or heating on the extraction of pectin from grapefruit peel

Accepted Manuscript Effects of ultrasound and/or heating on the extraction of pectin from grapefruit peel Yuting Xu, Lifen Zhang, Yakufu Bailina, Zhi Ge, Tian Ding, Xingqian Ye, Donghong Liu PII: DOI: Reference:

S0260-8774(13)00572-4 http://dx.doi.org/10.1016/j.jfoodeng.2013.11.004 JFOE 7626

To appear in:

Journal of Food Engineering

Received Date: Revised Date: Accepted Date:

31 August 2013 1 November 2013 5 November 2013

Please cite this article as: Xu, Y., Zhang, L., Bailina, Y., Ge, Z., Ding, T., Ye, X., Liu, D., Effects of ultrasound and/ or heating on the extraction of pectin from grapefruit peel, Journal of Food Engineering (2013), doi: http:// dx.doi.org/10.1016/j.jfoodeng.2013.11.004

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Effects of ultrasound and/or heating on the extraction of pectin from grapefruit peel

Yuting Xu a, Lifen Zhang b, Yakufu Bailina a, Zhi Ge a, Tian Ding a, Xingqian Ye

a,c

,

Donghong Liu a,c,*

a

College of Biosystems Engineering and Food Science, Zhejiang University,

Hangzhou, Zhengjiang 310058, PR China; b

College of Food Science and Technology, Henan University of Technology,

Zhengzhou, Henan 450001, PR China; c

Fuli Institute of Food Science, Zhejiang University, Hangzhou, Zhengjiang 310058,

PR China

* Corresponding author. Address: College of Biosystems Engineering and Food Science, Zhejiang University, 866 Yuhangtang Rd., Hangzhou, Zhejiang 310058, PR China. Tel.: +86 057188982169 Fax: +86 057188982144 E-mail: [email protected]

1

ABSTRACT Effects of ultrasound and/or heating on the yield of pectin, swelling behavior of material, and kinetics of pectin extraction from grapefruit peel were investigated. Several extraction parameters significantly affected the yield of pectin and swelling index (SI) of vegetal tissue. The optimal extraction conditions for ultrasound-assisted heating extraction (UAHE) selected through single-factor experiments were as follows: ultrasound power density 0.40 W/mL, duty cycle 50%, temperature 60°C, solid-liquid ratio 1/50 g/mL. Image studies showed that UAHE disrupted the vegetal tissue and significantly improved its swelling behavior. There existed significantly high correlations between tissue SI and pectin yield, indicating that the improvement of pectin extractability via disrupting vegetal tissue was the main mechanism for ultrasonic enhancement of extraction. A theoretical model, which could simultaneously describe the extractability, dissolution and degradation rates of pectin, and predict the maximal yield and the optimal time, was used to study the extraction kinetics when ultrasound and/or heating were applied. Yields of pectin extracted using UAHE (0.40 W/mL and 60°C), ultrasound-assisted extraction (UAE, 0.40 W/mL and 30°C), heating extraction (HE, 60°C), room temperature extraction (RE, 30°C) and conventional heating extraction (CHE, 80°C) within 60 min were monitored and analyzed by the model respectively.

The kinetics study showed that both heating

Abbreviations CHE, Conventional heating extraction; GPP, Grapefruit peel powder; HE, Heating extraction; RE, Extraction at room temperature; S/L, Solid-liquid ratio; SI, Swelling index; UAE, Ultrasound-assisted extraction; UAHE, Ultrasound-assisted heating extraction. 2

and ultrasound could significantly facilitate the extractability, dissolution and degradation of pectin, and there existed synergistic effect between them. Compared with CHE, UAHE significantly improved the extractability and extraction rate of pectin, leading to higher yield (26.74%) with shorter extraction time (51.79 min) and reduced temperature (60°C). These results suggested that UAHE could be an efficient technique for the extraction of pectin from plant materials. Key words: ultrasound; heating; pectin extraction; swelling; kinetics.

1. Introduction Pectin is a family of complex heteropolysaccharides consisting of a backbone of α-(1→4) galacturonic acid residues which are partially esterified with methyl alcohol or acetic acid at the carboxylic acid. As a structural component of plant cell wall, it is extensively distributed in the primary cell wall and middle lamella of all plant tissues (Ridley et al., 2001; Thakur et al., 1997). Pectin is widely used as gelling, stabilizing and thickening agents in food systems such as jams and jellies, confectionery, and fruit juice (Thakur et al., 1997; Zouambia et al., 2009). It is also shown to possess various pharmaceutical activities including healing wound (Hokputsa et al., 2004), inhibiting lipase activity (Edashige et al., 2008; Kumar and Chauhan, 2010), inhibiting growth and metastasis and inducing apoptosis of human cancer cell (Jackson et al., 2007; Nangia-Makker et al., 2002), as well as immunostimulating activity, anti-metastasis activity, anti-ulcer activity, cholesterol decreasing effect and so on (Yamada, 1996). 3

In the commercial extraction process, pectin is conventionally extracted using hot water (60-100°C) acidified with a mineral acid (such as sulfuric, phosphoric, nitric, hydrochloric or citric acid) within the pH 1.5-3 for 0.5-6 hours (Koubala et al., 2008). The process is time consuming and leads to pectin degradation, so the conventional heating extraction (CHE) has both quantitative and qualitative disadvantages for pectin extraction (Liu et al., 2006). To produce pectin with superior yield and quality, it is of great importance to explore novel methods or modification of the existing methods to avoid using extreme extraction conditions. As an applicable and innovative technology, the application of ultrasound-assisted extraction from plant material is widely published (Ebringerová and Hromádková, 2010; Esclapez et al., 2011; Vilkhu et al., 2008). Many studies have been carried out to extract pectin from apple pomace or grapefruit peel by ultrasound-assisted heating extraction (UAHE) (Bagherian et al., 2011; Panchev et al., 1988; 1994). All of these studies showed that UAHE increased yield and/or extraction rate as well as reduction in extraction time. However, there is no comprehensive study on the effects of UAHE conditions, especially the ultrasonic parameters, on pectin yield. Knowledge of the mechanism and kinetics of extraction process is generally needed for extraction process design and operating conditions optimization. Some researchers have studied the mechanism of ultrasound-assisted extraction through exploring the effect of ultrasound on the vegetal materials. These studies observed the disruption of cell structure of vegetal tissue exposed to ultrasound irradiation using scanning electron microscope (SEM) or light microscope (LM), and concluded that this 4

increases the accessibility of the solvent to the internal particle structure, thereby facilitating the release of the cell contents (Anese et al., 2013; Li et al., 2004; Supardan et al., 2011; Zhao et al., 2007). Moreover, it was noteworthy that ultrasound also enhanced tissue hydration swelling during steeping, which was deemed to be responsible for the increases of extraction yield in a short time (Toma et al., 2001). However, there was no available information about the relationship between tissue swelling behavior and pectin yield in the published studies. Exploring the correlation between them will present a new insight into the mechanism of the enhancement of pectin extraction. On the other hand, several empirical kinetics models have been developed for pectin extraction based on the hypothesized and simplified extraction mechanism. Panchev et al. (1989) presented a model to describe the kinetics of pectin extraction from apple pomace involving the dissolution of pectin from protopectin and the degradation of dissolved pectin. Cheung et al. (2012) evaluated the suitable kinetic models for ultrasound-assisted extraction of water-soluble polysaccharides from both the fruit body and mycelia form of medicinal fungi. These model equations, albeit empirical, are useful for design and optimization of the extraction processes and for analysis and understanding of the major factors and their effects. To the best of our knowledge, however, no previous studies have been documented on the kinetics models for the extraction of pectin using UAHE. The aim of this study was to explore the effects of UAHE parameters on pectin yield and swelling behavior of grapefruit peel, as well as the relationship between 5

them, to gain better understanding of the mechanism of UAHE. Meanwhile, the single and combined effects of ultrasound and heating on the kinetics of pectin extraction were investigated. In order to show the advantages of the new method, a comparison was also conducted between the effects of UAHE and CHE.

2. Materials and methods 2.1 Material and chemical reagents Fresh grapefruit (Citrus paradisi Macf.) were picked from Taizhou, China. The collected peels were pretreated according to Guo et al. (2012), with minor modifications. Firstly, they were soaked in a water bath at 90°C for 5 min to inactivate enzymes, then dried in an oven with air circulation at 50°C. The dried peels were grounded by an electric grinder (Qijian Q-250A3, Shanghai Bingdu Electrical Appliance Co., LTD, China) and passed through an 80 mesh sieve to obtain particles <180 μm. The samples were then vacuum-packed and stored inside desiccators until use. All chemicals used in the experiments were analytical grade and purchased from Sinopharm Chemical Reagent Co., LTD (Shanghai, China). 2.2 Experimental set-up and characterization of ultrasonic field Ultrasound was applied by a probe system (Sonics, VCX800, USA; 800 W, 20 kHz), which allows the probe tip to be changed. Two types of probes with different diameters, 13 mm and 25 mm, were used. 6

Deionized water with pH of 1.5 adjusted by 0.5 M HCl was used as the extraction solvent. Grapefruit peel powder (GPP) was mixed with extraction solvent (150 mL) in a 200 mL glass reactor with jacket for external circulation. The ultrasonic emitter was immersed 1.5 cm into the solution. During the experiments, the temperature was measured with a digital thermometer located in the center of the extraction vessel and held constantly at a desired value by a thermostatic bath that drove cooling water through jacketed reactor. Periodically agitation (10 s on: 50 s off) was conducted to keep the mixture evenly distributed. The reactor was sealed to prevent water leaks during sonication. A calorimetric procedure was used to determine the effective ultrasonic power transferred into the medium for every condition tested (Raso et al., 1999). For this purpose, the temperature of the solvent being sonicated was recorded versus time with a digital thermometer without controlling the temperature. Thus, using the temperature rise caused by cavitation, the ultrasonic power applied (P, W) was calculated as: P = (M·CP) ·(dT/dt), where M (kg) is the solvent mass, CP (J/kg °C) is the heat capacity and dT/dt is the slope of the logged temperature-time curve. The solvent used in all experiments was distilled water, which was thermostated at the starting temperature before starting any experiment. It was found that there existed a linear relationship between ultrasonic amplitude and the output power when the amplitude ranged from 30%-80% of the maximum (13 mm probe: 114 μm; 25 mm probe: 35 μm). For both probes, the actual output powers of ultrasound versus a series of amplitudes were determined to construct standard 7

curves (13 mm probe: y=85.05x-4.9233, R2=0.9981; 25 mm probe: y=118.65x-7.4783, R2=0.9961; y is the output power, x is the % of the maximal amplitude). Power density (D, W/mL) of ultrasound dissipated into the medium with volume V is given by D=P/V. For output power levels of 30, 40, 50, 60, 70 and 80 W, the calculated power densities were 0.20, 0.27, 0.33, 0.40, 0.47 and 0.53 W/mL, respectively. Power intensity (I, W/cm2) dissipated from a probe tip with radius r is given by I=P/πr2. 2.3 Extraction and purification of pectin 2.3.1 Extraction of pectin using UAHE Single-factor experiments were employed to determine the effects of different UAHE parameters including emitter surface (13 mm and 25 mm), power density (0.20, 0.27, 0.33, 0.40, 0.47 and 0.53 W/mL), duty cycle (33, 40, 50, 60, 70 and 80%), temperature (30, 40, 50, 60, 70 and 80°C) , solid-liquid ratio (S/L) (1/30, 1/40, 1/50, 1/60 and1/70 g/mL) and sonication time (10, 20, 30, 40, 50 and 60 min) on pectin yield and tissue swelling. According to the preliminary experiments (Bagherian et al., 2011; Guo et al., 2012; Pahchev et al., 1988), extraction temperature, S/L, and sonication time were initially fixed at 70°C, 1/50 g/mL and 30 min respectively. The probe with 25 mm diameter was applied in the studies on the latter 5 parameters. After extraction, the extraction mixture was quickly transferred into centrifugal tubes, the volumes measured (Vm), and placed in a cold-water bath for cooling. Then the mixture was centrifuged at 9000 rpm for 30 min under 4°C using a low-temperature centrifuge (HITACHI CF16RXII, Japan) and the volume of filtrate was measured and recorded as Vf. The filtrate was coagulated using equal volume of 8

95% ethanol and left for 2 h under 4°C. The coagulated pectin was separated by centrifuge and washed three times with 95% ethanol. It was dried at 40°C in a laboratory drier until its weight was constant; then the dried pectin weight was weighed with an analytical balance (Mettler Toledo GB204, Switzerland). The extraction yield of pectin was calculated from the initial volume of the extraction solvent according to Panchev et al. (1988). The swelling index (SI) of tissue residue was defined as the volume in milliliters occupied by 1g GPP according to Toma et al. (2001) with slight modification. The standing stage was replaced by centrifugation under the consistent conditions (centrifuging at 9000 rpm for 30 min under 4°C). SI was calculated as: SI=(Vm-Vf)/m, where m is the dry weight of GPP. All experiments were done in triplicate. 2.3.2 Extraction of pectin by different methods Five different extraction conditions as shown in Table 1 were used in the kinetics study. UAE and HE respectively stand for the individual application of ultrasound and heating, while UAHE was a combination of them. Power density and heating temperature, as well as other parameters, were set at the optimized level selected by the single-factor experiments as described in 2.3.1. RE was the extraction at room temperature, CHE was the conventional method for pectin extraction from orange peel, according to the method of Kratchanova et al. (2004) with slight modification. Extraction parameters of these two methods, except the extraction temperatures, were the same with HE. 2.4 Preparation of the samples for examination by SEM 9

The extraction residues were air-dried and then mounted onto a SEM specimen stub with a double-sided adhesive tape prior to coating. Specimens were coated with gold using a HITACHI E-1020 ion sputter (JEOL, Japan) and subsequently photographed using Scanning Electron Microscope Cambridge Stereoscan 260 coupled with energy dispersive X-ray microanalysis system (Leica, UK). 2.5 Extraction kinetics study 2.5.1 Theoretical development of mathematical models The extraction process of pectin from GPP involves two simultaneous transformations: the transformation of insoluble pectin (protopectin) into soluble pectin and diffusion of the pectin from plant tissues into the solution (rate constant k1), and the degradation of partial dissolved pectin (rate constant k2) (Panchev et al., 1989). In this study, the mathematical model for the extraction kinetics of pectin was described according to Panchev et al. (1989) and Cho et al. (2000) with some modifications as follows. The reaction scheme for pectin extraction is Protopectin→Dissolved pectin→Degraded pectin

(1)

Here the amount of protopectin that can be extracted from the material under some specified conditions after extraction for a time t was marked as z(t), the amount of the degraded pectin as q(t); and the dissolved pectin that will be obtained as y(t). Then we can get the following first-order reaction kinetics: dz(t)/dt=-k1·z(t)

(2)

dq(t)/dt=k2·y(t)

(3)

10

dy(t)/dt=k1·z(t) - k2·y(t)

(4)

PE was used to denote the percentage of the extractable pectin to the raw material under specified extraction conditions. Then, PE= z(t)+q(t) +y(t)

(5)

After solving the equations (2)-(5), the following equations describing the changes with time of protopectin z(t), the obtained pectin y(t), the degraded pectin q(t) and the total dissolved pectin p(t) can be obtained. z(t)= PE·exp(-k1·t) y(t)= PE·k1/( k2- k1)( exp(-k1·t)- exp(-k2·t))

(6) (7)

q(t)= PE·(1+ k2/( k1- k2)·( exp(-k1·t)+ k1/( k2- k1)·( exp(-k2·t)) (8) p(t)= y(t)+ q(t)= PE·(1-exp(-k1·t))

(9)

From the above equations, we can find out the time, tmax, that the pectin yield reached its maximum value, ymax. tmax=ln(k1/ k2)/ (k1- k2) ymax= PE·(k2/ k1) (k2/ k1)/(1-k2/ k1)

(10) (11)

The changes of reaction rates for these processes, Ry(t), Rp(t) and Rq(t) with time can be calculated using the following differential equations. Ry(t)=dy(t)/dt

(12)

Rq(t)=dq(t)/dt

(13)

Rp(t)=dp(t)/dt

(14)

2.5.2 Data acquisition and determination of model parameters The data of the pectin yield during different extraction duration extracted by 5 11

methods: UAHE, UAE, HE, RE and CHE, was used for studying the extraction kinetics. The kinetics curves were fitted for equation (7) by nonlinear regression analysis using Origin software (Version 8.5) and the parameters PE, k1 and k2 were calculated. Then the maximum value of pectin yield (ymax) and the moment of time (tmax) that the pectin yield reached ymax were calculated from equation (10) and (11). The apparent activation energy (Ea) of pectin dissolution and degradation was calculated from the logarithmic form of the Arrhenius equation by plotting lnk against 1/T: lnk=lnA-Ea/RT. Where A is the Arrhenius constant; R is the universal gas constant (8.314 J/mol K); T is the absolute temperature (K). 2.6 Statistical analysis and figure plotting All experiments were performed in triplicate. The data was analyzed using analysis of variance (ANOVA) by SPSS software (Version 16.0), and expressed as mean value ± standard deviation. The confidence level for statistical significance was set at a probability value of 0.05. The figures were plotted using Origin software (Version 8.5).

3. Results and discussion 3.1 Effects of extraction parameters on the yield of pectin The results of different extraction parameters on pectin yields were shown in Fig. 1. 3.1.1 Emitter surface Effects of different emitter surfaces were compared at three ultrasound output 12

power levels: 0.27, 0.33 and 0.40 W/mL (Fig. 1A). Duty cycle was set as 50% (2 s on: 2 s off). For both probes, the yields of pectin extracted under 0.40 W/mL were significantly higher than those extracted under 0.27 W/mL (p<0.05). More importantly, at the same power level, pectin yields extracted with 13 mm probe were significantly higher than those with 25 mm probe (p<0.05). It was because the ultrasound intensity emitted from the probe with smaller emitter surface (30.08-45.11 W/cm2) was much higher than that of the larger one (8.15-12.22 W/cm2). 3.1.2 Ultrasound power density The effect of power density on pectin yield was shown in Fig. 1B. With increasing power density from 0.20 to 0.40 W/mL, the pectin yield was significantly increased from 22.67% to 27.27% (p<0.05). This can be explained by the fact that the cavitation bubble collapse became more violent with amplitude or power increased, since the resonant bubble size is proportional to the amplitude of the ultrasonic wave (Brotchie et al., 2009; Merouani et al., 2013). However, when the power density became higher than 0.40 W/mL, a significant decrease of pectin yield showed up (p<0.05). It was probably because the cavitation activity was reduced at high bubble volume concentrations. On the one hand, a cloud of cavitation bubbles produced around the probe tip could screen and reduce the energy transmission into the reaction medium, which was considered as the “saturation effect” (Contamine et al., 1995). On the other hand, the increase of interbubble impacts would increase the probability of the bubbles deformation and their collapse in a nonspherical way, thus decreasing the energy efficiency of bubbles collapsing (Dezhkunov et al., 2005). In addition, 13

ultrasonic degradation of the extracted pectin would also be responsible for yield decrease, since the degradation effect on pectin increased with the increasing of ultrasound intensity (Zhang et al., 2013). 3.1.3 Duty cycle Fig. 1C showed the changes of pectin yield according to different duty cycles, when the total extraction time was fixed at 60 min. It can be seen that the pectin yield increased at first and then decreased with duty cycle ranged from 33% to 70%, and a peak was obtained at 50%. Similarly, Chavan and Singhal (2013) reported that a duty cycle of 50% was optimal for the extraction of bioactives from arecanut. The reason for this phenomenon was complicated. At lower duty cycle (33%), the reduction of pectin yield might be because the total sonication time (20 min) was relatively short, thus the cavitation effect was not sufficient to disrupt the raw material and facilitate the release of pectin. The decrease of pectin yield from 50% to 70% duty cycle could also be attributed to the “saturation effect” and bubble interactions as discussed above, because the pulse modulation of ultrasound decreased the concentration of cavitation bubbles with a simultaneous increase of the cavitation intensity produced by the collapsing bubbles comparing with high duty cycle or continuous irradiation (Francescutto et al., 1999). Besides, it was interesting that the pectin yield underwent a significant increase at 80% duty cycle, this was because the longer sonication time (48 min) made up for the loss of cavitation effect caused due to the “saturation effect” and interbubble impacts. To reduce the electrical energy consumption, 50% was selected as the optimal level of duty cycle. 14

3.1.4 Temperature Fig. 1D showed the effects of extraction temperature on the extraction yield of pectin using UAHE. The profile demonstrated that the pectin yield was significantly increased from 17.79% to 26.68% with the temperature from 30°C to 60°C. This increase was mainly due to the improvement of pectin solubility caused by temperature increase. However, the increase of pectin yield from 60°C to 70°C became not significant (p>0.05), then it slightly decreased with the temperature increased to 80°C. The decrease of pectin yield could partially be resulted from the decrease of power output since it has been reported that temperature significantly affects the ultrasonic power output. At ambient pressure, the output power was hardly affected in the range 20-70°C, however, when temperature became higher it decreased drastically and at 100°C no power output was detected (Raso et al., 1999). This is because the vapor pressure of the heated liquid is elevated, which allows cavitation to be achieved at lower ultrasound intensity, thus reduces the strength of shear forces in the vicinity of the bubble (Mason and Lorimer, 2002). On the other hand, thermal degradation of pectin at high temperature was also attributed for the decrease of pectin yield (Panchev et al., 1989). Considering the energy consumption, 60°C was selected as the optimal temperature for UAHE. 3.1.5 Solid-liquid ratio (S/L) Solid-liquid ratio (S/L) is a routine parameter that significantly affects the extraction efficiency. However, its effect on the yield of pectin is still unclear. Here the effect of the S/L (1/30-1/70 g/mL) on pectin yield was shown in Fig. 1E. As 15

shown in the figure, the yield of pectin significantly increased with the S/L decreased from 1/30 to 1/50 g/mL. Two factors might explain this phenomenon. Firstly, the higher the S/L was, the higher the concentration and viscosity of the extraction solvent would be. But the intensity of cavitation falls down with an increase in the viscosity of the medium, since the formation of cavitation requires the negative pressure in the rarefaction region of wave function overcome the natural cohesive forces. Therefore, cavitation is more difficult to produce in viscous liquids, where the cohesive forces are stronger (Gogate and Pandit, 2004; Majumdar et al., 1998). On the other hand, with the lower S/L, the ultrasound intensity imposed on the average vegetal tissue was higher, which was conducive of the fragmentation of raw material. For the same reasons, the degradation rate of polymers increased with a decrease of polymer concentration (Kanwal et al., 2000; Mohod and Gogate, 2011). This could explain the decline of pectin yield when the S/L decreased from 1/50 to 1/70 g/mL. Besides, the low content of raw material provided less protection for the dissolved pectin, which also facilitated the degradation of pectin. From the above results, it can be obtained that 1/50 g/mL was the best S/L for pectin extraction using UAHE. 3.2 The structural changes of GPP tissue using UAHE and CHE 3.2.1 Image study on tissue swelling during UAHE Some morphological changes of raw material after extraction were observed and significant differences were shown between UAHE and CHE. Fig. 2 showed the tissue morphology of extracted mixture using CHE and UAHE, before and after centrifugation. As shown in Fig. 2A, the solvent-material mixture extracted after CHE 16

was still a typical suspension system, which was easy to settle into layers after standing for a while. However, the mixture extracted by UAHE (Fig. 2B) became a relatively homogenous system, in which the vegetal tissue was significantly hydrated and swelled, distributed all across the system. Fig. 2C and Fig. 2D were the tissue residues after the pectin solution being separated through centrifugation under the same conditions. The residue of CHE was compact and absorbed little quantity of liquid, while that of UAHE represented obvious swelling behavior. Fig. 3 showed the SEM images of air-dried tissue residues of CHE and UAHE. Fig. 3A (with a magnification factor of 500 ×) and 3B (3000 ×) showed that the structure of GPP tissue was still complete and compact by heating extraction. However, the tissue extracted by UAHE, as shown in Fig. 3C (500 ×), was greatly disintegrated, the surface morphology was visibly changed. It was obvious that the tissue became porous and some microfractures and hollow openings were generated. Besides, from Fig. 3D (3000 ×), it can be seen that the tissue of UAHE became much looser than that extracted by CHE (see Fig. 3B). The porous and loosen structure of GPP tissue is caused by ultrasonic cavitation forces. The sudden collapse of cavitation bubbles released large amounts of energy. The temperature and the pressure at the moment of collapse have been estimated to be up to 5000 K and 2000 atmospheres at room temperature. When these bubbles collapse onto the surface of the plant tissue, the high pressure and temperature generate microjets directed towards the solid surface, which are capable of destroying the cell wall material (Chemat et al., 2011). 17

3.2.2 Relationship between tissue SI and pectin yield The effects of extraction parameters on the swelling behavior of GPP tissue were investigated and shown in Fig. 1. From the figure, it can be seen that SI presented similar changing trends with the changing of pectin yields. The correlation between tissue SI and pectin yield using different extraction conditions was analyzed using Bivariate analysis (see Table 1). The table showed that there existed high positive correlations between the two indexes, and the correlations were significant or very significant. It demonstrated that the more seriously the tissue swelled, the higher the pectin yield would be. From a microscopic point of view, the rehydration swelling process during the steeping stage of extraction, is the result of large number of hydroxyl groups taken up by the polysaccharide network consisted in cell wall and middle lamella (Toma et al., 2001). Polysaccharide network immersed in a solvent reaches a swelling equilibrium, in which the forces driving hydration and expansion of the network are balanced by elastic resistances to deformation provided by ionic and covalent bonds between macromolecules (MacDougall and Ring, 2003). In the process of UAHE, ultrasound irradiation could break the intermolecular and intramolecular covalent crosslinks of macromolecules in cell wall matrix (Hromádková et al., 2002; Sun et al., 2004) and release large amounts of free hydroxyl groups. This increased the driving forces, whilst decreasing the resistances, moving the equilibrium towards hydration swelling. Therefore, the SI value quantitatively reflected the disruption degree or the integrity of tissue under different extraction conditions. 18

On the other hand, the integrity of raw material directly determines the extractability of pectin. It has been reported that the particle size and porosity of material significantly influenced the extractability of analytes (Kosikova et al., 1990; Krogell et al., 2013). Ultrasound irradiation gave rise to the fragmentation of raw material and improved the porosity of GPP tissue, which facilitated the hydrolysis of protopectin inside the particles and internal mass transfer of pectin, in turn increased the extractability of pectin. Therefore, the significantly high correlations between tissue SI and pectin yield signified that improving the extractability of pectin through disrupting vegetal tissue was the main mechanism of the ultrasonic enhancement of pectin extraction. 3.3 Kinetics study for pectin extraction by different extraction methods 3.3.1 Changes of pectin yields during extraction using different methods As shown in Fig. 4A, the effects of different extraction methods were investigated in the time range from 10 to 60 min, with other parameters at the optimal level selected from the single-factor experiments above. The yields of pectin increased rapidly in the first 20 min, then the increasing rates gradually slowed down. However, pectin yields of UAHE and CHE showed significant decrease with time prolonging. This loss at the later stage of extraction was due to the degradation of pectin when exposed to combined ultrasound irradiation and heating. Differently, pectin yields of HE and UAE did not change, while that of RE was still increasing when extracting for 60 min. On the basis of the experimental date, the extractable pectin PE, rate constants k1 19

and k2, tmax and ymax were calculatedand presented in Table 3. The fitting curves and regression equations were shown in Fig. 4A. Statistical testing of the regression equations were performed by both residual analysis and ANOVA (Table 4). The residuals showed fairly random pattern with the increasing of extraction time, indicating that the model could provide a decent fit to the data. The values of the adjusted coefficient of determination (R2Adj) for the five extraction methods were approximate to 1, indicating a high degree of correlation between the experimental and predicted values. Besides, the model would be more significant if the F-value becomes greater and the p-value becomes smaller. Therefore, the high F-value and low p-value (<0.001) showed in Table 4 implied that the model was very significant. All these results suggested that the regression models were adequate for the predication of the yield of pectin within the time range employed. 3.3.2 The profile of extraction process Based on PE, k1 and k2, pectin yield y(t), quantity of dissolved pectin p(t), degraded pectin q(t), as well as obtaining rate Ry(t), dissolution rate Rp(t) and degradation rate Rq(t) for different extraction methods were calculated using equations (7), (8), (9), (12), (13) and (14). Their changes with extraction time were shown in Fig. 4. Fig. 4A showed the comparison between the predicted and experimental values for pectin yield, which showed that there were good agreements between them. From Fig. 4B, it can be seen that the quantity of dissolved pectin presented increasing trends, and infinitely approached to PE; Fig. 4C showed that the quantity of degraded pectin was increasing during the extraction time. As shown in Fig. 4E, Rp(t) decreased all 20

along the extraction duration, and infinitely approached to 0; while Rq(t) increased quickly at first, then slightly decreased (Fig. 4F). Since Ry(t) was calculated from Rp(t) minus Rq(t), Ry(t) presented decreasing trend with time (Fig. 4D). When Rp(t)>Rq(t), then Ry(t)>0, the pectin yield showed increasing trend; when Rp(t)=Rq(t), then Ry(t)=0, the pectin yield achieved the maximum; and when Rp(t)
Kratchanova et al., 2004; Luthria, 2008; Yapo, 2009). Thus the undissolved protopectin left in the material after extraction was inappropriately considered to be degraded, leading to the overestimation of k2 in the model of Panchev et al. (1989). The lower the extraction temperature was, the more k2 was overestimated, because the more pectin was not dissolved yet. Therefore, the modified model in the present study could more adequately describe the dissolution of pectin from protopectin and the degradation of dissolved pectin processes. More importantly, it introduced a new parameter, PE, to denote the extractability of pectin under specified extraction conditions. 3.3.4 Effects of ultrasound and/or heating on the kinetics Table 3 showed that PE, k1 and k2 of both HE and UAE were much higher than that of RE, in which neither heating nor ultrasound was applied. This indicated that both heating and ultrasound irradiation had significant effect on the improvement of extractability and dissolution rate of pectin. Meanwhile, they intensified the degradation of pectin, which was not beneficial for the extraction. To date, no report has explored their coupling relationship on the extraction of pectin yet. In this study, the combined/individual application of heating and ultrasound made it possible. Compared with RE, the enhancement of dissolution rate (k1) caused by UAHE (4.69 ×10-2 min-1), was much higher than the sum (1.91 ×10-2 min-1) of that caused by HE (0.83 ×10-2 min-1) and UAE (1.08 ×10-2 min-1). Besides, the enhancements of PE and k2 caused by the three different treatments also had the same relationship. The results indicated that there existed synergistic effect between 22

heating and ultrasound on the extractability, dissolution rate and degradation rate of pectin. This was because the protopectin hydrolysis enhanced by heating softened the GPP tissue, making it more susceptible to ultrasound fragmentation (Marsilio et al., 2000); on the other hand, ultrasound facilitated the penetration of hot extraction solvent, which could be testified by the enhancement of hydration swelling (Toma et al., 2001). However, different to our results, Zhang et al. (2013) utilized ultrasound to treat the pectin solution and reported that the degradation efficiency of ultrasound decreased with the increase of temperature. This difference was probably because it was difficult to study the depolymerization of pectin in the solid-liquid mixture, since the extraction system was much complicated (Panchev et al., 1989). The apparent activation energy (Ea) of pectin dissolution and degradation with/without ultrasound irradiation were compared (Table 5). As shown from the table, the Ea of pectin dissolution (Ea1) with ultrasound (18.62 kJ/mol) was higher than that without ultrasound (9.69 kJ/mol). This indicated that temperature had more influence on the dissolution rate of extraction methods with ultrasound, further confirming the synergistic effect between heating and ultrasound. On the contrary, the Ea of pectin degradation (Ea2) with ultrasound (7.78 kJ/mol) was lower than that without ultrasound (8.46 kJ/mol), which indicated that ultrasound could decrease the activation energy and accelerate the degradation of pectin. 3.3.5 Comparison between UAHE and CHE Comparing the extraction kinetics between UAHE and CHE, it was obvious that the pectin extractability and dissolution rate were significantly higher for the former, 23

while the degradation rate was at about the same level. The maximum yield of pectin (26.74%) was achieved at 51.79 min (sonication for 25.90 min) using UAHE, while for CHE, the maximum yield of pectin (23.44%) was achieved at 72.26 min. It can be concluded that the optimized UAHE significantly increased the yield of pectin by 14.08%, shortened the extraction time by 39.53% and reduced extraction temperature by 20°C. The results were constant with several published studies. Panchev et al. (1988) demonstrated that, compared with CHE (80 , 30min), continuous sonication (22kHz, 1-1.2 W/cm2) increased pectin yield by 21.3%; while pectin yields under intermittent sonication (2 min on; 3 min off) reached the maximum at 45 min, and were increased by 28% and 23% from different materials. In another study, the yield of pectin extracted from apple pressings gained an increase about 18% and the optimum ultrasonic time proved to be within 24-30 min using continuous sonication treatment (Panchev et al., 1994). These results demonstrated that UAHE is an effective, time and energy saving method for pectin extraction. However, there were also some limitations when using UAHE. The cost and bulkiness of equipment, noise generated by cavitation (Patist and Bates, 2002), metallic contamination from probe (Weiss et al., 2002) and alterations of analytes (Pingret et al., 2013) should be considered carefully in larger scale applications.

4. Conclusions This study showed that three ultrasonic parameters (emitter surface, power density, and duty cycle) and three routine parameters (temperature, S/L and extraction time) 24

have significant effects on pectin yield and GPP tissue swelling. The extraction parameters for UAHE were optimized as ultrasound power density 0.40 W/mL, duty cycle 50%, temperature 60°C, S/L 1/50 g/mL. The significantly high correlation between pectin yield and tissue swelling demonstrated that the main mechanism of the ultrasonic enhancement of pectin extraction was improving the extractability through disrupting vegetal tissue. The modified model adequately described the extraction kinetics involving the dissolution of protopectin and degradation of dissolved pectin. Pectin extractability, dissolution and degradation rates, the maximal yield and optimal extraction time could be obtained by modeling the extraction process of different methods. Both heating and ultrasound irradiation had significant effect on the improvement of extractability, dissolution rate and degradation rate of pectin, and there existed synergistic effect between ultrasound and heating on the extraction of pectin. Compared with CHE, the optimized UAHE significantly increased pectin yield, shortened extraction time, and reduced extraction temperature. All these results illustrated that UAHE was an innovative technology of high-efficiency and low energy consumption for pectin extraction from vegetal materials, despite some limitations that need to be considered for industrial application.

Acknowledgements This work was financially supported by National Natural Science Foundation of China (31371872 and 31171784). 25

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Figure captions

Fig. 1 Effects of ultrasound-assisted heating extraction (UAHE) parameters on pectin yield and tissue swelling index (SI). Means in the same plot with different letters are significantly different (p ≤ 0.05).

Fig. 2 Tissue morphology of extracted mixture using conventional heating method (CHE) and ultrasound-assisted heating extraction (UAHE), before and after centrifugation.

Fig. 3 SEM images of the structure of air-dried grapefruit peel powder (GPP) residues after

pectin

extraction

using

conventional

heating

method

(CHE)

and

ultrasound-assisted heating extraction (UAHE) (A. CHE, 500 ×; B. CHE, 3000 ×; C. UAHE, 500 ×; D. UAHE, 3000 ×).

Fig. 4 Effects of different extract methods on the kinetics of pectin extraction. A: the yield of pectin; B: dissolution quantity of pectin; C: degradation quantity of pectin; D: obtaining rate of pectin; E: dissolution rate of pectin; F: degradation rate of pectin. UAHE: Ultrasound-assisted heating extraction; UAE: Ultrasound-assisted extraction; HE: Heating extraction; RE: Extraction at room temperature; CHE: Conventional heating extraction. 34

35

36

37

38

Table 1 Essential extraction parameters for different methods used in single-factor experiments and kinetics study

Extraction

Power density

Heating

(W/mL)

temperature (°C)

UAHE

0.40

60

UAE

0.40

30

HE

/

60

RE

/

30

CHE

/

80

methods

UAHE: Ultrasound-assisted heating extraction; UAE: Ultrasound-assisted extraction; HE: Heating extraction; RE: Extraction at room temperature; CHE: Conventional heating extraction.

39

Table 2 Correlation analysis between pectin yield and tissue swelling index (SI) under different extraction conditions.

Extraction factors 2

R Significance

Power density 13 mm probe 0.989** <0.001

Duty cycle

Temperature

Solid-liquid ratio (S/L)

0.941** <0.001

0.894** <0.001

25 mm probe 0.686* 0.014

0.413 0.235

*. Correlation is significant at the 0.05 level; **. Correlation is significant at the 0.01 level.

40

Table 3 Kinetic parameters of pectin extraction from grapefruit peel using different extraction methods

Extraction methods

Extractable pectin PE (%)

Dissolution rate constant k1 (102min-1)

Degradation rate constant k2 (102min-1)

Optimal extraction time tmax (min)

Maximum yield ymax (%)

UAHE UAE HE RE CHE

29.25 20.41 21.66 12.96 26.48

7.43 3.82 3.57 2.74 4.80

0.173 0.131 0.142 0.105 0.169

51.79 91.38 93.99 123.79 72.26

26.74 18.11 18.96 11.38 23.44

UAHE: Ultrasound-assisted heating extraction; UAE: Ultrasound-assisted extraction; HE: Heating extraction; RE: Extraction at room temperature; CHE: Conventional heating extraction.

41

Table 4 Statistical analysis of the kinetic model for pectin extraction from grapefruit peel using different methods

Extraction time (min)

Differences between the measured and theoretical yields (%) UAHE UAE HE RE CHE

20 40 60 80 100 120

-0.032 0.032 0.186 -0.178 -0.214 0.203

0.195 -0.446 0.396 -0.026 0.143 -0.055

0.321 -0.735 0.727 -0.259 -0.019 0.016

0.300 -0.518 0.316 <0.001 -0.017 -0.022

0.298 -0.433 -0.164 0.443 0.189 -0.282

R2Adj F-value p-value

0.9996 32568.47 <0.0001

0.9977 5199.77 <0.0001

0.9937 1893.53 <0.0001

0.9934 1674.90 <0.0001

0.9979 6157.27 <0.0001

UAHE: Ultrasound-assisted heating extraction; UAE: Ultrasound-assisted extraction; HE: Heating extraction; RE: Extraction at room temperature; CHE: Conventional heating extraction.

42

Table 5 Apparent activation energy (Ea) of pectin dissolution (Ea1) and degradation (Ea2) with or without ultrasound

Apparent activation energy (kJ/mol)

With ultrasound

Without ultrasound

Ea1 Ea2

18.62 7.78

9.69 8.46

43

Research highlights 1. Pectin was affected by many parameters of ultrasound-assisted heating extraction. 2. Swelling behavior of vegetative material was closely correlated with pectin yield. 3. Both heating and ultrasound had significant effects on the extraction kinetics. 4. Heating and ultrasound showed a synergistic effect on pectin extraction. 5. It was more efficient in pectin extraction when combining heating with ultrasound.

44