1.55 mol/L and extraction time of 73 min. Figure 2 Response surface plots showing the operating parameter effects on antioxidant activity. (A) the antioxidant activity vs. HCl concentration and extraction time at fixed ethanol concentration of 63% and extraction temperature of 48 ºC; (B) the antioxidant activity vs. ethanol concentration and extraction temperature at fixed HCl concentration of 1.66 mol/L and extraction time of 66 min; (C) the antioxidant activity vs. extraction temperature and time at fixed HCl concentration of 1.66 mol/L and ethanol concentration of 63%. Figure 3 Response surface plots showing the operating parameter effects on anthocyanins. (A) the anthocyanins vs. HCl concentration and ethanol concentration at fixed extraction temperature of 41 ºC and time of 68 min; (B) the anthocyanins vs. HCl concentration and extraction time at fixed ethanol concentration of 61% and extraction temperature of 41 ºC; (C) the anthocyanins vs. ethanol concentration and extraction temperature at fixed HCl concentration of 1.72 mol/L and extraction time of 68 min.

22

(A)

(B)

(C) Fig. 1.Response surface plots of sugar beet molasses showing the effect of (A) HCl concentration and ethanol concentration; (B) HCl concentration and extraction time; (C) ethanol concentration and extraction temperature on total phenolic content.

28

(A)

(B)

(C) Fig. 2.Response surface plots of sugar beet molasses showing the effect of (A) HCl concentration and extraction time; (B) ethanol concentration and extraction temperature. (C) extraction temperature and time on antioxidant activity.

28

(A)

(B)

(C) Fig. 3.Response surface plots of sugar beet molasses showing the effect of (A) HCl concentration and ethanol concentration; (B) HCl concentration and extraction time; (C) ethanol concentration and extraction temperature on anthocyanins.

28

Table.1. Range of coded and actual values for central composite design

Independent variables

Code units

Coded levels −2

−1

0

1

2

HCl concentration (mol/L)

X1

0.8

1.2

1.6

2.0

2.4

Ethanol concentration (%, v/v)

X2

50

60

70

80

90

Extraction temperature (°C)

X3

20

30

40

50

60

Extraction time (min)

X4

30

45

60

75

90

23

Table.2. Total phenolic content, antioxidant activity, and anthocyanins of the extract of sugar beet molasses under different conditions of ultrasonic-assisted extraction based on a central composite design (CCD) for response surface analysis

Run

extraction conditions

analytical results

X1

X2

X3

X4

Total phenolic

Antioxidant

Anthocyanins

HCl

Ethanol

Extraction

Extraction

Yield (%)

content

activity

(mg/100g)

concentration

concentration

temperature

time

(mg GAE/g )

(mg TE/g)

(mol/L)

(%,v/v)

(°C)

(min)

1

1.2 (−1)

60 (−1)

30 (−1)

45 (−1)

22.23 ± 0.32

15.13 ± 0.08

15.38 ± 0.12

29.11 ± 0.12

2

2.0 (1)

60 (−1)

30 (−1)

45 (−1)

19.32 ± 0.21

15.88 ± 0.05

14.37 ± 0.03

30.84 ± 0.09

3

1.2 (−1)

80 (1)

30 (−1)

45 (−1)

5.51 ± 0.44

12.54 ± 0.11

14.14 ± 0.05

26.64 ± 0.13

4

2.0 (1)

80 (1)

30 (−1)

45 (−1)

15.20 ± 0.56

14.23 ± 0.02

12.89 ± 0.07

27.49 ± 0.11

5

1.2 (−1)

60 (−1)

50 (1)

45 (−1)

18.76 ± 0.17

14.11 ± 0.05

15.17 ± 0.06

29.04 ± 0.07

6

2.0 (1)

60 (−1)

50 (1)

45 (−1)

27.54 ± 0.84

16.87 ± 0.06

14.26 ± 0.05

30.75 ± 0.05

7

1.2 (−1)

80 (1)

50 (1)

45 (−1)

9.32 ± 1.21

12.78 ± 0.12

14.56 ± 0.07

27.72 ± 0.09

8

2.0 (1)

80 (1)

50 (1)

45 (−1)

28.31 ± 0.64

16.98 ± 0.13

14.78 ± 0.12

31.13 ± 0.15

9

1.2 (−1)

60 (−1)

30 (−1)

75 (1)

25.11 ± 0.84

16.08 ± 0.03

14.87 ± 0.05

30.07 ± 0.13

10

2.0 (1)

60 (−1)

30 (−1)

75 (1)

13.88 ± 0.53

13.54 ± 0.04

15.78 ± 0.08

29.77 ± 0.11

11

1.2 (−1)

80 (1)

30 (−1)

75 (1)

10.89 ± 0.21

13.14 ± 0.09

12.76 ± 0.12

27.54 ± 0.06

12

2.0 (1)

80 (1)

30 (−1)

75 (1)

6.71 ± 0.56

12.73 ± 0.12

12.45 ± 0.08

27.75 ± 0.07

13

1.2 (−1)

60 (−1)

50 (1)

75 (1)

29.13 ± 1.23

16.97 ± 0.02

14.67 ± 0.09

30.79 ± 0.16

14

2.0 (1)

60 (−1)

50 (1)

75 (1)

17.10 ± 0.53

14.87 ± 0.05

16.56 ± 0.04

29.78 ± 0.08

15

1.2 (−1)

80 (1)

50 (1)

75 (1)

20.18 ± 1.55

15.33 ± 0.04

16.34 ± 0.03

30.33 ± 0.07

16

2.0 (1)

80 (1)

50 (1)

75 (1)

22.33 ± 1.03

16.57 ± 0.08

15.35 ± 0.13

30.53 ± 0.13

17

0.8 (−2)

70 (0)

40 (0)

60 (0)

19.15 ± 0.45

15.89 ± 0.11

14.34 ± 0.05

29.62 ± 0.18

18

2.4 (2)

70 (0)

40 (0)

60 (0)

17.66 ± 0.67

15.55 ± 0.05

14.88 ± 0.08

30.29 ± 0.21

19

1.6 (0)

50 (−2)

40 (0)

60 (0)

18.36 ± 0.31

15.78 ± 0.06

14.71 ± 0.04

31.77 ± 0.07

20

1.6 (0)

90 (2)

40 (0)

60 (0)

16.41 ± 0.64

14.69 ± 0.04

15.89 ± 0.08

29.09 ± 0.08

21

1.6 (0)

70 (0)

20 (−2)

60 (0)

12.45 ± 0.73

13.05 ± 0.12

13.46 ± 0.02

28.03 ± 0.04

22

1.6 (0)

70 (0)

60 (2)

60 (0)

19.34 ± 0.43

15.56 ± 0.02

15.45 ± 0.13

30.44 ± 0.14

23

1.6 (0)

70 (0)

40 (0)

30 (−2)

11.23 ± 0.98

13.89 ± 0.07

13.88 ± 0.02

29.53 ± 0.12

24

1.6 (0)

70 (0)

40 (0)

90 (2)

15.45 ± 0.45

14.88 ± 0.04

14.23 ± 0.09

29.59 ± 0.16

25

1.6 (0)

70 (0)

40 (0)

60 (0)

25.71 ± 0.63

16.78 ± 0.06

15.14 ± 0.05

31.99 ± 0.18

26

1.6 (0)

70 (0)

40 (0)

60 (0)

26.32 ± 0.88

15.23 ± 0.15

16.01 ± 0.07

29.96 ± 0.09

27

1.6 (0)

70 (0)

40 (0)

60 (0)

25.94 ± 1.54

16.77 ± 0.05

16.33 ± 0.06

31.62 ± 0.13

28

1.6 (0)

70 (0)

40 (0)

60 (0)

26.75 ± 0.46

17.03 ± 0.04

16.88 ± 0.13

31.53 ± 0.08

29

1.6 (0)

70 (0)

40 (0)

60 (0)

26.39 ± 0.54

16.67 ± 0.02

17.34 ± 0.18

31.55 ± 0.04

30

1.6 (0)

70 (0)

40 (0)

60 (0)

25.76 ± 0.78

16.35 ± 0.05

16.87 ± 0.05

31.86 ± 0.11

All results are the means ± SD (n = 3). 24

Table.3. Analysis of variance (ANOVA) for the fitted quadratic polynomial model for optimization of extraction parameters

Source

Total phenolic content (R2=0.9158)

Antioxidant activity (R2=0.8826)

Anthocyanins (R2=0.9009)

SS

DF

MS

F-value

p-value

SS

DF

MS

F-value

p-value

SS

DF

MS

F-value

p-value

Model

55.00

14

3.93

11.65