A green and efficient deproteination method for polysaccharide from Meretrix meretrix Linnaeus by copper ion chelating aerogel adsorption

Journal of Cleaner Production 252 (2020) 119842

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A green and efficient deproteination method for polysaccharide from Meretrix meretrix Linnaeus by copper ion chelating aerogel adsorption Yong Zhu a, b, d, 1, Hailun Li c, 1, Jingrui Ma a, Tingting Xu a, b, Xinyu Zhou a, Shiqi Jia a, Jiaxin Zha b, Dengping Xue b, Weili Tao b, Qingping Xiong a, b, **, Jun Yuan a, b, *, Jing Chen a, b, *** a

Key Laboratory for Palygorskite Science and Applied Technology of Jiangsu Province, Huaiyin Institute of Technology, Huaian, 223003, China Jiangsu Key Laboratory of Regional Resource Exploitation and Medicinal Research, Huaiyin Institute of Technology, Huai’an, 223003, China Affiliated Huai’an Hospital of Xuzhou Medical University, Huai’an, 223002, Jiangsu, China d National & Local Joint Engineering Research Center for Mineral Salt Deep Utilization, Huaiyin Institute of Technology, Huaian, 223003, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 June 2019 Received in revised form 4 December 2019 Accepted 21 December 2019 Available online 23 December 2019

Polysaccharides (PMM) from Meretrix meretrix Linnaeus exhibit a variety of biological activities; however, conventional purification methods are costly, time consuming and introducing toxic agents. This paper aimed to develop a green and efficient method for the deproteinization of PMM by copper ion chelating aerogel (CCA) adsorption. First, CCA was successfully prepared by copper ion chelating composite aerogel (CA). Then, CCA adsorption was applied to remove protein mixed in PMM. The optimal parameter conditions of this deproteinization method were screened, using the deproteinization rate (Dr%), polysaccharide recovery rate (Rr%) and selectivity coefficient (Kc) as evaluation indicators, by single factor experiment. The optimal conditions were obtained as follows: CCA dose of 1.4%, pH value of 5.0 and adsorption temperature of 30
C. Under these conditions, equivalent Dr% (93.31 ± 1.33%), higher Rr% (93.34 ± 1.08%) and Kc (14.01 ± 0.46) were achieved compared with the latest reported deproteinization report. Finally, the effect of deproteinization between CCA adsorption and Sevag method, which is the most classical deproteinization method, were compared to verify the practicability of CCA adsorption. As a result, CCA adsorption not only showed much higher Rr% as well as Kc, but also would not cause any reagent contamination and environment pressure. In conclusion, CCA adsorption would be a green and highly efficient method with great potential in large-scale environmentally sustainable deproteinization of polysaccharides in the future. © 2019 Elsevier Ltd. All rights reserved.

Handling editor: Kathleen Aviso Keywords: Copper ion chelating aerogel Polysaccharides Deproteinization Meretrix meretrix linnaeus Adsorption

1. Introduction * Corresponding author. Key Laboratory for Palygorskite Science and Applied Technology of Jiangsu Province, Huaiyin Institute of Technology, Huaian, 23003, China. ** Corresponding author. Jiangsu Key Laboratory of Regional Resource Exploitation and Medicinal Research, Huaiyin Institute of Technology, Huai’an, 223003, China. *** Corresponding author. Key Laboratory for Palygorskite Science and Applied Technology of Jiangsu Province, Huaiyin Institute of Technology, Huaian, 223003, China. E-mail addresses: [email protected] (Y. Zhu), [email protected] (H. Li), [email protected] (J. Ma), [email protected] (T. Xu), [email protected] (X. Zhou), [email protected] (S. Jia), 13616184194@ sohu.com (J. Zha), [email protected] (D. Xue), [email protected] (W. Tao), [email protected] (Q. Xiong), [email protected] (J. Yuan), [email protected] (J. Chen). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jclepro.2019.119842 0959-6526/© 2019 Elsevier Ltd. All rights reserved.

Meretrix meretrix Linnaeus (M. meretrix), classified as genus clam, is an indispensable marine bivalve mainly distributed in the coastal areas of Asia (Dai et al., 2015). M. meretrix is edible and medical aquatic product since ancient times. More and more research suggested that M. meretrix possesses much biological activity, including anti-oxidant (Yuan et al., 2018), antitumor (Ferreira et al., 2015), and strengthen of immune systems (Wang et al., 2019), etc. Current research found that its pharmacological activity was close associated with the corresponding functional polysaccharide (Li et al., 2016). Thus, the polysaccharides (PMM) from M. meretrix arise as a hot topic in the field of bioactive macromolecules in recent years. At present, a series of mature preparation methods of PMM has been formed (Wang et al., 2018b, 2018c). Unfortunately,

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PMM always contains a large amount of protein due to the similar polarity and solubility between them. Large amounts of protein have a great effect on bioactivity evaluation and product development of PMM. Hence, it is necessary to adopt a suitable method to remove protein impurities in PMM solution. So far, a variety of methods had been applied to remove proteins from crude polysaccharides. Based on different principles, they can be mainly grouped into physical methods, chemical methods and biological methods. The physical methods remove proteins by physical means such as freeze-thaw deproteinization (Xiong et al., 2017) and adsorption deproteinization (Song et al., 2018). Enzymatic hydrolysis method is the most typical biological method (Zeng et al., 2019). Chemical method is to achieve the goal of protein removal through chemical reagents that induced protein denaturation, such as Sevag method (Sevag et al., 1938), salt fractionation (Chen and Huang, 2018) and trichloroacetic acid (TCA) (Gao and Huang, 2019). Among them, Sevag method is not only the most classical chemical deproteinization method but also the representative method of current deproteinization. However, the application of chemical method always introduces organic reagents, and the residue greatly limits the application of polysaccharide in food and medicine (Kong et al., 2015). Enzymatic hydrolysis method is barely applied in large-scale production because of its complicated process and considerable cost. In contrast, the adsorption method can effectively avoid these defects and have certain advantages in practicality and security. Therefore, physical deproteinization has a broad application prospect. In the existing adsorption deproteinization technology, deproteinization by immobilized metal affinity materials is a common method first cited by Porath et al. (Porath et al., 1975; Shi et al., 2019). Chelated metal ions and electron-donating groups (such as amino and hydroxyl groups located on the surface of the protein) interact to form new covalent bonds for the purpose of selectively capturing of proteins (Wang et al., 2018a). Related studies demonstrated that iminodiacetic acid (IDA) (An et al., 2017) and nitrilotriacetic acid (NTA) (Saini et al., 2018) were the most common chelate ligands. However, low specific surface area and functional group density greatly hindered their application in protein purification techniques. Up to now, there has been almost no report on deproteinization by metal chelated composite aerogel. Herein, silk fibroin/attapulgite composite aerogel (CA) was selected as the chelating ligand. As a low-density porous nanomaterial with high surface specific area and large pore volume, aerogel is a potentially excellent adsorbent material. (Tian et al., 2019). The unique crystal structure of attapulgite itself helps to form more pores, which makes up for the shortcomings of insufficient specific surface area as adsorbent material. Silk fibroin, natural high-molecular fibrin, has a large number of amino groups and hydroxyl groups on its surface, which could create coordination bonds with more metal ions. The introduction of silk fibroin would greatly compensate for the shortcomings of insufficient functional groups on the surface of adsorbent materials. Then CA was prepared by combining calcium ion crosslinking of sodium alginate with hydrogen bond between attapulgite. Finally, copper ions were applied as immobilized metal ions. The energy of one d-orbital, one s-orbital, and two p-orbitals in copper ions were similar and interfertile. These hybrid orbits were able to accommodate lone electrons of four ligands, thereby forming a new stable coordination key. Therefore, it could be assumed that copper ion chelating aerogel (CCA) can be used to selectively remove proteins in PMM with a harmless and efficient manner. In this paper, a green and efficient deproteinization method by CCA adsorption was established. For the first time, CA was prepared by crosslinking polymerization, which was chelated with copper

ions to obtain CCA. Then various factors affecting CCA deproteinization from PMM were systematically optimized by single factor experiments. In order to verify the practicability of CCA deproteinization method for PMM, a series of parameters of PMM solution before and after deproteinization were compared with that treated by the latest reported method and the most classic Sevag method. 2. Experimental section 2.1. Chemicals and materials Cocoons of Bombyx mori silkworm were purchased from Sichuan Province; Sodium alginate (1100 cps) was bought from Juda Algae Industry Group Co., Ltd. (Qingdao, China); Attapulgite was friendly provided by Zhongyuan New Material Technology Co., Ltd (Huaiyin, China). The fresh M. meretrix was purchased from Huaiyin Farmers Market (Jiangsu, China), identified by Professor Qingping Xiong, Huaiyin Institute of Technology, Huaiyin, Jiangsu. Monosaccharide standards were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The phenols (99.0%), sulfuric acid (98%, H2SO4), phosphoric acid (85%, H3PO4), Coomassie brilliant blue (CBB), ethanol (99.7%), chloroform (99.0%) and n-butyl alcohol (99.5%) were provided by Aladdin Biochemical Technology Co., Ltd. (Shanghai, PR China). The reagents for experiment were all analytical grade. The experimental water was deionized water. 2.2. Preparation of PMM The crude PMM solution was processed on the basis of the previous report with minor modifications (Xiong et al., 2017). In short, fresh M. meretrix was washed and heated in ethanol to remove the shell and internal organs. The flesh was pulverized with a masher, and the pulverized slurry was refluxed together with absolute ethanol in order to wipe off lipids and some colored substances. The minced meat was dried in an oven at 50
C and then extracted with deionized water in proportion of 1:25 (dried powder to deionized water, g/mL) for 3 h at 95
C. The extract was cooled down to room temperature and then centrifuged to obtain the supernatant clarified extract. The sediment was further extracted twice as described above. The resulting extract was uniformly mixed and stored at 4
C. 2.3. Preparation and characterization of CCA 2.3.1. Preparation of CCA The silk fibroin solution was prepared as previously described (Yang et al., 2004). In short, the silkworm cocoons were boiled by Na2CO3 (0.02 M) at 98
C for 30 min (3 times) in order to remove sericin, lipids and other impurities. The resulting silk fibers were washed and dried at 50
C. Then the silk fibroin solution (4%, w/w) was prepared by dissolving the degumming silk in LiBr solution (9.3 M) at 65
C for 1 h and stored at 4
C for use. Attapulgite powder and sodium alginate were dispersed respectively in deionized water under stirring at room temperature for 6 h. Then the two uniform slurries were mixed and stirred in a certain ratio to obtain attapulgite-alginate solution. The silk fibroin solution was added to attapulgite-alginate solution and uniformly mixed. In order to enhance the degree of crosslinking, the mixed solution was immersed in 0.2% glucose lactone solution for at least 12 h to become “green gel”. Then, the “green gel” was sufficiently desalted by immersed in flowing deionized water at room temperature for 3 days and freeze-dried to obtain aerogel named as CA. At last, CA (1 g) was immersed in 200 mL of CuSO4 (50 mM) and stirred at 45
C for 4 h. The obtained

Y. Zhu et al. / Journal of Cleaner Production 252 (2020) 119842

product was washed by deionized water to remove unreacted copper ions and dried at 40
C, yielding CCA. 2.3.2. SEM and EDS analysis The scanning electron microscope (SEM) (Se300N, Hitachi, Japan) was used to observe the microstructure of the samples. The sample element content was distinguished by a Quantax 400 energy dispersive spectrometer (EDS) (Bruker, Germany). In short, the sample to be tested was adhered to a metal scaffold with conductive silver glue and coated with a conductive gold film by an ion sputtering apparatus. And then the metal scaffold was placed into the scanning electron microscope equipped with the energy dispersive spectrometer to obtain SEM and EDS images. 2.3.3. Measurement of FT-IR Fourier transform infrared spectrometer (FT-IR) measurement of the samples was carried out according to previously reported method (Zhao et al., 2019). Briefly, 2 mg of sample to be tested was thoroughly ground to powder, and then mixed with 200 mg of KBr powder. The well-mixed powder was compressed into tablets and measured by FT-IR (Nicolet 5700, Thermo Fisher Scientific, USA) to obtain the spectra within the wavelength scope of 4000e400 cm1. 2.3.4. Determination of XRD The crystal structure analysis of the samples was carried out on an X-ray powder diffractometer (XRD) (D8 Advance, Bruker, Germany) equipped with GADDS two-dimensional detector. The analysis conditions were set to the scanning range of 5e40
, the scanning speed of 10
/min and the target material of Cu (40 mA and 40 kV). 2.4. Deproteinization by CCA adsorption method The CCA adsorption deproteinization process was carried out as follow: A dose of CCA was added to the crude PMM solution and adjusted to different pH values with 0.12 M of HCl and 0.12 M of NaOH. The suspension was shaken in a water bath thermostat oscillator for 2 h. Then, CCA was separated at room temperature by centrifuging the suspension at 5000 rpm for 8 min. Finally, CCA adsorption deproteinization parameters, such as dose of CCA (0.7, 1.4, 2.1, 2.8 and 3.5%), pH value (4.0, 5.0, 6.0, 7.0 and 8.0), adsorption temperature (20, 30, 40, 50 and 60
C) and adsorption time (5, 15, 30, 45, 60, 90 and 120 min), were systematically optimized. 2.5. Deproteinization by Sevag method Deproteinization by Sevag method was performed for comparison and carried out according to the previous report (Sevag et al., 1938). 90 mL PMM solution and 30 mL Sevag reagent (chloroform to n-butyl alcohol, v/v ¼ 5:1) were poured into a conical flask and stirred well for 30 min. The mixed solution was centrifuged to separate into three layers, which respectively were deproteinized PMM solution, denatured protein layer and residual Sevag reagent layer. The same ratio of Sevag reagent was added to the uppermost supernatant for repeated deproteinization until the intermediate denatured protein layer no longer appeared, while the other two layers were recycled. Finally, the supernatant was recovered and treated as PMM solution treated by Sevag method. 2.6. Deproteinization evaluation 2.6.1. Measurement of deproteinization ratio The deproteinization ratio of PMM solution was measured according to the traditional Bradford method (Bradford, 1976). In short, 1 mL PMM solution before and after deproteinization and

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5 mL CBB reagent were put into a 10 mL test tube and mixed thoroughly. The spectrum of 350e750 nm was measured by an ultraviolet and visible spectrophotometer (UV-2401 UVevis spectrophotometer, Shimadzu, Japan). The maximal absorbance value at 595 nm in the spectrum was selected to calculate the deproteinization ratio (Dr%) of PMM solution as follows:

Dr ð%Þ ¼

D0  D1  100% D0

(1)

where D0 and D1 are absorbance value (595 nm) of PMM solution colored by CBB reagent before and after deproteinization, respectively. 2.6.2. Measurement of polysaccharide recovery ratio of PMM The polysaccharide recovery ratio of PMM was measured according to the previously reported method (Dubois et al., 1956). In short, 0.5 mL PMM solution, 1 mL phenol and 5 mL sulfuric acid were sequentially added into a 10 mL test tube and thoroughly mixed. The spectrum of 350e750 nm was recorded by UV-2401 UVevis spectrophotometer. The maximal absorbance value at 490 nm in the spectrum was selected to calculate the polysaccharide recovery ratio (Rr%) of PMM solution as follows:

Rr ð%Þ ¼

P1  100% P0

(2)

where P0 and P1 are absorbance value (490 nm) of PMM solution colored by sulfuric acid-phenol reagent before and after deproteinization, respectively. 2.6.3. Coefficient of selectivity The coefficient of selectivity was selected to determine the selectivity of different deproteinization methods. The selectivity coefficient (Kc) formula was exhibited as below:

Kc ¼

Dr ð%Þ 100  Rr ð%Þ

(3)

where Dr (%) and Rr (%) represent respectively deproteinization ratio and polysaccharide recovery ratio of PMM. 2.6.4. UV and IR analysis The PMM solution before and after deproteinization were measured in wavelength range of 200e800 nm. In addition, the PMM solution before and after deproteinization were respectively colored by CBB and phenol-sulfuric acid, and the UVevis spectrogram were obtained in wavelength range of 350e750 nm. Finally, the Dr%, Rr% and Kc were computed based on the absorbance value at 595 nm and 490 nm, respectively. The FT-IR characterization of PMM samples before and after deproteinization were measured as follows: 2 mg of freeze-dried PMM sample before or after deproteinization was thoroughly ground to power, and then well-mixed with KBr powder (200 mg). The mixed powder was compressed into tablets and analyzed by the Nicolet 5700 FT-IR spectrometer to obtain the spectra within the wavelength scope of 4000e400 cm1. 2.6.5. Composition of monosaccharide Dried PMM sample (5 mg) was placed into a 10 mL ampoule bottle, and then 4 mL trifluoroacetic acid (TFA, 2 M) was added to hydrolyze at 120
C for 3 h (Jiang et al., 2015). The residual TFA was removed by adding 2 mL methanol and evaporating completely under vacuum with a rotary evaporator. Repeat the above steps three times. 5 mg inositolum (internal standard), 10 mg

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oxammonium hydrochloride and 0.6 mL pyridine were simultaneously added to the ampoule bottle containing hydrolysate for a preliminary reaction at 90
C for 40 min. Then 1 mL acetic anhydride reagent was added into the ampoule bottle for further react for 40 min. The final product was allowed to stand for cooling and filtered through a 0.45 mm organic filter to obtain the acetylated derivative of sample. Seven monosaccharide standards were all acetylated as described above. The gas chromatography of the acetylated derivative samples was measured by an Agilent 7890N gas chromatograph. All monosaccharide compositions of samples were determined by comparing the retention time of the gas chromatogram of the monosaccharide standards. 2.6.6. Analysis of Mw The average molecular weight (Mw) of PMM solution was measured by a size-exclusion high-performance liquid chromatography (HPGPC) equipped with a TSKgel G4000PWXL gel filtration chromatographic column (300 mm  7.8 mm  10 mm, Tosoh Corp., Japan). The mobile phase was 0.01 M Na2SO4 solution in PBS buffer (0.01 M, pH 6.8). The PMM solution (1 mg/mL) before and after deproteinization was dissolved in mobile phase and filtered by a 0.45 mm filter membrane. Chromatographic conditions established for the flow rate of 0.4 mL/min, injection volume of 20 mL and column temperature of 35
C. Five dextrans with different Mw (3.62, 12.6, 70.8, 126 and 289 kDa) were adopted to establish a calibration curve with retention time set as abscissa and logarithm of respective Mw as ordinate. The average Mw was obtained by taking the retention time of sample to be tested into the calibration curve. The regression equation of Mw was exhibited as follows:

  logMw ¼  0:2804X þ 7:5145 R2 ¼ 0:9987

(4)

where X represents the retention time of dextran (min). 2.7. Statistical analysis All experimental data were shown as mean values ± SD. SPSS version 20.0 (International Business Machines. New York, USA) was employed to do statistical analysis. Group comparisons were carried out by one-way ANOVA. The normally distributed data was compared in pairs by post hoc Tukey’s test. Meanwhile, the non-normally distributed data was evaluated with the Kruskal-Wallis test and Dunn-Bonferroni post hoc test. The differences between two groups were analyzed by Student’s t-test. It was considered statistically significant when values of P < 0.05 between the two groups.

vibration absorption peak, and interlayer H2O molecule bending vibration absorption peak (Frost et al., 2001). It could be seen that the overall skeleton structure of attapulgite was not destroyed. Different from the spectrum of attapulgite, the NeH stretching vibration peak of CA at 1534 cm1 further verified the preparation of CA. In the spectrum of CCA, the intensity of OeH vibrational absorption peak at 3402 cm1 showed a systematic decrease, reflecting that eOH participated in the process of copper ion chelation. The disappearance of the stretching vibration absorption peak of the carbonate at 1446 cm1 was attributed to the fact that the carbonate can be removed under weak acid conditions (CuSO4). The crystal structures of attapulgite, CA and CCA were studied by XRD. As shown in Fig. 1F, the diffraction peaks of attapulgite at 2q ¼ 8.5
, 13.9
, 16.4
, 19.9
, 27.6
and 35.3
were attributed to the diffraction peaks of the crystal plane (110), (200), (130), (040), (400) and (161) (Liu et al., 2013), respectively. The spectrum of CA showed the same pattern as that of attapulgite. In contrast, CCA spectra showed that the disappearance of a carbonate diffraction peak at 2q ¼ 30.9
was consistent with the infrared spectra. According to the analysis of the spectrum by Jade 6 software, diffraction peaks at 2q ¼ 13.9
, 16.6
, 22.8
, 34.5
and 35.6
corresponded to a mineral similar to Cu2SO4(OH)6, indicating that copper ions had been successfully chelated on CA. 3.2. Deproteinization by CCA adsorption method 3.2.1. Effect of dose of CCA on the deproteinization performance of CCA adsorption method Adsorbent dose is always a key factor affecting the deproteinization ratio during the adsorption process (Wei et al., 2017). The increasing dose of absorbent could directly improve the rate of deproteinization, and also lead to the loss of more polysaccharides (Li et al., 2019). In this paper, the effect of different CCA dose (0.7, 1.4, 2.1, 2.8 and 3.5%) was investigated at pH value of 7.0, adsorption temperature of 30
C for 120 min. As illustrated in Fig. 2, the value of Dr% was linearly going up with the increase of CCA dose from 0.7% to 3.5%. When the CCA dose was initially 0.7%, the value of Dr% was a minimum (33.35 ± 1.14%), while the CCA dose increased to 3.5%, Dr% reached the maximum of 90.03 ± 0.85%. The addition of more adsorbents would introduce more adsorption sites and channels, which could accommodate much more proteins and thus improve the deproteinization rate (Song et al., 2018). On the contrary, the values of Rr% presented a decreasing trend (from 96.43 ± 1.03% to 90.40 ± 0.78%). When the dose of CCA reached 1.4%, the selectivity coefficient (Kc) reached a maximum (12.96 ± 0.76). Therefore, the economical CCA dose for deproteinization in this experiment was 1.4%.

3. Results and discussion 3.1. Characterization of CCA In this study, the surface topography of attapulgite and CA was observed by SEM. As shown in Fig. 1A, attapulgite rod crystals tended to exist in the form of densely packed aggregates or crystal bundles, while CA (Fig. 1B) exhibited a porous network-like structure with pore size ranging from 1 to 5 mm. EDS analysis was applied to evaluate the immobilization of copper ions on CA. N and Cu element were found existed in the EDS spectra of CCA (Fig. 1D). These phenomena indicated that silk fibroin was compounded with attapulgite, and copper ions had been successfully immobilized on the surface of CA. Fig. 1E showed the FT-IR spectrum of attapulgite, CA and CCA. The strong peaks near 471 cm1, 1030 cm1 and 1654 cm1 were all subordinated to the characteristic peaks of attapulgite: SieOeAl vibration absorption peak, SieOeSi asymmetric stretching

3.2.2. Effect of adsorption temperature on the deproteinization performance of CCA adsorption method In general, the molecules would be more active when the temperature increases, making adsorption in a favorable direction. In this paper, the effect of temperature (20, 30, 40, 50 and 60
C) was studied at pH value of 7.0, dose of CCA 1.4% for 120 min. Fig. 3 clearly showed that, when the adsorption temperature rose from 20
C to 30
C, the values of Dr% significantly increased from 41.04 ± 1.09% to 53.00 ± 1.05% and then slowly increased with the increase of adsorption temperature from 30
C to 60
C. This phenomenon indicated that CCA adsorption process was endothermic. However, temperature had little effect on values of Rr%, rising from 20
C to 60
C with Rr% reducing from 96.12 ± 1.07% to 93.13 ± 0.86%. And Kc reached the maximum (13.38 ± 0.68) at adsorption temperature of 30
C. Thus, 30
C was selected for further experiments considering the economic optimization and the maximum Kc appearing at 30
C.

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Fig. 1. SEM images of attapulgite (A) and CA (B); EDS images of attapulgite (C) and CCA (D); FT-IR spectra of CCA (E); XRD spectra of CCA (F).

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Fig. 2. Effects of different doses on deproteinization efficiency: visual image (A) and UVevis Spectrogram (C) colored with CBB, visual image (B) and UVevis Spectrogram (D) colored with sulfuric acid-phenol, and the efficiency of selective deproteinization (E). Superscript a designates before deproteinization and b-f orderly represent dose of 0.7, 1.4, 2.1, 2.8 and 3.5%, respectively.

3.2.3. Effect of pH value on the deproteinization performance of CCA adsorption method It was turned out that the adsorption process is greatly influenced by pH value (Xu and Grassian, 2017), which could not only transform the surface charge of CCA and PMM, and but also influence the degree of functional group dissociation at CCA active sites. In this paper, the effect of pH value (4.0, 5.0, 6.0, 7.0 and 8.0) was investigated with adsorption temperature of 30
C, dose of CCA 1.4% for 120 min. We could find from Fig. 4, at pH of 4.0 and 5.0 which correspond to the isoelectric point of protein (pH ¼ 4e5), Dr% reached the highest values (94.63 ± 1.53% and 93.31 ± 1.33%). It could be inferred that the protein had the minimum solubility at its isoelectric point and was thus most easily adsorbed because of none influence by the same charge repulsion. However, the adsorption of polysaccharide by CCA was not significantly affected by pH value. At pH values of 4.0 and 8.0, the Rr% were minimums (88.43 ± 1.05%) and maximums (96.62 ± 0.92%), respectively. Kc of 14.01 ± 0.46 reached its maximum at pH value of 5.0. As a result, the pH value of 5.0 was selected for further experiments. 3.2.4. Effect of adsorption time on the deproteinization performance of CCA adsorption method The adsorption process always takes a period to achieve the

adsorption equilibrium. In this paper, the effect of time (5, 15, 30, 45, 60, 90 and 120 min) was studied at pH value of 5.0, adsorption temperature of 30
C and CCA dose of 1.4%. As shown in Fig. 5, we were surprised to find that the value of Dr% rose rapidly within 15 min and reached the maximum (93.12 ± 1.45%). With the further increase of adsorption time, the value of Dr% tended to be stable. The experimental phenomenon could be explained by the high binding affinity of the amino acid residue on surface of protein to the copper ions on CCA (Asadi et al., 2020). In summary, the optimal parameter conditions for CCA adsorption deproteinization of PMM were obtained as follows: CCA dose of 1.4%, pH value of 5.0, adsorption temperature of 30
C and equilibrium time of 15 min. The values of Dr%, Rr% and Kc were 93.31 ± 1.33%, 93.34 ± 1.08% and 14.01 ± 0.46, respectively. Compared with the latest reported deproteinization material (attapulgite removal of proteins from Arca granosa) (Li et al., 2019), from Table 1 we could clearly find that under the respective optimal adsorption conditions the values of Rr% were equivalent. However, CCA adsorption method not only consumed less adsorbent and greatly shortened the adsorption time, but also achieved higher Dr% and Kc. Thus CCA adsorption method is a more efficient deproteinization method compared with the latest report.

Y. Zhu et al. / Journal of Cleaner Production 252 (2020) 119842

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Fig. 3. Effects of temperatures on deproteinization efficiency: visual image (A) and UVevis Spectrogram (C) colored with CBB, visual image (B) and UVevis Spectrogram (D) colored with sulfuric acid-phenol, and the efficiency of selective deproteinization (E). Superscript a designates before deproteinization and b-f orderly represent temperatures of 20, 30, 40, 50 and 60
C, respectively.

3.3. Comparison of deproteinization performance between CCA adsorption and Sevag method To verify the practicability of CCA adsorption deproteinization method, a series of properties of PMM solution before and after deproteinization were compared, including visual images and UVevisible spectrogram before and after dyeing (colored by CBB and sulfuric acid-phenol, respectively), FT-IR spectrogram, HPGFC chromatogram, composition of monosaccharide, values of Dr%, Rr% and Kc. The results were shown in Table 2 and Fig. 6. It was observed from Fig. 6A and B that the deproteinization efficiency between CCA adsorption and Sevag method were almost identical (P > 0.05), but CCA adsorption method had obvious advantages (P < 0.05) in terms of polysaccharide recovery (Fig. 6C). Fig. 6D clearly showed the characteristic absorption peaks of PMM before and after deproteinization in the vicinity of 1024, 1223, 1405, 1550, 1647, 2929 and 3379 cm1 (Shi et al., 2016). The strong and wide peaks at 3379 and 1647 cm1 were respectively attributed to OeH stretching vibration and C]O characteristic vibration. The other strong peak near 1024 cm1 corresponded to the coupling of CeO and CeC stretching vibration. The results of FT-IR indicated that CCA adsorption method did not cause damage to the characteristic functional groups and skeleton structures of PMM.

As shown in Fig. 6E and F, CCA adsorption as well as Sevag method had not changed the monosaccharide composition (Glucose) of PMM. The retention time of original PMM solution and PMM solution treated by CCA adsorption and Sevag method were respectively 14.467, 14.468 and 14.469 min. Therefore, compared with Sevag method, CCA adsorption not only maintained the monosaccharide composition of PMM but also did not alter the average Mw of PMM to a larger trend. What’s more, Table 2 clearly explained that CCA adsorption significantly shortened deproteinization time and introduced none toxic agents that would cause environmental pollution compared to Sevag method. As a result, CCA adsorption was a green and practicability method for the deproteinization of PMM that could replace the most classical Sevag method. 4. Conclusions In this study, CCA adsorption deproteinization method for crude polysaccharides was successfully developed. First of all, CCA was originally prepared by copper ion chelation with composite aerogel as ligand. Then, single factor experiment system was used to optimize the parameters of CCA adsorption deproteinization for PMM. In comparison with the latest reported deproteinization method

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Fig. 4. Effects of different pH value on deproteinization efficiency: visual image (A) and UVevis Spectrogram (C) colored with CBB, visual image (B) and UVevis Spectrogram (D) colored with sulfuric acid-phenol, and the efficiency of selective deproteinization (E). Superscript a designates before deproteinization and b-f orderly represent pH value of 4.0, 5.0, 6.0, 7.0 and 8.0, respectively.

and the traditional Sevag method, CCA adsorption method was proven to be practical and high-efficient. As a result, the optimal CCA adsorption conditions were CCA dose 1.4%, adsorption temperature 30
C and pH value 5.0. Under these conditions, equivalent Dr% (93.31 ± 1.33%), higher Rr% (93.34 ± 1.08%) and Kc (14.01 ± 0.46) were achieved compared with the latest deproteinization report. Finally, compared with Sevag method, CCA method had no significant difference in Dr%, monosaccharide composition, characteristic groups and average molecular weight. And above all, CCA method reached higher Rr% (93.59 ± 0.85%) and Kc (14.59 ± 0.64) without any toxic reagent and environment pollution. Therefore, CCA method would be a green, high-efficient and practical method that replaces the other methods for large-scale and environmentally sustainable deproteinization of crude polysaccharides. Fig. 5. Effect of adsorption time on deproteinization efficiency.

Table 1 Comparison of CCA adsorption and attapulgite adsorption. Methods

Dose (%)

Temperature (
C)

pH value

Time (min)

Dr%

Rr%

Kc

CCA Attapulgite

1.4 3.8

30 60

5.0 7.0

15 136

93.31 ± 1.33% 94.89 ± 1.06%

93.34 ± 1.08% 87.07 ± 1.12%

14.01 ± 0.46 7.34 ± 0.52

Y. Zhu et al. / Journal of Cleaner Production 252 (2020) 119842

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Table 2 Comparison of CCA adsorption method and Sevag method. Methods Dr%

Rr%

Kc

Specific consumption of organic solvent (mL/1 mL)

Deproteinization time (h/batch) Average Mw (wDa)

Chloroform N-butyl alcohol CCA Sevag

93.50 ± 1.02% 93.59 ± 0.85%* 14.59 ± 0.64* 0.0 ± 0.00* 0.0 ± 0.00* 89.58 ± 2.15% 75.19 ± 2.83% 3.61 ± 0.62 2.22 ± 0.18 0.45 ± 0.09

Before deproteinization After deproteinization 0.25* 7.5

287.05

286.86 286.68

Data were presented as mean ± SD (n ¼ 3). Superscript “*” designate a significant difference compared with Sevag method (P < 0.01).

Fig. 6. Visual image and UVevis Spectrogram of PMM solution before and after deproteinization (A), colored with CBB reagent (B) and colored with phenol-sulfuric acid (C), FT-IR Spectrogram (D), High-performance liquid phase gel filtration chromatography (HPGFC) (E), Gas Chromatogram of monosaccharides (F). Ms represents monosaccharide standard. Superscript a is the PMM solution before deproteinization. Superscript b and c are PMM solutions treated by CCA adsorption and Sevag method, respectively.

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Y. Zhu et al. / Journal of Cleaner Production 252 (2020) 119842

Author contribution section 1. Study concept and design: Qingping Xiong, Jun Yuan and Jing Chen. 2. Study supervision: Qingping Xiong, Jun Yuan and Jing Chen. 3. Administrative, technical, or material support: Qingping Xiong, Jun Yuan and Jing Chen. 4. Drafting of the manuscript: Yong Zhu and Hailun Li. 5. Acquisition of data: Yong Zhu, Jingrui Ma, Xinyu Zhou, Shiqi Jia, Jiaxin Zha, Dengping Xue, Weili Tao. 6. Analysis and interpretation of data: Yong Zhu, Hailun Li, Tingting Xu. 7. Statistical analysis: Yong Zhu, Hailun Li, Qingping Xiong. 8. Language modification: Tingting Xu, Jun Yuan and Jing Chen. 9. Critical revision of the manuscript for important intellectual content: Yong Zhu, Hailun Li, Qingping Xiong, Jun Yuan and Jing Chen. Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements This work was partly supported by the Qing Lan Project of Jiangsu Province, Six Talent Peaks Project in Jiangsu Province (2017YY-003). References An, F., Wu, R., Li, M., Hu, T., Gao, J., Yuan, Z., 2017. Adsorption of heavy metal ions by iminodiacetic acid functionalized D301 resin: kinetics, isotherms and thermodynamics. React. Funct. Polym. 118, 42e50. Asadi, Z., Zarei, L., Golchin, M., Skorepova, E., Eigner, V., Amirghofran, Z., 2020. A novel Cu(II) distorted cubane complex containing Cu4O4 core as the first tetranuclear catalyst for temperature dependent oxidation of 3,5-di-tert-butyl catechol and in interaction with DNA & protein (BSA). Spectrochim. Acta A Mol. Biomol. Spectrosc. 227, e117593 https://doi.org/10.1016/j.saa.2019.117593. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Chen, L., Huang, G., 2018. Extraction, characterization and antioxidant activities of pumpkin polysaccharide. Int. J. Biol. Macromol. 118, 770e774. Dai, P., Huan, P., Wang, H., Lu, X., Liu, B., 2015. Characterization of a long-chain fatty acid-CoA ligase 1 gene and association between its SNPs and growth traits in the clam Meretrix meretrix. Gene 566, 194e200. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350e356. Ferreira, I.C., Heleno, S.A., Reis, F.S., Stojkovic, D., Queiroz, M.J., Vasconcelos, M.H., Sokovic, M., 2015. Chemical features of Ganoderma polysaccharides with antioxidant, antitumor and antimicrobial activities. Phytochemistry 114, 38e55. Frost, R.L., Locos, O.B., Ruan, H., Kloprogge, J.T., 2001. Near-infrared and mid-infrared spectroscopic study of sepiolites and palygorskites. Vib. Spectrosc. 27, 1e13. Gao, H., Huang, G., 2019. Preparation and antioxidant activity of carboxymethylated garlic polysaccharide. Int. J. Biol. Macromol. 121, 650e654. Jiang, C., Xiong, Q., Li, S., Zhao, X., Zeng, X., 2015. Structural characterization, sulfation and antitumor activity of a polysaccharide fraction from Cyclina sinensis. Carbohydr. Polym. 115, 200e206.

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