Chitooligosaccharide chromatography media based on polyacrylic acid gels, and preparation and use thereof

Abstract

The application discloses a chitosan oligosaccharide chromatographic separation medium based on polyacrylic acid gel and a preparation and application thereof, and relates to the technical field of polysaccharide separation and purification. The preparation method comprises the following steps: neutralizing acrylic acid to 50-90%, adding a crosslinking agent and an initiator to perform water solution polymerization, and obtaining a polyacrylic acid gel resin through post-treatment of the product, and then performing hydrochloric acid acidification, washing, drying and screening to obtain the chromatographic medium. The application also provides an application method of the medium: the medium is swelled and columned with hydrochloric acid of a certain concentration, chitosan oligosaccharide solution is loaded after balance, and then the separation is continued by using a hydrochloric acid solution for segmented elution, and different chitosan oligosaccharides are separated by being collected in segments. The medium preparation method is simple, the cost is low, the separation condition is mild, the activity of chitosan oligosaccharide can be effectively protected, the separation efficiency is high, and the medium has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide separation and purification technology, specifically to a separation medium for the efficient separation and purification of chitosan oligosaccharide (COS), particularly a hydrophilic adsorption separation medium with polyacrylic acid as the main functional group, its preparation method, and its application in COS purification. Background Technology

[0002] COS is an oligomer (usually referring to a degree of polymerization of 2 to 20) obtained by the degradation of chitosan. It has the advantages of good water solubility and high biological activity (such as antibacterial, anti-inflammatory, immunomodulatory, and plant growth promotion), and is widely used in medicine, food, cosmetics, agriculture and other fields.

[0003] However, the degradation products of chitosan are usually mixtures with varying degrees of polymerization and complex structures, containing monosaccharides, disaccharides, and even oligosaccharides and polysaccharides with higher degrees of polymerization. The bioactivity and function of COS with different degrees of polymerization vary significantly. Therefore, efficiently and cost-effectively separating and purifying COS with specific degrees of polymerization from mixtures is a key technological bottleneck for its high-value applications.

[0004] Currently, the main methods for separating and purifying COS include membrane separation, organic solvent precipitation, and chromatography. While membrane separation methods (such as ultrafiltration and nanofiltration) are simple to operate, their separation precision is limited, their effectiveness in separating components with similar molecular weights is limited, they are prone to clogging, their flux decreases rapidly, and it is difficult to precisely control the molecular weight distribution. Organic solvent precipitation is costly, and solvent residue issues affect product safety. Chromatography is the most effective method for separating and purifying oligosaccharides; however, commonly used gel filtration chromatographic media (such as the Sephadex series) are expensive, have poor mechanical strength, slow separation speed, and small throughput, making them unsuitable for large-scale preparation, and their separation effect on components with small molecular weight differences is poor. Ion exchange chromatography is also a commonly used chromatographic separation method, but traditional ion exchange media (such as styrene-divinylbenzene-based resins) are highly hydrophobic, which may lead to non-specific adsorption of hydrophilic molecules like COS, resulting in low recovery rates, unsatisfactory resolution, and the potential introduction of impurities during high-salt elution. Therefore, developing a novel chromatographic medium that is simple to prepare, low in cost, has high mechanical strength, and can efficiently separate COS under mild acidic conditions has significant industrial application value.

[0005] Polyacrylic acid is a strongly hydrophilic polyelectrolyte rich in carboxyl groups (-COOH). Its carboxyl groups can ionize (-COO₂) at appropriate pH levels. -It interacts with positively charged molecules (such as protonated amino groups of COS under acidic conditions) through ion exchange and hydrogen bonding. Its hydrophilic framework helps reduce non-specific adsorption of target analytes. Based on this, it can be prepared into a cross-linked polyacrylic acid gel, which is expected to provide a chromatographic separation medium with both hydrophilicity and ion exchange capabilities.

[0006] Currently, there is a lack of a dedicated separation medium that is optimized for the physicochemical properties of COS (hydrophilicity, positive charge, molecular size), and combines high adsorption capacity, high selectivity, good regenerability, easy operation, and controllable cost. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a COS chromatographic separation medium based on polyacrylic acid gel and its preparation method, as well as the application method of this medium in the separation and purification of COS mixtures, particularly suitable for the efficient separation and purification of high-purity COS components of specific molecular weights from enzymatic or acid hydrolysis products. The preparation method is simple, uses readily available raw materials, and is cost-effective; the prepared separation medium has good chemical stability and mechanical strength, and is particularly suitable for the efficient separation of COS under mild acidic conditions, which effectively protect the biological activity of COS.

[0008] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a method for preparing a chitosan oligosaccharide chromatographic separation medium based on polyacrylic acid gel, specifically comprising the following steps: (1) Place the acrylic monomer in an ice-water bath and add sodium hydroxide solution dropwise under stirring to make the degree of neutralization of acrylic acid reach 50% to 90% (that is, the amount of sodium hydroxide added is 50% to 90% of the amount of acrylic acid). (2) Add crosslinking agent N,N'-methylenebisacrylamide (MBA) and initiator potassium persulfate to the system obtained in step (1), mix evenly, then pass nitrogen gas to remove oxygen, and carry out polymerization reaction under constant temperature conditions to obtain polyacrylic acid gel; (3) The obtained polyacrylic acid gel was washed, dried and pulverized to obtain polyacrylic acid gel resin; (4) The polyacrylic acid gel resin is acidified with hydrochloric acid solution, then washed, dried, ground and sieved to obtain the polyacrylic acid gel chromatography separation medium.

[0009] Further, in step (2), the amount of crosslinking agent N,N'-methylenebisacrylamide added is 0.5% to 4.0% of the mass of acrylic acid monomer.

[0010] Further, in step (2), the amount of potassium persulfate initiator added is 0.5% to 2% of the mass of acrylic acid monomer.

[0011] Furthermore, in step (2), the polymerization reaction is carried out at a temperature of 30–80°C for 4–16 hours.

[0012] Furthermore, in step (4), the concentration of the hydrochloric acid solution is 0.1–2 mol / L.

[0013] Furthermore, in step (4), the acidification treatment time is 8 to 12 hours and the oscillation speed is 100 to 400 r / min.

[0014] Furthermore, in step (4), the mesh size of the sieve is 20 to 200 mesh.

[0015] Secondly, the present invention provides a polyacrylic acid gel chromatography medium obtained by the above preparation method, wherein the medium has polyacrylic acid as the backbone and carboxyl groups as the main functional groups, and can adsorb and separate chitosan oligosaccharides under acidic conditions.

[0016] Thirdly, the present invention provides the application of the polyacrylic acid gel chromatography medium in the separation and purification of chitosan oligosaccharides.

[0017] Furthermore, the application includes the following methods: Swelling: Add the polyacrylic acid gel chromatography medium to a 1-100 mmol / L hydrochloric acid solution and soak it to allow it to fully absorb water and swell. Column packing: Pour the swollen medium homogenate into the chromatography column and allow the medium to settle naturally (wet packing). Equilibration: Wash the chromatography column with 1-100 mmol / L hydrochloric acid solution until the column bed is compacted and stable; Sample loading: Add the chitosan oligosaccharide sample solution above the liquid surface of the chromatography column, and wait until the sample liquid surface is completely in the column bed; Elution and collection: Elute in fractions with 1–100 mmol / L hydrochloric acid solution, collect the eluent in fractions to obtain chitosan oligosaccharide with the target degree of polymerization; Furthermore, the chromatography column has an inner diameter of 1 cm and a length of 70 cm.

[0018] Furthermore, the concentration of the chitosan oligosaccharide sample solution is 10–200 mg / mL, and the loading volume is 1–10 mL.

[0019] Furthermore, the temperature of the eluent is 0–100°C, and the elution flow rate is 0.5–2.0 mL / min.

[0020] Furthermore, the obtained target degree of polymerization chitosan oligosaccharide includes one or more of the following: chitosan oligosaccharide monosaccharide GlcN, chitosan oligosaccharide disaccharide (GlcN)2, chitosan oligosaccharide trisaccharide (GlcN)3, chitosan oligosaccharide tetrasaccharide (GlcN)4, or chitosan oligosaccharide pentasaccharide (GlcN)5.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) Raw materials are readily available and the cost is low. The raw materials used in this invention—acrylic acid, crosslinking agent N,N'-methylenebisacrylamide, and initiator potassium persulfate—are all bulk chemical products, inexpensive, and widely available. The preparation process is a conventional aqueous solution polymerization, requiring no special equipment, and is simple and easy to scale up for production.

[0022] (2) Excellent dielectric properties By controlling the amount of crosslinking agent (0.5%–4.0% of the mass of acrylic monomer), the prepared polyacrylic acid gel resin exhibits suitable mechanical strength and good separation flow rate, strong acid resistance, and reusability. Scanning electron microscopy (SEM) image (…) Figure 1 The results show that the medium has a porous network structure, which is beneficial for the diffusion and mass transfer of chitosan oligosaccharide molecules.

[0023] (3) High separation efficiency This invention utilizes a cationic chromatographic medium with carboxyl groups as the main functional group, which can selectively interact with protonated chitosan oligosaccharides under acidic conditions through ion exchange and hydrogen bonding. Compared with conventional cationic chromatographic separation media, it exhibits higher mass transfer efficiency, less tailing, and higher resolution.

[0024] (4) High separation purity Using the method of this invention, chitosan oligosaccharide components with different degrees of polymerization can be efficiently separated from a mixture of chitosan oligosaccharides. The separation was performed by TLC (…). Figure 2 ) and HPLC Figure 3 The purity of each component was determined by testing and found to be: GlcN > 95%, (GlcN)2 > 85%, (GlcN)3 > 80%, (GlcN)4 > 80%, (GlcN)5 > 65% (see Table 1).

[0025] (5) Mild separation conditions The present invention performs separation under mild acidic conditions (1-100 mmol / L hydrochloric acid solution), with an elution temperature of 0-100℃ and an elution flow rate of 0.5-2.0 mL / min. These mild conditions effectively protect the bioactivity of chitosan oligosaccharides.

[0026] (6) It is easy to operate and has good reproducibility. The swelling, column packing, equilibration, sample loading, elution, and collection steps of this invention are simple to operate and require no complex equipment. The medium exhibits stable performance after acidification treatment and good batch-to-batch reproducibility.

[0027] (7) Good prospects for industrialization The method for preparing the medium in this invention is simple and inexpensive, with mild separation conditions, high separation efficiency, and the medium can be reused, showing good prospects for industrial application.

[0028] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0029] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0030] Figure 1 Scanning electron microscope (SEM) images of the polyacrylic acid gel chromatography medium prepared in Example 2 of this invention; wherein A is a SEM image magnified 5,000 times (scale bar 2 μm) and B is a SEM image magnified 50,000 times (scale bar 200 nm).

[0031] Figure 2 The thin-layer chromatography (TLC) chromatogram of the single degree of polymerization COS obtained in Example 3 of this invention; lane A represents glucosamine standard; lane B represents chitobiose standard; lane C represents chitosan 3-7 standard; lanes 1-48 represent the numbers of the eluent collected in chronological order.

[0032] Figure 3 Example 3 of the present invention shows the high performance liquid chromatography (HPLC) chromatograms of single degree of polymerization COS obtained by separation; wherein A is the HPLC chromatogram of GlcN, B is the HPLC chromatogram of (GlcN)2, C is the HPLC chromatogram of (GlcN)3, D is the HPLC chromatogram of (GlcN)4, and E is the HPLC chromatogram of (GlcN)5.

[0033] Figure 4 The following are thin-layer chromatography (TLC) chromatograms of single degree of polymerization (COS) obtained by separating the crosslinking agent at concentrations of 1% and 1.5% in Example 4 of this invention; wherein A represents the separation result with 1% crosslinking agent and B represents the separation result with 1.5% crosslinking agent; in the figure, lane A represents glucosamine standard, lane B represents chitobiose standard, lane C represents chitosan-7 sugar standard, and the remaining lanes represent the numbers of the eluents collected in chronological order.

[0034] Figure 5 The following is a thin-layer chromatography (TLC) chromatogram of single degree of polymerization (COS) obtained by separating the crosslinking agent at concentrations of 2.5% and 4% in Example 4 of this invention; wherein A represents the separation result with 2.5% crosslinking agent and B represents the separation result with 4% crosslinking agent; in the figure, lane A represents glucosamine standard, lane B represents chitobiose standard, lane C represents chitosan-7 sugar standard, and the remaining lanes represent the numbers of the eluents collected in chronological order. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0037] Example 1 Synthesis of polyacrylic acid gel Accurately measure 100 g of acrylic acid (purity ≥98%) and place it in a 500 mL beaker. Place the beaker in an ice-water bath. While magnetically stirring, slowly add a 30% (w / w) sodium hydroxide solution. Calculate and control the amount of sodium hydroxide added to achieve a neutralization degree of exactly 70% for the acrylic acid (i.e., the amount of NaOH added is 70% of the amount of acrylic acid). Transfer the 70% neutralized acrylic acid solution to a 1000 mL three-necked flask equipped with a stirrer, thermometer, and nitrogen inlet. Add 2.5 g of N,N'-methylenebisacrylamide (MBA, 2.5% of the mass of acrylic acid) and 0.5 g of potassium persulfate (KPS, 0.5% of the mass of acrylic acid) to the flask. Stir at room temperature for 30 minutes to ensure homogeneous mixing. Then continuously purge with high-purity nitrogen for 30 minutes to remove oxygen from the flask and proceed with the subsequent reaction under nitrogen protection. The three-necked flask was placed in a constant temperature water bath at 50°C and reacted for 8 hours. Stirring was used during the initial stage of the reaction, and stirring was stopped once the reaction solution began to solidify. After the reaction was complete, a blocky gel polymer was obtained. This was removed, cut into small pieces, and soaked, washed, and filtered in a large amount of distilled water. This process was repeated 5-6 times until the washing liquid was clear and transparent, completely removing unreacted monomers, initiators, oligomers, and other sol-like components. The washed gel was then placed in a 60°C oven and dried to constant weight. Finally, the dried gel blocks were pulverized into powder using a pulverizer to obtain polyacrylic acid gel resin raw powder.

[0038] Example 2: Acidification treatment of polyacrylic acid gel Take 50 g of the polyacrylic acid gel resin powder prepared in Example 1 and place it in a 2 L beaker. Add 1000 mL of 1 mol / L hydrochloric acid solution. Place the beaker on a magnetic stirrer, insert the rotor, and stir overnight (approximately 12 hours) at 200 rpm. After stirring, filter the mixture, discard the filtrate, and collect the filter cake.

[0039] The filter cake was repeatedly washed with deionized water until the pH of the washing solution was approximately neutral (pH 6.5-7.5). Then, the final washing solution was taken and tested with a 0.1 mol / L standard silver nitrate solution. If no white precipitate was formed, it indicated that no residual chloride ions were present.

[0040] After washing, the qualified resin is placed in a 60°C oven and dried to constant weight. After removal, it is pulverized with a high-speed blender and sieved through 60-mesh and 100-mesh standard sieves. Powder with a particle size of 60-100 mesh is collected to obtain the polyacrylic acid gel chromatography separation medium of the present invention, which is sealed and stored for later use.

[0041] The scanning electron microscope (SEM) image of the obtained polyacrylic acid gel chromatography medium is shown below. Figure 1 It has a porous network structure.

[0042] Example 3: Application of polyacrylic acid gel chromatography medium for the separation and purification of COS Weigh 10 g of the polyacrylic acid gel resin powder prepared in Example 2, add sufficient 5 mmol / L hydrochloric acid solution, and soak for 12 hours to allow it to fully swell. Using the wet packing method, slowly pour the swollen medium homogenate into a glass chromatography column (1 cm inner diameter, 70 cm length), open the column outlet, and allow the medium to settle naturally. After packing, connect a constant flow pump and flush the chromatography column with 5 mmol / L hydrochloric acid solution at a flow rate of 1.0 mL / min until the column bed height is stable and the column is compacted without air bubbles.

[0043] A COS solution with a concentration of 50 mg / mL was precisely prepared. The COS sample (molecular weight ≤1000 Da, prepared by enzymatic hydrolysis) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and was a mixture containing components with different degrees of polymerization, including glucosamine (GlcN), chitobiose ((GlcN)2), chitotriose ((GlcN)3), chitotetraose ((GlcN)4), and chitopentose ((GlcN)5), which served as the sample solution. 3 mL of the sample solution was accurately pipetted and slowly added above the liquid surface of the chromatography column. After the sample solution had completely entered the column bed, fractional elution was immediately performed with 5 mmol / L hydrochloric acid solution at a flow rate controlled at 0.5–1.0 mL / min. The eluent was monitored by thin-layer chromatography (TLC) or high-performance liquid chromatography (HPLC). When one major component was substantially eluted, the eluent was replaced with 10 mmol / L hydrochloric acid solution to continue elution, thus achieving fractional elution.

[0044] The eluent was collected using an automated fraction collector, set to collect one tube every 10 minutes. The collected eluent was then concentrated under reduced pressure at 55°C using a rotary evaporator, and finally analyzed by TLC (ThinPrecision Chromatography). Figure 2 ) and HPLC Figure 3Tracking and purity analysis were performed. The individual COS components with different degrees of polymerization were then collected and combined.

[0045] Results: Five chitosan oligosaccharide fractions with single degrees of polymerization were obtained through fractional elution and collection: GlcN, (GlcN)2, (GlcN)3, (GlcN)4, and (GlcN)5. The TLC chromatograms of each fraction are shown below. Figure 2 The HPLC detection results are shown in the figure. Figure 3 The purity of each component was calculated using the HPLC peak area normalization method, and the results are shown in Table 1.

[0046] Table 1 Purity of COS products with single degree of polymerization

[0047] Example 4: Effect of different crosslinking agent dosages on separation efficiency Following the method of Example 1, four types of gel media were prepared by adding MBA at concentrations of 1%, 1.5%, 2.5%, and 4.0% of the acrylic acid mass, respectively. These gel media were then treated according to the method of Example 2. 10 g of polyacrylic acid gel resin powder with each of the four crosslinking agent dosages was weighed and added to a sufficient amount of 10 mmol / L hydrochloric acid solution, allowing it to swell fully for 12 hours. Using a wet packing method, the swollen medium was slowly poured into a glass chromatography column (1 cm inner diameter, 70 cm length), and the column outlet was opened to allow the medium to settle naturally. After packing, a constant flow pump was connected, and the column was flushed with 10 mmol / L hydrochloric acid solution under a specific pump pressure until the column bed height was stable and the column was compacted without air bubbles.

[0048] A COS solution (commercial chitosan oligosaccharide, molecular weight ≤1000 Da, same as in Example 3) with a concentration of 50 mg / mL was precisely prepared as the sample solution. 3 mL of the sample solution was accurately pipetted and slowly added to the top of the chromatography column. After the sample solution had completely entered the column bed, fractional elution was immediately performed with 10 mmol / L hydrochloric acid solution. The elution flow rate was measured, and the time setting of the automatic fraction collector was adjusted to ensure that approximately one tube of eluent was collected every 10 mL. The collected eluent was concentrated under reduced pressure at 55°C using a rotary evaporator, and then analyzed by TLC (Temperature Computational Chromatography). Figure 4 and Figure 5 ) to conduct tracking and detection.

[0049] Results: The separation flow rates of gel resins with different degrees of crosslinking under the same pump pressure are shown in Table 2. The TLC chromatograms of the eluent components obtained from each tube are shown in Table 2. Figure 4 and Figure 5Gel resins with addition amounts ranging from 1% to 2.5% showed no significant difference in COS separation performance, with 4% showing the best separation effect. Under the same pump pressure, 2.5% gel resin exhibited the best separation flow rate, while 4% showed the lowest. Although PAA gel resin with 4.0% crosslinking agent added had a wider elution interval, its extremely low flow rate made it impractical. PAA gel resin with 2.5% crosslinking agent added maintained excellent separation while exhibiting the highest average flow rate. Therefore, 2.5% is the optimal crosslinking agent addition amount.

[0050] Table 2. Flow rates of COS separation by PAA gel resin at different degrees of crosslinking Collection time (min / tube) 20 15 10 30 Flow rate (mL / min) 0.2~0.3 0.3~0.4 0.6~0.9 0.01~0.2 The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a chitosan oligosaccharide chromatographic separation medium based on polyacrylic acid gel, characterized in that, Includes the following steps: (1) Place the acrylic monomer in an ice-water bath and add sodium hydroxide solution dropwise while stirring to achieve a neutralization degree of 50% to 90% for the acrylic acid; (2) Add crosslinking agent N,N'-methylenebisacrylamide and initiator potassium persulfate to the system obtained in step (1), mix evenly, then pass nitrogen gas to remove oxygen, and carry out polymerization reaction under constant temperature conditions to obtain polyacrylic acid gel; (3) The obtained polyacrylic acid gel was washed, dried and pulverized to obtain polyacrylic acid gel resin; (4) The polyacrylic acid gel resin is acidified with hydrochloric acid solution, then washed, dried, ground and sieved to obtain the polyacrylic acid gel chromatography separation medium.

2. The preparation method according to claim 1, characterized in that, In step (2), the amount of crosslinking agent N,N'-methylenebisacrylamide added is 0.5% to 4.0% of the mass of acrylic acid monomer; the amount of initiator potassium persulfate added is 0.5% to 2% of the mass of acrylic acid monomer.

3. The preparation method according to claim 1, characterized in that, In step (2), the polymerization reaction is carried out at a temperature of 30 to 80°C and for a reaction time of 4 to 16 hours.

4. The preparation method according to claim 1, characterized in that, In step (4), the concentration of the hydrochloric acid solution is 0.1-2 mol / L; the acidification treatment time is 8-12 hours, the oscillation speed is 100-400 r / min; and the sieve mesh size is 20-200 mesh.

5. The polyacrylic acid gel chromatography medium obtained by the preparation method according to any one of claims 1 to 4, characterized in that, The medium uses polyacrylic acid as its backbone and carboxyl groups as its main functional groups, enabling the adsorption and separation of chitosan oligosaccharides under acidic conditions.

6. The application of the polyacrylic acid gel chromatography medium of claim 5 in the separation and purification of chitosan oligosaccharides.

7. The application according to claim 6, characterized in that, Includes the following steps: Swelling: The polyacrylic acid gel chromatography medium is added to a 1-100 mmol / L hydrochloric acid solution and soaked to allow it to fully absorb water and swell; Column packing: Pour the swollen medium homogenate into the chromatography column and allow the medium to settle naturally; Equilibration: Wash the chromatography column with 1-100 mmol / L hydrochloric acid solution until the column bed is compacted and stable; Sample loading: Add the chitosan oligosaccharide sample solution above the liquid surface of the chromatography column, and wait until the sample liquid surface is completely in the column bed; Elution and collection: Elute in fractions with 1–100 mmol / L hydrochloric acid solution, collect the eluent in fractions to obtain chitosan oligosaccharides with the target degree of polymerization.

8. The application according to claim 7, characterized in that, The concentration of the chitosan oligosaccharide sample solution is 10–200 mg / mL, and the loading volume is 1–10 mL.

9. The application according to claim 7, characterized in that, The temperature of the eluent is 0–100℃, and the elution flow rate is 0.5–2.0 mL / min.

10. The application according to claim 6, characterized in that, The target degree of polymerization of chitosan oligosaccharides includes one or more of the following: chitosan oligosaccharide monosaccharide (GlcN), chitosan oligosaccharide disaccharide (GlcN)2, chitosan oligosaccharide trisaccharide (GlcN)3, chitosan oligosaccharide tetrasaccharide (GlcN)4, or chitosan oligosaccharide pentasaccharide (GlcN)5.

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