A water-soluble carboxymethyl curdlan of a specific molecular weight, and a method for preparing and use thereof

Abstract

The application discloses a water-soluble carboxymethyl curdlan with a specific molecular weight and a preparation method and application thereof, and belongs to the technical field of polysaccharide derivatization. The preparation method provided by the application comprises the following steps: suspending curdlan in an ethanol solution, adding a sodium hydroxide solution to perform alkalization, and then adding chloroacetic acid to perform etherification reaction; after the reaction is completed, the carboxymethyl curdlan intermediate is obtained through purification and drying; the carboxymethyl curdlan intermediate is prepared into a substrate solution, and a beta-glucanase is added to perform an enzymolysis reaction for 15-30 min; the enzyme is inactivated by heating, and then purification and drying are performed to obtain the product. Through the carboxymethylation modification combined with the time-limited enzymolysis strategy, the uniformity of the molecular weight is realized on the premise of maintaining the polysaccharide high molecular skeleton, so that the product has good water solubility and functional characteristics such as thickening and stability.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide derivatization technology, and in particular to a water-soluble carboxymethyl guar gum of a specific molecular weight, its preparation method, and its application. Background Technology

[0002] Glide gum is a microbial extracellular polysaccharide composed of glucose residues linearly linked by β-1,3-glycosidic bonds. The dense network of hydrogen bonds between its molecular chains assembles the polysaccharide chains into a compact triple helix structure, making glide gum extremely difficult to dissolve in room temperature water and common organic solvents, which severely limits its application in food processing and other fields.

[0003] To improve water solubility, gluconolactone and other chemical modifications can be used to chemically modify the polysaccharide, introducing hydrophilic groups to disrupt the triple helix structure and thus increase solubility. However, chemical modification only alters solubility and cannot effectively control the molecular weight of the polysaccharide; the modified products still have extremely high molecular weights and a wide distribution. To reduce molecular weight, existing technologies employ acid, alkali, or high-temperature thermal degradation methods, but the insolubility of gluconolactone causes the degradation reaction to occur only at heterogeneous interfaces, resulting in random glycosidic bond breakage and an extremely wide molecular weight distribution of the products. While enzymatic hydrolysis offers mild conditions and high specificity, the dense triple helix structure constitutes a physical barrier, making it difficult for enzyme molecules to effectively penetrate the polysaccharide particles, resulting in extremely low enzymatic hydrolysis efficiency.

[0004] Existing technologies have consistently struggled to balance the high molecular weight properties of polysaccharides with their water solubility and molecular weight uniformity. When the molecular weight is too high or the distribution is too wide, some macromolecular components in the product are difficult to dissolve completely; while when the molecular weight is too low, the polysaccharide chains are too short, resulting in the loss of the thickening and stabilizing functional properties necessary for high molecular weight polysaccharides, making it difficult to meet the performance requirements of high molecular weight raw materials in the food and daily chemical industries. Summary of the Invention

[0005] In view of this, the present invention provides a water-soluble carboxymethyl guar gum with a specific molecular weight, its preparation method and application. By combining carboxymethylation modification with a time-limited enzymatic hydrolysis strategy, the molecular weight is uniformly regulated while maintaining the polysaccharide polymer backbone, so that the product has both good water solubility and thickening and stabilizing functional properties.

[0006] In a first aspect, the present invention provides a method for preparing water-soluble carboxymethyl guar gum of a specific molecular weight, comprising the following steps: The gellan gum was suspended in an ethanol solution, alkalized by adding sodium hydroxide solution, and then etherified by adding chloroacetic acid. After the reaction was completed, the product was purified and dried to obtain a carboxymethyl curd gum intermediate. The carboxymethyl guar gum intermediate was prepared into a substrate solution, and β-glucanase was added to carry out an enzymatic hydrolysis reaction for 15-30 min. The enzyme is inactivated by heating, and after purification and drying, a water-soluble carboxymethyl guar gum with a specific molecular weight is obtained.

[0007] Preferably, the concentration of the sodium hydroxide solution is 5~15M, and the ratio of guar gum to sodium hydroxide solution is 1g : (0.2~2.5)mL.

[0008] Preferably, the alkalization temperature is 20~70℃ and the alkalization time is 20~60min.

[0009] Preferably, the molar ratio of chloroacetic acid to guar gum glucose units is (0.25~1.25):1.

[0010] Preferably, the etherification temperature is 20~80℃ and the etherification time is 2~6 h.

[0011] Preferably, the mass-volume concentration of the substrate solution is 3-8%.

[0012] Preferably, the temperature of the enzymatic hydrolysis reaction is 50~60℃, and the pH of the enzymatic hydrolysis reaction is 4.2~4.8.

[0013] Preferably, carboxymethyl guar gum of different molecular weights is obtained by adjusting the dosage of the β-glucanase: When the enzyme dosage was 0.05~0.1 U / g, a weight-average molecular weight of 0.336×10⁻⁶ was obtained. 6 ~0.350×10 6 g / mol of product; When the enzyme dosage was 2.0–4.0 U / g, a weight-average molecular weight of 0.148 × 10⁻⁶ was obtained. 6 ~0.233×10 6 g / mol of product; When the enzyme dosage was 6.0~10.0 U / g, a weight-average molecular weight of 0.072×10⁻⁶ was obtained. 6 ~0.107×10 6 g / mol of product.

[0014] Secondly, the present invention provides a water-soluble carboxymethyl guar gum of a specific molecular weight prepared by the above preparation method.

[0015] Thirdly, the present invention provides the application of the above-mentioned water-soluble carboxymethyl guar gum of a specific molecular weight in the preparation of food thickeners, stabilizers or texture modifiers.

[0016] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention modifies gellan gum sequentially through carboxymethylation and enzymatic hydrolysis. First, it disrupts the triple helix structure to improve water solubility and expose enzyme cleavage sites. Then, it limits the enzymatic hydrolysis time to a specific range, allowing the substrate molecular chain to be moderately sheared and avoiding complete degradation into oligosaccharide fragments. This achieves uniform molecular weight control while maintaining the high molecular weight properties of polysaccharides, endowing the product with good room temperature water solubility and thickening and stabilizing properties. It effectively overcomes the technical defects of existing technologies that are difficult to balance water solubility, molecular weight uniformity, and the integrity of the polymer backbone. Attached Figure Description

[0017] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0018] Figure 1 This illustrates the effect of different sodium hydroxide addition amounts on the degree of substitution in Example 1 of the present invention. Figure 2 This illustrates the effect of different alkalization temperatures on the degree of substitution in Example 1 of the present invention. Figure 3 This illustrates the effect of different etherification temperatures on the degree of substitution in Example 1 of the present invention. Figure 4 This illustrates the effect of different etherification times on the degree of substitution in Example 1 of the present invention. Figure 5 This illustrates the effect of different molar ratios of chloroacetic acid and guarana glucose units on the degree of substitution in Example 1 of the present invention. Figure 6 The effects of different enzymatic hydrolysis times and enzyme dosages on the weight-average molecular weight of carboxymethyl guar gum in Examples 7-9 of this invention; Figure 7 The Fourier transform infrared (FT-IR) spectra of natural curd gum and carboxymethyl curd gum intermediates with different degrees of substitution in Experimental Example 1 of this invention are shown. Figure 8 The X-ray diffraction (XRD) patterns of natural currant and carboxymethyl currant intermediates with different degrees of substitution in Experimental Example 2 of this invention are shown. Figure 9 The maximum absorption wavelength variation curves of natural curcumin and carboxymethyl curcumin intermediates with different degrees of substitution, mixed with Congo red, at different sodium hydroxide concentrations are shown in Experimental Example 3 of this invention. Figure 10 The thermogravimetric-differential thermogravimetric (TG-DTG) curves of natural curd gum and carboxymethyl curd gum intermediates with different degrees of substitution in Experimental Example 4 of this invention are shown. Detailed Implementation

[0019] 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.

[0020] Currant is a microbial extracellular polysaccharide composed of glucose residues linearly linked by β-1,3-glycosidic bonds. Its dense network of hydrogen bonds between molecular chains assembles the polysaccharide chains into a compact triple helix structure, making it extremely difficult to dissolve in room temperature water and common organic solvents, severely limiting its application in food processing and other fields. To address these problems, this invention provides a water-soluble carboxymethyl currant of a specific molecular weight, its preparation method, and its applications.

[0021] In a first aspect, the present invention provides a method for preparing water-soluble carboxymethyl guar gum of a specific molecular weight, comprising the following steps: The gellan gum was suspended in an ethanol solution, alkalized by adding sodium hydroxide solution, and then etherified by adding chloroacetic acid. After the reaction was completed, the product was purified and dried to obtain a carboxymethyl curd gum intermediate. The carboxymethyl guar gum intermediate was prepared into a substrate solution, and β-glucanase was added to carry out an enzymatic hydrolysis reaction for 15-30 min. The enzyme is inactivated by heating, and after purification and drying, a water-soluble carboxymethyl guar gum with a specific molecular weight is obtained.

[0022] In this invention, gellan gum is used as the starting material. Firstly, hydrophilic carboxymethyl groups are introduced into its molecular chain through carboxymethylation modification. The carboxymethylation modification includes two stages: alkalization and etherification. In the alkalization stage, gellan gum is treated with sodium hydroxide solution to fully swell its molecular chain and activate the hydroxyl groups. In the etherification stage, chloroacetic acid is added, and the chloroacetic acid undergoes a nucleophilic substitution reaction with the activated hydroxyl groups, introducing carboxymethyl groups onto glucose residues. The introduction of carboxymethyl groups increases the hydrophilicity of the molecular chain. Furthermore, the negatively charged carboxymethyl groups generate intermolecular electrostatic repulsion, effectively disrupting the original dense triple helix structure of gellan gum. This transforms the molecular chain from a tightly stacked rigid conformation to a relaxed, flexible, randomly coiled conformation, thereby significantly improving the water solubility of the product.

[0023] Subsequently, enzymatic hydrolysis was performed using carboxymethyl curdlan intermediate as a substrate. Because the triple helix structure was disrupted, the molecular chains were extended, and the β-1,3-glycosidic bonds, originally encapsulated by a hydrogen bond network, were fully exposed on the molecular chain surface. β-glucanase could effectively recognize and bind to the substrate, achieving specific cleavage. This invention controls the enzymatic hydrolysis reaction time to 15-30 min to ensure adequate cleavage of the substrate molecular chains. If the hydrolysis time is too short, the molecular weight reduction is insufficient, and the product still contains some large molecular components that are difficult to completely dissolve. In experiments, this invention found that after 15 min of enzymatic hydrolysis, the rate of molecular weight decrease in all dosage groups slowed significantly, entering a degradation plateau. Further extending the hydrolysis time to 30-60 min did not result in significant changes in molecular weight over time. This plateau is likely due to the electrostatic repulsion and steric hindrance effect created by the uniform distribution of carboxymethyl groups on the molecular chain, which forms a physical barrier. Once the exposed glycosidic bonds are cleaved, this steric barrier effectively slows down further enzyme attack on the internal glycosidic bonds. Based on the above observations, limiting the enzymatic hydrolysis time to 15–30 minutes precisely covers the optimal timeframe for the product to reach its target molecular weight and enter a stable state. Since there is no substantial degradation benefit beyond 30 minutes, this upper time limit prevents ineffective reaction processes, thus avoiding unnecessary energy waste in actual industrial scale-up and significantly shortening the production cycle. Simultaneously, avoiding prolonged incubation and mechanical stirring also helps maintain the batch-to-batch stability of the polysaccharide fragments' physicochemical properties and molecular weight distribution.

[0024] In some embodiments of the present invention, the concentration of the sodium hydroxide solution is 5-15 M, for example, it can be any single value or any two values ​​from 5M, 6M, 8M, 10M, 12M, and 15M, and is more preferably 10 M. Within this concentration range, the sodium hydroxide solution can effectively activate the hydroxyl groups on the gellan gum molecular chain, providing sufficient active sites for the subsequent etherification reaction. If the concentration is too low, alkalization is insufficient, the degree of hydroxyl activation is inadequate, and the degree of carboxymethyl substitution is low; if the concentration is too high, it may cause excessive degradation of the polysaccharide backbone or increase side reactions. The ratio of gellan gum to sodium hydroxide solution is 1 g : (0.2-2.5) mL, for example, it can be any single value or any two values ​​from 1 g : 0.2 mL, 1 g : 0.5 mL, 1 g : 1.0 mL, 1 g : 1.5 mL, 1 g : 2.0 mL, and 1 g : 2.5 mL, and is more preferably 1 g : 1.5 mL. The amount of sodium hydroxide used directly affects the alkaline environment of the reaction system and the degree of substrate activation. If the amount is too low, the alkalinity of the system is insufficient, resulting in low efficiency of carboxymethylation reaction. If the amount is too high, the excess alkali may cause degradation of polysaccharide chains and increase the burden of subsequent neutralization treatment.

[0025] In some embodiments of the present invention, the alkalization temperature is 20-70°C, for example, it can be any one value or a range of any two values ​​among 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C, and is more preferably 45°C. The alkalization time is 20-60 min, for example, it can be any one value or a range of any two values ​​among 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, and 60 min, and is more preferably 30 min. The control of alkalization temperature and time is of great significance in the carboxymethylation reaction. When the temperature is too low, the gellan gum molecular chains do not swell sufficiently, and the hydroxyl groups are difficult to expose effectively, affecting the efficiency of subsequent substitution reactions; when the temperature is too high, it may aggravate the degradation of the polysaccharide backbone. 45°C is the optimal alkalization temperature determined by the present invention after optimization. At this temperature, the degree of swelling of the gellan gum and the stability of the molecular chains are well balanced. An alkalization time of 20-60 minutes ensures that the gellan gum is fully activated under alkaline conditions. If the time is too short, the activation will be insufficient, and if the time is too long, it may cause unnecessary energy consumption and side reaction risks.

[0026] In some embodiments of the present invention, the molar ratio of chloroacetic acid to currant glucose units is (0.25~1.25):1, for example, it can be any single value or a range of any two values ​​from 0.25:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.25:1, and is more preferably 1:1. Chloroacetic acid is the carboxymethylating agent in the etherification reaction, and its amount directly determines the degree of carboxymethyl substitution in the product. When the amount of chloroacetic acid is too low, there are insufficient carboxymethyl groups available, the degree of substitution is low, and the improvement in the water solubility of the product is limited; when the amount is too high, the excess chloroacetic acid may lead to a decrease in the pH of the reaction system, an increase in side reactions, and when the carboxymethyl substitution is too high, the excessive negative charge on the molecular chain may affect the rheological properties of the product, while increasing the reagent cost. When the molar ratio is 1:1, the number of moles of chloroacetic acid and glucose units is roughly equal, which can ensure a high degree of substitution while avoiding reagent waste.

[0027] In some embodiments of the present invention, the etherification temperature is 20~80℃, for example, it can be any one value or a range of any two values ​​from 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, and 80℃, and is more preferably 55℃. The etherification time is 2~6 h, for example, it can be any one value or a range of any two values ​​from 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, and 6 h, and is more preferably 5 h. The etherification reaction is a process in which chloroacetic acid undergoes nucleophilic substitution with the activated hydroxyl group to form a carboxymethyl ether bond. This reaction requires a certain temperature and time to ensure that the substitution reaction proceeds fully. When the temperature is too low, the reaction rate is slow and the degree of substitution is low in the same amount of time; when the temperature is too high, the decomposition of chloroacetic acid is accelerated, the concentration of effective reactants is reduced, and the stability of the polysaccharide backbone decreases at high temperatures. Within 2-6 hours of etherification, the degree of substitution increases with the extension of reaction time. After 5 hours, the degree of substitution tends to reach equilibrium. Further extending the time has limited effect on increasing the degree of substitution and will increase energy consumption.

[0028] In the step "purification and drying after reaction to obtain carboxymethyl curdlan intermediate", the purification specifically includes: adjusting the pH of the reaction system to neutral with acid (such as 1 M hydrochloric acid solution) after the reaction to neutralize unreacted sodium hydroxide and terminate the reaction; subsequently washing the product repeatedly with 80% ethanol solution, anhydrous ethanol, and acetone to remove unreacted chloroacetic acid, sodium chloride, and other impurities, as well as residual organic solvents; redissolving the washed product in distilled water and transferring it to a dialysis bag with a molecular weight cutoff of 8000-14000 Da, dialyzing in deionized water for 36-60 h to further remove small molecule impurities; after dialysis, freezing-drying or vacuum drying the solution retained in the dialysis bag to obtain the purified carboxymethyl curdlan intermediate. The specific operating parameters for washing, dialysis, and drying can be adjusted according to actual conditions.

[0029] In some embodiments of the present invention, the mass-to-volume concentration of the substrate solution is 3-8%, for example, it can be any single value or a range of any two values ​​from 3%, 4%, 5%, 6%, 7%, to 8%, and is more preferably 5% (i.e., 5 g of the carboxymethyl curdlan intermediate per 100 mL of substrate solution). Substrate concentration is an important factor affecting the efficiency of the enzymatic hydrolysis reaction. If the concentration is too low, the number of substrate molecules per unit volume is small, the collision frequency between the enzyme and the substrate is low, the enzymatic hydrolysis efficiency is low, and a large amount of liquid needs to be processed in subsequent treatments, increasing operating costs. If the concentration is too high, the system viscosity increases, mass transfer resistance increases, enzyme diffusion and uniform substrate dispersion are restricted, and the enzymatic hydrolysis effect may decrease.

[0030] In some embodiments of the present invention, the temperature of the enzymatic hydrolysis reaction is 50-60°C, for example, any single value or a range of any two values ​​from 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, and 60°C, more preferably 55°C. The pH of the enzymatic hydrolysis reaction is 4.2-4.8, for example, any single value or a range of any two values ​​from 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, and 4.8, more preferably 4.5. The activity of β-glucanase is significantly affected by temperature and pH, and β-glucanase from different sources has different optimal reaction conditions. After long-term experimental exploration, the present invention has determined that 55°C and pH 4.5 are the best combination of enzymatic hydrolysis conditions. Under these conditions, the activity of β-glucanase is high, and it can achieve effective cleavage of the substrate within 15-30 minutes. Furthermore, the enzyme itself has good stability, which is beneficial for ensuring batch-to-batch consistency of the enzymatic hydrolysis reaction.

[0031] β-glucanase is the enzymatic hydrolysis reagent selected in this invention, capable of specifically recognizing and cleaving β-1,3-glycosidic bonds. Since gellan gum is entirely composed of β-1,3-glycosidic bonds, β-glucanase exhibits high substrate specificity. Compared to non-specific acid-base degradation methods, the specific cleavage of β-glucanase avoids random breakage of glycosidic bonds and destruction of monosaccharide rings, resulting in a mild and controllable degradation process and a narrower molecular weight distribution of the product. In this invention, the enzymatic hydrolysis reaction is terminated by heat inactivation of the enzyme. Specifically, after the set hydrolysis time has elapsed, the reaction system is placed in a boiling water bath at 100°C and heated for 5 minutes to denature and inactivate the β-glucanase, thereby terminating the enzymatic hydrolysis reaction. This heat inactivation method is simple to operate, low in cost, and can quickly terminate the reaction, preventing further degradation of the product due to residual enzyme activity during subsequent processing.

[0032] In some embodiments of the present invention, carboxymethyl guar gum of different molecular weights is obtained by adjusting the dosage of the β-glucanase: When the enzyme dosage was 0.05~0.1 U / g, a weight-average molecular weight of 0.336×10⁻⁶ was obtained. 6 ~0.350×10 6 g / mol of product; When the enzyme dosage was 2.0–4.0 U / g, a weight-average molecular weight of 0.148 × 10⁻⁶ was obtained. 6 ~0.233×10 6 g / mol of product; When the enzyme dosage was 6.0~10.0 U / g, a weight-average molecular weight of 0.072×10⁻⁶ was obtained. 6 ~0.107×10 6 g / mol of product.

[0033] In this invention, the enzyme dosage refers to the number of β-glucanase activity units (U / g) added per gram of substrate (carboxymethyl curdlan gum intermediate). Within the same enzymatic hydrolysis time (15-30 min), the enzyme dosage is a key regulatory parameter determining the molecular weight of the product. At lower enzyme dosages (0.05-0.1 U / g), the number of glycosidic bonds that can be cleaved per unit time is limited, the substrate undergoes only limited hydrolysis, and the degree of molecular chain cleavage is low. Therefore, the product molecular weight is maintained at a higher level (0.336 × 10⁻⁶). 6 ~0.350×10 6 When the enzyme dosage is appropriate (2.0~4.0 U / g), more glycosidic bonds are effectively cleaved, the substrate molecular chain is cut into fragments of appropriate length, and the molecular weight of the product is reduced to a moderate level (0.148×10⁻⁶ g / mol). 6 ~0.233×10 6 At higher enzyme doses (6.0–10.0 U / g), sufficient enzyme activity allows for more thorough cleavage of the substrate molecular chain within a limited time, reducing the product molecular weight to a lower level (0.072 × 10⁻⁶ g / mol). 6 ~0.107×10 6 (g / mol). By adjusting the dosage of the three different enzymes mentioned above, this invention can flexibly prepare a series of products covering a wide molecular weight range to meet the differentiated requirements of polysaccharide molecular weight for different application scenarios.

[0034] In the step "heating to inactivate enzymes, followed by purification and drying to obtain water-soluble carboxymethyl guar gum of a specific molecular weight", the purification specifically includes: after heating to inactivate the enzymes, cooling the reaction system to room temperature, centrifuging at 6000~10000 g for 10~30 min to remove any denatured enzyme protein aggregates and other insoluble substances that may be present in the system; collecting the supernatant, adding ethanol to the supernatant for alcohol precipitation (the amount of ethanol added should be such that the final ethanol concentration in the system reaches 70%~75%), so that the carboxymethyl guar gum precipitates out of the solution; collecting the precipitate, washing the precipitate repeatedly with anhydrous ethanol 4~6 times to further remove residual water and small molecule impurities; redissolving the washed precipitate in distilled water, and dialyzing again to remove residual ethanol and other small molecule impurities; finally, freeze-drying or vacuum drying to obtain the purified water-soluble carboxymethyl guar gum final product of a specific molecular weight. The combined use of alcohol precipitation and dialysis can effectively remove salts, oligosaccharide fragments, and residual organic solvents from the enzymatic hydrolysis system, ensuring the purity and quality of the final product. Freeze-drying or vacuum drying are gentle drying methods that protect the polysaccharide molecular chain structure and can avoid molecular chain aggregation or degradation that may be caused by high-temperature drying.

[0035] Secondly, the present invention provides a water-soluble carboxymethyl currant with a specific molecular weight prepared by the above-described preparation method. The degree of substitution of the carboxymethyl currant is 0.21 to 0.63.

[0036] The degree of substitution (DS) refers to the average number of hydroxyl groups substituted by carboxymethyl groups on each glucose unit, reflecting the grafting density of carboxymethyl groups on the molecular chain. Within the DS range of 0.21–0.63, an appropriate number of hydrophilic carboxymethyl groups are introduced into the molecular chain. When the DS is below 0.21, the introduced hydrophilic groups are insufficient to disrupt the triple helix structure, and the product's solubility in room temperature water remains low. When the DS reaches 0.63, the introduction of carboxymethyl groups is sufficient to disrupt the triple helix structure and impart sufficient electrostatic repulsion to the molecular chain to form a regular honeycomb-like three-dimensional network. The weight-average molecular weight M of the product is... w It is 0.072×10 6 ~0.350×10 6 g / mol.

[0037] In some embodiments of the present invention, the solubility of the carboxymethyl gellan gum in aqueous solution at room temperature is 12.50%~90.01%, and the absolute value of the Zeta potential is 12.60~56.67 mV. The significant increase in solubility is attributed to the introduction of carboxymethyl groups, which increases the hydrophilicity of the molecular chains, while electrostatic repulsion disrupts the tendency for aggregation between molecular chains. The increase in the absolute value of the Zeta potential indicates that the molecular chain surface carries a large number of negative charges, enhancing the electrostatic repulsion between molecular chains, which is beneficial for improving the dispersion stability in aqueous solution and preventing lateral aggregation and sedimentation of the molecular chains.

[0038] Thirdly, the present invention provides the application of the above-mentioned water-soluble carboxymethyl guar gum of a specific molecular weight in the preparation of food thickeners, stabilizers or texture modifiers.

[0039] The carboxymethyl cordanum is applied to food systems in the form of an aqueous solution, with a final concentration of 0.1% to 5% (w / v). For example, it can be any single value or a range of any two values ​​from 0.1%, 0.2%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, to 5.0%. When the concentration is below 0.1%, the carboxymethyl cordanum is unlikely to exert an effective thickening and stabilizing effect in the food system, and its improvement on the system's rheology and texture is not significant. When the concentration is above 5%, the excessively high viscosity of the system may affect the taste and processing suitability of the food, and increase raw material costs. In practical applications, those skilled in the art can flexibly adjust the amount added within the above range according to the specific type of food system and the expected thickening and stabilizing effect.

[0040] The ethanol solution, sodium hydroxide solution, β-glucanase and other reagents described in this invention can all be obtained through commercial channels. The β-glucanase is preferably an endoglucanase that can specifically cleave β-1,3-glycosidic bonds.

[0041] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0042] Example 1 This embodiment provides a single-factor optimization of the preparation conditions for carboxymethyl curdranol intermediate.

[0043] In this embodiment, the preparation method of the carboxymethyl chloroform gum intermediate is as follows: 6.0 g of curdlan gum was suspended in an ethanol solution, and sodium hydroxide solution was added. The mixture was stirred and alkalized for a set time at a predetermined temperature. Chloroacetic acid (the molar ratio to the curdlan gum glucose units was set according to the experimental design) was added, and the reaction was carried out at a set temperature for a set time for etherification. After the reaction was complete, the pH was adjusted to neutral using 1 M hydrochloric acid solution. The product was washed successively with 80% ethanol solution, anhydrous ethanol, and acetone, redissolved in distilled water, dialyzed for 48 h, and freeze-dried to obtain the carboxymethyl curdlan gum intermediate. Its degree of substitution was determined by conductivity titration.

[0044] (1) Effect of sodium hydroxide addition amount: With a fixed molar ratio of chloroacetic acid to gluconate glucose units of 1:1, an alkalization temperature of 45℃, an etherification temperature of 55℃, and an etherification time of 5 h, the effect of the amount of 10 M sodium hydroxide solution added (3~15 mL) on the degree of substitution was investigated, using 6 g of gluconate as a baseline. Figure 1 The results showed that the degree of substitution first increased and then decreased with the increase of sodium hydroxide addition, and the degree of substitution reached its peak when the addition amount was 9 mL.

[0045] (2) Effect of alkalization temperature: With a fixed molar ratio of chloroacetic acid to guar gum glucose units of 1:1, an addition of 9 mL of 10 M sodium hydroxide, an etherification temperature of 55 °C, and an etherification time of 5 h, the effect of alkalization temperature (20–70 °C) on the degree of substitution was investigated. Figure 2 The results showed that the degree of substitution was highest at an alkalization temperature of 45℃.

[0046] (3) Effect of etherification temperature: With a fixed molar ratio of chloroacetic acid to guar gum glucose units of 1:1, an addition of 9 mL of 10 M sodium hydroxide, an alkalization temperature of 45 °C, and an etherification time of 5 h, the effect of etherification temperature (20–80 °C) on the degree of substitution was investigated. Figure 3The results showed that the degree of substitution was highest at an etherification temperature of 55℃.

[0047] (4) Effect of etherification time: With a fixed molar ratio of chloroacetic acid to guar gum glucose units of 1:1, an addition of 9 mL of 10 M sodium hydroxide, an alkalization temperature of 45 °C, and an etherification temperature of 55 °C, the effect of etherification time (1–8 h) on the degree of substitution was investigated. Figure 4 The results showed that the degree of substitution increased with increasing reaction time before 5 h of etherification, and tended to reach equilibrium after 5 h.

[0048] (5) Effect of chloroacetic acid dosage: With a fixed addition of 9 mL of 10 M sodium hydroxide, an alkalization temperature of 45 °C, an etherification temperature of 55 °C, and an etherification time of 5 h, the effect of the molar ratio of chloroacetic acid to glucose units of gellan gum (0.1~1.8:1) on the degree of substitution was investigated. Figure 5 The results showed that the substitution effect was best when the molar ratio was 1:1, with a substitution degree of up to 0.63; excessive chloroacetic acid would exacerbate the degradation of the main chain and reduce the pH of the system.

[0049] Example 2 This embodiment provides a method for preparing a carboxymethyl curd gum intermediate (CM-Cur 4).

[0050] 6.0 g of curdlan gum was suspended in an ethanol solution, and 9 mL of 10 M sodium hydroxide solution was added. The mixture was stirred and alkalized at 45 °C for 30 min. 4.2 g of chloroacetic acid (the molar ratio of chloroacetic acid to curdlan gum glucose units was 1:1) was added, and the mixture was etherified at 55 °C for 5 h. After the reaction, the pH was adjusted to neutral using 1 M hydrochloric acid solution. The product was washed successively with 80% ethanol solution, anhydrous ethanol, and acetone, reconstituted in distilled water, and transferred to a dialysis bag with a molecular weight cutoff of 8000–14000 Da. Dialysis was performed in deionized water for 48 h. After freeze-drying under vacuum, a white, flocculent powder of carboxymethyl curdlan gum intermediate was obtained, named CM-Cur 4. Its degree of substitution was determined to be 0.63, its solubility in water at room temperature was 90.01%, and its weight-average molecular weight was 0.581 × 10⁻⁶. 6 g / mol.

[0051] Example 3 The only difference between this embodiment and Example 2 is that the molar ratio of chloroacetic acid to currant glucose unit is 0.25:1, resulting in a carboxymethyl currant intermediate with a degree of substitution of 0.21, denoted as CM-Cur 1.

[0052] Example 4 The only difference between this embodiment and Example 2 is that the molar ratio of chloroacetic acid to currant glucose unit is 0.50:1, resulting in a carboxymethyl currant intermediate with a degree of substitution of 0.33, denoted as CM-Cur 2.

[0053] Example 5 The only difference between this embodiment and Example 2 is that the molar ratio of chloroacetic acid to currant glucose unit is 0.75:1, resulting in a carboxymethyl currant intermediate with a degree of substitution of 0.45, denoted as CM-Cur 3.

[0054] Example 6 The only difference between this embodiment and Example 2 is that the molar ratio of chloroacetic acid to currant glucose unit is 1.25:1, resulting in a carboxymethyl currant intermediate with a degree of substitution of 0.50, denoted as CM-Cur 5.

[0055] Example 7 This embodiment provides a time-limited degradation method for preparing high molecular weight carboxymethyl guar gum using a bio-enzymatic method.

[0056] Using the carboxymethyl curazon intermediate CM-Cur 4 obtained in Example 2 as the substrate, a substrate solution with a mass-to-volume concentration of 5% (i.e., 5 g substrate per 100 mL of solution) was prepared. The pH of the system was adjusted to 4.5, and the solution was equilibrated in a 55°C water bath for 10 min. 0.05 U / g of β-glucanase was added according to the dry weight of the substrate to initiate an enzymatic hydrolysis reaction. Samples were taken at different reaction times. Immediately after the enzymatic hydrolysis reaction, the reaction system was placed in a 100°C boiling water bath for 5 min to inactivate the enzyme and terminate the reaction. After cooling to room temperature, the solution was centrifuged at 8000 g for 15 min. The supernatant was collected, and ethanol (final ethanol concentration 75%) was added to the supernatant for alcohol precipitation. The precipitate was collected and washed repeatedly with anhydrous ethanol 5 times. The washed precipitate was redissolved in distilled water, dialyzed again to remove residual small molecule impurities, and then freeze-dried to obtain high molecular weight carboxymethyl curazon. The molecular weights at different reaction times are shown below. Figure 6 As shown, the weight-average molecular weight of the product after the reaction was terminated at 5 minutes was determined to be 0.428 × 10⁻⁶. 6 The PDI was 1.297 g / mol; the weight-average molecular weight of the product after the reaction was terminated at 15 min was 0.350 × 10⁻⁶ g / mol. 6 The PDI was 1.397 g / mol; the weight-average molecular weight of the product stabilized at 0.336 × 10⁻⁶ after the reaction was terminated at 30 min. 6 The PDI was 1.294 g / mol; the weight-average molecular weight of the product after the reaction was terminated at 60 min was 0.337 × 10⁻⁶ g / mol. 6 g / mol, PDI is 1.313.

[0057] Example 8 The only difference between this embodiment and Example 7 is that 4.0 U / g of β-glucanase is added in this embodiment based on the dry weight of the substrate.

[0058] Molecular weight at different reaction times, such as Figure 6 As shown, the weight-average molecular weight of the product after the reaction was terminated at 5 minutes was determined to be 0.350 × 10⁻⁶. 6 g / mol, PDI 1.375; the weight-average molecular weight of the product terminated after 15 min was 0.170 × 10⁻⁶ g / mol. 6 g / mol, PDI 1.302; the weight-average molecular weight of the product stabilized at 0.148 × 10⁻⁶ g / mol after the reaction was terminated at 30 min. 6 The PDI was 1.552 g / mol; the weight-average molecular weight of the product after the reaction was terminated at 60 min was 0.147 × 10⁻⁶ g / mol. 6 g / mol, PDI is 1.308.

[0059] Example 9 The only difference between this embodiment and Example 7 is that 8.0 U / g of β-glucanase is added based on the dry weight of the substrate in this embodiment.

[0060] Molecular weight at different reaction times, such as Figure 6 As shown, the weight-average molecular weight of the product after the reaction was terminated at 5 minutes was determined to be 0.155 × 10⁻⁶. 6 The PDI was 1.228 g / mol; the weight-average molecular weight of the product after the reaction was terminated at 15 min was 0.078 × 10⁻⁶ g / mol. 6 The PDI was 1.639 g / mol; the weight-average molecular weight of the product stabilized at 0.072 × 10⁻⁶ g / mol after the reaction was terminated at 30 min. 6 The PDI was 1.309 g / mol; the weight-average molecular weight of the product after the reaction was terminated at 60 min was 0.071 × 10⁻⁶ g / mol. 6 g / mol, PDI is 1.181.

[0061] Test case 1. Fourier Transmission Infrared Spectroscopy (FT-IR) Analysis The natural curd gum and the carboxymethyl curd gum intermediates (CM-Cur 1~CM-Cur 5) prepared in Examples 2~6 were subjected to Fourier transform infrared (FT-IR) spectroscopy tests, such as... Figure 7 As shown, natural guar gum is at 3317 cm⁻¹. -1 A broad peak of OH stretching vibration was observed nearby. Compared with natural curdlan gum, the carboxymethyl modified samples showed a peak at 1607 cm⁻¹. -1 and 1418 cm -1 A pair of new peaks appeared, which were attributed to the carboxylate group (-COO).- The asymmetric and symmetric stretching vibration peaks of the product were observed, and the intensity of these two characteristic peaks gradually increased with increasing degree of substitution. These results indicate that carboxymethyl groups have been successfully grafted onto the gellan gum molecular chain, and the higher the degree of substitution, the greater the grafting density of carboxymethyl groups.

[0062] 2. X-ray diffraction (XRD) analysis XRD analysis was performed on natural currant and the carboxymethyl currant intermediate samples (CM-Cur 1~CM-Cur 5) prepared in Examples 2-6. Figure 8 As shown, the results indicate that natural currant exhibits distinct crystal diffraction peaks at 2θ of 11.8° and 29.6°, suggesting a highly ordered triple-helix crystal structure. For the carboxymethyl currant intermediate sample, these two characteristic diffraction peaks disappear, indicating that the introduction of carboxymethyl groups disrupts the original triple-helix crystal structure of currant and has a significant unwinding effect on the triple-helix conformation.

[0063] 3. Congo Red Experiment Natural curd gum and the carboxymethyl curd gum intermediates prepared in Examples 2-6 were taken separately and mixed with Congo red reagent (80 μmol / L). The maximum absorption wavelength λ was then measured in sodium hydroxide solutions of different concentrations (0-0.5 M). max The changes.

[0064] Figure 9 The results showed that natural currant gum, when combined with Congo red under alkaline conditions, exhibited λ max A significant red shift (from 496 nm to 512 nm) indicates that its triple helix structure remains intact under alkaline conditions, enabling it to form a stable complex with Congo red. With increasing carboxymethyl substitution, λ... max The redshift gradually decreases; when the degree of substitution reaches 0.49 or higher, λ max The absence of redshift indicates that the triple helix structure has unwound into a single helix or a random coil conformation. This experimental result, corroborated by XRD analysis, confirms the significant impact of carboxymethylation modification on the orderly-to-disorder transformation of the gellan triple helix structure.

[0065] 4. Thermogravimetric-Differential Thermogravimetric (TG-DTG) Analysis Natural curcumin and the carboxymethyl curcumin intermediates prepared in Examples 2-6 were taken and subjected to thermogravimetric analysis using a simultaneous thermal analyzer. The test conditions were: heating rate 10℃ / min, heating range from room temperature to 600℃, nitrogen atmosphere protection, and flow rate 20 mL / min.

[0066] Figure 10The results showed that the thermal degradation process of both natural currant and carboxymethyl modified samples exhibited three stages: the first stage was the weight loss of adsorbed water below 150℃; the second stage was the main thermal degradation stage of main chain scission at 200-400℃; and the third stage was the deep carbonization stage above 400℃. The maximum pyrolysis rate temperature T0 of natural currant in the second stage was [not specified]. max The temperature was 308℃. As the degree of carboxymethyl substitution increased, the temperature (T) of each modified sample increased. max Gradually decrease; when the degree of substitution reaches 0.63, T max The temperature dropped significantly to 240-250℃. These results indicate that the introduction of carboxymethyl groups weakened the hydrogen bond network between the gellan gum molecular chains, causing the rigid triple helix structure to unwind into a flexible, randomly coiled conformation, leading to a decrease in the thermal stability of the polysaccharide backbone.

[0067] 5. Determination of solubility and Zeta potential The solubility of CM-Cur was determined using the aluminum box constant weight method. A covered aluminum box was dried in an oven at 105°C until constant weight. After cooling in a desiccator for 30 min, the box was weighed, and this process was repeated until the difference in mass between two weighings did not exceed 2 mg. The CM-Cur sample was thoroughly dissolved in deionized water to obtain a 4% (w / v) sample solution, which was then centrifuged at 8000 g for 30 min at the same temperature. The supernatant was dried at 80°C for 12 h, and the final constant weight was retained. The solubility was calculated using the following formula:

[0068] Where W (g) is the weight of the dried supernatant; V (mL) is the volume of the supernatant; and C (g / mL) is the initial solution concentration.

[0069] The zeta potential (mV) of CM-Cur solution (1.0 mg / mL) was measured using a zeta potential and nanoparticle size analyzer after equilibration at 25 °C for 2 min.

[0070] The test results are shown in Table 1.

[0071] Table 1 Physicochemical parameters of Cur and CM-Cur samples with different degrees of substitution

[0072] Note: All data in the table are expressed as mean ± standard deviation. Different letters in the same column indicate significant differences (p < 0.05). Cur is not modified by carboxymethylation, and its DS is indicated by "-".

[0073] With increasing DS, the water solubility of CM-Cur was greatly improved, increasing from 12.50% in CM-Cur1 to 90.01% in CM-Cur4. Simultaneously, the negative charge density on the molecular surface increased significantly, with the Zeta potential decreasing from -12.60 mV in CM-Cur1 to -56.67 mV in CM-Cur4. The absolute value of the Zeta potential directly reflects the surface charge density of the particles; when the absolute value exceeds 30 mV, the system stabilizes due to electrostatic repulsion. The significant increase in the absolute value of the Zeta potential in the CM-Cur series samples is attributed to the successful grafting of numerous -COO- groups onto the polysaccharide backbone. The resulting strong intermolecular electrostatic repulsion effectively overcomes the tendency of polysaccharide chain aggregation, thus endowing it with excellent water solubility.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a water-soluble carboxymethyl guar gum of a specific molecular weight, characterized in that, Includes the following steps: The gellan gum was suspended in an ethanol solution, alkalized by adding sodium hydroxide solution, and then etherified by adding chloroacetic acid. After the reaction was completed, the product was purified and dried to obtain a carboxymethyl curd gum intermediate. The carboxymethyl guar gum intermediate was prepared into a substrate solution, and β-glucanase was added to carry out an enzymatic hydrolysis reaction for 15-30 min. The enzyme is inactivated by heating, and after purification and drying, a water-soluble carboxymethyl guar gum with a specific molecular weight is obtained.

2. The preparation method according to claim 1, characterized in that, The concentration of the sodium hydroxide solution is 5~15M, and the ratio of guar gum to sodium hydroxide solution is 1g : (0.2~2.5)mL.

3. The preparation method according to claim 1, characterized in that, The alkalization temperature is 20~70℃, and the alkalization time is 20~60min.

4. The preparation method according to claim 1, characterized in that, The molar ratio of chloroacetic acid to guar gum glucose units is (0.25~1.25):

1.

5. The preparation method according to claim 1, characterized in that, The etherification temperature is 20~80℃, and the etherification time is 2~6 h.

6. The preparation method according to claim 1, characterized in that, The mass-volume concentration of the substrate solution is 3-8%.

7. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis reaction is carried out at a temperature of 50-60℃ and a pH of 4.2-4.

8.

8. The preparation method according to claim 1, characterized in that, By adjusting the dosage of the β-glucanase, carboxymethyl guar gums of different molecular weights were obtained. When the enzyme dosage was 0.05~0.1 U / g, a weight-average molecular weight of 0.336×10⁻⁶ was obtained. 6 ~0.350×10 6 g / mol of product; When the enzyme dosage was 2.0–4.0 U / g, a weight-average molecular weight of 0.148 × 10⁻⁶ was obtained. 6 ~0.233×10 6 g / mol of product; When the enzyme dosage was 6.0~10.0 U / g, a weight-average molecular weight of 0.072×10⁻⁶ was obtained. 6 ~0.107×10 6 g / mol of product.

9. A water-soluble carboxymethyl guar gum of a specific molecular weight prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the specific molecular weight water-soluble carboxymethyl guar gum as described in claim 9 in the preparation of food thickeners, stabilizers or texture modifiers.

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