Composite probiotic for relieving constipation, and use thereof

Abstract

The present application provides a composite probiotic for relieving constipation, and a use thereof. The composite probiotic for relieving constipation is composed of a Bifidobacterium animalis subsp. lactis Bla36 strain with a preservation number of CGMCC No. 24029, a Lactobacillus rhamnosus LRa05 strain with a preservation number of CGMCC No. 24377, a Bacillus coagulans BC99 strain with a preservation number of CGMCC No. 19487, and a Bifidobacterium bifidum Bbi32 strain with a preservation number of CGMCC No. 16923. The four strains have a synergistic effect in relieving constipation symptoms.

Description

Compound probiotic for improving constipation and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotics, and relates to a compound probiotic for improving constipation and application thereof. BACKGROUND

[0002] Constipation is a common health problem that can be caused by a variety of factors, including improper diet, life stress, drug side effects, etc. Constipation problems can bring people discomfort and pain, seriously affecting people's quality of life, and can also cause other health problems, such as hemorrhoids, intestinal diseases, etc. Constipation is usually caused by slow intestinal peristalsis or water deficiency, and is characterized by persistent difficulty in defecation, low frequency, dry stool or incomplete defecation, frequent or chronic constipation, which can induce metabolic disorders and other related diseases, such as ischemic stroke, irritable bowel syndrome and coronary heart disease, with higher risk in elderly patients.

[0003] Probiotics are live microorganisms that are beneficial to the health of the host when given in sufficient amounts. Probiotics can relieve constipation through a variety of mechanisms, including regulating intestinal flora and fermentation, regulating the nervous system and the immune system. Constipation often leads to a disturbance of the intestinal microecology, resulting in an increase in the number of harmful bacteria and a decrease in the number of probiotics. Probiotics have the functions of maintaining intestinal flora balance, promoting intestinal peristalsis and improving constipation. Moreover, probiotics can also digest soluble dietary fiber in the human colon, thereby producing short-chain fatty acids, further promoting intestinal peristalsis, and helping to solve the problem of constipation. Therefore, by taking probiotics, it is helpful to restore the dominant position of probiotics in the intestine, so that the above two mechanisms can be effectively exerted.

[0004] Probiotics can also improve a series of problems caused by constipation, such as bloating, diarrhea, loss of appetite, intestinal indigestion, etc. Studies have shown that there are significant differences in the composition of intestinal flora between constipation patients and non-constipation patients. The composition of fecal microorganisms has been shown to be related to the colon ventilation time, while the composition of colon mucosa microorganisms has a direct relationship with constipation symptoms. Experiments have shown that after supplementing probiotics, the intestinal microbial community has undergone significant changes. This indicates that the use of probiotics may have an impact on the composition of certain microbial communities. Therefore, it is crucial to develop a probiotic product that can effectively prevent, alleviate or treat constipation. SUMMARY

[0005] The present application provides a compound probiotic for improving constipation and application thereof.

[0006] In a first aspect, the present application provides a composite probiotic for improving constipation, which is composed of Bifidobacterium animalis subsp. lactis BLa36 strain with a preservation number of CGMCC No. 24029, Lactobacillus rhamnosus LRa05 strain with a preservation number of CGMCC No. 24377, Bacillus coagulans BC99 strain with a preservation number of CGMCC No. 19487, and Bifidobacterium bifidum BBi32 strain with a preservation number of CGMCC No. 16923.

[0007] The present application develops a brand-new probiotic compounding method and a brand-new strategy for improving constipation, i.e. compounding Bifidobacterium animalis subsp. lactis BLa36 strain, Lactobacillus rhamnosus LRa05 strain, Bacillus coagulans BC99 strain and Bifidobacterium bifidum BBi32 strain, and finds that there is a potential interaction among the four strains, which can cooperate with each other and synergistically improve the effect of improving intestinal peristalsis and relieving constipation symptoms compared with the intervention method of missing any one of the strains, when the amount of the bacteria used is consistent. Specifically, (1) the levels of gastrointestinal peptides such as motilin, substance P and vasoactive intestinal peptide are significantly improved; (2) the intestinal propulsion rate is significantly improved; (3) the content level of short-chain fatty acids in the gastrointestinal tract is significantly improved. Therefore, the composite probiotic has a good prospect for use in preparing a drug for preventing, relieving or treating constipation. Meanwhile, the four strains are all probiotics, so that they are safe and less likely to cause dependence when used for preparing related efficacy products.

[0008] The preparation method of the composite probiotic can be achieved by using conventional technical methods in the art, which can be exemplarily as follows: after the four strains are activated, they are inoculated into culture media respectively for culture to obtain culture liquids; the culture liquids are centrifuged, and the bacterial bodies are resuspended to obtain bacterial suspensions; the four bacterial suspensions are mixed according to the proportion of viable bacterial count requirements, and then the composite probiotic is obtained. Alternatively, a protective agent is further added for freeze-drying to prepare a freeze-dried bacterial powder product.

[0009] Preferably, the culture medium comprises MRS culture medium.

[0010] Preferably, the MRS culture medium comprises, in terms of concentration, peptone 8-12 g / L, beef extract 8-12 g / L, glucose 15-25 g / L, sodium acetate 1-3 g / L, yeast powder 3-7 g / L, diammonium hydrogen citrate 1-3 g / L, K2PO4·3H2O 2-3 g / L, MgSO4·7H2O 0.05-0.2 g / L, MnSO4 0.01-0.1 g / L, Tween 80 0.5-2 mL / L, cysteine hydrochloride 0.1-1 g / L.

[0011] Preferably, the ratio of viable cell numbers of the BLa36 strain, the LRa05 strain, the BC99 strain and the BBi32 strain is (1-10):(1-10):(1-10):(1-10);

[0012] For example, the "1-10" can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, etc., and other specific point values within the numerical range can be selected, which will not be repeated here.

[0013] Based on the potential interaction relationship among the four strains, it is also found that when the four strains are used in combination in the above-mentioned specific viable cell number ratio, the effect of improving intestinal peristalsis and relieving constipation symptoms is more significant.

[0014] In a second aspect, the application provides a probiotic agent for improving constipation, wherein the strains in the probiotic agent comprise the complex probiotic bacteria of the first aspect.

[0015] Preferably, the viable cell content of the BLa36 strain, the LRa05 strain, the BC99 strain and the BBi32 strain in the probiotic agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g, for example, 1×10 8 CFU / mL (CFU / g), 2×10 8 CFU / mL (CFU / g), 5×10 8 CFU / mL (CFU / g), 8×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), etc., and other specific point values within the numerical range can be selected, which will not be repeated here.

[0016] Preferably, the dosage form of the probiotic agent comprises a lyophilized powder, a capsule, a tablet or a granule.

[0017] Preferably, the probiotic agent further comprises a lyophilization protective agent and / or an auxiliary additive.

[0018] Preferably, the lyophilization protective agent comprises any one or a combination of at least two of skimmed milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, acacia gum, sodium alginate, polyvinylpyrrolidone, sorbitol or xylitol.

[0019] Preferably, the auxiliary additive comprises any one or a combination of at least two of fructo-oligosaccharide, xylo-oligosaccharide, galacto-oligosaccharide, isomalto-oligosaccharide, soybean oligosaccharide, lentinan, polydextrose, alpha-lactalbumin or lactoferrin.

[0020] In a third aspect, the present application provides use of the composite probiotic according to the first aspect or the probiotic agent according to the second aspect in the preparation of a medicament for preventing, alleviating or treating constipation.

[0021] In a fourth aspect, the present application provides use of the composite probiotic according to the first aspect or the probiotic agent according to the second aspect in the preparation of a medicament for increasing the level of a gastrointestinal peptide.

[0022] In a fifth aspect, the present application provides use of the composite probiotic according to the first aspect or the probiotic agent according to the second aspect in the preparation of a medicament for increasing the level of a short-chain fatty acid in the gastrointestinal tract.

[0023] Preferably, the medicament further comprises a pharmaceutically acceptable excipient.

[0024] Preferably, the excipient comprises any one or a combination of at least two of a filler, a pH adjuster, an antioxidant, a binder, a wetting agent, a disintegrant, an emulsifier, a co-solvent, a solubilizer, an osmotic pressure regulator, a colorant, a bacteriostatic agent or a buffer.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The present application develops a brand-new probiotic compound method and a brand-new strategy for improving constipation, i.e., compound of Bifidobacterium animalis subsp. lactis BLa36 strain, Lactobacillus rhamnosus LRa05 strain, Bacillus coagulans BC99 strain and Bifidobacterium bifidum BBi32 strain. It is found that there is a potential interaction among the four strains, which can cooperate with each other and synergistically improve the effect of improving intestinal peristalsis and relieving constipation symptoms compared with the intervention method of missing any one strain, when the amount of bacteria used is consistent. Specifically, (1) the levels of gastrointestinal peptides such as motilin, substance P and vasoactive intestinal peptide are significantly improved; (2) the intestinal propulsion rate is significantly improved; (3) the content level of short-chain fatty acids in the gastrointestinal tract is significantly improved. Therefore, the compound probiotic bacteria have a good prospect for preparing drugs for preventing, relieving or treating constipation. At the same time, the four strains are all probiotics, so they are safe and less likely to cause dependence when used to prepare related efficacy products.

[0027] The classification name of the Bifidobacterium animalis subsp. lactis BLa36 strain involved in the present application is Bifidobacterium animalis subsp. lactis, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is December 2, 2021, the preservation number is CGMCC No. 24029, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0028] The classification name of the Lactobacillus rhamnosus LRa05 strain involved in the present application is Lactobacillus rhamnosus, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is January 24, 2022, the preservation number is CGMCC No. 24377, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0029] The classification name of the Bacillus coagulans BC99 strain involved in the present application is Bacillus coagulans, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is March 18, 2020, the preservation number is CGMCC No. 19487, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0030] The classification name of the Bifidobacterium bifidum strain involved in the present application is Bifidobacterium bifidum, the preservation unit is China General Microbiological Culture Collection Center, the preservation time is December 10, 2018, the preservation number is CGMCC No. 16923, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF DRAWINGS

[0031] Fig. 1 is a graph of the weight statistics of each group of mice.

[0032] Fig. 2 is a graph of the fecal water content statistics of each group of mice.

[0033] Fig. 3 is a graph of the intestinal propulsion rate statistics of each group of mice.

[0034] Fig. 4 is a graph of the first black statistics of each group of mice.

[0035] Fig. 5 is a graph of the intestinal acetic acid content statistics of each group of mice.

[0036] Fig. 6 is a graph of the intestinal butyric acid content statistics of each group of mice.

[0037] Fig. 7 is a graph of the serum motilin content statistics of each group of mice.

[0038] Fig. 8 is a graph of the serum P substance content statistics of each group of mice.

[0039] Fig. 9 is a graph of the serum vasoactive intestinal peptide content statistics of each group of mice. DETAILED DESCRIPTION

[0040] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application, and should not be regarded as specific limitations of the present application.

[0041] The classification name of the BLa36 strain involved in the following embodiments is Bifidobacterium animalis subsp. lactis, and the preservation number is CGMCC No. 24029.

[0042] The classification name of the LRa05 strain involved in the following embodiments is Lactobacillus rhamnosus, and the preservation number is CGMCC No. 24377.

[0043] The classification name of the BC99 strain involved in the following embodiments is Bacillus coagulans, and the preservation number is CGMCC No. 19487.

[0044] The classification name of the BBi32 strain involved in the following examples is Bifidobacterium bifidum, and the preservation number is CGMCC No. 16923.

[0045] The medium formula involved in the following examples is as follows:

[0046] Tryptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast powder 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, polysorbate 80 (Tween 80) 1 mL / L, cysteine amino acid salt 0.5 g / L.

[0047] The method for preparing the bacterial suspension involved in the following examples is as follows: inoculate the desired strain into liquid medium, cultivate at 37°C for 24 h for activation, continuously activate for 2 times to obtain an activation liquid; inoculate the activation liquid into liquid medium at an inoculation amount of 5% (v / v), cultivate at 37°C for 24 h to obtain a bacterial liquid; centrifuge the bacterial liquid at 4°C at 5000 rpm for 10 min, filter to obtain bacterial bodies, resuspend the bacterial bodies with PBS solution, and obtain the bacterial suspension.

[0048] The test result data is statistically analyzed using the ggplot2 of R language. Compared with the control group, ### represents p<0.001, ## represents p<0.01; compared with the model group, *** represents p<0.001, ** represents p<0.01, * represents p<0.05, and ns represents no significant difference.

[0049] Example

[0050] This example explores the effect of the composite probiotic bacteria involved in the present application on improving the constipation symptoms of mice:

[0051] (1) Test animals

[0052] Healthy female SPF Kunming mice were provided by Shanghai Experimental Animal Center, and the mice were given conventional granular rodent diet and pure water. All procedures involving mice comply with the guidelines provided by Shanghai Experimental Animal Care and Animal Experiment Center (license number 2023052101).

[0053] (2) Animal grouping, modeling and intervention method

[0054] 54 mice were randomly divided into 9 groups after adaptive feeding for 2 weeks, which were control group (CTL), model group (MG), test group 1 (BLa36+LRa05+BC99, the ratio of the number of viable bacteria of two bacteria was 5:1:1), test group 2 (BLa36+LRa05+BBi32, the ratio of the number of viable bacteria of two bacteria was 5:1:1), test group 3 (BLa36+BC99+BBi32, the ratio of the number of viable bacteria of two bacteria was 5:1:1), test group 4 (LRa05+BC99+BBi32, the ratio of the number of viable bacteria of two bacteria was 1:1:1), test group 5 (BLa36+LRa05+BC99+BBi32, the ratio of the number of viable bacteria of two bacteria was 5:1:1:1), test group 6 (animal bifidobacterium lactis ATCC700541+LRa05+BC99+BBi32, the ratio of the number of viable bacteria of two bacteria was 5:1:1:1), test group 7 (BLa36+LRa05+BC99+ Bacillus coagulans ATCC7050, the ratio of the number of viable bacteria of two bacteria was 5:1:1:1), and the total amount of bacteria liquid of each group of test groups 1-9 was 1×10 9 CFU / day / mouse.

[0055] Except for the control group, other groups were orally administered with loperamide (10 mg / kg) once a day for 14 consecutive days to induce and establish a constipation mouse model; the compound probiotic bacterial suspension was suspended in water, and test groups 1-9 were administered with the suspension once a day from the 15th day to the 28th day, and the control group and the model group were administered with normal saline at 10 mL / kg.

[0056] (3) Analysis of the body weight of mice

[0057] During the test, the body weight of mice was monitored every 7 days, and the statistical results of the body weight of mice in each group after the test were shown in FIG. 1. As shown in the figure, the body weight of mice in the model group increased slowly, and the body weight of mice in test group 5 (BLa36+LRa05+BC99+BBi32) increased significantly after the intervention of probiotics, which indicated that the compound probiotics involved in the present application could promote the growth of mice.

[0058] (4) Analysis of the water content in feces of mice

[0059] During the test, the feces of mice were collected. After collection, the wet weight (A) of feces was recorded, and the dry weight (B) of feces was recorded after drying in a drying oven for 3 h. The water content was calculated as follows: water content in feces (%) = (A-B) / A×100%. The results were shown in FIG. 2.

[0060] As can be seen from the figure, the fecal water content of the control group mice is always stable at about 68%, the fecal water content of the model group mice decreases rapidly compared with the control group, and then is stable at about 53.5%, after two weeks of probiotic intervention, the fecal water content of the mice in the test group increases to different degrees compared with the model group mice, and the effect of test group 5 (BLa36+LRa05+BC99+BBi32) is the best, which shows that the composite probiotic bacteria involved in the present application can restore the function of the intestinal tract.

[0061] (5) Small intestine propulsion rate analysis of mice

[0062] The intestinal transport rate (intestinal propulsion rate) is calculated as follows: intestinal transport rate (%) = C / D x 100% (C: length between pyloric sphincter and charcoal-stained intestinal end; D: full length of intestine (distance from pylorus to rectum). The results are shown in Figure 3.

[0063] As can be seen from Figure 3, the intestinal propulsion rate of the model group mice is significantly lower than that of the control group (p<0.01), while the intestinal propulsion rate of the mice after probiotic intervention is higher than that of the model group (p<0.01), and the effect of test group 5 (BLa36+LRa05+BC99+BBi32) is the best, close to that of the control group, which further proves that the composite probiotic bacteria preparation helps to restore the function of the intestinal tract.

[0064] (6) Analysis of the first black stool time of mice

[0065] During the test period, the color of the feces of each group of mice was observed, and the time of the first black stool was recorded. The time of the first black stool was obtained by subtracting the gavage time from the time of the first black stool, and the results are shown in Figure 4.

[0066] The time of black feces passing through the entire intestinal tract of the model group of constipated mice is significantly increased compared with the control group of normal mice (p<0.01), after probiotic intervention, the time of black feces of mice is significantly reduced (p<0.01), and the improvement effect of test group 5 (BLa36+LRa05+BC99+BBi32) is the best, which further shows that the composite probiotic bacteria have obvious effect on restoring the function of the intestinal tract, and can be applied in the preparation of drugs for preventing, relieving or treating constipation.

[0067] (7) Analysis of the content level of short-chain fatty acids in the feces of mice

[0068] During the experiment, the feces of mice in each group were collected at 0thweek and 4thweek. After vacuum freeze-drying, 25 mg of fecal sample was soaked in 500 μL of saturated NaCl solution for 30 min, vortexed and mixed, 20 μL of 10% H2SO4 solution was added for acidification, then 800 μL of anhydrous ether was added for extraction, centrifuged at 18000g for 15 min, the upper ether phase was taken and 250 mg of anhydrous Na2SO4 was added for drying, centrifuged at 18000g for 15 min, and the upper ether phase was taken as the mixed solution of short-chain fatty acids. The contents of short-chain fatty acids (acetic acid and butyric acid) in feces were analyzed by external standard method using GC-MS. The results are shown in Figures 5 and 6.

[0069] As can be seen from Figures 5 and 6, compared with the control group, the contents of acetic acid and butyric acid in the feces of constipated mice in the model group were significantly reduced, while the contents of acetic acid and butyric acid in the feces of mice in the probiotic intervention group were increased to different degrees compared with the model group, and the effect of test group 5 (BLa36+LRa05+BC99+BBi32) was the best, indicating that the compound probiotic bacteria can relieve constipation symptoms and regulate intestinal health by increasing the level of short-chain fatty acids.

[0070] (8) Analysis of the levels of gastrointestinal peptides in the serum of mice

[0071] The concentrations of motilin (MTL), substance P (SP) and vasoactive intestinal peptide (VIP) in the serum of mice in each group were determined by ELISA kit, and the detection results are shown in Figures 7, 8 and 9, respectively.

[0072] The results showed that compared with the normal mice in the control group, the levels of motilin, substance P and vasoactive intestinal peptide in the serum of constipated mice in the model group were significantly reduced, while the levels of motilin, substance P and vasoactive intestinal peptide in the serum of mice in the probiotic intervention group were increased to different degrees compared with the constipated mice in the model group, and the effect of test group 5 (BLa36+LRa05+BC99+BBi32) was the best, close to the normal value, proving that the compound probiotic bacteria can restore normal intestinal peristalsis and relieve constipation symptoms by regulating the level of gastrointestinal peptide hormones.

[0073] In summary, the compound probiotic bacteria involved in the present application have excellent effect of improving constipation symptoms, and the BLa36 strain, LRa05 strain, BC99 strain and BBi32 strain can cooperate and promote each other, and have synergistic effect on improving intestinal peristalsis and relieving constipation symptoms.

[0074] The applicant declares that the technical solutions of the present application are illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, the present application does not mean that it must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. fall within the protection scope and disclosure scope of the present application.

[0075] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

[0076] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable means without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A composite probiotic for improving constipation, which is composed of Bifidobacterium animalis subsp. lactis BLa36 strain with the preservation number of CGMCC No. 24029, Lactobacillus rhamnosus LRa05 strain with the preservation number of CGMCC No. 24377, Bacillus coagulans BC99 strain with the preservation number of CGMCC No. 19487 and Bifidobacterium bifidum BBi32 strain with the preservation number of CGMCC No. 16923.

2. The composite probiotic for improving constipation according to claim 1, wherein, The ratio of viable cell number of the BLa36 strain, the LRa05 strain, the BC99 strain and the BBi32 strain is (1-10) :(1-10) :(1-10) :(1-10). 3.A probiotic agent for improving constipation, wherein the strains include the composite probiotic of claim 1 or 2.

4. The constipation-improving probiotic agent according to claim 3, wherein The viable cell content of the BLa36 strain, the LRa05 strain, the BC99 strain and the BBi32 strain in the probiotic agent is not less than 1 x 10 8 CFU / mL or 1 x 10 8 CFU / g.

5. The constipation-improving probiotic agent according to claim 3, wherein The dosage form of the probiotic agent includes solution, lyophilized powder, capsule, tablet or granule.

6. The constipation-improving probiotic agent according to claim 3, wherein The probiotic agent further includes a lyophilization protective agent and / or an auxiliary additive.

7. The constipation-improving probiotic agent according to claim 6, wherein The lyophilization protective agent includes any one or a combination of at least two of skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, acacia gum, sodium alginate, polyvinylpyrrolidone, sorbitol or xylitol. 8.Use of the composite probiotic of claim 1 or 2 or the probiotic agent of any one of claims 3-7 in the preparation of a medicament for preventing, alleviating or treating constipation. 9.Use of the composite probiotic of claim 1 or 2 or the probiotic agent of any one of claims 3-7 in the preparation of a medicament for increasing the level of gastrointestinal peptides. 10.Use of the composite probiotic of claim 1 or 2 or the probiotic agent of any one of claims 3-7 in the preparation of a medicament for increasing the level of short-chain fatty acid content in the gastrointestinal tract.

Images