Modified extraction method of seaweed gracilaria lemaneiformis polysaccharide and method for preparing lutein coating by using seaweed gracilaria lemaneiformis polysaccharide

Abstract

The invention belongs to the technical field of algal polysaccharide extraction, and discloses a modification extraction method of algal asparagus polysaccharide and a method for preparing a lutein coating by using the algal asparagus polysaccharide. The modified extraction method comprises the following steps: extracting gracilaria lemaneiformis powder with a weak base solution, enabling lactide and polysaccharide to be subjected to grafting reaction through a redox initiation system under the assistance of ultrasonic waves, and then separating out a first polysaccharide component with high emulsibility and a second polysaccharide component with high gel property through graded alcohol precipitation. The two obtained functional components are used for coating xanthophyll, specifically, the first polysaccharide component emulsifies a xanthophyll oil phase to form a pre-emulsion, the second polysaccharide component is compounded with gelatin, a composite coacervate is formed on an oil drop interface by adjusting the pH value, and finally drying and curing are conducted. According to the method, two modified polysaccharides with definite functions can be synchronously obtained, the prepared xanthophyll coating is high in encapsulation efficiency, good in storage and processing stability and excellent in water dispersibility, and the problems that xanthophyll is unstable and difficult to apply are effectively solved.

Description

A method for modifying and extracting polysaccharides from the seaweed Gracilaria fusiforme and a method for preparing lutein-coated compounds from them. Technical Field

[0001] This invention belongs to the field of seaweed polysaccharide extraction technology, specifically relating to a modified extraction method for seaweed Gracilaria polysaccharide and a method for preparing lutein-coated compounds using it. Background Technology

[0002] Lutein is an important fat-soluble carotenoid with significant antioxidant, blue light filtering, and visual health protection functions. However, lutein itself has poor water solubility and is extremely sensitive to light, heat, and oxygen, making it highly susceptible to degradation during processing and storage, resulting in low bioavailability and limited efficacy. This severely restricts the application of lutein in functional foods, pharmaceuticals, and other fields. To improve the stability and application range of lutein, microencapsulation technology has become a key solution for the industry.

[0003] Currently, encapsulation systems based on natural polysaccharides have attracted much attention due to their high safety and mild conditions. Research primarily focuses on utilizing natural polysaccharides to crosslink through intermolecular forces, constructing hydrophilic networks or shells to effectively encapsulate, protect, and control the release of lutein. Examples include using sodium alginate and curdlan gum to form hydrophilic gel networks through ionic crosslinking to encapsulate lutein, and using chitosan and sodium alginate for layer-by-layer self-assembly to encapsulate lutein. The former achieves ionic crosslinking through ionic bonds, while the latter achieves electrostatic crosslinking through electrostatic interactions. Both can form hydrophilic gel or dense shell structures, effectively isolating lutein from oxygen, light, and heat to some extent. However, these two types of physical encapsulation systems have certain limitations. Specifically, firstly, the crosslinking in both methods occurs between hydrophilic polysaccharide chains, and the resulting barrier lacks effective hydrophobic interactions or chemical anchoring between the barrier and the hydrophobic lutein core. Therefore, the encapsulation remains within the scope of physical encapsulation, and the risk of core material migration and leakage still exists. Furthermore, the stability of the ionic cross-linked network and the integrity of its layer-by-layer assembly structure are highly dependent on the environmental ionic strength and pH. In the dynamic gastrointestinal environment, the swelling, dissociation, or recombination of the network is difficult to control precisely, posing a risk of unstable release kinetics and unsatisfactory targeting. Secondly, although the dense structures formed by the two cross-linking methods mentioned above can block some oxygen and light, the hydrophilic polysaccharide gel matrix itself is a good oxygen diffusion medium, and its oxygen blocking capacity has a physical upper limit, failing to provide fundamental protection for the highly oxidizable lutein.

[0004] To overcome this bottleneck, developing novel wall materials that combine good biocompatibility, excellent hydrophobic compatibility, and high barrier properties is crucial. Seaweed-derived polysaccharides, especially those from Gracilaria lemaneiformis, are considered a highly promising basic material due to their natural, edible nature and inherent gel-forming ability. The natural carboxyl and sulfate functional groups on the Gracilaria lemaneiformis polysaccharide molecular chain provide the structural basis for its functional modification. However, the strongly hydrophilic backbone structure of unmodified natural Gracilaria lemaneiformis polysaccharides makes it difficult to form a strong and dense encapsulation interface with hydrophobic cores such as lutein. If conventional extraction and encapsulation processes are used directly, the resulting products still struggle to overcome the aforementioned key challenges such as poor encapsulation and insufficient oxidative protection.

[0005] Therefore, how to precisely extract and structurally modify Gracilaria polysaccharides to directionally endow them with hydrophobic binding ability and enhance their barrier properties is a core issue that must be solved to transform it from a "potential raw material" into a "high-performance encapsulation wall material," a problem that existing technologies have not fully revealed. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a modified extraction method for Gracilaria fusiforme polysaccharides and a method for preparing lutein-coated compounds using them. The method provided by this invention modifies the Gracilaria fusiforme polysaccharides during the extraction process, directionally endowing them with hydrophobic binding capabilities. When used to coat lutein, it can form a tight hydrophobic interaction and chemical anchoring with lutein, which not only significantly reduces the risk of lutein migration and leakage from the core material; but also the coating layer formed by the modified polysaccharide has excellent oxygen barrier properties, fundamentally isolating lutein from the intrusion of oxygen, light, and heat, significantly improving the stability of lutein during processing and storage.

[0007] This invention provides a modified extraction method for polysaccharides from the seaweed Gracilaria.

[0008] Specifically, a method for modifying and extracting polysaccharides from the seaweed *Gnaphalium affine* includes the following steps: S1, mixing *Gnaphalium affine* powder with a weak alkaline solution for extraction; then adding a reducing agent, and simultaneously adding an oxidizing agent and lactide solution under ultrasonic treatment to carry out a modification reaction; after the reaction, adjusting the pH value, centrifuging to obtain a first supernatant; adding ethanol to the first supernatant until the ethanol volume percentage is 25%-35%, allowing it to settle and precipitate, then removing the precipitate to obtain a second supernatant; S2, adding ethanol to the second supernatant until the ethanol volume percentage is 50%-60%, allowing it to settle once, centrifuging to collect the precipitate and the third supernatant, washing and drying the obtained precipitate to obtain a first polysaccharide component; continuing to add ethanol to the third supernatant until the ethanol volume percentage is 75%-85%, allowing it to settle a second time, centrifuging to collect the precipitate, washing and drying the obtained precipitate to obtain a second polysaccharide component.

[0009] In some embodiments of the present invention, before mixing the Gracilaria zebrina powder with the weak alkaline solution in step S1, the Gracilaria zebrina powder is pretreated; the pretreatment process includes degreasing, decolorizing, and drying the pulverized algae powder. For example, 80%-90% ethanol solution is used for degreasing and decolorizing.

[0010] In some embodiments of the present invention, in step S1, the weak alkaline solution includes a sodium carbonate solution and / or a sodium bicarbonate solution, and the molar concentration of the weak alkaline solution is 0.1-0.5 mol / L.

[0011] In some embodiments of the present invention, in step S1, the ratio of the Gracilaria zebrina powder to the weak alkaline solution is 1:(20-40). It should be understood that the unit of the ratio in the present invention is g / mL.

[0012] In some embodiments of the present invention, in step S1, the extraction process is as follows: stirring and extracting at 45-65°C for 3-8 hours.

[0013] In some embodiments of the present invention, in step S1, the reducing agent includes at least one of sodium sulfite, sodium bisulfite, potassium bisulfite, and ascorbic acid; the molar concentration of the reducing agent in the reaction system is 1.0-3.0 mmol / L.

[0014] In some embodiments of the present invention, in step S1, the oxidant includes at least one of hydrogen peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate; the molar concentration of the oxidant in the reaction system is 1.0-3.0 mmol / L.

[0015] In some embodiments of the present invention, in step S1, the mass of lactide in the lactide solution is 8%-18% of the mass of the *Gnaphalium affine* powder; preferably, the mass of lactide in the lactide solution is 10%-15% of the mass of the *Gnaphalium affine* powder. It should be noted that when the *Gnaphalium affine* powder is pretreated, the mass of the *Gnaphalium affine* powder is calculated based on the mass of the pretreated *Gnaphalium affine* powder.

[0016] In some embodiments of the present invention, in step S1, the power of the ultrasonic wave is 200-500W and the frequency is 20-40kHz.

[0017] In some embodiments of the present invention, in step S1, the temperature of the modification reaction is 45-65°C, and the reaction time is 2-6 hours. Preferably, the temperature of the modification reaction is 50-60°C, and the reaction time is 3-5 hours.

[0018] In some embodiments of the present invention, in step S1, the pH value is adjusted using hydrochloric acid solution or citric acid solution to adjust the pH value to 6.5-7.5.

[0019] In some embodiments of the present invention, in step S1, the centrifugation speed is 4000-8000 r / min and the centrifugation time is 10-20 minutes.

[0020] In some embodiments of the present invention, in step S1, the temperature of the settling is room temperature (e.g., 0-25°C), and the settling time is 4-12 hours.

[0021] In some embodiments of the present invention, in step S2, the temperature of the first settling precipitation is 0-10°C, and the settling precipitation time is 8-15 hours.

[0022] In some embodiments of the present invention, in step S2, the temperature of the secondary settling precipitation is 0-10°C, and the time of the secondary settling precipitation is 8-15 hours.

[0023] In some embodiments of the present invention, in step S2, the process of washing the precipitate involves washing with anhydrous ethanol and acetone in sequence.

[0024] The present invention also provides a method for preparing lutein-coated compounds, comprising the following steps: (1) dissolving lutein in medium-chain triglycerides (MCT) to prepare a lutein oil solution, obtaining an oil phase; dissolving the first polysaccharide component prepared by the above modified extraction method in water to prepare a first polysaccharide component solution, obtaining an aqueous phase A; mixing the second polysaccharide component prepared by the above modified extraction method with edible gelatin at a mass ratio of (3-4):(1-2), dissolving in hot water to prepare a mixed solution of the second polysaccharide component and edible gelatin, obtaining an aqueous phase B; (2) dissolving the oil... Phase B is mixed with aqueous phase A, and after shearing and homogenization, a pre-emulsion is obtained; then, aqueous phase B is added to the pre-emulsion, and the pH of the mixture is adjusted to 4.0-4.5 to carry out a complex coagulation reaction to obtain a complex coagulated emulsion; finally, the complex coagulated emulsion is dried to obtain lutein-coated material; wherein, the mass ratio of the oil phase to aqueous phase A and aqueous phase B is 1:(3-5):(3-5); preferably, the mass ratio of the oil phase to aqueous phase A and aqueous phase B is 1:(4-5):(4-5).

[0025] In some embodiments of the present invention, in step (1), the mass percentage of lutein in the lutein oil solution is 5%-10%.

[0026] In some embodiments of the present invention, in step (1), the mass percentage of the first polysaccharide component in the first polysaccharide component solution is 1.0%-2.0%. Preferably, in step (1), the mass percentage of the first polysaccharide component in the first polysaccharide component solution is 1.0%-1.5%. Lower concentrations are beneficial for the amphiphilic molecules (the first polysaccharide component obtained after modification) to fully extend, and their hydrophobic PLA segments are more likely to anchor lutein in the oil phase.

[0027] In some embodiments of the present invention, in step (1), the total mass percentage of the second polysaccharide component and the edible gelatin in the mixed solution is 1.5%-2.5%. Preferably, in step (1), the total mass percentage of the second polysaccharide component and the edible gelatin in the mixed solution is 1.8%-2.2%. By pre-preparing the mixed solution of the second polysaccharide component and the edible gelatin and controlling the amounts of both, the negatively charged second polysaccharide component and the positively charged gelatin can be pre-mixed in the solution, which is beneficial for subsequent rapid electrostatic recombination.

[0028] In some embodiments of the present invention, in step (2), during the shearing process, the rotation speed is 6000-10000 rpm and the processing time is 1-5 min; the pressure of the homogenization process is 4-6 MPa, and the cycle is 2-3 times.

[0029] In some embodiments of the present invention, in step (2), the temperature of the composite coagulation reaction is 40-55°C and the time is 30-60 minutes.

[0030] In some embodiments of the present invention, in step (2), the drying is spray drying or freeze drying. The inlet air temperature of the spray dryer is 150-170°C, and the outlet air temperature is 80-90°C.

[0031] This invention involves chemically grafting and modifying Gracilaria polysaccharides during the extraction process, directionally grafting hydrophobic functional groups onto the Gracilaria polysaccharide molecular chains. Then, through fractional alcohol precipitation, a first polysaccharide component with high emulsifying properties and a second polysaccharide component with high gelling properties are obtained. In the process of encapsulating lutein, the first polysaccharide component acts as a functional aid and interface modifier. Its amphiphilic structure significantly reduces the interfacial tension between lutein and the aqueous polysaccharide phase, promoting uniform dispersion of the core material and stability in the early stages of encapsulation, and improving the overall processing rheology of the complex. The second polysaccharide component serves as the wall material matrix framework. Its deeply grafted hydrophobic segments provide strong hydrophobic interactions, tightly binding with the lutein core and forming a dense gel network, achieving physical barrier properties and improving the stability of the lutein-encapsulated material. In the process of encapsulating lutein, by controlling the ratio of the first polysaccharide component to the second polysaccharide component, as well as the amount of the second polysaccharide component and gelatin, a high encapsulation rate can be ensured to meet the requirements of rapid dispersion. At the same time, the risk of lutein migration and leakage in the core material can be significantly reduced, thus significantly improving the stability of lutein during processing and storage. Furthermore, the lutein-encapsulated material formed by encapsulating lutein with the modified and extracted Gracilaria polysaccharide of this invention exhibits strong stability, and its stability is no longer highly dependent on environmental ionic strength and pH. In the dynamic gastrointestinal environment, release kinetics can be precisely controlled to achieve a more ideal targeted release effect.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the process of preparing Gracilaria polysaccharide, the present invention extracts Gracilaria algae powder with a weak alkaline solution, and then, under the assistance of ultrasound, uses an oxidation-reduction initiation system to cause a grafting reaction between lactide and polysaccharide, and then separates the first polysaccharide component with high emulsification and the second polysaccharide component with high gelation by fractional alcohol precipitation. The present invention successfully prepares two functionally complementary modified polysaccharide components from Gracilaria simultaneously through an integrated process of extraction, grafting modification and fractional alcohol precipitation. This method realizes the functionalization and fractionation of raw materials in one step. The prepared first and second polysaccharide components have excellent solubility and dispersion properties, and the first polysaccharide component has strong emulsification stability; moreover, its extraction process is simple and efficient, opening up a new way for the high-value utilization of seaweed polysaccharide (Gracilaria polysaccharide).

[0033] (2) In the Gracilaria polysaccharide prepared by this invention, the first polysaccharide component has excellent emulsification stability (ESI value can reach more than 400 min), and can effectively form and stabilize nanoscale pre-emulsions; the second polysaccharide component has good gel-forming ability and solubility. Both components are optimized through experimental examples, and their performance is adjustable, providing ideal polysaccharide raw materials for constructing high-performance coating systems. By combining the two modified polysaccharides mentioned above with gelatin, lutein-coated products were prepared through pre-emulsification and electrostatic composite coagulation. The encapsulation rate of lutein was greater than 90%, reaching as high as 94%, which greatly reduced the processing loss of active ingredients. Under accelerated testing conditions (40℃, 75%RH, 6 months), the lutein retention rate remained above 87%, reaching as high as 92.3%, indicating that the coating layer has a very strong antioxidant and anti-migration barrier effect. Moreover, after heat treatment at 85℃ for 10 minutes, the loss rate of active ingredients was less than 10%, as low as 7.2%, proving that the coating system has strong processing tolerance. In addition, the obtained lutein-coated product is a free-flowing powder that can be rapidly dispersed in an aqueous phase within 30 seconds, making it convenient for use in various liquid foods, health products, and pharmaceuticals.

[0034] (3) This invention successfully transforms the polysaccharide of Gracilaria into a high-performance encapsulation wall material through precise extraction and structural modification. This effectively solves the key problems existing in the current lutein encapsulation technology, such as poor encapsulation, insufficient oxidative protection, and poor processing tolerance. It provides strong technical support for the widespread application of lutein in functional foods, pharmaceuticals and other fields. Detailed Implementation

[0035] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0036] Unless otherwise specified, the raw materials, reagents or apparatus used in the following examples and comparative examples are available from conventional commercial sources or can be obtained by existing known methods.

[0037] In the following examples and experimental cases, the test methods for polysaccharide yield, solubility and emulsification effect are as follows: (1) Yield: The mass of the first and second polysaccharide components were weighed after drying by weighing method, and the mass percentage relative to the pretreated algal powder was calculated.

[0038] (2) Solubility: Take 1g of sample and add it to 50mL of deionized water. Treat it under ultrasonic (power 300w) for 30s and observe whether it is completely dissolved, whether there is precipitation or turbidity.

[0039] (3) Emulsion stability: The emulsion stability index (ESI) is calculated by measuring the turbidity (absorbance) of the fresh emulsion and the emulsion after standing, and the emulsion stability is quantified. The higher the ESI value, the more stable the emulsion. The specific operation is as follows: A. Accurately weigh the first polysaccharide component and prepare a 1.0% (w / v) test solution with deionized water; B. Take 20 mL of test solution and mix it with 10 mL of soybean oil (oil-water volume ratio 1:2), and immediately homogenize it at 8000 rpm for 2 minutes to obtain the initial emulsion.

[0040] C. Absorbance measurement: A0: Immediately after homogenization, take 0.1 mL of emulsion from the bottom of the container, dilute it 100 times with 0.1% SDS solution, vortex to mix, and measure the absorbance at a wavelength of 500 nm.

[0041] A 30 The remaining emulsion was allowed to stand at 25°C. After 30 minutes, samples were taken from the same position at the bottom, diluted using the same method, and the absorbance was measured. This result was recorded as A. 30 .

[0042] Result calculation: According to the formula ESI 30 (min) = A0 × t / (A0 - A 30 Calculate ESI 30 .

[0043] The results are expressed as the average of three parallel experiments.

[0044] In the following application examples and comparative application examples, the test methods for the encapsulation efficiency, accelerated stability, solubility and processing tolerance of lutein coatings are as follows: (1) Encapsulation efficiency Among them, the total lutein content is determined by accurately weighing a certain mass (W1) of coating powder, ultrasonically crushing and completely dissolving it with dimethyl sulfoxide, filtering it, and then determining the total lutein content (M1) by high performance liquid chromatography.

[0045] Surface lutein content determination: Take another batch of the same mass (W2) of coated powder, and gently wash it three times with petroleum ether to dissolve only the unencapsulated lutein adsorbed on the surface. Collect the washing solution and determine the surface lutein content (M2) by HPLC.

[0046] Calculation: Encapsulation ratio = (M1 - M2) / M1 × 100%.

[0047] (2) Accelerating stability: The coated powder and the uncoated lutein raw material (as a control) were placed in open weighing bottles and placed in a constant temperature and humidity chamber with conditions set at 40±2℃ and relative humidity of 75%±5%.

[0048] Sampling and Measurement: Samples were taken at the end of month 0 and month 6 to measure the residual lutein content (M) in the coating. t(The total lutein content was determined according to the encapsulation rate test method described in (1) above).

[0049] Calculate retention rate and enhancement factor: Coating retention rate = M t / M0×100% (M0 is the initial content).

[0050] (3) Dispersion time test: Take 0.1g of coating powder, add 100mL of water at 25℃ and stir (200rpm), and record the time (seconds) required for the powder to be completely dispersed and for no obvious floating or clumping particles to appear on the liquid surface.

[0051] (4) Processing tolerance test method: The activity retention rate was determined after simulating mild heat treatment.

[0052] Heat treatment: Disperse the coating at 1% by mass in phosphate buffer (pH 7.0), place in a water bath, keep at 85±2℃ for 10 minutes, and immediately cool in an ice bath.

[0053] Content determination and calculation: Samples were taken before and after treatment to determine the lutein content.

[0054] Activity loss rate = [1 - (content after heat treatment / content before heat treatment)] × 100%.

[0055] Example 1: A modified extraction method for polysaccharides from the seaweed *Gracilaria fusiforme*, comprising the following steps: *Gracilaria fusiforme* dried powder is soaked in 85% ethanol for defatting and decolorization, filtered, and dried to obtain pretreated seaweed powder; 50g of pretreated seaweed powder and 0.2M sodium carbonate solution are added at a material-to-liquid ratio of 1:30 (g / mL); the mixture is heated to 60℃ and stirred for 5 hours, then 0.23g of sodium bisulfite (final concentration 1.5mmol / L) is added to initially mix with the polysaccharide system; then, under ultrasonic treatment, the mixture is extracted synchronously and slowly... Add 30 mL of 0.08 mol / L ammonium persulfate solution (final concentration 1.5 mmol / L) and 70 mL of lactide in acetone (concentration 0.5 mol / L, lactide added approximately 5 g, accounting for 10% of the pretreated algal powder). React at 60℃ with gentle stirring and ultrasound for 3.5 hours. After the reaction, cool the reaction system to room temperature, adjust the pH to 7.0 with dilute hydrochloric acid, centrifuge the reaction solution (8000 rpm, 5 min), and collect the supernatant. Add anhydrous ethanol to the supernatant to a final concentration of 30% (v / v), and allow to stand for 5 hours to precipitate. This step mainly precipitates and removes proteins, pigments, and some unreacted macromolecular impurities. Finally, centrifuge and discard the precipitate.

[0056] Fractional alcohol precipitation: In the first stage, highly emulsifiable components were collected. Anhydrous ethanol was added to the supernatant obtained after impurity removal to a concentration of 60% (v / v), and the mixture was allowed to stand at 4°C for 12 hours. The precipitate and supernatant were collected by centrifugation. The precipitate was washed sequentially with anhydrous ethanol and acetone, and then dried in a vacuum drying oven at 50°C to obtain the first polysaccharide component. In the second stage, highly gelatinous components were collected. Anhydrous ethanol was added to the supernatant obtained after the previous centrifugation to a concentration of 80% (v / v), and the mixture was allowed to stand at 4°C for 12 hours. The precipitate was collected by centrifugation. The precipitate was washed sequentially with anhydrous ethanol and acetone, and then dried in a vacuum drying oven at 50°C to obtain the second polysaccharide component.

[0057] Experimental Example 1: Based on Example 1, this study investigated the effects of lactide addition on the yield and solubility of the first and second polysaccharide components, as well as the emulsifying properties of the first polysaccharide component. The lactide addition amounts were 5%, 10%, 15%, 20%, and 25% of the mass of the *Gnaphalium affine* powder (corresponding to groups 1-1, 1-2, 1-3, 1-4, and 1-5 in Table 1, respectively), with the remaining steps the same as in Example 1.

[0058] Table 1. Effect of lactide addition on the yield and properties of polysaccharide components.

[0059] Table 1 shows that the yields of both the first and second polysaccharide components peaked around a 15% addition of lactide. Too low an addition (5%) resulted in insufficient grafting and a low yield of the modified product; too high an addition (25%) may trigger homopolymerization of lactide, producing byproducts insoluble in the alcohol precipitation system, thus decreasing the overall yield. Furthermore, the hydrophilicity of the polysaccharide decreased with the introduction of the hydrophobic segment (polylactic acid). At an addition of ≤15%, the amphiphilic structure still ensured good dispersion in water (forming micelles or a homogeneous solution); when the addition was ≥20%, excessive hydrophobicity led to decreased solubility, resulting in slight suspension or flocculation. The emulsification stability of the first polysaccharide component was optimal at a 15% addition. Moderate hydrophobic modification provided the polysaccharide with the best amphiphilic balance, enabling the formation of a strong and dense adsorption layer at the oil-water interface. If the amount added is too low, the hydrophobicity will be insufficient and the adsorption capacity will be weak; if it is too high, the molecules will be too hydrophobic, resulting in poor dispersibility in the aqueous phase, which will reduce its ability to effectively cover the oil droplet interface and reduce the stability of the emulsion.

[0060] Experimental Example 2, based on Example 1, investigated the effect of ethanol concentration (v / v) on the highly emulsifiable component (first polysaccharide component) collected during the first-stage alcohol precipitation. During the first-stage alcohol precipitation of the highly emulsifiable component, anhydrous ethanol was added to the supernatant obtained after impurity removal to ethanol concentrations of 40%, 45%, 50%, 55%, 60%, and 65% (v / v) (corresponding to groups 2-1, 2-2, 2-3, 2-4, 2-5, and 2-6 in Table 2, respectively). The remaining steps were the same as in Example 1.

[0061] Table 2. Effect of ethanol concentration during the first-stage alcohol precipitation on the yield and properties of polysaccharide components.

[0062] Table 2 shows a significant negative correlation between the yields of the first and second polysaccharide components. When the concentration of the first-stage alcohol precipitation is low (40%), a large number of target molecules are not precipitated and enter the supernatant, resulting in the lowest yield of the first component (2.2%) and the highest yield of the second component (6.7%). As the alcohol precipitation concentration increases, the yield of the first component increases and reaches a peak at 55%, while the yield of the second component continuously decreases. This indicates that the alcohol precipitation concentration effectively regulates the distribution of the product between the two functional components. When the concentration of the first-stage alcohol precipitation is 50%-60%, it can balance the yields of the first and second polysaccharide components and ensure the solubility of both and the emulsification stability of the first polysaccharide component.

[0063] Experimental Example 3, based on Example 1, investigated the effect of ethanol concentration (v / v) on the collection of highly gelatinous components (second polysaccharide components) by second-stage alcohol precipitation. During the second-stage alcohol precipitation process, anhydrous ethanol was added to the supernatant obtained after impurity removal to ethanol concentrations of 70%, 75%, 80%, 85%, and 90% (v / v) (corresponding to groups 3-1, 3-2, 3-3, 3-4, and 3-5 in Table 3, respectively), with the remaining steps being the same as in Example 1.

[0064] Table 3. Effect of ethanol concentration during the second-stage alcohol precipitation on the yield and properties of polysaccharide components.

[0065] Table 3 shows that the yield of the second polysaccharide component initially increased and then decreased with increasing alcohol precipitation concentration, reaching a peak at 80% ethanol concentration. As the alcohol precipitation concentration increased from 70% to 90%, the solubility of the precipitate gradually changed from clear and transparent to containing suspended matter. The component obtained at concentrations of 75%-85% could dissolve or disperse uniformly in water with a high yield. At this concentration, the polysaccharide component contained sufficient hydrophobic microdomains (possibly from lactide graft chains or intramolecular hydrophobic interactions) to drive intermolecular interactions and gel network formation, while maintaining good hydrophilicity to ensure dispersion and function in the aqueous phase. However, at excessively high concentrations (90%), the component became overly hydrophobic, and its solubility decreased significantly.

[0066] Example 2 differs from Example 1 in that the amount of lactide added is 15%, and during the first-stage alcohol precipitation to collect the highly emulsifiable component, anhydrous ethanol is added to the supernatant obtained after impurity removal until the ethanol concentration reaches 55%. The remaining process is the same as in Example 1. The yield and properties of the polysaccharide component are shown in Table 4.

[0067] Table 4. Yield and properties of polysaccharide components in Example 2

[0068] Comparative Example 1 differs from Example 1 in that ε-caprolactone is used instead of lactide, otherwise it is the same as Example 1.

[0069] Application Example 1: Lutein-coated material was prepared using the modified extraction method of Example 1 for Gracilaria polysaccharide. The specific steps are as follows: (1) Lutein was dissolved in medium-chain triglycerides (MCT) to prepare an 8% lutein oil solution, which is the oil phase; the first polysaccharide component was dissolved in deionized water to prepare a 1.2% solution, which is the aqueous phase A; the second polysaccharide component and edible gelatin were sampled at a mass ratio of 7:3, added to hot water at 60°C, stirred and dissolved, and a mixed solution with a total fraction of 2.0% was prepared, which is the aqueous phase B; this process allows the negatively charged second polysaccharide and the positively charged gelatin to be pre-mixed in the solution, which is beneficial for the subsequent rapid electrostatic recombination.

[0070] (2) The oil phase was slowly added to the aqueous phase A (mass ratio of oil phase to aqueous phase A is 1:4.5) under high-speed stirring. The mixture was first pre-emulsified using a high-speed shear mill (8000 rpm, 3 minutes), and then homogenized using a high-pressure homogenizer (5 MPa, 2 cycles) to prepare a nanoemulsion (target D50 < 500 nm). At this point, the first polysaccharide component was tightly adsorbed onto the surface of the oil droplets through hydrophobic interactions, forming a stable pre-emulsion. Under gentle stirring, the aqueous phase B (mass ratio of oil phase to aqueous phase B is 1:4.5) was slowly added to the pre-emulsion while maintaining the system temperature at 45°C. The pH of the mixture was slowly adjusted to 4.3 using dilute acid (1M HCl). At this point, the positively charged gelatin and the negatively charged second polysaccharide underwent electrostatic recombination. Simultaneously, the gelatin may also further cross-link with the first polysaccharide already adsorbed on the surface of the oil droplets through electrostatic and hydrophobic interactions, forming a dense composite aggregate layer around the oil droplets. Continue gentle stirring and maturation for 40 minutes to allow the coagulated layer to mature and stabilize, yielding a composite coagulated emulsion. Finally, spray dry the composite coagulated emulsion at an inlet air temperature of 160°C and an outlet air temperature of 85°C. Collect the powder from the bottom of the drying tower to obtain the lutein-coated product containing Gracilaria polysaccharides.

[0071] Application Example 2: The difference between this application example and Application Example 1 is that the lutein-coated compound is prepared using the Gracilaria polysaccharide prepared by the modified extraction method of Example 2. The rest is the same as in Application Example 1.

[0072] Application Example 3 differs from Application Example 1 in that the lutein-coated compound is prepared using the modified extraction method of groups 1-3 in Experiment Example 1. Otherwise, it is the same as Application Example 1.

[0073] Application Example 4 differs from Application Example 1 in that the lutein-coated compound is prepared using the modified extraction method of Gracilaria fusiforme prepared in Example 1. The mass ratio of oil phase to aqueous phase A and aqueous phase B is 1:4:5, and the rest is the same as in Application Example 1.

[0074] The difference between this application example and application example 1 is that the lutein-coated compound is prepared using the modified extraction method of Comparative Example 1. Otherwise, it is the same as application example 1.

[0075] The difference between Application Example 2 and Application Example 1 is that the lutein-coated compound is prepared using the modified extraction method of group 2-1 in Experiment Example 2, while the rest is the same as Application Example 1.

[0076] The difference between Application Example 3 and Application Example 1 is that the lutein-coated compound is prepared using the modified extraction method of groups 1-5 in Experiment Example 1. The rest is the same as Application Example 1.

[0077] The encapsulation efficiency, accelerated stability, solubility, and processing tolerance of the lutein coatings prepared in the above application examples and comparative application examples were tested. The results are shown in Table 5.

[0078] Table 5. Detection results of lutein-coated compounds prepared in the application examples and comparative application examples.

[0079] As shown in Table 5, the encapsulation efficiency of the lutein-coated material prepared by this invention is greater than 90%, reaching as high as 94.1%. After being stored at 40℃ and 75% relative humidity for 6 months, the lutein retention rate is greater than 87%, reaching as high as 92.3%. After treatment at 85℃ for 10 minutes, the activity loss rate is less than 10%, as low as 7.2%. Moreover, the lutein-coated material has good dispersibility, and all samples can be evenly dispersed within 30 seconds. The application comparison shows that when the extraction process of modified dragon's beard polysaccharide is changed, the prepared lutein-coated material exhibits varying degrees of decline in encapsulation efficiency, storage stability, redispersibility, and processing tolerance.

[0080] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for modifying and extracting polysaccharides from the seaweed Gracilaria, characterized in that, Includes the following steps: S1. Mix Gracilaria powder with a weak alkaline solution for extraction; then add a reducing agent, and simultaneously add an oxidizing agent and lactide solution under ultrasonic treatment to carry out a modification reaction; after the reaction is completed, adjust the pH value, centrifuge to obtain the first supernatant; add ethanol to the first supernatant to the volume percentage of ethanol to 25%-35%, let stand and remove the precipitate to obtain the second supernatant; S2. Add ethanol to the second supernatant to the volume percentage of ethanol to 50%-60%, let stand for precipitation once, centrifuge to collect the precipitate and the third supernatant, wash and dry the obtained precipitate to obtain the first polysaccharide component; continue to add ethanol to the third supernatant to the volume percentage of ethanol to 75%-85%, let stand for precipitation a second time, centrifuge to collect the precipitate, wash and dry the obtained precipitate to obtain the second polysaccharide component.

2. The modified extraction method according to claim 1, characterized in that, In step S1, the weak base solution includes sodium carbonate solution and / or sodium bicarbonate solution, and the molar concentration of the weak base solution is 0.1-0.5 mol / L.

3. The modified extraction method according to claim 2, characterized in that, In step S1, the ratio of the Gracilaria powder to the weak alkaline solution is 1:(20-40); the extraction process is as follows: stirring and extracting at 45-65℃ for 3-8 hours.

4. The modified extraction method according to any one of claims 1-3, characterized in that, In step S1, the reducing agent includes at least one of sodium sulfite, sodium bisulfite, potassium bisulfite, and ascorbic acid; the molar concentration of the reducing agent in the reaction system is 1.0-3.0 mmol / L; the oxidizing agent includes at least one of hydrogen peroxide, ammonium persulfate, potassium persulfate, and sodium persulfate; the molar concentration of the oxidizing agent in the reaction system is 1.0-3.0 mmol / L.

5. The modified extraction method according to any one of claims 1-3, characterized in that, In step S1, the lactide solution is an acetone solution of lactide, and the mass of lactide in the lactide solution is 8%-18% of the mass of the Gracilaria fusiforme powder.

6. The modified extraction method according to claim 5, characterized in that, In step S1, the temperature of the modification reaction is 45-65℃, and the time of the modification reaction is 2-6 hours.

7. A method for preparing a lutein-coated compound, characterized in that, The steps include: (1) dissolving lutein in medium-chain triglycerides to prepare a lutein oil solution, thus obtaining an oil phase; The first polysaccharide component prepared by the modified extraction method according to any one of claims 1-6 is dissolved in water to prepare a solution of the first polysaccharide component, and an aqueous phase A is obtained; the second polysaccharide component prepared by the modified extraction method according to any one of claims 1-6 is mixed with edible gelatin at a mass ratio of (3-4):(1-2), dissolved in water, and a mixed solution is prepared to obtain an aqueous phase B; (2) the oil phase is mixed with the aqueous phase A, and after shearing and homogenization, a pre-emulsion is obtained; then the aqueous phase B is added to the pre-emulsion, the pH value of the mixed system is adjusted to 4.0-4.5, and a composite coagulation reaction is carried out to obtain a composite coagulation emulsion; finally, the composite coagulation emulsion is dried to obtain lutein-coated material.

8. The preparation method according to claim 7, characterized in that, The mass ratio of the oil phase to the aqueous phase A and the aqueous phase B is 1:(3-5):(3-5).

9. The preparation method according to claim 8, characterized in that, In step (1), the mass percentage of lutein in the lutein oil solution is 5%-10%; the mass percentage of the first polysaccharide component in the first polysaccharide component solution is 1.0%-2.0%; and the total mass percentage of the second polysaccharide component and the edible gelatin in the mixed solution is 1.5%-2.5%.

10. The preparation method according to claim 7, characterized in that, In step (2), the temperature of the composite coagulation reaction is 40-55℃ and the time is 30-60 minutes.

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