Lotus seed oligosaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of natural product extraction and biological medicine, and relates to lotus seed oligosaccharide as well as a preparation method and application thereof. Raw lotus seeds or honey-fried lotus seeds are used as raw materials, and water extraction and alcohol precipitation are carried out. Effective components are extracted from raw lotus seeds and honey-fried lotus seeds, in-vivo experiments are carried out, and it is verified that the traditional Chinese medicine composition has the effects of improving immunity, reducing immunity, protecting intestinal barriers and adjusting intestinal flora and intestinal flora metabolism. In addition, the lotus seed oligosaccharide provided by the invention is convenient to obtain and safe, has universality, and is easy for large-scale production, so that the lotus seed oligosaccharide has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of natural product extraction and biomedicine, and relates to a lotus seed oligosaccharide, a preparation method thereof and an application thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily to be regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Immune deficiency refers to a state in which an individual's immune system function is reduced and cannot effectively resist the invasion of foreign pathogens or eliminate abnormal cells in the body. This state may be caused by various factors such as genetics, malnutrition, chronic diseases, long-term use of immunosuppressants, aging or psychological stress. Individuals with immune deficiency are more susceptible to infections by pathogens such as bacteria, viruses, fungi and parasites, and the condition is often more severe after infection, the recovery is slower, and long-term immune deficiency may lead to the occurrence of diseases such as chronic inflammation, autoimmune diseases and cancers. Frequent infections and chronic diseases will seriously affect the quality of life of individuals and increase the medical burden.

[0004] At present, most of the available drugs for enhancing immunity on the market have certain limitations, such as strong side effects and relatively single efficacy. Therefore, the development of immunomodulatory drugs or natural products with low toxicity and side effects and significant protective effects is of crucial significance for anti-immunosuppression and has also become one of the hotspots in the global scientific research field. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a lotus seed oligosaccharide, a preparation method thereof and an application thereof. The present invention extracts effective components from raw lotus seeds and roasted lotus seeds and conducts in-vivo experiments to verify that it has the effects of enhancing immunity, improving immune deficiency, protecting the intestinal barrier, and regulating the intestinal flora and intestinal flora metabolism. Based on the above research results, the present invention is completed.

[0006] To achieve the above technical purpose, the technical scheme adopted by the present invention is as follows:

[0007] In the first aspect, a lotus seed oligosaccharide is obtained by water extraction and alcohol precipitation using raw lotus seeds or roasted lotus seeds as raw materials.

[0008] Semen Nelumbinis is the dried and mature seed of Nelumbo nucifera Gaertn. of the Nymphaeaceae family, which has rich nutritional value and various pharmacological effects. In traditional Chinese medicine, Semen Nelumbinis is considered to have the effects of invigorating the spleen and stopping diarrhea, tonifying the kidney and astringing essence, and nourishing the heart and tranquilizing the mind. Research shows that Lotus Seed Oligosaccharides (LSO) is a natural functional oligosaccharide extracted from Semen Nelumbinis, which is composed of 2 - 10 monosaccharide molecules linked by glycosidic bonds. The monosaccharide components include glucose, mannose, fructose, and galactose, which have prebiotic activity to promote the growth of Lactobacillus acidophilus strains and can also increase the content of short-chain fatty acids. The research of this invention finds that both raw lotus seed oligosaccharides and roasted lotus seed oligosaccharides extracted from raw lotus seeds and roasted lotus seeds respectively have the effects of improving the immunity of rats, protecting the intestinal barrier, regulating the intestinal flora and intestinal flora metabolism. Among them, since roasted lotus seeds are the processed products formed by stir-frying raw lotus seeds, there are differences in the effects of the extracted raw lotus seed oligosaccharides and roasted lotus seed oligosaccharides. The roasted lotus seed oligosaccharides have better effects in improving immunity and protecting the intestinal barrier than the raw lotus seed oligosaccharides; the raw lotus seed oligosaccharides have better effects in regulating the intestinal flora and intestinal flora metabolism than the roasted lotus seed oligosaccharides.

[0009] Secondly, a preparation method of the above-mentioned lotus seed oligosaccharides is as follows:

[0010] Crush raw lotus seeds or roasted lotus seeds into powder.

[0011] Boil the powder with water for water extraction treatment, and separate the solid and liquid to obtain the supernatant.

[0012] Add ethanol to the supernatant for alcohol precipitation treatment, and separate the solid and liquid to obtain the precipitate.

[0013] Freeze-dry the precipitate to obtain the product.

[0014] In some embodiments, the powder is sieved through a 60 - 80 mesh sieve.

[0015] In some embodiments, the material-liquid ratio of the powder to water is 1:1 - 10 kg / L, preferably 1:1.5 - 5.5 kg / L.

[0016] In some embodiments, the time for water extraction treatment is 1 - 3 h, preferably 1.8 - 2.2 h.

[0017] In some embodiments, the number of times of water extraction treatment is 1 - 5 times, preferably 3 times.

[0018] In some embodiments, in the alcohol precipitation treatment system, the supernatant and 94 - 96 v% ethanol.

[0019] In some embodiments, the time for alcohol precipitation treatment is 10 to 40 h, preferably 23 to 25 h.

[0020] In some embodiments, protein removal is carried out by the Sevage method. Specifically, the protein is removed 5 to 8 times, preferably 7 to 8 times, using Sevage reagent (CH3Cl:BuOH = 4:1, v / v).

[0021] In some embodiments, the oligosaccharides are separated and purified by DEAE sepharose FF gel suspension ion exchange column chromatography and reverse phase silica gel column chromatography. Specifically, the elution separation salt concentrations are dH2O, 0.2 M NaCl, 0.5 M NaCl, and 1.0 M NaCl; the elution volume is 2 times, and the elution flow rate is 15 mL / min.

[0022] In the third aspect, an application of the above-mentioned lotus seed oligosaccharides in the preparation of a product for preventing and / or treating immune deficiency.

[0023] In some embodiments, the immune deficiency is induced by cyclophosphamide. The specific manifestations of preventing and / or treating immune deficiency are as follows: both raw lotus seed oligosaccharides and roasted lotus seed oligosaccharides can inhibit weight loss, improve the decrease in spleen index caused by cyclophosphamide, increase the contents of IgA, IgM, and IgG in serum, and increase the content of CD4 + CD8 + content. And the effect of roasted lotus seed oligosaccharides is better than that of raw lotus seed oligosaccharides.

[0024] In the fourth aspect, an application of the above-mentioned lotus seed oligosaccharides in the preparation of a product for preventing and / or treating intestinal barrier injury.

[0025] The specific manifestations of preventing and / or treating intestinal barrier injury are as follows: both raw / roasted lotus seed oligosaccharides can reduce the colon index, reduce the pathological damage of the colon, and increase the expression levels of tight junction proteins ZO-1 / Occludin / and Collagen IV. Roasted lotus seed oligosaccharides (especially the high-dose group) have a better effect on restoring the intestinal barrier than the raw lotus seed group.

[0026] In the fifth aspect, an application of the above-mentioned lotus seed oligosaccharides in the preparation of a product for preventing and / or treating intestinal flora disorder and intestinal flora metabolic disorder.

[0027] After induction with cyclophosphamide, the abundance of intestinal flora decreased. After administration of lotus seed oligosaccharides, the abundance of intestinal flora can be increased, and the effect of raw lotus seed oligosaccharides is better than that of roasted lotus seed oligosaccharides. At the phylum level, the abundance of Firmicutes decreased, the abundance of Bacteroidetes increased, and the ratio of Firmicutes to Bacteroidetes decreased.

[0028] The products of the present invention include but are not limited to foods and drugs.

[0029] The food mentioned herein can be understood in any edible form. For example, the food in the present invention includes ordinary food and special food. The special food in the present invention includes health food and formula food for special medical purposes; while ordinary food is relative to special food and is food suitable for everyone.

[0030] The medicine can be administered in unit dosage form, and the dosage form can be liquid dosage form or solid dosage form. The liquid dosage form can be true solution type, colloid type, particulate dosage form, emulsion dosage form, suspension dosage form. Other dosage forms such as tablets, capsules, dripping pills, aerosols, pills, powders, solution agents, emulsions, granules, suppositories, freeze-dried powder injections, inclusion compounds, landfill agents, patches and liniments, etc.

[0031] The medicine can be used for humans and non-human animals. The non-human animals can be birds and mammals, especially livestock and poultry animals.

[0032] In the present invention, the concept of "prevention and / or treatment" means any measure applicable to preventing and / or treating diseases related to cyclophosphamide-induced immunosuppression and intestinal flora disorder, or treating the diseases or symptoms manifested by such performance, or avoiding the recurrence of such diseases, such as recurrence after the end of the treatment period or treating the symptoms of the already occurring diseases, or pre-interventionally preventing or inhibiting or reducing the occurrence of such diseases or symptoms.

[0033] The present invention also provides a method for preventing and treating cyclophosphamide-induced immunosuppression and improving intestinal flora disorder, the method comprising administering the above-mentioned raw / roasted lotus seed oligosaccharide or product to a subject.

[0034] The subject can be a human or a non-human animal, preferably a human.

[0035] The beneficial effects of the present invention are as follows:

[0036] (1) The present invention discloses an extraction method of raw lotus seed and roasted lotus seed oligosaccharide, and the results show that it is successfully extracted from lotus seeds.

[0037] (2) The present invention discloses the immunomodulatory effects of raw lotus seed oligosaccharide and roasted lotus seed oligosaccharide. Specifically, the raw / roasted lotus seed oligosaccharide induces a rat immunosuppression model by cyclophosphamide (CTX) in in vivo experiments. The results show that the raw / roasted lotus seed oligosaccharide inhibits weight loss, improves the decrease in spleen index caused by cyclophosphamide, increases the contents of IgA, IgM and IgG in serum, and increases the CD4 + / CD8 + content. And the effect of roasted lotus seed oligosaccharide is better than that of raw lotus seed.

[0038] (3) The intestinal barrier protection effect of raw / roasted lotus seed oligosaccharides disclosed by the present invention is specifically manifested as: reducing the colon index, reducing colon pathological damage, and increasing the expression levels of tight junction proteins ZO-1 / Occludin / and Collagen IV. The roasted lotus seed oligosaccharide group has a better effect on restoring the intestinal barrier compared to the raw lotus seed group.

[0039] (4) The present invention discloses the intestinal flora regulation effect of raw / roasted lotus seed oligosaccharides. Specifically, the raw / roasted lotus seed oligosaccharides significantly regulate the intestinal flora disorder induced by cyclophosphamide in in vivo experiments. At the phylum level, the abundance of Firmicutes is reduced and the abundance of Bacteroidetes is increased. Moreover, the effect of raw lotus seed oligosaccharides on regulating intestinal flora is better than that of roasted lotus seed oligosaccharides.

[0040] (5) The raw / roasted lotus seed oligosaccharides disclosed by the present invention are widely applicable, easy to obtain, safe, have universality, and are easy to scale up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0042] Figure 1 Separation and purification curves and structures for Example 1 of the present invention; (A) Ion exchange column chromatography results, (B) Reverse-phase silica gel column chromatography elution curve of sample ULZ-E1, (C) HPLC chromatogram of sample ULZE1-F3, (D) Infrared spectrum of sample ULZE1-F3.

[0043] Figure 2 Separation and purification curves and structures for Example 1 of the present invention; (A) Ion exchange column chromatography results, (B) Reverse-phase silica gel column chromatography elution curve of sample RLZ-E1, (C) HPLC chromatogram of sample RLZE1-F2, (D) Infrared spectrum of sample RLZE1-F2.

[0044] Figure 3 Results graphs of the regulation of body weight, spleen index, and the contents of IgA, IgM, and IgG in serum of rats induced by CTX by the lotus seed oligosaccharides prepared in Example 1 of the present invention; wherein, (A) Body weight data, (B) Spleen index, (C) IgA content in rat serum, (D) IgM, (E) IgG.

[0045] Figure 4Measurement result diagram of the CD4+ / CD8+a of the immune ability of lotus seed oligosaccharides prepared in Example 1 of the present invention; (A) Control, (B) Model, (C) LLOS, (D) HLOS, (E) FLLOS, (F) FHLOS, (G) APS, (H) Comprehensive analysis of CD4+CD8+a.

[0046] Figure 5 Test result diagram of the protective effect of lotus seed oligosaccharides prepared in Example 1 of the present invention on the intestine; (A)-(H) Colon pathological sections; (A) Control, (B) Model, (C) LLOS, (D) HLOS, (E) FLLOS, (F) FHLOS, (G) APS, (H) Colon index.

[0047] Figure 6 Test result diagram of the protective effect of lotus seed oligosaccharides prepared in Example 1 of the present invention on the intestinal barrier; Histochemical localization of tight junction proteins ZO-1, Occludin and Collagen IV in the colon of each group.

[0048] Figure 7 Test result diagram of the protective effect of lotus seed oligosaccharides prepared in Example 1 of the present invention on the intestinal barrier; Localization analysis of tight junction proteins ZO-1, Occludin and Collagen IV in the colon of each group.

[0049] Figure 8 Test result diagram of the effect of lotus seed oligosaccharides prepared in Example 1 of the present invention on the intestinal microbial richness; Among them, (A) ASV Venn diagram, (B) Chao 1 index, (C) Shannon index, (D) Simpson index, (E) PCA analysis, (F) Top 10 microbial compositions at the phylum level, (G) Proportion of Firmicutes, (H) Proportion of Bacteroidetes, (I) Firmicutes / Bacteroidetes ratio. Detailed implementation manners

[0050] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with specific embodiments.

[0051] Example 1: Preparation of raw / roasted lotus seed oligosaccharides

[0052] 1. Preparation of raw lotus seed oligosaccharides

[0053] The raw / roasted lotus seed oligosaccharides are prepared through the following steps:

[0054] S1. The raw lotus seed samples after being dried at 50°C are pulverized with a high-speed pulverizer and sieved through 60 meshes (aperture 0.3 mm).

[0055] S2. Add water to the powder prepared in step S1 according to a solid-liquid ratio of 1:5 (kg / L), boil it, and then perform extraction for 2 hours to obtain an aqueous extract. Centrifuge to remove insoluble components and collect the supernatant. The number of times of boiling with water is 3 times.

[0056] S3. Add 95% ethanol to the supernatant obtained in step S2 according to a volume ratio of the supernatant to 95% ethanol of 1:4 for ethanol precipitation (stand for 24 hours). Collect the supernatant, remove alcohol, and after separation and purification, freeze-dry it to obtain raw lotus seed oligosaccharides, as Figure 1 shown.

[0057] Among them, the separation and purification include: first use the Sevage method to remove proteins (use Sevage reagent (CH3Cl:BuOH = 4:1, v / v) to remove proteins 8 times), and then use DEAE sepharose FF gel suspension ion exchange column chromatography and reverse-phase silica gel column chromatography to separate and purify oligosaccharides (the salt concentrations for elution and separation are dH2O, 0.2M NaCl, 0.5M NaCl, 1.0M NaCl; the elution volume is 2 times, and the elution flow rate is 15 mL / min).

[0058] 2. Preparation of roasted lotus seed oligosaccharides

[0059] The raw / roasted lotus seed oligosaccharides are prepared through the following steps:

[0060] S1. Crush the roasted lotus seed sample dried at 50°C with a high-speed crusher and sieve it through 60 meshes (pore diameter 0.3 mm).

[0061] S2. Add water to the powder prepared in step S1 according to a solid-liquid ratio of 1:5 (kg / L), boil it, and then perform extraction for 2 hours to obtain an aqueous extract. Centrifuge to remove insoluble components and collect the supernatant. The number of times of boiling with water is 3 times.

[0062] S3. Add 95% ethanol to the supernatant obtained in step S2 according to a volume ratio of the supernatant to 95% ethanol of 1:4 for ethanol precipitation (stand for 24 hours). Collect the supernatant, remove alcohol, and after separation and purification, freeze-dry it to obtain roasted lotus seed oligosaccharides, as Figure 2 shown.

[0063] Among them, the separation and purification include: first use the Sevage method to remove proteins (use Sevage reagent (CH3Cl:BuOH = 4:1, v / v) to remove proteins 8 times), and then use DEAE sepharose FF gel suspension ion exchange column chromatography and reverse-phase silica gel column chromatography to separate and purify oligosaccharides (the salt concentrations for elution and separation are dH2O, 0.2M NaCl, 0.5M NaCl, 1.0M NaCl; the elution volume is 2 times, and the elution flow rate is 15 mL / min).

[0064] The raw / processed lotus seed oligosaccharides prepared in this example were used in Examples 2-5.

[0065] Example 2: Treatment of cyclophosphamide-induced immunosuppression in rats with raw / processed lotus seed oligosaccharides

[0066] Seventy 4-week-old S / D rats (180±3 g) were randomly divided into 7 groups, namely the control group (Control), the model group (Model), the low-dose raw lotus seed oligosaccharide group (LLOS), the high-dose raw lotus seed oligosaccharide group (HLOS), the low-dose processed lotus seed oligosaccharide group (FLLOS), and the high-dose processed lotus seed oligosaccharide group (FHLOS). Except for the Control group, the remaining groups were intraperitoneally injected with 30 mg / kg of cyclophosphamide for 3 days, and the Control group was intraperitoneally injected with an equal amount of normal saline every day. From the 4th day, the LLOS group, HLOS group, FLLOS group, and FHLOS group were given drug gavage respectively. On the 15th day, all rats were euthanized, and blood, colon samples, and feces were collected. The samples after sampling were applicable to Examples 2-5.

[0067] The body weight, colon index, and the levels of IgA, IgM, and IgG in the serum were analyzed. The results showed that both raw / processed lotus seed oligosaccharides could effectively increase the body weight of cyclophosphamide-induced immunosuppressed rats, increase the spleen index, and improve the levels of IgA, IgM, and IgG in the serum. Moreover, the effect of processed lotus seed oligosaccharides was better than that of raw lotus seeds, and the FHLOS group had the best effect ( Figure 3 ).

[0068] Collect colon tissues, separate rat colon tissues, and immerse them in pre-cooled complete medium. Place the colon tissues between two layers of 300-mesh nylon mesh, immerse them in the complete medium, and laterally grind the colon along the same direction with the piston of a 5 mL syringe to free the colon epithelial cells. Filter the ground cell suspension through a 300-mesh nylon mesh, and collect the filtrate in a 15 mL centrifuge tube. Centrifuge, discard the supernatant, and retain the precipitate. Add red blood cell lysate to the precipitate, mix well, and incubate on ice for 5 min. Resuspend the cell precipitate with an appropriate amount of staining buffer, count under a microscope, and adjust the cell concentration to 1×10 6 cells / mL. Pipette 1×10 6Colon epithelial cells of a certain number were taken, 500 μL of Binding Buffer was added to suspend the cells, PE-labeled CD8 antibody and FITC-labeled CD4 antibody were added, and the cells were incubated in the dark at room temperature for 30 min. The supernatant was removed by centrifugation at 300 rpm. 1 mL of PBS buffer was added respectively and washed twice. 1% paraformaldehyde was used to make the volume up to 300 μL, and the cells were filtered through a 350-mesh filter and stored in the dark at 4°C. The ratios of CD4+ T cells and CD8+ T cells in each group of rats were detected by loading samples on a BD Calibur flow cytometer, and the CD4+ / CD8+ ratio was calculated. The results showed that after cyclophosphamide administration, the CD4+ / CD8+ ratio could be effectively reduced, and this symptom could be alleviated after feeding the medicine. Moreover, the effect of FHLOS was better than that of raw lotus seed oligosaccharide, and it showed a dose-dependent relationship( Figure 4 ).

[0069] Example 3: Intestinal barrier protection effect of raw / processed lotus seed oligosaccharide

[0070] Experimental method, animal model: The same as Example 2

[0071] Colon tissues were collected and stored in 4% paraformaldehyde fixative for more than 48 h. The fixed tissues were dehydrated by gradient, embedded in paraffin, sectioned into 5-μm-thick sections, stained with H&E and scored for pathological damage, and the morphological changes of liver tissues were observed under a microscope. The results of H&E staining showed that after modeling, goblet cells decreased and crypt structure was abnormal, specifically manifested as crypt atrophy. Compared with the blank group, it was alleviated after administration. Among them, the FHLOS group had the best effect( Figure 5 ).

[0072] The above-mentioned sections were used for localization analysis of tight junction proteins ZO-1, Occludin and Collagen IV in the colon of each group. The results of immunohistochemistry showed that both raw / processed lotus seed oligosaccharides could improve the tight junction of the colon in cyclophosphamide-induced immunosuppressed rats, and the effect of the FHLOS group was better than that of the other groups( Figures 6-7 ).

[0073] Example 4: Raw / processed lotus seed oligosaccharide can improve the intestinal microbiota disorder induced by cyclophosphamide Experimental method, animal model: The same as Example 2

[0074] 16S rRNA sequencing was performed on the feces of the rat colon. After 3 days of cyclophosphamide-induced immunosuppression, the colons of rats in each group were taken for studying the microbial regulatory effects of raw / processed lotus seed oligosaccharides. The extraction of colonic microbial DNA was carried out according to the kit instructions. The V3-V4 region was amplified, a library was constructed, and sequencing was performed on the Illumina NovaSeq platform. QIIME2 was used to analyze the alpha diversity of the intestinal microbiota. By analyzing LEfSe and DEseq2, the discriminant species between different ICR animal groups were revealed. Non-metric multidimensional scaling, partial least squares discriminant analysis, and principal coordinate analysis were used to analyze the flora structure. Microbiome phylogenetic analysis was performed using ggtree in the R package. MetaCyc and the enzyme database were used to target KEGG to analyze the functional differences of the rat microbiota.

[0075] The sequencing results showed that a total of 46 ASVs were obtained. There were 1105 ASVs in the Control group, 857 ASVs in the Model group, 1198 ASVs in the FLLOS group, 1066 ASVs in the FHLOS group, 1283 ASVs in the LLOS group, 1451 ASVs in the HLOS group, and 1043 ASVs in the APS group ( Figure 8 A). Alpha diversity analysis was used to calculate the species richness of the samples and the species diversity within each sample. Beta-diversity analysis was used to describe the microbial differences between groups, and the scatter plots between groups indicated differences in the microbial composition among the three groups ( Figure 8 B-E).

[0076] A detailed comparison was made of the bacteria with higher abundances among the groups at the phylum level. At the phylum level, after cyclophosphamide induction, the abundance of the intestinal flora decreased. After administration of the drugs, the abundance of the intestinal flora increased, and the effect of raw lotus seed oligosaccharides was better than that of processed lotus seed oligosaccharides. The abundance of Firmicutes decreased, the abundance of Bacteroidetes increased, and the ratio of Firmicutes to Bacteroidetes decreased ( Figure 8 F-I).

[0077] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A lotus seed oligosaccharide, characterized in that, It is obtained by taking raw lotus seeds or roasted lotus seeds as raw materials and through water extraction and alcohol precipitation.

2. A method for preparing lotus seed oligosaccharide according to claim 1, characterized in that the steps As follows: Crush raw lotus seeds or roasted lotus seeds into powder; Boil the powder with water for water extraction treatment, and perform solid-liquid separation to obtain the supernatant; Add ethanol to the supernatant for alcohol precipitation treatment, and perform solid-liquid separation to obtain the precipitate; Freeze-dry the precipitate to obtain the product.

3. The preparation method according to claim 2, characterized in that, The powder is sieved through a 60-80 mesh sieve; Or, the material-liquid ratio of the powder to water is 1:1-10, kg / L, preferably 1:1.5-5.5, kg / L.

4. The preparation method according to claim 2, characterized in that, The time for water extraction treatment is 1-3 h, preferably 1.8-2.2 h; Or, the number of times of water extraction treatment is 1-5 times, preferably 3 times.

5. The preparation method according to claim 2, characterized in that, The time for alcohol precipitation treatment is 10-40 h, preferably 23-25 h.

6. Use of the lotus seed oligosaccharide according to claim 1 in the preparation of a product for preventing and / or treating immune deficiency.

7. The application according to claim 6, characterized in that, The immune deficiency is induced by cyclophosphamide.

8. Use of the lotus seed oligosaccharide according to claim 1 in the preparation of a product for preventing and / or treating intestinal barrier damage.

9. Use of the lotus seed oligosaccharide according to claim 1 in the preparation of a product for preventing and / or treating intestinal flora disorder and intestinal flora metabolic disorder.

10. The application according to any one of claims 6 to 9, characterized in that, The product is a food or a drug.

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