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A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment
Journal of Cleaner Production xxx (2016) 1e10
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A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment Qingping Xiong a, b, c, 1, Song Huang a, b, 1, Jianhui Chen d, 1, Baolan Wang e, 1, Lian He f, Lei Zhang g, Shijie Li a, b, Jizhong Wang c, Jianguo Wu a, b, Xiaoping Lai a, b, *, Danyan Zhang a, b, ** a
School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, PR China Guangdong Provincial Key Laboratory of New Drug Development and Research of Chinese Medicine, Guangzhou 510006, Guangdong, PR China Key Laboratory for Medicinal Exploitation of Huai'an Regional Resource, College of Chemical Engineering, Huaiyin Institute of Technology, Huai'an 223003, Jiangsu, PR China d Affiliated Huai'an Hospital of Xuzhou Medical University, Huai'an 223002, Jiangsu, PR China e Huai'an First People's Hospital, Nanjing Medical University, Huai'an 223300, Jiangsu, PR China f Xinhua College, Sun Yat-sen University, Guangzhou 510520, Guangdong, PR China g Jiangsu Tasly Diyi Pharmaceutical Co., Ltd., Huai'an 223002, Jiangsu, PR 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 August 2016 Received in revised form 10 October 2016 Accepted 22 October 2016 Available online xxx
The purpose of this paper was to develop a green method for deproteinization of polysaccharide (PCC) from Cipangopaludina chinensis by freeze-thaw treatment (FTT). Based on single-factor experiments of FTT, firstly, the optimal deproteinization parameters of PCC were obtained by comparing their deproteinization ratio (Dr%), recovery ratio (Rr%) and selectivity coefficient (Kc) as follows: freezing temperature of 40 C, freezing time of 96 h, thawing temperature of 10 C and freeze-thaw cycles of 11 times. Under optimal conditions, The Dr%, Rr% and Kc of PCC were 87.10 ± 1.91%, 84.91 ± 2.45% and 5.77 ± 0.43, respectively. Then, the deproteinization performance of FTT for PCC under optimal conditions was compared with the most typical Sevage method. We found that there were no significant differences between FTT and Sevage method on the Dr%, monosaccharide composition, characteristic group and molecular weight degradation. However, compared with Sevage method, FTT not only had higher Rr% and Kc, but also were non-pollution. The results demonstrated that FTT would be a promising and green method for deproteinization of PCC. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Deproteinization Polysaccharides Cipangopaludina chinensis Freeze-thaw treatment Green method
1. Introduction Cipangopaludina chinensis (C. chinensis), widespread throughout the China and many other Asian countries (Li, 2012), is a common gastropod and invertebrate of viviparidae (Mccann, 2014). The flesh of C. chinensis is an important functional food supplement with remarkable efficacy for treatment of diabetes (Nurhasan et al., 2010), otitis media, urinary infection (Zhang, 2005) and liver
* Corresponding author. School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, PR China. ** Corresponding author. School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, PR China. E-mail addresses: [email protected] (X. Lai), [email protected] (D. Zhang). 1 These authors contributed equally to this paper.
diseases (Liu et al., 2013). Recent studies indicated that these pharmacological activities are closely related with its richness in polysaccharides (Yang et al., 2012). In our previous studies, for the first time, polysaccharide (PCC) was successfully extracted from C. chinensis with 8.7% yield (based on the dried flesh) (Jiang et al., 2013). Its structure characterization has been clearly identified (Shi et al., 2015). And the excellent immunostimulatory and antioxidant activities have also been confirmed (Xiong et al., 2013). Unfortunately, these studies employed the most typical Sevage deproteinization method of polysaccharides to remove protein impurities of PCC. The most typical Sevage method, consume a large amount of chloroform and n-butyl alcohol, resulting in a large number of toxic organic pollutants and difficulty in commercial production of PCC. In addition, their products are also not suitable for many applications in the food and pharmaceutical sectors due to high levels of toxic organic solvent residual. Therefore, it is
http://dx.doi.org/10.1016/j.jclepro.2016.10.125 0959-6526/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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indispensable and promising to develop a green deproteinization method for improving the quality of extracted PCC and protecting eco-environment. Freeze-thaw method, in the field of food science, refers to a way to extend the shelf life of food by freezing, storage and thawing rquez et al., 2015). It has been recognized as an excellent (Ma preservation method for maintaining microbiological stability of food (Zhao et al., 2013). Nevertheless, the freeze-thaw treatment (FTT) of protein-rich liquid food may cause complex changes of the buffer environment (Mun et al., 2008), including formation of icewater interfaces (Donsì et al., 2011), adsorption to container surfaces (Ghosh and Coupland, 2008), cryo-concentration of protein and solutes, pH changes, and phase separation (Zhao et al., 2015). Meanwhile, the intermolecular disulfide bonds among proteins with high hydrophobicity may be formed and exposed in the process of FTT (Wang et al., 2013). These changes could rapidly induce mass insoluble aggregation and precipitation of protein. Although this fact is unfavourable to the protein stability, it is exploited to removal protein impurities from products with unique superiority of green non-pollution. FTT could therefore be implemented as a green deproteinization strategy. However, to the best of our knowledge, there is no literature on this deproteinization method. In the present paper, for the first time, a green method based on FTT was developed for deproteinization of PCC. Various parameters influencing the deproteinization of PCC were optimized by singlefactor experiments of FTT. In order to evaluate the deproteinization effect of FTT for PCC, a series of performance parameters of PCC solutions before and after deproteinization was measured and compared with those of most typical Sevage method. The information in this study is significant for deproteinization of proteinrich polysaccharides. 2. Materials and methods 2.1. Materials and reagents The fresh C. chinensis was obtained from the Huaiyin Vegetable Product Market (Huaiyin, China) and identified by Prof. Danyan Zhang, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, PR China. Monosaccharide standards including rhamnose, arabinose, fucose, xylose, mannose, glucose and galactose were from Sigma Chemical Co. (St. Louis, MO, USA). The phenoles, trifluoroacetic acid (TFA), concentrated sulfuric acid (98%, H2SO4), chloroform and nbutyl alcohol of analytical grade were obtained from Shanghai Chemical Reagents Co., Ltd. (Shanghai, China). Dextrans with different molecular weight (Mw) were purchased from American Polymer Standards Co. (Colorado, USA). The kit of Coomassie brilliant blue (CBB) for protein determination was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Deionized water was prepared by a Milli-Q Water Purification system (Millipore, MA, USA). 2.2. Preparation of PCC solution The PCC solution was prepared according to our previously reported methods with some modifications (Jiang et al., 2013). Briefly, fresh C. chinensis was crushed to remove its shells. The flesh was stripped viscera and cleansed with cold water to thoroughly removal impurities, then smashed using a high speed disintegrator. The homogenate was refluxed with anhydrous ethanol to eliminate lipids and some colored materials, and air-dried at 50 C to a constant weight. The pre-treated powder form C. chinensis (50.0 g) was extracted with distilled water in a ratio of 1:25 (pre-treated powder to water, w/v, g/ml) for 3 h at 95 C. The extracting solution was
centrifuged at 5000 rpm for 20 min. The insoluble residue was repeated treatment two times as mentioned above. All supernatants were collected together, stirred uniformly and stored at 4 C, affording the PCC solution. 2.3. Deproteinization experiments of FTT for PCC solution The PCC solution (100 ml) was placed in a polyethylene bottle and frozen at a proper temperature. After a certain time of freezing, the frozen PCC solution was thawed with a designed test temperature. Subsequently, the above procedure of PCC solution was repeated under the same parameters. The PCC solution after multicycles of FTT was centrifuged at 4000 rpm for 30 min and then filtered through a 0.45 mm membrane filter. The filtrate was carefully collected, as PCC solution after FTT deproteinization. Using this way, the optimal deproteinization parameters of FTT, including freezing temperature, freezing time, thawing temperature and freeze-thaw cycles, were systematically investigated. 2.4. Deproteinization experiments of Sevage method for PCC solution Deproteinization experiments of Sevage method for PCC solution were carried out by the reported parameters (Sevag et al., 1938). 100 ml of PCC solution and 20 ml of Sevage reagent with the ratio of chloroform to n-butyl alcohol of 5:1 were added to a 250 ml round-bottomed flask. The mixed solution was continually stirred for 60 min and centrifuged at 4000 rpm for 30 min. As a result, the solution was divided into three layers, in order, PCC solution after deproteinization, denatured protein and redundant Sevage reagent from top to bottom. The supernatant was repeatedly deproteinized with the same amount of Sevage reagent to the absence of denatured protein layer. The supernatant after the last deproteinization was collected and passed through a 0.45 mm membrane filter. The filtrate was regarded as PCC solution after deproteinization with Sevage method. 2.5. Performance evaluation of deproteinization 2.5.1. Determination of deproteinization ratio The deproteinization ratio was determined as the follows method: 50 ml of PCC solution before or after deproteinization was loaded into a 10 ml Nessler tube. 3 ml of CBB agent (Blakesley and Boezi, 1977), a special chromogenic agent for protein (Sedmak and Grossberg, 1977), was added to this ones. After mixing the solution thoroughly and set aside for 30 min, the Spectrogram of the colored solution at 400e800 nm was determined by a UVevis Spectrophotometer (UV-2401 PC, Shimadzu, Japan). The maximum absorbance value in the spectrum, 595 nm, was applied to calculate deproteinization ratio. The deproteinization ratio was expressed as the following equation (Liu et al., 2010):
Dr ð%Þ ¼
AD0 AD1 100% AD0
(1)
where AD0 and AD1 are the maximum absorbance value of the sample solutions using CBB stainng before and after deproteinization, respectively. Dr % is deproteinization ratio. 2.5.2. Determination of recovery ratio of PCC The following method was used to measure the recovery ratio of PCC. 50 ml of PCC solution before or after deproteinization was added into a 10 ml Nessler tube, and then stained by the phenolsulfuric acid method (Dubois et al., 1956). Above mentioned UVevis spectrophotometer (UV-2401 PC) was employed to record
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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their Spectrogram at 350e800 nm. The maximum absorbance value in the spectrum, 490 nm, was picked to evaluate the recovery ratio of PCC according to the following equation (Liu et al., 2010):
Rr ð%Þ ¼
AR1 100% AR0
(2)
where AR0 and AR1 are the maximum absorbance value of the sample solutions with phenol-sulfuric acid reagent stainng before and after deproteinization, respectively. Rr % is recovery ratio of PCC. 2.5.3. Calculation of selectivity coefficient In order to evaluate selectivity deproteinization of each method, the selectivity coefficient (Kc) was calculated as follows:
Kc ¼
Dr ð%Þ 100 Rr ð%Þ
(3)
where Dr % and Rr % are deproteinization ratio and recovery ratio of PCC, respectively. Kc is selectivity coefficient.
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to determine Mw of the samples. The chromatographic separation was operated by a gel-filtration chromatographic column of TSKGEL G3000SWxl (300 mm 7.5 mm i.d., 5 mm diameter, Tosoh Corp., Japan) (Zhang et al., 2014). The mobile phase was 0.1 M Na2SO4 solution in PBS buffer (0.01 M, pH 6.8) and passed through a 0.22 mm membrane filter (Millipore, USA) prior to use (Liu et al., 2015). The chromatographic condition was set as flow rate of 0.8 ml/min, the injection volume of 10 ml and the column temperature of 25 C. Seven dextrans with different Mw (9.6, 21.1, 36.3, 72.7, 158.1, 344.8 and 714.5 kDa) were employed to establish a calibration curve by plotting the retention time against the logarithm of their respective Mw. The regression line for Mw was obtained as follows:
log Mw ¼ 0:0288X þ 5:3164 R2 ¼ 0:9985; n ¼ 7
(4)
where X is the retention time of dextrans (min). The retention time of the samples was substituted into the equation of the calibration curve to calculate their Mw. 2.6. Statistical analysis
2.5.4. Visual observation and photography A certain amount of these solution before and after deproteinization, such as unstained PCC solution, colored PCC solution using CBB agent in Section 2.5.1 or phenol-sulfuric acid method in Section 2.5.2, were placed in a 10 ml Nessler tube. A Sony DSC-W80 digital camera (Sony Corp., Park Ridge, NJ) was applied to photograph their visual characteristics on a black or white background. 2.5.5. Analysis of UVevis and IR spectra 200 ml of PCC solutions before or after deproteinization was put into a 10 ml Nessler tube and diluted to 5 ml with deionized water. UVevis Spectrogram of the diluted solution was recorded by a UV2401 PC UVevis spectrophotometer in the range of 200e800 nm. PCC solutions before or after deproteinization were concentrated to a proper volume and freeze-dried. 1 mg freeze-dried powder was mixed thoroughly with 100 mg KBr powder in a running infrared drying oven. The mixed powder pressed into a tablet and then placed in a Nicolet 6700 FT-IR Spectrometer (Thermo Co., USA) to record their IR Spectrogram in the frequency range of 4000e400 cm1 as reported method (Geng et al., 2009). 2.5.6. Identification of monosaccharide composition 5.0 mg freeze-dried powder in Section 2.5.5 was hydrolyzed with 4 ml of 2 M trifluoroacetic acid (TFA) at 120 C for 2 h (Souza et al., 2012). The redundant TFA was fully replaced by methanol and evaporated to dryness. The hydrolyzate was dissolved in 0.6 ml pyridine, and then transferred to a 3 ml reaction flask with 10 mg hydroxylamine hydrochloride and 5 mg inositol (internal standard). After reacting at 90 C for 30 min, 1.0 ml acetic anhydride was added into the flask to further react for 30 min. The reaction solution, cooling to room temperature, was the acetylated derivatives of the samples. All monosaccharide standards were also acetylated in the same way. The GC chromatogram of the acetylated samples was analyzed by a 7890N GC (Agilent Technologies, Santa Clara, CA, USA) equipped with flame ionization detector and a HP-5 fused silica capillary column (30 m 0.32 mm i.d. 0.25 mm) as the reported methods (Jiang et al., 2015). The monosaccharide composition of all samples was identified by the retention time of GC chromatogram from monosaccharide standards. 2.5.7. Measurement of Mw A size-exclusion HPLC chromatography (HPGPC) instrument (Agilent 1200, USA) with a refractive index detector (RID) was used
All data were expressed as mean ± standard deviation (SD) and evaluated by the Student's t-test and one-way analysis of variance followed using Duncan's multiple-range tests. The statistical analyses were performed by SPSS for Windows, Version16.0 (SPSS, Chicago, IL). The difference with P < 0.05 or P < 0.01 was considered to be statistically significant. 3. Results and discussion 3.1. Effect of freezing temperature on deproteinization efficiency of FTT The intermolecular disulfide bonds and hydrophobic interactions among proteins, resulting from stress cryoconcentration of protein and solutes in the unfrozen parts (Xu et al., 2015), participated in the formation of insoluble aggregates of proteins (Ghosh and Coupland, 2008). Freezing temperature can manipulate the speed of water crystallization and cryoconcentration, thereby affecting the stability of protein. Therefore, the effects of different freezing temperatures (20, 30, 40, 50, 60, 70, or 80 C) on deproteinization efficiency of FTT for PCC solution were evaluated under the conditions of freezing time of 72 h, thawing temperature of 20 C and freeze-thaw cycles of 9 times. As shown in Fig. 1. With the decrease in freezing temperature, the deproteinization ratio was raised, indicating lower temperature was advantageous to removal of protein impurities from PCC. However, when the freezing temperature was further lowered below 40 C, the changed trend of deproteinization ratio reached a relative steady-state (P > 0.05), whit the recovery ratio of PCC beginning to fall significantly (P < 0.01) as the freezing temperature further decreased. The highest selectivity coefficient of 5.21 appeared at 40 C. Hence, the freezing temperature of 40 C should be picked for further experiment. 3.2. Effect of freezing time on deproteinization efficiency of FTT Freezing time is an important factor that affects the deproteinization efficiency of FTT. It has been reported that increasing frozen storage time decreased the average particle size of protein (Wang et al., 2013), leading to a sharp increase in surface free energy and a rapid destabilization of protein. According to this theory,
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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Fig. 1. Effects of different freezing temperature on visual photograph (A), visual photograph (B) and Vis Spectrogram (C) after CBB staining, visual photograph (D) and Vis Spectrogram (E) after phenol-sulfuric acid staining, and deproteinization efficiency (F). BDP designate before deproteinization. I ~ VII orderly represent freezing temperature of 20, 30, 40, 50, 60, 70 and 80 C, respectively.
a longer freezing time is favored for deproteinization efficiency of FTT. However, the polysaccharides would suffer great losses with excessive extension of freezing time due to mass adsorption and embedding of protein precipitation. Thus, it is very necessary to investigate the effects of different freezing time on deproteinization
efficiency of FTT for PCC solution. In present paper, when other parameters were set as freezing temperature of 40 C, thawing temperature of 20 C and freeze-thaw cycles of 9 times, the deproteinization efficiency were evaluated at freezing time of 12, 24, 48, 72, 96, 120, 144 and 168 h, respectively. The result was
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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shown in Fig. 2. Although the deproteinization ratio increased as freezing time prolonged, the difference of deproteinization ratio was not significant in the range of 96e168 h (P > 0.05). These results suggested that the precipitation of protein has tended to the equilibrium at about 96 h. A slow and mild decrease in recovery
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ratio of PCC was noted with the extension of freezing time. However, after the freezing time was prolonged above 96 h, the downtrend of PCC recovery ratio became significant (P < 0.01). These results were consistent with the above theory. The selectivity coefficient increased first and then decreased at different freezing
Fig. 2. Effects of different freezing time on visual photograph (A), visual photograph (B) and Vis Spectrogram (C) after CBB staining, visual photograph (D) and Vis Spectrogram (E) after phenol-sulfuric acid staining, and deproteinization efficiency (F). BDP designate before deproteinization. I ~ VIII orderly represent freezing time of 12, 24, 48, 72, 96, 120, 144 and 168 h, respectively.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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time, reaching a maximum at 96 h. The results indicated that their deproteinization efficiency has attained an optimal state at 96 h. Thus, freezing time of 96 h was selected for the following experiments.
3.3. Effect of thawing temperature on deproteinization efficiency of FTT To investigate the effect of different thawing temperature on
Fig. 3. Effects of different thawing temperature on visual photograph (A), visual photograph (B) and Vis Spectrogram (C) after CBB staining, visual photograph (D) and Vis Spectrogram (E) after phenol-sulfuric acid staining, and deproteinization efficiency (F). BDP designate before deproteinization. I ~ VIII orderly represent thawing temperature of 4, 10, 20, 30, 40, 50, 60 and 70 C, respectively.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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deproteinization efficiency, the FTT for PCC solution was carried out using thawing temperature of 4, 10, 20, 30, 40, 50, 60 or 70 C, while other parameters were designed as freezing temperature of 40 C, freezing time of 96 h and freeze-thaw cycles of 9 times. As shown in Fig. 3, the deproteinization ratio had a decrease trendency with
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increasing the thawing temperature. When the thawing temperature was raised to more than 20 C, the changes in deproteinization ratio became significant (P < 0.01). PCC recovery ratio, in contrast, increased as thawing temperature raised, but insignificant difference after the thawing temperature reached 10 C. These results
Fig. 4. Effects of different freeze-thaw cycles on visual photograph (A), visual photograph (B) and Vis Spectrogram (C) after CBB staining, visual photograph (D) and Vis Spectrogram (E) after phenol-sulfuric acid staining, and deproteinization efficiency (F). BDP designate before deproteinization. I ~ VIII orderly represent freeze-thaw cycles of 1, 3, 5, 7, 9, 11, 13 and 15 times, respectively.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
Fig. 5. Visual photograph and UVevis Spectrogram (A), visual photograph and Vis Spectrogram after CBB staining (B), visual photograph and Vis Spectrogram after phenol-sulfuric acid staining (C), IR Spectrogram (D), HPGFC Chromatogram (E) and GC of monosaccharides (F) of PCC solutions before and after deproteinization. BDP designate before deproteinization. MS was monosaccharide standard. I and II represent after deproteinization using FTT and Sevage, respectively.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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can be explained that the high hydrophilic precipitate of the polysaccharides, which formed and maintained stable structures in the freezing process, turned into soluble aggregates and were easy to redissolve in thawing solvent at the lower thawing temperature. On the contrary, the aggregates of proteins become an insoluble hydrophobic precipitate by cooling and freezing processes due to the formation of denatured products and intermolecular disulfide bonds among proteins. Its dissolution requires providing a more vigorous thermal motion, caused by high thawing temperature. The fact indicated discrepancies of the solubility between polysaccharide and protein at lower thawing temperature was benefited for selective precipitation of proteins. However, the selectivity coefficient had a maximum value at the thawing temperature of 10 C. Therefore, the thawing temperature of 10 C was adopted in the following experiments to synthetically evaluate its effect on protein removal and polysaccharide retention. 3.4. Effect of freeze-thaw cycles on deproteinization efficiency of FTT Recent studies have shown that the growth of ice crystals during protein freezing could facilitate its structure opening and unfolding (Zhao et al., 2015). Changes in protein stability occurred during freeze-thaw cycles (Guo et al., 2011), as repeated FTT and reformation of ice crystals (Benjakul and Bauer, 2000) induced change in the secondary, tertiary and quaternary structure of protein (Zhang et al., 2011). When the number of freeze-thaw cycles increased, these changes could enhance the exposure of SH groups content and surface hydrophobicity (Noh et al., 2006), which are closely related with the formation of protein precipitation. In order to optimize freeze-thaw cycles, the FTT for PCC solution were performed using the freeze-thaw cycles of 1, 3, 5, 7, 9, 11, 13 or 15 times, accompanied by freezing temperature of 40 C, freezing time of 96 h and thawing temperature of 10 C. As shown in Fig. 4, an increasing number of freeze-thaw cycles were associated with higher deproteinization ratio and lower PCC recovery ratio. The increase in deproteinization ratio was insignificant after the freezethaw cycle reached 9 times, while the cycle number was 13 times when decrease in PCC recovery ratio became negligible. The selectivity coefficient increased at first with increasing number of freeze-thaw cycle and peaked at 11 times, where it began to decrease afterwards. The results suggested that the optimal freezethaw cycles should choose 11 times. 3.5. Comparison of deproteinization efficiency between FTT and Sevage method Based on the above experiments of influential factors, the optimal deproteinization parameters of FTT for PCC were summarized as follows: freezing temperature of 40 C, freezing time of 96 h, thawing temperature of 10 C and freeze-thaw cycles of 11 times. In order to confirm the dependability of the present method, the performance parameters of deproteinization for PCC solutions before and after deproteinization, including visual photograph,
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UVevis Spectrogram, visual photograph and Vis Spectrogram after CBB staining, visual photograph and Vis Spectrogram after phenolsulfuric acid staining, IR Spectrogram, HPGFC Chromatogram, GC of monosaccharides, Dr%, Rr%, Kc, consumption of organic solvent and Mw degradation, were compared with the most typical Sevage method. Qualitative deproteinization efficiency of different methods was shown in Fig. 5. The results of Fig. 5 A and Fig. 5 B could be obviously observed that both FTT and Sevage method had remarkable deproteinization for the PCC solutions, but there was no significant difference between them. FTT was obviously superior to Sevage method on the polysaccharide recovery (Fig. 5C). As shown in Fig. 5D, characteristic absorptions peaks of polysaccharides around 3280, 2932, 1662, 1545, 1448, 1242 and 1154 cm1 were clear for the samples before and after deproteinization of PCC (Shi et al., 2015). The strong and broad peaks around the 3280 cm1 and 1662 cm1 were OeH stretching vibrations and deformation vibration respectively. The peaks around 2932 cm1 were assigned to the CeH asymmetric stretching vibration. And the strong extensive absorption in the region of 900e1200 cm1 for coupled CeO and CeC stretching and CeOH bending vibrations. Besides, the absorption peaks toward about 1241 cm1 were corresponding to SeO asymmetry stretching vibrations, revealing PCC being sulfate radical. The characterizations of IR Spectroscopy demonstrated that the characteristic groups and molecular backbone of PCC were adequately retained after deproteinization using FTT and Sevage method. As shown in Fig. 5E and Table 1, Dr%, Rr%, Kc and change value of Mw in the present method were 87.10 ± 1.91%, 84.91 ± 2.45%, 5.77 ± 0.43 and 3.24 ± 0.47 kDa, respectively. Compared with the Sevage method, FTT has higher Rr% and Kc with similar Dr% and change value of Mw. Fig. 5F shown PCC and deproteinized PCCs by FTT and Sevage method were similar on the monosaccharide composition with a molar ratio of 5.289:0.279:0.036, 5.312:0.263:0.038, 5.255:0.272:0.035 for rhamnose, fucose and glucose, respectively. The result indicated molecular composition of PCC was not converted during the deproteinization process. Even more remarkable was the fact that the whole process of deproteinization in FTT did not use any toxic organic reagents. The above results demonstrated that FTT could replace the most typical Sevage method and represent a green method for deproteinization of PCC.
4. Conclusions Based on FTT, a novel green deproteinization method of PCC was successfully developed in this study. The deproteinization parameters of FTT for PCC were systematically optimized by single-factor experiments. The comparison between FTT and the most typical Sevage method was performed to validate the reliability and feasibility of FTT for PCC deproteinization. As a result, its optimal deproteinization parameters were gained as follows: freezing temperature of 40 C, freezing time of 96 h, thawing temperature of 10 C and freeze-thaw cycles of 11 times. The Dr%, Rr% and Kc of FTT for PCC deproteinization under optimal conditions were
Table 1 The comparison of different deproteinization methods. Methods
Sevage FTT
D r%
91.01 ± 2.61 87.10 ± 1.91
Rr%
68.99 ± 3.11 84.91 ± 2.45
Kc
b
a
2.94 ± 0.31 5.77 ± 0.43
Specific consumption of organic solvent (ml/1 ml)
Mw (kDa)
Chloroform
Before deproteinization
After deproteinization
Change value
90.03 ± 2.14
87.79 ± 1.21 86.76 ± 1.89
2.26 ± 0.31 3.24 ± 0.47
3.33 ± 0.14 0.00 ± 0.00
b
N-butyl alcohol 0.67 ± 0.07 0.00 ± 0.00
b
Data were presented as mean ± SD (n ¼ 3). Superscript a-b designate a significant differences.
a
P < 0.05 compared with FTT.
b
P < 0.01 compared with FTT.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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87.10 ± 1.91%, 84.91 ± 2.45% and 5.77 ± 0.43, respectively. The Dr%, monosaccharide composition, characteristic group and molecular weight degradation of FTT were similar to the most typical Sevage method. FTT had higher Rr% and Kc with green non-pollution. The results demonstrated that FTT would be an environmentally friendly method for deproteinization of PCC. As a promising novel deproteinization technology, it might be able to replace the most typical Sevage method to remove protein impurities of the polysaccharides for large-scale production, and be widely applied in food and medicine. However, some limitations should be mentioned in the present paper. In order to promote its application, the changes on the activity, structural characteristics and property of protein, caused by FTT, should been identified to further evaluate its detailed deproteinization mechanisms in later work. Acknowledgements This work was partly supported by the National Natural Science Foundation of China (No. 81503387), Natural Science Fund for Colleges and Universities in Jiangsu Province of China (No. 15KJB360002, No. 14KJB210002), Hong Kong, Macao, and Taiwan Science and Technology Cooperation Projects of Ministry of Science and Technology of China (No. 2014DFH30010), Qing Lan Project of Jiangsu Province of China, Science and Technology Program of Guangdong Province of China (No. 2013B090800052, No. 2013A022100002), Special Funds of Applied Science and Technology Research and Development of Guangdong Province of China (No. 2015B020234008) and Special Funds from Guangdong Province of China for the Construction of High Level University (Grant No. 2050205). References Benjakul, S., Bauer, F., 2000. Physicochemical and enzymatic changes of cod muscle proteins subjected to different freeze-thaw cycles. J. Sci. Food Agric. 80, 1143e1150. Blakesley, R.W., Boezi, J.A., 1977. A new staining technique for proteins in polyacrylamide gels using coomassie brilliant blue G250. Anal. Biochem. 82, 580e582. Donsì, F., Wang, Y., Huang, Q., 2011. Freeze-thaw stability of lecithin and modified starch-based nanoemulsions. Food Hydrocoll. 25, 1327e1336. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350e356. Geng, W., Nakajima, T., Takanashi, H., Ohki, A., 2009. Analysis of carboxyl group in coal and coal aromaticity by Fourier transform infrared (FT-IR) spectrometry. Fuel 88, 139e144. Ghosh, S., Coupland, J.N., 2008. Factors affecting the freeze-thaw stability of emulsions. Food Hydrocoll. 22, 105e111. Guo, Y.Y., Kong, B.H., Xia, X.F., Yang, Z., 2011. Effect of number of freeze-thaw cycles on physico-chemical characteristics of carp muscle. Food Sci. 32, 125e130. Jiang, C., Jiao, Y., Chen, X., Xia, L., Yan, W., Bo, Y., Xiong, Q., 2013. Preliminary characterization and potential hepatoprotective effect of polysaccharides from Cipangopaludina chinensis. Food Chem. Toxicol. 59, 18e25.
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. Li, K.J., 2012. Molecular analysis of intestinal bacterial communities in Cipangopaludina chinensis used in aquatic ecological restorations. Ecol. Eng. 39, 36e39. Liu, J., Luo, J., Yi, S., Hong, Y., Lu, Z., Zeng, X., 2010. A simple method for the simultaneous decoloration and deproteinization of crude levan extract from Paenibacillus polymyxa EJS-3 by macroporous resin. Bioresour. Technol. 101, 6077e6083. Liu, X.Y., Li, C.P., Wang, K.X., 2013. Study on polysaccharide of Cipangopaludina chinensis against HBV in vitro. China J. Chin. Mater. Med. 38, 879e883. Liu, Y., Liu, C., Jiang, H., Zhou, H., Li, P., Wang, F., 2015. Isolation, structural characterization and neurotrophic activity of a polysaccharide from Phellinus ribis. Carbohydr. Polym. 127, 145e151. rquez, A.L., Salvatore, G.N., Otero, R.G., Wagner, J.R., Palazolo, G.G., 2015. Impact Ma of freeze-thaw treatment on the stability of calcium-fortified soy beverages. LWT Food Sci. Technol. 62, 474e481. Mccann, M.J., 2014. Population dynamics of the non-native freshwater gastropod, Cipangopaludina chinensis (Viviparidae): a capture-mark-recapture study. Hydrobiologia 730, 17e27. Mun, S., Cho, Y., Decker, E.A., Mcclements, D.J., 2008. Utilization of polysaccharide coatings to improve freeze-thaw and freeze-dry stability of protein-coated lipid droplets. J. Food Eng. 86, 508e518. Noh, E.J., Kang, C., Hong, S.T., Yun, S.E., 2006. Freezing of soybeans influences the hydrophobicity of soy protein. Food Chem. 97, 212e216. Nurhasan, M., Maehre, H.K., Malde, M.K., Stormo, S.K., Halwart, M., James, D., Elvevoll, E.O., 2010. Nutritional composition of aquatic species in Laotian rice field ecosystems. J. Food Compos. Anal. 23, 205e213. Sedmak, J.J., Grossberg, S.E., 1977. A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250. Anal. Biochem. 79, 544e552. Sevag, M.G., Lackman, D.B., Smolens, J., 1938. Isolation of components of Streptococcal nucleoproteins in serologically active form. J. Biol. Chem. 124, 0425e0436. Shi, Y., Xiong, Q., Wang, X., Xia, L., Yu, C., Jie, W., Jing, Y., Zhao, X., Ying, X., Hao, C., 2015. Characterization of a novel purified polysaccharide from the flesh of Cipangopaludina chinensis. Carbohydr. Polym. 136, 875e883. Souza, B.W.S., Cerqueira, M.A., Bourbon, A.I., Pinheiro, A.C., Martins, J.T., Teixeira, J.A., Coimbra, M.A., Vicente, A.A., 2012. Chemical characterization and antioxidant activity of sulfated polysaccharide from the red seaweed Gracilaria birdiae. Food Hydrocoll. 27, 287e292. Wang, L., Chen, Y., Li, Z., 2013. The effects of freezing on soybean microstructure and qualities of soymilk. Food Eng. 116, 1e6. Xiong, Q., Jiao, Y., Zhao, X., Chen, X., Zhang, Q., Jiang, C., 2013. Purification, characterization and immunostimulatory activity of polysaccharide from Cipangopaludina chinensis. Carbohydr. Polym. 98, 217e223. Xu, D., Zhang, J., Cao, Y., Wang, J., Xiao, J., 2015. Influence of microcrystalline cellulose on the microrheological property and freeze-thaw stability of soybean protein hydrolysate stabilized curcumin emulsion. LWT Food Sci. Technol. 66, 590e597. Yang, S., Li, P., Mi, Z., 2012. LPS-induced TNFa factor (LITAF) in the snail Cipangopaludina chinensis : gene cloning and its apoptotic effect on NCI-H446 cells. Fish Shellfish Immun. 32, 268e272. Zhang, A., Qi, W., Singh, S.K., Fernandez, E.J., 2011. A new approach to explore the impact of freeze-thaw cycling on protein structure: hydrogen/deuterium exchange mass spectrometry (HX-MS). Pharm. Res. 28, 1179e1193. Zhang, K.H., 2005. Nutrifion and healthcare function of field-snail-meat as well as its processing technique. Food Mach. 21, 55. Zhang, X., Liu, L., Lin, C., 2014. Isolation, structural characterization and antioxidant activity of a neutral polysaccharide from Sisal waste. Food Hydrocoll. 39, 10e18. Zhao, J., Dong, F., Li, Y., Kong, B., Liu, Q., 2015. Effect of freezeethaw cycles on the emulsion activity and structural characteristics of soy protein isolate. Process Biochem. 50, 1607e1613. Zhao, L., Li, L., Liu, G.Q., Chen, L., Liu, X., Zhu, J., Li, B., 2013. Effect of freeze-thaw cycles on the molecular weight and size distribution of gluten. Food Res. Int. 53, 409e416.
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A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment Qingping Xiong a, b, c, 1, Song Huang a, b, 1, Jianhui Chen d, 1, Baolan Wang e, 1, Lian He f, Lei Zhang g, Shijie Li a, b, Jizhong Wang c, Jianguo Wu a, b, Xiaoping Lai a, b, *, Danyan Zhang a, b, ** a
School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, PR China Guangdong Provincial Key Laboratory of New Drug Development and Research of Chinese Medicine, Guangzhou 510006, Guangdong, PR China Key Laboratory for Medicinal Exploitation of Huai'an Regional Resource, College of Chemical Engineering, Huaiyin Institute of Technology, Huai'an 223003, Jiangsu, PR China d Affiliated Huai'an Hospital of Xuzhou Medical University, Huai'an 223002, Jiangsu, PR China e Huai'an First People's Hospital, Nanjing Medical University, Huai'an 223300, Jiangsu, PR China f Xinhua College, Sun Yat-sen University, Guangzhou 510520, Guangdong, PR China g Jiangsu Tasly Diyi Pharmaceutical Co., Ltd., Huai'an 223002, Jiangsu, PR 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 August 2016 Received in revised form 10 October 2016 Accepted 22 October 2016 Available online xxx
The purpose of this paper was to develop a green method for deproteinization of polysaccharide (PCC) from Cipangopaludina chinensis by freeze-thaw treatment (FTT). Based on single-factor experiments of FTT, firstly, the optimal deproteinization parameters of PCC were obtained by comparing their deproteinization ratio (Dr%), recovery ratio (Rr%) and selectivity coefficient (Kc) as follows: freezing temperature of 40 C, freezing time of 96 h, thawing temperature of 10 C and freeze-thaw cycles of 11 times. Under optimal conditions, The Dr%, Rr% and Kc of PCC were 87.10 ± 1.91%, 84.91 ± 2.45% and 5.77 ± 0.43, respectively. Then, the deproteinization performance of FTT for PCC under optimal conditions was compared with the most typical Sevage method. We found that there were no significant differences between FTT and Sevage method on the Dr%, monosaccharide composition, characteristic group and molecular weight degradation. However, compared with Sevage method, FTT not only had higher Rr% and Kc, but also were non-pollution. The results demonstrated that FTT would be a promising and green method for deproteinization of PCC. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Deproteinization Polysaccharides Cipangopaludina chinensis Freeze-thaw treatment Green method
1. Introduction Cipangopaludina chinensis (C. chinensis), widespread throughout the China and many other Asian countries (Li, 2012), is a common gastropod and invertebrate of viviparidae (Mccann, 2014). The flesh of C. chinensis is an important functional food supplement with remarkable efficacy for treatment of diabetes (Nurhasan et al., 2010), otitis media, urinary infection (Zhang, 2005) and liver
* Corresponding author. School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, PR China. ** Corresponding author. School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, PR China. E-mail addresses: [email protected] (X. Lai), [email protected] (D. Zhang). 1 These authors contributed equally to this paper.
diseases (Liu et al., 2013). Recent studies indicated that these pharmacological activities are closely related with its richness in polysaccharides (Yang et al., 2012). In our previous studies, for the first time, polysaccharide (PCC) was successfully extracted from C. chinensis with 8.7% yield (based on the dried flesh) (Jiang et al., 2013). Its structure characterization has been clearly identified (Shi et al., 2015). And the excellent immunostimulatory and antioxidant activities have also been confirmed (Xiong et al., 2013). Unfortunately, these studies employed the most typical Sevage deproteinization method of polysaccharides to remove protein impurities of PCC. The most typical Sevage method, consume a large amount of chloroform and n-butyl alcohol, resulting in a large number of toxic organic pollutants and difficulty in commercial production of PCC. In addition, their products are also not suitable for many applications in the food and pharmaceutical sectors due to high levels of toxic organic solvent residual. Therefore, it is
http://dx.doi.org/10.1016/j.jclepro.2016.10.125 0959-6526/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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indispensable and promising to develop a green deproteinization method for improving the quality of extracted PCC and protecting eco-environment. Freeze-thaw method, in the field of food science, refers to a way to extend the shelf life of food by freezing, storage and thawing rquez et al., 2015). It has been recognized as an excellent (Ma preservation method for maintaining microbiological stability of food (Zhao et al., 2013). Nevertheless, the freeze-thaw treatment (FTT) of protein-rich liquid food may cause complex changes of the buffer environment (Mun et al., 2008), including formation of icewater interfaces (Donsì et al., 2011), adsorption to container surfaces (Ghosh and Coupland, 2008), cryo-concentration of protein and solutes, pH changes, and phase separation (Zhao et al., 2015). Meanwhile, the intermolecular disulfide bonds among proteins with high hydrophobicity may be formed and exposed in the process of FTT (Wang et al., 2013). These changes could rapidly induce mass insoluble aggregation and precipitation of protein. Although this fact is unfavourable to the protein stability, it is exploited to removal protein impurities from products with unique superiority of green non-pollution. FTT could therefore be implemented as a green deproteinization strategy. However, to the best of our knowledge, there is no literature on this deproteinization method. In the present paper, for the first time, a green method based on FTT was developed for deproteinization of PCC. Various parameters influencing the deproteinization of PCC were optimized by singlefactor experiments of FTT. In order to evaluate the deproteinization effect of FTT for PCC, a series of performance parameters of PCC solutions before and after deproteinization was measured and compared with those of most typical Sevage method. The information in this study is significant for deproteinization of proteinrich polysaccharides. 2. Materials and methods 2.1. Materials and reagents The fresh C. chinensis was obtained from the Huaiyin Vegetable Product Market (Huaiyin, China) and identified by Prof. Danyan Zhang, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, PR China. Monosaccharide standards including rhamnose, arabinose, fucose, xylose, mannose, glucose and galactose were from Sigma Chemical Co. (St. Louis, MO, USA). The phenoles, trifluoroacetic acid (TFA), concentrated sulfuric acid (98%, H2SO4), chloroform and nbutyl alcohol of analytical grade were obtained from Shanghai Chemical Reagents Co., Ltd. (Shanghai, China). Dextrans with different molecular weight (Mw) were purchased from American Polymer Standards Co. (Colorado, USA). The kit of Coomassie brilliant blue (CBB) for protein determination was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Deionized water was prepared by a Milli-Q Water Purification system (Millipore, MA, USA). 2.2. Preparation of PCC solution The PCC solution was prepared according to our previously reported methods with some modifications (Jiang et al., 2013). Briefly, fresh C. chinensis was crushed to remove its shells. The flesh was stripped viscera and cleansed with cold water to thoroughly removal impurities, then smashed using a high speed disintegrator. The homogenate was refluxed with anhydrous ethanol to eliminate lipids and some colored materials, and air-dried at 50 C to a constant weight. The pre-treated powder form C. chinensis (50.0 g) was extracted with distilled water in a ratio of 1:25 (pre-treated powder to water, w/v, g/ml) for 3 h at 95 C. The extracting solution was
centrifuged at 5000 rpm for 20 min. The insoluble residue was repeated treatment two times as mentioned above. All supernatants were collected together, stirred uniformly and stored at 4 C, affording the PCC solution. 2.3. Deproteinization experiments of FTT for PCC solution The PCC solution (100 ml) was placed in a polyethylene bottle and frozen at a proper temperature. After a certain time of freezing, the frozen PCC solution was thawed with a designed test temperature. Subsequently, the above procedure of PCC solution was repeated under the same parameters. The PCC solution after multicycles of FTT was centrifuged at 4000 rpm for 30 min and then filtered through a 0.45 mm membrane filter. The filtrate was carefully collected, as PCC solution after FTT deproteinization. Using this way, the optimal deproteinization parameters of FTT, including freezing temperature, freezing time, thawing temperature and freeze-thaw cycles, were systematically investigated. 2.4. Deproteinization experiments of Sevage method for PCC solution Deproteinization experiments of Sevage method for PCC solution were carried out by the reported parameters (Sevag et al., 1938). 100 ml of PCC solution and 20 ml of Sevage reagent with the ratio of chloroform to n-butyl alcohol of 5:1 were added to a 250 ml round-bottomed flask. The mixed solution was continually stirred for 60 min and centrifuged at 4000 rpm for 30 min. As a result, the solution was divided into three layers, in order, PCC solution after deproteinization, denatured protein and redundant Sevage reagent from top to bottom. The supernatant was repeatedly deproteinized with the same amount of Sevage reagent to the absence of denatured protein layer. The supernatant after the last deproteinization was collected and passed through a 0.45 mm membrane filter. The filtrate was regarded as PCC solution after deproteinization with Sevage method. 2.5. Performance evaluation of deproteinization 2.5.1. Determination of deproteinization ratio The deproteinization ratio was determined as the follows method: 50 ml of PCC solution before or after deproteinization was loaded into a 10 ml Nessler tube. 3 ml of CBB agent (Blakesley and Boezi, 1977), a special chromogenic agent for protein (Sedmak and Grossberg, 1977), was added to this ones. After mixing the solution thoroughly and set aside for 30 min, the Spectrogram of the colored solution at 400e800 nm was determined by a UVevis Spectrophotometer (UV-2401 PC, Shimadzu, Japan). The maximum absorbance value in the spectrum, 595 nm, was applied to calculate deproteinization ratio. The deproteinization ratio was expressed as the following equation (Liu et al., 2010):
Dr ð%Þ ¼
AD0 AD1 100% AD0
(1)
where AD0 and AD1 are the maximum absorbance value of the sample solutions using CBB stainng before and after deproteinization, respectively. Dr % is deproteinization ratio. 2.5.2. Determination of recovery ratio of PCC The following method was used to measure the recovery ratio of PCC. 50 ml of PCC solution before or after deproteinization was added into a 10 ml Nessler tube, and then stained by the phenolsulfuric acid method (Dubois et al., 1956). Above mentioned UVevis spectrophotometer (UV-2401 PC) was employed to record
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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their Spectrogram at 350e800 nm. The maximum absorbance value in the spectrum, 490 nm, was picked to evaluate the recovery ratio of PCC according to the following equation (Liu et al., 2010):
Rr ð%Þ ¼
AR1 100% AR0
(2)
where AR0 and AR1 are the maximum absorbance value of the sample solutions with phenol-sulfuric acid reagent stainng before and after deproteinization, respectively. Rr % is recovery ratio of PCC. 2.5.3. Calculation of selectivity coefficient In order to evaluate selectivity deproteinization of each method, the selectivity coefficient (Kc) was calculated as follows:
Kc ¼
Dr ð%Þ 100 Rr ð%Þ
(3)
where Dr % and Rr % are deproteinization ratio and recovery ratio of PCC, respectively. Kc is selectivity coefficient.
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to determine Mw of the samples. The chromatographic separation was operated by a gel-filtration chromatographic column of TSKGEL G3000SWxl (300 mm 7.5 mm i.d., 5 mm diameter, Tosoh Corp., Japan) (Zhang et al., 2014). The mobile phase was 0.1 M Na2SO4 solution in PBS buffer (0.01 M, pH 6.8) and passed through a 0.22 mm membrane filter (Millipore, USA) prior to use (Liu et al., 2015). The chromatographic condition was set as flow rate of 0.8 ml/min, the injection volume of 10 ml and the column temperature of 25 C. Seven dextrans with different Mw (9.6, 21.1, 36.3, 72.7, 158.1, 344.8 and 714.5 kDa) were employed to establish a calibration curve by plotting the retention time against the logarithm of their respective Mw. The regression line for Mw was obtained as follows:
log Mw ¼ 0:0288X þ 5:3164 R2 ¼ 0:9985; n ¼ 7
(4)
where X is the retention time of dextrans (min). The retention time of the samples was substituted into the equation of the calibration curve to calculate their Mw. 2.6. Statistical analysis
2.5.4. Visual observation and photography A certain amount of these solution before and after deproteinization, such as unstained PCC solution, colored PCC solution using CBB agent in Section 2.5.1 or phenol-sulfuric acid method in Section 2.5.2, were placed in a 10 ml Nessler tube. A Sony DSC-W80 digital camera (Sony Corp., Park Ridge, NJ) was applied to photograph their visual characteristics on a black or white background. 2.5.5. Analysis of UVevis and IR spectra 200 ml of PCC solutions before or after deproteinization was put into a 10 ml Nessler tube and diluted to 5 ml with deionized water. UVevis Spectrogram of the diluted solution was recorded by a UV2401 PC UVevis spectrophotometer in the range of 200e800 nm. PCC solutions before or after deproteinization were concentrated to a proper volume and freeze-dried. 1 mg freeze-dried powder was mixed thoroughly with 100 mg KBr powder in a running infrared drying oven. The mixed powder pressed into a tablet and then placed in a Nicolet 6700 FT-IR Spectrometer (Thermo Co., USA) to record their IR Spectrogram in the frequency range of 4000e400 cm1 as reported method (Geng et al., 2009). 2.5.6. Identification of monosaccharide composition 5.0 mg freeze-dried powder in Section 2.5.5 was hydrolyzed with 4 ml of 2 M trifluoroacetic acid (TFA) at 120 C for 2 h (Souza et al., 2012). The redundant TFA was fully replaced by methanol and evaporated to dryness. The hydrolyzate was dissolved in 0.6 ml pyridine, and then transferred to a 3 ml reaction flask with 10 mg hydroxylamine hydrochloride and 5 mg inositol (internal standard). After reacting at 90 C for 30 min, 1.0 ml acetic anhydride was added into the flask to further react for 30 min. The reaction solution, cooling to room temperature, was the acetylated derivatives of the samples. All monosaccharide standards were also acetylated in the same way. The GC chromatogram of the acetylated samples was analyzed by a 7890N GC (Agilent Technologies, Santa Clara, CA, USA) equipped with flame ionization detector and a HP-5 fused silica capillary column (30 m 0.32 mm i.d. 0.25 mm) as the reported methods (Jiang et al., 2015). The monosaccharide composition of all samples was identified by the retention time of GC chromatogram from monosaccharide standards. 2.5.7. Measurement of Mw A size-exclusion HPLC chromatography (HPGPC) instrument (Agilent 1200, USA) with a refractive index detector (RID) was used
All data were expressed as mean ± standard deviation (SD) and evaluated by the Student's t-test and one-way analysis of variance followed using Duncan's multiple-range tests. The statistical analyses were performed by SPSS for Windows, Version16.0 (SPSS, Chicago, IL). The difference with P < 0.05 or P < 0.01 was considered to be statistically significant. 3. Results and discussion 3.1. Effect of freezing temperature on deproteinization efficiency of FTT The intermolecular disulfide bonds and hydrophobic interactions among proteins, resulting from stress cryoconcentration of protein and solutes in the unfrozen parts (Xu et al., 2015), participated in the formation of insoluble aggregates of proteins (Ghosh and Coupland, 2008). Freezing temperature can manipulate the speed of water crystallization and cryoconcentration, thereby affecting the stability of protein. Therefore, the effects of different freezing temperatures (20, 30, 40, 50, 60, 70, or 80 C) on deproteinization efficiency of FTT for PCC solution were evaluated under the conditions of freezing time of 72 h, thawing temperature of 20 C and freeze-thaw cycles of 9 times. As shown in Fig. 1. With the decrease in freezing temperature, the deproteinization ratio was raised, indicating lower temperature was advantageous to removal of protein impurities from PCC. However, when the freezing temperature was further lowered below 40 C, the changed trend of deproteinization ratio reached a relative steady-state (P > 0.05), whit the recovery ratio of PCC beginning to fall significantly (P < 0.01) as the freezing temperature further decreased. The highest selectivity coefficient of 5.21 appeared at 40 C. Hence, the freezing temperature of 40 C should be picked for further experiment. 3.2. Effect of freezing time on deproteinization efficiency of FTT Freezing time is an important factor that affects the deproteinization efficiency of FTT. It has been reported that increasing frozen storage time decreased the average particle size of protein (Wang et al., 2013), leading to a sharp increase in surface free energy and a rapid destabilization of protein. According to this theory,
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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Fig. 1. Effects of different freezing temperature on visual photograph (A), visual photograph (B) and Vis Spectrogram (C) after CBB staining, visual photograph (D) and Vis Spectrogram (E) after phenol-sulfuric acid staining, and deproteinization efficiency (F). BDP designate before deproteinization. I ~ VII orderly represent freezing temperature of 20, 30, 40, 50, 60, 70 and 80 C, respectively.
a longer freezing time is favored for deproteinization efficiency of FTT. However, the polysaccharides would suffer great losses with excessive extension of freezing time due to mass adsorption and embedding of protein precipitation. Thus, it is very necessary to investigate the effects of different freezing time on deproteinization
efficiency of FTT for PCC solution. In present paper, when other parameters were set as freezing temperature of 40 C, thawing temperature of 20 C and freeze-thaw cycles of 9 times, the deproteinization efficiency were evaluated at freezing time of 12, 24, 48, 72, 96, 120, 144 and 168 h, respectively. The result was
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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shown in Fig. 2. Although the deproteinization ratio increased as freezing time prolonged, the difference of deproteinization ratio was not significant in the range of 96e168 h (P > 0.05). These results suggested that the precipitation of protein has tended to the equilibrium at about 96 h. A slow and mild decrease in recovery
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ratio of PCC was noted with the extension of freezing time. However, after the freezing time was prolonged above 96 h, the downtrend of PCC recovery ratio became significant (P < 0.01). These results were consistent with the above theory. The selectivity coefficient increased first and then decreased at different freezing
Fig. 2. Effects of different freezing time on visual photograph (A), visual photograph (B) and Vis Spectrogram (C) after CBB staining, visual photograph (D) and Vis Spectrogram (E) after phenol-sulfuric acid staining, and deproteinization efficiency (F). BDP designate before deproteinization. I ~ VIII orderly represent freezing time of 12, 24, 48, 72, 96, 120, 144 and 168 h, respectively.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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time, reaching a maximum at 96 h. The results indicated that their deproteinization efficiency has attained an optimal state at 96 h. Thus, freezing time of 96 h was selected for the following experiments.
3.3. Effect of thawing temperature on deproteinization efficiency of FTT To investigate the effect of different thawing temperature on
Fig. 3. Effects of different thawing temperature on visual photograph (A), visual photograph (B) and Vis Spectrogram (C) after CBB staining, visual photograph (D) and Vis Spectrogram (E) after phenol-sulfuric acid staining, and deproteinization efficiency (F). BDP designate before deproteinization. I ~ VIII orderly represent thawing temperature of 4, 10, 20, 30, 40, 50, 60 and 70 C, respectively.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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deproteinization efficiency, the FTT for PCC solution was carried out using thawing temperature of 4, 10, 20, 30, 40, 50, 60 or 70 C, while other parameters were designed as freezing temperature of 40 C, freezing time of 96 h and freeze-thaw cycles of 9 times. As shown in Fig. 3, the deproteinization ratio had a decrease trendency with
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increasing the thawing temperature. When the thawing temperature was raised to more than 20 C, the changes in deproteinization ratio became significant (P < 0.01). PCC recovery ratio, in contrast, increased as thawing temperature raised, but insignificant difference after the thawing temperature reached 10 C. These results
Fig. 4. Effects of different freeze-thaw cycles on visual photograph (A), visual photograph (B) and Vis Spectrogram (C) after CBB staining, visual photograph (D) and Vis Spectrogram (E) after phenol-sulfuric acid staining, and deproteinization efficiency (F). BDP designate before deproteinization. I ~ VIII orderly represent freeze-thaw cycles of 1, 3, 5, 7, 9, 11, 13 and 15 times, respectively.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
Fig. 5. Visual photograph and UVevis Spectrogram (A), visual photograph and Vis Spectrogram after CBB staining (B), visual photograph and Vis Spectrogram after phenol-sulfuric acid staining (C), IR Spectrogram (D), HPGFC Chromatogram (E) and GC of monosaccharides (F) of PCC solutions before and after deproteinization. BDP designate before deproteinization. MS was monosaccharide standard. I and II represent after deproteinization using FTT and Sevage, respectively.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
Q. Xiong et al. / Journal of Cleaner Production xxx (2016) 1e10
can be explained that the high hydrophilic precipitate of the polysaccharides, which formed and maintained stable structures in the freezing process, turned into soluble aggregates and were easy to redissolve in thawing solvent at the lower thawing temperature. On the contrary, the aggregates of proteins become an insoluble hydrophobic precipitate by cooling and freezing processes due to the formation of denatured products and intermolecular disulfide bonds among proteins. Its dissolution requires providing a more vigorous thermal motion, caused by high thawing temperature. The fact indicated discrepancies of the solubility between polysaccharide and protein at lower thawing temperature was benefited for selective precipitation of proteins. However, the selectivity coefficient had a maximum value at the thawing temperature of 10 C. Therefore, the thawing temperature of 10 C was adopted in the following experiments to synthetically evaluate its effect on protein removal and polysaccharide retention. 3.4. Effect of freeze-thaw cycles on deproteinization efficiency of FTT Recent studies have shown that the growth of ice crystals during protein freezing could facilitate its structure opening and unfolding (Zhao et al., 2015). Changes in protein stability occurred during freeze-thaw cycles (Guo et al., 2011), as repeated FTT and reformation of ice crystals (Benjakul and Bauer, 2000) induced change in the secondary, tertiary and quaternary structure of protein (Zhang et al., 2011). When the number of freeze-thaw cycles increased, these changes could enhance the exposure of SH groups content and surface hydrophobicity (Noh et al., 2006), which are closely related with the formation of protein precipitation. In order to optimize freeze-thaw cycles, the FTT for PCC solution were performed using the freeze-thaw cycles of 1, 3, 5, 7, 9, 11, 13 or 15 times, accompanied by freezing temperature of 40 C, freezing time of 96 h and thawing temperature of 10 C. As shown in Fig. 4, an increasing number of freeze-thaw cycles were associated with higher deproteinization ratio and lower PCC recovery ratio. The increase in deproteinization ratio was insignificant after the freezethaw cycle reached 9 times, while the cycle number was 13 times when decrease in PCC recovery ratio became negligible. The selectivity coefficient increased at first with increasing number of freeze-thaw cycle and peaked at 11 times, where it began to decrease afterwards. The results suggested that the optimal freezethaw cycles should choose 11 times. 3.5. Comparison of deproteinization efficiency between FTT and Sevage method Based on the above experiments of influential factors, the optimal deproteinization parameters of FTT for PCC were summarized as follows: freezing temperature of 40 C, freezing time of 96 h, thawing temperature of 10 C and freeze-thaw cycles of 11 times. In order to confirm the dependability of the present method, the performance parameters of deproteinization for PCC solutions before and after deproteinization, including visual photograph,
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UVevis Spectrogram, visual photograph and Vis Spectrogram after CBB staining, visual photograph and Vis Spectrogram after phenolsulfuric acid staining, IR Spectrogram, HPGFC Chromatogram, GC of monosaccharides, Dr%, Rr%, Kc, consumption of organic solvent and Mw degradation, were compared with the most typical Sevage method. Qualitative deproteinization efficiency of different methods was shown in Fig. 5. The results of Fig. 5 A and Fig. 5 B could be obviously observed that both FTT and Sevage method had remarkable deproteinization for the PCC solutions, but there was no significant difference between them. FTT was obviously superior to Sevage method on the polysaccharide recovery (Fig. 5C). As shown in Fig. 5D, characteristic absorptions peaks of polysaccharides around 3280, 2932, 1662, 1545, 1448, 1242 and 1154 cm1 were clear for the samples before and after deproteinization of PCC (Shi et al., 2015). The strong and broad peaks around the 3280 cm1 and 1662 cm1 were OeH stretching vibrations and deformation vibration respectively. The peaks around 2932 cm1 were assigned to the CeH asymmetric stretching vibration. And the strong extensive absorption in the region of 900e1200 cm1 for coupled CeO and CeC stretching and CeOH bending vibrations. Besides, the absorption peaks toward about 1241 cm1 were corresponding to SeO asymmetry stretching vibrations, revealing PCC being sulfate radical. The characterizations of IR Spectroscopy demonstrated that the characteristic groups and molecular backbone of PCC were adequately retained after deproteinization using FTT and Sevage method. As shown in Fig. 5E and Table 1, Dr%, Rr%, Kc and change value of Mw in the present method were 87.10 ± 1.91%, 84.91 ± 2.45%, 5.77 ± 0.43 and 3.24 ± 0.47 kDa, respectively. Compared with the Sevage method, FTT has higher Rr% and Kc with similar Dr% and change value of Mw. Fig. 5F shown PCC and deproteinized PCCs by FTT and Sevage method were similar on the monosaccharide composition with a molar ratio of 5.289:0.279:0.036, 5.312:0.263:0.038, 5.255:0.272:0.035 for rhamnose, fucose and glucose, respectively. The result indicated molecular composition of PCC was not converted during the deproteinization process. Even more remarkable was the fact that the whole process of deproteinization in FTT did not use any toxic organic reagents. The above results demonstrated that FTT could replace the most typical Sevage method and represent a green method for deproteinization of PCC.
4. Conclusions Based on FTT, a novel green deproteinization method of PCC was successfully developed in this study. The deproteinization parameters of FTT for PCC were systematically optimized by single-factor experiments. The comparison between FTT and the most typical Sevage method was performed to validate the reliability and feasibility of FTT for PCC deproteinization. As a result, its optimal deproteinization parameters were gained as follows: freezing temperature of 40 C, freezing time of 96 h, thawing temperature of 10 C and freeze-thaw cycles of 11 times. The Dr%, Rr% and Kc of FTT for PCC deproteinization under optimal conditions were
Table 1 The comparison of different deproteinization methods. Methods
Sevage FTT
D r%
91.01 ± 2.61 87.10 ± 1.91
Rr%
68.99 ± 3.11 84.91 ± 2.45
Kc
b
a
2.94 ± 0.31 5.77 ± 0.43
Specific consumption of organic solvent (ml/1 ml)
Mw (kDa)
Chloroform
Before deproteinization
After deproteinization
Change value
90.03 ± 2.14
87.79 ± 1.21 86.76 ± 1.89
2.26 ± 0.31 3.24 ± 0.47
3.33 ± 0.14 0.00 ± 0.00
b
N-butyl alcohol 0.67 ± 0.07 0.00 ± 0.00
b
Data were presented as mean ± SD (n ¼ 3). Superscript a-b designate a significant differences.
a
P < 0.05 compared with FTT.
b
P < 0.01 compared with FTT.
Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125
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Q. Xiong et al. / Journal of Cleaner Production xxx (2016) 1e10
87.10 ± 1.91%, 84.91 ± 2.45% and 5.77 ± 0.43, respectively. The Dr%, monosaccharide composition, characteristic group and molecular weight degradation of FTT were similar to the most typical Sevage method. FTT had higher Rr% and Kc with green non-pollution. The results demonstrated that FTT would be an environmentally friendly method for deproteinization of PCC. As a promising novel deproteinization technology, it might be able to replace the most typical Sevage method to remove protein impurities of the polysaccharides for large-scale production, and be widely applied in food and medicine. However, some limitations should be mentioned in the present paper. In order to promote its application, the changes on the activity, structural characteristics and property of protein, caused by FTT, should been identified to further evaluate its detailed deproteinization mechanisms in later work. Acknowledgements This work was partly supported by the National Natural Science Foundation of China (No. 81503387), Natural Science Fund for Colleges and Universities in Jiangsu Province of China (No. 15KJB360002, No. 14KJB210002), Hong Kong, Macao, and Taiwan Science and Technology Cooperation Projects of Ministry of Science and Technology of China (No. 2014DFH30010), Qing Lan Project of Jiangsu Province of China, Science and Technology Program of Guangdong Province of China (No. 2013B090800052, No. 2013A022100002), Special Funds of Applied Science and Technology Research and Development of Guangdong Province of China (No. 2015B020234008) and Special Funds from Guangdong Province of China for the Construction of High Level University (Grant No. 2050205). References Benjakul, S., Bauer, F., 2000. Physicochemical and enzymatic changes of cod muscle proteins subjected to different freeze-thaw cycles. J. Sci. Food Agric. 80, 1143e1150. Blakesley, R.W., Boezi, J.A., 1977. A new staining technique for proteins in polyacrylamide gels using coomassie brilliant blue G250. Anal. Biochem. 82, 580e582. Donsì, F., Wang, Y., Huang, Q., 2011. Freeze-thaw stability of lecithin and modified starch-based nanoemulsions. Food Hydrocoll. 25, 1327e1336. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350e356. Geng, W., Nakajima, T., Takanashi, H., Ohki, A., 2009. Analysis of carboxyl group in coal and coal aromaticity by Fourier transform infrared (FT-IR) spectrometry. Fuel 88, 139e144. Ghosh, S., Coupland, J.N., 2008. Factors affecting the freeze-thaw stability of emulsions. Food Hydrocoll. 22, 105e111. Guo, Y.Y., Kong, B.H., Xia, X.F., Yang, Z., 2011. Effect of number of freeze-thaw cycles on physico-chemical characteristics of carp muscle. Food Sci. 32, 125e130. Jiang, C., Jiao, Y., Chen, X., Xia, L., Yan, W., Bo, Y., Xiong, Q., 2013. Preliminary characterization and potential hepatoprotective effect of polysaccharides from Cipangopaludina chinensis. Food Chem. Toxicol. 59, 18e25.
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Please cite this article in press as: Xiong, Q., et al., A novel green method for deproteinization of polysaccharide from Cipangopaludina chinensis by freeze-thaw treatment, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.10.125