Tumor inhibition probiotic functional beverage based on Xu5P enhancer and preparation method thereof

Abstract

The invention provides a tumor inhibition probiotic functional beverage based on an Xu5P enhancer and a preparation method of the tumor inhibition probiotic functional beverage, and relates to the technical field of biological medicines. Comprising the following raw materials in parts by weight: 10-20% of an Xu5P improver, 0.5-2.0% of a probiotic strain, 1-5% of an anti-tumor plant extract, 2-5% of functional cellulose, 0.1-1.0% of vitamins, 0.1-1.0% of a mineral additive, 0.1-2.0% of a polyphenol compound, 1-3% of a plant fermentation product, 0.5-3.0% of a plant-derived active compound, 1-3% of prebiotics, 1-3% of traditional Chinese medicine components and the balance of water. The Xu5P has no obvious influence on the migration and clone formation ability of tumor cells, but by applying the Xu5P or an accelerant thereof in a tumor mouse model, the metastasis of mouse tumors can be obviously reduced, and the CD8 + T cell infiltration and tumor killing function can be improved. The research finds that a scientific basis is provided for tumor treatment, especially tumor metastasis inhibition treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a tumor-suppressing probiotic functional beverage based on a Xu5P enhancer and a preparation method thereof. Background Art

[0002] Tumors have become one of the most important diseases threatening human health and life. Although surgical resection, chemotherapy, radiotherapy, targeted therapy and other treatment methods have brought certain effects to the treatment of tumors, the prognosis of most tumor patients is still poor due to the existence of tumor metastasis. The development of tumor microenvironment and immune checkpoint inhibitors has brought new opportunities for the treatment of various tumors, but tumor immunotherapy also has shortcomings. Except for certain diseases (melanoma, non-small cell lung cancer, colon cancer and other tumors), most tumors do not respond well to immune checkpoint inhibitors. For example, in renal cell carcinoma, there is usually extensive CD8+T cell infiltration in the microenvironment, but most of them have lost the ability to kill tumor cells. If a drug is found to restore the function of CD8+T cells, it will have an epoch-making impact on the treatment of tumors and tumor metastasis.

[0003] Xu5P (Xylulose 5-phosphate) is a metabolic intermediate that plays an important role in the sugar metabolism of organisms, especially in the pentose phosphate pathway (PPP). Although the function of Xu5P in sugar metabolism has been studied, there are relatively few studies on its relationship with tumors and tumor metastasis, so there may be certain defects or research gaps: There are relatively few direct studies on Xu5P and tumors and tumor metastasis. Therefore, there is a lack of effective drugs to inhibit tumor metastasis in the existing technology. Summary of the invention

[0004] 1. Technical issues to be solved

[0005] In view of the deficiencies of the prior art, the present invention provides a tumor-suppressing probiotic functional beverage based on Xu5P enhancer and a preparation method thereof, which solves the problem of lack of effective drugs for inhibiting tumor metastasis in the prior art.

[0006] (II) Technical solution

[0007] To achieve the above purpose, injection of Xu5P into mice can significantly inhibit the growth and metastasis of tumor cells in mice. Therefore, the present invention provides the following technical solution: a tumor-suppressing probiotic functional beverage based on Xu5P enhancer, comprising the following raw materials in parts by weight: 10-20% Xu5P enhancer, 0.5-2.0% probiotic strain, 1-5% anti-tumor plant extract, 2-5% functional cellulose, 0.1-1.0% vitamins, 0.1-1.0% mineral additives, 0.1-2.0% polyphenol compounds, 1-3% plant fermentation products, 0.5-3.0% plant-derived active compounds, 1-3% prebiotics and 1-3% traditional Chinese medicine ingredients, and the balance is water.

[0008] Preferably, the probiotic strain is selected from modified Lactobacillus acidophilus or Bifidobacterium; and the anti-tumor plant extract is selected from any one of green tea extract, curcumin and garlic extract.

[0009] Preferably, the functional cellulose is selected from oligosaccharides or arabinose; the vitamins are selected from any two of vitamin B group, vitamin C and vitamin D; and the added minerals are selected from any 3-6 of calcium, iron, magnesium, zinc, copper, manganese, selenium, potassium, sodium and iodine.

[0010] Preferably, the polyphenol compound is selected from any one of catechins, anthocyanins and tannic acid; the plant fermentation product is selected from fermented soybean extract or fermented black wolfberry; the prebiotic is selected from inulin or oligofructose; and the traditional Chinese medicine ingredient is selected from Ganoderma lucidum extract or Astragalus extract.

[0011] Preferably, the Xu5P enhancer includes Xu5P and / or Xu5P promoter, and the Xu5P enhancer has the function of increasing the level of Xu5P in the body, which means that after the administration of the Xu5P enhancer, the level of Xu5P in the body can be increased compared with not administering the Xu5P enhancer.

[0012] Preferably, the Xu5P promoter for the purpose includes Xu5P synthesis promoter and Xu5P absorption promoter, which increase the Xu5P level by regulating the Xu5P metabolic pathway.

[0013] Preferably, the tumor-suppressing probiotic functional beverage is used to enhance CD8+T cells.

[0014] Preferably, the preparation method of the tumor-suppressing probiotic functional beverage based on Xu5P enhancer comprises the following steps:

[0015] S1. Mixing solid ingredients: pour the probiotic strains, anti-tumor plant extracts, functional cellulose, vitamins, mineral additives, polyphenol compounds, plant fermentation products, plant-derived active compounds, prebiotics and traditional Chinese medicine ingredients into a clean mixing container in sequence and mix them evenly;

[0016] S2. Dissolving and mixing liquid components: dissolving Xu5P enhancer in water at a ratio of 10-20% and stirring evenly by a stirring device, and then gradually adding the mixed solid components to the liquid and stirring evenly to ensure that all components are completely dissolved;

[0017] S3. Adjust pH: Use acidic or alkaline substances to adjust the pH of the beverage to ensure that it is suitable for the growth of probiotics. The pH value is usually between 4.0-5.5.

[0018] S4. Homogenization: The mixed liquid is homogenized by a homogenization device to ensure uniform distribution and stability of all ingredients in the beverage;

[0019] S5. Sterilization: The mixed liquid is subjected to pasteurization or other suitable sterilization methods to ensure the safety and stability of the beverage. Note that the sterilization process should not destroy the activity of the probiotics;

[0020] S6. Packaging and storage: The prepared beverage shall be divided into appropriate containers and stored under sealed conditions, ensuring that the beverage maintains appropriate temperature and humidity during storage to maintain its quality and activity.

[0021] Preferably, in the step S2, if Xu5P itself is used, it needs to be dissolved in water or other suitable solvents to ensure that its concentration in the final beverage reaches 10-20%;

[0022] In the step S2, if Xu5P accelerator is used, it is necessary to ensure that the total amount of the accelerator in the final beverage accounts for 10-20%.

[0023] (III) Beneficial effects

[0024] The invention provides a tumor-suppressing probiotic functional beverage based on a Xu5P enhancing agent and a preparation method thereof.

[0025] It has the following beneficial effects:

[0026] 1. The present invention can improve intestinal health and promote the normal functioning of the immune system through the combination of probiotics and prebiotics. In particular, probiotics can help enhance the function of CD8+T cells, which is crucial for the recognition and attack of anti-tumor cells. The anti-tumor plant extracts in the beverage are rich in antioxidants and anti-inflammatory ingredients, which help to slow down or inhibit the development of tumors. Xu5P enhancers may further enhance the effects of these anti-tumor ingredients by regulating the level of Xu5P in the body.

[0027] 2. The present invention can improve intestinal microecology, promote the growth of beneficial bacteria, maintain intestinal barrier function, prevent and relieve intestinal problems such as constipation through functional cellulose, probiotics and prebiotics; vitamins, minerals and plant fermentation products provide the nutrients needed by the body, help maintain healthy physiological functions and metabolic processes; polyphenol compounds and plant-derived active compounds have strong antioxidant and anti-inflammatory effects, help protect cells from oxidative stress and inflammatory damage, thereby supporting physical health and preventing diseases.

[0028] 3. The present invention may promote the body's metabolism and energy level by regulating the Xu5P metabolic pathway through the addition of Xu5P enhancer, thereby improving the overall health status and quality of life; in general, this beverage may help improve immune function, anti-tumor, improve intestinal health, provide comprehensive nutrition, and enhance the body's overall metabolic and antioxidant capabilities by combining multiple beneficial ingredients.

[0029] 4. The present invention has no significant effect on the migration and cloning ability of tumor cells by Xu5P itself, but the administration of Xu5P or its promoter in the tumor mouse model can significantly reduce the metastasis of mouse tumors and increase CD8+T cell infiltration and tumor killing function. The above research findings provide a scientific basis for the treatment of tumors, especially the treatment of inhibiting tumor metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 (A) Mouse lung metastasis tumor imaging image of Example 3 of the present invention; (B) HE staining image of mouse lung metastasis tumor tissue; (C) statistical image of mouse lung metastasis lesion area;

[0031] Figure 2 (A) Schematic diagram of XYLB catalyzing the conversion of xylulose into Xu5P according to Example 4 of the present invention; (B) Western blot detection of the knockdown effect of XYLB; (C) Schematic diagram of the effect of XYLB knockdown on the cloning ability of Renca cells; (D) Schematic diagram of the effect of XYLB knockdown on the migration ability of Renca cells; (E) Detection diagram of the content of metabolite Xu5P in tumor cells after XYLB knockdown;

[0032] Figure 3 (A) HE staining results of Example 5 of the present invention show that XYLB knockdown promotes the progression of lung metastasis of Renca tumor cells in mice; (B) Schematic diagram of the level of Xu5P in tumor tissues of XYLB knockdown mice; (CF) Graphs showing the effects of different immune cell elimination on the response of mice to XYLB knockdown tumor cell lines;

[0033] Figure 4(A) The ratio of CD8+CD44+T cells and CD8+CD25+T cells before and after Xu5P stimulation in Example 6 of the present invention; (B) The ratio of Ki67+ cells, IFNγ+T cells, and TNFα+T cells in CD8+T cells before and after Xu5P stimulation;

[0034] Figure 5 (A) Experimental flow chart of killing Renca-GFP-OVA cells by OT-1 mouse CD8+T cells; (B) Ratio diagram of killing Renca-GFP-OVA cells; (CD) Ratio diagram of TFN-γ and TNF-α positive cells in OT-1 mouse CD8+T cells according to Example 7 of the present invention. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Embodiment 1:

[0037] The embodiment of the present invention provides a tumor-suppressing probiotic functional beverage based on Xu5P enhancer, comprising the following raw materials in parts by weight: 10% Xu5P enhancer, 0.5% probiotic strain, 1% anti-tumor plant extract, 2% functional cellulose, 0.1% vitamins, 0.1% mineral additives, 0.1% polyphenol compounds, 1% plant fermentation products, 0.5% plant-derived active compounds, 1% prebiotics and 1% traditional Chinese medicine ingredients, and the balance is water.

[0038] The probiotic strain is selected from modified Lactobacillus acidophilus or Bifidobacterium; the anti-tumor plant extract is selected from any one of green tea extract, curcumin and garlic extract; the functional cellulose is selected from oligosaccharides or arabinose; the vitamins are selected from any two of vitamin B group, vitamin C and vitamin D; the mineral addition is selected from any three of calcium, iron, magnesium, zinc, copper, manganese, selenium, potassium, sodium and iodine; the polyphenol compound is selected from any one of catechins, anthocyanins and tannic acid; the plant fermentation product is selected from fermented soybean extract or fermented black wolfberry; Prebiotics are selected from inulin or oligofructose; Chinese medicinal ingredients are selected from Ganoderma lucidum extract or Astragalus extract; Xu5P enhancers include Xu5P and / or Xu5P promoters, and the Xu5P enhancers used have the function of increasing the level of Xu5P in the body, which means that after the administration of the Xu5P enhancer, the level of Xu5P in the body can be increased compared with that without the administration of the Xu5P enhancer; the Xu5P promoters used include Xu5P synthesis promoters and Xu5P absorption promoters, which increase the level of Xu5P by regulating the Xu5P metabolic pathway.

[0039] Application of tumor suppressive probiotic functional beverage in enhancing CD8+T cells.

[0040] The preparation method of a tumor-suppressing probiotic functional beverage based on Xu5P enhancer comprises the following steps:

[0041] S1. Mixing solid ingredients: pour the probiotic strains, anti-tumor plant extracts, functional cellulose, vitamins, mineral additives, polyphenol compounds, plant fermentation products, plant-derived active compounds, prebiotics and traditional Chinese medicine ingredients into a clean mixing container in sequence and mix them evenly;

[0042] S2. Dissolving and mixing liquid components: dissolving Xu5P enhancer in water at a ratio of 10% and stirring evenly by a stirring device, and then gradually adding the mixed solid components to the liquid and stirring evenly to ensure that all components are completely dissolved;

[0043] S3. Adjust pH: Use acidic or alkaline substances to adjust the pH of the beverage to ensure that it is suitable for the growth of probiotics. The pH value is usually more suitable at 4.0.

[0044] S4. Homogenization: The mixed liquid is homogenized by a homogenization device to ensure uniform distribution and stability of all ingredients in the beverage;

[0045] S5. Sterilization: The mixed liquid is subjected to pasteurization or other suitable sterilization methods to ensure the safety and stability of the beverage. Note that the sterilization process should not destroy the activity of the probiotics;

[0046] S6. Packaging and storage: The prepared beverage shall be divided into appropriate containers and stored under sealed conditions, ensuring that the beverage maintains appropriate temperature and humidity during storage to maintain its quality and activity.

[0047] In step S2, if Xu5P itself is used, it needs to be dissolved in water or other suitable solvents to ensure that its concentration reaches 10% in the final beverage;

[0048] In step S2, if Xu5P accelerator is used, it must be ensured that its total amount in the final beverage accounts for 10%;

[0049] The chemical structure of Xu5P is as follows:

[0050]

[0051] In this embodiment, the combination of probiotics and prebiotics can improve intestinal health and promote the normal functioning of the immune system. In particular, probiotics can help enhance the function of CD8+T cells, which is crucial for the recognition and attack of anti-tumor cells. The anti-tumor plant extracts (such as green tea extract, curcumin or garlic extract) in the beverage are rich in antioxidants and anti-inflammatory ingredients, which help to slow down or inhibit the development of tumors. Xu5P enhancers may further enhance the effects of these anti-tumor ingredients by regulating the level of Xu5P in the body. Functional cellulose (such as oligosaccharides or arabinose) as well as probiotics and prebiotics can improve intestinal microecology, promote the growth of beneficial bacteria, maintain intestinal barrier function, and prevent and relieve intestinal problems such as constipation. Vitamins (such as vitamin B The ingredients of this beverage include vitamins (such as vitamins A, B, C, D), minerals (such as calcium, iron, zinc, etc.) and plant fermentation products, which provide the nutrients needed by the body to help maintain healthy physiological functions and metabolic processes; polyphenols (such as catechins, anthocyanins or tannic acid) and plant-derived active compounds have strong antioxidant and anti-inflammatory effects, which help protect cells from oxidative stress and inflammatory damage, thereby supporting physical health and preventing diseases; the addition of Xu5P enhancers may promote the body's metabolism and energy levels by regulating the Xu5P metabolic pathway, thereby improving overall health and quality of life; in general, this beverage, by combining a variety of beneficial ingredients, may help improve immune function, fight tumors, improve intestinal health, provide comprehensive nutrition, and enhance the body's overall metabolic and antioxidant capabilities.

[0052] Embodiment 2:

[0053] The embodiment of the present invention provides a tumor-suppressing probiotic functional beverage based on Xu5P enhancer, comprising the following raw materials in parts by weight: 20% Xu5P enhancer, 2.0% probiotic strain, 5% anti-tumor plant extract, 5% functional cellulose, 1.0% vitamins, 1.0% mineral additives, 2.0% polyphenol compounds, 3% plant fermentation products, 3.0% plant-derived active compounds, 3% prebiotics and 3% traditional Chinese medicine ingredients, and the balance is water.

[0054] The probiotic strain is selected from modified Lactobacillus acidophilus or Bifidobacterium; the anti-tumor plant extract is selected from any one of green tea extract, curcumin and garlic extract; the functional cellulose is selected from oligosaccharides or arabinose; the vitamins are selected from any two of vitamin B group, vitamin C and vitamin D; the mineral addition is selected from any six of calcium, iron, magnesium, zinc, copper, manganese, selenium, potassium, sodium and iodine; the polyphenol compound is selected from any one of catechins, anthocyanins and tannic acid; the plant fermentation product is selected from fermented soybean extract or fermented black wolfberry; Prebiotics are selected from inulin or oligofructose; Chinese medicinal ingredients are selected from Ganoderma lucidum extract or Astragalus extract; Xu5P enhancers include Xu5P and / or Xu5P promoters, and the Xu5P enhancers used have the function of increasing the level of Xu5P in the body, which means that after the administration of the Xu5P enhancer, the level of Xu5P in the body can be increased compared with that without the administration of the Xu5P enhancer; the Xu5P promoters used include Xu5P synthesis promoters and Xu5P absorption promoters, which increase the level of Xu5P by regulating the Xu5P metabolic pathway.

[0055] Application of tumor suppressive probiotic functional beverage in enhancing CD8+T cells.

[0056] The preparation method of a tumor-suppressing probiotic functional beverage based on Xu5P enhancer comprises the following steps:

[0057] S1. Mixing solid ingredients: pour the probiotic strains, anti-tumor plant extracts, functional cellulose, vitamins, mineral additives, polyphenol compounds, plant fermentation products, plant-derived active compounds, prebiotics and traditional Chinese medicine ingredients into a clean mixing container in sequence and mix them evenly;

[0058] S2. Dissolving and mixing liquid components: dissolving Xu5P enhancer in water at a ratio of 20% and stirring evenly by a stirring device, then gradually adding the mixed solid components to the liquid and stirring evenly to ensure that all components are completely dissolved;

[0059] S3. Adjust pH value: Use acidic or alkaline substances to adjust the pH value of the beverage to ensure that it is suitable for the growth of probiotics. The pH value is usually between 5.5.

[0060] S4. Homogenization: The mixed liquid is homogenized by a homogenization device to ensure uniform distribution and stability of all ingredients in the beverage;

[0061] S5. Sterilization: The mixed liquid is subjected to pasteurization or other suitable sterilization methods to ensure the safety and stability of the beverage. Note that the sterilization process should not destroy the activity of the probiotics;

[0062] S6. Packaging and storage: The prepared beverage shall be divided into appropriate containers and stored under sealed conditions, ensuring that the beverage maintains appropriate temperature and humidity during storage to maintain its quality and activity.

[0063] In step S2, if Xu5P itself is used, it needs to be dissolved in water or other suitable solvents to ensure that its concentration reaches 20% in the final beverage;

[0064] In step S2, if Xu5P accelerator is used, it must be ensured that its total amount in the final beverage accounts for 20%;

[0065] The chemical structure of Xu5P is as follows:

[0066]

[0067] In this embodiment, the combination of probiotics and prebiotics can improve intestinal health and promote the normal functioning of the immune system. In particular, probiotics can help enhance the function of CD8+T cells, which is crucial for the recognition and attack of anti-tumor cells. The anti-tumor plant extracts (such as green tea extract, curcumin or garlic extract) in the beverage are rich in antioxidants and anti-inflammatory ingredients, which help to slow down or inhibit the development of tumors. Xu5P enhancers may further enhance the effects of these anti-tumor ingredients by regulating the level of Xu5P in the body. Functional cellulose (such as oligosaccharides or arabinose) as well as probiotics and prebiotics can improve intestinal microecology, promote the growth of beneficial bacteria, maintain intestinal barrier function, and prevent and relieve intestinal problems such as constipation. Vitamins (such as vitamin B The ingredients of this beverage include vitamins (such as vitamins A, B, C, D), minerals (such as calcium, iron, zinc, etc.) and plant fermentation products, which provide the nutrients needed by the body to help maintain healthy physiological functions and metabolic processes; polyphenols (such as catechins, anthocyanins or tannic acid) and plant-derived active compounds have strong antioxidant and anti-inflammatory effects, which help protect cells from oxidative stress and inflammatory damage, thereby supporting physical health and preventing diseases; the addition of Xu5P enhancers may promote the body's metabolism and energy levels by regulating the Xu5P metabolic pathway, thereby improving overall health and quality of life; in general, this beverage, by combining a variety of beneficial ingredients, may help improve immune function, fight tumors, improve intestinal health, provide comprehensive nutrition, and enhance the body's overall metabolic and antioxidant capabilities.

[0068] Embodiment three:

[0069] like Figure 1 As shown, the present invention provides Xu5P for the progression of Renca tumor cell lung metastasis in mice:

[0070] Six-week-old male BALB / c mice were injected with 1×106 Renca cells (200ul) through the tail vein to establish a mouse lung metastasis model. The mice were randomly divided into two groups, one group was intravenously injected with 20mg / kg Xu5P once every three days, and the other group was given the same volume of PBS as a control. Two weeks later, the mice were killed under anesthesia, and the lung tissues were fixed with 4% paraformaldehyde (PFA), embedded in paraffin according to the conventional process, and then paraffin sections were taken for H&E staining and statistical analysis.

[0071] The experimental results are as follows Figure 1 As shown in the mouse lung metastasis model, after treatment with 20 mg / kg Xu5P, it was found that Xu5P could significantly inhibit the number and size of metastatic foci in lung tissue ( Figure 1 AC), indicating that supplementation of Xu5P can effectively inhibit the process of tumor lung metastasis.

[0072] Embodiment 4:

[0073] like Figure 1 and Figure 2 As shown, the present invention provides that knocking down xylulokinase (XYLB) in Renca tumor cells has no significant effect on tumor cell clone formation and migration:

[0074] The main source of Xu5P in the body is xylulose phosphorylation by xylulokinase (XYLB), so its level is regulated by the expression level of XYLB ( Figure 2 A). In Renca cells, a cell line with stable knockdown of XYLB was constructed using shRNA and lentiviral vectors, and the expression level of XYLB was verified by Western blot to determine the knockdown effect ( Figure 2 B).

[0075] The effect of XYLB expression level on Renca cell migration ability was studied by clone formation and Transwell assay. 1000 XYLB stable knockdown and control Renca cells were inoculated in 6-well plates, cultured for 10 days, fixed with 4% paraformaldehyde solution and stained with crystal violet, the number of clones was counted and photographed to evaluate the clone formation ability of different cells. 5×104 XYLB stable knockdown and control Renca cells resuspended in serum-free medium were added to the upper chamber of Transwell, and medium containing 15% FBS was added to the lower chamber for 48 hours. The chamber was removed, fixed with 4% paraformaldehyde solution and stained with crystal violet, then the cells in the chamber were wiped clean with a cotton swab, photographed under a microscope, and the migration ability of different cells was evaluated.

[0076] like Figure 2 As shown in Figure 2, in Renca cells, after knocking down the expression of XYLB using shRNA, the level of Xu5P in the cells was significantly decreased ( Figure 2 E), but the clone formation ability and migration ability of knockdown cells were not affected. Comprehensive comparison of the effects of Xu5P on tumor metastasis ability in vivo and in vitro ( Figure 1 , Figure 2 ), indicating that Xu5P does not affect the metastatic ability of tumor cells themselves, but may inhibit tumor metastasis by regulating the body's anti-tumor immunity.

[0077] Embodiment five:

[0078] like Figure 3 As shown, the present invention provides CD8+T cells to determine the response of mice to XYLB knockdown tumor cell lines:

[0079] Six-week-old male BALB / c mice and BALB / c-Nude mice were injected with 1×106 Renca-shNT cells or Renca-shXylb cells (200ul) through the tail vein to establish a mouse tumor lung metastasis model. For BALB / c mice given Renca-shNT cells or Renca-shXylb cells, they were divided into three groups and given 200μg IgG, anti-CD8 antibody or anti-CD4 antibody once every three days. After 2 weeks, the mice were killed under anesthesia, and the lung tissues were fixed with 4% paraformaldehyde (PFA), embedded in paraffin according to the conventional process, and then paraffin sections were taken for H&E staining and statistical analysis. For mice given IgG, lung lesions were taken and local Xu5P levels were detected by mass spectrometry.

[0080] The results are as follows Figure 3As shown in A, Xylb knockdown in tumor cells can significantly promote the growth of tumor cells in BALB / c mice, and Xylb knockdown can indeed reduce the level of Xu5P in tumor lesions ( Figure 3 B), the results indicate that the reduction of Xu5P level mediated by Xylb knockdown can promote tumor growth and metastasis. Conversely, as shown in Example 1, the increase of Xu5P level can inhibit tumor growth and metastasis.

[0081] In nude mice lacking T cells, the number of metastatic foci caused by Xylb knockdown Renca tumor cells was close to that of normal Renca tumor cells, indicating that the phenomenon of promoting tumor cell growth caused by Xylb knockdown is mediated by T cells ( Figure 3 C). To further investigate whether the effect is mediated by CD4+T cells or CD8+T cells, anti-CD8 antibodies or anti-CD4 antibodies were used to eliminate CD8+T cells and CD4+T cells in BALB / c mice, respectively. Figure 3 As shown in DF, compared with the control IgG, the number of lung metastases caused by Xylb-knockdown Renca tumor cells in mice injected with anti-CD4+ antibodies was significantly greater than that of normal Renca tumor cells, while the metastatic foci of Xylb-knockdown Renca tumor cells in mice injected with anti-CD8+ antibodies were similar to those of normal Renca tumor cells, indicating that CD8+T cells mediated the tumor-promoting effect caused by Xylb knockdown in tumor cells, which indirectly suggests that the anti-tumor effect mediated by Xu5P may be caused by regulating CD8+T cells.

[0082] Embodiment six:

[0083] like Figure 4 As shown, the present invention provides Xu5P stimulation to promote mouse CD8+T cell activation and cytotoxic factor expression:

[0084] Six-week-old male BALB / c mice were prepared, and primary mouse CD8+T cells were extracted using a kit and cultured. The cells were divided into two groups, 50 μM Xu5P was added to one group, and the same volume of PBS was given to the other group as a control. Flow cytometry analysis was performed after 24-48 hours of Xu5P stimulation. The expression of CD44 molecules on the surface of CD8+T cells is related to the formation of effector T cells, effector memory T cells, and central memory T cells, while the expression of CD25 molecules on the surface of CD8+T cells is related to the formation of effector T cells, both of which are cell surface markers of CD8+T cell activation.

[0085] like Figure 4As shown in the figure, the function of CD8+T cells in the Xu5P-treated group and the untreated group was detected, and it was found that Xu5P could significantly increase the proportion of CD8+CD44+T cells and CD8+CD25+T cells. In addition, the proportion of Ki67+, TNFα+, and IFNγ+ cells in CD8+T cells increased, indicating that Xu5P can promote the proliferation of CD8+T cells and promote the expression of tumor-killing cytokines. The above results all indicate that Xu5P can activate the anti-tumor function of CD8+T cells.

[0086] Embodiment seven:

[0087] like Figure 5 As shown, the present invention provides that Xu5P supplementation can restore the response of mice to XYLB knockdown tumor cell lines:

[0088] Six-week-old male OT-1 mice were prepared, and primary mouse CD8+T cells were extracted using a kit. After cytokine stimulation and activation, they were divided into four groups and incubated with Renca-shNT-GFP-OVA cells+PBS, Renca-shXylb-GFP-OVA cells+PBS, Renca-shXylb-GFP-OVA cells+rXylb+PBS, and Renca-shXylb-GFP-OVA cells+Xu5P, respectively. The experimental process is shown in 5A. The proportion of tumor cell death was detected, and the proportion of IFN-γ and TNF-α positive cells in CD8+T cells was analyzed by flow cytometry.

[0089] Tumor cell death Figure 5 As shown in B, the cell death rate of Xylb knockdown was significantly reduced, indicating that Xylb knockdown inhibited the killing ability of CD8+T cells, while re-expression of recombinant Xylb protein could restore the killing ability of CD8+T cells. In addition, the addition of Xu5P could also restore the killing rate of CD8+T cells against tumor cells. Figure 5 As shown in CD, low expression of Xylb in tumor cells reduced the levels of cytokine IFNγ and TNFα in CD8+T cells, while recombinant expression of Xylb in tumor cells or additional supplementation of Xu5P could reverse the decrease in cytokine IFNγ and TNFα levels. The above results indicate that supplementation of Xu5P can reverse tumor escape caused by Xylb knockdown and enhance the body's anti-tumor ability.

[0090] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Tumor-suppressing probiotic functional beverage based on Xu5P enhancer, Features: The invention comprises the following raw materials in parts by weight: 10-20% of Xu5P enhancing agent, 0.5-2.0% of probiotic strain, 1-5% of anti-tumor plant extract, 2-5% of functional cellulose, 0.1-1.0% of vitamins, 0.1-1.0% of mineral additives, 0.1-2.0% of polyphenol compounds, 1-3% of plant fermentation products, 0.5-3.0% of plant-derived active compounds, 1-3% of prebiotics and 1-3% of traditional Chinese medicine ingredients, and the balance is water.

2. The tumor-suppressing probiotic functional beverage based on Xu5P enhancer according to claim 1, Features: The probiotic strain is selected from modified Lactobacillus acidophilus or Bifidobacterium; the anti-tumor plant extract is selected from any one of green tea extract, curcumin and garlic extract.

3. The tumor-suppressing probiotic functional beverage based on Xu5P enhancer according to claim 1, Features: The functional cellulose is selected from oligosaccharides or arabinose; the vitamins are selected from any two of vitamin B group, vitamin C and vitamin D; and the added minerals are selected from any 3-6 of calcium, iron, magnesium, zinc, copper, manganese, selenium, potassium, sodium and iodine.

4. The tumor-suppressing probiotic functional beverage based on Xu5P enhancer according to claim 1, Features: The polyphenol compound is selected from any one of catechins, anthocyanins and tannic acid; the plant fermentation product is selected from fermented soybean extract or fermented black wolfberry; the prebiotic is selected from inulin or oligofructose; and the traditional Chinese medicine ingredient is selected from ganoderma lucidum extract or astragalus extract.

5. The tumor-suppressing probiotic functional beverage based on Xu5P enhancer according to claim 1, Features: The Xu5P enhancer includes Xu5P and / or Xu5P promoter. The Xu5P enhancer has the function of enhancing the level of Xu5P in the body, which means that after the Xu5P enhancer is applied, the level of Xu5P in the body can be enhanced compared with that without the application of the Xu5P enhancer.

6. The tumor-suppressing probiotic functional beverage based on Xu5P enhancer according to claim 5, Features: The Xu5P promoter for the purpose includes Xu5P synthesis promoter and Xu5P absorption promoter, and the Xu5P level is increased by regulating the Xu5P metabolic pathway.

7. The tumor-suppressing probiotic functional beverage based on Xu5P enhancer according to claim 1, Features: The application of the tumor suppressive probiotic functional beverage in enhancing CD8+T cells.

8. Preparation method of tumor-suppressing probiotic functional beverage based on Xu5P enhancer, Features: The following steps are involved: S1. Mixing solid ingredients: pour the probiotic strains, anti-tumor plant extracts, functional cellulose, vitamins, mineral additives, polyphenol compounds, plant fermentation products, plant-derived active compounds, prebiotics and traditional Chinese medicine ingredients into a clean mixing container in sequence and mix them evenly; S2. Dissolving and mixing liquid components: dissolving Xu5P enhancer in water at a ratio of 10-20% and stirring evenly by a stirring device, and then gradually adding the mixed solid components to the liquid and stirring evenly to ensure that all components are completely dissolved; S3. Adjust pH: Use acidic or alkaline substances to adjust the pH of the beverage to ensure that it is suitable for the growth of probiotics. The pH value is usually between 4.0-5.

5. S4. Homogenization: The mixed liquid is homogenized by a homogenization device to ensure uniform distribution and stability of all ingredients in the beverage; S5. Sterilization: The mixed liquid is subjected to pasteurization or other suitable sterilization methods to ensure the safety and stability of the beverage. Note that the sterilization process should not destroy the activity of the probiotics; S6. Packaging and storage: The prepared beverage shall be divided into appropriate containers and stored under sealed conditions, ensuring that the beverage maintains appropriate temperature and humidity during storage to maintain its quality and activity.

9. The method for preparing the tumor-suppressing probiotic functional beverage based on Xu5P enhancer according to claim 8, Features: In the step S2, if Xu5P itself is used, it needs to be dissolved in water or other suitable solvents to ensure that its concentration in the final beverage reaches 10-20%; In the step S2, if Xu5P accelerator is used, it is necessary to ensure that the total amount of the accelerator in the final beverage accounts for 10-20%.

10. The tumor-suppressing probiotic functional beverage based on Xu5P enhancer according to claim 1, Features: The tumor-suppressing probiotic functional beverage can also be applied to fermented foods, nutritional supplements, energy bars, cereals, bread, baked foods, soups, sauces and ice cream.

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