Microbial composition for regulating intestinal inflammation and preparation method thereof

Abstract

The invention discloses a microbial composition for regulating intestinal inflammation and a preparation method thereof, relates to the technical field of probiotics, and provides a microbial composition which contains lactobacillus delbrueckii subsp. Bulgaricus, lactobacillus plantarum, lactobacillus casei and lactobacillus mucus fermentation and is used for regulating intestinal inflammation. According to the present invention, the intestinal flora environment can be effectively improved, the intestinal digestion and absorption ability can be enhanced, the intestinal inflammation level can be reduced, and the strains used in the present invention are probiotics, such that the product safety is high, the application range is wide, and the preparation method can be used for preparing various products for preventing and improving intestinal inflammation.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, specifically a microbial composition for regulating intestinal inflammation and its preparation method. Background Technology

[0002] The human gut is home to a large number of different types of bacteria, which are key components of the gut microbiota ecosystem. The gut microbiota system is also the largest and most complex microbial metabolic system in the human body. Gut microbiota are closely related to human health, helping to aid digestion and absorption and inhibiting pathogens and toxins. However, with the overuse of antibiotics in modern life, gut microbiota imbalance has become increasingly common in the population. Long-term use of antibiotics can kill beneficial gut bacteria along with pathogens, disrupting the balance of the gut microbiota, causing dysbiosis, and leading to discomfort such as diarrhea, bloating, and abdominal pain.

[0003] Currently, there are two main approaches to regulating and treating gut microbiota imbalance: antibiotic regulation and probiotic regulation. Probiotic preparations are widely used to treat related problems. They improve the balance of the human gut microbiota through beneficial active microorganisms that can colonize the human body and alter the composition of the microbiota in specific parts of the host.

[0004] However, current probiotic supplements have significant shortcomings: the types of bacteria are relatively limited, and even if they can colonize the intestines, they can only increase the number of ingested probiotics, with limited regulatory effects on the overall structure of the gut microbiota. Moreover, these probiotics are affected by stomach acid and bile, resulting in a low survival rate after entering the intestines, ultimately leading to a significant decrease in regulatory effects. Summary of the Invention

[0005] The purpose of this invention is to provide a microbial composition for regulating intestinal inflammation and a method for preparing the same, in order to solve the problems raised in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a microbial composition for regulating intestinal inflammation, having the following technical features: the microbial composition for regulating intestinal inflammation is composed of a compound probiotic agent, a protectant, and natural polysaccharides;

[0007] The compound probiotic agent is composed of Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Lactobacillus plantarum MCA-3066, Lactobacillus casei LPM-0188 and Lactobacillus fermentum LAF-1387.

[0008] The Lactobacillus delbrueckii subsp. bulgaricus RHM-1068 is deposited under the CGMCC No. 24013.

[0009] The Lactobacillus plantarum MCA-3066 has the accession number CGMCC No. 24489;

[0010] The Lactobacillus casei LPM-0188 has the accession number CGMCC No. 23959;

[0011] The Lactobacillus mucinus LAF-1387 culture was deposited under the accession number CGMCC No. 24056.

[0012] Furthermore, the total live bacteria count of the compound probiotic agent is 1×10⁻⁶. 6 ~10 12 CFU / mL.

[0013] Furthermore, in the compound probiotic agent, based on the proportion of live bacteria, the ratio of Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Lactobacillus plantarum MCA-3066, Lactobacillus casei LPM-0188 to Lactobacillus fermentum LAF-1387 is (1.5~2.5):(2.5~3):(1.5~2):(2.5~3).

[0014] Furthermore, the protective agent is a composition of cellulose and sorbitol in a mass ratio of 1:(0.2~0.8).

[0015] Furthermore, the natural polysaccharide is fructooligosaccharide.

[0016] Furthermore, by weight, the microbial composition for treating intestinal inflammation comprises a compound probiotic agent, a protective agent, and a natural polysaccharide in a mass ratio of 10:(1~3):(2~5.5).

[0017] Furthermore, a method for preparing a microbial composition for regulating intestinal inflammation includes the following steps:

[0018] S1. Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Lactobacillus plantarum MCA-3066, Lactobacillus casei LPM-0188 and Lactobacillus fermentum LAF-1387 were inoculated and cultured separately. After the culture was completed, they were mixed in proportion to obtain a compound probiotic agent.

[0019] S2. Weigh the compound probiotic agent, protectant and natural polysaccharide separately, mix them, and stir at 100~300 rpm until they are evenly mixed to obtain a microbial composition for regulating intestinal inflammation.

[0020] Furthermore, the microbial composition for regulating intestinal inflammation can be used to prepare intestinal regulation products.

[0021] Furthermore, the gut health products include pharmaceuticals and beverages.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] This invention provides a microbial composition containing *Lactobacillus delbrueckii* subsp. bulgaricus, *Lactobacillus plantarum*, *Lactobacillus casei*, and *Lactobacillus fermentum* for regulating intestinal inflammation. This composition can effectively improve the intestinal flora environment, enhance intestinal digestive and absorptive capacity, and reduce intestinal inflammation levels.

[0024] The *Lactobacillus delbrueckii* subsp. bulgaricus strain, as a probiotic, can ferment digestive products in the human gut, producing large amounts of lactic acid and short-chain fatty acids. This lowers the intestinal pH, inhibiting the proliferation of pathogenic bacteria such as *Escherichia coli*, reducing inflammation induced by pathogenic bacteria, and maintaining intestinal flora balance. Furthermore, *Lactobacillus delbrueckii* subsp. bulgaricus has high gastric acid tolerance, which helps other probiotics colonize the gut, providing a stable growth environment for them. To further enhance the gastric acid tolerance of the probiotics, this invention also uses a protectant and natural polysaccharides added to the composition. The cellulose and natural polysaccharides can coat the probiotics in gastric acid, reducing the impact of gastric acid on the probiotics and improving their colonization efficiency in the gut.

[0025] The plant lactobacillus contained in this invention can secrete substances such as bacteriocins and organic acids, which can directly inhibit the growth of pathogenic bacteria such as Helicobacter pylori and Shigella, while reducing the excessive proliferation of conditionally pathogenic bacteria in the intestine. The casei bacillus and fermentative mucoid lactobacillus can further enhance the intestinal immune level, activate immune signaling pathways, thereby promoting the secretion of anti-inflammatory molecules and inhibiting inflammation.

[0026] Furthermore, the strains used in this invention are all probiotics, ensuring high product safety and a wide range of applications. They can be used in the preparation of various products for preventing and improving intestinal inflammation. Detailed Implementation

[0027] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1. Identification of the bacterial strain;

[0029] The *Lactobacillus delbrueckii* subsp. bulgaricus RHM-1068 described in this invention was identified as *Lactobacillus delbrueckii* subsp. bulgaricus, belonging to the Lactobacillus group, based on colony characteristics and 16S rDNA sequence. The *Lactobacillus delbrueckii* subsp. bulgaricus described in this invention is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 1, 2021. Its classification and naming are: *Lactobacillus delbrueckii* subsp. bulgaricus. The 16S rDNA sequence of RHM-1068 is shown below:

[0030]

[0031] The remaining bacterial strains in this invention, including *Lactobacillus plantarum* MCA-3066, are deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number CGMCC No. 24489; deposit date: March 9, 2022; and classification name: *Lactobacillus plantarum*.

[0032] The Lactobacillus casei LPM-0188 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 23959; deposit date: November 24, 2021; classification and name: Lactobacillus casei.

[0033] The Lactobacillus fermentum LAF-1387 described above is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession number: CGMCC No. 24056; deposit date: December 8, 2021; classification and name: Lactobacillus fermentum.

[0034] Example 2. Preparation of compound probiotic agent A;

[0035] Lactobacillus delbrueckii subsp. bulgaricus RHM-1068 was inoculated into MRS medium and cultured at 37°C for 18 hours. This process was repeated three times for activation. The activated strain was then inoculated into fresh MRS medium at a concentration of 2.5% and cultured at 37°C until the logarithmic growth phase, yielding a bacterial concentration of 1×10⁻⁶. 10 CFU / mL of Lactobacillus delbrueckii subsp. bulgaricus RHM-1068 bacterial suspension;

[0036] Following the above method, the bacterial concentration was sequentially cultured to obtain a concentration of 1×10⁻⁶. 10 After CFU / mL of Lactobacillus plantarum MCA-3066, Lactobacillus casei LPM-0188, and Lactobacillus fermentum LAF-1387 bacterial suspensions;

[0037] The four bacterial solutions were calculated according to the proportion of live bacteria. The compound probiotic agent A was obtained by mixing Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Lactobacillus plantarum MCA-3066, Lactobacillus casei LPM-0188 and Lactobacillus fermentum LAF-1387 in a ratio of 1.5:3:1.5:3.

[0038] Example 3. Preparation of compound probiotic agent B;

[0039] Compared with Example 2, this example increases the amount of Lactobacillus delbrueckii subsp. bulgaricus RHM-1068 bacterial culture added;

[0040] The four bacterial solutions were calculated according to the proportion of live bacteria. The compound probiotic agent B was obtained by mixing the following bacterial solutions: Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Lactobacillus plantarum MCA-3066, Lactobacillus casei LPM-0188 and Lactobacillus fermentum LAF-1387 in a ratio of 2.5:3:1.5:3.

[0041] Example 4. Preparation of a microbial composition for regulating intestinal inflammation;

[0042] Weigh 10 parts of compound probiotic agent A, 1 part of protectant and 2 parts of natural polysaccharide by weight, mix them and stir at 300 rpm until they are evenly mixed to obtain a microbial composition for regulating intestinal inflammation.

[0043] The protective agent is a mixture of cellulose and sorbitol in a mass ratio of 1:0.2, and the natural polysaccharide is fructooligosaccharide.

[0044] Example 5. Preparation of a microbial composition for regulating intestinal inflammation;

[0045] Weigh out 10 parts of compound probiotic agent B, 1 part of protectant and 2 parts of natural polysaccharide by weight, mix them and stir at 300 rpm until they are evenly mixed to obtain a microbial composition for regulating intestinal inflammation.

[0046] The protective agent is a mixture of cellulose and sorbitol in a mass ratio of 1:0.2, and the natural polysaccharide is fructooligosaccharide.

[0047] Example 6. Preparation of a microbial composition for regulating intestinal inflammation;

[0048] Compared with Example 4, this example increases the amount of protective agent added;

[0049] Weigh out 10 parts of compound probiotic agent A, 3 parts of protectant and 2 parts of natural polysaccharide by weight, mix them and stir at 300 rpm until they are evenly mixed to obtain a microbial composition for regulating intestinal inflammation.

[0050] The protective agent is a mixture of cellulose and sorbitol in a mass ratio of 1:0.2, and the natural polysaccharide is fructooligosaccharide.

[0051] Example 7. Preparation of a microbial composition for regulating intestinal inflammation;

[0052] Compared with Example 5, this example increases the amount of protective agent added;

[0053] Weigh out 10 parts of compound probiotic agent B, 3 parts of protectant and 2 parts of natural polysaccharide by weight, mix them and stir at 300 rpm until they are evenly mixed to obtain a microbial composition for regulating intestinal inflammation.

[0054] The protective agent is a mixture of cellulose and sorbitol in a mass ratio of 1:0.2, and the natural polysaccharide is fructooligosaccharide.

[0055] Comparative Example 1. Preparation of compound probiotic agent C;

[0056] The bacterial solution was calculated according to the proportion of live bacteria, and a compound probiotic agent C was obtained by mixing Lactobacillus plantarum MCA-3066, Lactobacillus casei LPM-0188 and Lactobacillus fermentum LAF-1387 in a ratio of 3:1.5:3.

[0057] Comparative Example 2. Preparation of a microbial composition for regulating intestinal inflammation;

[0058] Weigh 10 parts of compound probiotic agent C, 1 part of protectant and 2 parts of natural polysaccharide by weight, mix them and stir at 300 rpm until they are evenly mixed to obtain microbial composition A for regulating intestinal inflammation.

[0059] The protective agent is a mixture of cellulose and sorbitol in a mass ratio of 1:0.2, and the natural polysaccharide is fructooligosaccharide.

[0060] Tests: The experimental animals were male SD mice, 20 mice per group, with a single mouse weight of 150±20g. The experimental sample was prepared according to the ratio of microbial composition for regulating intestinal inflammation to formula milk powder at a mass ratio of 5:95. During feeding, 10g of experimental sample was mixed with 75g of warm water and administered by gavage at a ratio of 20mL / kg for 20 days. After 20 days, the mice were tested.

[0061] Another control group was set up, which was fed pure formula milk powder without the addition of the microbial composition used to regulate intestinal inflammation;

[0062] The test results are shown in Table 1 below;

[0063] Table 1.

[0064]

[0065] As shown in Table 1 above, the data from Examples 4 and 5 and Comparative Example 2 indicate that increasing the amount of *Lactobacillus delbrueckii* subsp. bulgaricus RHM-1068 further improved the food utilization rate in mice. This demonstrates that *Lactobacillus delbrueckii* subsp. bulgaricus RHM-1068 has a significant advantage in maintaining intestinal flora balance. *Lactobacillus delbrueckii* subsp. bulgaricus can ferment digestive products in the intestine, producing large amounts of lactic acid and short-chain fatty acids, thereby lowering the intestinal pH and inhibiting the proliferation of pathogenic bacteria such as *Escherichia coli*, reducing inflammation induced by pathogenic bacteria, and thus maintaining intestinal flora balance. Furthermore, *Lactobacillus delbrueckii* subsp. bulgaricus has high gastric acid tolerance, which can assist other probiotics in successfully colonizing the intestine, thus providing a stable growth environment for other bacterial species. Examples 4 and 6, and Examples 5 and 7 show that adding a protectant further improved the success rate of probiotic colonization in the intestine.

[0066] Another experiment was set up, and mice were fed according to the experimental pattern of Example 5 and pure formula milk powder. After the mice were sacrificed on the 7th day, some colon RNA was extracted and the expression of TNF-α and IL-1β was detected by RT-qPCR. The detection results are shown in Table 2 below.

[0067] Table 2.

[0068]

[0069] As can be seen from the table above, in Example 5, which used the probiotic composition, the levels of inflammatory factors, TNF-α and IL-1β, were all lower than those in the pure milk powder control group. This means that the microbial composition prepared in this invention has a significant inhibitory effect on intestinal inflammation.

[0070] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microbial composition for modulating inflammation in the gut, characterized in that: The microbial composition for regulating intestinal inflammation is composed of a composite probiotic agent, a protective agent, and a natural polysaccharide; The composite probiotic agent is composed of Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Lactobacillus plantarum MCA-3066, Lactobacillus casei LPM-0188, and Lactobacillus muci-nosus fer-mentum LAF-1387; The Lactobacillus delbrueckii subsp. bulgaricus RHM-1068 has a preservation number of CGMCC No. 24013; The Lactobacillus plantarum MCA-3066 has a preservation number of CGMCC No. 24489; The Lactobacillus casei LPM-0188 has a preservation number of CGMCC No. 23959; The Lactobacillus muci-nosus fer-mentum LAF-1387 has a preservation number of CGMCC No. 24056.

2. The microbial composition for use in the regulation of intestinal inflammation according to claim 1, characterized in that: The total viable count of the complex probiotic agent is 1 x 10 6 ~10 12 CFU / mL.

3. The microbial composition for use in the regulation of intestinal inflammation according to claim 1, characterized in that: In the composite probiotic agent, the quantity ratio of the Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, the Lactobacillus plantarum MCA-3066, the Lactobacillus casei LPM-0188, and the Lactobacillus muci-nosus fer-mentum LAF-1387 is (1.5-2.5):(2.5-3):(1.5-2):(2.5-3) according to the proportion of viable bacteria.

4. The microbial composition for use in the regulation of intestinal inflammation according to claim 1, characterized in that: The protective agent is a composition of cellulose and sorbitol in a mass ratio of 1:(0.2-0.8).

5. The microbial composition for use in the regulation of intestinal inflammation according to claim 1, characterized in that: The natural polysaccharide is polyfructose.

6. The microbial composition for use in the regulation of intestinal inflammation according to claim 1, characterized in that: In the microbial composition for regulating intestinal inflammation, the mass ratio of the composite probiotic agent, the protective agent, and the natural polysaccharide is 10:(1-3):(2-5.5) by weight.

7. A method for preparing the microbial composition for regulating intestinal inflammation according to any one of claims 1 to 6, characterized by, The method comprises the following steps: S1. Lactobacillus delbrueckii subsp. bulgaricus RHM-1068, Lactobacillus plantarum MCA-3066, Lactobacillus casei LPM-0188, and Lactobacillus muci-nosus fer-mentum LAF-1387 are inoculated and cultured respectively, and after the culture is completed, they are mixed in proportion to obtain a composite probiotic agent; S2. The composite probiotic agent, the protective agent, and the natural polysaccharide are weighed respectively, mixed, and then stirred uniformly at a rotation speed of 100-300 rpm to obtain the microbial composition for regulating intestinal inflammation.

8. Use of the microbial composition for regulating intestinal inflammation according to any one of claims 1-6 in the preparation of an intestinal regulation product.

9. Use according to claim 8, characterized in that, The intestinal regulation product includes a medicament and a beverage.

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