Brönsted acidic ionic liquid based ultrasound-microwave synergistic extraction of pectin from pomelo peels

International Journal of Biological Macromolecules 94 (2017) 309–318

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Brönsted acidic ionic liquid based ultrasound-microwave synergistic extraction of pectin from pomelo peels Zaizhi Liu a , Lu Qiao b , Fengjian Yang a , Huiyan Gu c , Lei Yang a,∗ a b c

Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China College of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou 450008, China School of Forestry, Northeast Forestry University, Harbin 150040, China

a r t i c l e

i n f o

Article history: Received 23 June 2016 Received in revised form 24 September 2016 Accepted 11 October 2016 Keywords: Brönsted acidic ionic liquid Pectin Pomelo Ultrasound-microwave synergistic extraction Response surface methodology

a b s t r a c t 3-Methyl-1-(4-sulfonylbutyl) imidazolium hydrogensulfate, [HO3 S(CH2 )4 mim]HSO4 , was applied as an extractant in an ultrasound-microwave synergistic extraction approach to substitute conventional solvent for the extraction of pectin from the albedo part of pomelo peels. The analysis of variance (ANOVA) test and response surface method were employed for the optimization of the extraction conditions. A pectin yield of 328.64 ± 4.19 mg/g was achieved using the obtained optimal conditions, which was significantly higher than yields of conventional methods with reference solvents. Pectin samples extracted with [HO3 S(CH2 )4 mim]HSO4 and hydrochloric acid solutions were tested by ANOVA and showed no significant differences in total carbohydrate content and degree of esterification; while galacturonic acid content was significantly different for the pectin from each extraction solvents. The differences revealed from images of atomic force microscopy and scanning electron microscope, Fourier transform infrared spectroscopy, and thermogravimetric analysis suggested the physiochemical properties of pectin could be affected by the extraction solvent. The [HO3 S(CH2 )4 mim]HSO4 proved to be a promising alternative to conventional solvents and the proposed method is efficient for the extraction of pectin from the albedo of pomelo peels. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Ionic liquids are a group of non-molecular ionic chemicals and are comprising organic and inorganic ions [1]. Ionic liquids have attracted increasing attention for a variety of processes because of their excellent physicochemical parameters [2,3]. Ionic liquids have been successfully used to dissolve cellulose effectively [4]. Meanwhile, their extensive applications are also reflected in natural products separation science, including the extraction of flavonoids [5], volatile components [6], and coumarins [7]. Task-specific Brönsted acidic imidazole ionic liquids, such as 3-methyl-1-(4-sulfonylbutyl) imidazolium hydrogen sulfate ([HO3 S(CH2 )4 mim]HSO4 ) with two acidic sites, have been designed primary as an alternative to traditional mineral acids (e.g. H2 SO4 and HCl) for catalytic applications [8,9]. Very recently, [HO3 S(CH2 )4 mim]HSO4 ] has been demonstrated as an excellent solvent with good acidic effects with a promising future for the preparation of natural products, such as flavonoid glycosides [10] and phenolic acids [11].

∗ Corresponding author. E-mail address: [email protected] (L. Yang). http://dx.doi.org/10.1016/j.ijbiomac.2016.10.028 0141-8130/© 2016 Elsevier B.V. All rights reserved.

Pomelo (belonging to Rutaceae family) is a native citrus species of Southeast Asia that has great economic value and is cultivated and consumed worldwide [12]. Large amounts of waste products (albedo and flavedo) are produced every year because of high consumption of pomelo. The albedo part of pomelo peel has been regarded as a potential source of soluble dietary fibres as it is rich in pectin [13,14]. Pectin is a complex polysaccharide primary contained in the cell walls of many fruits and vegetables. Pectin has gelling and stabilizing characteristics and is used commercially as a food additive for the following applications: thickener (for jams, jellies and confectionery production), emulsifier, and stabilizing agent (in acidified milk beverages) [15]. Pectin also possesses a variety of health benefits, such as cholesterol-lowering properties [16], immunostimulating activity [17], and antipancreatic cancer activity [18]. Industrial extraction techniques for the production of pectin are generally carried out using acidic conditions (inorganic acids solution, pH 1.5–3.0) at high temperature [19], which can give rise to negative environmental impacts and high corrosion damage to equipment. Enzymolysis seems to be a good alternative to traditional extraction methods [20]. However, this approach has some limitations because enzymatic specificity is a disadvantageous factor and could increase the cost of large-scale industrial production.

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To our best knowledge, there is only one reported application of nonacidic ionic liquids to extract pectin from lemon peels [21]. Extraction solvents with low pH are indispensable for the extraction of pectin [22]. The insoluble pectin constituents can be directly contacted and hydrolyzed into soluble pectin under acidic conditions and thus released and dissolved from plant tissues [22–24]. We were interested in the potential of [HO3 S(CH2 )4 mim]HSO4 as solvent to extract pectin from the albedo part of pomelo peels and intended to take advantage of the good solvent effect and intrinsic acidic property of this SO3H-functionalized ionic liquid. Ultrasound-microwave synergistic extraction (UME) processing has developed as a novel technique in the field of separation science in recent years [25,26]. The outstanding performance of UME is mainly ascribed to the benefits of both microwave and ultrasonic irradiation. The benefits of microwave irradiation are: first, microwave energy can accelerate water permeability to capillaries and the water absorption ability of the plant material, thus improving extraction efficiency [27]; second, the special heating mechanism heats the entire material simultaneously in a short time; third, microwaves can induce dipole rotation of molecules and migration of dissolved ions, which is considered to be responsible for the disruption of hydrogen bonds and thus break down of the plant cell walls [28]. A previous study has reported microwave energy can be efficiently absorbed and transferred by ionic liquids [29]. Ultrasonication provides three beneficial aspects, cavitation, the mechanical function, and thermal effect, which are maximally utilized in the extraction process and giving rise to relatively high extraction efficiency. In the present study, a Brönsted acidic ionic liquid based ultrasound-microwave synergistic extraction (BUME) technique was proposed for the extraction of pectin from the albedo part of pomelo peels. The response surface method (RSM) was used to evaluate the importance of three major variables to optimize the extraction conditions of pectin. Conventional heat reflux extraction (HRE) method and traditional solvents were investigated and compared with BUME. In addition, the physicochemical properties of the pectin samples that were extracted with HO3 S(CH2 )4 mim]HSO4 aqueous solution and hydrochloric acid solution using UME were investigated. 2. Experimental 2.1. Plant material Fresh pomelo fruit were purchased from Hada fruit market (Harbin, China) in November 2015. The pomelo were cultivated in Guanxi (Fujian province, China), and authenticated by Prof. Baojiang Zheng of the College of Life Science, Northeast Forestry University, China. Fresh samples of albedo were cut into lumps with a knife, dried in a shaded place at 25 ◦ C for 7 days, then powered into a 40–60 mesh and stored in a desiccator for the future experiments. 2.2. Solvents and chemicals [HO3 S(CH2 )4 mim]HSO4 and 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) were bought from Chengjie Chemical Co. Ltd. (Shanghai, China) and used without purification. Other chemicals were of analytical grade. Water applied in the experiments was purified by a reverse osmosis Milli-Q (Millipore, Bedford, MA, USA) instrument. 2.3. Apparatus An UWave-1000 ultrasound-microwave synergy extraction system (XTrust, Shanghai, China) laboratory-scale apparatus operating at a frequency of 2450 Hz was employed in BUME, UME and

microwave assisted extraction (MAE) procedures for the extraction of pectin from the albedo part of pomelo peels. Microwave energy transmitted to the reactor could be adjusted through a power feedback/control. Maximum microwave irradiation power output was 700 W. Ultrasonic power was uncontrolled and fixed at 50 W (40 kHz). An infrared temperature sensor was used to detect the temperature of the reaction cavity (57 cm × 51 cm × 52 cm). 2.4. BUME procedure The extraction procedure is presented in Fig. 1. Dried albedo powder 1 g and a proper volume of [HO3 S(CH2 )4 mim]HSO4 aqueous solution were mixed in a flask and then treated in the extraction system. The supernatant was isolated from insoluble residue after extraction using four-layer filter paper and then cooled to 25 ◦ C. Crude extracts were precipitated by adding ethanol to a proportion of 80% (v/v) and standing overnight at 4 ◦ C. Floccus precipitates were gathered by centrifugation (8000 × g for 30 min at 20 ◦ C), washed three times with dehydrated alcohol and dried in a freezer dryer. Gravimetric analysis was carried out to calculate pectin yield. Dried pectin samples were redissolved in pure water and detected by a UV–vis Spectra (UV-2550; Shimadzu, Kyoto, Japan) to evaluate the amount of residual [HO3 S(CH2 )4 mim]HSO4 [30]. No absorbance at 211 nm was detected in pectin samples, demonstrating the improved extraction method is reliable. Concentrations of [HO3 S(CH2 )4 mim]HSO4 , liquid–solid ratio, microwave irradiation power, and extraction time were investigated with single factor experiments for selecting suitable ranges for subsequent optimization experiments. 2.5. Optimization of BUME procedure with RSM A Box–Behnken design of RSM was employed to predict the optimal pectin extraction conditions in regard to three factors, which include extraction time, liquid–solid ratio, and microwave irradiation power. The factorial design is composed of 17 runs (12 factorial runs and 5 center runs). Pectin yield was set for the response to combine the independent factors (Table 1). Experiments were performed at random to minimize the impacts of unforeseen variability in the determined responses. Each factorial run was conducted in triplicate and the average values were presented as actual values. The interactions between the responses and the three independent variables were evaluated by the generalized form of a quadratic equation as following: Y = ˇ0 +

3  i=1

ˇi Xi +

3  i=1

ˇii Xi 2 +

3 2  

ˇij Xi Xj

(1)

i=1 j=i+i

where Y is the estimated response; the regression coefficients for the intercept, square, linearity and interaction are expressed as ˇ0 , ˇi , ˇii , and ˇij , respectively; and the three independent factors are presented as X1 , X2 , and X3 . 2.6. Comparison of BUME with reference method and solvents HRE was investigated as the reference pectin extraction method. Conditions for HRE were: hydrochloric acid solution (pH 2.5), 1000 W of heating power, 180 min of extraction time, and a liquid–solid ratio of 27 mL/g. Pure water, hydrochloric acid solution, and Na2 SO4 (10 mM) were used as reference extraction solvents with UME under the optimal conditions obtained for the experimental design to identify the prominently effect of an [HO3 S(CH2 )4 mim]HSO4 aqueous solution on the extraction of pectin. MAE was performed with 1.0 M [Bmim]Cl as described in previous study [21] to compare with the improved method.

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Fig. 1. Process for the extraction of pectin.

2.7. Pectin characterization Phenol-sulfuric acid photometric and m-hydroxydiphenyl methods were performed as described in a previous study [31] for each of the pectin extract from different extraction solvents to determine the total carbohydrate and galacturonic acid content, respectively. The titrimetric method and was applied to determine the degree of esterification (DE) [32]. The Coomassie brilliant blue staining method was carried out to evaluate the protein content of the pectin extrcts [33].

2.8. FTIR analysis Pectin samples (7.5 mg) extracted with 10 mM [HO3 S(CH2 )4 mim]HSO4 and hydrochloric acid solution were separately mixed with KBr (200 mg), powdered and then pressed by the addition of a 5 tons load for 8 min to form disks. FTIR spectra of pectin samples were recorded between wavelengths of 4000–400 cm−1 at a resolution of 5 cm−1 over 32 scans with a MAGNA-IR560 E.S.P (Nicolet, USA).

2.9. Thermogravimetric (TG) analysis TG analysis was conducted with a thermogravimetrical analyser (STA 6000-SQ8, PerkinElmer, USA). Each sample was subjected to heating from 40 to 800 ◦ C at a rate of 10 ◦ C/min under 20 mL/min nitrogen flow. Samples (approximately 5 mg) were placed in open

aluminum pans and the detailed percentage weight loss was recorded. 2.10. Atomic force microscopy (AFM) Pectin samples were observed on an AFM instrument (NSC15, MikroMasch, Wilsonville, OR, USA). Each pectin sample of 1 mg was dissolved in 10 mL Milli-Q water and diluted to 10 ␮g/mL. A 2-mL sample of the diluted solution was dropped onto a freshly-cleaved mica sheet and air-dried for 20 min. Pectin molecules adhered on the mica surface were imaged in the tapping mode at a scan rate of 0.5–1 Hz after drying in a desiccator overnight. The imaging was repeated several times to collect morphology images (512 × 512pixel resolution) using AFM tips (NSC15, MikroMasch, Wilsonville, OR, USA) with a force constant in the range 20–75 N/m. 2.11. Scanning electron microscopy (SEM) Characterization of the pectin powder morphology was observed on a SEM (SEM, Quanta 200, FEI, USA). Each sample was stuck to stubs with double faced adhesive tape and then sputtercovered with a gold-palladium layer (5–10 nm; 10 mA; 30 s) at ambient temperature before imaging. 2.12. Statistical analysis Box–Behnken design of RSM was achieved in the “Design Expert” software version 8.0 (Stat-Ease, Minneapolis, USA). Analy-

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Table 1 Experimental design matrix to screen for variables that determine the pectin yield and ANOVA results.a No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

BBD experiments X1

X2

X3

10 (0) 10 (0) 10 (0) 15 (1) 5 (−1) 15 (1) 15 (1) 15 (1) 5 (−1) 10 (0) 5 (−1) 10 (0) 10 (0) 10 (0) 10 (0) 10 (0) 5 (−1)

20 (−1) 20 (−1) 30 (1) 30 (1) 30 (1) 25 (0) 20 (−1) 25 (0) 20 (−1) 25 (0) 25 (0) 25 (0) 25 (0) 25 (0) 25 (0) 30 (1) 25 (0)

540 (1) 230 (−1) 230 (−1) 385 (0) 385 (0) 230 (−1) 385 (0) 540 (1) 385 (0) 385 (0) 540 (1) 385 (0) 385 (0) 385 (0) 385 (0) 540 (1) 230 (−1)

Y Predicted

Determined

291.66 282.92 298.74 329.54 310.95 321.82 323.17 299.34 297.94 317.05 302.51 317.05 317.05 317.05 317.05 295.19 274.84

290.62 282.37 299.78 330.31 308.59 320.01 325.53 298.02 297.17 323.18 304.32 310.31 313.54 312.42 325.81 295.74 276.16

ANOVA Source

Sum of squares

Degree of freedom

Mean square

F-value

p-value

Modelb X1 X2 X3 X1 X2 X1 X3 X2 X3 X1 2 X2 2 X3 2 Residual Lack of fit Pure error Cor total

3726.48 959.88 187.5 13.47 11.02 628.76 37.76 36 88.17 1743.42 218.61 25.11 193.5 3945.09

9 1 1 1 1 1 1 1 1 1 7 3 4 16

414.05 959.88 187.5 13.47 11.02 628.76 37.76 36 88.17 1743.42 31.23 8.37 48.37

13.26 30.74 6 0.43 0.35 20.13 1.21 1.15 2.82 55.82

0.0013** 0.0009*** 0.0441* 0.5324 0.5711 0.0028** 0.3079 0.3186 0.1368 0.0001***

0.17

0.9094

Credibility analysis of the regression equations Standard deviation

Mean

Coefficient of variation%

R2

Adjust R2

Predicted R2

Adequacy precision

5.59

306.7

1.82

0.9446

0.8733

0.82

12.76

a b * ** ***

The results were obtained with Design Expert 8.0 software (Stat-Ease, Minneapolis, USA). X1 is extraction time (min), X2 is the liquid–solid ratio (mL/g), X3 is the microwave irradiation power (W) and Y is pectin yield, mg/g. p < 0.05, significant. p < 0.01, highly significant. p < 0.001, extremely significant.

sis of variance (ANOVA) was used to evaluate the significance of the differences with respect to pectin yield. All the experiments were conducted in triplicate and the mean values of results are presented as yield ± SD.

3.1. Single factor BUME experiments

ally decreased. Based on a previously reported research [34], the viscosity of solvents enhances with an increase of ionic liquids concentration. The increase viscosity at high [HO3 S(CH2 )4 mim]HSO4 concentrations likely impedes the extraction solvent penetration into the plant materials and the diffusion of the analyte into solvent, resulting in a decreased yield. Similar results were also observed in the previous studies [35]. A 10 mM [HO3 S(CH2 )4 mim]HSO4 concentration was chosen for use in the RSM optimization.

3.1.1. Effect of [HO3 S(CH2 )4 mim]HSO4 concentration Albedo powder (1 g) was blended with 25 mL of [HO3 S(CH2 )4 mim]HSO4 aqueous solutions at different concentrations and extracted for 10 min at a microwave irradiation power of 385 W to obtain the satisfactory concentration of extraction solvent for the BUME method. The highest yield of pectin was achieved at 10 mM [HO3 S(CH2 )4 mim]HSO4 (Fig. 2a). The yield significantly increased with [HO3 S(CH2 )4 mim]HSO4 concentration between 2.5 mM and 10 mM, indicating that low concentrations of [HO3 S(CH2 )4 mim]HSO4 aqueous solutions were insufficiently acidic to efficiently extract pectin; but further increasing the concentration range from 10 to 50 mM, the pectin yield gradu-

3.1.2. Effect of microwave irradiation power Extraction procedures were performed at different levels of microwave irradiation power at atmospheric pressure for 10 min to estimate the influence of this parameter on pectin yield. The liquid–solid ratio and HO3 S(CH2 )4 mim]HSO4 concentration were fixed at 25 mL/g and 10 mM, respectively. The yield was maximum at 385 W (Fig. 2b), and decreased dramatically at greater microwave irradiation power. Microwave irradiation power of 230–540 W was chosen for further optimization to obtain high yield and minimize energy consumption.

3. Results and discussion

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Fig. 2. Effects of [HO3 S(CH2 )4 mim]HSO4 concentration (a), microwave irradiation power(b), liquid-solid ratio (c) and extraction time (d) on the yield of pectin from the albedo of pomelo peels. Each value is expressed as a mean (triplicate) ± standard deviation (SD). Bars annotated with the same letters are not significantly different at the 5% (P < 0.05) level according to ANOVA.

3.1.3. Effect of liquid–solid ratio Liquid–solid ratio determines the contact area of liquid with solid and influences the pectin yield. The influence of liquid volume to solid ratio on pectin yield was evaluated to simplify the extraction procedure and minimize solvent consumption. [HO3 S(CH2 )4 mim]HSO4 concentration, microwave irradiation power and extraction time were fixed at 10 mM, 385 W and 10 min, respectively. A low liquid–solid ratio led to incomplete immersion of raw materials in solvent (Fig. 2c). High liquid–solid ratio required large solvent consumption and caused high acidity, which was unbeneficial for the extraction of pectin. Therefore, 20–30 mL/g was chosen as the liquid–solid ratio range for the further optimization.

3.1.4. Effect of extraction time Extraction time usually has a great influence on the yield of analytes. BUME procedures were conducted at 385 W with a liquid–solid ratio of 25 mL/g for different extraction times (2.5, 5, 10, 15 and 20 min) to find an appropriate extraction time to obtain a high pectin yield. Pectin yield improved significantly with increasing extraction time from 2.5 min to 10 min, with slight improvement for longer extraction times (Fig. 2d). A 5–15-min extraction time range was used for further optimization experiments.

3.2. Further optimization by RSM The RSM was employed to further investigate the interactions among three different factors (X1 : extraction time, X2 : liquid–solid ratio and X3 : microwave irradiation power). The factors X1 , X1 X3 , X2 2 , X3 2 were significant, with “Prob > F” values lower than 0.05 (Table 1). The high model F-value (14.11) and low P-value (0.0011) implied the model fit is significant [36]. The high R2 (0.9446) and adj-R2 (0.8733) values clearly indicated the precision of developed model for the interactions between the response values and independent factors. A coefficient of variation of 1.82% demonstrated the high accuracy and dependability of the extraction experiments [37]. The signal to noise ratio of 13.26 for response indicated a favorable coordination for the developed model. The final equation to predict pectin yield was given by RSM as follows, Yield of pectin (mg/g) = −77.57 + 7.74X 1 + 12.31X 2 + 0.92X 3 − 0.06X 1 X 2 − 0.02X 1 X 3 − 0.04X 2 X 3 + 0.12X 1 2 − 0.18X 2 2 − 8.47 × 10−4 X 3 2

(2)

3D surface charts were built using Eq. (2) to identify the optimal extraction conditions. Fig. 3a is the response surface for a

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Fig. 4. Performance of [HO3 S(CH2 )4 mim]HSO4 recycle number on the yield of pectin. Each value is expressed as a mean (triplicate) ± standard deviation (SD). Results with the same letters are not significantly different at the 5% (P < 0.05) level according to ANOVA.

fixed microwave irradiation power of 385 W. The yield of pectin increased with the liquid–solid ratio and increasing extraction time. The highest yield was achieved after 15 min of microwave irradiation with a liquid–solid ratio of 27 mL/g. Fig. 3b shows the interrelations between microwave irradiation power and extraction time at a liquid–solid ratio of 25 mL/g. The yield of pectin improved dramatically with increasing extraction time for a set microwave irradiation power and peaked at approximately 15 min. A quadratic impact was observed for the effect of microwave irradiation power on pectin yield and a satisfactory yield was obtained around 360 W, with a sharp decline in yield observed at higher wattage. Fig. 3c displays the influence of the microwave irradiation power and liquid–solid ratio on pectin yield for a set extraction time of 10 min. A linear effect and a striking quadratic effect on the pectin yield were observed for the liquid–solid ratio and microwave irradiation power, respectively. The optimum conditions estimated by the RSM software were: liquid–solid ratio, 27 mL/g; extraction time, 15 min; and microwave irradiation power, 357 W. The pectin yield was predicted to be 332.78 mg/g using these conditions. Verification experiments were performed thrice using optimal conditions with a slightly modification to the microwave irradiation power (360 W) and the pectin yield achieved was 328.64 ± 4.19 mg/g.

3.3. Performance of recovered [HO3 S(CH2 )4 mim]HSO4 in subsequent extractions Residues from the extraction were isolated by filtration and could be used for the preparation of bio-fuels or other chemicals. Vacuum distillation was applied to recover [HO3 S(CH2 )4 mim]HSO4 from the residue solutions and reused without further purification. The [HO3 S(CH2 )4 mim]HSO4 could be recycled five times with more than 90% of the optimum pectin yield achieved for the fifth cycled (Fig. 4).

Fig. 3. Response surface and contour plots for the effects of variables on the extraction yields of pectin. (a) Extraction time and liquid–solid ratio (1 g dried sample was mixed with 10 mM [HO3 S(CH2 )4 mim]HSO4 and extracted at a fixed microwave irradiation power of 385 W with Brönsted acidic ionic liquidbased ultrasound-microwave synergistic extraction (BUME)), (b) extraction time

and microwave irradiation power (1 g dried sample was mixed with 25 mL 10 mM [HO3 S(CH2 )4 mim]HSO4 and extracted with BUME), (c) liquid–solid ratio and microwave irradiation power (1 g dried sample was mixed with 10 mM [HO3 S(CH2 )4 mim]HSO4 and then extracted 10 min with BUME).

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Table 2 Comparison of Brönsted acidic ionic liquid-based ultrasound-microwave synergistic extraction (BUME) with ultrasound-microwave synergistic extraction (UME), microwave assisted extraction (MAE) and heat reflux extraction (HRE) methods, mean ± S.D. (n = 3). Methodsa

Solvent

Extraction time (min)

Pectin yield (mg/g)

BUME HRE UME UME UME MAEb

10 mM [HO3 S(CH2 )4 mim]HSO4 Hydrochloric acid solution Pure water Hydrochloric acid solution 10 mM Na2 SO4 1.0 M [Bmim]Cl

15 180 15 15 15 9.6

328.64 ± 6.11a 197.24 ± 5.71c 90.50 ± 4.16d 269.31 ± 4.86b 84.71 ± 3.25d 210.39 ± 5.82c

Each value is expressed as a mean (triplicate) ± standard deviation (SD). The same letters in the column are not significantly different at the 5% (P < 0.05) level according to ANOVA. a 1 g dried sample was mixed with 27 mL of different solutions and then extracted with corresponding methods. b Experiment was carried out at as described by Huang et al. [21].

3.4. Comparison of BUME with traditional extraction method and solvents The BUME procedure had a higher yield than that of HRE (Table 2). The specific mechanisms of ultrasonic and microwave irradiation used in the BUME guarantee a relatively higher yield than HRE. The [HO3 S(CH2 )4 mim]HSO4 has an excellent influence to improve the yield compared with the selected solvents. Comparisons between [HO3 S(CH2 )4 mim]HSO4 , [Bmim]Cl, inorganic salts (Na2 SO4 ) and pure water demonstrated that the improvement is a solvent effect, not a salt effect. Our [HO3 S(CH2 )4 mim]HSO4 system provided a higher yield than that of 1.0 M [Bmim]Cl [21]. The pectin yield of hydrochloric acid solution (pH 2.5) was significantly higher than that of pure water (P < 0.05). The significant differences of yields between [HO3 S(CH2 )4 mim]HSO4 , [Bmim]Cl and hydrochloric acid solutions suggest that synergistic acid and solvent effects

gave rise to the remarkable enhancement of the pectin yield by [HO3 S(CH2 )4 mim]HSO4 .

3.5. Pectin characterization The physiochemical characterizations of pectin powders extracted with HO3 S(CH2 )4 mim]HSO4 (10 mM) and hydrochloric acid solution (pH 2.5) using UME are listed in Table 3. The total carbohydrate was similar for the two experiments with 52.05 ± 3.25% pectin extracted with HO3 S(CH2 )4 mim]HSO4 solution and 52.44 ± 2.71% for that extracted with hydrochloric acid solution. There was no significant difference in the DE values of the two pectin samples (P > 0.05) and both could be classified as high methoxyl pectin. The pectin extracted with HO3 S(CH2 )4 mim]HSO4 solution gave a slightly lower DE value (56.55 ± 2.80%) than that from the hydrochloric acid solution

Fig. 5. FTIR spectra, thermogravimetric (TG) and derivative thermogravimetric (DTG) analysis of pomelo pectin extracts. FTIR spectra of pectin extracted with [HO3 S(CH2 )4 mim]HSO4 aqueous solution (a) and hydrochloric acid solution (b); TG curves of pectin extracted with [HO3 S(CH2 )4 mim]HSO4 aqueous solution (c-␣) and hydrochloric acid solution (c-␤); DTG curves of pectins extracted with [HO3 S(CH2 )4 mim]HSO4 aqueous solution (d-␣) and hydrochloric acid solution (d-␤).

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Table 3 Chemical and physicochemical properties of pectin extracted from albedo of pomelo peels. Extraction solventsa

10 mM [HO3 S(CH2 )4 mim]HSO4

Hydrochloric acid solution

Total carbohydrate (%) Degree of esterification (%) Galacturonic acid (%) Protein (%)

52.05 ± 3.25a 56.55 ± 2.80a 66.14 ± 3.98b Trb

52.44 ± 2.71a 62.40 ± 3.59a 75.59 ± 2.11a Tr

Each value is expressed as a mean (triplicate) ± standard deviation (SD). The same letters in each column are not significantly different at the 5% (P < 0.05) level according to ANOVA. a Extraction processes were performed using the optimal extraction conditions for each solvent. The extraction conditions were: 10 mM [HO3 S(CH2 )4 mim]HSO4 , liquid–solid ratio 27 mL/g, extraction time 15 min, and microwave irradiation power 357 W. b Tr means trace.

(62.40 ± 3.59%). All pectin extracts meet the requirements of provisions of the Food and Agriculture Organization (FAO) and European Union (EU), which stipulate the amount of galacturonic acid contained in ‘pectin’ should be greater than 65% [38]. The galacturonic acid content was 66.14 ± 3.98% for pectin extracted with HO3 S(CH2 )4 mim]HSO4 aqueous solution and 75.59 ± 2.11% for that extracted with hydrochloric acid solution. The galacturonic acid content of pectin extracted with HO3 S(CH2 )4 mim]HSO4 is approximate to that reported for a previous pomelo pectin extraction [39]. The relatively low galacturonic acid content of pectin extract with HO3 S(CH2 )4 mim]HSO4 aqueous solution is probably ascribable to the unique properties of HO3 S(CH2 )4 mim]HSO4 in the BUME extraction process. The content of protein in pectin extracted with

different solvents was trace, which indicated that the two pectin extracts are in the range of FAO standards [40].

3.6. FTIR and TG analysis Fig. 5a and b show the FTIR spectra of the two pectin samples that were extracted using HO3 S(CH2 )4 mim]HSO4 aqueous solution (a) and pH 2.5 hydrochloric acid solution (b). Both pectin samples demonstrated strong absorption bands around 3460, 2940, 1750 and 1650 cm−1 , which corresponded well with previously reported research [41]. Some structural differences between the two pectin samples can be inferred from the spectra because of the relatively different infrared absorption intensities for the 1750 and 1650 cm−1 bands [42]. DE was also obtained from the relative percentage of the absorption band area at 1750 cm−1 over the sum of absorption bands areas at 1750 and 1650 cm−1 . The DE value of pectin obtained from the proposed method was slightly lower than that of extracted with hydrochloric acid solution, which was probably because the protonated groups of the hydrochloric acid solution sample could weaken the absorption band around 1750 cm−1 and strengthen the band around 1650 cm−1 [39]. The [HO3 S(CH2 )4 mim]HSO4 is a strong proton donor ionic liquid in the extraction process, which combines with functionalized butyl sulfonic chain and the greater Brönsted acidity hydrogensulfate (HSO4 ) anion [43]. The TG and derivative thermogravimetric (DTG) analysis were used to determine the inorganic composition content of the pectin samples the degradation temperatures (Fig. 5c and d). Both pectin extracts presented three step thermal degradation characteristics

Fig. 6. Atomic force microscopy and scanning electron microscope images of pectins. Atomic force microscopy images of pectin extracted with [HO3 S(CH2 )4 mim]HSO4 aqueous solution (a) and hydrochloric acid solution (b); Scanning electron microscope images of pectin extracted with [HO3 S(CH2 )4 mim]HSO4 aqueous solution (c) and hydrochloric acid solution (d).

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and the pectin extracted with [HO3 S(CH2 )4 mim]HSO4 (Fig. 5c-␣) demonstrated a lower weight loss compared with that extracted with hydrochloric acid solution (Fig. 5c-␤). Two notable inflection points were observed in the DTG curves for both samples and occurring for the pectin extracted with hydrochloric acid solution around 10 ◦ C earlier than for that from [HO3 S(CH2 )4 mim]HSO4 . The inflection point occurring below 100 ◦ C is the loss of polar micromolecules (e.g. water and ethanol). This was more facile for pectin extracted with [HO3 S(CH2 )4 mim]HSO4 than for that extracted with hydrochloric acid solution, which indicated the former possesses a certain extent of hydrophobicity. The second inflection was caused by the degradation of macromolecules which generally occurred higher than 100 ◦ C. The relatively higher degradation temperature of the pectin extracted with [HO3 S(CH2 )4 mim]HSO4 (Fig. 5d-␣) compared with pectin extracted by hydrochloric acid solution (Fig. 5d-␤) suggested the former has higher thermostability.

3.7. AFM and SEM AFM images were obtained of the pomelo pectin extracted with different solvents under the optimal extraction conditions (Fig. 6a and b). Pectin extracted with [HO3 S(CH2 )4 mim]HSO4 (Fig. 6a) has an obviously network structure with dense and unequal in size ellipse areas interspersed throughout the image, which is similar to that observed for orange pectin [44]. The sample extracted with hydrochloric acid solution exhibited a relatively sparse nonnetwork structure with a large amount of kinked branches diffused in the whole image (Fig. 6b). Fig. 6c and d show the SEM images of the two pectin samples. The pectin extracted with [HO3 S(CH2 )4 mim]HSO4 possessed rather compact, rough and hard structures, with many irregular fragmental particles of varying size and form stuck on the surface (Fig. 6c). In contrast, the pectin extracted with hydrochloric acid solution (Fig. 6d) displayed smooth, puffy and soft folds with fewer sporadic particles stuck on the surface. The distinct characters of the pectin surface morphologies observed on the two pectin samples could be ascribed to the extraction ability differences of the two solvents.

4. Conclusion In this study, a BUME approach was developed for the extraction of pectin from the albedo part of pomelo peels. This paper mainly focused on proposing an efficient separation technique for the extraction of pectin from pomelo peels. The ANOVA test and a multivariate study based on RSM were applied to evaluate the influences of three major variables that can potentially influence the performance of BUME. The optimum conditions were 10 mM [HO3 S(CH2 )4 mim]HSO4 , 15 min of extraction time, 360 W of microwave irradiation power, and 27 mL/g of liquid–solid ratio. Pectin yield was 328.64 ± 4.19 mg/g using the optimum conditions. The developed method is efficient compared with the reference method and solvents trialed. The high pectin yield obtained from the recycled [HO3 S(CH2 )4 mim]HSO4 indicated the proposed double acidic site ionic liquid is capable of multiple pectin extractions. The total carbohydrate content and DE of pectin extracted with [HO3 S(CH2 )4 mim]HSO4 aqueous solution were not significantly different to those of pectin extracted with hydrochloric acid solution. However, the differences of pectin galacturonic acid content, FTIR and TG analysis, and SEM and AFM images suggested that the physiochemical properties of pectin can be affected by extraction solvents. Investigations concentrating on the detailed characterization of pectin extracted with [HO3 S(CH2 )4 mim]HSO4 aqueous solution await further studies.

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