A probiotic agent for enhancing intestinal immunity and its application

Abstract

The present invention relates to a probiotic agent for enhancing intestinal immunity and its application. The strains in the probiotic agent for enhancing intestinal immunity include Lactobacillus rhamnosus LRa66 strain and Bifidobacterium longum subsp. infantis BI03 strain. There are potential interactions between the two strains, which can cooperate with each other and synergistically enhance the efficacy of enhancing intestinal immunity. Specifically, it is reflected in: improving the related symptoms of the diarrhea mouse model; improving the related symptoms of the constipation mouse model; significantly enhancing the immune function of mice. When the amount of bacteria used is the same, compared with the single LRa66 strain or the single BI03 strain, the compounding of the two bacteria significantly improves the above-mentioned efficacy.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotic agents, and relates to a probiotic agent for enhancing intestinal immunity and its application. Background Art

[0002] Dysbiosis generally refers to a state in which the proportion among various strains in the normal flora of a certain part of the body changes significantly beyond the normal range, and the resulting disease is dysbiosis or flora alternation. When the dynamic balance of the intestinal flora is disrupted, the body's metabolism is affected, and a series of pathophysiological changes will occur in the body. The disorder of the dynamic balance of the intestinal flora will lead to the proliferation of pathogenic bacteria, stimulate the intestinal mucosal barrier, damage the body's immune system, and cause a decline in immune function. Moreover, the release of a large number of pathogenic bacteria increases the endotoxin in the body's intestine, destroys the intestinal mucosal barrier, and leads to intestinal dysfunction.

[0003] Probiotics have the potential to promote human intestinal health. The probiotic market is growing rapidly. The gut microbiota may play a role in the development of metabolic diseases such as obesity, diabetes, and inflammatory bowel disease, and the administration of probiotics has been proven to improve the prognosis and management of these diseases. Therefore, it is very meaningful to develop more probiotic strategies that can be used to maintain intestinal health and increase intestinal immunity. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a probiotic agent for enhancing intestinal immunity and its application, specifically providing a probiotic agent for enhancing intestinal immunity and its application in the preparation of products for enhancing intestinal immunity, as well as its application in the preparation of products for preventing, alleviating or treating constipation, and its application in the preparation of products for preventing, alleviating or treating diarrhea.

[0005] To achieve the purpose of this invention, the following technical solutions are adopted:

[0006] In the first aspect, the present invention provides a probiotic agent for enhancing intestinal immunity. The strains in the probiotic agent for enhancing intestinal immunity include Lactobacillus rhamnosus LRa66 strain with the preservation number of CGMCC No. 24282 and Bifidobacterium longum subsp. infantis BI03 strain with the preservation number of CGMCC No. 24473.

[0007] The present invention has developed a brand-new probiotic compounding method, which combines Lactobacillus rhamnosus LRa66 strain and Bifidobacterium longum subsp. infantis BI03 strain. It is found that there is a potential interaction between the two, and they can cooperate with each other to synergistically enhance the efficacy of improving intestinal immunity, specifically manifested in: (1) improving the related symptoms of diarrhea mouse models; (2) improving the related symptoms of constipation mouse models; (3) significantly enhancing the immune function of mice. When the amount of bacteria used is the same, compared with a single LRa66 strain or a single BI03 strain, the compounding of the two bacteria significantly improves the above-mentioned efficacy. Therefore, this probiotic agent provides a new strategy for preventing and treating intestinal-related diseases. Since both Lactobacillus rhamnosus LRa66 strain and Bifidobacterium longum subsp. infantis BI03 strain are probiotics, they have high safety and are not likely to produce resistance when used in the preparation of related efficacy products.

[0008] Preferably, the viable count ratio of the Lactobacillus rhamnosus LRa66 strain to the Bifidobacterium longum subsp. infantis BI03 strain is 1:10 - 10:1, such as 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0009] Preferably, in the probiotic agent, the total viable count is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g, such as 2×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), 1×10 13CFU / mL (CFU / g), etc.; other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0010] Preferably, the dosage form of the probiotic agent includes solution, freeze-dried powder, capsule, tablet or granule.

[0011] Preferably, the probiotic agent further includes a freeze-drying protectant and / or a functional adjuvant.

[0012] Preferably, the freeze-drying protectant includes any one or a combination of at least two of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol;

[0013] The functional adjuvant includes any one or a combination of at least two of fructooligosaccharide, galactooligosaccharide, xylooligosaccharide, isomaltooligosaccharide, soybean oligosaccharide, inulin, spirulina, arthrospira, polysaccharide-krestin, stachyose, polydextrose, α-lactalbumin or lactoferrin.

[0014] Preferably, the probiotic agent is a freeze-dried powder, which is prepared by the following preparation method:

[0015] The LRa66 strain and the BI03 strain are respectively inoculated into a culture medium for activation and fermentation culture to obtain a fermentation broth; the fermentation broth is centrifuged respectively, mixed with a freeze-drying protectant and then freeze-dried to obtain LRa66 bacterial powder and BI03 bacterial powder; the LRa66 bacterial powder and the BI03 bacterial powder are mixed according to the viable count ratio to obtain the probiotic agent for enhancing intestinal immunity.

[0016] In a second aspect, the present invention provides the application of the probiotic agent described in the first aspect in the preparation of a product for enhancing intestinal immunity.

[0017] Preferably, the product includes drugs, foods and health products.

[0018] Preferably, the product further includes excipients.

[0019] The excipients include any one or a combination of at least two of excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizing agents, solubilizers, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants, bacteriostatic agents or buffers.

[0020] In a third aspect, the present invention provides the application of the probiotic agent described in the first aspect in the preparation of a product for preventing, alleviating or treating constipation.

[0021] Preferably, the product includes drugs.

[0022] Preferably, the product further includes excipients.

[0023] The adjuvants include any one or a combination of at least two of excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizing agents, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants, bacteriostatic agents or buffers.

[0024] Fourthly, the present invention provides an application of the probiotic agent described in the first aspect in the preparation of a product for preventing, alleviating or treating diarrhea.

[0025] Preferably, the product includes medicines.

[0026] Preferably, the product further includes adjuvants.

[0027] The adjuvants include any one or a combination of at least two of excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizing agents, osmotic pressure regulators, coating materials, colorants, pH regulators, antioxidants, bacteriostatic agents or buffers.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention develops a brand-new probiotic compounding method, compounding Lactobacillus rhamnosus LRa66 strain and Bifidobacterium longum subsp. infantis BI03 strain, and finds that there are potential interactions between the two, which can cooperate with each other and synergistically enhance the efficacy of enhancing intestinal immunity, specifically reflected in: (1) improving the related symptoms of a diarrhea mouse model; (2) improving the related symptoms of a constipation mouse model; (3) significantly improving the immune function of mice. When the amount of bacteria used is the same, compared with a single LRa66 strain or a single BI03 strain, the compounding of the two bacteria significantly improves the above-mentioned efficacy. Therefore, the probiotic agent provides a new strategy for preventing and treating intestinal-related diseases. Since Lactobacillus rhamnosus LRa66 strain and Bifidobacterium longum subsp. infantis BI03 strain are both probiotics, they are highly safe and not likely to produce resistance when used in the preparation of related efficacy products.

[0030] The taxonomic naming of the LRa66 strain involved in the present invention is Lactobacillus rhamnosus. The preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation time is January 10, 2022, and the preservation number is CGMCC No. 24282. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0031] The taxonomic naming of the BI03 strain involved in the present invention is Bifidobacterium longum subsp. infantis. The preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation time is March 7, 2022, and the preservation number is CGMCC No. 24473. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a statistical result graph of the serum total protein levels of each group of mice;

[0033] Figure 2 It is a statistical result graph of the serum albumin levels of each group of mice;

[0034] Figure 3 It is a statistical result graph of the IL-1β levels in the colon tissues of each group of mice;

[0035] Figure 4 It is a statistical result graph of the TNF-α levels in the colon tissues of each group of mice;

[0036] Figure 5 It is a statistical result graph of the IL-10 levels in the colon tissues of each group of mice;

[0037] Figure 6 It is a statistical result graph of the fecal water content of each group of mice;

[0038] Figure 7 It is a statistical result graph of the intestinal propulsion rate of each group of mice. DETAILED DESCRIPTION OF THE INVENTION

[0039] The technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0040] The culture medium formula involved in the following embodiments is as follows:

[0041] MRS culture medium (g / L): 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, K2 PO 4 ·3H 2 O 2.6 g / L, MgSO 4 ·7H 2 O 0.1 g / L, MnSO 4 0.05 g / L, Tween 80 1 mL / L, cysteine hydrochloride 0.5 g / L.

[0042] The taxonomic nomenclature of the LRa66 strain involved in the following examples is Lactobacillus rhamnosus, the preservation time is January 10, 2022, and the preservation number is CGMCC No. 24282.

[0043] The taxonomic nomenclature of the BI03 strain involved in the following examples is Bifidobacterium longum subsp. infantis, the preservation time is March 7, 2022, and the preservation number is CGMCC No. 24473.

[0044] The method for preparing the bacterial suspension involved is as follows: inoculate the required strain into MRS liquid medium, culture at 37 °C for 18 h for activation, and activate continuously for 2 times to obtain the activated solution; inoculate the activated solution into MRS liquid medium at an inoculation amount of 2% (v / v), culture at 37 °C for 24 h to obtain the bacterial solution; centrifuge the bacterial solution at 5000 rpm at 4 °C for 5 min, filter to obtain the bacterial cells, and resuspend the bacterial cells with physiological saline for standby.

[0045] Example 1

[0046] The improvement effect of the probiotic agent on the diarrhea mouse model:

[0047] (1) Test animals: SPF-grade male ICR mice (48, purchased from Shanghai Laboratory Animal Center), with a body weight of 18 - 22 g. These mice were raised in a controlled environment, the room temperature was maintained at 22 ± 2 °C, the humidity was 55% ± 5%, and they followed a 12 h light / dark cycle. They could eat and drink freely.

[0048] (2) Animal grouping: After the above mice were adaptively fed for 1 week, the mice were randomly divided into 6 groups (8 mice in each group): normal control group (AD group), model control group (MC group), LRa66 group (denoted as S1 group), BI03 group (denoted as S2 group), compound bacteria group 1 (LRa66 + BI03 group, the viable bacteria ratio is 1:1, denoted as S3 group), compound bacteria group 2 (commercially available Lactobacillus rhamnosus ATCC7469 + BBr60 group, the viable bacteria ratio is 1:1, denoted as S4 group).

[0049] (3) Animal modeling and intervention method:

[0050] Except for the AD group, mice in other groups were gavaged with sodium ampicillin at a dose of 22.4 g / kg once a day for 3 consecutive days. After the modeling was completed. Each probiotic intervention group gavaged the mice with a probiotic suspension (the amount of bacteria given was 10 7 CFU / mouse), once a day for 14 consecutive days; the AD group and the MC group were gavaged with an equal volume of normal saline every day.

[0051] (4) Index analysis:

[0052] (4.1) Detection of serum total protein and albumin levels:

[0053] One hour after the last administration, blood was collected from the eye, left standing for about 1 hour, centrifuged (3000 r / min) for 10 minutes, and then the serum was separated. ELISA kits were used to measure the levels of total protein and albumin in the serum samples.

[0054] The results are as Figure 1 and Figure 2 shown. Compared with the AD group, the levels of total protein and albumin in the serum of the MC group showed a downward trend. Compared with the MC group, the levels of total protein and albumin in each probiotic intervention group showed an upward trend, indicating that the probiotic agent involved in the present invention has a good effect on alleviating the reduction of protein levels caused by diarrhea.

[0055] (4.2) Detection of inflammatory cytokine levels in colon tissue:

[0056] Colon tissues of mice in each group were collected, colon homogenates were prepared, supernatants were separated, and ELISA kits were used to measure the levels of IL-1β, IL-10, and TNF-α in the colon tissues.

[0057] The results are as Figures 3 - 5 shown. Compared with the AD group, the levels of IL-1β and TNF-α in the colon tissues of the MC group showed an upward trend, and the level of IL-10 showed a downward trend. Compared with the MC group, the levels of IL-1β and TNF-α in each probiotic intervention group showed a downward trend, and the level of IL-10 showed an upward trend. It shows that the probiotic agent involved in the present invention has a good effect on regulating the levels of intestinal inflammatory factors in diarrhea mice.

[0058] Example 2

[0059] Improvement effect of probiotic agent on constipation mouse model:

[0060] (1) Experimental animals: SPF-grade Kunming mice (48, purchased from Shanghai Laboratory Animal Center) were raised in a controlled environment with the room temperature maintained at 22 ± 2°C and the humidity at 55% ± 5%, following a 12 h light / dark cycle. They had free access to food and water.

[0061] (2) Animal grouping: After 1 week of adaptive feeding of the mice as described above, the mice were randomly divided into 6 groups (8 mice in each group): normal control group (CTL group), model control group (STC group), LRa66 group (denoted as S1 group), BI03 group (denoted as S2 group), compound bacteria group 1 (LRa66 + BI03 group, the viable bacteria count ratio is 2:1, denoted as S3 group), compound bacteria group 2 (commercially available Lactobacillus rhamnosus ATCC7469 + BBr60 group, the viable bacteria count ratio is 2:1, denoted as S4 group).

[0062] (3) Animal modeling and intervention method:

[0063] Except for the CTL group, the mice in other groups were intragastrically administered loperamide hydrochloride at a dose of 3 mg / kg until the end of the experiment, once a day. At the same time, the probiotic intervention groups were intragastrically administered probiotic suspension (the dose of bacteria is 10 9 CFU / mouse), once a day. The CTL group and the STC group were intragastrically administered an equal volume of normal saline every day. The experiment was carried out for 4 weeks.

[0064] (4) Index analysis:

[0065] (4.1) Detection of fecal water content:

[0066] After the experiment ended, the feces of the mice in each group were collected. Immediately after collection, the wet weight of the feces was recorded. After drying in an oven for 3 h, the dry weight of the feces was recorded. The formula for calculating the fecal water content (%) is: fecal water content (%) = (wet weight of feces - dry weight of feces) / wet weight of feces × 100%.

[0067] The results are as Figure 6 shown. It can be seen from the figure that the probiotic agent involved in the present invention can reverse the decrease in fecal water content in constipated mice and can significantly improve constipation.

[0068] (4.2) Gastrointestinal motility evaluation:

[0069] After 4 weeks of intervention, the mice were fasted overnight with free access to water. After intragastrically administering ink for 25 minutes, they were immediately sacrificed by cervical dislocation. The sacrificed mice were placed supine on a wooden board and fixed with nails. The abdominal skin and muscle layers of the mice were gradually cut open with surgical scissors, and the greater omentum and fat were separated and removed with forceps to expose the abdominal organs. The stomach was found in the upper left abdominal cavity of the mice. The gastroesophageal junction was cut at the pylorus, the stomach was lifted, and the mesentery was gradually cleaned downward to separate the intestine. The connection between the small intestine and the colon was cut at the ileocecal part. The separated intestinal tube was placed on a tray, and the small intestine was gently pulled into a straight line. 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 measured as the "ink propulsion length". The intestinal propulsion rate was calculated according to the following formula: intestinal propulsion rate (%) = ink propulsion length (cm) / total length of the small intestine (cm) × 100%.

[0070] The results are as Figure 7 shown. As can be seen from the figure, the probiotic agent involved in the present invention can reverse the decrease in the intestinal propulsion rate of constipated mice and can significantly improve constipation.

[0071] Example 3

[0072] Effect of probiotic agent on enhancing the immune function of mice:

[0073] (1) Test animals: SPF-grade BALB / c mice, female, weighing 18 - 22 g (40 mice) were raised in a controlled environment with the room temperature maintained at 22 ± 2 °C and the humidity at 55% ± 5%, following a 12 h light / dark cycle. They had free access to food and water.

[0074] (2) Animal grouping: After 1 week of adaptive feeding of the above mice, the mice were randomly divided into 5 groups (8 mice in each group): normal control group (CTL group), LRa66 group (denoted as S1 group), BI03 group (denoted as S2 group), compound bacteria group 1 (LRa66 + BI03 group, the viable bacteria count ratio was 2:1, denoted as S3 group), compound bacteria group 2 (commercially available Lactobacillus rhamnosus ATCC7469 + BBr60 group, the viable bacteria count ratio was 2:1, denoted as S4 group).

[0075] (3) Animal modeling and intervention method:

[0076] Each mouse in the normal control group was intragastrically administered 0.2 mL of 12% skim milk per day; each probiotic intervention group was intragastrically administered 0.2 mL of a bacterial suspension with a total concentration of 10 8 CFU / mL per day. In the above intervention groups, the strains were added and dissolved in 12% skim milk. The gavage was performed for 4 weeks, and the body weight was measured once a week. 5 days before sacrifice, 0.5 mL of 2% (V / V) sheep red blood cells (SRBC) were intraperitoneally injected for sensitization.

[0077] (4) Index analysis:

[0078] Detection of the degree of delayed type hypersensitivity (DTH):

[0079] 4 days after the intraperitoneal injection of sheep red blood cells in mice, that is, 1 day before sacrifice, the thickness of the left hind paw was measured with a vernier caliper. 20 μL of 20% (V / V) SRBC was subcutaneously injected at the measurement site for each mouse, and the thickness of the left hind paw was measured again 24 h later. The average value was taken after measuring 3 times at the same site. The reaction degree of DTH was represented by the difference in paw thickness before and after injection. The results are shown in Table 1.

[0080] Table 1

[0081] Group Toe thickness difference CTL group 0.23±0.87 S1 group 0.82±0.56 S2 group 0.76±0.45 S3 group 0.87±0.13 S4 group 0.36±0.19

[0082] As can be seen from the data results in Table 1, compared with the normal control group, the mice intervened with probiotics all showed thickening of the toe thickness. In this experiment, the hind toes of the mice were attacked with an antigen (sheep red blood cells), and the change in the swelling thickness of the skin after receiving the same antigen stimulation reflected the intensity of the delayed hypersensitivity reaction, reflecting the T cell immune response of the body. The delayed hypersensitivity reaction of the mice marked by the change in toe thickness was enhanced, fully proving that the probiotic agent involved in the present invention can promote the cellular immune response of the body.

[0083] The applicant declares that the present invention illustrates the technical solution of the present invention through the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0084] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0085] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any suitable manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A probiotic for improving intestinal immunity, characterized in that: The strain in the probiotic for improving intestinal immunity is Lactobacillus rhamnosus with a preservation number of CGMCC No. 24282 ( Lactobacillus rhamnosus )LRa66 strain and Bifidobacterium longum subsp. infantis with the deposit number of CGMCC No.24473 ( Bifidobacterium longum subsp. infantis ) BI03 strain; The Lactobacillus rhamnosus ( Lactobacillus rhamnosus )LRa66 strain and Bifidobacterium longum subsp. infantis ( Bifidobacterium longum subsp. infantis ) The ratio of the number of viable cells of the BI03 strain was 1:5-5:1; In the probiotics, the total number of viable bacteria is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

2. The probiotic for improving intestinal immunity according to claim 1, characterized in that: The dosage form of the probiotics includes solution, lyophilized powder, capsule, tablet or granule.

3. The probiotic for improving intestinal immunity according to claim 1, characterized in that: The probiotics further include a freeze-drying protectant and / or a functional aid.

4. The probiotic for improving intestinal immunity according to claim 3, characterized in that: The lyoprotectant includes any one or a combination of at least two of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol; The functional additives include any one of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrospira, versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin, or a combination of at least two of them.

5. The probiotic for improving intestinal immunity according to claim 1, characterized in that: The probiotic is a freeze-dried powder, which is prepared by the following preparation method: The LRa66 strain and the BI03 strain were inoculated into a culture medium for activation and fermentation to obtain a fermentation broth; the fermentation broth was centrifuged, mixed with a lyophilization protective agent, and then freeze-dried to obtain LRa66 bacterial powder and BI03 bacterial powder; The LRa66 bacterial powder and the BI03 bacterial powder are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent for improving intestinal immunity.

6. Use of the probiotic according to any one of claims 1 to 5 in the preparation of a product for improving intestinal immunity.

7. Use of the probiotic according to any one of claims 1 to 5 in the preparation of a medicine for preventing, alleviating or treating constipation.

8. Use of the probiotic according to any one of claims 1 to 5 in the preparation of a medicine for preventing, alleviating or treating diarrhea.

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