A probiotic agent containing Bifidobacterium animalis subsp. lactis C-1 for regulating the intestinal environment and its application

Abstract

The present invention relates to a probiotic agent for regulating the intestinal environment and its application. The strains in the probiotic agent for regulating the intestinal environment include Bifidobacterium animalis subsp. Bifidobacterium animalis subsp. lactis C-1 strain and Lactobacillus gasseri Lactobacillus gasseri CKCC 1913 strain; there is a synergistic relationship between the two bacteria, which can regulate the intestinal microecological environment, relieve intestinal injury and improve the intestinal barrier function by mechanisms such as reducing the levels of inflammatory factors, balancing the content of gastrointestinal regulatory peptides, and promoting intestinal peristalsis. Therefore, this probiotic agent is suitable for preparing preparations with the efficacy of preventing, improving or treating constipation and other inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial culture, and relates to a probiotic agent for regulating the intestinal environment and its application. Background Art

[0002] A wide variety of microorganisms inhabit the gastrointestinal tract of healthy people, and these microorganisms are called the intestinal flora. The intestinal flora is combined in a certain proportion, and the bacteria restrict and depend on each other, forming an ecological balance in terms of quality and quantity. Once the internal and external environment of the body changes, such as changes in diet, use of antibiotic drugs, changes in age, intestinal immune dysfunction, etc., sensitive intestinal bacteria are inhibited, and the uninhibited bacteria take the opportunity to multiply, resulting in dysbiosis and causing symptoms of constipation in people.

[0003] Probiotics are defined as "live microorganisms", which have a health impact on the host when ingested in sufficient amounts. The intestine is the most important organ for the digestion and absorption of nutrients in humans and other animals, and its health level determines the overall health of the body. Bifidobacterium animalis ( Bifidobacterium ) is one of the dominant bacteria in the intestines of humans and many mammals, and plays an important role in aspects such as the nutrition of the body and the prevention of intestinal diseases. For example: Bifidobacterium animalis can quickly colonize the intestinal mucosa, form a beneficial bacteria barrier, densely form a bacterial film on the intestinal mucosa, so that pathogenic bacteria cannot colonize; it can also produce acetic acid, lactic acid, etc. to regulate the pH value of the intestinal environment and inhibit saprophytic bacteria; produce bacteriocin-like proteins, which have a certain bactericidal effect; Bifidobacterium animalis can decompose conjugated bile acids into free bile acids, and this kind of acid has a stronger inhibitory effect on bacteria. It can be seen that Bifidobacterium animalis is an important physiological bacterium in the intestines of humans and animals, participating in a series of physiological processes such as immunity, nutrition, digestion, and protection, and playing an important role.

[0004] Therefore, it is of great significance to develop a microbial preparation product containing probiotics that can effectively regulate the intestinal microecological environment and promote intestinal health. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a probiotic agent for regulating the intestinal environment and its application.

[0006] To achieve the purpose of this invention, the following technical solutions are adopted by the present invention:

[0007] In the first aspect, the present invention provides a Bifidobacterium animalis subsp. lactis C-1 for regulating the intestinal environment, and the taxonomic nomenclature of the Bifidobacterium animalis subsp. lactis C-1 is Bifidobacterium animalis subsp. lactis , with the preservation number of GDMCC No: 64543, and the preservation date of April 22, 2024.

[0008] A novel Bifidobacterium animalis subsp. lactis strain for regulating the intestinal environment was isolated from milk base and preserved, named Bifidobacterium animalis subsp. lactis C-1 strain. This strain can balance the levels of gastrointestinal regulatory peptides and reduce the contents of inflammatory factors such as tumor necrosis factor-α and interleukin-1β, thereby improving the intestinal microecological environment and enhancing intestinal health. Therefore, the Bifidobacterium animalis subsp. lactis C-1 strain can be used to prepare drugs with the effects of improving, preventing or treating constipation and other related intestinal diseases or other inflammatory diseases.

[0009] In a second aspect, the present invention provides a culture of Bifidobacterium animalis subsp. lactis C-1 as described in the first aspect. The culture is prepared by the following method: inoculating Bifidobacterium animalis subsp. lactis C-1 into a culture medium and culturing at 35 - 38 °C for 22 - 26 h.

[0010] Among them, the above "35 - 38 °C" can be, for example, 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C, 37.5 °C, 38 °C, etc. Other specific point values within this numerical range can be selected and will not be elaborated here one by one.

[0011] The above "22 - 26 h" can be, for example, 22 h, 22.5 h, 23 h, 23.5 h, 24 h, 24.5 h, 25 h, 25.5 h, 26 h, etc. Other specific point values within this numerical range can be selected and will not be elaborated here one by one.

[0012] In a third aspect, the present invention provides a probiotic agent for regulating the intestinal environment. The strains in the probiotic agent for regulating the intestinal environment include the Bifidobacterium animalis subsp. lactis C-1 strain as described in the first aspect.

[0013] Preferably, in the probiotic agent, the viable count of Bifidobacterium animalis subsp. lactis C-1 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g, such as 1×10 9 CFU / mL (CFU / g), 2×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 8×10 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), etc. Other specific point values within this numerical range can be selected and will not be elaborated here one by one.

[0014] Preferably, the strains in the probiotic agent for regulating the intestinal environment further include Lactobacillus gasseri Lactobacillus gasseri CKCC 1913, with the preservation number of CGMCC No. 23175 and the preservation date of August 23, 2021.

[0015] The present invention also creatively discovers that the above-mentioned Lactobacillus gasseri Lactobacillus gasseri strain CKCC 1913 can be compounded with Bifidobacterium animalis subsp. Bifidobacterium animalis subsp. lactis strain C-1 to have a more excellent effect in regulating the intestinal environment than a single bacterial agent or other compounding methods, indicating that strain CKCC 1913 and strain C-1 have a synergistic effect in regulating the intestinal microecological environment, reducing the level of inflammatory factors, and balancing the content of gastrointestinal regulatory peptides.

[0016] Preferably, the ratio of the viable count of Bifidobacterium animalis subsp. C-1 to that of strain CKCC 1913 is 1:10 - 10:1, for example, it can be 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 5:1, 6:1, 8:1, 10:1, etc. Other specific point values within this numerical range can be selected and will not be elaborated here one by one.

[0017] Preferably, the dosage form of the probiotic agent includes solution, lyophilized powder, capsule, tablet or granule.

[0018] Preferably, the probiotic agent further includes a protective agent.

[0019] Preferably, the protective agent 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.

[0020] Fourthly, the present invention provides an application of Bifidobacterium animalis subsp. C-1 as described in the first aspect, or the culture as described in the second aspect, or the probiotic agent as described in the third aspect in the preparation of a preparation with the efficacy of preventing, alleviating or treating constipation.

[0021] Fifthly, the present invention provides an application of Bifidobacterium animalis subsp. C-1 as described in the first aspect, or the culture as described in the second aspect, or the probiotic agent as described in the third aspect in the preparation of a preparation with the efficacy of preventing, alleviating or treating inflammatory diseases.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] A new strain of *Bifidobacterium animalis* subsp. *lactis* that regulates the intestinal environment was isolated from milk base and preserved in the present invention, named as *Bifidobacterium animalis* subsp. *lactis* C-1 strain. This strain can balance the levels of gastrointestinal regulatory peptides and reduce the contents of inflammatory factors such as tumor necrosis factor-α and interleukin-1β, thereby improving the intestinal microecological environment and enhancing intestinal health. Therefore, the *Bifidobacterium animalis* subsp. *lactis* C-1 strain can be used to prepare drugs with the effects of improving, preventing or treating constipation and other related intestinal diseases or other inflammatory diseases.

[0024] The taxonomic name of the C-1 strain involved in the present invention is Bifidobacterium animalis subsp. lactis , the preservation unit is the Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC No: 64543, the preservation date is April 22, 2024, and the preservation address is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou;

[0025] The taxonomic name of the CKCC 1913 strain involved in the present invention is *Lactobacillus gasseri* Lactobacillus gasseri , the preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, the preservation number is CGMCC No. 23175, the preservation date is August 23, 2021, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the Drawings

[0026] Figure 1 It is a statistical result chart of the fecal water content of each group of mice;

[0027] Figure 2 It is a statistical result chart of the first black edge excretion time and small intestine transit rate of each group of mice;

[0028] Figure 3 It is a statistical result chart of the content levels of gastrointestinal regulatory peptides in each group of mice;

[0029] Figure 4 It is a statistical result chart of the content levels of each short-chain fatty acid in the fecal samples of each group of mice;

[0030] Figure 5 It is a statistical result chart of the histopathological observation of the colon sections of the mice in the S1 group and the S7 to S9 groups;

[0031] Figure 6 It is a statistical result chart of the protein expression levels in the colon tissues of each group of mice;

[0032] Figure 7 It is a statistical result chart of the content levels of each cytokine in the colon tissues and serum samples of each group of mice. Detailed Embodiments

[0033] The technical solution of the present invention will be further described below by 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 on the present invention.

[0034] The strain information involved below is as follows:

[0035] ① The taxonomic name of the C-1 strain involved below is Bifidobacterium animalis subsp. lactis , and the deposit number is GDMCC No: 64543;

[0036] ② The taxonomic name of the CKCC 1913 strain involved below is Lactobacillus gasseri Lactobacillus gasseri , and the deposit number is CGMCC No. 23175;

[0037] ③ The CICC 24210 strain involved below is Bifidobacterium animalis subsp. CICC 24210.

[0038] The culture media and their formulations involved below are as follows:

[0039] ① MRS liquid medium: 10.0 g of beef extract, 20.0 g of glucose, 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of sodium chloride, 2.0 g of diammonium hydrogen citrate, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 1.0 g of Tween-80, made up to 1000 mL with distilled water, pH 6.2 - 6.4.

[0040] MRS solid medium: 10.0 g of beef extract, 20.0 g of glucose, 10.0 g of peptone, 5.0 g of yeast extract, 5.0 g of sodium chloride, 2.0 g of diammonium hydrogen citrate, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 1.0 g of Tween-80, 2% agar powder, made up to 1000 mL with distilled water, pH 6.2 - 6.4.

[0041] ② TPY liquid medium: Weigh 26.4 g of commercially available TPY medium powder, made up to 1000 mL with distilled water,

[0042] TPY solid medium: Weigh 26.4 g of commercially available TPY medium powder, made up to 1000 mL with distilled water, 2% agar powder.

[0043] The preparation methods of the bacterial suspension and bacterial powder involved below are as follows: After activating the strain, inoculate it into the culture medium for cultivation respectively to obtain the culture solution; centrifuge the culture solution, resuspend the bacterial cells to obtain the bacterial suspension, or further add a cryoprotectant for freeze-drying to prepare the freeze-dried bacterial powder product.

[0044] The MTL, GAS, VIP, SS and ELISA kits involved below are all purchased from Nanjing Herbogen Biotechnology Co., Ltd.

[0045] Example 1

[0046] In this example, a strain of Bifidobacterium animalis subsp. lactis that regulates the intestinal environment was isolated and screened, and the steps are as follows:

[0047] (1) Select samples isolated from milk-based products, dilute them 10-fold with normal saline with a mass concentration of 0.9% for 3 times, coat them on the solid medium, and after culturing at 37°C for 48 h, pick out the single colonies with different morphologies and colors, perform three-zone streaking on the MRS medium, invert the plate, and culture anaerobically at 37°C for 48 h. Pick out the single colonies after purification twice, expand the culture in the liquid medium at 37°C, and then preserve them with glycerol with a mass concentration of 35% and store them in a -80°C refrigerator. Example

[0048] In this example, morphological identification and 16S rRNA molecular biological identification were carried out on the strain screened in Example 1, and the steps are as follows:

[0049] (1) Morphological identification:

[0050] Inoculate the strain into the TPY medium, and after culturing anaerobically at 37°C for 48 h, observe it under the microscope. After smear and Gram staining, it was observed under the microscope that: the Gram staining was positive, and the strain morphology was short rod-shaped, milky white, semicircular convex, with a smooth and moist surface and neat edges.

[0051] (2) 16S rRNA molecular biological identification:

[0052] Take out the strain stored at -80°C, inoculate it into a centrifuge tube containing 20 mL of TPY liquid medium at a ratio of 2% (v / v), culture it at 37°C for 24 h and then perform centrifugal separation, centrifuge at 8000 rpm for 10 min, remove the supernatant, and collect the bacterial cells. Extract the genome of the strain, add universal bacterial primers for PCR amplification, and send the amplified product to a sequencing company for sequencing identification. After sequencing analysis of the strain, its 16S rRNA sequence is shown in SEQ ID No:1. Compare the sequenced sequence with the nucleic acid sequence in GeneBank, and the result shows that the strain is Bifidobacterium animalis subsp. lactis.

[0053] SEQ ID No:1:

[0054]

[0055] Based on the results of 16S rRNA molecular biological identification and morphological identification in Example 2, it was confirmed that the strain belongs to Bifidobacterium animalis subsp. lactis, named Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis , strain C-1.

[0056] Test Example 1

[0057] This test example tested the acid tolerance ability of strain C-1, and the specific steps were as follows:

[0058] Strain activation: The strain was inoculated into MRS liquid medium at an inoculation amount of 4% (v / v), and anaerobically cultured at 37 °C for 48 h to obtain C-1 seed liquid.

[0059] Take the C-1 seed liquid and inoculate it into MRS liquid medium with a pH of 4.0 at an inoculation amount of 4% (v / v), place it in a 37 °C incubator for constant temperature anaerobic incubation for 4 h, take samples for viable cell counting (N1), and use the measured value at 0 h as the blank control (N0). The survival rate of the strain can reach 54%, indicating that this strain has good acid tolerance ability.

[0060] Strain survival rate (%) = N1 / N0 × 100%

[0061] Among them, N1 represents the number of viable cells in the strain system after treatment, CFU / mL; N0 represents the initial number of viable cells in the strain system, that is, the number of viable cells measured at 0 h, CFU / mL.

[0062] Test Example 2

[0063] This test example explored the effects of strain C-1 on various indicators of mice with intestinal environment disorder (constipation) model, and the specific steps were as follows:

[0064] (1) Experimental animals: 6-week-old SPF-grade male BALB / c mice, with a body weight of 19 ± 1 g, breeding environment: temperature 25 °C, humidity: 50%, lighting 12 h. Adaptively breed with basic mouse food for one week (free access to food and water).

[0065] (2) Intervention method:

[0066] During the experiment, each group of mice had free access to food and water.

[0067] After one week of adaptive feeding, 64 mice were randomly selected and gavaged with loperamide hydrochloride at 20 mg / kg for one week to establish a constipation model. They were randomly divided into 8 groups: group S1 (C-1 bacterial suspension), group S2 (CKCC 1913 bacterial suspension), group S3 (C-1 bacterial suspension + CKCC 1913 bacterial suspension, viable bacteria ratio 10:1), group S4 (C-1 bacterial suspension + CKCC 1913 bacterial suspension, viable bacteria ratio 1:1), group S5 (C-1 bacterial suspension + CKCC 1913 bacterial suspension, viable bacteria ratio 1:10), group S6 (CICC 24210 bacterial suspension + CKCC 1913 bacterial suspension, viable bacteria ratio 10:1), group S7 as the positive control group (1 g / ml lactulose oral liquid), group S8 as the model group (normal saline), and group S9 as the blank group (normal non-modeled mice, normal saline). There were 8 mice in each group. The total viable bacteria in each of groups S1 - S6 were 1×10 9 CFU / mL. Each mouse in each group was gavaged once a day, and the gavage volume of the bacterial suspension / lactulose oral liquid / normal saline was 0.2 mL each time. The experiment was conducted for 4 weeks. The first week was for adaptive feeding, and the subsequent three weeks were for gavage treatment.

[0068] (3)Detection and analysis of related indicators

[0069] (a)Fecal water content

[0070] On the 14th and 28th days of the experiment, the mice were transferred to clean empty cages respectively, and fecal samples were collected for 3 h. During the collection period, the number of fecal pellets of each mouse was recorded, and they were separately placed into EP tubes and weighed for wet weight and dry weight to measure the fecal water content. The fecal water content was calculated by the following formula, and the measurement results are as Figure 1 shown.

[0071] Fecal water content (%) = (fecal wet weight - fecal dry weight) / fecal wet weight × 100%

[0072] Fecal water content is one of the important indicators reflecting the degree of constipation, and it is also used to determine whether the model is successfully established by measuring the fecal water content. Therefore, the fecal water content was measured on the 14th day (the 7th day of gavage) of the mouse experiment. From the results in the figure, it can be seen that compared with the blank group of normal non-modeled mice, the fecal water content of the mice in the model group decreased significantly. It indicates that after gavage with loperamide hydrochloride, the fecal water content of the mice decreased, suggesting that the mice in the model group had dry and hard feces and the constipation model was successfully established. Both C-1 and CKCC 1913 strains can increase the fecal water content of mice and improve constipation symptoms, and the effect of the C-1 bacterial suspension is the best. In addition, it was unexpectedly found that when the two strains of C-1 and CKCC 1913 were used in combination, the effect of improving constipation symptoms in mice was better.

[0073] (b)Defecation time of the first black feces, small intestine transit rate, and number of fecal pellets in constipated mice

[0074] Measurement of the defecation time of the first black feces: After the end of the 4th week of the experiment, each group of mice was orally administered 0.2 mL of 20% ink suspension, and then placed individually in a clean cage. The time at this moment was recorded as the starting time. After the mice excreted the first black feces, the corresponding time was recorded as the ending time, and the interval between the two times was the defecation time of the first black feces;

[0075] Measurement of the small intestine transit rate: After the end of the 4th week of the experiment, the mice were fasted overnight without water deprivation. 30 minutes after intragastric administration of 20% ink, they were immediately sacrificed by cervical dislocation. The sacrificed mice were placed supine on a wooden board and fixed with nails. The stomach was found in the upper left abdominal cavity of the mice, and the gastroesophageal junction was cut at the pylorus. The intestine was separated, and the connection between the small intestine and the colon was cut. The separated intestinal tube was placed on a tray, and the small intestine was gently straightened. The length of the intestinal tube was measured as the "total length of the small intestine", and the distance from the pylorus to the ink front was the "small intestine transit rate". The ink transit rate was calculated according to the following formula.

[0076] Small intestine transit rate (%) = Ink propulsion length (cm) / Total length of small intestine (cm) × 100%

[0077] The above test and calculation results are as Figure 2 shown;

[0078] Measurement of the number of fecal pellets: After the end of the 4th week of the experiment, each mouse in each group was placed individually in a cage, and feces were collected for 5 h. The number of fecal pellets of each mouse was recorded, which was the number of fecal pellets in 5 h. The results are shown in Table 1.

[0079] Table 1

[0080]

[0081] From Table 1 and the relevant test results in Figure 2 , it can be seen that the defecation time of the first black feces in S8 (model group) mice was significantly increased compared with that in S9 (control group), and the small intestine transit rate was significantly decreased. The number of fecal pellets, that is, the defecation frequency, is an important indicator reflecting the health status of the individual digestive system. Less fecal output is an important manifestation of constipation. The fecal output of S8 (model group) mice was significantly lower than that of S9 (control group). It can be seen that the constipation mouse model was successfully constructed.

[0082] After intragastric administration of C-1 bacteria and CKCC 1913 strains, the defecation time of the first black feces could be shortened, the small intestine transit rate and fecal output could be increased, and the effect of C-1 bacteria suspension was the best. In addition, it was unexpectedly found that when C-1 and CKCC 1913 strains were used in combination, intestinal peristalsis could be promoted, and the effect of improving mouse constipation symptoms was better.

[0083] (c)Analysis of the content level of gastrointestinal regulatory peptides

[0084] At the end of the 4th week of the experiment, the mice were sacrificed, blood was collected from the eye sockets, and the blood samples were allowed to stand for 30 min and then centrifuged to collect serum samples.

[0085] An ELISA kit was used to measure the content of gastrointestinal regulatory peptides in the serum, including the excitatory gastrointestinal regulatory peptides motilin (MTL) and gastrin (GAS), as well as the inhibitory gastrointestinal regulatory peptides vasoactive intestinal peptide (VIP) and somatostatin (SS). The test method was referred to the kit instruction manual, and the results are as Figure 3 shown.

[0086] It can be seen from the statistical results in the figure that the levels of excitatory neurotransmitters (MTL and GAS) in the serum of mice in S8 (model group) were significantly decreased compared with those in S9 (control group), while the levels of inhibitory neurotransmitters (VIP and SS) were significantly increased. This indicates that loperamide hydrochloride has an inhibitory effect on MTL and GAS in constipated mice and a promoting effect on VIP and SS. It can be seen that constipation can lead to changes in gastrointestinal active peptides, inhibit the movement of intestinal muscles, and thus weaken the intestinal peristalsis ability.

[0087] In the groups treated with the bacterial suspensions of strain C-1 and CKCC 1913, the levels of various gastrointestinal regulatory peptides in the serum of mice were effectively improved, and the effect of the C-1 bacterial suspension was the best. In addition, it was unexpectedly found that when the two strains of C-1 and CKCC 1913 were used in combination, the improvement effect of the related peptide index levels was better.

[0088] (d)Analysis of the content level of short-chain fatty acids

[0089] In the fourth week of the experiment, the feces of each group of mice were collected, and the content of short-chain fatty acids (SCFAs) in the feces of mice was quantitatively analyzed by gas chromatography (GC). Accurately weigh 20 mg of the fecal samples of each group of mice into a centrifuge tube, immerse them in saturated NaCl solution, mash them until there are no obvious lumps, add 20 μL of 10% H 2 SO 4 solution for acidification, and then add 500 μL of anhydrous ether for extraction. Centrifuge the extracted mixture at 18000 g for 10 min, and transfer the supernatant to a centrifuge tube containing 0.25 g of anhydrous Na 2 SO 4 for drying; after standing for 30 min, centrifuge at 12000 g for 5 min, and transfer the upper ether phase to a sample bottle for quantitative analysis. The results are as Figure 4 shown.

[0090] From the quantitative statistical results in the figure, it can be seen that the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid in the feces of S8 (model group) mice were significantly lower than those of S9 (control group). In the groups treated with the bacterial suspensions of C-1 bacteria and CKCC 1913 strains, the levels of various short-chain fatty acids in the feces of mice were increased to varying degrees, and the promotion effect of the C-1 bacterial suspension was the best. In addition, it was unexpectedly found that when the two bacteria, C-1 and CKCC 1913 strains, were used in combination, the promotion effect of related indicators was better.

[0091] (e) Histopathological observation of colon sections

[0092] At the end of the 4th week of the experiment, the mice were sacrificed, and the colon tissues of the mice in groups S1, S7, S8 and S9 were collected. After being rinsed clean with physiological saline, they were fixed overnight in a 10% (v / v) paraformaldehyde solution. Paraffin sections were made using paraffin embedding technology. After dewaxing with xylene, hematoxylin-eosin (H&E) staining was performed, and the histopathological changes of the colon sections were observed through an optical microscope. The results are as Figure 5 shown.

[0093] From the staining results in the figure, it can be seen that the colon tissues of the mice in S9 (control group) were normal, the colon wall was intact, the crypt differentiation was normal, and the goblet cells were arranged neatly without damage or folds; in S8 (model group), the goblet cells were incomplete, the colon wall was damaged, and the thickness of the muscularis mucosa was thinned; after intervention with the C-1 bacterial suspension, the damage induced by constipation could be effectively alleviated, and the thickness of the colon mucosa could be increased.

[0094] (f) Analysis of protein expression levels in colon tissues

[0095] At the end of the 4th week of the experiment, the mice were sacrificed, and the colon tissues of the mice in each group were collected. According to the operation instructions of the RNA Easy Fast Animal Tissue / Cell RNA Extraction Kit, the RNA in the colon tissues of the mice was extracted. After reverse transcription into cDNA using the reverse transcription (ABScriptIII RT Master Mix for qPCR) kit, real-time fluorescence quantitative PCR was performed using the kit (2X Universal SYBRGreen Fast qPCR Mix) to analyze the expression levels of mucin-2 (MUC-2), zonula occludens protein-1 (ZO-1), claudin-1 and occludin. The results are as Figure 6 shown.

[0096] It can be seen from the detection and statistical results in the figure that the expression levels of mucin, zonula occludens protein, claudin, and occludin in the colon of S8 (model group) mice were all significantly reduced. Due to the destruction of the intestinal mucosal layer caused by constipation, the subsequent inflammatory reaction affects the related proteins between epithelial cells, thereby changing the intestinal permeability. In the groups treated with the C-1 bacteria and CKCC 1913 bacterial suspensions, the expression of the four proteins showed an upward trend to varying degrees, and the C-1 bacterial suspension had the best improvement effect. In addition, it was unexpectedly found that when the C-1 and CKCC 1913 strains were used in combination, the improvement effect of the relevant indicators was better.

[0097] (g)Analysis of inflammatory marker levels

[0098] The real-time fluorescence quantitative PCR analysis method was used to detect the inflammatory factors in the colon tissue, and the detection indicators included tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10);

[0099] The ELISA kit was used to detect the levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10) in the serum of mice;

[0100] The above detection results are all as Figure 7 shown.

[0101] IL-1β and TNF-α are two highly efficient pro-inflammatory factors that play important roles in the inflammatory reaction. Among them, IL-1β can guide inflammatory cells into the lesion site, promote the expression of vascular leukocyte adhesion molecules, and cause an inflammatory reaction; while TNF-α mainly induces other inflammatory factors in a variety of cells at the initial stage of inflammation, producing an important synergistic effect; IL-10 is a multi-cell source and multi-functional cytokine that plays an important regulatory role in the immune system. It can regulate cell growth and differentiation and participate in inflammatory and immune responses.

[0102] From the detection and statistical results in the figure, it can be seen that the relative expression levels of IL-1β and TNF-α in the colon tissue and serum of S8 (model group) mice are significantly increased compared with those of S9 (control group), while the relative expression level of the anti-inflammatory factor IL-10 is significantly decreased. It can be seen that the intestinal environment of constipated mice is imbalanced and an inflammatory response occurs. After intragastric administration of C-1 bacteria and CKCC 1913 strains, the relative expression levels of IL-1β and TNF-α in the colon can be reduced to varying degrees, and the relative expression level of IL-10 can be up-regulated, reducing the damage of inflammatory cells to intestinal tissues. Moreover, the effect of the C-1 bacteria suspension is the best. In addition, it was unexpectedly found that when C-1 and CKCC 1913 strains are used in combination, the release of inflammatory factors in the intestine can be reduced, and the effect of relieving intestinal injury is better.

[0103] The applicant declares that the technical solution of the present invention is illustrated by 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.

[0104] 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.

[0105] In addition, it should be noted that, in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A probiotic for regulating the intestinal environment, characterized in that: The strains in the probiotic for regulating the intestinal environment include Bifidobacterium animalis subsp. lactis and Bifidobacterium spp. Bifidobacterium animalis subsp. lactis C-1 strain and Lactobacillus gasseri Lactobacillus gasseri CKCC 1913 strain; The preservation number of the animal Bifidobacterium lactis subspecies C-1 is GDMCC No: 64543, and the preservation date is April 22, 2024; the preservation number of the Lactobacillus gasseri CKCC 1913 is CGMCC No. 23175, and the preservation date is August 23, 2021.

2. The probiotic for regulating the intestinal environment according to claim 1, characterized in that: In the probiotic, the viable count of the animal Bifidobacterium lactis subspecies C-1 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

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

4. The probiotic for regulating the intestinal environment according to claim 1, characterized in that: The probiotics also include a protective agent; The protective agent includes any one of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol, or a combination of at least two thereof.

5. Use of the probiotic according to any one of claims 1 to 4 in the preparation of a preparation for preventing, alleviating or treating constipation.

6. Use of the probiotic according to any one of claims 1 to 4 in balancing gastrointestinal regulating peptides.

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