CN118370775A - Application of bacillus coagulans BC07 in preparing health care product with digestion promoting/constipation relieving functions or constipation improving medicine - Google Patents

Abstract

The use of Bacillus coagulans BC07 in the preparation of health products with digestive/laxative functions or drugs for improving constipation belongs to the field of microbial technology. The present invention provides the use of Bacillus coagulans BC07 in the preparation of health products with digestive/laxative functions or drugs for improving constipation or inhibitors of Porphyromonas gingivalis/Bacteroides fragilis/Streptococcus pyogenes. Bacillus coagulans BC07 can effectively relieve constipation, improve the small intestinal propulsion rate of constipated mice, reduce the time of the first black stool, significantly increase the water content of feces, reduce the content of inhibitory peptide neurotransmitter VIP, increase the level of excitatory peptide neurotransmitter MTL, significantly increase the level of anti-inflammatory factors (IL-10), reduce the content of pro-inflammatory factors (TNF-α), and increase the relative abundance of beneficial bacterial genera such as Mycobacterium and Lachnospira, and significantly reduce the relative abundance of harmful bacterial genera such as Acinetobacter and Pseudomonas.

Description

Use of Bacillus coagulans BC07 in the preparation of digestion-promoting/laxative health products or constipation-improving drugs

Technical Field

The present invention belongs to the technical field of microorganisms, and in particular relates to the use of Bacillus coagulans BC07 in preparing health products with digestion-promoting/laxative functions or medicines for improving constipation.

Background technique

Indigestion is a common gastrointestinal disease in clinical practice, which is mainly manifested by a group of clinical syndromes with postprandial fullness, early satiety, upper abdominal pain, and burning sensation in the upper abdomen. Excessive mental stress, irregular diet, decreased digestive function in the elderly, slowed peristalsis of the stomach and duodenum, rough, cold, greasy food, and indigestible food may all cause indigestion. At present, the pathogenesis of indigestion is not fully understood, and treatment methods are mostly empirical. There is no method or drug that has a definite effect on all patients, and there is a lack of objective and reliable criteria for judging the effect.

In modern society, due to various environmental, genetic, social and economic reasons, the incidence of constipation is gradually increasing. Long-term constipation will seriously affect normal work and life. At present, most of the clinical use of rhubarb, laxatives, etc. to relieve constipation. Long-term use of constipation drugs may cause abdominal distension, diarrhea and various complications. Based on the pathogenesis of constipation, the study found that the intestinal flora of constipated patients is disordered, suggesting that we can achieve the purpose of relieving constipation by regulating the disordered intestinal flora in constipated patients.

Bacillus coagulans is a lactic acid-producing bacillus with promising probiotic properties.

The Bacillus coagulans BC07 strain is a strain of Bacillus coagulans reported in Chinese invention patent ZL202211141056.X, which can effectively relieve diarrhea and inhibit diarrhea-related pathogens.

However, the development of uses of Bacillus coagulans BC07 is quite limited, and there is an urgent need to develop new uses of Bacillus coagulans BC07 in other aspects in the art.

Summary of the invention

In order to expand more new uses of Bacillus coagulans BC07, the present invention provides uses of Bacillus coagulans BC07 in preparing health products with digestion-promoting/laxative functions or medicines for improving constipation.

The technical solution of the present invention is as follows:

Use of Bacillus coagulans BC07 in preparing health products with digestion-promoting/laxative functions or medicines for improving constipation or inhibitors of Porphyromonas gingivalis/Bacteroides fragilis/Streptococcus pyogenes.

The digestive/laxative functions are selected from: producing proteases or cellulases, tolerating gastric juice/bile salts, increasing stool water content, reducing the time to pass the first black stool, increasing small intestinal propulsion rate, regulating peptide neurotransmitter content, increasing anti-inflammatory factor levels, reducing pro-inflammatory factor levels, and increasing intestinal flora index.

Improvement of constipation is selected from: production of protease or cellulase, tolerance of gastric juice/bile salts, increase of stool water content, reduction of time to first black stool, increase of small intestinal propulsion rate, regulation of peptide neurotransmitters, increase of anti-inflammatory factor levels, reduction of pro-inflammatory factor levels, increase of intestinal flora index, and improvement of intestinal microbial flora diversity.

Modulating peptide neurotransmitters is selected from: reducing the content of inhibitory peptide neurotransmitter VIP and/or increasing the content of excitatory peptide neurotransmitter MTL.

The anti-inflammatory factor is selected from: IL-10.

The pro-inflammatory factor is selected from: TNF-α.

Intestinal flora indexes include: ACE index and Simpson index.

To improve the diversity of intestinal microbial flora, the abundance of Alistipes and Lachnospiraceae (NK4A136) increased, and the abundance of Acinetobacter and Pseudomonas decreased.

The diameter of the inhibition zone of Bacillus coagulans BC07 against Porphyromonas gingivalis is 25 mm.

The diameter of the inhibition zone of Bacillus coagulans BC07 against Bacteroides fragilis was 22 mm;

Preferably, the diameter of the inhibition zone of Bacillus coagulans BC07 against Streptococcus pyogenes is 23 mm.

The beneficial effects of the present invention are as follows:

The present invention has discovered new uses of Bacillus coagulans BC07 strain in promoting digestion, relieving constipation, and improving constipation, and further expanded the antibacterial spectrum of BC07 strain. The present invention has proved through a large number of experiments that BC07 strain has superior protease activity and good cellulase activity, and based on these activities, it can effectively relieve constipation, improve the small intestinal propulsion rate of constipated mice, reduce the time of the first black stool, significantly increase the water content of feces, reduce the content of inhibitory peptide neurotransmitter VIP, increase the level of excitatory peptide neurotransmitter MTL, significantly increase the level of anti-inflammatory factor (IL-10), reduce the content of pro-inflammatory factor (TNF-α), and increase the relative abundance of beneficial bacteria such as Alternaria and Lachnospiraceae_NK4A136 (Lachnospiraceae), and significantly reduce the relative abundance of harmful bacteria such as Acinetobacter and Pseudomonas. At the same time, BC07 strain can effectively inhibit pathogens such as Porphyromonas gingivalis, Bacteroides fragilis, and Streptococcus pyogenes. The deposit information of BC07 strain is as follows:

Deposit number: CCTCC NO: M 2022195;

Taxonomic nomenclature: Bacillus coagulans BC07;

Deposit date: March 4, 2022;

Depository: China Center for Type Culture Collection;

Deposit address: Wuhan University, Wuhan, China.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the results of the test of the resistance of Bacillus coagulans BC07 to artificial gastric juice and bile salt solution in Experimental Example 1 of the present invention.

FIG. 2 is a diagram showing the results of the digestive enzyme test of Bacillus coagulans BC07 of Experimental Example 3 of the present invention; wherein A is a photo of a protease degradation circle, and B is a photo of a cellulase degradation circle.

FIG3 is a bar graph showing the water content of feces of mice in Experimental Example 4 of the present invention.

FIG. 4 is a bar graph showing the time of first black stool in the mouse experiment of Experimental Example 4 of the present invention.

FIG. 5 is a bar graph of the small intestinal propulsion rate of mice in Experimental Example 4 of the present invention.

FIG. 6 is a bar graph of neurotransmitters in mouse serum in Experimental Example 4 of the present invention.

FIG. 7 is a bar graph of mouse serum cellular immune factors in Experimental Example 4 of the present invention.

FIG8 is a bar graph showing the results of α-diversity analysis of the intestinal flora of mice in Experimental Example 4 of the present invention.

FIG. 9 is a graph showing the species abundance analysis results of the mouse experimental intestinal flora in Experimental Example 4 of the present invention.

The meanings of the labels on the abscissas of Figures 3 to 9 are listed as follows: NC refers to the blank control group, MC refers to the model group, BC07 refers to the Bacillus coagulans group, and BC07P refers to the Bacillus coagulans + inulin group.

Detailed ways

The present invention is further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

1. Sources of biological materials

(I) The Bacillus coagulans BC07 ( Bacillus coagulans ) used in Examples 1-4 of the present invention is a strain recorded in Chinese invention patent ZL202211141056.X, and its deposit number is CCTCC NO: M 2022195.

(II) Porphyromonas gingivalis (ATCC-BAA-308), Streptococcus pyogenes (ATCC19615), and Bacteroides fragilis (ATCC25285) used in Experimental Example 2 of the present invention were purchased from Guangdong Provincial Microbiological Culture Collection Center.

(III) The mice used in Experiment 3 of the present invention were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., and the experimental animal welfare ethics review number is: Safety Assessment Center Animal (Fu) No. 202310248.

2. Reagents and consumables

(I) The proportion of the Bacillus coagulans culture medium used in the following experimental examples is: 0.1 g glucose, 5 g yeast extract powder, 10 g peptone, 5 g sodium chloride, add water to make up to 1000 mL, adjust the pH to 7.0, and pack into 500 mL Erlenmeyer flasks, 300 mL per bottle. According to the amount of each bottle, weigh 18 g/L agar powder into the Erlenmeyer flask, plug it with cotton, wrap it with kraft paper, and sterilize it at 121 ℃ for 20 minutes. When the sterilized culture medium cools to 45~50 ℃, pour it into a plate culture medium.

(ii) MRS medium: peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, K ₂ HPO ₄ 2 g, sodium citrate 2 g, Mg SO ₄ ·7H ₂ O 0.2 g, sodium acetate 5.0 g, MnSO ₄ ·4H ₂ O 0.2 g, Tween 80 1 mL, 1000 mL distilled water. Sterilize at 121 ℃ for 20 min.

Protease detection plate: 15 g skim milk powder, 2 g yeast powder, 10 g NaCl, 20 g agar, 1000 mL distilled water, pH 7.0-7.2.

Cellulase detection plate: 10 g sodium carboxymethyl cellulose, 1.5 g potassium dihydrogen phosphate, 2.5 g peptone, 0.5 g yeast powder, 2.5 g disodium hydrogen phosphate, 15 g agar, 1000 mL distilled water, pH 7.0-7.2.

Culture medium for Porphyromonas gingivalis, Streptococcus pyogenes, and Bacteroides fragilis: TSA culture medium was purchased from Qingdao Haibo. Solid culture medium used TSA + 5% defibrinated sheep blood.

Unless otherwise specified, all kinds of reagents and consumables used in the embodiments and experimental examples of the present invention are commercially available, and the relevant experimental steps are common operations in the field and have technical meanings that can be conventionally understood by those skilled in the art.

All embodiments of the present invention provide the use of BC07 in preparing a health product with digestion-promoting/laxative function or a medicine for improving constipation or an inhibitor of Porphyromonas gingivalis/Bacteroides fragilis/Streptococcus pyogenes.

In a specific embodiment, the digestion/laxative function is selected from: producing proteases or cellulases, tolerating gastric juice/bile salts, increasing fecal water content, reducing the time to excrete the first black stool, increasing small intestinal propulsion rate, regulating peptide neurotransmitter content, increasing anti-inflammatory factor levels, reducing pro-inflammatory factor levels, and increasing intestinal flora index.

In other embodiments, improving constipation is selected from: producing proteases or cellulases, tolerating gastric juice/bile salts, increasing stool water content, reducing the time to excrete the first black stool, increasing small intestinal propulsion rate, regulating peptide neurotransmitters, increasing the level of anti-inflammatory factors, reducing the level of pro-inflammatory factors, increasing the intestinal flora index, and increasing the diversity of intestinal microbial flora.

In some embodiments, modulating a peptide neurotransmitter is selected from: decreasing the level of an inhibitory peptide neurotransmitter VIP and/or increasing the level of an excitatory peptide neurotransmitter MTL.

In a specific embodiment, the anti-inflammatory factor is selected from: IL-10.

In other specific embodiments, the pro-inflammatory factor is selected from: TNF-α.

In a preferred embodiment, the intestinal flora index includes: ACE index and Simpson index.

In a further embodiment, improving the diversity of the intestinal microbial flora is selected from: increasing the abundance of Alistipes and Lachnospiraceae ( Lachnospiraceae_NK4A136 ), and decreasing the abundance of Acinetobacter and Pseudomonas .

In a preferred embodiment, the diameter of the inhibition zone of BC07 against Porphyromonas gingivalis is 25 mm.

In some embodiments, the inhibition zone diameter of BC07 against Bacteroides fragilis is 22 mm;

Preferably, the diameter of the inhibition zone of BC07 against Streptococcus pyogenes is 23 mm.

Experimental Example 1: Gastrointestinal fluid tolerance test of Bacillus coagulans BC07

Simulate artificial gastric juice: prepare PBS solution, add 0.3% pepsin, adjust the pH value to 2.5 with 1 mol/L HCl, and filter with a 0.22 μm microporous filter membrane to sterilize after fully dissolving.

Simulated artificial intestinal fluid: prepare PBS solution, add 0.1% trypsin and then add 0.3% ox bile salt, adjust the pH value to 8.0 with 0.1 mol/L NaOH, filter and sterilize with 0.22 μm microporous filter membrane after fully dissolving, and set aside.

Among them, survival rate%=Nt/N0×100%,

Where N0 represents the number of viable bacteria before treatment (CFU/mL), and Nt represents the number of viable bacteria after treatment (CFU/mL).

The experimental results are shown in Figure 1. After BC07 was incubated at 37 °C for 3 h under gastric juice pH 2.5 conditions, the survival rate of live bacteria of Bacillus coagulans BC07 was as high as 67%. After being treated with 0.3% bile salt conditions for 3 h, the survival rate of live bacteria was as high as 99%, indicating that Bacillus coagulans BC07 has a strong ability to pass through the gastrointestinal tract and can maintain a high survival rate in the body, thereby exerting a probiotic effect.

Experimental Example 2: Experiment on the inhibition of pathogens by Bacillus coagulans BC07

Preparation of Bacillus coagulans BC07 bacterial liquid: Bacillus coagulans BC07 was inoculated into liquid MRS culture medium at a certain ratio and cultured at 42°C for 24 h to obtain BC07 bacterial liquid.

Preparation of indicator bacterial suspension: Activate the indicator strains on the corresponding plate culture medium, culture at 37 ℃ for 24 h, and pick the bacterial moss to prepare a bacterial suspension of 10 ⁸ CFU/mL in physiological saline.

Antibacterial experiment: The pathogen solid culture medium containing 1.5% agar was dissolved by heat and cooled to about 50 ℃, mixed with the pathogen suspension in a certain proportion, and the concentration of the bacterial solution was adjusted (10 ⁶ CFU/mL), and then quickly poured into the plate with the Oxford cup placed in advance. After the culture medium was cooled and solidified, the Oxford cup was taken out, and 200 μL of antagonistic bacterial solution (10 ⁸ CFU/mL) was added to each well. 200 μL of sterile MRS liquid culture medium was used as a blank control. After standing for a few minutes, it was placed in a 37 ℃ constant temperature incubator and cultured for 48-72 h. The diameter of the inhibition zone was measured with a vernier caliper. Three replicates were performed for each bacterium. The results are shown in Table 1.

Table 1. Antagonistic ability to pathogens

Pathogen name Diameter of inhibition zone (mm) Porphyromonas gingivalis 25±0.5 Bacteroides fragilis 22±0.4 Streptococcus pyogenes 23±0.3 MRS 0

The results are shown in Table 1. Bacillus coagulans BC07 has a strong inhibitory ability against Porphyromonas gingivalis, and can also inhibit the growth of Bacteroides fragilis and Streptococcus pyogenes. This shows that BC07 has a positive effect in antagonizing pathogenic bacteria and maintaining the health of the body.

Experimental Example 3: Digestive enzyme experiment of Bacillus coagulans BC07

1. Protease Experiment

The BC07 strain was activated as in Experimental Example 2, the bacterial solution concentration was adjusted to 10 ⁹ CFU/mL, 3 μL was spotted on the protease detection plate, and cultured at 42°C for 24 h. The experimental results are shown in Figure 2A. A significant protein degradation zone appeared around the BC07 bacteria, indicating that Bacillus coagulans BC07 can produce protease and has the ability to degrade protein, thereby promoting the body's digestion of protein in food and improving the absorption of small peptides and amino acids.

2. Cellulase production experiment

The BC07 strain was activated as in Experimental Example 2, and the bacterial solution concentration was adjusted to 10 ⁹ CFU/mL. 3 μL was spotted on the cellulose production test plate. After culturing at 42 °C for 24 h, the cellulase production culture plate was stained with 0.1% Congo red solution for 15 min, the stain was discarded and 1 mol/L NaCl solution was added for decolorization 2 to 3 times, and the size of the transparent hydrolysis circle around the bacteria was observed. The results are shown in Figure 2B. An obvious transparent circle appeared around the BC07 bacteria, indicating that Bacillus coagulans BC07 can produce cellulase, which helps to decompose cellulose in the intestines and promote the digestion and absorption of cellulose in dietary ingredients. The water produced by decomposing cellulose has the effect of softening stool and improving constipation.

Table 2. Digestive enzyme test

Pathogen name Transparent ring diameter (mm) Protease degradation ability 20±0.3 Cellulase degradation ability 15±0.5

In summary, as shown in Table 2, Bacillus coagulans BC07 has excellent protease activity, good cellulase activity, and has the potential to promote the digestion and absorption of nutrients in the gastrointestinal tract.

Experimental Example 4: Experiment on improving constipation in mice with Bacillus coagulans BC07 and inulin

1. Preparation of bacterial suspension and materials

Preparation of Bacillus coagulans BC07 bacterial suspension: The activated BC07 was inoculated into a solid culture medium of Bacillus coagulans and cultured at 42°C for 72 h. The bacterial cells were collected to prepare a bacterial suspension of 10 ⁹ CUF/mL and stored in a -80°C refrigerator for later use.

Preparation of Bacillus coagulans BC07 inulin solution: Prepare 2.5% inulin solution and place it in a 4 ℃ refrigerator away from light. When using, add Bacillus coagulans BC07 to make the final concentration of 10 ⁹ CUF/mL.

Preparation of ink: Mix 100 g of gum arabic with 800 mL of water, boil until the solution becomes transparent, add 50 g of activated carbon, boil three times, wait for the solution to cool, add water to make up to 1000 mL, store in a refrigerator at 4 ℃, and shake well before use.

2. Animal Experiment Design

BALB/c male mice (20-22 g) aged 6-8 weeks were housed in an environment with a room temperature of 25 °C±2 °C, a humidity of 50±5%, and a 12-h light-dark cycle. After a 7-day adaptation period, mice were randomly divided into 4 groups, including a blank control group (NC group, gavage with sterile saline), a model group (MC group, gavage with loperamide hydrochloride + sterile saline), a Bacillus coagulans group (BC07 group), and a Bacillus coagulans + inulin group (BC07P). The intervention started on the first day after the end of the adaptation period. The NC group and the MC group were gavaged with saline, the BC07 group was gavaged with Bacillus coagulans BC07 solution, and the BC07P group was gavaged with inulin solution containing BC07, which lasted for 14 days. On days 15-17, loperamide hydrochloride was used to establish the model. The MC group, BC07 group, and BC07P group were gavaged with loperamide hydrochloride at a dose of 10 mg/kg.bw, and the NC group was gavaged with normal saline. 4 hours later, the NC group and MC group were gavaged with normal saline, the BC07 group was gavaged with BC07 bacterial solution, and the BC07P group was gavaged with BC07 inulin solution.

During the experiment, mice in each group were fed with standard feed. On the 18th day, the time of the first black stool excretion of mice was observed and the feces of mice were collected. On the 19th day, eye blood was collected and the mice were killed. Intestinal transit was observed and cecal and colon samples were collected.

3. Index determination

(1) Determination of fecal water content

On day 17, each mouse was transferred to a clean empty cage, and fecal samples were collected for 3 h. The number of fecal particles of each mouse was recorded during the collection period, and the feces were placed in EP tubes to weigh the wet weight and dry weight of the feces to determine the moisture content.

Fecal moisture content (%) = (fecal wet weight - fecal dry weight) / fecal wet weight × 100%

(2) Determination of the time of first black stool

On the 18th day (after fasting for 18 h), mice in the NC group were gavaged with normal saline, and mice in the other groups were gavaged with loperamide hydrochloride. One hour later, mice in the NC and MC groups were gavaged with Chinese ink, mice in the BC07 group were gavaged with Chinese ink containing 10 ⁹ CFU/mL of Bacillus coagulans BC07, and mice in the BC07P group were gavaged with Chinese ink containing 10 ⁹ CFU/mL of Bacillus coagulans and 2.5% inulin. Each mouse was transferred to a clean empty cage and provided with feed and water. The timing started from the gavage with Chinese ink, and the time of the first black stool of each mouse was recorded. The time of the first black stool of the last mouse in the model group was taken as the termination time.

(3) Determination of small intestinal propulsion rate

On the 19th day of gavage (after 18 h of fasting), mice in the NC group were gavaged with normal saline, and mice in other groups were gavaged with loperamide hydrochloride. One hour later, the NC and MC groups were gavaged with ink, the BC07 group was gavaged with ink containing 10 ⁹ CFU/mL of Bacillus coagulans BC07, and the BC07P group was gavaged with ink containing 10 ⁹ CFU/mL of Bacillus coagulans and 2.5% inulin. Mice were killed 20 minutes later, the abdominal cavity was opened, the mesentery was separated, and the intestinal tube from the upper end to the pylorus and the lower end to the ileum and cecum was cut. The small intestine was gently pulled into a straight line, and the length of the intestinal tube was measured as the total length of the small intestine, and the length from the pylorus to the front of the ink was the ink propulsion length. Calculation of small intestinal propulsion rate:

Small intestine propulsion rate (%) = (ink propulsion length (cm))/(total small intestine length (cm)) × 100%

(4) Mouse serum biochemical analysis

ELISA kits were used to detect the levels of gastrointestinal regulatory neurotransmitters related to constipation in mouse serum: mouse motilin (MTL); vasoactive intestinal peptide (VIP); inflammatory factors interleukin IL-10 and TNF-α.

(5) Determination of mouse intestinal flora

The Qubit dsDNA HS detection kit was used to extract microbial genomic DNA from the cecal feces of each group of mice, and the DNA concentration of the samples was detected using the Qubit dsDNA HS detection kit and the Qubit 4.0 fluorometer. The V3 ~ V4 hypervariable regions of all bacteria in the samples were sequenced by 16S rDNA on the Illumina novaseq 6000 sequencing platform, and bioinformatics was analyzed using Biomarker Cloud to determine the characteristics of the intestinal microbiome.

4. Results Analysis

(1) Effects of Bacillus coagulans on fecal water content and intestinal motility in mice

Fecal water content is an important indicator for evaluating fecal quality. Low fecal water content causes dry stool and constipation. The experimental results are shown in Figure 3. Compared with the model group, the fecal water content of the BC07 group and the BC07P group increased significantly, which improved the stool quality and reduced the possibility of constipation.

The time to the first black stool and the small intestinal propulsion rate reflect the motility of the entire intestine. In the intestinal motility inhibition model, as shown in Figures 4 and 5, the time to the first black stool in the model group was significantly prolonged and the small intestinal propulsion rate was significantly reduced compared with the blank group, indicating that the model was successfully established. Compared with the model group, the time to the first black stool in the BC07 group and the BC07P group was significantly reduced (P < 0.05). At the same time, the small intestinal propulsion rate in the BC07 group was higher than that in the model group, and the BC07P group was significantly higher than that in the model group (P < 0.05). This shows that Bacillus coagulans BC07 can promote intestinal motility and accelerate defecation, and the effect of BC07 combined with inulin on improving defecation is more significant.

(2) Mouse serum biochemical analysis

Figure 6 shows the effects of the two intervention groups on serum neurotransmitters in constipated mice. The inhibitory peptide neurotransmitter VIP has the effect of relaxing the gastrointestinal tract and gastrointestinal sphincter, and is an important factor in producing descending inhibition, which leads to slow transmission; the excitatory peptide neurotransmitter MTL affects the transport of water and electrolytes, can promote gastric contraction and small intestinal segmental movement, accelerate intestinal transit time, and increase colon movement. Compared with the constipation model group induced by loperamide, the BC07 group and the BC07P group reduced the content of the inhibitory peptide neurotransmitter VIP and increased the level of the excitatory peptide neurotransmitter MTL, and Bacillus coagulans BC07 showed significance (P < 0.05).

By measuring the levels of pro-inflammatory factors (TNF-α) and anti-inflammatory factors (IL-10) in the serum of constipated mice, the effect of BC07 on the levels of inflammatory factors in the serum of constipated mice was quantitatively studied. As shown in Figure 7, the anti-inflammatory factor (IL-10) of the constipated mice in the model group was significantly lower than that in the control group (P < 0.01), and the pro-inflammatory factor (TNF-α) was significantly higher than that in the control group (P < 0.001), indicating that the serum cellular inflammatory factors of constipated mice were affected. After intervention, BC07 significantly increased the level of anti-inflammatory factors (IL-10) (P < 0.05) and reduced the content of pro-inflammatory factors (TNF-α). BC07 combined with inulin treatment also showed the same effect.

(3) Analysis of mouse intestinal flora

As shown in Figure 8, compared with the control group, the ACE index and Simpson index of the α-diversity of intestinal microorganisms in the model group mice were decreased, indicating that constipation caused a decrease in the α-diversity index of the intestinal flora in mice; compared with the model group, the ACE index and Simpson index of the BC07 group and BC07P group were increased, indicating that Bacillus coagulans BC07 and BC07 combined with inulin intervention would promote the increase of the α-diversity index of the intestinal flora in mice, proving that supplementing BC07 or BC07 inulin mixture can improve the richness and community diversity of intestinal microbial flora.

Figure 9 shows the species abundance bar graph at the genus level in different groups. OTU cluster analysis found that compared with the control group, the relative abundance of Lachnospiraceae_NK4A136 and Alternaria in the model group decreased, while the relative abundance of harmful bacteria genera Acinetobacter and Pseudomonas increased (P < 0.01). The relative abundance of beneficial bacteria genera Alternaria and Lachnospiraceae_NK4A136 (Lachnospiraceae) increased in the two intervention groups. The relative abundance of harmful bacteria genera Acinetobacter (P < 0.01) and Pseudomonas (P < 0.001) decreased significantly. Alternaria helps human digestion, nutrient absorption and regulation. Lachnospiraceae_NK4A136 Lachnospiraceae is an important member of the Lachnospiraceae family and belongs to the Firmicutes phylum. It can hydrolyze starch and ferment sugars to produce short-chain fatty acids such as butyrate to provide energy sources for the host. Acinetobacter, a Gram-negative bacterium, can colonize human skin, wounds, respiratory tract and gastrointestinal tract, and can also exist in oral biofilms, which can easily cause pneumonia after being inhaled into the lower respiratory tract. Pseudomonas is associated with intestinal barrier dysfunction and infection, which may lead to sepsis and multiple organ dysfunction syndrome. In addition, Pseudomonas is also related to the occurrence of inflammatory bowel disease, and higher abundance has been detected in the intestines of IBD patients. In addition, Pseudomonas can cause an imbalance in the intestinal flora and affect the progress of disease treatment.

The above shows that functional constipation is closely related to intestinal dysfunction. Supplementation of exogenous probiotics can effectively regulate intestinal function. Both BC07 and BC07 combined with inulin can relieve constipation, mainly showing different advantages. BC07 restores the content of MTL and VIP and upregulates the level of anti-inflammatory cytokine IL-10 in serum, thereby repairing the inflammatory response caused by loperamide hydrochloride and promoting gastrointestinal motility. BC07 combined with inulin has significant effects in shortening the time of first feces excretion in mice, promoting small intestinal transit, and increasing the apparent physiological content of feces. At the same time, BC07 and BC07 combined with inulin can improve the intestinal flora structure of constipated mice, enhance the richness and diversity of intestinal flora, increase the abundance of beneficial bacteria in the intestine, prevent the growth of harmful bacteria, and promote intestinal health.

Claims (10)

1. The application of the bacillus coagulans BC07 in preparing health care products with digestion promoting/bowel relaxing functions or constipation improving medicines or Porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitors is characterized in that the preservation number of the bacillus coagulans BC07 is CCTCC NO: m2022195.

2. Use of bacillus coagulans BC07 according to claim 1, for the preparation of health products for promoting digestion/relaxing the bowels or medicines for improving constipation or porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitors, characterized in that the digestion/relaxing functions are selected from the group consisting of: producing protease or cellulase, tolerating gastric juice/bile salts, increasing fecal moisture content, decreasing head set black stool time, increasing small intestine propulsion, modulating peptide neurotransmitter content, increasing anti-inflammatory factor levels, decreasing pro-inflammatory factor levels, increasing intestinal flora index.

3. Use of bacillus coagulans BC07 according to claim 1, for the preparation of health products for promoting digestion/relaxing the bowels or medicines for improving constipation or porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitors, characterized in that the improvement of constipation is selected from: producing protease or cellulase, tolerating gastric juice/bile salts, increasing fecal moisture content, reducing head set black stool time, increasing small intestine propulsion, modulating peptide neurotransmitters, increasing anti-inflammatory factor levels, reducing pro-inflammatory factor levels, increasing intestinal microbiota index, and increasing intestinal microbiota community diversity.

4. Use of bacillus coagulans BC07 according to claim 2 or 3, for the preparation of health products for promoting digestion/relaxing the bowels or of drugs for improving constipation or of porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitors, characterized in that the regulator peptide neurotransmitters are chosen from: lowering the level of the inhibitory peptide neurotransmitter VIP and/or increasing the level of the excitatory peptide neurotransmitter MTL.

5. Use of bacillus coagulans BC07 according to claim 2 or 3, for the preparation of health products for promoting digestion/relaxing the bowels or medicines for improving constipation or porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitors, characterized in that said anti-inflammatory factor is selected from the group consisting of: IL-10.

6. Use of bacillus coagulans BC07 according to claim 2 or 3, for the preparation of health products for promoting digestion/relaxing the bowels or medicines for improving constipation or porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitors, characterized in that said pro-inflammatory factor is selected from the group consisting of: TNF-alpha.

7. Use of bacillus coagulans BC07 according to claim 2 or 3, for the preparation of health products for promoting digestion/relaxing the bowels or medicines for improving constipation or porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitors, characterized in that the intestinal flora index comprises: ACE index, simpson index.

8. Use of bacillus coagulans BC07 according to claim 2 or 3, for the preparation of health products for promoting digestion/relaxing the bowels or medicines for improving constipation or porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitors, characterized in that increasing the intestinal microbiota community diversity is selected from: the abundance of Acremonium (ALISTIPES) and Mollum (Lachnospiraceae _NK4A 136) increases, and the abundance of Acinetobacter (Acinetobacter) and Pseudomonas (Pseudomonas) decreases.

9. Use of bacillus coagulans BC07 according to any one of claims 1-3 for the preparation of a digestion promoting/bowel relaxing functional health product or a constipation improving drug or a porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitor, wherein the diameter of the zone of inhibition of porphyromonas gingivalis by bacillus coagulans BC07 is 25 mm.

10. The use of bacillus coagulans BC07 according to any one of claims 1-3 for the preparation of a digestion promoting/bowel relaxing functional health product or a constipation improving drug or a porphyromonas gingivalis/bacteroides fragilis/streptococcus pyogenes inhibitor, wherein the diameter of the zone of inhibition of bacillus coagulans BC07 against bacteroides fragilis is 22 mm;

and/or the diameter of a bacteriostasis zone of the bacillus coagulans BC07 for inhibiting the streptococcus pyogenes is 23 mm.