Optimization of Circulating Extraction of Polysaccharides from

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Optimization of Circulating Extraction of Polysaccharides from Gracilaria Lemaneiformis using Pulsed Electrical Discharge Ting Ju, Yong Deng, and Jun Xi ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06183 • Publication Date (Web): 31 Dec 2018 Downloaded from http://pubs.acs.org on January 7, 2019

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ACS Sustainable Chemistry & Engineering

Title: Optimization of Circulating Extraction of Polysaccharides from Gracilaria Lemaneiformis using Pulsed Electrical Discharge Authors: Ting Ju, Yong Deng, Jun Xi*

Address: School of Chemical Engineering, Sichuan University, Chengdu 610065, China

*Corresponding author: Jun Xi

Address: School of Chemical Engineering, Sichuan University, Chengdu 610065, China Tel: +86 28 65292503; Fax: +86 28 65292503 E-mail address: [email protected] (J Xi)

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ABSTRACT Gracilaria lemaneiformis (GL) is considered as a valuable plant because of its high content of polysaccharides. The purpose of this study was to optimize and validate the circulating extraction of polysaccharides from GL using pulsed electrical discharge (PED) through single factor analysis. The maximal yield of polysaccharides (164.34±4.62 mg/g) was obtained under the optimal operating conditions: 24 min extraction time, 250 mL/min flow rate of materials, 50 mL/g liquid to solid ration, 13 kV voltage, and distilled water was considered as the solvent. Compared with continuous PED and reflux extraction, circulating PED had the highest yield, shortest extraction time and lowest specific energy input, while the monosaccharide compositions of polysaccharides were mainly composed of galactose and glucose with slight xylose, fucose, mannose and arabinose. These results indicated that circulating PED method was time-saving and energy-saving for economical extraction of polysaccharides from plant materials and presented prospective in large-scale application. Keywords:

Circulating

extraction,

Pulsed

electrical

discharge,

Polysaccharides.


Gracilaria

lemaneiformis,

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INTRODUCTION Gracilaria lemaneiformis (GL, Figure 1) is a member of the Gracilariaceae family and possesses a predominant medicinal value and excellent taste, because it contains a large number of nutrient substances, such as polysaccharides, protein, carotene, and so on.1–3 The polysaccharides are the polymers composed of at least 10 monosaccharides linked in union by glycosidic bonds and largely contained in multifarious creatures, plants, mushrooms and bacteria.4 Recently, the polysaccharides have been given more and more attention by many people due to their outstanding bioactivities and correspondingly low toxicity.5 Furthermore, lots of studies have reported that the polysaccharides have multiply biological benefits including antitumor, antioxidant, hypoglycaemic, hypolipidaemic and anti-inflammatory activities.6–10 Therefore, it is particularly urgent to explore an extraction method to improve the polysaccharides yield of GL for its application in health food, cosmetic and pharmaceutical areas. Over the past decades, the reflux extraction (RE) was the conventional technology used to extract polysaccharides from plant materials, because of its prominent advantages, such as, simple operation, less cost, etc. However, this method always led to the high temperature, long

time dissipation and

low yield.11,12 With the development of science and technology, some emerging and environmental-friendly methods including microwave-assisted extraction (MAE),7 ultrasonic-assisted extraction (UAE),13 ultrasound-assisted enzymatic extraction (PAE)

15

14

and pectinase-assisted extraction

were reported to extract polysaccharides to get higher extraction efficiency. Although the

development of promising extraction technologies is widely spread, the industrial application of them still have a long way to go. MAE is the most developing emerging method in extraction polysaccharides. By affecting the movement of molecules in solvents, MAE produced highly localized



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temperature that leads to selective migration of target substances from the material to the solvents, which would reduce both extraction time and solvent consumption.16 Nevertheless, the high local temperature causes the limitation of MAE applications in industrial. The mechanism of UAE is mainly attributed to the cavitation, which leads to collapsing of material matrix and raises the rate of recovery of target substances from material matrix to solvent. This technology is rapid, simple, less expensive and high extraction efficiency within a minimum time.17 But the enormous noise pollution produced in extraction process is the biggest problem of this extraction method and needs to be solved. The PAE, a green-chemistry technique, uses enzyme as a catalyst to destroy the cytoderm of plant materials, which would reduce the mass transfer resistance.15 Although PAE method has the advantages of high quality product, gentle reaction and high extraction efficiency, the disadvantages of the huge cost and long extraction time can not be ignored. In recent years, pulsed electrical discharge (PED), an efficient, non-thermal and promising technology, has gradually replaced some thermal and trivial extraction methods.18,19 During the extraction process, a plasma channel is formed in water when high voltage is exerted between the positive and negative electrodes. With the continuous development of plasma channel from one electrode to another one, the arc discharge occurs which usually accompanies with bright UV light, strong shock wave, cavitation and some oxidizing substances. The UV light and shock waves would cause the deformation of cell and mechanical destruction of cell membranes to reinforce the mass transfer processes.18 So far, there are two kinds of the developed PED systems, that is, batch extraction and continuous extraction. The batch PED method (Figure 2) had been proved to possess lots of merits of lower temperature, less organic solvent consumption and had been explored by many researchers. 19–22

It usually had two steps during the process. The first step was PED pre-treatment (Figure 2a): the


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materials would be treated for a few minutes in the “needle to plane electrodes” treatment chamber by PED. Because of the defined space, the materials being handled are immobilized and the interaction between discharge energy and materials is rather limited. The second step was diffusion process and shown in Figure 2b: the round incubator shaker was employed to place the treated materials and performed at a high Reynolds number (>104) for 60 min to make further extraction. Owing to the unique advantages, PED was widely used as a pre-treatment technique for the extraction of various effective substances, such as polyphenols,19 lignans,23 protein.24 Unfortunately, its extraction process was intermittent with a low capacity treatment chamber and long extraction time,18 and was unable to achieve industrial production. However, the continuous treatment chamber which would enable the extraction process in continuous pattern was expected in industries application. Thus the continuous PED extraction system with a “converged electric field type” treatment chamber had been devised by our research group and had been successfully optimized and presented a higher extraction efficiency of phenolic compounds from pomegranate peel.25 As shown in Figure 3a, the system contained three parts of a PED generator, a continuous treatment system and a data acquisition system. As the most important part in the continuous treatment system, the “converged electric field type” treatment chamber (Figure 3b) was consisted by a pair of parallel stainless steel mesh electrodes and an insulating plate with a small hole. It had much larger electric field intensity than the “needle to plane electrodes” treatment chamber in the discharge region, because the continuous structure could concentrate more electric field lines together. The distance between two stainless steel mesh electrodes was 1 mm to form a small treatment region, which indicated the system would present a small flow rate of materials and lead to the clogging and the accumulation of heat quantity. Moreover, based on the continuous extraction device, the samples cannot be completely treated by the discharge because



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the frequency of PED was limited. Thus, it is necessary to invent a device to solve these problems of low flow rate, clogging and insufficiency process of materials. In this study, a circulating PED extraction system of GL polysaccharides had been successfully developed. Thus, the goal of the present work is to validate and optimize the circulating extraction of polysaccharides from GL using PED. The effects of the extraction time, flow rate of materials, liquid-solid ratio and voltage amplitude on the yield of polysaccharides extracted from GL through the single factor analysis were investigated. Then, in order to evaluate the extraction efficiency of the proposed circulating PED method, the extraction yield, energy input and monosaccharide compositions of extracted polysaccharides of circulating PED, continuous PED and RE were also compared.

MATERIALS AND METHODS Materials and Chemicals. Sun-dried GL was provided by the market located in Shandong province of China, and dried at 60 °C for 4 h in a hot oven and grounded into powder by a laboratory grinder (KC-1000, Beijing Kaichuangtonghe Technology Development Co., Ltd., Beijing, China). The powders were stored in a brown bottle after passing through a 40 mesh sieve for RE and circulating PED extraction and a 100 mesh sieve for continuous PED extraction. Then 95% ethanol (1:20 g/mL, w/v) was added in a Soxhlet system to eliminate the monosaccharides at 60 °C for 2 h, and then filtered, dried and embed in a brown glass bottle and placed in a vacuum dryer until used. UV-Vis spectrophotometer (751-GW) was from Shanghai Analytical Instrument Overall Factory (Shanghai, China). The HPLC system used was a LabAlliance 340 HPLC system (SSI, USA) equipped with a 2000ES evaporative light scattering detector (Alltech, USA) and prevail carbohydrate ES


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analysis column (5 μm, 4.6 × 250 mm, Alltech, USA). Distilled water used in the experiment was provided by a Milli-Q system (Millipore, USA). The monosaccharide standards of galactose, glucose, mannose, arabinose, xylose and fucose, pharmaceutical grade standard (purity > 99.9%), were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Glutaraldehyde, phenol and sulfuric acid were analytical reagent grade chemicals and obtained from Chengdu Kelong Chemical Reagent Co., Ltd., Chengdu, China. Circulating PED Extraction System. As shown in Figure 4(a), the circulating PED extraction system was composed of a tapered recirculation tank, a screw pump with adjustable flow rate (Shanghai Nuoni Light Industrial Machinery Co., Ltd., Shanghai, China), a“needle to ring electrodes” treatment chamber and PED generator (Shanghai Xuji Electric Co., Ltd., Shanghai, China), and the total working volume was 1100 mL. The rectangular wave, 0-20 kV voltage and 0-50 Hz frequency could be available by the PED generator. The typical voltage (red curve) and current curves (black curve) during an electrical discharge were shown in Figure 4(b). The tapered recirculation tank was consisted of an electric mixer (200 rpm, Jintan Hongke Instrument Factory, Jintan, China) and a conical tank to enhance the processed times of materials. The materials were inputted from the entrance and homogeneous mixed by electric mixer. Then the screw pump transferred the materials to treatment chamber to suffer discharge treatment. The transparent cylindrical treatment chamber was made of the polycarbonate for convenient viewing and a photograph of intense UV light occurring in the treatment region at optimal conditions was shown in Figure 4(c). It can be seen from Figure 4(d) that the “needle to ring electrodes” was consisted of a needle electrode and a ring electrode. The grounded needle electrode can be vertically adjusted to determine the needle electrode in the center of the ring electrode and its diameter was 0.67 mm. The ring electrode was tightly compressed though the



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polycarbonate wall and connected by a stainless steel bar as a leading-out electrode for being exerted high voltage. The bore diameter of the ring electrode was fixed at 4 mm to obtain a treatment region allowing large size particles to pass through, which would bring large flow rate of materials (250 mL/min) and prevent clogging. In other words, the circulating PED extraction, which was equivalent to combination of two steps of the batch extraction, not only improved the treatment capacity and reduced the extraction time, but also simplified the extraction process. The influence factors including the extraction time (8, 16, 24, 32 min), flow rate of materials (150, 200, 250, 300 mL/min), liquid-solid ratio (30, 50, 70, 90 mL/g) and voltage amplitude (9, 11, 13, 15 kV) were studied to optimize the polysaccharides extraction from GL. The distilled water was considered as the solvent because of its good solubility of polysaccharides and low electrical conductivity. Accurately weighed a certain amount of dried GL powder, and mixed with 1100 mL distilled water to acquire the required liquid-solid ratio. The mixed solution was poured into the tapered tank with various flow rates and treated with different voltage amplitude, and the frequency of discharge was 3 Hz. After extraction, the obtained treated samples were gathered by beakers and centrifuged at 4000 × g for 10 minutes to take the supernatant for the yield analysis. Control Extraction Methods. Conventional RE was performed according to the method of Ye et al.26 with some modifications. 22 g dried GL powder was dissolved in 1100 mL distilled water to form the 50: 1 mL/g liquid-solid ratio, and the mixed solution was placed in round-bottomed flask and incubated at 90 °C for 5 h. The continuous PED extraction was carried out according to the report of Xi et al.25 with tiny changes. 1100 mL mixture continuously was introduced into the treatment chamber and operated at the preliminary optimized extraction conditions with 40 mL/g liquid-solid ratio, 14 mL/min flow rate of


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materials and 11 kV voltage amplitude. The photograph of UV light appearing in the treatment region at optimal voltage was shown in Figure 3c and the discharge frequency was 2.8 Hz. Then the treated samples were operated as described above. Determination of the Extraction Yield of Polysaccharides. The extraction yield of polysaccharides was determined according to the phenol-sulfuric acid method27 with some modifications. Briefly, 1 mL of centrifuged sample solution was pipetted into a colorimetric tube, and 1 mL of distilled water was added to dilute sample solution, then 1 mL of 5 % phenol was added. Then 5 mL of concentrated sulfuric acid was added uniformly to avoid higher temperature in the tube. The tubes were allowed to shaken and placed for 30 minutes in a water bath at 25 °C. Finally, the absorbance of the reacted characteristic yellow-orange color was determined at 490 nm by UV-Vis spectrophotometer (751-GW). Then the polysaccharides yield was calculated through following formula: The polysaccharides yield (mg/g) = C·D/M

(1)

Where, C (mg/mL) was the concentration of the sample that calculated by the glucose standard curve y=8.4025x+0.22608, R2=0.99. D was the dilution multiple of the extraction solution and M (g) was the total mass of sample in the experiment. Each experiment was repeated three times. Calculation of Input Energy. The data of discharge voltage and current were collected (Figure 4b) by the voltage sensors (Wuhan Huatian Electric Power Automation CO., LTD., Wuhan, China) and a Rogowski transformer (Hubei Tianrui Electronic CO., LTD., Hubei, China) with an oscilloscope (Shenzhen Zhiyong Electronics Co., Ltd., Shenzhen, China). The total dissipated energy per pulse Wi (kJ) during the whole duration time T was calculated using the following equation: 28 𝑇

𝑊𝑖 = ∫0𝑈(𝑡)𝐼(𝑡)𝑑𝑡


(2)


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Where, U(t) was the discharge voltage and I(t) was the discharge current. In order to evaluate the capability of the device more accurate，the specific energy input 𝑊𝑠 (kJ/kg) was defined by equation (3): 𝑛

𝑊𝑠 =

∑𝑖 = 1𝑊𝑖

(3)

𝑚

Where, m (kg) was the total mass of the sample, n is the number of discharge and can be calculated by equation (4): (4)

𝑛=𝑡×𝑓 Where, t (s) was the total treatment time, f (Hz) was the frequency of discharge.

For traditional RE, the total dissipated energy 𝑊𝐻 was obtained by the following equation (5):29 𝑊𝐻 =

𝐶 ∙ 𝑚𝑠𝑜𝑙𝑣𝑒𝑛𝑡 ∙ ∆𝑇 𝑚𝑠𝑜𝑙𝑢𝑡𝑒

(5)

Where, C was specific heat capacity of water (estimated as 4.2 kJ/kg K), ∆𝑇 (∆𝑇 = 65𝐾) was the increased temperature in the experiment, 𝑚𝑠𝑜𝑙𝑣𝑒𝑛𝑡 was the mass of distilled water and 𝑚𝑠𝑜𝑙𝑢𝑡𝑒 was the mass of GL powder. Analysis of Monosaccharide Composition of the Extracted Polysaccharides. The effect of circulating PED extraction on the monosaccharide composition of the extracted polysaccharides were analyzed by a LabAlliance 340 HPLC system (SSI, USA) equipped with a 2000ES evaporative light scattering detector and prevail carbohydrate ES analysis column (5 μm, 4.6 × 250 mm, Alltech, USA) at 30 °C through following produces.30 Briefly, the supernatant obtained from circulating PED, continuous PED and RE methods was removed protein by using Sevage method three times. Dialyzed against distilled water and evaporated to a suitable concentration, then added fourfold volume of anhydrous ethanol and cultivated at 4 °C for 24 h to get precipitate, then freeze-dried at −50 °C for 48 h to get purified polysaccharides. The purified polysaccharides (10 mg) were reacted with 2 mL of 2


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mol/L trifluoroacetic acid (TFA) in ampoule bottle at 120 °C for 4 h. The remaining TFA in solution was entirely removed by adding methanol for three times and evaporated to dryness. Finally, the dried hydrolysate was diluted with distilled water to 5 mL for analyzing with HPLC. The column was using acetonitrile and distilled water (85: 15, v/v) to elute at a flow rate of 1.0 mL/min and the injection volume was 20 μL. Similarly, six monosaccharide standards including fucose, xylose, arabinose, mannose, glucose, and galactose were operated in the same way.

RESULTS AND DISCUSSION Optimization of the Circulating PED Extraction. The influence factors of the circulating PED extraction including extraction time, flow rate of materials, liquid-solid ratio and voltage amplitude had the prominent effects on the yield of the polysaccharides from GL. The results were shown in Figure 5. Effect of the Extraction Time. The extraction time was one of the most important factors that influence the polysaccharides yield, and many studies had demonstrated that the yield of the target product could be improved at a proper extraction time.31 As aforementioned, the whole process of circulating PED extraction included two steps: (1) discharge treatment in the treatment chamber by PED; (2) diffusion process in the tapered recirculation tank. Therefore, an enough extraction time is crucial. To investigate the effect of extraction time on the polysaccharides yield, different extraction time (8, 16, 24, 32 min) was carried out based on the other conditions which were flow rate of 250 mL/min, liquid-solid ratio of 50 mL/g and voltage of 13 kV. As can be seen in the Figure 5(a), the yield of polysaccharides increased drastically from 122.45±5.74 to 164.34±4.62 mg/g with the extraction time



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increased from 8 to 24 min. The reason was that enough time could guarantee the completion of the extraction process, and longer extraction time could make the diffusion process more sufficient and dissolve still more of target substances.32 Meanwhile, as the flow rate of materials was keep constant, the increase of extraction time meant more number of discharge treatment and circulating times and hence increased the polysaccharides yield. In contrast, the extraction yield slightly decreased as the extraction time continued up to 32 min, which might be because the mass transfer equilibrium has been reached, and the solution of polysaccharide has higher viscosity. The longer extraction time of polysaccharide might result in higher viscosity and lower fluidity, which will decrease the content of targeted polysaccharides.32 Another reason was that the prolonged extraction time would lead to the concurrent extraction of other dissolved impurities (protein, phenolic compounds, etc.), which would hindered with the dissolution of polysaccharides as well .33 Thus the yield of polysaccharide was decreased rather than increased. The results showed the importance of extraction time on the polysaccharides yield. Thence, the optimal extraction time was 24 min. Effect of the Flow Rate. To assess the impact of flow rate on the yield of polysaccharides, the experiments were performed under various flow rates (150, 200, 250, 300 mL/min) and the other operation conditions applied were extraction time of 24 min, liquid to solid ration of 50 mL/g and extraction voltage of 13 kV. As can be seen from Figure 5(b), the polysaccharides yield increased from 93.53 ± 5.16 to 164.34 ± 4.62 mg/g with the increase of flow rate from 150 to 250 mL/min and received the maximum yield of 250 mL/min. The phenomenon could be illustrated by the fact that the increased flow rate resulted in a decrease of the thickness of the boundary layer and more extracts can be released.25 On the other hand, higher flow rate of materials in the circulating PED extraction would correspond to more number of discharge treatment and circulating times, which would increase the


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polysaccharides yield. Nevertheless, with the flow rate maintaining increasing, a slight decrease of the yield was observed. The reason was that higher linear velocity of the solution lead to insufficient extraction in the boundary layer between the solute and solvent phases.34 Hence, 250 mL/min flow rate of materials was selected. Effect of the Liquid-solid Ratio. In the case of a certain solute, small volume of solvent possible cause insufficient dissolution, while a large amount of solvent may lead to the unnecessary waste and a greater cost. Thereby, when the other conditions were adjusted at 250 mL/min flow rate, 24 minutes extraction time and 13 kV voltages, the various liquid-solid ratios of 40, 50, 60 and 70 mL/g were measured to choose an appropriate value and the results were shown in Figure 5(c). The yield of polysaccharides was promoted with the liquid-solid ratio increased from 40 to 50 mL/g, and then had a slight decrease with the liquid-solid ratio further increased. The mass transfer principle states that the diffusion rate is directly proportional to concentration gradient which increases at higher liquid-solid ratio. An explanation for this phenomenon was that higher liquid-solid ratio corresponding to higher concentration difference between the inside and outside of the cell, which had higher diffusion rate and increased the power of penetration of distilled water into the matrix and more polysaccharides would be dissolved in solvent.32However, with the increase of the liquid-solid ratio, the diffusion distance of intra-tissue was raised and the release resistance of the extraction process was ascended as well, which may lead to a slight decrease of polysaccharides yield. Furthermore, the increased liquid-solid ratio may reduce the dispersion of discharge energy insolvent, which causes that raw materials would be processed by less discharge energy, thus the yield of polysaccharides was decreased

17

So, 50 mL/g

was the optimum liquid-solid ratio in the experiments. Effect of the Voltage Amplitude. When the “needle to ring electrodes” were exerted a high



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voltage, the electrical breakdown occurred in water, producing some chemical reactions near the discharge plasma, strong shock waves and UV light, which acted on the materials and had a great influence on the mass transfer process between solutions and solvents.28 Thus, the voltage parameter played the most important role in the extraction process and a proper voltage was necessary. The effect of the voltage on the yield of polysaccharides was studied at 9, 11, 13 and 15 kV, meanwhile, the flow rate, liquid-solid ratio and extraction time of materials were set to 250 mL/min, 50 mL/g and 24 min, respectively. As shown in the Figure 5(d), the yield of polysaccharides was increased when raising the voltage up to 13 kV, while above this value, a negative effect on the polysaccharides yield was observed. Therefore, the voltage of 13 kV was considered as the optimal in the present assay. The results showed that voltage amplitude had a prodigious effect on the yield of polysaccharides. During the extraction process, a plasma channel is formed in water when high voltage is exerted between the positive and negative electrodes. With the continuous development of plasma channel, arc discharge occurs, which usually accompanies with bright UV light, strong shock wave, cavitation and some oxidizing substances. The enhancement of the extraction yield before 13 kV might be because the shock waves can mechanically destroyed cell membranes and induced turbulence of fluid. Higher voltage amplitude lead to a stronger effect on the sample solution, thus, the process of polysaccharides extraction had been enhanced.18 However, the yield of polysaccharide decreased when the voltage amplitude was over 13 kV, which might be due to the higher voltage amplitude would generate more free radicals during discharge to disrupt target extracts.25 In conclusion, the optimal conditions were 24 min extraction time, 250 mL/min flow rate of materials, 50 mL/g liquid to solid ration and 13kV voltage. The verification test was carried out for three times and the yield of polysaccharides was 164.34 ± 4.62 mg/g. Previous study had extracted


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13.5 g/100g (135 mg/g) pectin from sugar beet pulp by the batch PED under the conditions of 40 kV voltage and 100 number of pulses. The 63.3 min extraction time of this study contained 200 s discharge pretreatment and 60 min acidified water extraction.35 These results indicated that the circulating PED preformed higher extraction efficiency. Comparison of Circulating Extraction with Control Methods. RE was the traditional technology with convenience and safety, which have developed for a long time to decoct Chinese herbal plant. Due to a great requirement of high yield and industrialization, the continuous PED was produced to extract polysaccharides in our study. Thus, to explore the superiority of the circulating PED extraction, RE and continuous PED were chosen for comparison in terms of the extraction yield of polysaccharides. As shown in Table 1, the polysaccharides yields of RE, continuous PED and circulating PED under optimal conditions were 140.19 ± 1.23, 147.94 ± 2.95, 164.34 ± 4.62 mg/g, respectively. It could be obviously found that the yield of circulating PED extraction were 24.15 and 16.4 mg/g higher than those of RE and continuous PED extraction. On the other hand, 79 min and 5 h extraction time were cost for continuous PED and RE, which extremely exceeded that of circulating PED extraction (24 min). The total specific energy input was calculated and the results were shown in Table 1. The 752.88 kJ/kg total specific energy input of continuous PED extraction was over two times than that of circulating PED extraction (343.64 kJ/kg), but the extraction yield of circulating PED extraction was 11% more than that of continuous PED. This could be explained from the structure of the extraction device and electrode: (1) Treating the same volume of sample solution, the extremely small discharge region of continuous PED extraction would result in long treatment time. According to the equation (4), the long treatment time lead to large number of discharge, thus considerable energy was wasted.



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(2) Due to the limitation of discharge frequency of continuous PED, partial substance of the sample solution was not treated by discharge, while the circulating PED device could overcome this shortcoming and gained a higher yield of polysaccharides. Therefore, the circular treatment had an important function in guaranteeing the sufficient contact between discharge energy and materials. (3) According to the formula 𝑃0 = 𝐾 ∙ 𝑊𝛼𝑖 reported by Boussetta et al.,20 the parameters of K (m-1) and α are relying on the geometry of inter-electrode geometry and distance between the needle electrode and pressure sensor. The peak voltage 𝑃0 has the proportional relationship with energy input per pulse Wi. Therefore, the circulating PED possessed higher peak voltage 𝑃0 because its energy input per pulse of 1.75 kJ was higher than that of continuous PED (1.56 kJ). Thus, when solid particles were subjected to shock wave, they could be destroyed more easily in the higher voltage environment. The energy input of RE was calculated by equation (5), and the value of 13650 kJ/kg was far exceeded that of circulating PED. Moreover, the energy used to maintain the temperature had not been included because of the heat dissipation. The reason for this phenomenon was that lots of energy had been absorbed by water to rise temperature of water, which exactly led to an enormous unavoidable energy waste.34 In a word, compared with RE and continuous PED extraction, the circulating PED extraction was time-saving, energy-saving and could take a distinct enhancement on the polysaccharides yield, which supposed to be a promising extraction method in industry. The compositional monosaccharides of extracted polysaccharides were investigated by HPLC and compared with standard monosaccharides. As depicted in Figure 6, the peaks of polysaccharides from 1 to 6 were identified as mannose, xylose, arabinose, glucose, galactose and fucose, respectively. As shown in Figure 6(b) and (c), the monosaccharide compositions of continuous PED and RE were similar with circulating PED, which pointed out that the PED treatment did not lead to great effect on


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the monosaccharide compositions of polysaccharides. As shown in Table 1, the molar ratio percentage of mannose, xylose, arabinose, glucose, galactose and fucose for circulating PED were 5.95, 8.29, 5.87, 21.07, 41.84 and 10.02%, respectively, and those of continuous PED were 5.82, 8.05, 6.90, 20.89, 37.89 and 10.45%, respectively. It could be concluded that the galactose was the major composition of polysaccharides obtained by circulating and continuous PED. This results varied in the content of galactose (87.49%) according to the research of Liu et al.35 The reasons for the differences of the galactose and glucose contents were as followed: (1) The differences of cultivation surroundings and extraction methods; (2) The carbonization of galactose because of the long hydrolyzation time; (3) The accumulation of component including glucose during the purification.3 In this work, a circulating PED extraction system was developed and optimized to extract polysaccharides from GL. The experimental conditions were optimized by single factor experiment and 164.34 ± 4.62 mg/g maximum yield of polysaccharides was obtained under the optimum conditions: 24 min extraction time, 250 mL/min flow rate of materials, 50 mL/g liquid to solid ration and 13kV voltage. Through HPLC analysis, polysaccharides extracted by the circulating PED method consisted of mannose, xylose, arabinose, glucose, galactose and fucose, whose yield was higher than those of continuous PED and RE techniques. Therefore, the circulating PED method did not cause substance changes in the monosaccharide compositions. Compared with the continuous PED and RE, the circulating PED had the highest extraction yield, shortest extraction time and lowest specific energy input. Therefore, the circulating PED system provided an effective and environmental technology for large-scale extraction of polysaccharides from plant materials.



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AUTHOR INFORMATION Corresponding Author Phone: +86 28 85405209. Fax: +86 28 85405209. E-mail address: [email protected]. Funding Sources This work is financially supported by the National Natural Science Foundation of China (No. 21376150), Postdoctoral Science Foundation of China (No. 2013M530400, 2014T70871) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20100181120076). Notes The authors declare no competing financial interest.

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Page 24 of 30

DOI. 10.1021/acs.jafc.6b01086.

Figure 1. Gracilaria lemaneiformis

Needle electrode Solvent Materials Plane electrode

PED Generator

Grounded

(a) PED pre-treatment

(b) Diffusion

Figure 2. Schematic diagram of the batch PED extraction system with two steps of PED pre-treatment process (a) and diffusion process (b).


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Treatment chamber

PED generator

Oscilloscope

M

Ground Flow meter

Untreated product

Cooling system

Peristaltic pump

Treated product

(a) Outlet Treatment region Electrode Insulating plate with a hole Electrode

Inlet

(b)

(c)

Figure 3. Schematic diagram of the continuous PED extraction system (a); the “converged electric field type” treatment chamber (b); a photograph of UV light appearing in the treatment region at 11 kV (c).



Needle electrode Ring electrode

Discharge region

12

600

8

400

4

200

0

0

-4

0

5

10

15

20

Current, A

Inlet

Voltage, kV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 30

-200

Duration time, us

Outlet

(a)

(b)

Needle electrode Ring electrode

Inlet

Discharge region

(c)

(d)

Figure4. The schematic diagram of the circulating PED extraction system (a): (I) PED generator, (II) the electric mixer, (III) the “needle to ring electrodes” treatment chamber, (IV) the tapered recirculation tank, (V) the screw pump. The typical voltage and current curve during an electrical Outlet discharge (b). The photograph of UV light appearing in the treatment region at optimal conditions (c).

The needle to ring electrodes (d).


Page 27 of 30

180

(a) The yield of polysaccharides (mg/g)

The yield of polysaccharides (mg/g)

180

150

120

90

60

30

0

8

16

24

150

120

90

60

30

0

32

(b)

150

Extraction time (min)

120

90

60

30

300

(d) 150

120

90

60

30

0

40

250

180

(c)

150

0

200

Flow rate of materials (mL/min)


180


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

60

70

Liquid to solid rate (mL/g)

9

11

13

15

Voltage amplitude (kV)

Figure 5. The effect of extraction time (a), flow rate of materials (b), liquid–solid ratio (c), and voltage amplitude (d) on the yield of polysaccharides from GL.



60

(a)

5 50

mVdits

4 6

2

40

1

3

30

20 7

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

(b)

5 50

mVdits

4 6 40

2

3

1 30

20

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes 60

(c) 50

mVdits

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 30

40

5

2

1

6

4

30

3 20 7

8

9

10

11

12

13

14

15

16

17

18

19

20

Minutes

Figure 6. The monosaccharides of polysaccharides extracted from circulating PED (a), continuous PED (b), and RE (c). The peaks: (1) Mannose, (2) Xylose, (3) Arabinose, (4) Glucose, (5) Galactose,


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(6) Fucose.

Table 1. The comparison of three extraction methods on extraction yield, extraction conditions, input energy and monosaccharide composition. Method

RE

Extraction yield (mg/g)

140.19±1.23

Continuous PED 147.94±2.95

Circulating PED 164.34±4.62

Extraction time (min)

300

79

24

Temperature (°C)

90

25

25

Energy input per pulse (Wi, kJ)

/

1.56

1.75

Total specific energy input (Ws, kJ/kg)

13650

752.88

343.64

Mannose

10.23

5.82

5.95

Xylose

12.85

8.05

8.29

Arabinose

1.02

6.90

5.87

Glucose

17.08

20.89

21.07

Galactose

25.83

37.89

41.84

Fucose

18.04

10.45

10.02

Monosaccharide compositions (%)



TOC Graphic: A circulating PED extraction was developed and evaluated with continuous PED and RE method, which indicated that the circulating PED extraction was better.

GL powder

Reflux extraction

GLP solution

Calculated input energy

Calculated yield of GLP

Purification of polysaccharides

Analysis monosaccharide compounds


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Optimization of Circulating Extraction of Polysaccharides from

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