Bacterial strain for promoting intestinal barrier repair and nutrient absorption and application thereof

Abstract

The invention provides a bacterial strain for promoting intestinal barrier repair and nutrient absorption and application of the bacterial strain. The preservation number of bifidobacterium animalis subsp. Lactis BL-11 is CGMCC (China General Microbiological Culture Collection Center) No.20847. The bacterial strain can effectively improve the intestinal mucosa barrier structure of a mouse with colitis, promote the length of intestinal villi, increase the intestinal absorption area, promote goblet cells to secrete mucus, increase the thickness of intestinal mucosa, enhance the expression of tight junction proteins ZO-1 and Occludin of intestinal epithelial cells, reduce the levels of proinflammatory factors TNF-alpha and IL-6, improve the anti-inflammatory factor IL-10, improve the anti-inflammatory activity of the mouse with colitis and improve the anti-inflammatory activity of the mouse with colitis. Therefore, intestinal and body inflammation is relieved, and intestinal barrier repair is promoted. In addition, the homeostasis maintenance of the BL-11 strain on the intestinal barrier can also promote the absorption of nutrient substances (such as calcium).

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a probiotic for promoting intestinal barrier repair and nutrient absorption and an application thereof. Background Art

[0002] The intestinal barrier is an important protective barrier in the human digestive tract, mainly composed of intestinal epithelial cells, mucosal layers and intestinal microbial communities. The epithelial cell layer forms a physical barrier to prevent the passage of macromolecules and microorganisms; the mucus layer mainly provides a living environment for symbiotic microorganisms; the intestinal microbial community regulates the immune response, promotes the digestion and absorption of nutrients, and prevents the colonization of pathogenic microorganisms through interactions with the host. Functionally, the intestinal barrier not only has a physical barrier function to prevent harmful substances and pathogenic microorganisms from entering the body, but also plays a key role in the absorption of nutrients. The nutrient absorption function undertaken by the intestinal barrier is extremely important for maintaining the health of the body. The intestinal barrier ensures that the body obtains adequate nutrition by regulating the selective permeability of nutrients, relying on the metabolic effects of intestinal microorganisms, maintaining the integrity of tight junctions, and relying on the support of the immune system.

[0003] Intestinal nutrient malabsorption may cause a variety of developmental abnormalities, especially during childhood, when adequate absorption of nutrients is essential for the normal development of various body systems. Inadequate nutrient absorption can lead to some important problems: ① Growth retardation: Inadequate absorption of nutrients such as protein, carbohydrates, and fat in the body will affect the child's physical growth and development rate. ② Bone dysplasia: Lack of adequate calcium, vitamin D, and other minerals may lead to bone development problems such as osteoporosis and rickets, making the bone structure fragile and prone to fractures and deformities. ③ Affected intellectual development: Lack of nutrients such as vitamin B12 and folic acid may lead to stunted development of the nervous system, thereby affecting children's intellectual development and learning ability. ④ Decreased immune function: Malnutrition leads to a low immune system function, increasing the risk of infection and disease, especially for children, which may lead to repeated infections and health problems. ⑤ Digestive system problems: Digestive problems including diarrhea, constipation, and bloating further affect the absorption of nutrients and the health of the digestive system. ⑥ Anemia and blood problems: Anemia caused by lack of iron, vitamin B12, etc. may affect oxygen delivery and blood function, thereby affecting overall health and development. ⑦ Vision and visual development problems: Lack of nutrients such as vitamin A may lead to night blindness and other visual problems, affecting the normal development of the visual system.

[0004] Probiotics refer to active microorganisms that are beneficial to the host's health after a certain amount of ingestion. Common species include lactic acid bacteria and bifidobacteria. However, the effects of existing probiotics in promoting intestinal barrier repair and calcium absorption need to be improved. In particular, it is necessary to develop probiotic strains that have the functions of promoting intestinal barrier repair and calcium absorption, so as to promote the repair and maintenance of intestinal barrier function and prevent and treat nutrient malabsorption. Summary of the invention

[0005] The present invention provides a kind of animal Bifidobacterium lactis subspecies BL-11 strain, which can effectively improve the intestinal mucosal barrier structure of colitis mice, promote the length of intestinal villi, increase the intestinal absorption area, promote goblet cells to secrete mucus, increase the thickness of intestinal mucosa, enhance the expression of tight junction proteins ZO-1 and Occludin of intestinal epithelial cells, reduce the level of pro-inflammatory factors TNF-α and IL-6, and increase the anti-inflammatory factor IL-10, thereby reducing intestinal and body inflammation and promoting the repair of intestinal barrier. In addition, the BL-11 strain can also promote the absorption of nutrients (such as calcium) by maintaining the steady state of intestinal barrier.

[0006] One aspect of the present invention provides a strain of Bifidobacterium animalis subsp. lactis BL-11, and the deposit number of the Bifidobacterium animalis subsp. lactis BL-11 is CGMCC No.20847.

[0007] Another aspect of the present invention provides a bacterial agent, wherein the bacterial agent comprises the Bifidobacterium animalis subsp. lactis.

[0008] Another aspect of the present invention provides the use of the animal Bifidobacterium lactis subsp. or the bacterial agent in a product for improving the disease activity index of enteritis.

[0009] Another aspect of the present invention provides the use of the Bifidobacterium animalis subsp. lactis or the bacterial agent in a product for improving or repairing the intestinal mucosal barrier.

[0010] Another aspect of the present invention provides the use of the animal Bifidobacterium lactis subspecies or the bacterial agent in a product for promoting the length of intestinal villi and increasing the intestinal absorption area.

[0011] Another aspect of the present invention provides the use of the animal Bifidobacterium lactis subspecies or the bacterial agent in a product for promoting goblet cells to secrete mucus and increase the thickness of intestinal mucosa.

[0012] Another aspect of the present invention provides the use of the animal Bifidobacterium lactis subspecies or the bacterial agent in the preparation of anti-inflammatory products, wherein the product is used to reduce pro-inflammatory factors and increase the level of anti-inflammatory factors in individuals with enteritis.

[0013] Another aspect of the present invention provides the use of the animal Bifidobacterium lactis subspecies or the bacterial agent in a product for promoting the absorption of nutrients, preferably, the nutrient is calcium. Another aspect of the present invention provides the use of the animal Bifidobacterium lactis subspecies or the bacterial agent in any of the above items, wherein the number of viable cells of the animal Bifidobacterium lactis subspecies in the drug is 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g. Another aspect of the present invention provides a method for preparing a product composition, the method comprising: adding the Bifidobacterium animalis subsp. lactis or the bacterial agent to a product matrix to obtain the product composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is shown that the animal Bifidobacterium lactis subspecies BL-11 strain in the present invention promotes the maintenance and repair of the intestinal barrier in colitis model mice. (A) Mouse disease activity index DAI. (B) Ileal H&E staining and Alcian blue staining images; (C) Alcian blue staining quantitative statistics; (D) Ileal villus length statistics; (E) Intestinal tight junction protein ZO-1 and tight junction protein (Occludin) mRNA expression levels in ileal tissue; (F) Serum inflammatory factor levels. n=10, ns>0.05; (vs normal) *p<0.05, **p<0.01, ***p<0.001; (vs model) #p<0.05, ##p<0.01, ###p<0.001.

[0015] Figure 2 It is shown that the animal Bifidobacterium lactis subspecies BL-11 strain in the present invention promotes calcium absorption in mice. (A) Statistics of weight gain in mice; (B) Blood calcium level in mice; (C) Femoral calcium content in mice. n=10, ns>0.05; (vs normal) *p<0.05, **p<0.01, ***p<0.001; (vs model) #p<0.05, ##p<0.01, ###p<0.001. Example

[0016] Example 1: Evaluation of the repair effect of Bifidobacterium animalis subsp. lactis BL-11 on the intestinal mucosal barrier

[0017] The probiotic strain BL-11 described in the present invention is isolated from breast milk of healthy women and has been preserved in the General Microbiology Center of China Microorganism Culture Collection Administration on October 10, 2020, with the preservation number: CGMCC No. 20847. The original species name is Bifidobacterium lactis, and the species name has now been changed to Bifidobacterium anlmalissubsp.lactis (refer to the announcement issued by the National Health Commission on the update of the "List of Bacteria that can be used in Food" and the "List of Bacteria that can be used in Infant Food").

[0018] In the experiment, the frozen BL-11 strain was activated and subcultured for two generations, then inoculated into MRS liquid culture medium at 2-4%, cultured continuously for 16h-24h, and centrifuged (6000r / min, 10min, 4°C). The obtained supernatant was the fermentation supernatant of the BL-11 strain. The bacterial sludge was washed twice with PBS, and then the bacterial count was adjusted to 5×10 with sterile PBS solution. 9 CFU / mL was used for subsequent animal experiments.

[0019] 1.1 Experimental methods

[0020] (1) Establishment of colitis model:

[0021] Thirty 6-8 week old male C57BL / 6 mice (purchased from Vital River) were purchased, weighing about 20-25 g. After one week of adaptive feeding, the mice were randomly divided into 3 groups, 10 mice in each group, and grouped into normal group (NC), model group (Model), and model + BL-11 group (Model + BL-11). The model mice (model group, model + BL-11 group) continuously drank water containing 2.5% DSS (dextran sulfate sodium) for 7 days to induce an acute enteritis model. The mice in the normal group drank ordinary water.

[0022] (2) BL-11 probiotic intervention:

[0023] After DSS-induced enteritis, the BL-11 intervention group mice were given probiotics by gavage, 10^9 CFU / day / mouse, for 4 weeks. The remaining mice were gavaged with an equal volume of normal saline once a day for 4 weeks.

[0024] (3) Index detection:

[0025] ① Clinical scoring: The weight changes, stool consistency, and bleeding of mice were recorded throughout the experiment to evaluate the symptoms of enteritis.

[0026] ② Histological analysis: After the experiment, the mice were killed, and the ileum tissues were collected. H&E staining and Alcian blue staining were performed according to routine procedures to observe the structure of the intestinal mucosal barrier.

[0027] ③ Gene expression of tight junction proteins: The RNA of ileum tissue was extracted by Trizol method, and then the RNA of extracted tissue was reverse transcribed into cDNA using a reverse transcription kit, and then amplified using the reverse transcribed cDNA as a template according to the Real-time-PCR instructions.

[0028] ④ Levels of inflammatory factors: After the experiment, the mouse serum was separated, and the ELISA method was used to determine the levels of TNF-α, IL-6 and IL-10 in the mouse serum and colon tissue homogenate to evaluate the inflammatory response.

[0029] 1.2 Experimental results

[0030] ①Clinical scoring

[0031] According to the phenotype of the mice, the weight recovery rate of the mice in the enteritis model group after probiotic intervention was significantly faster than that in the model group, the stool consistency and bleeding were also alleviated, and the comprehensive disease score DAI was significantly reduced (model vs model + BL: 5.8 vs 4.1) ( Figure 1 ).

[0032] ② Histological analysis

[0033] HE staining results showed that the ileum tissue of the model group mice was obviously loose, broken, and disordered, with ulcers and inflammatory damage. After BL-11 probiotic intervention, the ileum mucosal structure was intact, the epithelial cells were tightly arranged, the inflammatory cell infiltration was reduced, the villi were neatly arranged, complete and dense, and the intestinal mucosal damage was significantly alleviated ( Figure 1 B); The villi length was measured and statistically analyzed. The results showed that the average villi length of the model group was about 118 μm, which was much lower than the villi length of normal mice (163 μm). After BL-11 strain treatment, the villi of the model mice became longer, with a length of about 142 μm ( Figure 1 D). The intestinal mucus layer plays an important role in maintaining the intestinal barrier, and is home to a large number of intestinal microorganisms that provide nutrition for their growth. The goblet cells of the mucus layer can produce mucin, which provides a nutrient source for the growth of some beneficial bacteria. After staining the ileum tissue with Alcian blue, it was found that compared with the normal group of mice, the mucin in the model group mice was significantly reduced (blue). After intervention with BL-11 probiotics, the amount of mucin was restored ( Figure 1 B); quantitative statistics of staining showed that the average relative area of ​​Alcian blue staining in normal mice was about 4.2%, and that in the model group was 1.2%. The relative area of ​​Alcian blue staining in the BL-11 probiotic intervention group increased to 3.3%, and the overall trend was consistent with the staining results ( Figure 1 C).

[0034] ③Expression analysis of tight junction proteins

[0035] ZO-1 and Occludin are intestinal tight junction proteins, and their expression levels can reflect whether the permeability of the intestinal barrier is abnormal. The detection of the mRNA levels of ZO-1 and Occludin in the ileum tissue showed that compared with the normal group of mice, the expression of tight junction proteins in the ileum of the model group mice was significantly downregulated to about half of that of the normal group, while the mRNA levels of these two genes were significantly increased after BL-11 intervention, and the relative expression levels were 0.78 and 0.67 respectively, which was consistent with the structural change trend of the ileum tissue ( Figure 1 E). These results indicate that in colitis mice, the intestinal barrier is damaged and the permeability increases due to the decreased expression of tight junction proteins in intestinal epithelial cells. The probiotic BL-11 intervention can upregulate the expression level of tight junction proteins, effectively enhance the tight junctions of intestinal epithelial cells, and thus improve the intestinal barrier damage.

[0036] ④ Levels of inflammatory factors

[0037] Damage to the intestinal barrier structure can lead to inflammation. ELISA detection of pro-inflammatory and anti-inflammatory factors in mouse serum and intestinal tissue samples revealed that ( Figure 1 F), the average values ​​of pro-inflammatory factors TNF-α and IL-6 in the serum of normal mice were about 286ng / L and 178ng / L, compared with the levels of TNF-α and IL-6 in the model group, which were significantly increased to 421ng / L and 279ng / L, respectively; while the level of anti-inflammatory factor IL-10 was significantly lower than that of normal mice (204ng / L vs 293ng / L). When BL-11 probiotics were gavaged into colitis mice, the levels of the above inflammatory factors were improved. Compared with the model group, the levels of TNF-α and IL-6 decreased (TNF-α: 421ng / L vs 364ng / L; IL-6: 279ng / L vs 210ng / L), while the content of IL-10 increased significantly (204ng / L vs 252ng / L), indicating that the intervention mice showed significant relief of inflammation. According to the above results, the animal Bifidobacterium lactis subspecies BL-11 of the present invention can alleviate the DSS-induced colitis phenotype, enhance the expression of tight junction proteins ZO-1 and Occludin in intestinal epithelial cells, reduce the levels of proinflammatory factors TNF-α and IL-6, and increase the anti-inflammatory factor IL-10, thereby alleviating intestinal and body inflammation, improving the intestinal mucosal structural damage in colitis model mice, and promoting the repair of the intestinal barrier.

[0038] Example 2: Evaluation of the effect of animal Bifidobacterium lactis subspecies BL-11 on promoting calcium absorption

[0039] This example is used to illustrate the promoting effect of animal Bifidobacterium lactis subspecies BL-11 on calcium absorption in mice.

[0040] 2.1 Experimental methods

[0041] 40 mice aged 6-8 weeks were purchased. After one week of adaptive feeding, the mice were randomly divided into 4 groups: normal control group (normal group), model group, experimental group (model + BL-11 group) and positive group (model + calcium carbonate group), with 10 mice in each group. Except for the normal control mice fed with regular feed, the mice in the other groups were given low-calcium feed. At the same time, the mice in the experimental group were gavaged with BL-11 probiotic live bacteria once a day, 10^9 CFU / day / mouse; the positive group was gavaged with calcium carbonate at a dose of 0.33g / kg / day, and the normal control group and the model group were gavaged with an equal volume of normal saline. The entire experimental period lasted for 4 weeks.

[0042] 2.2 Experimental testing

[0043] ① Growth monitoring: Weigh the mice every week and observe the growth and activity of mice in each group.

[0044] ② Serum calcium determination: After the mice were fasted overnight, blood was collected from the mouse eye sockets, and the serum was separated and stored at -80°C. The blood calcium content was determined according to the instructions of the blood calcium detection kit.

[0045] ③ Bone calcium determination: After the experiment, the mice in each group were killed, the femurs on both sides of the mice were completely removed, the soft tissue and cartilage were carefully removed, and then the bone calcium content was determined using the atomic absorption spectrometry (GB / T5009.92).

[0046] 2.3 Experimental Results

[0047] ① Growth experiment

[0048] During the entire experiment, the mice grew well, moved normally, had shiny fur, and showed no abnormalities. Figure 2 As shown in A, except that the weight gain of mice in the low calcium model group was slightly lower than that in the normal control group, there was no significant difference in the weight gain of mice in other groups.

[0049] ② Serum calcium content determination

[0050] Figure 2B shows that in the normal control group, the blood calcium content mean value is about 2.4mmo / L. Compared with the normal control, the serum calcium content of the low calcium model group mice is significantly reduced (2.27mmol / L), and the blood calcium content of the experimental group (2.33mmol / L) and the positive group mice (2.57mmol / L) processed by probiotics BL-11 is significantly higher than that of the low calcium model group, indicating that both treatments can improve the calcium content supplemented to low calcium mice. Wherein, in this experiment, the blood calcium content can exceed the normal group mice after calcium carbonate treatment, indicating that the calcium carbonate of this dosage is effective for supplementing calcium; the blood calcium concentration of low calcium mice after BL-11 intervention in the present embodiment can rise to the value between the low calcium model group and the normal control group, indicating that gavage BL-11 probiotics has the effect of improving blood calcium content, but because it does not contain calcium itself, the effect will be weaker than the positive control.

[0051] ③ Determination of femoral calcium content

[0052] The results of bone calcium content determination are as follows Figure 2 As shown in Figure C, in the normal control group, the average calcium content of the femur was about 146 mg / g, and the bone calcium content of the model group (136 mg / g) after low calcium modeling was lower than that of the normal group; while the bone calcium content of mice in the experimental group and the positive control group treated with BL-11 strain was higher than that of the low calcium model group, 140 mg / g and 149 mg / g respectively. Consistent with the trend of serum calcium test results, the bone calcium content increased after the intervention of probiotics BL-11, but was weaker than that of the normal control group, while the bone calcium level of mice in the positive group was slightly higher than that of the normal group. These results indicate that BL-11 probiotics can improve the absorption of bone calcium and promote bone development.

[0053] Example 3: Effect of Bifidobacterium animalis subspecies lactis BL-11 on the absorption of nutrients by adolescents

[0054] This example is used to evaluate the effect of animal Bifidobacterium lactis subsp. BL-11 on calcium absorption in adolescents.

[0055] Through volunteer recruitment, 18 adolescents (10-18 years old) with representative gender, age and nutritional status were selected, and the participants were divided into two groups: 10 in the intervention group (5 males and 5 females) and 8 in the placebo group (3 males and 5 females). The experimental group took a specific dose of probiotic BL-11 powder daily, while the placebo group took a placebo with the same appearance and taste without probiotics. The entire intervention period was set to about 10 weeks, during which the participants' dietary records were collected regularly. A baseline assessment was conducted before the intervention, including the eating habits, body composition (such as weight, height) and serum calcium levels of all participants. After the intervention, the serum calcium levels of all participants were reassessed.

[0056] The promoting effect of BL-11 on calcium absorption is shown in Table 2. The baseline levels of blood calcium in the placebo group and the intervention group were 8.92 mg / dL and 9.02 mg / dL, respectively. After 10 weeks of probiotic BL-11 intervention, the vitamin D level in the intervention group was significantly higher than that in the placebo group (9.77 mg / dL vs 9.16 mg / dL), and the blood calcium in the intervention group increased; this indicates that BL-11 can increase the calcium content in adolescents and promote the intestinal absorption of nutrients.

[0057] Table 2 Blood calcium levels of adolescents in this example

[0058]

[0059] In summary, these results show that BL-11 probiotics not only promote the repair and improvement of the intestinal barrier, but also can improve the body's absorption of calcium. The intestinal barrier itself is responsible for the absorption of nutrients. Therefore, the animal Bifidobacterium lactis subspecies BL-11 in this embodiment helps to maintain the homeostasis of the intestinal barrier and can be used to promote the exploration and development of new probiotic products such as intestinal mucosal repair, homeostasis maintenance, and calcium absorption promotion.

Claims

1. A strain of Bifidobacterium animalis subsp. lactis BL-11, characterized in that: The preservation number of the animal Bifidobacterium lactis subspecies BL-11 is CGMCC No.20847.

2. A bacterial agent, characterized in that The bacterial agent comprises the animal Bifidobacterium lactis subsp.

3. Use of the animal Bifidobacterium lactis subsp. lactis described in claim 1 or the bacterial agent described in claim 2 in a product for improving the disease activity index of enteritis.

4. Use of the animal Bifidobacterium lactis subsp. lactis according to claim 1 or the bacterial agent according to claim 2 in a product for improving or repairing the intestinal mucosal barrier.

5. Use of the animal Bifidobacterium lactis subsp. according to claim 1 or the bacterial agent according to claim 2 in products for promoting the length of intestinal villi and increasing the intestinal absorption area.

6. Use of the animal Bifidobacterium lactis subsp. lactis described in claim 1 or the bacterial agent described in claim 2 in a product for promoting goblet cells to secrete mucus and increase the thickness of intestinal mucosa.

7. Use of the animal Bifidobacterium lactis subsp. according to claim 1 or the bacterial agent according to claim 2 in the preparation of anti-inflammatory products, characterized in that: The product is used to reduce the level of pro-inflammatory factors and increase the level of anti-inflammatory factors in individuals with enteritis.

8. Use of the Bifidobacterium animalis subsp. lactis according to claim 1 or the bacterial agent according to claim 2 in a product for promoting absorption of nutrients, preferably, the nutrient is calcium.

9. Use of the animal Bifidobacterium lactis subsp. lactis according to claim 1 or the preparation according to claim 2 or the bacterial agent according to claim 2 in any one of claims 3 to 8, characterized in that: The viable count of animal Bifidobacterium lactis subspecies in the drug is 1×10 3 -1×10 14 CFU / mL or 1×10 3 -1×10 14 CFU / g.

10. A method for preparing a product composition, characterized in that: The method comprises: adding the animal Bifidobacterium lactis subsp. animalis described in claim 1 or the bacterial agent described in claim 2 to a product matrix to obtain the product composition.

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