阅读说明：本技术 一种减轻化疗副作用的中药益生菌发酵液制剂 (Traditional Chinese medicine probiotic fermentation liquor preparation for relieving side effects of chemotherapy ) 是由 袁杰力 李明 杨永亮 王斌 于 2021-09-17 设计创作，主要内容包括：本发明公开了一种用于减轻化疗副作用的中药益生菌发酵液制剂,是将长双歧杆菌、发酵乳杆菌和嗜酸乳杆菌添加到含有灵芝和莱菔子水提液的MRS肉汤培养基中增殖发酵获得,MRS肉汤培养基中灵芝浓度为16.6～66.4mg/mL,莱菔子浓度为0.5～2g/mL；添加益生菌菌量比为长双歧杆菌：发酵乳杆菌：嗜酸乳杆菌=1：1：1,使混合后每种益生菌在添加中药的MRS肉汤培养基中初始CFU/mL=1～2*10～(6),进行增殖并发酵中药至发酵液中总CFU/mL=3～5*10～(9)；这是一种能够有效减轻病人因化疗产生副作用的中药益生菌发酵液制剂。(The invention discloses a traditional Chinese medicine probiotic fermentation broth preparation for relieving side effects of chemotherapy, which is obtained by adding bifidobacterium longum, lactobacillus fermentum and lactobacillus acidophilus into an MRS broth culture medium containing water extract of lucid ganoderma and radish seed for proliferation and fermentation, wherein the concentration of the lucid ganoderma in the MRS broth culture medium is 16.6-66.4 mg/mL, and the concentration of the radish seed is 0.5-2 g/mL; the probiotic bacteria are added in a ratio of bifidobacterium longum: lactobacillus fermentum: lactobacillus acidophilus = 1:1:1, mixing each probiotic in MRS broth culture medium added with traditional Chinese medicines, wherein the initial CFU/mL = 1-2 x 10 6 Proliferating and fermenting the traditional Chinese medicine until the total CFU/mL = 3-5 x 10 in the fermentation liquor 9 (ii) a The drug can effectively relieve the side effect of chemotherapyA medicinal probiotic fermented liquid preparation.)

1. A traditional Chinese medicine probiotic fermentation liquor preparation for relieving side effects of chemotherapy is characterized in that: the bifidobacterium longum, lactobacillus fermentum and lactobacillus acidophilus are added into an MRS broth culture medium containing water extract of ganoderma lucidum and radish seed for proliferation and fermentation, wherein the concentration of the ganoderma lucidum in the MRS broth culture medium is 16.6-66.4 mg/mL, and the concentration of the radish seed is 0.5-2 g/mL; the probiotic bacteria are added in a ratio of bifidobacterium longum: lactobacillus fermentum: lactobacillus acidophilus = 1:1:1, mixing each probiotic in MRS broth culture medium added with traditional Chinese medicinesInitial CFU/mL = 1-2 x 10⁶Proliferating and fermenting the traditional Chinese medicine until the total CFU/mL = 3-5 x 10 in the fermentation liquor⁹。

2. The traditional Chinese medicine probiotic fermented liquid preparation for alleviating the side effects of chemotherapy according to claim 1, characterized in that: bifidobacterium longum, Lactobacillus fermentum, and Lactobacillus acidophilus, respectively in MRS broth culture medium to obtain bacterial liquid.

3. The traditional Chinese medicine probiotic fermented liquid preparation for alleviating the side effects of chemotherapy according to claim 1, characterized in that: the three probiotics are simultaneously added into MRS broth culture medium added with traditional Chinese medicines for proliferation and fermentation.

4. The traditional Chinese medicine probiotic fermented liquid preparation for alleviating the side effects of chemotherapy according to claim 1, characterized in that: adding lactobacillus fermentum into MRS broth containing Chinese medicinal materials, culturing at 37 deg.C for 12 hr, adding Bifidobacterium longum and Lactobacillus acidophilus, and performing proliferation fermentation.

Technical Field

The invention relates to a traditional Chinese medicine probiotic fermentation liquor preparation prepared by fermenting two traditional Chinese medicines by using specific probiotics, in particular to a traditional Chinese medicine probiotic fermentation liquor preparation for relieving side effects of chemotherapy.

Background

Chemotherapy is one of the most effective means of treating cancer at present. However, some chemotherapy drugs (such as cyclophosphamide CTX) can kill tumor cells and damage normal cells, especially hematopoietic stem cells and intestinal mucosa cells which grow faster, causing side effects. The more serious clinical manifestations are nausea and vomiting, emaciation, hypoimmunity, inflammatory reaction and the like caused by gastrointestinal flora disorder. On the other hand, chemotherapy can seriously damage the intestinal flora structure of a patient, and the flora diversity and the abundance of beneficial bacteria are reduced. Thereby aggravating mucosal inflammation and gastrointestinal reaction. Meanwhile, as the intestinal flora is highly involved in drug metabolism and active substance conversion, flora disturbance will affect the utilization degree of some oral drugs, especially Chinese medicinal formulas. The existing research shows that the ganoderma lucidum has the effects of enhancing the immunity of organisms, inhibiting the release of histamine from cells and enhancing the functions of various organs; the Raphani semen has functions of enhancing ileal rhythmic contraction and enhancing mechanical digestion. The effects of the two medicinal and edible materials can not be realized without the decomposition, transformation, structural modification and the like of the intestinal probiotics. The bifidobacterium can effectively protect the intestinal mucosa barrier and inhibit the permeability increase of the intestinal mucosa; the lactobacillus can regulate cellular immunity, recover intestinal flora and relieve diarrhea of patients. However, the efficacy of the probiotics on the fermentation and transformation of medicinal and edible materials in the medical field, particularly on the aspect of relieving side effects after chemotherapy, is not developed and utilized.

Disclosure of Invention

The invention provides a traditional Chinese medicine probiotic fermentation broth preparation capable of effectively relieving side effects generated by chemotherapy of patients in order to solve the defects of the existing clinical chemotherapy technology.

The technical solution of the invention is as follows: a traditional Chinese medicine probiotic fermentation liquor preparation for relieving side effects of chemotherapy is characterized in that: the bifidobacterium longum, lactobacillus fermentum and lactobacillus acidophilus are added into an MRS broth culture medium containing water extract of ganoderma lucidum and radish seed for proliferation and fermentation, wherein the concentration of the ganoderma lucidum in the MRS broth culture medium is 16.6-66.4 mg/mL, and the concentration of the radish seed is 0.5-2 g/mL; the probiotic bacteria are added in a ratio of bifidobacterium longum: lactobacillus fermentum: lactobacillus acidophilus ═ 1:1:1, mixing each probiotic in MRS broth culture medium added with traditional Chinese medicines, wherein the initial CFU/mL is 1-2 x 10⁶Proliferating and fermenting the traditional Chinese medicine until the total CFU/mL is 3-5 x 10 in the fermentation liquor⁹。

Further, bifidobacterium longum, lactobacillus fermentum and lactobacillus acidophilus are respectively obtained into bacterial liquids in MRS broth culture media.

Further, the three probiotics can be simultaneously added into MRS broth culture medium added with traditional Chinese medicines for proliferation and fermentation, and the best mode is that the lactobacillus fermentum is firstly added into the MRS broth culture medium containing traditional Chinese medicines, cultured for 12h at 37 ℃, added with bifidobacterium longum and lactobacillus acidophilus, and then subjected to proliferation and fermentation.

Compared with the prior art, the invention has the following advantages:

the traditional Chinese medicine probiotic fermentation liquor preparation for relieving the side effect of chemotherapy is prepared from edible traditional Chinese medicines, is wide in application range, easy to obtain, safe and free of toxic and side effects. All strains are from human bodies, and the safety is high through screening and formula verification, and the clinical application is guaranteed. It has obvious effects of relieving intestinal flora imbalance and intestinal mucosa barrier damage caused by chemotherapy, improving intestinal microecological imbalance, enhancing intestinal mucosa barrier function, and improving immunity. Therefore, the traditional Chinese medicine probiotic fermentation liquor preparation is used as an enteral nutrition preparation, can provide enteral nutrition for chemotherapy patients, can also adjust intestinal flora, is widely used, can be used as an auxiliary treatment, can greatly relieve the pain of the patients in the chemotherapy process, and improves the life quality of the patients.

Detailed Description

The following will explain specific embodiments of the present invention

The traditional Chinese medicine probiotic fermentation liquid preparation is prepared by carrying out propagation fermentation on Bifidobacterium longum (Bifidobacterium longum), Lactobacillus fermentum (Lactobacillus fermentum) and Lactobacillus acidophilus (Lactobacillus acidophilus) in an MRS broth culture medium added with water extract of lucid ganoderma and radish seed, wherein the concentration of the lucid ganoderma added in the MRS broth culture medium is 16.6-66.4 mg/mL, and the concentration of the radish seed is 0.5-2 g/mL; the mixed probiotics is bifidobacterium longum (Bi)Bacillus longum), Lactobacillus fermentum (Lactobacillus fermentum) and Lactobacillus acidophilus (Lactobacillus acidophilus), respectively taking three bacterial liquids from an MRS broth culture medium to the MRS broth culture medium added with traditional Chinese medicines, wherein the quantity ratio of the added probiotics is Bifidobacterium longum: lactobacillus fermentum: lactobacillus acidophilus ═ 1:1:1, mixing each probiotic in MRS broth culture medium added with traditional Chinese medicines, wherein the initial CFU/mL is 1-2 x 10⁶Then, multiplying and fermenting the traditional Chinese medicine, and finally, adding 3-5 x 10 of total CFU/mL in the fermentation liquor⁹。

Example 1

Taking Bifidobacterium longum (Bifidobacterium longum), Lactobacillus fermentum (Lactobacillus fermentum) and Lactobacillus acidophilus (Lactobacillus acidophilus) bacterial liquid from MRS broth culture medium, adding 0.5g/mL Raphani semen and 66.4mg/mL Ganoderma MRS broth culture medium simultaneously, wherein the initial CFU/mL of each probiotic is 2-10⁶Proliferating probiotics and fermenting the traditional Chinese medicine, proliferating and fermenting for 24h in a constant temperature box at 37 ℃, and counting the viable bacteria (CFU/mL) ═ 5.0 x 10⁹。

Example 2

Equal amounts of Bifidobacterium longum (Bifidobacterium longum), Lactobacillus fermentum (Lactobacillus fermentum) and Lactobacillus acidophilus (Lactobacillus acidophilus) are taken from MRS broth culture medium, and 2g/mL of semen Raphani and 16.6mg/mL of Ganoderma MRS broth culture medium are added to the broth culture medium, wherein the initial CFU/mL of each probiotic is 1.5-10⁶Proliferating probiotics and fermenting traditional Chinese medicine, proliferating and fermenting in a thermostat at 37 ℃ for 36h, and counting viable bacteria (CFU/mL) ═ 3.9 × 10⁹。

Embodiment 3

Taking Lactobacillus fermentum (Lactobacillus fermentum) liquid from MRS broth culture medium, adding 1g/mL Raphani semen, 33.2mg/mL Ganoderma MRS broth culture medium, and adding initial CFU/mL to 1 × 10⁶After culturing at 37 ℃ for 12h, a strain of Bifidobacterium longum (Bifidobacterium longum) and Lactobacillus acidophilus (Lactobacillus acidophilus) was added to the MRS broth, and the initial CFU/mL of Bifidobacterium longum and Lactobacillus acidophilus were 1 × 10⁶Continuously performing propagation fermentation for 24h in a constant temperature box at 37 ℃, and counting the viable bacteria (CFU/mL) -4.2 x 10⁹。

The following are experiments on the growth conditions of probiotics and animal experiments on the traditional Chinese medicine probiotic fermentation broth. The animal experiment is a model experiment.

The growth condition of the probiotics in the culture medium added with the traditional Chinese medicine is researched:

the bifidobacterium longum, the lactobacillus acidophilus and the lactobacillus fermentum are independently cultured in an MRS broth culture medium and added into the MRS broth culture medium according to the bacterial quantity of 1:1:1 for mixed culture, viable bacteria count is carried out on the MRS agar culture medium every 12 hours, the growth condition of each bacterium is calculated (shown in table 1), the growth speed of the mixed bacterial liquid and the growth speed of a single probiotic bacterium tend to be consistent, and the 12 h-36 h is a stable period. Preparing MRS broth culture medium added with traditional Chinese medicines according to 1g/mL of semen raphani and 33.2mg/mL of lucid ganoderma concentration, separately culturing bifidobacterium longum, lactobacillus acidophilus and lactobacillus fermentum in the MRS broth culture medium added with traditional Chinese medicines and mixing and culturing the bifidobacterium longum, the lactobacillus acidophilus and the lactobacillus fermentum in the MRS broth culture medium added with traditional Chinese medicines according to the bacterial quantity of 1:1:1, counting live bacteria on an MRS agar plate every 12h, calculating the growth condition of each bacterium (shown in table 2), finding that the time for the bifidobacterium longum and the lactobacillus acidophilus to enter a plateau phase is 24 h-36 h, the time for the lactobacillus fermentum to enter the plateau phase is about 36h, growing longer bifidobacterium and slightly slower lactobacillus acidophilus because the growth speed of the probiotics in the MRS broth culture medium added with traditional Chinese medicines is slower than that the probiotics are fermented in the common MRS broth culture medium during the growth process, in order to explore the influence of the side effects of the fermented components on the chemotherapy, animal model experiments were conducted for further studies by controlling single variables.

Study of animal models of chemotherapy:

adding Raphani semen and Ganoderma into MRS broth culture medium at a concentration of 1g/mL Raphani semen and 33.2mg/mL Ganoderma to obtain Chinese medicinal (GS) group. Adding equal amounts of Bifidobacterium longum (Bifidobacterium longum), Lactobacillus fermentum (Lactobacillus fermentum) and Lactobacillus acidophilus (Lactobacillus acidophilus) into MRS broth, culturing at 37 deg.C for 12 hr, and counting total viable bacteria (CFU/mL) ═ 4.0 ═ 10%⁹And is probiotic (Pro) group. Taking Lactobacillus fermentum (Lactobacillus fermentum) liquid from MRS broth culture medium,adding radish seed 1g/mL, Ganoderma lucidum MRS broth 33.2mg/mL, initial CFU/mL-1 × 10⁶After culturing at 37 ℃ for 12h, a strain of Bifidobacterium longum (Bifidobacterium longum) and Lactobacillus acidophilus (Lactobacillus acidophilus) was added to the MRS broth, and the initial CFU/mL of Bifidobacterium longum and Lactobacillus acidophilus was 1 × 10⁶And continuously performing propagation fermentation for 24 hours in a constant temperature oven at 37 ℃, wherein the viable count CFU/mL is 4.2 x 109, and the strain is a traditional Chinese medicine probiotic fermentation broth (GD + Pro) group (example 3). The method comprises the steps of randomly dividing SPF-level BALB/c mice into five groups, namely a normal group (CON), a Cyclophosphamide (CTX) group and a normal saline group, a Cyclophosphamide (CTX) group and a traditional Chinese medicine (GS) group, a Cyclophosphamide (CTX) group and a probiotic (Pro) group, and a Cyclophosphamide (CTX) group and a traditional Chinese medicine probiotic fermentation liquor (GS + Pro) group. GS group is prepared from Raphani semen 10g/kg, Ganoderma 3.32g/kg, and Pro group is prepared from CFU 10⁹Per mL, GS + Pro group is 10g/kg radish seed, 3.32g/kg glossy ganoderma, CFU 10⁹the/mL continuous gavage is carried out for 7 days, and the CON group and the CTX group are drenched with physiological saline with the same volume.

And 2, injecting cyclophosphamide into the abdominal cavity of the CTX group, the GS group, the Pro group and the GS + Pro group according to 100mg/kg for 3 consecutive days, and injecting equal volume of normal saline into the abdominal cavity of the CON group. While maintaining the gavage. After modeling was completed, 3 mice were sacrificed in each group, and spleen, serum, colon contents and colon were retained for further experiments.

The GS, Pro, and GS + Pro groups were gavaged continuously with the same dose, and the remaining mice were sacrificed 4 days after treatment, and the spleen, serum, colon contents, and colon were collected for further experiments.

The condition of gnawing kaolin recorded in the modeling process (see table 3) is recorded, and the fact that on the 1 st day of modeling, except CON group, other groups are increased, but the gnawing amount of GS group, Pro group and GS + Pro group is obviously lower than that of CTX group, and the pica is less in symptom than that of CTX group.

And (3) taking the colon for H & E staining, and performing pathological scoring (see table 4), wherein the pathological scoring of the colon is lower than that of the CTX group when the modeling is finished and the treatment is finished, the pathological scoring of the colon is lower than that of the GS group when the modeling is finished, the CTX group is different from the GS group, the Pro group and the GS + Pro group when the modeling is finished (P is less than 0.05), and the CTX group is different from the Pro group when the treatment is finished (P is less than 0.05). Spleens were weighed and calculated as spleen index g/body weight g 100% (see table 5), and at the end of treatment, the Pro group and GS + Pro group were found to be different (P < 0.05), especially the GS + Pro group, compared to the CTX group (P < 0.001). After the modeling is finished, the total lymphocytes of the CTX group, the GS group, the Pro group and the GS + Pro group are remarkably reduced, B (CD19+) cells are remarkably increased, and T (CD3+) cells are remarkably increased, and the increase is most remarkable as CD4+ T cells. After treatment is finished, total lymphocytes of the GS group, the Pro group and the GS + Pro group are remarkably recovered, the GS group is different from the CTX group (P is less than 0.05), the GS + Pro group is obviously different from the CTX group (P is less than 0.01), T (CD3+) cells of the GS group, the Pro group and the GS + Pro group are reduced, and the GS + Pro group is obviously different from the T cells, CD4+ T cells and CD8+ T cells of the CTX group (P is less than 0.01).

Table 1: growth of individual probiotics in MRS broth

Table 2: adding 1g/mL radish seed and 33.2mg/mL ganoderma MRS broth culture medium, and controlling the growth of each probiotic:

TABLE 3 animal models to establish values of Kaolin gnaw by mice

Note: the kaolin values are the average values in g of kaolin eaten by each mouse per day per group.

TABLE 4 animal model Experimental Colon pathology scores

Note: compared with the normal group, the ratio of a to P is less than 0.05, the ratio of b to P is less than 0.01, compared with the model group, the ratio of c to P is less than 0.05, and the ratio of d: p is less than 0.01

TABLE 5 animal model experiment spleen index

Note: compared with the normal group, the ratio of a to P is less than 0.05, the ratio of b to P is less than 0.01, and the ratio of c: p is less than 0.001, compared with the model group, d is P less than 0.05, e: p is less than 0.01, f is P less than 0.001

TABLE 6 flow cytometry detection of splenic lymphocytes and their subpopulations at the end of modeling

Note: compared with the normal group, the ratio of a to P is less than 0.05, the ratio of b to P is less than 0.01, compared with the model group, the ratio of c to P is less than 0.05, and the ratio of d: p is less than 0.01. The values in the table are all percentages (%).

TABLE 5 flow cytometry detection of spleen lymphocytes and subpopulations thereof at the end of treatment

Note: compared with the normal group, the ratio of a to P is less than 0.05, the ratio of b to P is less than 0.01, compared with the model group, the ratio of c to P is less than 0.05, and the ratio of d: p is less than 0.01. The values in the table are all percentages (%).