Bifidobacterium longum probiotics combined with qi-yang-blood nourishing food-medicine homologous composition, preparation method and application in regulating immunity, inhibiting tumor cell proliferation and reducing tumor-induced inflammation

Abstract

Disclosed in the application are a Bifidobacterium longum probiotic combined with a qi-tonifying and blood-nourishing medicinal and edible homologous composition, a preparation method thereof and application thereof in regulating immunity, inhibiting tumor cell proliferation and reducing tumor-induced inflammation, including Bifidobacterium longum subsp. infantis NKU FB3-14 and the qi-tonifying and blood-nourishing medicinal and edible homologous composition; the qi-tonifying and blood-nourishing medicinal and edible homologous composition comprises, by weight, 5-30 parts of ginseng, 5-20 parts of longan arillus, 20-50 parts of astragalus root, 10-50 parts of polygonatum, 5-20 parts of poria cocos, 5-15 parts of mulberry, 5-20 parts of licorice, 10-30 parts of orange peel, 5-15 parts of citrus medica, 5-20 parts of double-petaled rose flower and 3-10 parts of hawthorn. The probiotic preparation and the medicinal and edible homologous material have a synergistic effect, and the ability to inhibit the proliferation level of liver tumor cells H22 in vitro is determined.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a Bifidobacterium longum probiotic combined with a qi-tonifying and blood-nourishing food-medicine homologous composition, a preparation method thereof and application thereof in regulating immunity, inhibiting tumor cell proliferation and reducing tumor-induced inflammation. BACKGROUND

[0002] Qi and blood deficiency syndrome is a syndrome name in traditional Chinese medicine, which is caused by deficiency of qi and blood, and is characterized by fatigue, shortness of breath, pale or yellowish complexion, dizziness, pale lips, heart palpitations, insomnia, and weak pulse. The causes are mainly due to postnatal disorders, prolonged illness, or prolonged illness without recovery, loss of blood and consumption of qi, such as in the later stage of consumptive diseases such as tumors, or surgery, radiotherapy and chemotherapy, which consume body energy and damage the digestive system, leading to intestinal flora disorder and immune function disorder. Therefore, diet and food-medicine homologous theory can be used for conditioning, and attention should be paid to tonifying and invigorating.

[0003] Traditional Chinese medicine has had the theory of food-medicine homology since ancient times. Modern detection technology has proved that food-medicine homology substances are rich in compounds that maintain health effects, and also contain proteins, vitamins, trace elements and other nutrients, which have good tonifying effect on weak people. In recent years, with the continuous deepening of research on probiotics, postbiotics and other related research, many studies have confirmed that they have good improvement effect on the absorption of nutrients and the regulation of intestinal flora. Appropriate supplementation of high-quality probiotics and postbiotics can be used as one of the daily maintenance strategies for people with body weakness caused by various factors. Modern research has proved that through previous optimization and screening, probiotics and postbiotics combined with food-medicine homology substances have more abundant material basis and more extensive action targets, and have the potential of 1+1>2 for maintaining immune homeostasis.

[0004] Therefore, it is of high economic value and social significance to develop a postbiotic compound food-medicine homology substance product that follows the compatibility theory, has rich material composition, is safe and non-toxic to the human body, can be consumed for a long time, and is helpful for preventing and improving qi and blood deficiency. SUMMARY

[0005] Therefore, the present application aims to provide a Bifidobacterium longum probiotic combined with a qi-tonifying and blood-nourishing food-medicine homologous composition, a preparation method thereof and application thereof in regulating immunity, inhibiting tumor cell proliferation and reducing tumor-induced inflammation, so as to solve at least one technical problem in the background art.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0007] A qi-nourishing and blood-nourishing medicine-food homologous composition is applied to food, and the qi-nourishing and blood-nourishing medicine-food homologous composition comprises, by weight, 5-30 parts of ginseng, 5-20 parts of longan arillus, 20-50 parts of astragalus root, 10-50 parts of polygonatum, 5-20 parts of poria cocos, 5-15 parts of mulberry, 5-20 parts of licorice, 10-30 parts of orange peel, 5-15 parts of citrus medica, 5-20 parts of heavy-petaled rose, and 3-10 parts of hawthorn.

[0008] The qi-nourishing and blood-nourishing medicine-food homologous composition is applied to health-care food,

[0009] The ginseng in the present application is artificially planted for five years or less, has the effects of tonifying primordial qi, tonifying the spleen and lung, promoting saliva secretion, soothing the nerves and improving intelligence, can improve the immune function of the body, enhance the disease resistance, promote metabolism, relieve fatigue, and reduce the generation of free radicals.

[0010] The longan arillus has the effects of tonifying the heart and spleen, nourishing blood and soothing nerves, promoting growth and development, and enhancing physical fitness, can reduce the generation of free radicals, improve the immune function of cells, and has the effects of anti-anxiety and endocrine regulation.

[0011] The astragalus root has the effects of invigorating the spleen, tonifying the center, raising yang and lifting depression, benefiting the defensive system and stabilizing the exterior, diuresis, and promoting toxin elimination and tissue regeneration, can significantly improve the non-specific immune function, has the effects of promoting or enhancing humoral immunity and cellular immunity, and has the effects of anti-aging, anti-mutation, liver protection, anti-fatigue, anti-hypoxia, anti-cold, anti-radiation, tumor inhibition, and anti-pulmonary arterial hypertension.

[0012] The polygonatum has the effects of nourishing yin and moistening the lung, tonifying the spleen and invigorating qi, and nourishing the kidney and filling essence, has the effects of resisting pathogenic microorganisms, reducing blood lipids, delaying aging, and other effects, and has influences on the cardiovascular system, immune function, cyclic nucleotide content, and blood sugar.

[0013] The poria cocos has the effects of promoting water excretion and swelling, promoting the spleen, and soothing the heart, can significantly increase the serum IgG content, has the effects of promoting cellular immunity and humoral immunity, and has the effects of reducing blood sugar, resisting tumors, resisting pathogenic microorganisms, and promoting hematopoietic function.

[0014] The mulberry has the effects of nourishing yin and tonifying blood, promoting saliva secretion and moistening dryness, can promote the maturation of T cells, thereby restoring the function of aging T cells, has the effects of promoting humoral immune function in young mice and promoting the growth of granulocyte macrophage cells, and has the effect of preventing leukopenia caused by cyclophosphamide.

[0015] The licorice has the effects of tonifying qi and the center, moistening the lung and relieving cough, clearing heat and detoxifying, relieving acute pain, and regulating the medicinal properties, can promote the weight increase of part of the immune organs, enhance the activity of natural killer cells, and regulate the immune response of macrophages.

[0016] Orange peel has the effects of regulating qi and invigorating the spleen, drying dampness and reducing phlegm, and has the effects of removing oxygen free radicals and resisting lipid peroxidation, and has the effects of promoting bile secretion and reducing serum cholesterol.

[0017] Citrus medica has the effects of soothing the liver and relieving depression, regulating qi and harmonizing the center, drying dampness and reducing phlegm, and has the effects of resisting inflammation and resisting viruses.

[0018] Double rose has the effects of soothing the liver and relieving depression, promoting blood circulation and relieving pain, and can promote the blood flow velocity of arterioles and improve microcirculation disorders.

[0019] Hawthorn has the effects of digesting and removing food accumulation, promoting blood circulation and removing blood stasis, and has the effects of improving appetite, promoting blood circulation, enhancing immune response, resisting tumors, etc.

[0020] In summary, the medicine-food homologous combination provided by the present application, which comprises ginseng, longan arillus, astragalus root, polygonatum, poria cocos, mulberry fruit, licorice, orange peel, citron, double rose and hawthorn, can not only benefit qi and blood, regulate immune response, enhance energy metabolism, etc., but also avoid adverse reactions to the recipients, and is beneficial to popularization.

[0021] The long bifidobacterium metagenome combined with the qi and blood nourishing medicine-food homologous composition comprises the long bifidobacterium infantis subsp. NKUFB3-14 and the above-mentioned qi and blood nourishing medicine-food homologous composition, the long bifidobacterium infantis subsp. (Bifidobacterium longum subsp. infantis) NKU FB3-14 is preserved in the China General Microbiological Culture Collection Center, No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 25762, and the preservation date is September 21, 2022.

[0022] The long bifidobacterium infantis subsp. NKU FB3-14 disclosed in the present application has been disclosed in Chinese patent CN202310453211.X, and the test proves that the long bifidobacterium infantis subsp. NKU FB3-14 is a safe strain and has good acid resistance, bile salt resistance and DPPH clearance capacity, and can be used for human and animal bodies.

[0023] The present application also provides a preparation method of the long bifidobacterium metagenome combined with the qi and blood nourishing medicine-food homologous composition described in the above technical solution, comprising the following steps:

[0024] S1: preparing the long bifidobacterium infantis subsp. NKU FB3-14 into long bifidobacterium infantis subsp. NKU FB3-14 inactivated bacterial suspension;

[0025] S2: mixing ginseng, longan arillus, astragalus, polygonatum, poria cocos, mulberry, liquorice, orange peel, citron, double rose flower, hawthorn and the like extracts in proportion to obtain a mixture, filtering, homogenizing and constant volume to prepare a qi and blood nourishing medicine and food homologous composition;

[0026] S3: mixing the long bifidobacterium infantis subsp. NKU FB3-14 inactivated bacterial body resuspension in step S1 and the qi and blood nourishing medicine and food homologous composition in step S2 to prepare a long bifidobacterium postbiotic combined qi and blood nourishing medicine and food homologous composition.

[0027] Further, the preparation of long bifidobacterium infantis subsp. NKU FB3-14 inactivated bacterial body resuspension in step S1 includes the following steps:

[0028] A1: inoculating long bifidobacterium infantis subsp. NKU FB3-14 into bifidobacterium liquid medium for fermentation culture to obtain a fermentation culture solution;

[0029] A2: centrifuging the fermentation culture solution obtained in step A1 to separate the solid and liquid to obtain long bifidobacterium infantis subsp. NKU FB3-14 fermentation supernatant and long bifidobacterium infantis subsp. NKU FB3-14 bacterial body;

[0030] A3: resuspending the long bifidobacterium infantis subsp. NKU FB3-14 bacterial body obtained in step A2 and inactivating to obtain long bifidobacterium infantis subsp. NKU FB3-14 inactivated bacterial body resuspension.

[0031] The concentration of long bifidobacterium infantis subsp. NKU FB3-14 in the fermentation culture solution, the heat-inactivated bacterial body resuspension and the heat-inactivated fermentation culture solution is preferably 1×10 8 ~ 2×10 9 CFU / mL, further preferably 1×10 9 ~ 2×10 9 CFU / mL, more preferably 2×10 9 ~ 3×10 9 CFU / mL. The resuspension agent of the bacterial body resuspension and the heat-inactivated bacterial body resuspension is preferably bifidobacterium liquid medium, more preferably BS liquid medium.

[0032] Further, the ginseng, longan arillus, astragalus, polygonatum, poria cocos, mulberry, orange peel, citron, double rose flower and hawthorn water extracts in step S2 are mixed in proportion, and after adding drinking water and homogenizing, the mixture is filtered, and the mixture is placed in a rotary evaporator to constant volume to 0.8-1.2g / mL of crude drug amount;

[0033] And / or, the mass / volume ratio of the mixture to drinking water is 4-6.

[0034] Further, the volume ratio of the Bifidobacterium longum infantis NKU FB3-14 inactivated bacterial suspension in step S3 to the qi-tonifying and blood-nourishing food composition is 1:0.8-1.2.

[0035] The weight parts of the food composition are screened, and the final combination weight parts are determined by optimizing the proliferation level inhibition ability of the liver tumor cells H22. The optimal ratio of the metagenomic preparation to the food composition is 1:1, and the optimal ratio of the metagenomic preparation to the food composition is determined by measuring the proliferation level inhibition ability of the liver tumor cells H22 in vitro.

[0036] The above-mentioned Bifidobacterium longum metagenomic preparation combined with the qi-tonifying and blood-nourishing food composition is applied to food.

[0037] The above-mentioned Bifidobacterium longum metagenomic preparation combined with the qi-tonifying and blood-nourishing food composition is applied to health food.

[0038] The above-mentioned Bifidobacterium longum metagenomic preparation combined with the qi-tonifying and blood-nourishing food composition is applied to the preparation of one or more drugs for regulating immunity, inhibiting tumor cell proliferation, and reducing tumor-induced inflammation.

[0039] Further, the drug for improving tumor-induced immune disorders is a drug for inhibiting the growth of liver tumor cells.

[0040] Compared with the prior art, the Bifidobacterium longum metagenomic preparation combined with the qi-tonifying and blood-nourishing food composition, the preparation method thereof, and the application thereof in regulating immunity, inhibiting tumor cell proliferation, and reducing tumor-induced inflammation have the following advantages:

[0041] 1. The metagenomic preparation and the food composition have a synergistic effect, and the proliferation level inhibition ability of the liver tumor cells H22 in vitro is measured. The metagenomic preparation provides a good internal environment for the qi-tonifying and blood-nourishing composition to play a role by regulating the intestinal flora and the immune system. The qi-tonifying and blood-nourishing composition enhances the overall immune function of the body by tonifying qi and nourishing blood, and further amplifies the immune regulation effect of the metagenomic preparation.

[0042] 2. The metagenomic preparation and the food composition can be used as an auxiliary treatment method for tumor patients to improve immune disorders and enhance anti-tumor immune response.

[0043] 3. The Bifidobacterium longum metagenomic preparation combined with the qi-tonifying and blood-nourishing food composition regulates the immune system through multiple targets and multiple pathways, improves tumor-induced immune disorders, and has significant immune enhancement and anti-tumor potential. This combined application provides a new idea and method for tumor treatment and immune regulation. BRIEF DESCRIPTION OF DRAWINGS

[0044] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. The embodiments of the present application illustrated in the drawings are presented by way of example or for purpose of illustration and not limitation.

[0045] Figure 1 The schematic diagram of the inhibition effect of the water extract of different phagohormetic compositions described in embodiment 2 of the present application on the proliferation rate of H22 cells;

[0046] Figure 2 The schematic diagram of the inhibition effect of different ratios of the phagohormetic compound described in embodiment 2 of the present application on the proliferation of H22 cells;

[0047] Figure 3 The schematic diagram of the inhibition effect of FB3-14 live bacteria group, FB3-14 phagohormetic group, phagohormetic compound group, and compound group described in embodiment 2 of the present application on the proliferation of H22 cells;

[0048] Figure 4 The comparison diagram of the appearance of tumor tissues of each intervention group described in embodiment 3 of the present application (each intervention group is model group, FB3-14 group, compound group, and compound group, respectively);

[0049] Figure 5 The comparison diagram of the weight and volume of tumor tissues of each intervention group described in embodiment 3 of the present application (a is the comparison diagram of the weight of tumor tissues of each intervention group, b is the comparison diagram of the volume of tumor tissues of each intervention group, and each intervention group is model group, FB3-14 group, compound group, and compound group, respectively);

[0050] Figure 6 The comparison diagram of the histopathology of tumor tissues of each intervention group described in embodiment 3 of the present application (a is model group, b is FB3-14 group, c is compound group, and d is compound group);

[0051] Figure 7 The expression level of serum inflammatory factors TNF-α and IFN-β and the proportion change of spleen lymphocytes of each group described in embodiment 4 of the present application (a is the proportion change diagram of serum inflammatory factor TNF-α, b is the proportion change diagram of the expression level of IFN-β, and c is the proportion change diagram of spleen lymphocytes);

[0052] Figure 8 The expression level diagram of NF-κB, P53, and P38 proteins in tumor tissues of each group described in embodiment 4 of the present application (a is the development diagram after incubation with secondary antibody at room temperature for 2 h, and b is the comparison analysis diagram of the gray value of the band using Image J analysis software);

[0053] Figure 9ALT level change graph of serum of each group described in Embodiment 5 of the present application (each group is blank group, model group, FB3-14 group, compound group and complex group respectively);

[0054] Figure 10 AST level change graph of serum of each group described in Embodiment 5 of the present application (each group is blank group, model group, FB3-14 group, compound group and complex group respectively);

[0055] Figure 11 Urea nitrogen level change graph of serum of each group described in Embodiment 5 of the present application (each group is blank group, model group, FB3-14 group, compound group and complex group respectively). DETAILED DESCRIPTION

[0056] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0057] The present application will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0058] Embodiment 1: Preparation of Bifidobacterium longum subsp. infantis NKU FB3-14 probiotic preparation

[0059] Bifidobacterium longum subsp. infantis NKU FB3-14 is inoculated into BS liquid culture medium at a volume fraction of 5%, and after inoculation, it is cultured in an anaerobic incubator at 37°C for 20-24h, until the OD value of the culture solution is 1.5, and the bacterial solution concentration is close to 1×10 10 CFU / mL. The bacterial solution concentration is adjusted to 2-3×10 9 CFU / mL using BS liquid culture solution. The mixed solution is centrifuged at 8000g for 5min to separate the fermentation supernatant and the bacterial body precipitate. The bacterial body precipitate is resuspended to obtain a bacterial body resuspension. The fermentation culture solution, the fermentation supernatant and the bacterial body resuspension are heated in a water bath at 85°C for 15min to obtain the Bifidobacterium longum subsp. infantis NKU FB3-14 probiotic form as heat-killed bacterial body resuspension.

[0060] BS liquid culture medium: 10.0g / L proteose peptone, 5.0g / L liver extract powder, 3.0g / L beef extract powder, 5.0g / L yeast extract powder, 8.0g / L pancreatic casein peptone, 0.5g / L soluble starch, 1g / L sodium chloride, 1g / L potassium hydrogen phosphate, 1g / L potassium dihydrogen phosphate, 10g / L glucose, 0.01g / L ferric sulfate heptahydrate, 0.005g / L manganese sulfate, 0.5g / L L-cysteine.

[0061] In the embodiment of the present application, the BS liquid medium can be replaced by a BS solid medium, and the BS solid medium comprises 10.0 g / L of proteose peptone, 5.0 g / L of liver extract powder, 3.0 g / L of beef extract powder, 5.0 g / L of yeast extract powder, 8.0 g / L of pancreatic casein peptone, 0.5 g / L of soluble starch, 1 g / L of sodium chloride, 1 g / L of potassium phosphate dibasic, 1 g / L of potassium phosphate monobasic, 10 g / L of glucose, 0.01 g / L of ferric sulfate heptahydrate, 0.005 g / L of manganese sulfate, 0.5 g / L of L-cysteine, and 20 g / L of agar.

[0062] Example 2: Preparation method optimization of probiotic composition of Bifidobacterium longum subsp. infantis NKU FB3-14

[0063] H22 is a suspension of liver tumor cells, which is cultured in a 1640 complete medium containing 10% FBS in a 5% CO2, 37℃ saturated incubator, and the culture solution is replaced every 3 days. After the cells are collected and centrifuged, the supernatant is discarded, and the cells are subcultured. The logarithmic growth phase cells are configured into a cell suspension of 1*10 7 / mL, and the cell suspension is directly injected into the abdominal cavity of a mouse at 0.1 mL by using a syringe. After 7 days, the first generation ascites is collected. After centrifugal washing with PBS for three times, the cell suspension is prepared again, and the cell suspension is injected into the abdominal cavity of a new mouse. After 7 days, the second generation ascites is collected, and the ascites is centrifugally washed and inoculated into a 96-well plate.

[0064] The full formula composition comprises 8 parts of ginseng, 8 parts of longan arillus, 30 parts of astragalus, 30 parts of polygonatum, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of orange peel, 10 parts of citrus medica, 10 parts of double-petaled rose, and 5 parts of hawthorn.

[0065] The ginseng-removed composition comprises 8 parts of longan arillus, 30 parts of astragalus, 30 parts of polygonatum, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of orange peel, 10 parts of citrus medica, 10 parts of double-petaled rose, and 5 parts of hawthorn.

[0066] The longan arillus-removed composition comprises 8 parts of ginseng, 30 parts of astragalus, 30 parts of polygonatum, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of orange peel, 10 parts of citrus medica, 10 parts of double-petaled rose, and 5 parts of hawthorn.

[0067] The astragalus-removed composition comprises 8 parts of ginseng, 8 parts of longan arillus, 30 parts of polygonatum, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of orange peel, 10 parts of citrus medica, 10 parts of double-petaled rose, and 5 parts of hawthorn.

[0068] The polygonatum-removed composition comprises 8 parts of ginseng, 8 parts of longan arillus, 30 parts of astragalus, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of orange peel, 10 parts of citrus medica, 10 parts of double-petaled rose, and 5 parts of hawthorn.

[0069] Prepare the full formula composition, the ginseng-free composition, the longan aril-free composition, the radix astragali-free composition, the rhizoma polygonati-free composition, and the poria-free composition water extract, respectively, 6 intervention holes in each group, add the filtered water extract according to the intervention volume of 20%, terminate the intervention after 24 h of culture, and evaluate the inhibition effect of the water extract on the proliferation level of H22 cells by using the CCK-8 detection kit, and the results are shown in Table 1. Figure 1

[0070] Prepare the Bifidobacterium longum subsp. infantis NKU FB3-14 postbiotic and the homoeopathy water extract compound composition with the compound ratio of 1:1, 1:2, and 2:1, respectively, add the filtered water extract to the 96-well plate according to the intervention volume of 20%, terminate the intervention after 24 h of H22 intervention, and evaluate the inhibition effect of the water extract on the proliferation level of H22 cells by using the CCK-8 detection kit, and the results are shown in Table 2. Figure 2

[0071] Figure 1 Figure 2 The results show that the inhibition effect on H22 cells of the homoeopathy compound formula after removing ginseng, longan aril, radix astragali, rhizoma polygonati, and poria is significantly decreased, and the inhibition effect on H22 cells of the Bifidobacterium longum subsp. infantis NKU FB3-14 postbiotic / homoeopathy compound composition with the compound ratio of 1:1 is the most significant.

[0072] Prepare the corresponding intervention according to the preparation method of the FB3-14 live bacteria and the Bifidobacterium longum subsp. infantis NKU FB3-14 postbiotic (FB3-14 postbiotic) in Example 1, prepare the corresponding intervention according to the optimized homoeopathy compound and the postbiotic / homoeopathy compound ratio, and detect the inhibition rate of each group of intervention on H22 cells by using the CCK-8 kit after 24 h of intervention on H22 cells according to the method described above.

[0073] The compound group is prepared by using the homoeopathy compound formula and the postbiotic / homoeopathy compound ratio of 1:1 to prepare the corresponding intervention;

[0074] The FB3-14 live bacteria group is prepared by using only the FB3-14 live bacteria to intervene;

[0075] The FB3-14 postbiotic group is prepared by using only the Bifidobacterium longum subsp. infantis NKU FB3-14 postbiotic to intervene;

[0076] The homoeopathy compound group is prepared by using only the homoeopathy compound formula to intervene.

[0077] Figure 3 The results show that, compared with the compound group, the inhibition rate of the FB3-14 live bacteria group, the FB3-14 postbiotic group, and the homoeopathy compound group on H22 cells is significantly decreased, which indicates that, compared with each single group, the compound group has the most obvious effect on the inhibition of H22 cells. ​​​

[0078] Example 3: Application of NKU FB3-14 postbiotic combined with a food-medicine homology composition in a tumor-bearing mouse model

[0079] H22 tumor-bearing mouse model was established, and blank control group, model group, FB3-14 group, compound group and compound combination group were set up. After 1 week of acclimatization, the blank control group was not treated at all. The model group, FB3-14 group, compound group and compound combination group were given physiological saline, FB3-14 post-biotic, water extract of food and medicine homology, and FB3-14 post-biotic + water extract of food and medicine homology, respectively, for 4 weeks of preventive intervention. After culturing H22 cells, cell suspension was prepared and injected into the right forelimb axilla of other mice (Balb / c mice) except the blank control group. The intervention continued for 2 weeks. After the end of the intervention, serum, tumor tissue, spleen tissue, liver tissue, kidney tissue, colon tissue and cecal contents of each group of mice were collected and stored at -80℃ for later use.

[0080] The preparation methods of the components of the above-mentioned gavage intervention are the same as those in Examples 1 and 2.

[0081] The effects of different intervention groups on tumor tissue weight, volume, and histopathological analysis in H22 tumor-bearing mice are as follows: Figures 4-6 As shown, the intervention in the model group was an equal dose of physiological saline, the intervention in the FB3-14 group was FB3-14 post-biotic, the intervention in the compound group was a food-medicine homology composition, and the compound combination group was a compound food-medicine homology composition of FB3-14 post-biotic.

[0082] Depend on Figures 4-6 It can be seen that after intervention for 4 weeks before modeling and 2 weeks after modeling, the weight and volume of tumor tissue in each intervention group decreased. HE histopathological staining results showed that compared with the model group, the local inflammatory infiltration of tumor tissue in each intervention group was alleviated and the cytoplasm-nucleus ratio was improved. Necrotic cells were visible in the compound group and the compound preparation group. Compared with the FB3-14 group and the compound group, the reduction in tumor tissue weight and volume in the compound preparation group was more significant (* indicates p < 0.05 compared with the model group, ** indicates p < 0.01 compared with the model group, # indicates p < 0.05 compared with the compound preparation group, ## indicates p < 0.01 compared with the compound preparation group).

[0083] Example 4: Effects of NKU FB3-14 postbiotic combined with a food-medicine homology composition on immune regulation in tumor-bearing mouse models.

[0084] 1) The combination of post-biotic and food-derived ingredients has an effect on serum TNF-α and IFN-β, and T lymphocytes (CD4+) in spleen tissue. + T cells and CD8 + The influence of the proportion of T cells.

[0085] After the end of the intervention, the whole blood of the mice was collected, centrifuged at 3000r for 15min, and the supernatant was aliquoted and stored; the expression levels of TNF-α and IFN-β in the serum were detected by ELISA kit, the standard was centrifuged at 10000xg for 1min, 1mL of standard & sample diluent was added to the freeze-dried standard, the cap was tightened, and it was left to stand for 10min, and then it was inverted several times until it was fully dissolved, and then it was mixed gently to avoid foaming, and then 1000pg / mL of standard working solution was prepared, and then it was diluted by the required multiple, and the following concentrations were prepared: 1000, 500, 250, 125, 62.5, 31.25, 15.63, 0pg / mL. The standard wells, blank wells and sample wells were set. 100μL of the diluted standard was added to the standard wells, 100μL of the standard & sample diluent was added to the blank wells, and 100μL of the sample to be tested was added to the rest of the wells. The enzyme-labeled plate was covered with film, and incubated at 37℃ for 90min. 100μL of biotinylated antibody working solution was added to each well, and incubated at 37℃ for 1h. 350μL of washing solution was added to each well, soaked for 1min, and then the washing solution was removed, and the plate was washed for 3 times. 100μL of HRP enzyme conjugate working solution was added to each well, the enzyme-labeled plate was covered with film, and incubated at 37℃ for 30min. After removing the working solution, the plate was washed for 5 times, 90μL of substrate solution (TMB) was added to each well, and incubated at 37℃ for 15min. 50μL of stop solution was added to each well to terminate the reaction. The optical density (OD value) of each well was immediately measured at 450nm wavelength by the enzyme-labeled instrument.

[0086] The mice were sacrificed by cervical dislocation, immersed in 75% ethanol, and the spleen was removed in a clean bench. Mouse lymphocyte separation medium was added to a 35mm culture dish, ground, and then the filter screen was washed with a Pasteur pipette for 3 times. The spleen cell suspension was immediately transferred to a 15mL centrifuge tube, and RPMI 1640 medium was added. Centrifugation was performed at room temperature and 800g for 30min, and after centrifugation, the lymphocyte layer (middle cloud layer) was aspirated. 2mL of red blood cell lysis solution was added to each tube, and lysis was performed for 2-3min, followed by centrifugation at 500g for 5min, and the supernatant was discarded. 10mL of RPMI 1640 medium was added to each tube, and washed by inversion. After centrifugation at room temperature, the cells were resuspended in culture medium, counted, and an appropriate number of cells were collected for antibody incubation. After the completion of antibody incubation, the cells were washed, resuspended, and detected by machine.

[0087] The detection results are shown in Table 1. Figure 7 As shown in Table 1, the ELISA detection results showed that the content of TNF-α and IFN-β in the serum of mice inoculated with H22 tumor cells increased significantly, and the content of each intervention group decreased, and the content of the compound group decreased more significantly; the flow cytometry detection results showed that the proportion of CD4 + T cells in the spleen of mice inoculated with H22 tumor cells increased significantly, and compared with the model group, the proportion of CD4 +T cell ratio decreased, and the compound group and the compound group decreased more significantly, while CD8 + T cell ratio changed without statistical difference, suggesting that the effect of the composition on spleen lymphocytes was mainly on CD4 + T is the main (* indicates p<0.05 compared with the model group, ** indicates p<0.0 compared with the model group).

[0088] 2) The effect of the combination of postbiotics and food-herbal composition on NF-κB, P53 and P38 related proteins in tumor tissue.

[0089] Take the mouse tumor tissue, rinse with pure water, and dry. Weigh 100mg of tissue sample per tube, add 1000ul of RIPA lysis buffer, use homogenizer, lyse for 30min on ice, centrifuge at 12000rpm / 10min at 4℃, add appropriate amount of protein buffer, heat at 99℃ for 5min. After cooling to room temperature, the protein sample is directly loaded into the SDS gel loading hole for electrophoresis, then transferred to membrane, blocked, and 1:2000 concentration of NF-κB, P53, P38 and internal reference antibody is configured for primary antibody incubation, 4℃ incubation overnight, secondary antibody incubation, room temperature incubation for 2h, then development, and then use Image J analysis software to compare and analyze the band gray value, and the analysis result is as follows Figure 8 .

[0090] As shown in Figure 8 , compared with the model group, the expression level of NF-κB protein in FB3-14 group and compound group decreased significantly, the expression level of P53 protein in FB3-14 group, compound group and compound group increased significantly, and the expression level of P38 protein in compound group and compound group decreased significantly, suggesting that different interventions were involved in the regulation of proteins related to inflammation, tumor cell proliferation and metastasis in tumor tissue, and the effect of the compound group was more significant.

[0091] Example 5: Effect of NKU FB3-14 postbiotic combined with food-herbal composition on liver and kidney function of tumor-bearing model mice

[0092] The kit was used to detect the levels of ALT, AST and urea nitrogen in the serum of each group of mice, and the effect of each group of interventions on the liver and kidney function of tumor-bearing model mice after 6 weeks of oral intervention was evaluated, and the results are shown in Figures 9-11 As shown in

[0093] In summary, the Bifidobacterium longum postbiotic combined with Qi and blood-herbal composition can be used to adjust one or more of the applications of immune regulation, improvement of tumor-induced immune disorders.

[0094] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A probiotic composition of Bifidobacterium longum subsp. infantis NKU FB3-14 combined with a qi-tonifying and blood-nourishing food-medicinal composition, characterized in that: the probiotic composition of Bifidobacterium longum subsp. infantis NKU FB3-14 combined with a qi-tonifying and blood-nourishing food-medicinal composition is composed of Bifidobacterium longum subsp. infantis NKU FB3-14 and a qi-tonifying and blood-nourishing food-medicinal composition; the Bifidobacterium longum subsp. infantis NKU FB3-14 is preserved in the China General Microbiological Culture Collection Center, No. 3, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC No. 25762 and a preservation date of September 21, 2022; the qi-tonifying and blood-nourishing food-medicinal composition is composed of 5-30 parts of ginseng, 5-20 parts of longan arillus, 20-50 parts of astragalus, 10-50 parts of polygonatum, 5-20 parts of poria cocos, 5-15 parts of mulberry, 5-20 parts of licorice, 10-30 parts of orange peel, 5-15 parts of citrus medica, 5-20 parts of heavy-petaled rose, and 3-10 parts of hawthorn; the volume ratio of the inactivated Bifidobacterium longum subsp. infantis NKU FB3-14 cell suspension to the qi-tonifying and blood-nourishing food-medicinal composition is 1:0.8-1.2; the preparation method of the inactivated Bifidobacterium longum subsp. infantis NKU FB3-14 cell suspension is to prepare the inactivated Bifidobacterium longum subsp. infantis NKU FB3-14 cell suspension from Bifidobacterium longum subsp. infantis NKU FB3-14; and the preparation method of the qi-tonifying and blood-nourishing food-medicinal composition is to mix the raw materials of ginseng, longan arillus, astragalus, polygonatum, poria cocos, mulberry, licorice, orange peel, citrus medica, heavy-petaled rose, and hawthorn in a certain proportion to obtain a mixture, and then to filter and homogenize the mixture to a constant volume to obtain the qi-tonifying and blood-nourishing food-medicinal composition. The preparation method comprises the following steps: S1: preparing the inactivated Bifidobacterium longum subsp. infantis NKU FB3-14 cell suspension from Bifidobacterium longum subsp. infantis NKU FB3-14; S2: mixing the raw materials of ginseng, longan arillus, astragalus, polygonatum, poria cocos, mulberry, licorice, orange peel, citrus medica, heavy-petaled rose, and hawthorn in a certain proportion to obtain a mixture, and then filtering and homogenizing the mixture to a constant volume to obtain the qi-tonifying and blood-nourishing food-medicinal composition; S3: mixing the inactivated Bifidobacterium longum subsp. infantis NKU FB3-14 cell suspension in step S1 with the qi-tonifying and blood-nourishing food-medicinal composition in step S2 to obtain the probiotic composition of Bifidobacterium longum subsp. infantis NKU FB3-14 combined with a qi-tonifying and blood-nourishing food-medicinal composition. The preparation of the inactivated Bifidobacterium longum subsp. infantis NKU FB3-14 cell suspension in step S1 comprises the following steps:

2. The preparation method of the Bifidobacterium longum probiotics combined with the qi-tonifying and blood-nourishing food-medicinal isogenic composition of claim 1, characterized in that: A1: inoculating Bifidobacterium longum subsp. infantis NKU FB3-14 into a Bifidobacterium liquid culture medium for fermentation culture to obtain a fermentation culture solution; A2: centrifuging the fermentation culture solution obtained in step A1 to separate the solid and liquid to obtain Bifidobacterium longum subsp. infantis NKU FB3-14 cells; A3: resuspending the Bifidobacterium longum subsp. infantis NKU FB3-14 cells obtained in step A2 and inactivating the resuspended cells to obtain the inactivated Bifidobacterium longum subsp. infantis NKU FB3-14 cell suspension. Step S2 comprises adding drinking water to the food-medicinal composition mixture for homogenization, placing the mixture in a rotary evaporator for constant volume, and constant volume to 0.8-1.2 g / mL of crude drug amount.

3. The preparation method of the Bifidobacterium longum probiotics combined with the qi-tonifying and blood-nourishing food homologous composition according to claim 2, characterized in that: ​ ​ ​ ​ 4. The preparation method of the Bifidobacterium longum probiotics combined with the qi-tonifying and blood-nourishing food homologous composition according to claim 2, characterized in that: The concentration of the long Bifidobacterium infantis NKU FB3-14 inactivated bacterial body resuspension solution in step A3 is 1×10 8 ~2×10 9 CFU / mL.

5. The preparation method of the Bifidobacterium longum probiotics combined with the qi-tonifying and blood-nourishing food homologous composition according to claim 2, characterized in that: The concentration of the long Bifidobacterium infantis NKU FB3-14 inactivated bacterial body resuspension solution in step A3 is 1×10 9 ~2×10 9 CFU / mL.

6. The preparation method of the Bifidobacterium longum probiotics combined with the qi-tonifying and blood-nourishing food homologous composition according to claim 2, characterized in that: ​ The mixture is mixed with drinking water in a mass / volume ratio of 4-6.

7. The Bifidobacterium longum probiotic combined with the qi-tonifying and blood-nourishing food-medicinal isogenic composition of claim 1 or the Bifidobacterium longum probiotic combined with the qi-tonifying and blood-nourishing food-medicinal isogenic composition prepared by the method of any one of claims 2-6 for preparing food.

8. The Bifidobacterium longum probiotic combined with the qi-tonifying and blood-nourishing food-medicinal isogenic composition of claim 1 or the Bifidobacterium longum probiotic combined with the qi-tonifying and blood-nourishing food-medicinal isogenic composition prepared by the method of any one of claims 2-6 for preparing health food.

9. The use of the Bifidobacterium longum probiotic combined with the qi-tonifying and blood-nourishing food-medicinal isogenic composition prepared by the method of any one of claims 1-6 in the preparation of a medicament for regulating immunity, inhibiting tumor cell proliferation, and / or reducing tumor-induced inflammation.

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