Lycium barbarum polysaccharide, and preparation method and application thereof

Abstract

The application discloses a Chinese wolfberry polysaccharide and a preparation method and application thereof. The polysaccharide is a uniform component, has an average molecular weight of 26.231 kDa, presents an irregular spherical polymer structure, contains alpha and beta glycosidic bonds, has a clear monosaccharide composition and proportion, has good thermal stability, has an antioxidant capacity and cell safety. The polysaccharide is prepared by a method combining water extraction, alcohol precipitation, DEAE-52 column and Sephadex G-150 column purification, and the parameters are clear and repeatable. The polysaccharide can be applied to the fields of food, medicine and cosmetics as a functional active ingredient, and can also be added into milk to prepare functional dairy products. The polysaccharide has a clear structure, stable performance and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically to a wolfberry polysaccharide, its preparation method, and its application. Background Technology

[0002] Oxidative stress is a core driver of various chronic pathological processes, including aging, neurodegenerative diseases (such as Alzheimer's), cardiovascular diseases, diabetic complications, and liver damage. Therefore, screening natural products for active ingredients that can efficiently scavenge free radicals, enhance the activity of endogenous antioxidant enzymes, and protect cells from oxidative damage has become an important direction in the research and development of functional foods and drugs. In recent years, with the continuous development of research on functional foods and natural active ingredients, natural polysaccharides derived from plants, fungi, and medicinal and edible resources have attracted widespread attention due to their good biocompatibility, safety, and various biological activities. Polysaccharides possess multiple functions such as antioxidation, immunomodulation, anti-inflammation, and cell protection, and have promising development prospects in functional foods, nutritional health products, and dairy products. Among them, natural polysaccharides with antioxidant activity can scavenge excess free radicals in the body and reduce oxidative stress damage, thereby improving the functional properties and nutritional value of products. Therefore, developing novel natural polysaccharides with well-defined structures, stable activity, and suitability for food systems has significant application prospects.

[0003] Ningxia wolfberry (Lycium barbarum) is rich in various active ingredients such as polysaccharides, flavonoids, and betaine. Among them, wolfberry polysaccharides (LBPs) have become an important research focus in natural functional factor research due to their antioxidant, immunomodulatory, and cell-protective biological activities. The bioactivity of wolfberry polysaccharides is closely related to their structural characteristics, such as molecular weight, monosaccharide composition, glycosidic bond type, and spatial conformation. However, due to differences in raw material sources, extraction and purification processes, and separation conditions, existing wolfberry polysaccharides generally suffer from complex compositions, high structural heterogeneity, and significant differences in activity. Meanwhile, there is a lack of research on the interaction between polysaccharides and key components in milk, such as whey protein and casein, which limits their application in functional dairy products. Therefore, developing a novel wolfberry polysaccharide with a well-defined structure, good antioxidant activity, and compatibility with dairy products is of great significance for expanding the application of wolfberry active ingredients in dairy products. Summary of the Invention

[0004] The purpose of this invention is to provide a structurally well-defined and stable Lycium barbarum polysaccharide LBP-1, its preparation method, and its applications. By clarifying the molecular weight, structure, composition, and key performance data of the polysaccharide, a uniform and stable polysaccharide product is provided. A standardized separation and purification process is employed to achieve efficient preparation of the polysaccharide, ensuring product purity and performance. Simultaneously, its applications in the food, pharmaceutical, cosmetic, and dairy product fields are expanded, addressing the problems of unclear structure, unstable performance, and limited application scenarios of existing Lycium barbarum polysaccharides, thereby enhancing the application value and practicality of the polysaccharide.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a Lycium barbarum polysaccharide, wherein the Lycium barbarum polysaccharide is a homogeneous polysaccharide component LBP-1 with an average molecular weight of 26.231 kDa, exhibiting an irregular spherical polymeric structure, and containing both α-glycosidic bonds and β-glycosidic bonds in the molecular chain.

[0006] Furthermore, the wolfberry polysaccharide is a non-starch polysaccharide with a total sugar content ≥93.86%, a furfural content of 8.05%, a protein content ≤0.34%, and a Zeta potential of -29.89±1.1mV.

[0007] Furthermore, the monosaccharide composition of the wolfberry polysaccharide includes arabinose, glucose, xylose, mannose and galacturonic acid, and the molar percentage of each monosaccharide is: arabinose 0.5%, glucose 95.39%, xylose 1.3%, mannose 1.5% and galacturonic acid 1.3%.

[0008] Furthermore, the wolfberry polysaccharide exhibits thermal stability within the temperature range of 30℃ to 200℃.

[0009] Furthermore, the Lycium barbarum polysaccharide, within a concentration range of 0.25 mg / mL to 8.00 mg / mL, can scavenge DPPH free radicals, ABTS free radicals, and hydroxyl free radicals, and possesses Fe... 3+ Restorative ability.

[0010] Furthermore, the wolfberry polysaccharide exhibits ≥90% cell viability in Caco-2 cells at concentrations ranging from 25 μg / mL to 1000 μg / mL, with CAT enzyme activity of 5.19 U / mg prot at 800 μg / mL and SOD enzyme activity of 32.34 U / mg prot at 200 μg / mL.

[0011] This invention also provides a method for preparing Lycium barbarum polysaccharides, comprising the following steps: The crude polysaccharide of Ningxia wolfberry was obtained by water extraction and alcohol precipitation. The crude polysaccharide of Lycium barbarum was obtained by collecting the target component by DEAE-52 cellulose anion exchange column chromatography, and then purified by Sephadex G-150 gel column chromatography to collect the main peak. The collected main peak component was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed for 48h~72h. After freeze drying, the Lycium barbarum polysaccharide was obtained.

[0012] Furthermore, the DEAE-52 cellulose anion exchange column is eluted with a gradient of 0~0.5 mol / L NaCl solution, and the mobile phase of the Sephadex G-150 gel column is sodium chloride solution.

[0013] This invention also provides an application of Lycium barbarum polysaccharide in the field of functional active ingredients in functional foods, pharmaceuticals, or cosmetics.

[0014] Furthermore, the application involves adding the wolfberry polysaccharide as a functional ingredient to milk, where the monosaccharide component of the wolfberry polysaccharide forms a stable complex with whey protein and casein in milk, for the preparation of functional milk products.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: The Lycium barbarum polysaccharide LBP-1 provided by this invention has a clear structure and stable performance. Its average molecular weight, spatial structure and glycosidic bond type have been clarified, and key parameters such as monosaccharide composition and ratio and thermal stability temperature range have been defined. This solves the problems of unclear structure and ambiguous performance of existing Lycium barbarum polysaccharides, and provides a clear basis for the identification, separation and application of polysaccharides, ensuring the uniformity and stability of polysaccharide products.

[0016] The preparation method of this invention has standardized steps and clear parameters. By using DEAE-52 anion exchange column in combination with Sephadex G series gel column and dialysis purification process, the efficient preparation of Lycium barbarum polysaccharides is achieved. The operation is repeatable and easy to scale up, which solves the problems of cumbersome preparation process and insufficient product purity in the existing process, and improves the efficiency and stability of polysaccharide preparation.

[0017] The Lycium barbarum polysaccharide of this invention has a wide range of applications, and can be used as a functional active ingredient in functional foods, pharmaceuticals, and cosmetics. In particular, it can be added to milk to prepare functional dairy products. By clarifying its interaction with milk proteins, molecular-level support is provided for the practical application of the polysaccharide, expanding its application scope and enhancing its application value and market potential. (See attached figures.) Figure 1 This is a schematic diagram of the extraction, separation, and purification process of the novel Lycium barbarum polysaccharide in this invention; Figure 2Separation and purification of crude polysaccharide LBP-C from Lycium barbarum; (A) Elution curve of LBP-C on DEAE-52 column; (B) Curve obtained by Sephadex G-150 column chromatography of LBP-1; (C) Curve obtained by Sephadex G-150 column chromatography of LBP-2; (D) UV-Vis spectra of LBP-1 and LBP-2; (E) Absolute molecular weight analysis diagram of LBP-1; (F) Absolute molecular weight analysis diagram of LBP-2; Figure 3 The images show the morphological characteristics of the two polysaccharides; A, C, and E are the SEM and AFM structures of LBP-1, and B, D, and F are the SEM and AFM structures of LBP-2. Figure 4 Structural analysis of two fractionated polysaccharides; (A) Congo red experiment; (B) X-ray diffraction; (C) Thermogravimetric analysis of LBP-1; (D) Thermogravimetric analysis of LBP-2; (E) Differential scanning calorimetry; (F) Infrared analysis of LBP-1; (G) Infrared analysis of LBP-2; (H) Monosaccharide composition analysis of LBP-1; Figure 5 Evaluation of the in vitro antioxidant activity of two polysaccharides; (A) DPPH radical scavenging ability; (B) ABTS radical scavenging ability; (C) hydroxyl radical scavenging rate; (D) Fe 3+ Restorative ability; Figure 6 The effects of Lycium barbarum polysaccharide LBP-1 on oxidative stress in Caco-2 cells; (A) cck8 of LBP-1; (B) H2O2 oxidative stress model; (C) cell survival rate after oxidative damage; (D) CAT; (E) SOD; (F) MDA; Figures 7-12 The interaction between Lycium barbarum polysaccharide LBP-1 and various protein components (α-LA, β-LG, αS1-CN, αS2-CN, β-CN, κ-CN) in milk was analyzed based on molecular docking; A is glucose, B is arabinose, C is xylose, D is mannose, and E is galacturonic acid. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0019] This invention isolates and purifies two homogeneous polysaccharide fractions, LBP-1 and LBP-2, from Ningxia wolfberry through distribution purification and water extraction with alcohol precipitation. LBP-1 has a molecular weight of 26.231 kDa and its monosaccharide composition includes arabinose (Ara), glucose (Glc), xylose (Xyl), mannose (Man), and galacturonic acid (GalA), with a molar percentage of 0.5%:95.39%:1.3%:1.5%:1.3%. This structure differs significantly from previously reported wolfberry polysaccharides, classifying it as a novel polysaccharide. Stability studies show that LBP-1 does not undergo depolymerization due to glycosidic bond breakage within the temperature range of 30℃ to 200℃, exhibiting good thermal stability and suitability for high-temperature environments such as food processing. In vitro activity evaluation results show that LBP-1 has significant scavenging ability against DPPH, ABTS, and hydroxyl radicals, and exhibits strong Fe2+ scavenging activity. 3+ Reducing capacity. Cell experiments further confirmed that LBP-1 was non-cytotoxic to Caco-2 cells within a concentration range of 25–1000 μg / mL, and effectively protected Caco-2 cells from H2O2-induced oxidative stress damage by enhancing catalase (CAT) and superoxide dismutase (SOD) activities and reducing malondialdehyde (MDA) content. Molecular simulations showed that the monosaccharide components of the novel polysaccharide could stably bind to key proteins in milk. The novel Lycium barbarum polysaccharide LBP-1 provided by this invention has a well-defined structure, good stability, and excellent antioxidant activity, and has the potential to be used as a functional component in milk.

[0020] Example 1 1. Method 1.1 Extraction and purification of Lycium barbarum polysaccharides Goji berries were dried at 60℃, pulverized, and sieved through a 120-mesh sieve to improve raw material uniformity and subsequent extraction efficiency. They were then defatted with petroleum ether and impurities were removed with ethanol to reduce the impact of lipids and pigments on polysaccharide extraction and purification. The pretreated goji berry powder was added to ultrapure water at a specific material-to-liquid ratio and extracted with hot water at 80℃ to fully dissolve the water-soluble polysaccharides. The extract was collected by centrifugation and precipitated with 80% ethanol to promote polysaccharide precipitation. Proteins were then removed using Sevage reagent, and finally, the crude goji berry polysaccharide, denoted as LBP-C, was obtained by freeze-drying. The crude polysaccharide LBP-C was further purified using a DEAE-52 cellulose anion exchange column. Since negatively charged polysaccharides bind to the anion exchange medium, while neutral or weakly charged components preferentially elute, preliminary separation of different polysaccharide components was achieved. The target fraction obtained by ion exchange chromatography was then dialyzed in a dialysis bag with a molecular weight cutoff of 3500 Da to remove salt ions and small molecule impurities, thereby improving polysaccharide purity. The dialyzed sample was further purified using a Sephadex G-150 gel column. This method separates molecules based on size differences, with larger molecules eluting preferentially while smaller molecules enter the gel channels and elute more slowly, resulting in polysaccharide fractions with a more uniform molecular weight distribution. Finally, the purified polysaccharide fractions were freeze-dried to obtain two homogenized Lycium barbarum polysaccharide fractions, LBP-1 and LBP-2, for subsequent structural characterization and functional activity studies. The content of the eluted polysaccharides was quantified by phenol-sulfuric acid determination and spectrophotometry, and the fractions with higher purity were selected and named LBP-1 and LBP-2 for further analysis. The extraction process of Lycium barbarum polysaccharides can be found in [reference needed]. Figure 1 The extraction and purification process was repeated three times, and both LBP-1 and LBP-2 polysaccharide components were successfully obtained, proving that these two polysaccharide components were not obtained randomly, but could be obtained stably and repeatedly.

[0021] Optionally, sodium chloride can be used as the mobile phase for gel column purification.

[0022] Optionally, dialysis can be performed by placing the sample in a dialysis bag with a molecular weight cutoff of 3500 Da for 48 to 72 hours.

[0023] 1.2 Determination of molecular weight The molecular weights of the two components were determined using a gel permeation chromatography-differential-multi-angle laser light scattering (GPS-MS) system. The liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS II (Wyatt technology, CA, USA). Briefly, the sample was dissolved in a 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, filtered through a 0.45 mm filter, and then analyzed. Elution was performed using Ohpak SB-805 HQ (300 × 8 mm) and Ohpak SB-803 HQ (300 × 8 mm) gel size exclusion columns in tandem. The elution program was set as follows: column temperature 45℃, injection volume 100mL, mobile phase A (0.02% NaN3, 0.1M NaNO3), flow rate 0.6mL / min, elution gradient: isocratic for 75min.

[0024] 1.3 Determination of chemical composition, potential and ultraviolet scanning analysis Total sugar content in samples (LBP-1 and LBP-2) was determined using the phenol-sulfuric acid method. Galacturonic acid content was measured using the sulfuric acid-carbazole method. Protein content was quantified using the Bradford method (Beyotime Biotechnology, Shanghai, China). Iodine and potassium iodide (I2-KI) were used to determine whether the polysaccharide was a starch-based polysaccharide. Sample solutions (prepared at a concentration of 1 mg / mL) were scanned using a UV spectrophotometer (Purchip Technology T9CS, China) with the wavelength range set to 200-600 nm. Potential was measured using a laser particle size zeta potential meter.

[0025] 1.4 Scanning electron microscopy (SEM) of polysaccharide components A scanning electron microscope (Nava NanoSEM 450) was used to observe the structure of the polysaccharide. The purified polysaccharide sample was fixed on the stage with conductive adhesive, and excess powder was gently blown away with a bulb syringe. Then, gold was sputtered onto the sample, and finally, the morphology of the sample was observed at an accelerating voltage of 10 kV and a magnification of 1000×.

[0026] 1.5 Atomic Force Microscope (AFM) The molecular morphology of the polysaccharide was determined using an Oxford Cypher VRS atomic force microscope. Two fractionated components were dissolved in ultrapure water to a concentration of 10 μg / mL and filtered through a 0.45 mm pinhole filter. A suitable sample was then deposited onto a mica sheet, dried at room temperature, and scanned. The probe used was an AC160TS-R3; probe frequency: 300 kHz; probe elastic modulus: 26 N / m; tip radius: 7 nm; morphology resolution: 256 × 256; scan frequency: 2.44 Hz.

[0027] 1.6 Congo Red Test Two fractionated solutions (2 mg / mL) were mixed with Congo red solution (80 mmol / L) in 0–0.5 mol / L NaOH solution. The maximum absorption wavelength was measured using a UV-Vis spectrophotometer (Purchip T9CS, China) at 400–660 nm. A redshift of the maximum absorption wavelength indicates the presence of a triple helix structure.

[0028] 1.7 X-ray diffraction (XRD) The crystal structures of the two freeze-dried fractions were observed using an X-ray powder diffractometer (D8 DISCOVER A25, Bruker, Germany). The parameters were set as Cu-Ka (40kV, 40mA), the scanning range was 2q = 5-70°, the scanning step size was 0.02°, and the scanning speed was 0.2° / min.

[0029] 2. Results Table 1 shows the chemical composition analysis and molecular weight differences of the two fractionated polysaccharides.

[0030] 2.1 Isolation and purification of Lycium barbarum polysaccharides LBP-C was purified by DEAE-52 cellulose ion exchange column chromatography and eluted with 0-0.5 mol / L NaCl solution, yielding three independent elution peaks, such as... Figure 2 As shown in A, B, and C, these are defined as LBP-1, LBP-2, and LBP-3, respectively. Due to the low elution recovery rate of LBP-3, which was insufficient for the experimental baseline, only LBP-1 and LBP-2 were subsequently collected. The eluents from the same peak position were also collected, concentrated, and further purified using gel chromatography. During chromatography, substances with larger molecular weights cannot enter the gel particles and therefore elute at a faster rate, while substances with smaller molecular weights are blocked by the gel and elute at a slower rate. This method is widely used for the purification of biomolecules. Therefore, to obtain a homogeneous fraction, purification was performed using a Sephadex G-150 column. LBP-1 and LBP-2 were further collected, dialyzed, and lyophilized for subsequent analysis.

[0031] 2.2 Chemical composition of the two fractionated components As shown in Table 1, the total sugar content of crude LBP-C, LBP-1, and LBP-2 was 74.17%, 93.86%, and 99.22%, respectively; the furfural content was 7.04%, 8.05%, and 14.44%, respectively; and the protein content was 2.79%, 0.34%, and 0.52%, respectively. The negative I2-KI test indicates that the isolated polysaccharides are non-starch polysaccharides. Figure 2 UV spectroscopy revealed that after purification with DEAE-52 and Sephadex G-150, proteins that were not removed by the sevage method could be further removed from LBP-C. LBP-C (-23.64±1.17mV), LBP-1 (-29.89±1.1mV), and LBP-2 (-36.40±1.39mV) all exhibited negative zeta potentials, indicating that the purified polysaccharide possessed better structural stability.

[0032] 2.3 Molecular weights of the two fractionated components As shown in Table 1, the average molecular weights (Mw) of LBP-1 and LBP-2 are 26.231 kDa and 13.146 kDa, respectively, consistent with the molecular weight distribution of natural polysaccharides. The polydispersity indices (Mw / Mn, PDI) of the two polysaccharide fractions are 1.894 and 1.229, respectively, indicating a relatively uniform molecular weight distribution. Figure 2 E and F, LS detected that the scattered light intensity peaks of LBP-1 molecules were scattered and fluctuated greatly, suggesting that they may be aggregated in aqueous solution. In contrast, LBP-2 exhibited a single, sharp, and symmetrical peak.

[0033] 2.4 Morphological characteristics of the two polysaccharides like Figure 3 As shown, the two polysaccharides (LBP-1 and LBP-2) exhibit significant morphological differences at a magnification of 1000×. LBP-1 shows large aggregates of spherical particles with some adhesion and a smooth surface. In contrast, LBP-2 shows fragmented, fibrous structures with attached small spherical particles, an irregular morphology, dispersed distribution, and circular pores. Furthermore, AFM images show that LBP-1 mainly exists as irregular spherical aggregates rather than single chains, while the AFM images of LBP-2 show a relatively smooth polysaccharide surface, smaller particle size, and a more uniform structural distribution.

[0034] 2.5. Conformation and Crystal Structure of the Two Polysaccharides like Figure 4 As shown in Figure A, no red shift was detected in the Congo red complexes of LBP-1 and LBP-2 at any concentration of NaOH, indicating that neither polysaccharide contains a triple helix conformation. Figure 4XRD analysis of B showed relatively diffuse diffraction peaks near 2q = 20.0°, indicating an amorphous structure. The similarity in the amorphous structures of the two polysaccharides suggests that the separation and purification process did not alter their amorphous nature. Figure 4 The infrared spectra of C and D show that LBP-1 and LBP-2 are located in the typical protein absorption region (1651 cm⁻¹). -1 and 1555cm 1 No obvious absorption bands were observed, indicating that the protein content in the two purified polysaccharides was extremely low. A distinct absorption pattern was observed in the polysaccharide-specific fingerprint region (1300-900 cm⁻¹), with a peak at 1147 cm⁻¹. -1 and 1154cm 1 The absorption peaks observed at 835.3 cm⁻¹ indicate the presence of pyranose rings in both polysaccharides. Furthermore, the two polysaccharides exhibit absorption peaks at 835.3 cm⁻¹, respectively. 1 and 908.2cm 1 The presence of peak values ​​indicates that α- and β-glycosidic bonds may exist in the structure of the polysaccharide.

[0035] Example 2 1. Method 1.1 Thermogravimetric Analysis (TGA) The thermal stability of the two polysaccharides was evaluated using a Themys One thermogravimetric analyzer (French) under a nitrogen atmosphere. 5 mg of each polysaccharide was weighed into a crucible. The nitrogen flow rate was maintained at 20 mL / min, and the temperature was increased from 30 °C to 800 °C at a rate of 10 °C / min. The derivative form of the TGA (DTG) was obtained by taking the first derivative of the TGA curve.

[0036] 1.2 Differential Scanning Calorimetry (DSC) Polysaccharide samples were characterized using a Discovery DSC250 differential scanning calorimeter. A suitable sample was placed in a sealed aluminum dish, and nitrogen gas was introduced at a rate of 50 mL / min under a nitrogen atmosphere. The temperature was set from 30°C to 300°C. A blank crucible was used as a reference to eliminate potential interference factors.

[0037] 1.3 DPPH, ABTS, and hydroxyl radical (·OH) scavenging activity The antioxidant capacity of DPPH, ABTS, and hydroxyl radicals in samples (0.25-8.00 mg / mL) was determined according to the method reported by Liu et al.

[0038] 1.4 Fe ion reduction LBP (1 mg / mL) solution was mixed with 2 mL of phosphate buffer (0.2 mol / L, pH 6.6) and 2 mL of 1% potassium ferricyanide. The mixture was heated in a 50°C water bath for 20 min. Then, 2 mL of 10% ferric chloride (FeCl3) was added, and the solution was centrifuged at 4000 g / min for 10 min. Next, 2.5 mL of 10% FeCl3 was added, and the mixture was centrifuged at 4000 g / min for 10 min. Then, 2.5 mL of the supernatant was collected, and 2.5 mL of distilled water and 1 mL of 0.1% FeCl3 were added and mixed. The sample was allowed to stand for 10 min. Finally, the absorbance of the reaction solution was measured at 700 nm, using distilled water as a blank for zeroing. A higher A700 value indicates a higher reducing power of the sample.

[0039] 1.5 Determination of Monosaccharide Components The monosaccharide composition of sample LBP-1 was determined using an ICS-5000 HPAEC system equipped with a Carbopac™ PA-20 analytical column (3 mm × 150 mm; Dionex) and an electrochemical detector. Briefly, 5 mg of the polysaccharide sample was hydrolyzed with 2 M trifluoroacetic acid (TFA) at 120 °C for 3 h. The hydrolysate was evaporated to dryness under a nitrogen stream and then reconstituted in ultrapure water for high-performance anion exchange chromatography (HPAEC) analysis of the monosaccharide composition. Elution was performed at a flow rate of 0.3 mL / min, an injection volume of 25 µL, and a column temperature of 30ºC. The elution gradients are as follows: 0 min A / B / C (98.8:1.2:0, V / V), 18 min A / B / C (98.8:1.2:0, V / V), 20 min A / B / C (30:70:0, V / V), 30 min A / B / C (30:70:0, V / V), 30.1 min A / B / C (0:0:100, V / V), 46 min A / B / C (0:0:100, V / V), 46.1 min A / B / C (0:100:0, V / V), 50 min A / B / C (0:100:0, V / V), 50.1 min Phase A / Phase B / Phase C (98.8:1.2:0, V / V), 80 min. (Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaOAc).

[0040] 2. Results 2.1 TGA Analysis like Figure 4As shown in E and F, LBP-1 exhibited approximately 12% weight loss during the initial heating phase (30–150 °C). A second phase of weight loss occurred between 150 °C and 800 °C, with a weight reduction of 62.85%, and the dTG curve clearly showed the point of maximum weight loss at 194 °C. LBP-2 was observed to have three phases of weight loss throughout the process. In the initial phase (30 °C–198.67 °C), LBP-2's loss was attributed to moisture evaporation. Significant weight loss, exceeding 50% of the initial weight, occurred between 200 °C and 342.85 °C, attributed to thermal degradation of glycosidic bonds and depolymerization of the polysaccharide backbone. Weight loss observed between 350 and 477 °C corresponded to polymer carbonization. During the high-temperature carbonization phase (480 °C–800 °C), the rate of weight loss gradually slowed and eventually stabilized, indicating near-complete carbonization. The residual weight order at 800 °C was: LBP-1 > LBP-2, indicating that LBP-1 possessed the highest carbonization capacity. The temperature at which the mass of two polysaccharides is reduced by 50% (T) 50 All of them show good thermal stability.

[0041] 2.2 DSC Analysis like Figure 4 As shown in Figure G, the DSC thermograms of both LBP-1 and LBP-2 exhibit two exothermic peaks. The first peak occurs at 76.6℃ and 88.6℃, respectively, while the second peak occurs at 257.5℃ and 239.3℃. The second endothermic peak indicates that the depolymerization is caused by the breaking of glycosidic bonds. Importantly, the second endothermic peak temperature of LBP-1 is higher than that of LBP-2, indicating that the polysaccharide structure of LBP-1 is more stable. In summary, both DSC and TGA suggest that the two polysaccharides possess good thermal stability, broadening their application prospects in future food processing.

[0042] 2.3 Monosaccharide composition of the novel Lycium barbarum polysaccharide LBP-1 like Figure 4 As shown in H, monosaccharide composition analysis revealed that LBP-1 is composed of arabinose (Ara), glucose (Glc), xylose (Xyl), mannose (Man), and galacturonic acid (GalA), with a molar percentage of 0.5%: 95.39%: 1.3%: 1.5%: 1.3%.

[0043] 2.4 Comparison of the in vitro antioxidant activities of the two polysaccharides like Figure 5 As shown, using vitamin C (Vc) as a positive control, the scavenging ability of different LBP components within the concentration range of 0.25 mg / mL to 8 mg / mL increased sharply with increasing concentration. However, at the same concentration, LBP-1 showed significantly stronger scavenging rates for various antioxidant indicators than LBP-2 and LBP-C. This indicates that LBP-1 has better antioxidant activity.

[0044] Example 3 1. Method 1.1 Cell Culture Caco-2 colon cancer cells were maintained in high-glucose medium (DMEM) supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin at 37°C and 5% CO2. When the cell density reached 70%-80%, the cells were digested with 0.25% trypsin and passaged at a ratio of 1:2.

[0045] 1.2 Cell viability determination Caco-2 cell density was adjusted to 1×10⁻⁶. 5 Cells were seeded at 100 mL / well in 96-well plates and cultured with different concentrations of LBP-1 (25, 50, 100, 200, 400, 600, 800, 1000 mg / mL) for 24 h, with six replicates. Then, 10 mL of CCK-8 solution was added to each well, and the absorbance at 450 nm was measured after 1.5 h. The cell viability of Caco-2 cells under H2O2-induced oxidative stress was also evaluated using the above method. The experiment was divided into three groups: control group (normal Caco-2 cells), model group (Caco-2 cells with H2O2-induced oxidative damage), and treatment group (cells pretreated with different concentrations of LBP-1 for 24 h and then exposed to H2O2 at the same concentration).

[0046] 1.3 Establishment of an H2O2-induced oxidative stress model in Caco-2 colon cancer cells A Caco-2 oxidative stress model was established and induced. H2O2 was sequentially diluted in DEME at concentrations from 0 to 800 nm, and viability was assessed using a microplate at 450 nm by CCK-8 assay.

[0047] 1.4 ROS Level Cells were loaded at 2×10 5Cells were seeded per well in a 6-well plate with a total culture medium volume of 2 mL. When the cells reached approximately 60%-70% confluence, different concentrations of LBP-1 were added. After treatment, the culture medium was discarded, and the cells were washed twice with PBS. Then, 1 mL of 10 mmol / L LCFH-DA was added, and the cells were incubated at 37°C for 20 min. Cells were washed three times with serum-free medium for 5 min each time to prevent background fluorescence interference. Finally, 1 mL of PBS was added for imaging. The excitation wavelength was set to 488 nm, and the emission wavelength to 525 nm. The fluorescence intensity was positively correlated with the intracellular ROS level. Quantitative analysis of the fluorescence images was performed using Image-J software. Five fields of view were randomly selected from each well, and the average fluorescence intensity was measured. By comparing the fluorescence intensity of different treatment groups, the effect of LBP-1 on H2O2-induced intracellular ROS levels in Caco-2 cells was evaluated.

[0048] 1.5, MDA, SOD and CAT levels Caco-2 cells were seeded into 6-well plates (1×10⁻⁶ cells per well). 6 Cells were exposed to different concentrations of LBP-1 (50, 100, 200, 400, 600, 800 mg / mL) and then washed with PBS and added to lysis buffer. The proteins were collected by centrifugation, and the activities of catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured according to the kit manufacturer's protocol.

[0049] 2. Results 2.1. Protection of Caco-2 colon cancer cells by LBP-1 against H2O2-induced oxidative damage. like Figure 6As shown in A, B, and C, cell viability remained above 90% at all tested concentrations (25–1000 mg / mL), indicating that LBP-1 is safe as a natural polysaccharide from a food source. Within the LBP-1 concentration range of 400–1000 mg / mL, cell survival increased with increasing concentration, suggesting that LBP-1 may promote cell proliferation. Oxidative stress models of cells under different H2O2 concentrations (100–800 mM) were established. Compared to the control group, Caco-2 cells exposed to 500 mM H2O2 for 1.5 h showed a cell viability decrease to 58.88%, close to the half-inhibitory concentration. Therefore, 500 mM H2O2 was selected as the optimal concentration for inducing cell damage and used to detect the protective effect of LBP-1 against H2O2-induced oxidative damage in Caco-2 colon cancer cells in the oxidative model. The effect of different concentrations (50, 100, 200, 400, 600, 800 mg / mL) of LBP-1 on the survival rate of Caco-2 cells treated with H2O2. A significant difference in survival rate was observed between the Model group and the Control group. p <0.05), the polysaccharide treatment group showed that cell survival rate increased with increasing polysaccharide concentration, indicating that LBP-1 has a protective effect against cellular oxidative stress.

[0050] 2.2 Effects of LBP-1 on CAT, SOD, and MDA in Caco-2 colon cancer cells like Figure 6 As shown in D, E, and F, compared with the Control group, the CAT and SOD activities in the Model group were significantly decreased ( p <0.05%, MDA content increased significantly ( p The concentration <0.05 indicates increased cellular sensitivity to oxidative stress. In contrast, the LBP-1 treatment group potently enhanced the activity of key enzymes in a dose-dependent manner. For CAT, LBP-1 significantly increased CAT enzyme activity to approximately 5.19 (U / mg prot) at 800 mg / mL. LBP-1 reduced SOD enzyme activity to 32.34 (U / mg prot) at 200 mg / mL. Furthermore, SOD content decreased significantly with increasing LBP-1 dosage, indicating that the novel Lycium barbarum polysaccharide LBP-1 can protect against H2O2-induced oxidative stress in Caco-2 cells by enhancing CAT and SOD activities and reducing MDA content, thereby improving cell survival and demonstrating potential as a novel functional ingredient for the food, pharmaceutical, and cosmetic industries.

[0051] Example 4 1. Method Molecular docking of bovine whey proteins (α-LA, B-LG) and caseins (S1-CN, αS2-CN, β-CN, and κ-CN) with monosaccharide components of novel polysaccharides (glucose (Glc), arabinose (Ara), xylose (Xyl), mannose (Man), and galacturonic acid (GalA)) was performed using AutoDock Vina software (version 1.5.7). First, water molecules were removed from the protein files. Then, polar hydrogen atoms and Gasteiger charges were added to balance the protein file charges, and a box was established in the pocket structure of the active region. Ligand optimization was then performed. After docking, the result with the highest absolute binding energy was selected, and three-dimensional interaction analysis was performed using Pymol software.

[0052] 2. Results like Figures 7-12 As shown, this invention, based on the monosaccharide composition of LBP-1, utilizes molecular docking to analyze the interaction mechanisms between various monosaccharide components in LBP-1 and various subtypes of casein and whey protein, providing a molecular-level mechanism for the application of Lycium barbarum polysaccharides in milk. Various caseins can form stable complexes with the five monosaccharides, demonstrating a basis for molecular interactions. The interaction mechanism is primarily hydrogen bonding, supplemented by hydrophobic interactions and van der Waals forces, with multiple residues synergistically enhancing binding stability. Key residues: αS1-CN is mainly composed of Phe, Gln, Ala, and His; αS2-CN is mainly composed of Tyr and Val; β-CN is mainly composed of Pro, Met, and Asn; κ-CN is mainly composed of Lys, Ser, Gln, and Pro. α-LA and β-LG in milk whey protein can form stable bindings with the five monosaccharides, exhibiting high absolute binding energies and reasonable conformations, demonstrating a basis for molecular interactions. α-LA primarily utilizes hydrogen bonding with polar amino acids (Ser, Asp, His); β-LG relies on a synergistic effect of polar / nonpolar residues, with both hydrogen bonding and hydrophobic interactions playing a role. Ligand differences: Galacturonic acid and mannose exhibit multi-site binding, short interaction distances, and high stability in all four casein proteins, making them the optimal binding ligands; glucose, arabinose, and xylose can also form effective binding bonds, indicating that whey proteins have a broad-spectrum binding capacity for natural monosaccharides.

[0053] In summary, the Lycium barbarum polysaccharide LBP-1 provided by this invention has a molecular weight of 26.231 kDa and a monosaccharide composition containing arabinose, glucose, etc., with molar proportions of 0.5%, 95.39%, 1.3%, 1.5%, and 1.3%, respectively, making it a novel structural polysaccharide. Its preparation process is simple, feasible, scalable, and reproducible. It exhibits good thermal stability and antioxidant function, protecting H2O2-induced Caco-2 cells by enhancing CAT and SOD activity and reducing MDA content. It can also form stable bonds with whey protein and casein in milk. This invention clarifies the key parameters of the polysaccharide, solving the problems of ambiguous structure and limited application of existing Lycium barbarum polysaccharides. It can be widely used in the food, pharmaceutical, and cosmetic fields, and can also be used to develop functional dairy products. Furthermore, molecular docking clarifies its interaction mechanism with milk components, providing a molecular basis for its application, indicating broad application prospects.

Claims

1. A Lycium barbarum polysaccharide, characterized in that, The wolfberry polysaccharide is a homogeneous polysaccharide component LBP-1 with an average molecular weight of 26.231 kDa. It has an irregular spherical polymer structure and contains both α-glycosidic bonds and β-glycosidic bonds in its molecular chain.

2. The Lycium barbarum polysaccharide according to claim 1, characterized in that, The wolfberry polysaccharide is a non-starch polysaccharide with a total sugar content ≥93.86%, furfural content 8.05%, protein content ≤0.34%, and zeta potential -29.89±1.1mV.

3. The Lycium barbarum polysaccharide according to claim 1, characterized in that, The monosaccharide composition of the Lycium barbarum polysaccharide includes arabinose, glucose, xylose, mannose and galacturonic acid, with the following molar percentages: arabinose 0.5%, glucose 95.39%, xylose 1.3%, mannose 1.5% and galacturonic acid 1.3%.

4. The Lycium barbarum polysaccharide according to claim 1, characterized in that, The wolfberry polysaccharide exhibits thermal stability within the temperature range of 30℃ to 200℃.

5. The Lycium barbarum polysaccharide according to claim 1, characterized in that, The Lycium barbarum polysaccharide, within a concentration range of 0.25 mg / mL to 8.00 mg / mL, can scavenge DPPH free radicals, ABTS free radicals, and hydroxyl free radicals, and possesses Fe... 3+ Restorative ability.

6. The Lycium barbarum polysaccharide according to claim 1, characterized in that, The Lycium barbarum polysaccharide exhibits ≥90% cell viability in Caco-2 cells within a concentration range of 25 μg / mL to 1000 μg / mL, with CAT enzyme activity of 5.19 U / mg prot at 800 μg / mL and SOD enzyme activity of 32.34 U / mg prot at 200 μg / mL.

7. A method for preparing a Lycium barbarum polysaccharide according to any one of claims 1 to 6, characterized in that, Includes the following steps: The crude polysaccharide of Ningxia wolfberry was obtained by water extraction and alcohol precipitation. The crude polysaccharide of Lycium barbarum was obtained by collecting the target component by DEAE-52 cellulose anion exchange column chromatography, and then purified by Sephadex G-150 gel column chromatography to collect the main peak. The collected main peak component was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed for 48h~72h. After freeze drying, the Lycium barbarum polysaccharide was obtained.

8. The method for preparing Lycium barbarum polysaccharide according to claim 7, characterized in that, The DEAE-52 cellulose anion exchange column was eluted with a gradient of 0~0.5 mol / L NaCl solution, and the mobile phase of the Sephadex G-150 gel column was sodium chloride solution.

9. The application of the Lycium barbarum polysaccharide according to any one of claims 1 to 6 in the field of functional active ingredients in functional foods, pharmaceuticals, or cosmetics.

10. The application according to claim 9, characterized in that, The application involves adding the wolfberry polysaccharide as a functional ingredient to milk. The monosaccharide component of the wolfberry polysaccharide forms a stable complex with whey protein and casein in milk, which is used to prepare functional milk products.

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