Preparation of water-soluble uniform heteropolysaccharides from highland barley and its new application in promoting expression of small intestinal mucosa β-galactosidase

Abstract

This invention discloses a method for preparing a water-soluble homogeneous heteropolysaccharide from highland barley and its novel application in promoting the expression of β-galactosidase in the small intestinal mucosa, belonging to the field of polysaccharide preparation and application technology. This invention provides a method for preparing the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley, involving extraction, alcohol precipitation, decolorization, and deproteinization of highland barley raw materials, followed by separation and purification using anion exchange column chromatography and gel column chromatography. The water-soluble homogeneous heteropolysaccharide HBP-1a was successfully isolated from the highland barley polysaccharide. This invention is the first to discover that the water-soluble homogeneous heteropolysaccharide HBP-1a can effectively promote the expression of β-galactosidase in the small intestinal mucosa and can be used to improve symptoms related to lactose intolerance diarrhea. This invention explores a new use for the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley, broadening its application fields and possessing broad prospects and market value.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide preparation and application technology, and in particular to the preparation of a water-soluble homogeneous heteropolysaccharide from highland barley and its novel application in promoting the expression of β-galactosidase in the small intestinal mucosa. Background Technology

[0002] Lactose intolerance is a common digestive disorder in certain populations, particularly newborns and infants. Statistics show that approximately 90% of Asians have experienced lactose intolerance, with an incidence rate as high as 87% in children aged 3-13 years. This condition primarily arises after weaning in mammals, when the activity of β-galactosidase in the small intestinal mucosa gradually weakens under genetic regulation, leading to the incomplete breakdown of ingested lactose. Undigested lactose enters the large intestine and is fermented by intestinal microorganisms, producing gases such as hydrogen, carbon dioxide, and methane, as well as short-chain fatty acids. This increases intestinal osmotic pressure, causing fluid and electrolyte accumulation, resulting in typical clinical manifestations such as bloating, abdominal pain, and diarrhea. These symptoms can have a particularly lasting impact on the growth, development, and overall health of infants and children.

[0003] Currently, the main methods to alleviate lactose intolerance symptoms include: (1) choosing lactose-free diets to replace dairy products and avoiding lactose intake can effectively solve lactose intolerance. However, lactose not only provides energy for the body, but is also an excellent prebiotic and structural sugar for the brain development of infants and young children, and plays an important physiological role in calcium absorption. Refusing lactose intake may cause more serious nutritional deficiencies and diseases; (2) using exogenous β-galactosidase to hydrolyze lactose in dairy products and reduce the level of lactose intake. However, such products are expensive, have strict production conditions, and taste sweet, making it difficult to satisfy most consumers; (3) supplementing with exogenous β-galactosidase, which can effectively hydrolyze ingested dietary lactose. However, it is expensive and the activity of β-galactosidase will be lost after entering the digestive tract, reducing the effect; (4) taking prebiotics, which can regulate the intestinal flora and increase the proliferation of colonic bacteria that can metabolize lactose, thereby reducing the accumulation of lactose in the colon and alleviating lactose intolerance symptoms. However, it has the disadvantages of slow onset, general effect, and high cost. Furthermore, the above methods can only temporarily relieve lactose intolerance symptoms and cannot solve the problem at its root.

[0004] Current research in this field has utilized the prebiotic properties of polysaccharides to promote lactose fermentation efficiency in the colon by regulating gut microbiota composition, or to create a suitable environment for enzymes by alleviating inflammation and oxidative stress, thereby promoting enzyme activity and alleviating lactose intolerance symptoms to some extent. For example, the literature (Lu Yujia, Mao Kaiwen, Zhong Geng. In vitro study on the effects of konjac glucomannan and oxidized konjac glucomannan on probiotic growth and lactose metabolism [J]. Food Industry Technology, 2020, 41(11):7.DOI:10.13386 / j.issn1002-0306.2020.11.050.) used in vitro fermentation to explore the promoting effect of different doses of konjac glucomannan and oxidized konjac glucomannan on lactose metabolism in milk powder, providing a convenient and novel method for alleviating lactose intolerance. This study found that milk powder with added konjac glucomannan or oxidized konjac glucomannan can promote the growth of beneficial bacteria such as lactic acid bacteria in the intestines. By enhancing the activity of lactase in the cecum and ileum, it reduces the lactose content in the milk powder and promotes lactose metabolism. Chinese invention patent CN113693237A discloses a natural solid intervention agent for lactose intolerance diarrhea, composed of lotus proanthocyanidins and carboxymethyl poria cocos polysaccharide. This intervention agent enhances the body's ability to metabolize dietary lactose by activating intestinal lactase activity, while also improving intestinal health, gut microbiota, and providing a favorable digestive environment, thus intervening in lactose intolerance diarrhea and related symptoms.

[0005] However, the regulation of gut microbiota or enzyme activity by directly consuming prebiotics or utilizing the prebiotic properties of polysaccharides still depends on the types and quantities of probiotics and enzymes in the gut. Significant differences exist among different populations, leading to large individual variations in effectiveness. These methods require first promoting the growth and reproduction of probiotics, then generating benefits from them, and creating a suitable environment for enzymes to enhance activity. This is a multi-step, indirect process, and growth and activity are easily affected by various factors such as diet, medication, and health status, leading to fluctuations or interruptions in effectiveness. In summary, existing methods generally suffer from slow onset and limited effectiveness. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a method for preparing a water-soluble homogeneous heteropolysaccharide from highland barley, wherein a specific water-soluble homogeneous heteropolysaccharide (HBP-1a) is isolated from a wide variety of water-soluble heteropolysaccharides (HBP). This water-soluble homogeneous heteropolysaccharide is then used to promote the expression of β-galactosidase in the small intestinal mucosa, thereby increasing both β-galactosidase activity and its mRNA expression level, thus significantly increasing the ability to hydrolyze lactose.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows: In a first aspect of the present invention, a method for preparing the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley is provided, comprising the following steps: (1) The defatted barley raw material is mixed with water and extracted at a certain temperature. After the extraction is completed, the supernatant is collected by centrifugation. (2) The supernatant obtained was subjected to alcohol precipitation, and after the precipitation was completed, it was centrifuged and the precipitate was collected; the precipitate was washed and dried to obtain barley water-soluble crude polysaccharide; (3) Dissolve barley water-soluble crude polysaccharide in water, and after decolorization, deproteinization and drying, obtain decolorized and deproteinized barley water-soluble heteropolysaccharide HBP; (4) The barley water-soluble heteropolysaccharide HBP was dissolved in water and purified by anion exchange column chromatography. After loading the sample, it was eluted with water and sodium chloride aqueous solution of different concentrations. The eluents were collected and combined. After concentration, dialysis and drying, barley water-soluble heteropolysaccharide HBP-1 was obtained. (5) Dissolve barley water-soluble heteropolysaccharide HBP-1 in water, purify by gel column chromatography, elute with water after loading, collect and combine the eluent, and dry to obtain barley water-soluble homogeneous heteropolysaccharide HBP-1a.

[0008] Preferably, in step (1), the extraction temperature is 60~80 ℃ and the extraction time is 5~6 h.

[0009] Those skilled in the art can select the appropriate defatting method and type of barley raw material in step (1) based on actual conditions or needs. For example, powdered barley raw material helps increase the contact area for defatting and extraction. Technicians first grind the dried barley into powder, and then add a solvent that meets food and pharmaceutical safety standards (such as ethanol) to complete the defatting. In the extraction process, the parameters that play a dominant role in optimization are temperature and time. Under the guidance of the above-mentioned preferred parameters, technicians can add an appropriate amount of water according to the actual production scale, for example, to complete hot water extraction at a material-to-liquid ratio of 1:10 g / L or other ratios.

[0010] Preferably, in step (2), the alcohol precipitation treatment uses ethanol, the treatment temperature is 0~4 ℃, and the final concentration of ethanol in the alcohol precipitation treatment is 80 vol.%~90 vol.%.

[0011] Based on the principle of complying with the hygiene and safety standards of food or medicine, and to ensure that the solvent residue in the product meets the national standards, ethanol is the preferred solvent type in step (2). Compared with methanol and isopropanol, ethanol is safer, easier to recover, and easier to remove residues, making it a particularly suitable raw material choice for alcohol precipitation. After alcohol precipitation, the precipitate is washed with ethanol and dried to easily obtain the target barley water-soluble crude polysaccharide.

[0012] After decolorization and deproteinization, the water-soluble crude polysaccharide from highland barley is better able to remove interference from pigments and proteins, which can then be processed using subsequent anion exchange column chromatography and gel column chromatography. Various methods exist for decolorization and deproteinization. Those skilled in the art can use adsorbents commonly found in the food and pharmaceutical fields, such as activated carbon, for decolorization, or methods such as enzymatic methods, trichloroacetic acid methods, salting-out methods, hydrochloric acid methods, and Sevage extraction methods for deproteinization.

[0013] Preferably, in step (4), the anion exchange column chromatography uses a DEAE-Fast flow anion exchange column; after loading the sample, gradient elution is performed with water and 0.3~1.0 mol / L sodium chloride aqueous solution, respectively, at a flow rate of 0.8~1.0 mL / min, and 8~10 mL of eluent is collected from each tube. The eluent is collected according to the elution curve, and after merging, concentration, dialyzing and drying, barley water-soluble heteropolysaccharide HBP-1 is obtained.

[0014] In the food and pharmaceutical fields, the DEAE-Fast flow (or DEAE Sepharose Fast Flow) anion exchange chromatography column is a weak anion exchange chromatography medium widely used for the purification of biological macromolecules. It is suitable for the separation and purification of negatively charged molecules such as proteins and nucleic acids. In step (4), after loading the barley water-soluble heteropolysaccharide HBP solution onto the anion exchange chromatography column, gradient elution can be performed using water (considered as a 0 mol / L sodium chloride aqueous solution), 0.3 mol / L, 0.5 mol / L, and 1.0 mol / L sodium chloride aqueous solutions, respectively. Under the preferred parameter range, barley water-soluble heteropolysaccharide HBP-1 can be obtained.

[0015] Preferably, in step (5), the gel column chromatography uses a Sephadex G-200 gel column; after loading the sample, water is used for elution, and 8~10 mL of eluent is collected from each tube at a flow rate of 0.8~1.0 mL / min. The eluent is collected and combined according to the elution curve, and dried to obtain barley water-soluble homogeneous heteropolysaccharide HBP-1a.

[0016] Similarly, the Sephadex G-200 gel chromatography column is a conventional medium for gel filtration chromatography, designed to separate biomolecules based on molecular size. Here, "G" represents the gel type, and "200" indicates its exclusion limit, i.e., the smallest molecular weight that cannot enter the internal pores of the gel. Under the aforementioned preferred type, it can efficiently and rapidly separate large and small molecules in the barley water-soluble heteropolysaccharide HBP-1, and perform fine separation by size within its effective range, thereby obtaining the homogeneous barley water-soluble heteropolysaccharide HBP-1a.

[0017] In a second aspect of the present invention, a water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley is provided, which is prepared using the preparation method of the first aspect of the present invention.

[0018] Preferably, the weight-average molecular weight of the barley water-soluble homogeneous heteropolysaccharide HBP-1a is 4.3~6.7×10⁻⁶. 4 Da; its monosaccharide composition includes glucose, arabinose, and xylose, and it has β-(1,3)-glucosidic bonds and β-(1,4)-glucosidic bonds; its composition includes the structural units shown below: .

[0019] More preferably, the molar ratio of glucose, arabinose and xylose is 6~7:6~7:1.

[0020] More preferably, the content ratio of the β-(1,4)-glucosidic bond to the β-(1,3)-glucosidic bond is 2.5~3:1.

[0021] In a third aspect of the invention, the application of the barley water-soluble homogeneous heteropolysaccharide HBP-1a of the second aspect of the invention is provided, including: using it as a raw material to prepare at least one of foods, health products, and pharmaceuticals that alleviate lactose intolerance.

[0022] In the aforementioned applications, besides the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley used as a raw material, other safe and applicable excipients may be included in the corresponding food, health product, or pharmaceutical formulations, depending on actual needs. The forms of food, health product, or pharmaceutical formulations containing the water-soluble homogeneous heteropolysaccharide HBP-1a are diverse, such as capsules, tablets, pills, granules, or oral liquids. Excipient types include one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, preservatives, and pH adjusters. Those skilled in the art can select according to actual needs.

[0023] Based on the above technical solutions, the design concept and principle of this invention are as follows: The safety and gut health-enhancing effects of barley polysaccharides have been well-studied, demonstrating antioxidant, anti-inflammatory, immune-boosting, and gut microbiota-regulating effects in the gut. Barley polysaccharides are derived from barley (a type of barley plant in the genus *Haloxylon* of the Poaceae family). Hordeum vulgare var. coeleste A type of polysaccharide extracted from L., whose physicochemical and structural properties vary with different extraction processes.

[0024] Traditional methods of improving the microbial community environment with polysaccharides suffer from drawbacks such as low precision in regulation and long processing time. Polysaccharides promote enzyme activity by targeting pre-existing enzyme protein molecules. Their main principles include: allowing polysaccharides to bind to enzymes as effectors, altering their spatial conformation to make the active site more efficient; improving the microenvironment (such as pH and ionic strength) to allow the enzyme to work under optimal conditions; or stabilizing the enzyme structure, acting as a protectant to prevent inactivation. The core effect is to improve the catalytic efficiency of existing enzyme molecules, but the duration is short, the effect is slow, and the results are often less than expected.

[0025] This invention employs a specific preparation process to successfully isolate and extract a water-soluble homogeneous heteropolysaccharide, HBP-1a, from highland barley. In its application, it was discovered for the first time that HBP-1a can increase the mRNA expression of β-galactosidase in Caco-2 cells (with a growth rate of up to 130%), thereby increasing its ability to hydrolyze lactose. Simultaneously, in a high-lactose diet-induced diarrhea model in mice, HBP-1a also significantly promoted the mRNA expression of β-galactosidase in the small intestine of mice (up to 238%), reducing the diarrhea rate from 87% to 9% and the diarrhea index from 3.89 to 0.56. Within 3 days of exposure to a high-lactose diet, the lactose intolerance-related symptoms in the tested mice were significantly improved.

[0026] Unlike existing technologies that use polysaccharides to regulate gut microbiota and promote enzyme activity, the core concept of this invention lies in the fact that the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley, while possessing prebiotic properties and enzyme-enhancing functions, can significantly increase the mRNA expression of β-galactosidase, thus optimizing both the quantity and activity of the corresponding enzyme. When the expression of the subject's own β-galactosidase mRNA is enhanced, lactose metabolism is correspondingly improved, thereby solving the technical problems of traditional methods, such as the need to introduce exogenous prebiotics and limited enzyme activity optimization. Based on the above principles and test results, this scheme directly regulates the expression of the subject's own β-galactosidase gene, increasing the endogenous synthesis of intestinal lactose hydrolase, thereby replacing the indirect pathway of traditional regulatory methods and providing a novel strategy for fundamentally solving lactose intolerance.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects: This invention provides a method for preparing the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley. The method involves extracting, precipitating, decolorizing, and deproteinizing the highland barley raw material, followed by separation and purification using anion exchange column chromatography and gel column chromatography. The water-soluble homogeneous heteropolysaccharide HBP-1a was successfully isolated from the highland barley polysaccharide. This invention is the first to discover that the water-soluble homogeneous heteropolysaccharide HBP-1a can effectively promote the expression of β-galactosidase in the small intestinal mucosa and can be used to improve symptoms related to lactose intolerance diarrhea. This invention explores a new application for the water-soluble homogeneous heteropolysaccharide HBP-1a, broadening its application fields and possessing broad prospects and market value. Attached Figure Description

[0028] Figure 1 DEAE-Fast flow elution curve of HBP, a water-soluble heteropolysaccharide from highland barley; Figure 2 The Sephadex G-200 elution curve of HBP-1, a water-soluble heteropolysaccharide from highland barley; Figure 3 The image shows the UV scan analysis of HBP-1a, a water-soluble heteropolysaccharide from highland barley. Figure 4 A schematic diagram showing the high performance liquid chromatography (HPLC) purity results of HBP-1a, a water-soluble heteropolysaccharide from highland barley. Figure 5 The monosaccharide composition spectrum of HBP-1a, a water-soluble heteropolysaccharide from highland barley, was determined. Figure 6 Total ion chromatogram of methylated sugar alcohol acetate derivatives of the water-soluble heteropolysaccharide HBP-1a from highland barley; Figure 7 HBP-1a, a water-soluble heteropolysaccharide from highland barley 1 H NMR spectrum; Figure 8 HBP-1a, a water-soluble heteropolysaccharide from highland barley 13 C NMR spectrum; Figure 9 Correlation spectroscopy (COSY) of HBP-1a, a water-soluble heteropolysaccharide from highland barley. Figure 10 Heteronuclear singular quantum correlation (HSQC) spectrum of HBP-1a, a water-soluble heteropolysaccharide from highland barley. Figure 11 Heteronuclear multiple bond correlation spectroscopy (HMBC) of HBP-1a, a water-soluble heteropolysaccharide from highland barley. Figure 12 The structural formula of HBP-1a, a water-soluble heteropolysaccharide from highland barley; Figure 13 The effect of barley water-soluble heteropolysaccharide HBP-1a on the diarrhea index in lactose-intolerant mice; Figure 14 Effects of barley water-soluble heteropolysaccharide HBP-1a on the morphology and structure of jejunal tissue in lactose-intolerant diarrhea mice; Figure 15 Effects of barley water-soluble heteropolysaccharide HBP-1a on jejunal villus height and crypt depth in lactose-intolerant diarrhea mice; Figure 16 The effect of barley water-soluble heteropolysaccharide HBP-1a on the amount of residual lactose in the feces of lactose-intolerant diarrhea mice; Figure 17 The effects of barley water-soluble heteropolysaccharide HBP-1a on ileal mucosal β-galactosidase activity and mRNA expression. Detailed Implementation

[0029] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0030] Example 1 This embodiment provides a method for preparing the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley, the steps of which are as follows: (1) Hot water extraction: Dry the barley, grind it into powder, pass it through a 60-mesh sieve, add anhydrous ethanol at a material-to-liquid ratio of 1:5 g / mL to defatt the barley, and then dry it to obtain defatted barley powder; then, weigh 5.0 g of defatted barley powder, add distilled water at a material-to-liquid ratio of 1:10 g / mL, extract at 70 ℃ for 6 h, and centrifuge to obtain the supernatant; (2) Alcohol precipitation: Add anhydrous ethanol to the supernatant obtained in step (1) to make the final ethanol concentration 85%, let it stand overnight at 4 ℃, centrifuge to collect the precipitate, wash it 3 times with anhydrous ethanol to remove organic reagents, freeze dry to obtain barley water-soluble crude polysaccharide. (3) Decolorization and deproteinization: Dissolve the barley water-soluble crude polysaccharide obtained in step (2) in distilled water, add activated carbon for decolorization, add Sevage reagent for deproteinization, freeze dry, and obtain decolorized and deproteinized barley water-soluble heteropolysaccharide HBP; (4) DEAE-Fast flow anion exchange column chromatography: Take 100 mg of the barley water-soluble heteropolysaccharide HBP obtained in step (3), dissolve it completely in 2 mL of distilled water and filter it through a 0.45 μm microporous membrane. Slowly load the filtrate onto the inner wall of the top of the chromatography column (5 cm × 50 cm). Subsequently, perform gradient elution with distilled water and 0.3 mol / L, 0.5 mol / L, and 1.0 mol / L sodium chloride aqueous solutions, respectively, at a flow rate of 1.0 mL / min. Collect 10 mL of eluent from each tube and elute according to the following... Figure 1 The elution curves shown indicate that the eluents eluted with distilled water were collected, combined, and concentrated. Subsequently, the concentrated solution was placed in a 3500 Da dialysis bag and dialyzed in distilled water for 72 h. After dialysis, the solution was freeze-dried to obtain barley water-soluble heteropolysaccharide HBP-1. (5) Sephadex G-200 gel column chromatography: Dissolve 20 mg of HBP-1 obtained in step (4) in 10 mL of distilled water and filter through a 0.45 μm microporous membrane. Slowly load the filtrate onto the inner wall of the top of the chromatography column (5 cm × 50 cm). Then, elute with distilled water at a flow rate of 1.0 mL / min. Collect 10 mL of eluent from each tube and process according to the following... Figure 2 The eluents from tubes 21 to 25 were collected and combined, and after freeze-drying, the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley was obtained.

[0031] This embodiment illustrates a typical process for extracting water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley. With other optimized parameters or raw material selections in this invention, those skilled in the art can also extract water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley to achieve the purpose of this invention.

[0032] Example 2 This embodiment provides a water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley, prepared using the method described in Example 1.

[0033] This study characterized the physicochemical properties and structure of HBP-1a, a water-soluble homogeneous heteropolysaccharide from highland barley, including the following: (1) Determination of the content of water-soluble heteropolysaccharide HBP-1a in highland barley: The polysaccharide content in the water-soluble heteropolysaccharide HBP-1a from highland barley was determined using the phenol-sulfuric acid method. The determined polysaccharide content in HBP-1a reached 96.49%.

[0034] (2) Ultraviolet full-band scanning of barley water-soluble heteropolysaccharide HBP-1a: The water-soluble heteropolysaccharide HBP-1a from highland barley was prepared to a concentration of 1.0 mg / mL using distilled water. Using distilled water as a blank control, the sample solution was scanned at 200–400 nm using a UV-Vis spectrophotometer to obtain the UV spectrum of the sample. The results are as follows: Figure 3 As shown. Figure 3 The water-soluble heteropolysaccharide HBP-1a from highland barley did not show obvious absorption peaks at 260 nm and 280 nm, indicating that the extracted product does not contain nucleic acid and protein impurities and has high purity.

[0035] (3) Determination of the molecular weight of barley water-soluble heteropolysaccharide HBP-1a: The water-soluble heteropolysaccharide HBP-1a from highland barley was dissolved in 0.1 mol / L sodium nitrate containing 0.05% sodium azide, at a concentration of 1.0 mg / mL. The solution was then filtered through a 0.22 nm membrane filter and placed in a sample vial for analysis.

[0036] Chromatographic method: A Waters high-performance liquid chromatograph was used with a polymer-based water-soluble column (SB-806HQ (Shodex OHpak, 8 mm × 300 mm) for detection. The mobile phase was 0.1 mol / L sodium nitrate after degassing and membrane treatment. The injection volume was 200 μL, the flow rate was 0.4 mL / min, the column temperature was 40 ℃, and the dn / dc value was 0.148 mL / g.

[0037] Test results are as follows Figure 4 As shown. The results indicate that the molecular weight of the barley water-soluble heteropolysaccharide HBP-1a is 6.7 × 10⁻⁶. 4 The fact that the spectral signal peak is a single and symmetrical narrow peak indicates that it is a homogeneous polysaccharide.

[0038] (4) Determination of the monosaccharide composition of the water-soluble heteropolysaccharide HBP-1a from highland barley: Preparation of sample solution: Weigh 5.0 mg of barley water-soluble heteropolysaccharide HBP-1a, add 4 mL of trifluoroacetic acid solution (4 mol / L), seal, and place in a 100 ℃ water bath for hydrolysis for 3 h. After removal, repeatedly evaporate the acid with methanol. Finally, add 5 mL of distilled water to dissolve, transfer to a chromatographic vial for analysis.

[0039] Chromatographic method: An ICS-3000 Dionex ion chromatograph equipped with a pulsed amperometric detector was used. The chromatographic column consisted of a CarboPac™ PA1 analytical column (4 × 250 mm) and a guard column (4 × 50 mm). The column temperature was 30 ℃, and the injection volume was 20 μL. Mobile phase A was 20 mmol / L sodium hydroxide solution, and mobile phase B was ultrapure water. The flow rate of the mobile phase was 0.6 mL / min. Simultaneously, corresponding monosaccharide standards were determined, and a concentration-peak area standard curve was established.

[0040] like Figure 5 As shown, the results of the monosaccharide composition determination indicate that the water-soluble heteropolysaccharide HBP-1a from highland barley is composed of glucose, xylose, and arabinose. The concentration was calculated using the standard curves of the corresponding monosaccharide standards, and the molar ratio of glucose:arabinose:xylose was calculated to be 6.9:6.1:1 based on the relative molecular mass.

[0041] (5) Total ion chromatogram of methylated sugar alcohol acetate derivatives of barley water-soluble heteropolysaccharide HBP-1a: Weigh 15 mg of the sample and add 10 mL of dry dimethyl sulfoxide, stirring for 3 h until fully dissolved. Then add 200 mg of dry sodium hydroxide powder, seal, and stir for 2–3 h. Next, slowly add 1 mL of iodomethane reagent under ice-water bath conditions and react for 1 h, followed by slow addition of 1 mL of iodomethane and reacting for 1.5 h. Finally, add 3 mL of distilled water to terminate the reaction. Then, extract with an equal volume of chloroform, collect the lower extract, and wash three times with distilled water. Combine the extracts and pass them through an anhydrous sodium sulfate column to remove water.

[0042] Subsequently, 4 mL of trifluoroacetic acid solution (4 mol / L) was added to the fully methylated sample, and the mixture was sealed and placed in a 100 °C water bath for hydrolysis for 3 h. After removal, the sample was repeatedly evaporated with methanol to remove acid. The sample was completely dissolved in 3 mL of distilled water, and 60 mg of sodium borohydride was added for reduction. The mixture was stirred at room temperature for 12 h, and glacial acetic acid was added dropwise until no bubbles were generated in the solution. The solution was then evaporated to dryness under reduced pressure. Finally, 2 mL of acetic anhydride and 2 mL of pyridine were added to the resulting powder, and the reaction was carried out at 100 °C for 2 h. The mixture was evaporated to dryness under reduced pressure, and acetic anhydride and pyridine were removed using methanol. The partially methylated sugar alcohol acetate was redissolved in dichloromethane and filtered through a 0.22 μm filter membrane for analysis.

[0043] Chromatographic method: Glycosidic bond composition was determined using gas chromatography-mass spectrometry (GC-MS) with an HP-5 capillary column (30 m × 0.25 mm × 0.25 μm). High-purity helium was used as the carrier gas. The column flow rate was 1 mL / min. The injection volume was 1.0 μL, the split ratio was 1:1, and the injection port temperature was set to 260 ℃. The initial temperature was 60 ℃, increased to 80 ℃ at 5 ℃ / min and held for 1 min, then increased to 220 ℃ at 10 ℃ / min and held for 1 min, and finally increased to 300 ℃ at 20 ℃ / min and held for 1 min. The ion source temperature was set to 300 ℃, the electron collision energy to 70 eV, the interface temperature to 300 ℃, the solvent delay time to 5.0 min, and the mass scan range to m / z 30–450 amu.

[0044] like Figure 6The results showed that the sugar units of the barley water-soluble heteropolysaccharide HBP-1a are mainly linked through 1,4-xylp (pyranose residues linked by β-(1,4)-glycosidic bonds), 1,4-glcp (pyranose glucose residues linked by β-(1,4)-glycosidic bonds), and 1,3-glcp (pyranose glucose residues linked by β-(1,3)-glycosidic bonds). Based on the ratio of the peak areas representing 1,4-glcp and 1,3-glcp, the ratio of β-(1,4)-glucosidic bonds to β-(1,3)-glucosidic bonds was calculated to be 2.7.

[0045] (6) NMR analysis of water-soluble heteropolysaccharide HBP-1a from highland barley: 50 mg of the water-soluble heteropolysaccharide HBP-1a from highland barley was dissolved in 0.5 mL of D2O. One-dimensional NMR was measured using a 600 MHz Bruker nuclear magnetic resonance spectrometer. 1 H NMR, 13 C NMR and two-dimensional nuclear magnetic resonance (COSY, HSQC, HMBC).

[0046] Depend on Figure 7 of 1 ¹H NMR revealed the presence of several anomers in the range of 3.7–4.2 ppm, indicating that it exists in more than one configuration. Figure 8 of 13 C NMR and Figure 10 Based on the HSQC spectrum, combined with the monosaccharide composition and methylation results, it can be seen that the α-Araf-(1→) residues can be divided into three cases, labeled A, B, and C respectively. Further combining... Figure 9 COSY and Figure 11The HMBC spectrum assigned anodic carbon signals to 107.57 ppm, 108.33 ppm, and 109.35 ppm. Following this, the remaining carbon and hydrogen signals were assigned based on the carbon signals in the non-anodic carbon regions of the HSQC spectrum; for example, H1 / H2 in residue A was located at 5.34 / 3.88 ppm. Based on the monosaccharide composition, methylation results, NMR results and references ([1] Sun Y, Cui SW, Gu X, et al. Isolation and structural characterization of water unextractable arabinoxylans from Chinese black-grained wheat bran [J]. CarbohydratePolymers, 2011, 85(3): 615-621; [2] Kang J, Guo Q, Shi Y. NMR and methylation analysis of hemicellulose purified from corn bran [J]. Food Hydrocolloids, 2019, 94: 613-621; [3] Revanappa SB, Nandini CD, Salimath P V. Structural variations of arabinoxylans extracted from different wheat ( Triticum aestivum cultivars in relation to chapati -quality [J]. Food Hydrocolloids, 2015, 43: 736-742; [4] Yadav MP, Kale MS, Hicks KB, et al. Isolation, characterization and the functional properties of cellulosic arabinoxylanfiber isolated from agricultural processing by-products, agricultural residues and energy crops [J]. Food Hydrocolloids, 2017, 63: 545-551.), the main structural units of barley water-soluble heteropolysaccharide HBP-1a are as follows Figure 12 As shown.

[0047] Example 3 This embodiment studied the effects of barley water-soluble heteropolysaccharide HBP-1a on β-galactosidase activity and mRNA expression in Caco-2 cells. This embodiment can be regarded as an application embodiment to confirm the efficacy of barley water-soluble heteropolysaccharide HBP-1a.

[0048] Caco-2 cell line was purchased from Wuhan Pronosei Life Sciences Co., Ltd. Caco-2 cells were cultured in DMEM medium at 37 ℃ in a 5% CO2 incubator. When the cell density reached 90%, cells were digested and passaged using trypsin solution, and cells in the logarithmic growth phase were harvested. Experimental groups included: a blank control group (lactose-free, HBP-1a-free), a lactose group (20 µg / mL lactose), a sample control group (50 µg / mL HBP-1a), and a sample group (20 µg / mL lactose + 50 µg / mL HBP-1a). Cells were grown at a rate of 1 × 10⁶ cells / mL. 4 The samples were seeded at a density of 100 cells / mL in 96-well plates. After 24 h of adhesion, the samples were treated with different groups as described above for 24 h, and relevant indicators were measured. Simultaneously, referring to the preparation method in Example 1, the HBPs obtained under different preparation conditions (HBP-A, HBP-B, HBP-C), as well as HBP-1, HBP-2, HBP-3, and HBP-4 (see Example 1) were also measured. Figure 1 The promoting effect of ).

[0049] The effects of different sample treatments on β-galactosidase activity and mRNA expression in Caco-2 cells are shown in Table 1.

[0050] Table 1: Effects of different sample treatments on β-galactosidase activity and mRNA expression in Caco-2 cells

[0051] The β-galactosidase activity of untreated Coca-2 cells (blank control group) was 2.62 au; the expression level of β-actin (internal control) was 15.64 au. As shown in Table 1, compared to the blank control group, administration of lactose alone or HBP-1a induced upregulation of β-galactosidase mRNA expression and enzyme activity in Caco-2 cells. This indicates that HBP-1a may promote the expression of β-galactosidase in intestinal mucosal cells, thereby promoting the digestion of dietary lactose and intervening in lactose intolerance and related symptoms.

[0052] Table 2 shows the key preparation parameters for HBP (HBP-A, HBP-B, HBP-C), as well as the HBP-1, HBP-2, HBP-3, and HBP-4 groups, and their effects on the molecular weight, monosaccharide composition, and glycosidic bond composition of HBP.

[0053] Table 2: Effects of key preparation parameters on HBP molecular weight, monosaccharide composition, and glycosidic bond composition

[0054] In the table, "\" represents not adding anything.

[0055] As shown in Table 2, according to the aforementioned preferred conditions, in the preparation of barley water-soluble heteropolysaccharide HBP, the extraction temperature is 60~80 ℃, the extraction time is 5~6 h, and the final ethanol concentration during alcohol precipitation is 80%~90%. To illustrate the specific function of the preferred parameters in the process, the effects of different extraction temperatures, extraction times, and the final ethanol concentration during alcohol precipitation on the structural parameters of the obtained HBPs (HBP-A, HBP-B, HBP-C) were measured as above. The results showed that when the three key extraction parameters were within the optimal range, the monosaccharide composition and the ratio of β-(1,4)- to β-(1,3)-glucosidic bonds in HBP were close to those in HBP-1a, and were within the range of the optimal monosaccharide composition and the ratio of β-(1,4)- to β-(1,3)-glucosidic bonds in HBP-1a, i.e., composed of glucose, arabinose, and xylose in a molar ratio of 6~7:6~7:1; containing both β-(1,4)-glucosidic bonds and β-(1,3)-glucosidic bonds, with a ratio of 2.5~3:1.

[0056] Furthermore, according to the aforementioned preferred conditions, in the preparation of the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley, the eluent eluted with distilled water was collected based on the elution curve of the DEAE-Fast flow anion exchange chromatography column. Also based on the elution curve ( Figure 1 The structural parameters of samples in different eluents were determined, namely HBP-1 (eluted with distilled water), HBP-2 (eluted with 0.3 M NaCl), HBP-3 (eluted with 0.5 M NaCl), and HBP-4 (eluted with 1.0 M NaCl). The results showed that only the monosaccharide composition and the ratio of β-(1,4)- to β-(1,3)-glucosidic bonds in HBP-1 were similar to those in HBP-1a.

[0057] To verify the effect of the process of the present invention on the effect, this embodiment tested the effect of different HBP samples on β-galactosidase activity and mRNA expression in Caco-2 cells, and the results are shown in Table 3.

[0058] Table 3: Effects of different HBP samples on β-galactosidase activity and mRNA expression in Caco-2 cells

[0059] The β-galactosidase activity of Coca-2 cells treated with HBP was 4.83 au; the relative expression level of β-galactosidase mRNA, used as an internal control, was 27.62. Table 3 shows that the promoting effect of HBP was more significant than that of HBP-A, HBP-B, and HBP-C, indicating that appropriate extraction temperature, extraction time, and final ethanol concentration during precipitation may be key parameters for further preparation of high-efficiency HBP-1a. Furthermore, the promoting effect of HBP-1 was superior to that of HBP-2, HBP-3, and HBP-4, suggesting that collecting the distilled water elution fraction during HBP purification via a DEAE-Fast flow anion exchange chromatography column is a crucial step in preparing high-efficiency HBP-1a, rather than a routine procedure in this field.

[0060] Example 4 This embodiment studied the intervention effect of barley water-soluble heteropolysaccharide HBP-1a on lactose intolerance diarrhea. This embodiment can be regarded as an application embodiment to confirm the efficacy of barley water-soluble heteropolysaccharide HBP-1a.

[0061] Fifty-six healthy male ICR mice aged 3 weeks were purchased from Wuhan Luobin Life Science Technology Co., Ltd. After one week of acclimatization, the mice were randomly divided into 7 groups (n=8): blank control group (C), model group (M), low-dose HBP-1a group (L, 100 mg / kg), medium-dose HBP-1a group (MI, 200 mg / kg), high-dose HBP-1a group (H, 400 mg / kg), sample control group (S, 400 mg / kg), and positive control group (galactooligosaccharide, G, 200 mg / kg). After the acclimatization period, mice were administered samples by gavage for 21 consecutive days. Mice in groups C and M were administered sterile saline (0.2 mL) daily by gavage; mice in groups L, MI, H, and S were administered different sample solutions (0.2 mL) according to their respective doses. All groups were fed a basal diet during this period. From day 22 to 24, gavage was stopped. Mice in groups C and S continued to be fed a normal basal diet, while mice in groups M, L, MI, H and G were fed a high-lactose diet. The nutritional composition of the two diets is shown in Table 4, in order to construct a lactose intolerance model and evaluate the effects of different treatments on related indicators.

[0062] Table 4: Composition of basal feed and high-lactose feed

[0063] (1) Effect of barley water-soluble heteropolysaccharide HBP-1a on diarrhea index in lactose-intolerant mice: Changes in the diarrhea index in mice as follows Figure 13 As shown in the figure, the model group exhibited significant diarrhea after lactose gavage. The diarrhea index was 3.89 on day 1, 3.07 on day 2, and 2.76 on day 3, remaining at a high level overall, with a total diarrhea rate of 87%, indicating that lactose induction successfully established a lactose intolerance model. Compared with the model group, the diarrhea severity in mice was alleviated after intervention with barley water-soluble heteropolysaccharide HBP-1a. Specifically, the diarrhea index in the high-dose group was 2.05 on day 1, 1.75 on day 2, and decreased to 0.56 on day 3, with an overall diarrhea rate of 9%, significantly better than the commonly used prebiotic GOS. Overall, all doses of barley water-soluble heteropolysaccharide HBP-1a could reduce the diarrhea index to some extent, with the high-dose group showing a more significant improvement.

[0064] (2) Effects of barley water-soluble heteropolysaccharide HBP-1a on the morphology and structure of jejunal tissue in lactose-intolerant diarrhea mice: β-galactosidase is mainly found on the surface of the villi of the small intestinal mucosa. Figure 14 The (A) blank control group, (B) sample control group, (C) model group, (D) high-dose group, and (E) galactooligosaccharide group showed that the jejunal histology of the blank control group and sample control group was intact, with normal crypt structure and regular brush-like arrangement of villi; while the model group showed obvious tissue damage, villi atrophy, and deepened crypts. For the high-dose group and galactooligosaccharide group, the intervention of the sample effectively improved these pathological changes, and the tissue structure was more intact and healthy than that of the model group, which may explain why the high-dose group mice had milder diarrhea symptoms.

[0065] (3) Effects of barley water-soluble heteropolysaccharide HBP-1a on jejunal villus height and crypt depth in lactose-intolerant diarrhea mice: Villus height and crypt depth are key indicators for evaluating intestinal digestive and absorptive function and mucosal health, and are closely related to normal lactose metabolism. Figure 15The results showed that the villus height in the blank control group was 0.296 μm and the crypt depth was 0.085 μm, indicating intact intestinal structure and well-developed villi. In contrast, the villus height in the model group mice decreased significantly to 0.203 μm, while the crypt depth increased to 0.120 μm, indicating that diarrhea led to villus atrophy and crypt deepening, resulting in significant damage to the intestinal structure. Under the influence of HBP-1a, the villus height in the high-dose group mice reached 0.369 μm, while the crypt depth decreased to 0.109 μm, which was better than the model group. This indicates that high-dose treatment can effectively promote villus repair and improve intestinal structure, with better effects than the commonly used commercial prebiotic GOS. Notably, in the sample control group (treated with HBP-1a but without modeling), the villus height and crypt depth of the mice were both better than the blank group, indicating that HBP-1a is beneficial for strengthening intestinal structure and improving digestion and absorption.

[0066] (4) Effect of barley water-soluble heteropolysaccharide HBP-1a on the amount of residual lactose in the feces of lactose-intolerant diarrhea mice: like Figure 16 As shown, the residual lactose in the feces of the model group was 43.49 mg / mL, significantly higher than that of other groups, indicating that the mice in this group had a lower ability to digest lactose, possibly due to lower expression and activity of β-galactosidase. Compared with the model group, the residual lactose content in the feces of the high-dose group was significantly reduced, at only 3.26 mg / mL, indicating that undigested lactose in the intestine was significantly reduced after HBP-1a intervention. This confirms that the water-soluble heteropolysaccharide HBP-1a from highland barley can effectively intervene in diarrhea caused by lactose intolerance by promoting the body's ability to digest lactose.

[0067] (5) Effects of barley water-soluble heteropolysaccharide HBP-1a on ileal mucosal β-galactosidase activity and expression: lactase, etc. Figure 17 As shown in Figure A, the model group had the lowest β-galactosidase activity, significantly lower than the blank control group, indicating that lactose intolerance diarrhea damages the intestinal structure, leading to a further decrease in β-galactosidase activity. Furthermore, the β-galactosidase activity in the sample control group (treated with HBP-1a but without modeling) was higher than that in the blank control group, indicating that the barley water-soluble heteropolysaccharide HBP-1a can enhance β-galactosidase activity. In the high-dose barley polysaccharide group, β-galactosidase activity was significantly increased compared to the model group, approaching that of the blank, disease-free group, indicating that intervention with the barley water-soluble heteropolysaccharide HBP-1a can, to some extent, restore intestinal β-galactosidase activity in diarrheal mice, thereby enhancing the body's ability to hydrolyze lactose and significantly reducing intestinal lactose residue.

[0068] Simultaneously, the relative expression of β-galactosidase mRNA in the ileal mucosa of different groups of mice was measured. For example... Figure 17As shown in Figure B, the relative expression level in the blank control group was 0.433, in the sample control group it was 0.764, in the model group it decreased to 0.119, while in the high-dose treatment group it significantly increased to 0.402. The relative expression level in the model group was significantly lower than that in the blank control group and the sample control group, indicating that the construction of the lactose intolerance model led to the inhibition of β-galactosidase gene transcription, affecting lactose digestion and causing diarrhea. The sample control group had the highest relative expression level, indicating that HBP-1a can increase the expression of β-galactosidase in the intestinal mucosa. In addition, compared with the model group, the relative expression level in the high-dose treatment group recovered and approached the level of the blank control group. These results collectively suggest that HBP-1a can effectively promote β-galactosidase gene expression, thereby improving lactose digestibility.

[0069] (6) Effects of different HBP samples on diarrhea index and ileal mucosal β-galactosidase activity and expression in lactose-intolerant mice: This embodiment measured the effects of different HBP samples described in Embodiment 3 of the invention on the diarrhea index and ileal mucosal β-galactosidase activity and expression in lactose-intolerant mice, and the results are shown in Table 5.

[0070] Table 5: Effects of different HBP samples on diarrhea index and ileal mucosal β-galactosidase activity and expression in lactose-intolerant mice

[0071] The β-galactosidase activity in the small intestine of mice fed HBP was measured to be 0.21 au. In the detection of relative β-galactosidase mRNA expression, the expression level of β-actin (used as an internal control) was above 25.46. These results indicate that HBP and HBP-1 exhibit relatively good anti-diarrheal effects and the ability to promote β-galactosidase activity and expression, further demonstrating the criticality of the optimal preparation parameters for HBP-1a described in the invention.

[0072] In summary, the results from the Caco-2 assay and mouse experiments demonstrate that HBP-1a not only strengthens the integrity and health of the intestinal structure, but also innovatively enhances β-galactosidase activity and mRNA expression in the ileal mucosa. While the exact mechanism by which HBP-1a promotes its expression is not yet fully understood, it is speculated that this may be closely related to the metabolites produced by the fermentation of this large molecule by colonic microbiota. Short-chain fatty acids, organic acids, oligosaccharides, and other polysaccharide secondary metabolites may act on intestinal epithelial cells, regulating their expression of β-galactosidase, ultimately alleviating lactose intolerance symptoms.

[0073] In summary, this invention provides a specific method for preparing the water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley. The method involves extracting, precipitating, decolorizing, and deproteinizing the highland barley raw material, followed by separation and purification using anion exchange column chromatography and gel column chromatography. The water-soluble homogeneous heteropolysaccharide HBP-1a was successfully isolated from highland barley polysaccharides. This invention is the first to discover that the water-soluble homogeneous heteropolysaccharide HBP-1a can effectively promote the expression of β-galactosidase in the small intestinal mucosa and can be used to improve symptoms related to lactose intolerance diarrhea. This invention explores a new application for the water-soluble homogeneous heteropolysaccharide HBP-1a, broadening its application fields and possessing broad prospects and market value.

[0074] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley, characterized in that, Includes the following steps: (1) The defatted barley raw material is mixed with water and extracted at a certain temperature. After the extraction is completed, the supernatant is collected by centrifugation. (2) The supernatant obtained was subjected to alcohol precipitation, and after the precipitation was completed, it was centrifuged and the precipitate was collected; the precipitate was washed and dried to obtain barley water-soluble crude polysaccharide; (3) Dissolve barley water-soluble crude polysaccharide in water, and after decolorization, deproteinization and drying, obtain decolorized and deproteinized barley water-soluble heteropolysaccharide HBP; (4) The barley water-soluble heteropolysaccharide HBP was dissolved in water and purified by anion exchange column chromatography. After loading the sample, it was eluted with water and sodium chloride aqueous solution of different concentrations. The eluents were collected and combined. After concentration, dialysis and drying, barley water-soluble heteropolysaccharide HBP-1 was obtained. (5) Dissolve barley water-soluble heteropolysaccharide HBP-1 in water, purify by gel column chromatography, elute with water after loading, collect and combine the eluent, and dry to obtain barley water-soluble homogeneous heteropolysaccharide HBP-1a.

2. The method of claim 1, wherein the preparation of the water-soluble homogeneous heteropolysaccharide HBP-1a from Highland Barley is characterized by: In step (1), the extraction temperature is 60~80 ℃ and the extraction time is 5~6 h.

3. The method of claim 1, wherein the preparation of the water-soluble homogeneous heteropolysaccharide HBP-1a from Highland Barley is characterized by: In step (2), the alcohol precipitation treatment uses ethanol, the treatment temperature is 0~4 ℃, and the final concentration of ethanol in the alcohol precipitation treatment is 80 vol.%~90 vol.%.

4. The method of claim 1, wherein the preparation of the water-soluble homogeneous heteropolysaccharide HBP-1a from Highland Barley is characterized by: In step (4), the anion exchange column chromatography was performed using a DEAE-Fast flow anion exchange column. After loading the sample, gradient elution was performed with water and 0.3~1.0 mol / L sodium chloride aqueous solution, respectively, at a flow rate of 0.8~1.0 mL / min. 8~10 mL of eluent was collected from each tube. The eluent was collected according to the elution curve, combined, concentrated, dialyzed, and dried to obtain barley water-soluble heteropolysaccharide HBP-1.

5. The method of claim 1, wherein the preparation of the water-soluble homogeneous heteropolysaccharide HBP-1a from Highland Barley is characterized by: In step (5), the gel column chromatography uses a Sephadex G-200 gel column; after loading the sample, water is used for elution, and 8~10 mL of eluent is collected from each tube at a flow rate of 0.8~1.0 mL / min. The eluent is collected and combined according to the elution curve, and dried to obtain barley water-soluble homogeneous heteropolysaccharide HBP-1a.

6. A water-soluble homogeneous heteropolysaccharide HBP-1a from highland barley, characterized in that: It is prepared by any one of the preparation methods described in claims 1 to 5.

7. The water-soluble homogeneous heteropolysaccharide HBP-1 a according to claim 6, characterized by: The weight average molecular weight of the highland barley water-soluble uniform heteropolysaccharide HBP-1a is 4.3-6.7*10 4 Da; the monosaccharide composition thereof includes glucose, arabinose and xylose, and has beta-(1,3)-glucoside bonds and beta-(1,4)-glucoside bonds; the composition thereof includes the structural unit as shown in the following formula: 。 8. The water-soluble homogeneous heteropolysaccharide HBP-1a according to claim 7, characterized by: The molar ratio of glucose, arabinose, and xylose is 6~7:6~7:

1. 9.The HBP-1a of claim 7, wherein the HBP-1a is a homogeneous heteropolysaccharide. The content ratio of the β-(1,4)-glucosidic bond to the β-(1,3)-glucosidic bond is 2.5~3:

1.

10. Use of the water-soluble homogeneous heteropolysaccharide HBP-1a of claim 6-9, characterized in that, include: It is used as a raw material in the preparation of at least one of the following: foods, health products, and medicines that alleviate lactose intolerance.

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