Purification of DP 6 to 8 chitooligosaccharides by nanofiltration from the prepared chitooligosaccharides syrup
© Dong et al.; licensee Springer. 2014
Received: 13 July 2014
Accepted: 14 October 2014
Published: 19 November 2014
Chitooligosaccharides (COS) with degrees of polymerization (DP) 6 to 8 are degraded from chitosan, which possess excellent bioactivities. However, technologies that could purify them from hydrolysis mixtures in the narrow DP range (984 to 1,306 Da) are absent. The objective of this research is to purify DP 6 to 8 COS by nanofiltration on the basis of appropriate adjustments of the feed condition.
Syrup containing DP 6 to 8 COS at different concentrations (19.0 to 46.7 g/L) was prepared. A commercial membrane (QY-5-NF-1812) negatively charged was applied. Experiments were carried out in full recycle mode, so that the observed COS retentions were investigated at various transmembrane pressures (6.0 to 20.0 bar), temperatures (10°C to 50°C), and pHs (5.0 to 9.0). Then, the feasibility of separation of DP 6 to 8 COS was further studied by concentration ratio under optimum conditions.
The results indicate that the purification of DP 6 to 8 COS by nanofiltration NF is feasible. It was found that the permeate flux was 95.0 L/(m2 h) at 10.0 bar, while it reached to 140.0 L/(m2 h) at 20.0 bar, and it increased with feed temperature, but the membrane pores were also swelled by heating and led to an irreversible wastage of target oligomers. Additionally, the retention behaviors of chitooligosaccharides are significantly influenced by pH.
KeywordsNanofiltration Chitooligosaccharides Degree of polymerization Separation MALDI-TOF-MS
According to many previous reports, COS possess a series of attractive bioactive properties, including antibacterial , anticoagulant , antimicrobial , antioxidant , anticancer , hypolipidemic , and immune-stimulating  effects. Based on these excellent advantages mentioned before, COS are responsible for practical applications in beverage processing , functional ingredients , and biomedicines , which are different from chitosan and chitin except for their contributions on food packaging . Nowadays, hybrid enzymatic hydrolysis has become the ideal technology for COS preparation due to its high efficiency and little structural modification . Unfortunately, the products after enzymatic degradation were just intermediate ones . Suitable methods should be used to separate target COS from mixtures coexisting in the solution, such as the high molecular weight of chitosan, proteins (enzymes), and inorganic salts.
Conventionally, COS is purified by various chromatographies. Fan et al. succeeded in obtaining COS by macroporous resins from fermentation broth, and the productivity of target products could go up to 90% (w/w) under optimum conditions . Meanwhile, Cabrera and Custem reported that the concentrations of COS could be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF-MS) . Although the chromatography technology is feasible to remove irrelevant components and refine COS in terms of DP, it is only accepted in laboratory analysis but far from scale-ups. In practice, several negative problems inevitably appear during continuous using of chromatography, including the demands on cleaning and regeneration of column packing, adsorption capacities of COS, and relatively high production cost ,.
Recently, there is a growing attention on nanofiltration (NF) applications, especially for COS preparation. Kim et al. reported that several instruments were used to prepare COS, the details of which could be summarized in immobilized enzymatic columns and ultrafiltration (UF) membrane reactors . Han et al. demonstrated the desalination feasibility of NF-40 membrane for chitobiose solution. Under acidic conditions, the interception of solutes ranks as chitobiose > glucosamine > Na+ > H+. Furthermore, the steric and electrostatic effects are inferred according to the sequence. Han et al. also studied the influence of three membranes (DL, DK, NTR-7540) on COS separation and drew identical conclusions . In a word, NF has been proved to be an effective technology for the separation and purification of COS.
Chitosan was originally hydrolyzed by enzymes, which resulted in the coexistence of various DP COS, glucosamines, and salts. It is evident that there is a great difference in molecular weight among the solutes. Also, the wider ranges of hydrolysis products make it difficult to improve the purity and yield of COS. For instance, DP 6 to 8 COS (984 to 1,306 Da) plays an important role in cancer curing . Therefore, this study is to investigate the separation performance of DP 6 to 8 COS at different solution properties and present a promising purification technology by NF.
The raw syrup containing DP 6 to 8 COS was made from chitosan by enzymatic hydrolysis. Chitosan was supplied by Yunzhou Biochemistry Co., Ltd. (Shandong, China). The enzyme mixture (chitosanase from Streptomyces griseus - EC 126.96.36.199; cellulase from Trichoderma - EC 188.8.131.52) was obtained from Golden-Shell Biochemical Co. (Zhejiang, China). DP 2 to 8 COS and glucosamine standards were purchased from Huicheng Biochemical Co., Ltd. (Shanghai, China). All chemicals used in the NF operation and high-performance liquid chromatography (HPLC) analysis were analytical grade or chromatographic grade, respectively. Deionized water (conductivity <3.0 μs/cm) for membrane cleaning was produced by ion exchange.
2.2 Preparation of chitooligosaccharide syrup
Chitosan (91.5% degree of deacetylation) was dissolved in acetic acid (1%, w/v) with stirring, and pH was adjusted to 5.3, and then kept at 45°C. The chitosan concentration was 5% (w/v). Combined enzymes (75 U/g) based on chitosan were added into the chitosan solution and hydrolyzed for 6 h, and then, the hydrolysis was terminated by immersion in a boiling water bath. Finally, a UF membrane module (QY-3-UF-1812, AMFOR Inc., Newport Beach, US) was applied to remove enzymes at 50°C. After cooling to ambient temperature, the syrup was diluted to various concentrations for NF separation.
Information of NF membrane module
Filtration area (Am)
0°C to 60°C
4 to 12
2.4 Membrane permeate flux
where Jv is the average permeate flux of the membrane [L/(m2 h)]; Vp is the volume of permeate accumulated in testing time (L); Am is the effective areas of membrane (m2); t is the testing time (h).
2.5 Observed retention ratio
where Robs is the observed retention ratio of solute (%); Cp is the concentration of solute in the permeate (mol/L); Cb is the concentration of solute in the feed (mol/L).
2.6 Full recycle mode of NF
Initially, 5.0 L raw syrup was added, and the recycling flow rate was adjusted to 360 L/h, which represented the optimum rotation condition for the working pump. The effects of transmembrane pressure (TMP), operation temperature, and pH on NF performance were successively carried out. All the permeate were flowed back to the feed tank, while the Jv and retention behaviors of DP 6 to 8 COS at different concentrations were measured after the renewed conditions were stable for 15 min. It is worth being noted that two of the parameters mentioned must be constant when the third is variable. During the process, the temperature was controlled at 50°C ± 2°C by a heat exchanger surrounding the feed tank, except for the experiments to investigate the effects of temperature on the NF retention on DP 6 to 8 COS.
2.7 Purification of DP 6 to 8 chitooligosaccharides
Purification experiments for DP 6 to 8 COS were executed in batch mode. Under the optimized conditions obtained from the preliminary experiments, the concentrate stream was circulated back to the feed tank, whereas the permeate was collected individually. Considering the practical capacity of the feed tank, 7.0 L of diluted syrup (Cb = 19.0 g/L) was added firstly. Every 1.0 L of extra diluted syrup should be supplied as soon as the permeate was equally removed. Certainly, the systematic temperature during NF was maintained by cooling water. After adding all the syrup (16.0 L), the process was terminated until the volume of permeate reached 14.0 L (2.0 L syrup left in the tank). The effect of the concentration ratio on the purity of DP 6 to 8 COS was confirmed by flux and rejections.
2.8 HPLC analysis
The concentrations of glucosamine and DP 2 to 8 COS were analyzed by an HPLC system (Shimadzu 10A, Shimadzu, Kyoto, Japan) equipped with a high-performance sugar column (Shodex Asahipak NH2P-50 4E, Shodex, Kyoto, Japan) and an RI detector. The mobile phase consists of methyl cyanide and pure water with the ratio of 70:30 (v/v). The column temperature was maintained at 30°C, and the flow velocity was kept at 1.0 mL/min. All the solutes were measured in the form of single-arranged peaks. In general, the glucosamine was firstly eluted, and then, the dimer and trimer were sequentially characterized due to the adsorption strength difference to the stationary phase. The distributions of COS were quantified by integrating peak areas.
2.9 MALDI-TOF-MS analysis
MALDI-TOF-MS analysis of COS was carried out using Shimadzu AXIMA Performance matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Shimadzu, Kyoto, Japan). All spectra were measured in the reflector mode by external calibration. The laser was scanned at a scale from 500 to 1,500 Da. An aqueous solution of 2,5-dihydroxybenzoic acid (DHB, 100 mg/mL) was used as the matrix.
2.10 Statistical methods
All parameters and experimental results were obtained with means ± SD of parallel tests. The data was analyzed by Statistix 9.0 software (Analytical Software, Tallahassee, FL, USA).
3 Results and discussion
3.1 Characterization of chitooligosaccharide syrup
As the raw material for NF process, the COS syrup was defined as the permeate of UF that was pretreated after enzymatic hydrolysis. Summarized by the previous tests, 10.0 L of chitosan hydrolysates were prepared and purified by UF, while a 2.0-L concentrate and 8.0-L permeate were separately bulked at each concentration level due to the cross-flow filtration mode.
T he component analysis of raw materials for the NF process
DP 6 to8 chitooligosaccharidesa(g/L)
1.4 ± 0.3
0.4 ± 0.1
9.6 ± 1.2
2.7 ± 0.2
0.9 ± 0.1
19.0 ± 1.5
3.3 ± 0.4
1.4 ± 0.2
28.3 ± 1.7
2.9 ± 0.3
1.8 ± 0.2
37.6 ± 2.4
1.4 ± 0.3
2.3 ± 0.4
46.7 ± 2.1
In addition, with the increment of crude concentration (Cchitosan), the contents of glucosamine (Cs) and DP 6 to 8 COS (CDP 6 to 8) were also dramatically increased. For example, Cg and CDP 6 to 8 at Cchitosan = 2.0% were 0.9 and 19.0 g/L, respectively, while those at Cchitosan = 5.0% changed to 2.3 and 46.7 g/L, respectively. The tendency indicates that the preparation and separation process of DP 6 to 8 COS was rarely affected by the syrup concentration. According to the data listed in Table 2, the conclusion could be drawn that the purification of hydrolysates by the UF membrane was highly performed. What is more important, the excellent elimination of unexpected impurities may greatly benefit for the next NF treatments.
3.2 Effects of transmembrane pressure on permeate flux
Conversely, Jv decreased with the increment of concentration of DP 6 to 8 COS. It could be well explained by the interaction of TMP and the accumulation effects. On the one hand, the solutes would be greatly accumulated by the driving pressure, which is beneficial for the formation of cake layer around the boundary of the membrane. As a result, the membrane pore would be partially blocked and result in a decrement of Jv. On the other hand, the concentration polarization was also exacerbated with the increase of the sugar concentration. When the osmotic pressure was 16.0 bar, Jv declined from 108.0 to 67 L/(m2 h), although the concentration of filtrated syrup ascended from 19.0 to 46.7 g/L.
3.3 Effects of TMP on Robsof DP 6 to 8 COS
Additionally, the Robs of DP 6 to 8 COS increased with TMP, which indicate that the concentration polarization was a subordinate factor for the separation conditions in this case. As an alkaline molecule, the rejection properties of COS are significantly affected by the Donnan effects . When the concentration was kept at 28.3 g/L, the Robs of solute decreased with the decrease of the pressure (6.0 to 16.0 bar). According to the Donnan theory, dielectric exclusion impels the increasing sugar accumulation, which is responsible for the considerable Robs of DP 6 to 8 COS. Therefore, it could be seen that the dropping of observed retention at a high-pressure range (18.0 to 20.0 bar) was similar. The corresponding results were of the same order with reported phenomena by Zhang . Impressive retention proportions of DP 6 to 8 COS at all selected concentrations and applied pressures were performed.
3.4 Effects of operation temperature on permeate flux
To be detailed, the membrane pores were stretched by structural modification when thermal expansion was interposed by external temperature. At the same moment, the increment of temperature meant for a decline of solution viscosity but a rise both in Reynolds number (Re) and mass transfer coefficient (k). Followed by the rules analyzed above, all solutes in the process preferred to move to the bulk part of the syrups, which reduced the rate of concentration polarization and thus improved the membrane flux.
However, excessive temperature was negative to preserve the stability of DP 6 to 8 COS. Because of the special structures that the amino group locates on C-2 sites in every monomer unit linked by β (1 → 4) glycosidic bonds, the velocity of the Maillard reaction was motivated by high temperature. Certainly, further studies are needed to understand the mechanism of the Maillard reaction and its derivative products in permeate during the filtration process.
3.5 Effects of operation temperature on Robsof DP 6 to 8 COS
The retention manners of DP 6 to 8 chitooligosaccharides in different concentrations of syrup are shown in Figure 5, as the temperature varied from 10°C to 50°C. As expected, the retentions of DP 6 to 8 chitooligosaccharides decreased with the increment of temperature. All curves coincide with the conclusion drawn from Figure 5. That is, the thermo swelling of membrane structures promoted more DP 6 to 8 chitooligosaccharides permeated through the NF membrane from the concentrate side. Specifically, the Robs of DP 6 to 8 chitooligosaccharides was 0.912 in 20°C, whereas it diminished to 0.895 at 50°C with the concentration of 37.6 g/L. The transformation was apparently introduced that the Robs of solutes decreased with a boosting temperature that forced the fluid properties of chitooligosaccharide syrup to be a Newtonian liquid and hence resulted in a decrease in viscosity.
3.6 Effects of pH on Robsof COS
3.7 Purification of DP 6 to 8 COS
Moreover, the Robs of DP 6 to 8 COS went up when the circulating flux decreased (Figure 9). The result was principally accredited to the synergy with the DSPM and steric hindrance pore (SHP) effects. On the one hand, the anions preferred to be repelled while the cations were attracted to the NF membrane. The unique property transported the amino groups, which are commonly sensed in COS, to the permeate. Ultimately, the molecules containing NH4+ were separated from the syrup because of the electric attraction. On the other hand, the Robs of DP 6 to 8 COS was up to 1.0 after the concentration ratio increased to 8.0. The MWCO of the applied membrane is 500 Da, while the molecular weights of hexamer, heptamer, and octamer are 984, 1,145 and 1,306, respectively. There is a conspicuous difference in the sizes between membrane pores and DP 6 to 8 COS. Therefore, the observed retention behaviors illustrated that DP 6 to 8 COS were rejected sterically and a promising purity (82.2%) was eventually achieved via the NF purification designed.
3.8 MALDI-TOF-MS analysis of chitooligosaccharides
In this study, the separation behavior of COS syrups, which were enriched by different concentrations of DP 6 to 8 COS, was investigated via a bench-scale NF process.
One negatively charged membrane with MWCO of 500 Da was selected to purify DP 6 to 8 COS. During the full recycle mode, the experimental results indicated that the retentions of these components increased with pressure before 16.0 bar. Also, the operation temperature was optimized. Although the circulating flux could be improved by elevating temperature, a greater wastage of DP 6 to 8 COS was irreversibly formed during the process as well. In addition, the effects of pH on Robs of COS were compared. The HPLC profiles illustrated that the Robs of COS within DP ≤4 in alkali conditions was significantly higher than that in acidic environment. This phenomenon could be explained by structural curling and sterical overlap due to hydrogen bonds. However, the mechanisms should be discussed in further researches.
Under the optimum conditions (TMP = 16.0 bar, T = 40°C, and pH = 5.0), the purification of DP 6 to 8 COS was carried out. It was found that the membrane could support a reluctant flux after the concentration ratio was over 6.0 in the syrup with the concentration of 19.0 g/L DP 6 to 8 COS. MALDI-TOF mass spectrum confirmed that DP 6 to 8 COS were dominant in the final products, and the purity was up to 82.2% (w/w) according to HPLC profiles. As a conclusion, the NF system equipped with a selected membrane module is a promising approach in the purification of DP 6 to 8 COS from specific syrups.
This work is financially supported by the National Natural Science Foundation of China (No. 31371725 and No. 31101381). Also, the authors are grateful to the Fundamental Research Funds for the Central Universities.
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