Ulva sulfated polysaccharide with high gel strength, hydrogel and preparation method and application of ulva sulfated polysaccharide and hydrogel
Ulva sulfated polysaccharides with high molecular weight and high sulfate content were prepared by two-step enzymatic method, and the problem of poor gelation performance of Ulva polysaccharides was solved through metal ion cross-linking technology, and hydrogels with high gel strength and high energy storage modulus were achieved, and its application in biological preparations and food fields was broadened.
Patent Information
- Application Number
- CN202510210279.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-25
- Publication Date
- 2025-06-10
AI Technical Summary
In the prior art, Ulva polysaccharide has poor gelation performance, resulting in limited application in the fields of medicines, food, tissue engineering, etc.
Ulva polysaccharide was prepared by two-step enzymatic method. By controlling the molecular weight and sulfate content of the polysaccharide, Ulva sulfated polysaccharide with high gel strength, and crosslinking by adding metal ions to prepare a high-performance hydrogel.
The high gel strength of Ulva sulfated polysaccharide and the high energy storage modulus of hydrogels have been achieved, which has significantly improved its application potential in the fields of biological agents and food.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of biological products, and particularly relates to an ulva sulfate polysaccharide with high gel strength, a hydrogel, and a preparation method and application thereof. Background Art
[0002] Ulva is a green alga with rich resources, and its cell wall contains abundant sulfated polysaccharides. This sulfated polysaccharide is mainly composed of rhamnose, glucose, glucuronic acid, and xylose, and about 4% of the xylose part contains sulfate groups. With its various biological activities such as antioxidant, anti-tumor, antiviral, and improving intestinal health, and its rich resource volume, it has gradually become a hot spot in functional foods and health products.
[0003] Gelation is one of the commonly used methods to improve the texture of foods. The three-dimensional network structure that plays a key role can not only enhance the support and mechanical strength of materials but also capture a large amount of water molecules and bioactive substances inside. The biopolymers used to develop gelling agents in the food field mainly include proteins and polysaccharides, which is attributed to their non-toxic characteristics, long molecular chains, and rich active groups. Polysaccharide hydrogels have been widely developed in the fields of tissue engineering, biomedicine, and food due to their non-toxicity, high bioavailability, and biodegradability. For example, in the food field, gelation is an effective method to protect nutrients from being corroded and enzymatically hydrolyzed by the strong acid environment of the stomach to the greatest extent. Many studies have shown that ulva polysaccharides are macromolecular substances with poor gel performance, which greatly limits their applications in the fields of medicine, food, tissue engineering, agriculture, etc. Kidgell et al. (Carbohydrate Polymers, 2021) previously reported that different types of ulva polysaccharides form weak gels with divalent metal ions in the presence of borate, and the storage modulus is distributed between 0.1 - 74.2 Pa. The long-chain structure and high occupancy rate of sulfate groups are the key factors restricting the gelation characteristics of ulva polysaccharides. Specifically, the molecular entanglement of long molecular chains and the high charge density caused by the high sulfate group content reduce the non-covalent crosslinking between polysaccharide chains.
[0004] Currently, the extraction of ulva polysaccharides mostly uses traditional hot water and acid-base extraction methods. The molecular weight and sulfate group content of the obtained polysaccharides are easily interfered by the combined action of multiple components, showing the characteristics of low viscosity and weak gel. Therefore, preparing an ulva sulfate polysaccharide with high gel strength is the key issue to broaden its application fields. Summary of the Invention
[0005] To solve the deficiencies in the above-mentioned background technology, the purpose of the present invention is to provide a Ulva sulfated polysaccharide with high gel strength, a hydrogel, and its preparation method and application. The present invention obtains a polysaccharide structure of Ulva suitable for preparing a high-strength hydrogel through a two-step enzymatic hydrolysis method, solving the problems of low viscosity, poor gel strength, and limited application of Ulva polysaccharide.
[0006] To achieve the above-mentioned invention purpose, the following technical solutions are specifically adopted: The present invention provides a Ulva sulfated polysaccharide with high gel strength, the molecular weight of the Ulva sulfated polysaccharide is 300 kDa to 900 kDa, and the sulfate group content is 3% to 20%; the Ulva sulfated polysaccharide contains a disaccharide repeating unit of ΔUA-(1→4)-α-L-Rha3S-(1→4)-β-D-Xyl-(1→4)-α / β-L-Rha3S; the chemical structural formula of the disaccharide repeating unit of the Ulva sulfated polysaccharide is as follows: 。
[0007] The present invention also provides a hydrogel prepared from the Ulva sulfated polysaccharide, and the gel strength of the hydrogel is 0.5 to 2.0 N, and the storage modulus is 40 to 1400 Pa.
[0008] The present invention also provides a preparation method of the hydrogel, and the specific operation steps are as follows: S1: Prepare the Ulva sulfated polysaccharide; S2: Prepare the Ulva sulfated polysaccharide into a polysaccharide solution; S3: Hydrate the polysaccharide solution at a certain temperature and then adjust the pH to an alkaline condition, add a metal coordination ion, stir at a high temperature, and then cool to form a hydrogel.
[0009] Further, the preparation method of the Ulva sulfated polysaccharide in the S1 step is as follows: (1) Dry the Ulva raw material and then crush it, mix the obtained Ulva fragments with water, and perform extraction under heating conditions; (2) Add ethanol precipitation to the extraction solution in the step (1), collect the precipitated precipitate, and obtain a crude polysaccharide after drying; (3) Prepare the crude polysaccharide into a polysaccharide solution, add Ulva polysaccharide lyase to the solution, and perform enzymatic hydrolysis under heating conditions to obtain a primary enzymatically hydrolyzed polysaccharide solution, and terminate the enzymatic reaction at a high temperature; (4) Add sulfatase to the primary enzymatically hydrolyzed polysaccharide solution, and perform enzymatic hydrolysis under heating conditions to obtain a secondary enzymatically hydrolyzed polysaccharide solution, and terminate the enzymatic reaction at a high temperature; (5) Dialyze and dry the secondary enzymatically hydrolyzed polysaccharide solution to obtain the Ulva sulfated polysaccharide.
[0010] Further, the mass-to-volume ratio of the Ulva lactuca fragments to water in step (1) is 1:20 to 30 (w / v), the heating temperature is 90°C to 95°C, and the extraction time is 100 to 150 min.
[0011] In the present invention, the unit of mass w in w / v is g, and the unit of volume v is mL for calculation.
[0012] Further, the mass addition amount of the Ulva lactuca polysaccharide lyase in step (3) accounts for 0.4% to 2% (w / v) of the volume of the polysaccharide solution, the enzymatic hydrolysis heating temperature is 30°C to 40°C, and the enzymatic hydrolysis time is 5 to 30 min.
[0013] Further, the mass addition amount of the sulfatase in step (4) accounts for 0.2% to 2% (w / v) of the volume of the primary enzymatically hydrolyzed polysaccharide solution, the enzymatic hydrolysis heating temperature is 30°C to 50°C, and the enzymatic hydrolysis time is 5 to 30 min.
[0014] Further, the molecular weight cut-off of the dialysis bag used in dialysis in step (5) is 2 kDa.
[0015] Further, the mass concentration of the polysaccharide solution in step S2 is 1% to 3% (w / v); in step S3, the pH is adjusted to 8.0 to 10.0, and the metal coordination ion is Al 3+ 、Fe 3+ or Cr 3+ or at least one of them, and the molar concentration of the metal coordination ion is 100 to 300 mM.
[0016] The present invention also provides the application of the Ulva lactuca sulfated polysaccharide or the hydrogel in the preparation of a biological agent with high gel strength.
[0017] Further, the relative molecular weight of the Ulva lactuca sulfated polysaccharide is >500 kDa, and the sulfate group content is 9.5% to 12%.
[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a preparation method of Ulva lactuca sulfated polysaccharide and its hydrogel. Its extraction method is green and efficient, the preparation process is simple and feasible, and it saves costs, which has reference significance for the large-scale production of related products in the later stage.
[0019] The present invention provides a hydrogel prepared from Ulva lactuca sulfated polysaccharide, whose gel hardness and rheological storage modulus have obvious performance improvements. The gel strength of the hydrogel is as high as 1.53 N, and the storage modulus is as high as 1396.6 Pa.
[0020] The ulva pertusa sulfated polysaccharide hydrogel prepared by the present invention realizes the intermolecular crosslinking of polysaccharide and metal ions by adding trivalent metal ions into the gel network. Among them, the UEP-2Fe hydrogel has the best gel strength, which is significantly better than the ulva pertusa sulfated polysaccharide hydrogels prepared with other metal ions.
[0021] The relatively high molecular weight (>500 kDa) and appropriate sulfate group content (9.5% - 12%) of the ulva pertusa sulfated polysaccharide described in the present invention are beneficial to the gel hardness of the polysaccharide hydrogel. Description of the Drawings
[0022] Figure 1 It is the appearance diagram of the hydrogels prepared by UEP-1, UEP-2, UEP-3, UEP-4 and UEP-5 provided by the embodiments of the present invention; Figure 2 It is the gel strength of the hydrogels prepared by UEP-1, UEP-2, UEP-3, UEP-4 and UEP-5 provided by the embodiments of the present invention under different metal ion conditions; Figure 3 It is the rheological behavior of the hydrogels prepared by UEP-1, UEP-2, UEP-3, UEP-4 and UEP-5 provided by the embodiments of the present invention under different metal ion conditions; Figure 4 It is the gel strength of the hydrogels prepared by UEP-1, UEP-2, UEP-3, UEP-4 and UEP-5 provided by the embodiments of the present invention under Fe 3+ conditions; Figure 5 It is the gel strength of the hydrogels prepared by UEP-1, UEP-2, UEP-3, UEP-4 and UEP-5 provided by the embodiments of the present invention under Fe 3+ conditions; Figure 6 It is the monosaccharide composition map of UEP-1 provided by the embodiments of the present invention; Figure 7 It is the monosaccharide composition map of UEP-2 provided by the embodiments of the present invention; Figure 8 It is the monosaccharide composition map of UEP-3 provided by the embodiments of the present invention; Figure 9 It is the monosaccharide composition map of UEP-4 provided by the embodiments of the present invention; Figure 10 It is the monosaccharide composition map of UEP-5 provided by the embodiments of the present invention. Detailed Embodiments
[0023] To better illustrate the purpose, technical solution and advantages of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the scope of protection of the present invention.
[0024] For those not specified in the embodiments of the present invention, they are carried out according to conventional conditions or conditions recommended by the manufacturer.
[0025] In the embodiments of the present invention, for the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained by purchasing in the market.
[0026] Example 1: Preparation of Ulva lactuca sulfated polysaccharide with high gel strength 1. Dry the Ulva lactuca raw material and then crush it. Mix 100 g of the obtained Ulva lactuca fragments with 2 L of water, and the mass-volume ratio of the Ulva lactuca fragments to water is 1:20 (w / v). After mixing, extract at 90 °C for 100 minutes. 2. Add ethanol precipitation to the extract, collect the precipitated precipitate, and obtain the crude polysaccharide after drying. 3. Prepare 500 mL of a polysaccharide solution with 5 g of the crude polysaccharide. Add 2 g of Ulva lactuca polysaccharide lyase to the solution. The proportion of Ulva lactuca polysaccharide lyase in the crude polysaccharide solution is 0.4% (w / v, the unit of w is g, and the unit of v is mL). Enzymatically hydrolyze at 30 °C for 5 minutes to obtain a primary enzymatically hydrolyzed polysaccharide solution, and terminate the enzyme reaction at 90 °C. 4. Add 1 g of sulfatase to the primary enzymatically hydrolyzed polysaccharide solution. The mass-volume ratio of sulfatase in the primary polysaccharide solution is 0.2% (w / v). Enzymatically hydrolyze at 30 °C for 5 minutes to obtain a secondary enzymatically hydrolyzed polysaccharide solution, and terminate the enzyme reaction at 90 °C. 5. Dialyze and dry the secondary enzymatically hydrolyzed polysaccharide solution to obtain Ulva lactuca sulfated polysaccharide with high gel strength (UEP-1).
[0027] The molecular weight of UEP-1 was determined by high performance liquid chromatography and differential refractive index detector. The sulfate group content of UEP-1 was determined by ion chromatography. The monosaccharide composition of UEP-1 was determined by PMP pre-column derivation method, using high performance liquid chromatography and ultraviolet detector. The results showed that the basic chemical composition of UEP-1 was shown in Table 1. It was a complex polysaccharide mainly composed of rhamnose, accompanied by glucose, glucuronic acid and xylose, with a molecular weight of 563.2 kDa and a sulfate group content of 15.45%.
[0028] Table 1 Chemical composition of UEP-1 prepared in this example
[0029] Example 2: Preparation of Ulva lactuca sulfated polysaccharide with high gel strength of the present invention 1. Dry the Ulva lactuca raw material and then crush it. Mix 100 g of the obtained Ulva lactuca fragments with 2.5 L of water. The mass-volume ratio of the Ulva lactuca fragments to water is 1:25 (w / v). After mixing, extract at 95 °C for 120 minutes; 2. Add ethanol to the extract for precipitation, and collect the precipitated matter obtained after drying to obtain the crude polysaccharide; 3. Prepare 5 g of the crude polysaccharide into a 500 mL polysaccharide solution. Add 4 g of Ulva lactuca polysaccharide lyase to the solution. The proportion of the Ulva lactuca polysaccharide lyase in the crude polysaccharide solution is 0.8% (w / v). Enzymatically hydrolyze at 35 °C for 10 minutes to obtain the first-stage enzymatically hydrolyzed polysaccharide solution, and terminate the enzyme reaction at 95 °C under high temperature; 4. Add 3 g of sulfatase to the first-stage enzymatically hydrolyzed polysaccharide solution. The mass-volume ratio of the sulfatase in the first-stage polysaccharide solution is 0.6% (w / v). Enzymatically hydrolyze at 35 °C for 10 minutes to obtain the second-stage enzymatically hydrolyzed polysaccharide solution, and terminate the enzyme reaction at 95 °C under high temperature; 5. Dialyze and dry the second-stage enzymatically hydrolyzed polysaccharide solution to obtain the Ulva lactuca sulfated polysaccharide with high gel strength (UEP-2).
[0030] Use high performance liquid chromatography and a differential refractive index detector to measure the molecular weight of UEP-2. Use ion chromatography to measure the sulfate group content of UEP-2. Use the PMP pre-column derivation method, and use high performance liquid chromatography and an ultraviolet detector to measure the monosaccharide composition of UEP-2. The results show that the basic chemical composition of UEP-2 is shown in Table 2. It is a complex polysaccharide mainly composed of rhamnose, accompanied by glucose, glucuronic acid and xylose, with a molecular weight of 684.2 kDa and a sulfate group content of 11.78%.
[0031] Table 2 Chemical composition of UEP-2 prepared in this example
[0032] Example 3: Preparation of Ulva lactuca sulfated polysaccharide with high gel strength of the present invention 1. Dry the Ulva lactuca raw material and then crush it. Mix 100 g of the obtained Ulva lactuca fragments with 3 L of water. The mass-volume ratio of the Ulva lactuca fragments to water is 1:30 (w / v). After mixing, extract at 95 °C for 150 minutes; 2. Add ethanol to the extract for precipitation, and collect the precipitated matter obtained after drying to obtain the crude polysaccharide; 3. Prepare 500 mL of a polysaccharide solution by dissolving 5 g of crude polysaccharide. Add 5 g of Ulva lactuca polysaccharide lyase to the solution, with the proportion of Ulva lactuca polysaccharide lyase in the crude polysaccharide solution being 1% (w / v). Enzymatically hydrolyze at 40 °C for 20 minutes to obtain a primary enzymatically hydrolyzed polysaccharide solution, and terminate the enzymatic reaction at 90 °C under high temperature. 4. Add 5 g of sulfatase to the primary enzymatically hydrolyzed polysaccharide solution, with the mass-to-volume ratio of sulfatase in the primary polysaccharide solution being 1% (w / v). Enzymatically hydrolyze at 40 °C for 20 minutes to obtain a secondary enzymatically hydrolyzed polysaccharide solution, and terminate the enzymatic reaction at 95 °C under high temperature. 5. Dialyze and dry the secondary enzymatically hydrolyzed polysaccharide solution to obtain high-gel-strength Ulva lactuca sulfated polysaccharide (UEP-3).
[0033] Determine the molecular weight of UEP-3 using high-performance liquid chromatography and a differential refractive index detector, determine the sulfate group content of UEP-3 using ion chromatography, and determine the monosaccharide composition of UEP-3 using the PMP pre-column derivation method, high-performance liquid chromatography, and an ultraviolet detector. The results show that the basic chemical composition of UEP-3 is as shown in Table 3, which is a complex polysaccharide mainly composed of rhamnose, accompanied by glucose, glucuronic acid, and xylose, with a molecular weight of 454.1 kDa and a sulfate group content of 8.24%.
[0034] Table 3 Chemical composition of UEP-3 prepared in this example
[0035] Example 4: Preparation of high-gel-strength Ulva lactuca sulfated polysaccharide of the present invention 1. Dry the Ulva lactuca raw material and then crush it. Mix the obtained 100 g of Ulva lactuca fragments with 2.5 L of water, with the mass-to-volume ratio of Ulva lactuca fragments to water being 1:25 (w / v). After mixing, extract at 90 °C for 120 minutes. 2. Add ethanol precipitation to the extract, collect the precipitated matter, and obtain crude polysaccharide after drying. 3. Prepare 500 mL of a polysaccharide solution by dissolving 5 g of crude polysaccharide. Add 10 g of Ulva lactuca polysaccharide lyase to the solution, with the proportion of Ulva lactuca polysaccharide lyase in the crude polysaccharide solution being 2% (w / v). Enzymatically hydrolyze at 35 °C for 30 minutes to obtain a primary enzymatically hydrolyzed polysaccharide solution, and terminate the enzymatic reaction at 95 °C under high temperature. 4. Add 10 g of sulfatase to the primary enzymatically hydrolyzed polysaccharide solution, with the mass-to-volume ratio of sulfatase in the primary polysaccharide solution being 2% (w / v). Enzymatically hydrolyze at 50 °C for 10 minutes to obtain a secondary enzymatically hydrolyzed polysaccharide solution, and terminate the enzymatic reaction at 90 °C under high temperature. 5. Dialyze and dry the secondary enzymatically hydrolyzed polysaccharide solution to obtain high-gel-strength Ulva lactuca sulfated polysaccharide (UEP-4).
[0036] The molecular weight of UEP-4 was determined by high performance liquid chromatography and a differential refractive index detector. The sulfate group content of UEP-4 was determined by ion chromatography. The monosaccharide composition of UEP-4 was determined by pre-column derivation with PMP, using high performance liquid chromatography and an ultraviolet detector. The results showed that the basic chemical composition of UEP-4 was as shown in Table 4, which was a complex polysaccharide mainly composed of rhamnose, accompanied by glucose, glucuronic acid and xylose, with a molecular weight of 357.5 kDa and a sulfate group content of 6.52%.
[0037] Table 4 Chemical composition of UEP-4 prepared in this example
[0038] Example 5: Preparation of Ulva lactuca sulfated polysaccharide with high gel strength of the present invention 1. The Ulva lactuca raw material was dried and then pulverized. 100 g of the obtained Ulva lactuca fragments were mixed with 3 L of water, and the mass-volume ratio of the Ulva lactuca fragments to water was 1:30 (w / v). After mixing, it was extracted at 95 °C for 150 minutes; 2. Ethanol was added to the extract for precipitation, and the precipitated precipitate was collected and dried to obtain a crude polysaccharide; 3. 5 g of the crude polysaccharide was formulated into a 500 mL polysaccharide solution. 5 g of Ulva lactuca polysaccharide lyase was added to the solution, and the proportion of the Ulva lactuca polysaccharide lyase in the crude polysaccharide solution was 1% (w / v). It was enzymatically hydrolyzed at 35 °C for 10 minutes to obtain a primary enzymatically hydrolyzed polysaccharide solution, and the enzymatic reaction was terminated at 90 °C; 4. 3 g of sulfatase was added to the primary enzymatically hydrolyzed polysaccharide solution, and the mass-volume ratio of the sulfatase in the primary polysaccharide solution was 0.6% (w / v). It was enzymatically hydrolyzed at 40 °C for 30 minutes to obtain a secondary enzymatically hydrolyzed polysaccharide solution, and the enzymatic reaction was terminated at 90 °C; 5. The secondary enzymatically hydrolyzed polysaccharide solution was dialyzed and dried to obtain Ulva lactuca sulfated polysaccharide with high gel strength (UEP-5).
[0039] The molecular weight of UEP-5 was determined by high performance liquid chromatography and a differential refractive index detector. The sulfate group content of UEP-5 was determined by ion chromatography. The monosaccharide composition of UEP-5 was determined by pre-column derivation with PMP, using high performance liquid chromatography and an ultraviolet detector. The results showed that the basic chemical composition of UEP-5 was as shown in Table 5, which was a complex polysaccharide mainly composed of rhamnose, accompanied by glucose, glucuronic acid and xylose, with a molecular weight of 825.8 kDa and a sulfate group content of 9.63%.
[0040] Table 5 Chemical composition of UEP-5 prepared in this example
[0041] Example 6: Preparation of Ulva lactuca sulfated polysaccharide hydrogel with high gel strength in the present invention The polysaccharides in this example are UEP-1 prepared in Example 1 and UEP-3 prepared in Example 3.
[0042] The above-mentioned Ulva lactuca sulfated polysaccharides were respectively prepared into 50 mL solutions containing 0.5 g of polysaccharide. The prepared mass concentration of Ulva lactuca sulfated polysaccharide was 1% (w / v). After hydration at 4 °C, the pH was adjusted to 8.0, and 0.6 mL of Al with a molar concentration of 100 mM was added. 3+ After high-temperature stirring, it was cooled to form a gel. Crosslinked to form a gel, named UEP-1Al and UEP-3Al.
[0043] Example 7: Preparation of Ulva lactuca sulfated polysaccharide hydrogel with high gel strength in the present invention The polysaccharides in this example are UEP-2 prepared in Example 2 and UEP-4 prepared in Example 4.
[0044] The above-mentioned Ulva lactuca sulfated polysaccharides were respectively prepared into 50 mL solutions containing 1 g of polysaccharide. The prepared mass concentration of Ulva lactuca sulfated polysaccharide was 2% (w / v). After hydration at 4 °C, the pH was adjusted to 9.0, and 0.6 mL of Fe with a molar concentration of 200 mM was added. 3+ After high-temperature stirring, it was cooled to form a gel. Crosslinked to form a gel, named UEP-2Fe and UEP-4Fe.
[0045] Example 8: Preparation of Ulva lactuca sulfated polysaccharide hydrogel with high gel strength in the present invention The polysaccharides in this example are UEP-2 prepared in Example 2 and UEP-5 prepared in Example 5.
[0046] The above-mentioned Ulva lactuca sulfated polysaccharides were respectively prepared into 50 mL solutions containing 1.5 g of polysaccharide. The prepared mass concentration of Ulva lactuca sulfated polysaccharide was 3% (w / v). After hydration at 4 °C, the pH was adjusted to 10.0, and 0.6 mL of Cr with a molar concentration of 300 mM was added. 3+ After high-temperature stirring, it was cooled to form a gel. Crosslinked to form a gel, named UEP-2Cr and UEP-5Cr.
[0047] Example 9: Appearance morphology of Ulva lactuca sulfated polysaccharide hydrogel with high gel strength in the present invention The gels in this example are UEP-1Al and UEP-3Al prepared in Example 6, UEP-2Fe and UEP-4Fe prepared in Example 7, and UEP-2Cr and UEP-5Cr prepared in Example 8.
[0048] The formed gel samples were photographed at a certain angle. The results are shown in Figure 1 .
[0049] The results are as Figure 1 shown, and all polysaccharides can form stable solid hydrogels under the crosslinking of trivalent metal ions.
[0050] Example 10: Gel hardness of the Ulva pertusa sulfated polysaccharide hydrogel with high gel strength of the present invention The gels in this example use UEP-1Al and UEP-3Al prepared in Example 6, UEP-2Fe and UEP-4Fe prepared in Example 7, and UEP-2Cr and UEP-5Cr prepared in Example 8.
[0051] The gel strength of the hydrogel was tested using a TA-XT texture analyzer (Stable Micro Systems Co, UK) at room temperature. A P / 0.5 cylindrical probe (radius 5 mm) was pressed down at a constant speed of 1 mm / s until fracture, and the hardness value of the gel at fracture was obtained.
[0052] The results are as Figure 2 shown. The gel hardness of the hydrogels prepared according to Example 6, Example 7, and Example 8 is greater than 0.7 N, indicating that the hydrogels prepared in the present invention have good mechanical properties, especially the hydrogels formulated with Ulva pertusa sulfated polysaccharides UEP-2 and UEP-5. Among them, the UEP-2Fe hydrogel has the strongest ability to resist external forces (1.53 N), which is 109.6% higher than the weakest UEP-3Al (0.73 N). Therefore, the present invention can realize the preparation of Ulva pertusa sulfated polysaccharides with high gel strength and establish a stable hydrogel system crosslinked by metal ions.
[0053] Example 11: Rheological behavior of the Ulva pertusa sulfated polysaccharide hydrogel with high gel strength of the present invention The gels in this example use UEP-1Al and UEP-3Al prepared in Example 6, UEP-2Fe and UEP-4Fe prepared in Example 7, and UEP-2Cr and UEP-5Cr prepared in Example 8.
[0054] A series of rheological properties of the hydrogel were measured using a controlled stress rheometer (AR2000EX) at room temperature. The sample was placed on a 20 mm stainless steel parallel plate and a frequency sweep was performed with an angular frequency change of 1 - 100 rad / s.
[0055] The results are as Figure 3As shown, the storage modulus (G') of the hydrogels prepared according to Example 6, Example 7, and Example 8 is greater than the loss modulus (G''), indicating that the elastic characteristics of the hydrogels prepared in the present invention are greater than the viscous characteristics, and they possess the rheological properties of solid gels. The UEP-2Fe hydrogel exhibits higher storage modulus (1396.9 Pa) and loss modulus (217.1 Pa) during structural damage, followed by UEP-2Cr and UEP-5Cr, and UEP-3Al has the weakest rheological properties (G' = 44.1 Pa). Therefore, the ulva pertusa sulfated polysaccharide hydrogel prepared in the present invention has excellent rheological behavior and broad development prospects in the fields of food, agriculture, tissue engineering, biopharmaceuticals, biomaterials, etc.
[0056] Example 12: Preparation of the ulva pertusa sulfated polysaccharide hydrogel with high gel strength in the present invention In this example, the ulva pertusa sulfated polysaccharides used are UEP-1 prepared in Example 1, UEP-2 prepared in Example 2, UEP-3 prepared in Example 3, UEP-4 prepared in Example 4, and UEP-5 prepared in Example 5.
[0057] The above ulva pertusa sulfated polysaccharides were respectively prepared into 50 mL solutions containing 1 g of polysaccharide. The prepared ulva pertusa sulfated polysaccharide had a mass concentration of 2% (w / v). After hydration at 4 °C, the pH was adjusted to 9.0, and 0.6 mL of Fe with a molar concentration of 200 mM was added. 3+ After high-temperature stirring, it was cooled to form a gel. Cross-linking to form a gel, named UEP-1Fe, UEP-2Fe, UEP-3Fe, UEP-4Fe, and UEP-5Fe, to explore the influence of structural differences on the preparation of hydrogels from ulva pertusa sulfated polysaccharides.
[0058] Example 13: Gel hardness of the ulva pertusa sulfated polysaccharide hydrogel with high gel strength in the present invention The gels in this example are UEP-1Fe, UEP-2Fe, UEP-3Fe, UEP-4Fe, and UEP-5Fe prepared in Example 12.
[0059] The gel strength test of the hydrogel was carried out at room temperature using a TA-XT texture analyzer (Stable Micro Systems Co, UK). A P / 0.5 cylindrical probe (radius 5 mm) was used for the downward pressure test at a constant speed of 1 mm / s until fracture, and the hardness value of the gel at fracture was obtained.
[0060] The results are as Figure 4As shown in the figure, the gel hardness of the hydrogels prepared according to Example 12 is all greater than 0.7 N, indicating that the hydrogels prepared in the present invention have good mechanical properties, especially UEP-2 and UEP-5. Due to the small difference in the monosaccharide composition of the five groups of polysaccharides, it can be known that the factors causing the large difference in the gel strength of the hydrogels are the molecular weight and the content of sulfate groups of the ulva sulfated polysaccharide. Among them, the UEP-2Fe hydrogel has the strongest ability to resist external forces (1.53 N), and the corresponding molecular weight and sulfate group content of UEP-2 are 684.2 kDa and 11.78%. Followed by the UEP-5Fe hydrogel (1.24 N), and the corresponding molecular weight and sulfate group content of UEP-5 are 825.8 kDa and 9.63%. In contrast, the UEP-4Fe hydrogel (0.77 N) has the weakest hardness. Combining the experimental data analysis, the weakness of the UEP-4Fe hydrogel is because UEP-4 has a smaller molecular weight (357.5 kDa) and a lower content of sulfate groups (6.52%). Therefore, the experimental data show that the relatively high molecular weight (>500 kDa) and appropriate sulfate group content (9.5%-12%) of the ulva sulfated polysaccharide described in the present invention are beneficial to the gel hardness of the polysaccharide hydrogel.
[0061] Example 14: Rheological Behavior of the Ulva Sulfated Polysaccharide Hydrogel with High Gel Strength of the Present Invention The gels in this example use UEP-1Fe, UEP-2Fe, UEP-3Fe, UEP-4Fe and UEP-5Fe prepared according to Example 12.
[0062] A series of rheological properties of the hydrogel were measured using a controlled stress rheometer (AR2000EX) at room temperature. The sample was placed on a 20 mm stainless steel parallel plate, and a frequency sweep was performed with an angular frequency change of 1-100 rad / s.
[0063] The results are as Figure 5 shown. The storage modulus (G') of the hydrogels prepared according to Example 12 is greater than the loss modulus (G''), indicating that the elastic characteristics of the hydrogels prepared in the present invention are greater than the viscous characteristics, and they have the rheological characteristics of solid gels. The UEP-2Fe hydrogel shows higher storage modulus (1396.9 Pa) and loss modulus (217.1 Pa) during the structural destruction, followed by the UEP-5Fe hydrogel. The two ulva polysaccharides, UEP-2 and UEP-5, have larger molecular weights and moderate sulfate group contents, which are more conducive to intermolecular interactions and hydrogen bond connections. UEP-4Fe has the weakest rheological properties (G' < 100 Pa), which is attributed to the shorter polysaccharide chain and insufficient sulfate group content of UEP-4 polysaccharide. Therefore, through experimental verification, it is further confirmed that the molecular weight and sulfate group of the ulva sulfated polysaccharide prepared in the present invention are the key factors affecting the hydrogel strength.
[0064] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.
Claims
1. A sulfated polysaccharide of Ulva glabra with high gel strength, characterized in that: The molecular weight of the Ulva sulfated polysaccharide is 300 kDa to 900 kDa, the sulfate content is 3% to 20%, and the Ulva sulfated polysaccharide contains a disaccharide repeating unit of ΔUA-(1→4)-α-L-Rha3S-(1→4)-β-D-Xyl-(1→4)-α / β-L-Rha3S; the chemical structure of the disaccharide repeating unit of the Ulva sulfated polysaccharide is as follows: 。 2. A hydrogel prepared using the sulfated Ulva polysaccharide according to claim 1, characterized in that: The hydrogel has a gel strength of 0.5 to 2.0 N and a storage modulus of 40 to 1400 Pa.
3. The method for preparing the hydrogel according to claim 2, characterized in that: The preparation method comprises the following steps: S1: preparing the Ulva sulfated polysaccharide; S2: preparing the sulfated Ulva polysaccharide into a polysaccharide solution; S3: After the polysaccharide solution is hydrated at a certain temperature, the pH is adjusted to an alkaline condition, and a metal The coordinated ions are stirred at high temperature and then cooled to form a hydrogel.
4. The method for preparing the hydrogel according to claim 3, characterized in that: The preparation method of Ulva sulfated polysaccharide in step S1 is: (1) drying the raw material of Ulva and then crushing it, mixing the obtained Ulva fragments with water and then leaching them under heating conditions; (2) adding ethanol to the extract in step (1) for precipitation, collecting the precipitate and drying it to obtain crude polysaccharide; (3) preparing the crude polysaccharide into a polysaccharide solution, adding ulva polysaccharide lyase to the solution, performing enzymatic hydrolysis under heating conditions to obtain a primary enzymatic polysaccharide solution, and terminating the enzyme reaction at high temperature; (4) adding sulfatase to the primary enzymatically hydrolyzed polysaccharide solution, performing enzymatic hydrolysis under heating conditions to obtain a secondary enzymatically hydrolyzed polysaccharide solution, and terminating the enzyme reaction at high temperature; (5) The secondary enzymatic polysaccharide solution is dialyzed and dried to obtain Ulva sulfated polysaccharide.
5. The method for preparing the hydrogel according to claim 4, characterized in that: The mass volume ratio of the Ulva fragments to water in step (1) is 1:20-30 w / v, the heating temperature is 90°C-95°C, and the extraction time is 100-150 min.
6. The method for preparing the hydrogel according to claim 4, characterized in that: In the step (3), the mass addition amount of the Ulva polysaccharide lyase accounts for 0.4% to 2% w / v of the volume of the polysaccharide solution, the enzymatic heating temperature is 30° C. to 40° C., and the enzymatic hydrolysis time is 5 to 30 min.
7. The method for preparing the hydrogel according to claim 4, characterized in that: The mass addition amount of sulfatase in step (4) is 0.2% to 2% w / v of the volume of the primary enzymatic polysaccharide solution, the enzymatic heating temperature is 30°C to 50°C, and the enzymatic time is 5 to 30 min.
8. The method for preparing the hydrogel according to claim 4, characterized in that: The mass concentration of the polysaccharide solution in step S2 is 1% to 3% w / v; the pH in step S3 is adjusted to 8.0 to 10.0, and the metal coordination ion is Al 3+ , Fe 3+ or Cr 3+ At least one of the metal coordination ions has a molar concentration of 100 to 300 mM.
9. Use of the Ulva sulfated polysaccharide according to claim 1 or the hydrogel according to claim 2 in preparing a biological agent with high gel strength.
10. The use according to claim 9, characterized in that: The relative molecular weight of the Ulva sulfated polysaccharide is greater than 500 kDa, and the sulfate content is 9.5% to 12%.