Bifidobacterium longum metaplasia combined qi-reinforcing and blood-nourishing medicinal and edible composition and preparation method thereof, and application of composition in aspects of regulating immunity, inhibiting tumor cell proliferation and relieving tumor-induced inflammations of bifidobacterium longum metaplasia combined qi-reinforcing and blood-nourishing medicinal and edible composition

Abstract

The invention discloses a bifidobacterium longum metaplasm combined qi-reinforcing and blood-nourishing medicinal and edible composition as well as a preparation method and application thereof in the aspects of regulating immunity, inhibiting tumor cell proliferation and relieving tumor-induced inflammation. The bifidobacterium longum metaplasm combined qi-reinforcing and blood-nourishing medicinal and edible composition comprises bifidobacterium longum subsp.infantis NKU FB3-14 and a qi-reinforcing and blood-nourishing medicinal and edible composition, the invention relates to a medicinal and edible composition capable of reinforcing qi and nourishing blood. The medicinal and edible composition comprises the following components in parts by weight: 5-30 parts of ginseng, 5-20 parts of longan aril, 20-50 parts of astragalus membranaceus, 10-50 parts of rhizoma polygonati, 5-20 parts of poria cocos, 5-15 parts of mulberries, 5-20 parts of liquorice, 10-30 parts of orange peel, 5-15 parts of citron, 5-20 parts of roses with double petals and 3-10 parts of hawthorns. According to the present invention, the metagen preparation and the medicinal and edible material have the synergistic effect, and the proliferation level inhibition ability on the liver tumor cell H22 in vitro is determined;

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a postbiotic of Bifidobacterium longum combined with a medicated diet homologous composition for supplementing qi and nourishing blood, its preparation method, and its applications in regulating immunity, inhibiting the proliferation of tumor cells, and alleviating tumor-induced inflammation. Background Art

[0002] Qi and blood deficiency syndrome, a name of a traditional Chinese medicine syndrome, refers to the deficiency of qi and blood and the lack of nourishment of the body. Common symptoms include fatigue, shortness of breath and reluctance to speak, pale or sallow complexion, dizziness and vertigo, pale lips and nails, palpitations and insomnia, pale tongue with weak pulse, etc. Its causes are mostly due to acquired disorders, long-term illness without proper care or prolonged illness without recovery, blood loss and qi consumption, such as in the late stage of consumptive diseases like tumors, or after surgery, radiotherapy, chemotherapy, etc., which consume the body's energy and damage the digestive system, inducing health problems such as intestinal flora disorders and immune function disorders. It can be regulated through diet and the theory of medicated diet homologous substances, and attention should be paid to strengthening the healthy qi and tonifying deficiency and dietary therapy.

[0003] Since ancient times, traditional Chinese medicine has had the theory of medicated diet homologous. Modern detection techniques have proven that medicated diet homologous substances are rich in compounds with health-maintaining effects, and at the same time contain various nutrient components such as proteins, vitamins, and trace elements, which have good tonifying effects on weak people. In recent years, with the continuous in-depth research on probiotics, postbiotics, etc., a number of studies have confirmed that they have good improvement effects on the absorption of nutrients, the regulation of intestinal flora, etc. Appropriate supplementation of high-quality probiotics and postbiotics can be used as one of the daily maintenance strategies for people with physical weakness caused by various factors. Modern research has shown that through preliminary optimization and screening, probiotics, postbiotics combined with medicated diet homologous substances have a richer material basis and more extensive action targets, and have the potential of 1 + 1 > 2 for maintaining the immune homeostasis of the body.

[0004] Therefore, developing a product of postbiotic compounded with medicated diet homologous substances that follows the compatibility theory, has a rich combination of substances, is safe and non-toxic to the human body, can be eaten for a long time, and helps prevent and improve qi and blood deficiency has high economic value and social significance. Summary of the Invention

[0005] In view of this, the present invention aims to provide a postbiotic of Bifidobacterium longum combined with a medicated diet homologous composition for supplementing qi and nourishing blood, its preparation method, and its applications in regulating immunity, inhibiting the proliferation of tumor cells, and alleviating tumor-induced inflammation, so as to solve at least one technical problem in the background art.

[0006] To achieve the above object, the technical solution of the present invention is realized as follows:

[0007] An qi-supplementing and blood-nourishing food-medicinal homologous composition is applied to food, including the qi-supplementing and blood-nourishing food-medicinal homologous composition. The qi-supplementing and blood-nourishing food-medicinal homologous composition includes, by weight components, 5-30 parts of ginseng, 5-20 parts of longan aril, 20-50 parts of astragalus membranaceus, 10-50 parts of polygonatum sibiricum, 5-20 parts of poria cocos, 5-15 parts of mulberry, 5-20 parts of licorice root, 10-30 parts of tangerine peel, 5-15 parts of citron, 5-20 parts of rosa rugosa cv. Plena, and 3-10 parts of hawthorn.

[0008] The above qi-supplementing and blood-nourishing food-medicinal homologous composition is applied to health food.

[0009] In the present invention, the ginseng is sourced from artificially cultivated ginseng of five years old or less, which has the effects of greatly supplementing primordial qi, invigorating the spleen and benefiting the lung, promoting the production of body fluid, calming the mind and improving intelligence, etc. It can improve the body's immune function, enhance disease resistance, promote metabolism, relieve fatigue, and reduce the generation of free radicals.

[0010] Longan aril has the effects of nourishing the heart and spleen, nourishing blood and calming the mind, promoting growth and development, enhancing physical fitness, reducing the generation of free radicals, improving cellular immune function, and at the same time having certain anti-anxiety and endocrine-regulating effects.

[0011] Astragalus membranaceus has the effects of invigorating the spleen and replenishing the middle-jiao, lifting yang and raising prolapse, benefiting qi and strengthening the exterior, promoting diuresis, and inducing granulation for discharging toxin. It can significantly improve non-specific immune function, and has promoting or enhancing effects on humoral immunity, cellular immunity, etc. It also has effects such as anti-aging, anti-mutation, liver protection, anti-fatigue, anti-hypoxia, anti-cold, anti-radiation, tumor inhibition, and anti-pulmonary artery hypertension.

[0012] Polygonatum sibiricum has the effects of nourishing yin and moistening the lung, invigorating the spleen and replenishing qi, and nourishing the kidney and filling essence. It has effects such as anti-pathogenic microorganism, reducing blood lipid, delaying aging and other effects, and has an impact on the cardiovascular system, immune function, cyclic nucleotide content, and blood sugar.

[0013] Poria cocos has the effects of inducing diuresis for removing edema, promoting diuresis and percolating dampness, invigorating the spleen, and calming the mind. It can significantly increase the content of serum IgG, has the effects of promoting cellular immunity and humoral immunity, and also has effects such as reducing blood sugar, anti-tumor, anti-pathogenic microorganism, and promoting hematopoiesis function.

[0014] Mulberry has the effects of nourishing yin and tonifying blood, promoting the production of body fluid and moistening dryness. It can promote the maturation of T cells, thus restoring the function of aging T cells, has a promoting effect on the humoral immune function of young mice, has a promoting effect on the growth of granulocyte progenitor cells, and also has the effect of preventing leukopenia caused by cyclophosphamide.

[0015] Licorice root has the effects of invigorating qi and replenishing the middle-jiao, moistening the lungs and relieving cough, clearing heat and detoxifying, relieving spasm and pain, and harmonizing the properties of medicines. It can promote the weight increase of some immune organs, enhance the activity of natural killer cells, and can regulate the immune response of macrophages.

[0016] Tangerine peel has the effects of regulating qi and strengthening the spleen, drying dampness and resolving phlegm, has the functions of scavenging oxygen free radicals and anti-lipid peroxidation, and has the functions of promoting bile secretion and reducing serum cholesterol.

[0017] Citron has the effects of soothing the liver and relieving depression, regulating qi and harmonizing the middle, drying dampness and resolving phlegm, and has the functions of anti-inflammatory, anti-viral, etc.

[0018] Double-petal rose has the effects of soothing the liver and relieving depression, promoting blood circulation to remove stasis, and can promote the blood flow velocity of microarteries and improve microcirculation disorders.

[0019] Hawthorn has the effects of promoting digestion and resolving food stagnation, promoting qi circulation and removing stasis, and at the same time has the functions of improving appetite, promoting blood circulation, enhancing immune response, anti-tumor, etc.

[0020] In summary, the combination of medicine and food proposed in the present invention, including a variety of combinations of medicine and food such as ginseng, longan pulp, astragalus membranaceus, polygonatum sibiricum, poria cocos, mulberry, licorice, tangerine peel, citron, double-petal rose, hawthorn, etc., can not only replenish qi and nourish blood, regulate immune response, enhance the body's energy metabolism, etc., but also avoid adverse reactions to the recipients and is conducive to promotion.

[0021] The postbiotics of Bifidobacterium longum subsp. infantis combined with the medicine and food homologous composition for replenishing qi and nourishing blood includes Bifidobacterium longum subsp. infantis NKUFB3-14 and the above-mentioned medicine and food homologous composition for replenishing qi and nourishing blood. Bifidobacterium longum subsp. infantis NKU FB3-14 is preserved in the China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number is: CGMCC No. 25762, and the preservation date is September 21, 2022.

[0022] The Bifidobacterium longum subsp. infantis NKU FB3-14 described in the present invention has been disclosed in Chinese Patent CN202310453211.X, and it has been proved by experiments that the Bifidobacterium longum subsp. infantis NKU FB3-14 is a safety strain and has good acid tolerance, bile salt tolerance and DPPH scavenging ability, and can be used in humans and animals.

[0023] The present invention also provides a preparation method of the postbiotics of Bifidobacterium longum subsp. infantis combined with the medicine and food homologous composition for replenishing qi and nourishing blood, including the following steps:

[0024] S1: Prepare the inactivated cell suspension of Bifidobacterium longum subsp. infantis NKU FB3-14;

[0025] S2: Mix the extracts of ginseng, longan pulp, astragalus root, polygonatum rhizome, poria cocos, mulberry fruit, liquorice root, tangerine peel, citron fruit, double-petal rose flower, hawthorn fruit, etc. in proportion to obtain a mixture, filter and homogenize the mixture to a fixed volume to prepare a qi-tonifying and blood-nourishing medicine and food homology composition;

[0026] S3: Mix the heat-inactivated cell suspension of Bifidobacterium infantis NKU FB3-14 in step S1 with the qi-tonifying and blood-nourishing medicine and food homology composition in step S2 to prepare a postbiotic combined qi-tonifying and blood-nourishing medicine and food homology composition.

[0027] Furthermore, the preparation of the heat-inactivated cell suspension of Bifidobacterium infantis NKU FB3-14 in step S1 includes the following steps:

[0028] A1: Inoculate Bifidobacterium infantis NKU FB3-14 into a bifidobacterium liquid medium for fermentation culture to obtain a fermentation culture solution;

[0029] A2: Centrifuge the fermentation culture solution obtained in step A1 to separate the solid and liquid phases to obtain the fermentation supernatant of Bifidobacterium infantis NKU FB3-14 and the cells of Bifidobacterium infantis NKU FB3-14;

[0030] A3: Resuspend and inactivate the cells of Bifidobacterium infantis NKU FB3-14 obtained in step A2 to obtain a heat-inactivated cell suspension of Bifidobacterium infantis NKU FB3-14.

[0031] In the fermentation culture solution, heat-inactivated cell suspension and heat-inactivated fermentation culture solution of Bifidobacterium infantis NKU FB3-14 of the present invention, the concentration of Bifidobacterium infantis NKU FB3-14 is preferably 1×10 8 ~2×10 9 CFU / mL, more preferably 1×10 9 ~2×10 9 CFU / mL, and even more preferably 2×10 9 ~3×10 9 CFU / mL. The resuspending agent of the cell suspension and heat-inactivated cell suspension of the present invention preferably includes a bifidobacterium liquid medium, and more preferably a BS liquid medium.

[0032] Furthermore, in step S2, the water extracts of ginseng, longan pulp, astragalus root, polygonatum rhizome, poria cocos, mulberry fruit, tangerine peel, citron fruit, double-petal rose flower, hawthorn fruit are mixed in proportion, added with drinking water and homogenized thoroughly, then filtered, and the mixture is placed in a rotary evaporator to be fixed to a crude drug amount of 0.8 - 1.2 g / mL;

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

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

[0035] The weight parts of the medicated and food homologous combination described in the present invention are screened, and the final combined weight parts are determined by optimizing the inhibitory ability on the proliferation level of liver tumor cells H22. The optimal ratio of the postbiotic preparation to the medicated and food homologous material described in the present invention is 1:1, and the optimal ratio of the postbiotic preparation to the medicated and food homologous composition is determined by measuring the inhibitory ability on the proliferation level of liver tumor cells H22 in vitro.

[0036] The above-mentioned combination of Bifidobacterium longum postbiotic and the medicated and food homologous composition for supplementing qi and nourishing blood is applied to food.

[0037] The above-mentioned combination of Bifidobacterium longum postbiotic and the medicated and food homologous composition for supplementing qi and nourishing blood is applied to health food.

[0038] The above-mentioned combination of Bifidobacterium longum postbiotic and the medicated and food homologous composition for supplementing qi and nourishing blood is used in the preparation of one or more medicaments for regulating immunity, inhibiting the proliferation of tumor cells, and reducing tumor-induced inflammation.

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

[0040] Compared with the prior art, the combination of Bifidobacterium longum postbiotic and the medicated and food homologous composition for supplementing qi and nourishing blood, its preparation method, and its application in regulating immunity, inhibiting the proliferation of tumor cells, and reducing tumor-induced inflammation have the following advantages:

[0041] 1. The postbiotic preparation and the medicated and food homologous material described in this application have a synergistic effect. By measuring the inhibitory ability on the proliferation level of liver tumor cells H22 in vitro, the postbiotic provides a good internal environment for the medicated and food homologous composition to play a role by regulating the intestinal flora and the immune system. The medicated and food homologous composition enhances the overall immune function of the body by supplementing qi and nourishing blood, and further amplifies the immune regulation effect of the postbiotic.

[0042] 2. The postbiotic preparation and the medicated and food homologous material described in this application can be used as an adjuvant treatment means for tumor patients to improve immune disorder and enhance the anti-tumor immune response.

[0043] 3. The combination of Bifidobacterium longum postbiotic and the medicated and food homologous composition for supplementing qi and nourishing blood described in this application regulates the immune system through multiple targets and multiple pathways, improves tumor-induced immune disorder, and has significant immune enhancement and anti-tumor potential. This combined application provides new ideas and methods for tumor treatment and immune regulation. Description of the Drawings

[0044] The accompanying drawings that form 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 of the present invention. In the accompanying drawings:

[0045] Figure 1 Schematic diagram of the inhibitory effect of aqueous extracts of different medicine-food homologous compositions described in Example 2 of the present invention on the proliferation rate of H22 cells;

[0046] Figure 2 Schematic diagram of the inhibitory effect of different ratios of postbiotics / medicine-food homologous compound pairs described in Example 2 of the present invention on the proliferation of H22 cells;

[0047] Figure 3 Schematic diagram of the inhibitory effect of the FB3-14 live bacteria group, FB3-14 postbiotics group, medicine-food homologous compound prescription group, and compound group described in Example 2 of the present invention on the proliferation of H22 cells;

[0048] Figure 4 Comparison diagram of the appearance of tumor tissues of each intervention group described in Example 3 of the present invention (each intervention group is the model group, FB3-14 group, compound prescription group, and compound group);

[0049] Figure 5 Comparison diagram of the weight and volume of tumor tissues of each intervention group described in Example 3 of the present invention (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 the model group, FB3-14 group, compound prescription group, and compound group);

[0050] Figure 6 Comparison diagram of the histopathology of tumor tissues of each intervention group described in Example 3 of the present invention (a is the model group, b is the FB 3-14 group, c is the compound prescription group, and d is the compound group);

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

[0052] Figure 8 Diagram of the expression levels of NF-κB, P53, and P38 proteins in tumor tissues of each group described in Example 4 of the present invention (a is the developed image after secondary antibody incubation at night and incubation at room temperature for 2 h, and b is the diagram of the comparative analysis of the band gray value using Image J analysis software);

[0053] Figure 9The change graph of ALT levels in the sera of each group described in Example 5 of the present invention (each group is the blank group, the model group, the FB3-14 group, the compound group, and the compounded group);

[0054] Figure 10 The change graph of AST levels in the sera of each group described in Example 5 of the present invention (each group is the blank group, the model group, the FB3-14 group, the compound group, and the compounded group);

[0055] Figure 11 The change graph of blood urea nitrogen levels in the sera of each group described in Example 5 of the present invention (each group is the blank group, the model group, the FB3-14 group, the compound group, and the compounded group). Detailed implementation manners

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

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

[0058] Example 1: Preparation of the postbiotic preparation of Bifidobacterium longum subsp. infantis NKU FB3-14

[0059] Inoculate Bifidobacterium longum subsp. infantis NKU FB3-14 into BS liquid medium at an inoculation amount of 5% by volume. After inoculation, anaerobically culture in a constant temperature incubator at 37°C for 20 - 24 h. When the OD value of the culture broth reaches 1.5, the bacterial concentration is close to 1×10 10 CFU / mL. Use BS liquid culture broth to adjust the bacterial concentration to 2 - 3×10 9 CFU / mL as the fermentation culture broth. Centrifuge the mixture at 8000g for 5 min to separate the fermentation supernatant and the bacterial cell precipitate. Resuspend the bacterial cell precipitate to obtain a bacterial cell resuspension. Heat the fermentation culture broth, the fermentation supernatant, and the bacterial cell resuspension in a water bath at 85°C for 15 min to obtain a postbiotic form of Bifidobacterium longum subsp. infantis NKU FB3-14 as a heat-inactivated bacterial cell resuspension.

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

[0061] In the embodiments of the present invention, the BS liquid medium can be replaced with a BS solid medium. The BS solid medium: peptone 10.0 g / L, liver extract powder 5.0 g / , beef extract powder 3.0 g / L, yeast extract powder 5.0 g / L, tryptone 8.0 g / L, soluble starch 0.5 g / L, sodium chloride 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1 g / L, glucose 10 g / L, ferric sulfate heptahydrate 0.01 g / L, manganese sulfate 0.005 g / L, L-cysteine 0.5 g / L, agar 20 g / L.

[0062] Example 2: Optimization of the preparation method of postbiotics combined with medicated and edible homologous compositions of Bifidobacterium longum subsp. infantis NKU FB3-14

[0063] H22 is a suspension of liver tumor cells, cultured in a complete 1640 medium containing 10% FBS in an incubator with 5% CO2 and 37 °C saturation. The culture medium is changed once every 3 days. After collecting the cells, the supernatant is centrifuged and discarded for subculture. Logarithmic growth phase cells are used to prepare a cell suspension of 1*10 7 / mL, and 0.1 mL of the cell suspension is directly injected into the abdominal cavity of mice with a syringe. After 7 days, the first-generation ascites is collected. After centrifugal washing with PBS three times, a new cell suspension is prepared and injected into the abdominal cavity of new mice. After 7 days, the second-generation ascites is collected, centrifuged and washed, and then inoculated into a 96-well plate.

[0064] The full-formula composition is 8 parts of ginseng, 8 parts of longan pulp, 30 parts of astragalus membranaceus, 30 parts of polygonatum sibiricum, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of tangerine peel, 10 parts of citron, 10 parts of rosa rugosa cv. Plena, and 5 parts of hawthorn.

[0065] The composition without ginseng is 8 parts of longan pulp, 30 parts of astragalus membranaceus, 30 parts of polygonatum sibiricum, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of tangerine peel, 10 parts of citron, 10 parts of rosa rugosa cv. Plena, and 5 parts of hawthorn.

[0066] The composition without longan pulp is 8 parts of ginseng, 30 parts of astragalus membranaceus, 30 parts of polygonatum sibiricum, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of tangerine peel, 10 parts of citron, 10 parts of rosa rugosa cv. Plena, and 5 parts of hawthorn.

[0067] The composition without astragalus membranaceus is 8 parts of ginseng, 8 parts of longan pulp, 30 parts of polygonatum sibiricum, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of tangerine peel, 10 parts of citron, 10 parts of rosa rugosa cv. Plena, and 5 parts of hawthorn.

[0068] The composition without polygonatum sibiricum is 8 parts of ginseng, 8 parts of longan pulp, 30 parts of astragalus membranaceus, 10 parts of poria cocos, 10 parts of mulberry, 10 parts of licorice, 20 parts of tangerine peel, 10 parts of citron, 10 parts of rosa rugosa cv. Plena, and 5 parts of hawthorn.

[0069] The water extracts of the full-formula composition, the composition without ginseng, the composition without longan aril, the composition without astragalus membranaceus, the composition without polygonatum sibiricum, and the composition without poria cocos were prepared respectively. There were 6 intervention wells in each group. The filtered water extract was added according to 20% of the intervention volume. After culturing for 24 h, the intervention was terminated. The CCK-8 detection kit was used to evaluate the inhibitory effect of the water extract on the proliferation level of H22 cells. The results are as Figure 1 shown.

[0070] Compositions with the compound ratio of probiotics of bifidobacterium infantis NKU FB3-14 and the water extract of medicine and food homology of 1:1, 1:2, and 2:1 were prepared respectively. After filtration, they were added to a 96-well plate according to 20% of the intervention volume. After intervening on H22 for 24 h, the intervention was terminated. The CCK-8 detection kit was used to evaluate the inhibitory effect of the water extract on the proliferation level of H22 cells. The results are as Figure 2 shown.

[0071] Figure 1 , Figure 2 The results showed that after removing ginseng, longan aril, astragalus membranaceus, polygonatum sibiricum, and poria cocos from the formula of medicine and food homology, the inhibitory effect on H22 cells decreased significantly. When the ratio of probiotics of bifidobacterium infantis NKU FB3-14 to the composition of medicine and food homology was 1:1, the inhibitory effect on H22 cells was the most significant.

[0072] The corresponding intervention substances were prepared according to the preparation methods of FB3-14 live bacteria and probiotics of bifidobacterium infantis NKU FB3-14 (FB3-14 probiotics) in Example 1. The corresponding intervention substances were prepared according to the optimized formula of medicine and food homology and the compound ratio of probiotics to the composition of medicine and food homology. After intervening on H22 cells for 24 h according to the method described above, the CCK-8 kit was used to detect the inhibition rate of each intervention substance on H22 cells.

[0073] The compound group was prepared by preparing the corresponding intervention substance according to the ratio of 1:1 of the formula of medicine and food homology and the compound ratio of probiotics to the composition of medicine and food homology for intervention;

[0074] The FB3-14 live bacteria group was intervened only with FB3-14 live bacteria;

[0075] The FB3-14 probiotics group was intervened only with probiotics of bifidobacterium infantis NKU FB3-14;

[0076] The medicine and food homology compound group was intervened only with the formula of medicine and food homology.

[0077] Figure 3 The results showed that compared with the compound group, the inhibition rates of the FB3-14 live bacteria group, the FB3-14 probiotics group, and the medicine and food homology compound group on H22 cells decreased significantly, indicating that compared with each single-use group, the compound group had the most obvious inhibitory effect on H22 cells.

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

[0079] An H22 tumor-bearing mouse model was established, and blank group, model group, FB3-14 group, compound group and compounded group were set up. After 1 week of adaptive feeding, no treatment was given to the blank group throughout the process. The model group, FB3-14 group, compound group and compounded group were respectively given normal saline, FB3-14 postbiotics, water extract of medicine and food homology, and FB3-14 postbiotics + water extract of medicine and food homology, 0.2 mL per mouse, for preventive intervention for 4 weeks. After culturing H22 cells, a cell suspension was prepared, and the cell suspension was injected subcutaneously into the right anterior axilla of other mice (Balb / c mice) except the blank group, and the intervention continued for 2 weeks. After the end, the serum, tumor tissue, spleen tissue, liver tissue, kidney tissue, colon tissue and cecal content of each group of mice were collected and stored at -80 °C for later use.

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

[0081] The effects of the intervention substances of different groups on the tumor tissue weight, volume and histopathological analysis of H22 tumor-bearing mice are as Figures 4 - 6 shown. The intervention substance of the model group is equal-dose normal saline, the intervention substance of the FB3-14 group is FB3-14 postbiotics, the intervention substance of the compound group is the medicine and food homology composition, and the compounded group is the FB3-14 postbiotics compounded with the medicine and food homology composition.

[0082] From Figures 4 - 6 it can be seen that after 4 weeks before modeling + 2 weeks after modeling intervention, the tumor tissue weight and volume of each intervention group have decreased. The HE histopathological staining results show that compared with the model group, the local inflammatory infiltration of the tumor tissue in each intervention group has been alleviated, the quality-nucleus ratio has been improved, and necrotic cells can be seen in the compound group and the compounded group; compared with the FB3-14 group and the compound group, the tumor tissue weight and volume of the compounded group have decreased more significantly (* 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 compounded group, ## indicates p < 0.01 compared with the compounded group).

[0083] Example 4: Effects of NKU FB3-14 postbiotics combined with medicine and food homology composition on immune regulation of tumor-bearing model mice

[0084] 1) Effects of postbiotics combined with medicine and food homology composition on TNF-α, IFN-β in serum and the proportions of T lymphocytes (CD4 + T cells and CD8 + T cells) in spleen tissue.

[0085] After the intervention, collect the whole blood of the mice, centrifuge at 3000 r for 15 min, take the supernatant, aliquot and store for later use; use an ELISA kit to detect the expression levels of TNF-α and IFN-β in the serum. Centrifuge the standard product at 10000×g for 1 minute, add 1 mL of standard product & sample diluent to the lyophilized standard product, tighten the tube cap, let it stand for 10 minutes, invert several times, and after it is fully dissolved, gently mix well to avoid foaming, to prepare a standard working solution of 1000 pg / mL, and then perform serial dilutions as needed to prepare the following concentrations: 1000, 500, 250, 125, 62.5, 31.25, 15.63, 0 pg / mL. Set the standard wells, blank wells and sample wells respectively. Add 100 μL of serially diluted standard product to the standard wells, add 100 μL of standard product & sample diluent to the blank wells, and add 100 μL of the sample to be tested to the remaining wells. Cover the ELISA plate with a film and incubate at 37 °C for 90 minutes. Add 100 μL of biotinylated antibody working solution to each well and incubate at 37 °C for 1 hour. Add 350 μL of washing solution to each well, soak for 1 minute and then remove the washing solution, repeat the plate washing 3 times. Add 100 μL of HRP enzyme conjugate working solution to each well, cover the ELISA plate with a film and incubate at 37 °C for 30 minutes. After removing the working solution, wash the plate 5 times, add 90 μL of substrate solution (TMB) to each well and incubate at 37 °C in the dark for 15 minutes. Add 50 μL of stop solution to each well to terminate the reaction. Immediately measure the optical density (OD value) of each well with an ELISA reader at a wavelength of 450 nm.

[0086] Decapitate the mice and soak them in 75% ethanol, and take out the spleens of the mice in a biosafety cabinet. Add mouse lymphocyte separation solution to a 35 mm culture dish, grind, and then use a Pasteur pipette to suck the separation solution and rinse the filter screen 3 times. Immediately transfer the separation solution containing spleen cells to a 15 mL centrifuge tube, and add an appropriate amount of RPMI 1640 medium. At room temperature, centrifuge at 800 g for 30 min with a horizontal rotor. After centrifugation, aspirate the lymphocyte layer (the middle cloudy layer). Add 2 mL of red blood cell lysate to each tube, lyse for 2 - 3 minutes, then centrifuge at 500 g for 5 min, and discard the supernatant. Add 10 mL of RPMI 1640 medium to each tube and invert to wash. After centrifugation at room temperature, resuspend the cells with the culture medium, count, and collect an appropriate number of cells for antibody incubation. After the corresponding antibody incubation is completed, wash and resuspend, and then perform the detection on the machine.

[0087] The detection results are as Figure 7 shown. The ELISA detection results show that after subcutaneous inoculation with H22 tumor cells, the contents of TNF-α and IFN-β in the serum of mice increased significantly, and the contents in each intervention group decreased to varying degrees, with the compound group showing a more significant decrease; the flow cytometry detection results show that after subcutaneous inoculation with H22 tumor cells, the proportion of CD4 + T cells in the spleens of mice increased significantly. Compared with the model group, the CD4 +The proportion of T cells decreased, and the decreasing trend was more significant in the compound group and the compound preparation group. However, there was no statistically significant difference in the proportion change of CD8 + T cells, suggesting that the effect of this composition on spleen lymphocytes was mainly on CD4 + T (* indicates p < 0.05 compared with the model group, ** indicates p < 0.0 compared with the model group).

[0088] 2) The postbiotics combined with the medicine and food homologous composition on related proteins such as NF-κB, P53, and P38 in tumor tissues.

[0089] Take the tumor tissues of mice, rinse them with pure water and then blot them dry. Weigh 100 mg of tissue samples in each tube, add 1000 μl of RIPA lysis buffer, homogenize using a homogenizer, lyse on ice for 30 min, centrifuge at 4°C at 12000 rpm / 10 min, take the supernatant, add an appropriate amount of protein buffer, and heat at 99°C for 5 min. After cooling to room temperature, directly load the protein samples into the loading wells of the SDS gel for electrophoresis. After that, transfer the membrane and block it. Prepare primary antibodies with a concentration of 1:2000 for NF-κB, P53, P38, and the internal reference antibody for primary antibody incubation. Incubate overnight at 4°C and then perform secondary antibody incubation. Incubate at room temperature for 2 h and then develop the film. After that, use the Image J analysis software to compare and analyze the gray values of the bands. The analysis results are as Figure 8 .

[0090] As Figure 8 shown, compared with the model group, the expression level of NF-κB protein in the FB3-14 group and the compound preparation group decreased significantly. The expression levels of P53 protein in the FB3-14 group, the compound group, and the compound preparation group increased significantly. The expression levels of P38 protein in the compound group and the compound preparation group decreased significantly, suggesting that different intervention substances participated in the regulation of proteins related to inflammation, tumor cell proliferation, and metastasis in tumor tissues, and the compound preparation group had a more significant effect.

[0091] Example 5: Effects of the postbiotics of Bifidobacterium longum NKU FB3-14 combined with the medicine and food homologous composition on the liver and kidney functions of tumor-bearing model mice

[0092] Use a kit to detect the levels of ALT, AST, and urea nitrogen in the serum of mice in each group, and evaluate the effects of the intervention substances in each group on the liver and kidney functions of tumor-bearing model mice after 6 weeks of oral intervention. The results are as Figures 9 - 11 shown. Compared with the blank group and the model group, there were no significant differences in the levels of serum ALT, AST, and urea nitrogen in the intervention groups, indicating that this composition had high safety.

[0093] In summary, the postbiotics of Bifidobacterium longum combined with the medicine and food homologous composition for replenishing qi and nourishing blood of the present invention can be used in the application of one or more drugs for regulating immunity and improving tumor-induced immune disorders.

[0094] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composition of medicine and food homologous for supplementing qi and nourishing blood is applied to food, and is characterized in that: The medicated and edible homologous composition for supplementing qi and nourishing blood comprises, by weight components: 5-30 parts of ginseng, 5-20 parts of longan aril, 20-50 parts of astragalus membranaceus, 10-50 parts of polygonatum sibiricum, 5-20 parts of poria cocos, 5-15 parts of mulberry, 5-20 parts of liquorice, 10-30 parts of tangerine peel, 5-15 parts of citron, 5-20 parts of rosa rugosa cv. Plena, and 3-10 parts of hawthorn; Preferably, the above medicated and edible homologous composition for supplementing qi and nourishing blood is applied to health food.

2. Postbiotics of Bifidobacterium longum combined with a medicinal and edible homologous composition for supplementing qi and nourishing blood, characterized in that: It includes bifidobacterium longum subsp. infantis NKU FB3-14 and the medicated and edible homologous composition for supplementing qi and nourishing blood as described in claim 1. Bifidobacterium longum subsp. infantis NKU FB3-14 is deposited in the China General Microbiological Culture Collection Center, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC No. 25762, and the deposit date is September 21, 2022.

3. The preparation method of the postbiotic of Bifidobacterium longum combined with the medicated and food homologous composition for supplementing qi and nourishing blood, characterized in that: It includes the following steps: S1: Prepare the inactivated cell suspension of bifidobacterium longum subsp. infantis NKU FB3-14. S2: Mix the raw materials such as ginseng, longan aril, astragalus membranaceus, polygonatum sibiricum, poria cocos, mulberry, liquorice, tangerine peel, citron, rosa rugosa cv. Plena, and hawthorn in proportion to obtain a mixture, and then filter, homogenize and make up the volume to prepare the medicated and edible homologous composition for supplementing qi and nourishing blood. S3: Mix the inactivated cell suspension of bifidobacterium longum subsp. infantis NKU FB3-14 in step S1 with the medicated and edible homologous composition for supplementing qi and nourishing blood in step S2 to obtain the postbiotics of bifidobacterium longum combined with the medicated and edible homologous composition for supplementing qi and nourishing blood.

4. The preparation method of the postbiotic combined with qi - tonifying and blood - nourishing medicated and edible homologous composition of Bifidobacterium longum, characterized in that: The preparation of the inactivated cell suspension of bifidobacterium longum subsp. infantis NKU FB3-14 in step S1 includes the following steps: A1: Inoculate bifidobacterium longum subsp. infantis NKU FB3-14 into the bifidobacterium liquid medium for fermentation culture to obtain the fermentation culture solution. A2: Centrifuge the fermentation culture solution obtained in step A1 to separate the solid and liquid phases to obtain the cells of bifidobacterium longum subsp. infantis NKU FB3-14. A3: Resuspend and inactivate the cells of bifidobacterium longum subsp. infantis NKU FB3-14 obtained in step A2 to obtain the inactivated cell suspension of bifidobacterium longum subsp. infantis NKU FB3-14.

5. The preparation method of the postbiotic of Bifidobacterium longum combined with the medicated and edible homologous composition for supplementing qi and nourishing blood, as claimed in claim 4, wherein: The concentration of the inactivated cell suspension of Bifidobacterium longum subsp. infantis NKU FB3-14 in step A3 is 1×10 8 ~2×10 9 CFU / mL.

6. The preparation method of the postbiotic combined with qi - tonifying and blood - nourishing medicated and edible homologous composition of Bifidobacterium longum subsp. longum as claimed in claim 4, wherein: In step A3, the concentration of the inactivated cell suspension of Bifidobacterium longum subsp. infantis NKU FB3-14 is 1×10 9 ~2×10 9 CFU / mL.

7. The preparation method of the postbiotic of Bifidobacterium longum combined with the medicated and edible homologous composition for supplementing qi and nourishing blood according to claim 3, characterized in that: In step S2, after the extracts are mixed in proportion, filtration, homogenization and volume determination are carried out, including adding drinking water to the mixture of medicated and edible homologous extracts for homogenization, and placing the mixture in a rotary evaporator to make up the volume to a crude drug amount of 0.8-1.2 g / mL. The mass-volume ratio of the mixture to drinking water is 4-6.

8. The preparation method of the postbiotic of Bifidobacterium longum combined with the medicated and food homologous composition for supplementing qi and nourishing blood, according to claim 3, is characterized in that: In step S3, the volume ratio of the inactivated cell suspension of bifidobacterium longum subsp. infantis NKU FB3-14 to the medicated and edible homologous composition for supplementing qi and nourishing blood is 1:0.8-1.

2.

9. The postbiotics of bifidobacterium longum combined with the medicated and edible homologous composition for supplementing qi and nourishing blood as described in claim 2 or the postbiotics of bifidobacterium longum combined with the medicated and edible homologous composition for supplementing qi and nourishing blood prepared by the preparation method as described in any one of claims 3-8 is applied to food; Preferably, the above medicated and edible diet is applied to health food.

10. Use of the composition of postbiotics of Bifidobacterium longum combined with medicated and edible homologous compositions for supplementing qi and nourishing blood prepared by the preparation method of the composition of postbiotics of Bifidobacterium longum combined with medicated and edible homologous compositions according to any one of claims 3-8 in the preparation of one or more medicaments for regulating immunity, inhibiting the proliferation of tumor cells, and reducing tumor-induced inflammation.

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