Compound probiotic for preventing and treating colitis, and use thereof

Abstract

Provided in the present application are a compound probiotic for preventing and treating colitis, and the use thereof. The compound probiotic for preventing and treating colitis consists of a Pediococcus acidilactici PA53 strain and a Lactobacillus helveticus LH76 strain. The two probiotics can cooperate with each other, boost each other, and achieve a synergistic effect in terms of preventing and treating colitis, which is specifically exhibited in alleviating the weight loss caused by colitis, reducing the disease activity index, restoring colon length, ameliorating inflammatory factor dysregulation and oxidative stress response caused by colitis, and increasing the content of short-chain fatty acids in the intestines. In a case where the same bacterial dosage is used, the compounding of the two strains exhibits significantly improved efficacy in preventing and treating colitis compared with strain interventions using either strain alone.

Description

A compound probiotic for the prevention and treatment of colitis and its application Technical Field

[0001] This application belongs to the field of microbial technology and relates to a compound probiotic for the prevention and treatment of colitis and its application. Background Technology

[0002] Colitis, especially ulcerative colitis, is a chronic nonspecific inflammatory disease with causes that extend deep into the colonic mucosa and its submucosa. Factors such as immune abnormalities, genetic predisposition, and intestinal flora imbalance play key roles in its pathogenesis.

[0003] Treatment for colitis primarily focuses on managing the condition to reduce the risk of colorectal cancer. Probiotics, as a class of live microorganisms that can play a positive role in the human body, have shown great potential in the treatment of colitis. Probiotics play a crucial role in colitis treatment through a series of complex mechanisms. First, they can competitively inhibit the colonization of pathogenic bacteria on the intestinal epithelium, effectively reducing the damage these pathogens cause to the intestinal mucosa. Second, probiotics can significantly improve the intestinal microecological environment, increasing the number of beneficial bacteria while reducing the proportion of harmful bacteria, thereby effectively alleviating the intestinal flora imbalance problem in colitis patients. Furthermore, probiotics can stimulate the intestinal mucosal immune system, enhance local immunity, and strengthen the body's defense against pathogenic bacteria. Finally, probiotics can also promote the proliferation and repair of intestinal mucosal cells, thereby alleviating the intestinal inflammatory symptoms in colitis patients.

[0004] The application of probiotics in the prevention and treatment of colitis is still in its early stages, and strategies are still limited. Therefore, developing more probiotic agents to improve colitis could provide new ideas and methods for its treatment. Summary of the Invention

[0005] This application provides a compound probiotic for the prevention and treatment of colitis and its application.

[0006] In a first aspect, this application provides a compound probiotic for the prevention and treatment of colitis, wherein the compound probiotic for the prevention and treatment of colitis is composed of Pediococcus acidilactici PA53 strain with accession number CGMCC No.18798 and Lactobacillus helveticus LH76 strain with accession number CGMCC No.18796.

[0007] The Pediococcus acidilactici PA53 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 4, 2019, with accession number CGMCC No. 18798, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The Lactobacillus helveticus LH76 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 4, 2019, with accession number CGMCC No. 18796. The address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] This application creatively develops a novel probiotic combination method and a novel strategy for preventing and treating colitis, namely, combining *Pediococcus acidilactici* PA53 strain and *Lactobacillus helveticus* LH76 strain. It was found that the two strains can cooperate and promote each other, synergistically enhancing their effects in preventing and treating colitis. Specifically, this is manifested in: (1) alleviating weight loss caused by colitis, reducing the disease activity index, and restoring colon length; (2) improving the imbalance of inflammatory factors and oxidative stress caused by colitis; and (3) increasing the content of short-chain fatty acids in the intestines of colitis-affected mice. With consistent bacterial counts, compared with intervention methods lacking either strain, the combination of the two strains significantly improved the efficacy in preventing and treating colitis. Furthermore, both strains are probiotics, ensuring high product safety and minimizing the risk of resistance.

[0010] Preferably, the ratio of viable cells of the PA53 strain to the LH76 strain is 1:5-5:1, such as 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0011] Secondly, this application provides a probiotic agent for preventing and treating colitis, wherein the strain in the probiotic agent is the compound probiotic described in the first aspect.

[0012] Preferably, the viable bacterial content of the PA53 strain and the LH76 strain in the probiotic agent is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL, for example 1×10 9 CFU / g (CFU / mL), 1×10 10 CFU / g (CFU / mL), 5×1010 CFU / g (CFU / mL), 1×10 11 CFU / g (CFU / mL), 3×10 11 CFU / g (CFU / mL), 5×10 11 CFU / g (CFU / mL), 1×10 12 CFU / g (CFU / mL), 1×10 13 CFU / g (CFU / mL), etc., and other specific point values ​​within this range can be selected, which will not be elaborated here.

[0013] Preferably, the dosage form of the probiotic agent includes solutions, lyophilized powders, capsules, tablets, or granules. The dosage form of the probiotic agent involved in this application is not limited, including the most commonly used solutions, lyophilized powders, or further prepared capsules, tablets, or granules.

[0014] Preferably, the probiotic agent is in the form of a solution, which is prepared by the following method:

[0015] PA53 and LH76 strains were inoculated into culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged and resuspended in solvent to obtain PA53 and LH76 bacterial suspensions. The PA53 and LH76 bacterial suspensions were mixed according to the live bacteria ratio to obtain the probiotic agent.

[0016] Preferably, the probiotic agent is in the form of a lyophilized powder, which is prepared by the following method:

[0017] PA53 and LH76 strains were inoculated into culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged, mixed with a protectant, and then freeze-dried to obtain PA53 and LH76 bacterial powders. The PA53 and LH76 bacterial powders were mixed according to the live bacteria ratio to obtain the probiotic agent.

[0018] Thirdly, this application provides the use of the compound probiotics described in the first aspect or the probiotic agents described in the second aspect in the preparation of medicaments for the prevention, improvement or treatment of colitis.

[0019] Preferably, the drug further contains excipients.

[0020] Preferably, the excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0021] Compared with the prior art, this application has the following advantages:

[0022] This application creatively develops a novel probiotic combination method and a novel strategy for preventing and treating colitis, namely, combining *Pediococcus acidilactici* PA53 strain and *Lactobacillus helveticus* LH76 strain. It was found that the two strains can cooperate and promote each other, synergistically enhancing their effects in preventing and treating colitis. Specifically, this is manifested in: (1) alleviating weight loss caused by colitis, reducing the disease activity index, and restoring colon length; (2) improving the imbalance of inflammatory factors and oxidative stress caused by colitis; and (3) increasing the content of short-chain fatty acids in the intestines of colitis-affected mice. With consistent bacterial counts, compared with intervention methods lacking either strain, the combination of the two strains significantly improved the efficacy in preventing and treating colitis. Furthermore, both strains are probiotics, ensuring high product safety and minimizing the risk of resistance. Attached Figure Description

[0023] Figure 1 shows the changes in body weight of mice in each group during the modeling period.

[0024] Figure 2 shows the statistical results of colon length in each group of mice after the experiment.

[0025] Figure 3 shows the statistical results of interleukin-6 levels in the colon tissue of mice in each group.

[0026] Figure 4 shows the statistical results of interleukin-10 levels in the colon tissue of mice in each group.

[0027] Figure 5 shows the statistical results of interleukin-1β levels in the colon tissue of mice in each group.

[0028] Figure 6 shows the statistical results of tumor necrosis factor-α levels in the colon tissue of mice in each group.

[0029] Figure 7 shows the statistical results of myeloperoxidase activity in the colon tissue of mice in each group.

[0030] Figure 8 shows the statistical results of superoxide dismutase activity in the colon tissue of mice in each group.

[0031] Figure 9 shows the statistical results of malondialdehyde content in the colon tissue of mice in each group. Detailed Implementation

[0032] The technical solution of this application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely to help understand this application and should not be regarded as specific limitations on this application.

[0033] The culture medium formulations involved in the following examples are as follows:

[0034] MRS medium: peptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 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, Tween 80 1 mL / L, cysteine ​​hydrochloride 0.5 g / L.

[0035] The PA53 strain involved in the following examples is classified as Pediococcus acidilactici, deposited on November 4, 2019, with accession number CGMCC No. 18798.

[0036] The LH76 strain involved in the following examples is classified as Lactobacillus helveticus, deposited on November 4, 2019, with accession number CGMCC No. 18796.

[0037] The following method involves the preparation of bacterial suspension: the desired bacterial strain is inoculated into liquid culture medium and activated by culturing at 37°C for 24 hours. This activation is repeated twice to obtain an activated solution. The activated solution is inoculated into liquid culture medium at an inoculation rate of 5% (v / v) and cultured at 37°C for 24 hours to obtain a bacterial suspension. The bacterial suspension is centrifuged at 5000 rpm for 10 minutes at 4°C, filtered, and the bacterial cells are obtained. The bacterial cells are resuspended in PBS solution to obtain the final product.

[0038] The experimental results were statistically analyzed using ggplot2 in R. Compared with the control group, ### represents p<0.001, ## represents p<0.01, and # represents p<0.05; compared with the model group, *** represents p<0.001, ** represents p<0.01, and * represents p<0.05. NS represents no significant difference.

[0039] Example

[0040] This embodiment investigates the ability of compound probiotics to improve symptoms in a mouse model of colitis:

[0041] (1) Experimental animals: Healthy male C57BL / 6 mice, 7 weeks old (64 mice), were raised in a controlled environment with room temperature maintained at 22-24℃ and humidity at 50-60%, following a 12h light / dark cycle. They were allowed to eat and drink freely.

[0042] (2) Animal grouping: After acclimatization feeding for 1 week, mice were randomly divided into 8 groups of 8 mice each: control group, model group, PA53 group (intervention with PA53 bacterial solution), LH76 group (intervention with LH76 bacterial solution), PA53+LH76 group 1 (intervention with PA53 and LH76 bacterial solutions in combination, with a live bacteria ratio of 1:1), PA53+LH76 group 2 (intervention with PA53 and LH76 bacterial solutions in combination, with a live bacteria ratio of 5:1), PA53+LH76 group 3 (intervention with PA53 and LH76 bacterial solutions in combination, with a live bacteria ratio of 1:5), and ATCC8042+ATCC15009 group (intervention with commercially available Pediococcus lactis ATCC8042 and commercially available Lactobacillus helveticus ATCC15009 bacterial solutions in combination, with a live bacteria ratio of 1:1).

[0043] (3) Animal modeling and intervention methods:

[0044] Throughout the experiment, mice in each probiotic intervention group were fed 0.2 mL daily of a 12% skim milk powder solution containing probiotics (1 × 10⁻⁶). 9 Mice in the control and model groups were fed 0.2 mL of 12% skim milk powder solution daily (CFU / day / mice). Control group mice had free access to water throughout the experiment, while mice in the model and probiotic intervention groups had free access to water for the first 7 days, and then from day 8 to day 14 were fed drinking water containing 3.0% sodium dextran sulfate (DSS) as in the same procedure.

[0045] (4) Indicator Analysis:

[0046] (4.1) Changes in mouse body weight:

[0047] The weight of the mice was measured daily from day 1 to day 7 of the modeling process (i.e., day 8 to day 14 of the experiment). The results are shown in Figure 1. The weight of the mice in the model group showed a significant downward trend. The intervention of each probiotic could delay and reduce the weight loss of the mice to varying degrees. In particular, the combined intervention of PA53 strain and LH76 strain had a more significant effect.

[0048] (4.2) Changes in the Disease Activity Index (DAI) in mice:

[0049] The Disease Activity Index (DAI) score is an important indicator for evaluating colonic injury. The DAI scoring standard is as follows: a comprehensive score is given for three factors: percentage weight loss, stool viscosity, and rectal bleeding. The total score of these three results is then calculated to obtain the DAI value. Specifically, the percentage weight loss is scored as follows: 0 points for no change in weight, 1-5% for a loss of 1 point, 5-10% for a loss of 2 points, 10-15% for a loss of 3 points, and more than 15% for a loss of 4 points; stool viscosity is scored as follows: 0 points for normal, 2 points for loose stool, and 4 points for diarrhea; and rectal bleeding is scored as follows: 0 points for normal, 2 points for positive occult blood, and 4 points for overt bleeding.

[0050] The results are shown in Table 1. Compared with the control group, the DAI score of mice in the model group was significantly higher. However, probiotic intervention in each group significantly reversed this trend, and the combined intervention of PA53 strain and LH76 strain showed a better reversal effect.

[0051] Table 1

[0052] (4.3) Mouse colon length:

[0053] After the experiment, the mice were sacrificed and the colon length of each group was recorded. The results are shown in Figure 2. Compared with the control group, the colon length of the model group mice was significantly shortened. After intervention with probiotics in each group, the shortening of colon length in mice with colitis could be improved, and the effect of restoring colon length in the PA53+LH76 group was closer to that in the control group.

[0054] (4.4) Changes in inflammatory markers in mice:

[0055] Mouse colon tissue was collected, homogenized, and centrifuged. The supernatant was collected, and the levels of colonic inflammatory factors were measured using a commercial kit. The results are shown in Figures 3-6. Compared with the control group, the concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the colon of the model group mice were significantly increased, while the concentration of interleukin-10 (IL-10) was significantly decreased. Probiotic intervention significantly reduced the concentrations of TNF-α, IL-6, and IL-1β in the colon and significantly increased the concentration of IL-10. Furthermore, compared with single probiotic intervention, the probiotic intervention using PA53 and LH76 was more beneficial for balancing colonic inflammatory factors. These results indicate that the compound probiotics involved in this application can promote the secretion of anti-inflammatory cytokines and inhibit the secretion of pro-inflammatory cytokines, thereby improving colitis.

[0056] (4.5) Changes in enzymes related to oxidative stress in mice:

[0057] Mouse colonic tissue was collected, homogenized, and centrifuged. The supernatant was collected, and the activities of colonic oxidative stress-related enzymes were measured using a commercial kit. The results, shown in Figures 7-9, indicate that myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were significantly increased in the colon of mice treated with DSS, suggesting an exacerbation of oxidative stress. However, compared to the model group, probiotic intervention significantly reduced MPO activity and MDA levels in the mouse colon, demonstrating the significant effect of probiotics in alleviating oxidative stress. Further analysis revealed that superoxide dismutase (SOD) activity in the colonic tissue of the model group was significantly lower than that of the control group, indicating that the antioxidant capacity of these mice was suppressed in response to oxidative stress. However, probiotic intervention significantly increased SOD activity in DSS-induced colitis tissue, demonstrating that probiotics can enhance the body's antioxidant capacity. Furthermore, compared to intervention with a single probiotic, the combined use of PA53 and LH76 probiotics showed a more significant effect in regulating oxidative stress.

[0058] (4.6) Changes in short-chain fatty acids:

[0059] Cecal contents were collected from mice in each group, and the concentrations (μmol / g) of various short-chain fatty acids were determined by gas chromatography-mass spectrometry (GC-MS). The results are shown in Table 2. Compared with the control group, the concentrations of acetic acid, propionic acid, butyric acid, and isobutyric acid in the model group were significantly reduced. However, probiotic intervention significantly increased the concentrations of acetic acid, propionic acid, butyric acid, and isobutyric acid in the cecum of colitis mice, especially the combined intervention of PA53 and LH76 strains, which showed the best effect.

[0060] Table 2

[0061] The applicant declares that this application illustrates the technical solution of this application through the above embodiments, but this application is not limited to the above embodiments, that is, it does not mean that this application must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent substitutions of the raw materials of this application's products, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this application.

[0062] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application.

[0063] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

Claims

1. A compound probiotic for the prevention and treatment of colitis, comprising Pediococcus acidilactici PA53 strain with accession number CGMCC No.18798 and Lactobacillus helveticus LH76 strain with accession number CGMCC No.18796.

2. The compound probiotic for preventing and treating colitis according to claim 1, wherein, The ratio of viable cells of the PA53 strain to that of the LH76 strain is 1:5-5:

1.

3. A probiotic agent for preventing and treating colitis, wherein the strain is the compound probiotic described in claim 1 or 2.

4. The probiotic agent according to claim 3, wherein, The viable bacterial content of the PA53 strain and LH76 strain in the probiotic agent is not less than 1×10⁻⁶. 9 CFU / g or 1×10 9 CFU / mL.

5. The probiotic agent according to claim 3, wherein, The dosage forms of the probiotic agent include solutions, lyophilized powders, capsules, tablets, or granules.

6. The probiotic agent according to claim 5, wherein, The probiotic agent is in the form of a solution, which is prepared by the following method: PA53 and LH76 strains were inoculated into culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged and resuspended in solvent to obtain PA53 and LH76 bacterial suspensions. The PA53 and LH76 bacterial suspensions were mixed according to the live bacteria ratio to obtain the probiotic agent.

7. The probiotic agent according to claim 5, wherein, The probiotic agent is in the form of a lyophilized powder, which is prepared by the following method: PA53 and LH76 strains were inoculated into culture medium and activated and fermented sequentially to obtain fermentation broth. The fermentation broth was centrifuged, mixed with a protectant, and then freeze-dried to obtain PA53 and LH76 bacterial powders. The PA53 and LH76 bacterial powders were mixed according to the live bacteria ratio to obtain the probiotic agent.

8. The use of the compound probiotic of claim 1 or 2 or the probiotic agent of any one of claims 3-7 in the preparation of a medicament for the prevention, improvement or treatment of colitis.

9. The application according to claim 8, wherein, The drug also contains excipients.

10. The application according to claim 9, wherein, The excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

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