Probiotic agent for regulating intestinal environment and application thereof

Abstract

The invention relates to a probiotic agent for regulating an intestinal environment and an application of the probiotic agent. The bacterial strains in the probiotic agent for regulating the intestinal environment comprise a Bifidobacterium animalis subsp. Lactis C-1 bacterial strain and a Lactobacillus gasseri CKCC 1913 bacterial strain, and the bacterial strains in the probiotic agent for regulating the intestinal environment comprise a lactobacillus plantarum subsp. Lactis C-1 bacterial strain and a lactobacillus gasseri CKCC 1913 bacterial strain, a lactobacillus plantarum subsp. Lactis CKCC 1913 bacterial strain, a lactobacillus plantarum subsp. Lactis CKCC 1913 bacterial strain and a lactobacillus plantarum subsp. Lactis CKCC 1913 bacterial strain. A synergistic interaction relationship exists between the two bacteria, so that the intestinal micro-ecological environment can be regulated, the intestinal injury can be relieved and the intestinal barrier function can be improved by reducing the level of inflammatory factors, balancing the content of gastrointestinal regulatory peptides, promoting intestinal peristalsis and other action mechanisms. Therefore, the probiotic agent is suitable for preparing a preparation with the efficacy of preventing, improving or treating constipation and other inflammatory diseases.

Description

Technical Field

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

[0002] A wide variety of microorganisms live in the gastrointestinal tract of healthy people. These microorganisms are called intestinal flora. Intestinal flora is composed in a certain proportion, and each bacteria restricts and depends on each other, forming an ecological balance in quality and quantity. However, once the internal and external environment of the body changes, such as changes in diet, use of antibiotics, age changes, intestinal immune dysfunction, etc., sensitive intestinal bacteria are suppressed, and the unsuppressed bacteria take the opportunity to multiply, thus causing flora imbalance and causing constipation symptoms.

[0003] Probiotics are defined as "live microorganisms" that, when ingested in sufficient quantities, have a health impact on the host. The intestine is the most important organ for the digestion and absorption of nutrients in humans and other animals, and its health determines the overall health of the body. Bifidobacterium ) is one of the dominant bacteria in the intestines of humans and many mammals, and plays an important role in the body's nutrition and prevention of intestinal diseases. For example: Animal Bifidobacterium can quickly colonize in the intestinal mucosa, forming a barrier of beneficial bacteria, densely covering the intestinal mucosa to form a biofilm, making it impossible for pathogenic bacteria to colonize; it can also produce acetic acid and lactic acid to regulate the pH value of the intestinal environment and inhibit saprophytic bacteria; it produces bacteriocin-like proteins, which have a certain bactericidal effect; Animal Bifidobacterium can decompose coupled bile acids into free bile acids, which have a stronger inhibitory effect on bacteria. It can be seen that Animal Bifidobacterium is an important physiological bacteria 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] In view of the deficiencies of the prior art, the object of the present invention is to provide a probiotic agent for regulating the intestinal environment and its application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an animal Bifidobacterium lactis subspecies C-1 for regulating the intestinal environment, wherein the classification of the animal Bifidobacterium lactis subspecies C-1 is named Bifidobacterium animalis subsp. lactis , the deposit number is GDMCC No: 64543, and the deposit date is April 22, 2024.

[0007] The present invention separates and preserves a new strain of animal Bifidobacterium lactis subspecies that regulates the intestinal environment from milk base, and names it as animal Bifidobacterium lactis subspecies C-1 strain. The strain can balance the level of gastrointestinal regulatory peptides, reduce the content of inflammatory factors such as tumor necrosis factor-α, interleukin-1β, etc., thereby improving the intestinal microecological environment and improving the health of the intestine. Therefore, the animal Bifidobacterium lactis subspecies C-1 strain can be used to prepare a drug that has the effect of improving, preventing or treating constipation and other related intestinal diseases or other inflammatory diseases.

[0008] In a second aspect, the present invention provides a culture of Bifidobacterium animalis subsp. lactis C-1 as described in the first aspect, wherein 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.

[0009] Among them, the above-mentioned "35-38℃" can be, for example, 35℃, 35.5℃, 36℃, 36.5℃, 37℃, 37.5℃, 38℃, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here.

[0010] The above-mentioned "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 the numerical range can be selected, and they will not be described one by one here.

[0011] In a third aspect, the present invention provides a probiotic for regulating the intestinal environment, wherein the strain in the probiotic for regulating the intestinal environment comprises the Bifidobacterium animalis subsp. lactis C-1 strain as described in the first aspect.

[0012] Preferably, 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, for example 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 described here one by one.

[0013] Preferably, the strains in the probiotics for regulating the intestinal environment also include Lactobacillus gasseri Lactobacillus gasseri CKCC 1913, the deposit number is CGMCC No. 23175, and the deposit date is August 23, 2021.

[0014] The present invention also creatively discovered that the above-mentioned Lactobacillus gasseri Lactobacillus gasseri CKCC 1913 strain can bind to Bifidobacterium animalis subsp. Bifidobacterium animalis subsp. lactis The compound of C-1 strain has a better effect in regulating the intestinal environment than single bacterial agents or other compound methods, indicating that CKCC 1913 bacteria and C-1 strain have synergistic effects in regulating the intestinal microecological environment, reducing the level of inflammatory factors and balancing the content of gastrointestinal regulatory peptides.

[0015] Preferably, the ratio of the viable counts of animal Bifidobacterium lactis subsp. C-1 and 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 they will not be described one by one here.

[0016] Preferably, the dosage form of the probiotic includes solution, lyophilized powder, capsule, tablet or granule.

[0017] Preferably, the probiotic further comprises a protective agent.

[0018] Preferably, the protective agent comprises 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.

[0019] In a fourth aspect, the present invention provides a use of the animal Bifidobacterium lactis subsp. C-1 as described in the first aspect, the culture as described in the second aspect, or the probiotic agent as described in the third aspect in the preparation of a preparation having the efficacy of preventing, alleviating or treating constipation.

[0020] In a fifth aspect, the present invention provides a use of the animal Bifidobacterium lactis subsp. C-1 as described in the first aspect, the culture as described in the second aspect, or the probiotic agent as described in the third aspect in the preparation of a preparation having the efficacy of preventing, alleviating or treating inflammatory diseases.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention separates and preserves a new strain of animal Bifidobacterium lactis subspecies that regulates the intestinal environment from milk base, and names it as animal Bifidobacterium lactis subspecies C-1 strain. The strain can balance the level of gastrointestinal regulatory peptides, reduce the content of inflammatory factors such as tumor necrosis factor-α, interleukin-1β, etc., thereby improving the intestinal microecological environment and improving the health of the intestine. Therefore, the animal Bifidobacterium lactis subspecies C-1 strain can be used to prepare a drug that has the effect of improving, preventing or treating constipation and other related intestinal diseases or other inflammatory diseases.

[0022] The classification of the C-1 strain involved in the present invention is named Bifidobacterium animalis subsp. lactis The depository is Guangdong Microbiological Culture Collection Center, the deposit number is GDMCC No: 64543, the deposit date is April 22, 2024, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; The CKCC 1913 strain involved in the present invention is classified as Lactobacillus gasseri Lactobacillus gasseri The depository is the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit number is CGMCC No. 23175, the deposit date is August 23, 2021, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the statistical result of fecal water content of each group of mice; Figure 2 This is the statistical result of the first black-border excretion time and small intestinal running rate of each group of mice; Figure 3 This is a statistical result diagram of the gastrointestinal regulatory peptide content levels in each group of mice; Figure 4 This is a statistical result chart of the content levels of various short-chain fatty acids in the fecal samples of each group of mice; Figure 5 This is a graph showing the statistical results of histopathological observation of colon sections of mice in groups S1 and S7 to S9; Figure 6 This is the statistical result diagram of the expression level of each protein in the colon tissue of each group of mice; Figure 7 This is a statistical graph of the levels of various cytokines in the colon tissue and serum samples of each group of mice. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0025] The following bacterial species information is: ① The classification and nomenclature of the C-1 strains involved below are Bifidobacterium animalis subsp. lactis , the deposit number is GDMCC No: 64543; ② The CKCC 1913 strain involved below is named as Lactobacillus gasseri Lactobacillus gasseri , the deposit number is CGMCC No. 23175; ③ The CICC 24210 strain involved in the following is Bifidobacterium animalis subspecies lactis CICC 24210.

[0026] The culture medium and its formula involved below are: ① MRS liquid medium: beef extract 10.0 g, glucose 20.0 g, peptone 10.0 g, yeast extract 5.0 g, sodium chloride 5.0 g, diammonium hydrogen citrate 2.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, Tween-80 1.0 g, distilled water to 1000 mL, pH 6.2-6.4.

[0027] MRS solid medium: beef extract 10.0 g, glucose 20.0 g, peptone 10.0 g, yeast extract 5.0 g, sodium chloride 5.0 g, diammonium hydrogen citrate 2.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, Tween-80 1.0 g, 2% agar powder, distilled water to 1000 mL, pH 6.2-6.4.

[0028] ② TPY liquid medium: weigh 26.4 g of commercially available TPY medium powder and dilute to 1000 mL with distilled water. TPY solid medium: weigh 26.4 g of commercially available TPY medium powder, dilute to 1000 mL with distilled water, and add 2% agar powder.

[0029] The preparation method of bacterial suspension and bacterial powder involved below is: after activating the strain, inoculate it into culture medium for cultivation to obtain culture solution; centrifuge the culture solution, resuspend the bacteria to obtain bacterial suspension, or further add protective agent for freeze drying to obtain freeze-dried bacterial powder product.

[0030] The MTL, GAS, VIP, SS and ELISA kits mentioned below were purchased from Nanjing Herb Source Biotechnology Co., Ltd. Example 1

[0031] In this example, a strain of Bifidobacterium animalis lactis subsp. lactis that regulates the intestinal environment was isolated and screened, and the steps were as follows: (1) Select samples separated from milk base, dilute them 10 times with 0.9% saline, dilute them 3 times, spread them on solid culture medium, culture them at 37℃ for 48 hours, pick out single colonies with different morphology and color, draw three lines in MRS culture medium, invert the plate, and culture them anaerobically at 37℃ for 48 hours. Pick out single colonies purified twice, expand them with liquid culture medium at 37℃, preserve them with 35% glycerol, and freeze them in a -80℃ refrigerator. Example 2

[0032] In this example, the strains screened in Example 1 were subjected to morphological identification and 16S rRNA molecular biological identification, and the steps were as follows: (1) Morphological identification: The strain was inoculated into TPY medium, cultured anaerobically at 37°C for 48 h, and then observed under a microscope. After smear and Gram staining, it was observed that the Gram staining was positive, the strain was short rod-shaped, milky white, with semicircular protrusions, smooth and moist surface, and neatly edged colonies.

[0033] (2) 16S rRNA molecular biological identification: The strain stored at -80℃ was taken out and inoculated into a centrifuge tube containing 20mL TPY liquid medium at a ratio of 2% (v / v). After culturing at 37℃ for 24 hours, it was centrifuged and centrifuged at 8000 rpm for 10 minutes. The supernatant was removed and the bacteria were collected. The genome of the strain was extracted, and bacterial universal primers were added for PCR amplification. The amplified product was sent to a sequencing company for sequencing identification. The strain was sequenced and analyzed, and its 16S rRNA sequence was shown in SEQ ID No: 1. The sequence obtained by sequencing was compared with the nucleic acid sequence in GeneBank, and the results showed that the strain was animal Bifidobacterium lactis subsp.

[0034] SEQ ID No: 1:

[0035] Based on the results of 16S rRNA molecular biological identification and morphological identification in Example 2, it was confirmed that the strain belonged to Bifidobacterium animalis subsp. lactis and was named Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis , C-1 strain.

[0036] Test Example 1 This test example tests the acid resistance of strain C-1. The specific steps are as follows: Activation of strains: The strain was inoculated into MRS liquid culture medium at an inoculum size of 4% (v / v), and cultured anaerobically at 37°C for 48 h to obtain C-1 seed solution.

[0037] The C-1 seed solution was inoculated at a 4% inoculation rate (v / v) into MRS liquid culture medium with a pH of 4.0, and incubated in a 37°C incubator for 4 h. Samples were taken for live bacteria count (N1), and the 0 h measurement value was used as the blank control (N0). The strain survival rate was as high as 54%, indicating that this strain had good acid resistance.

[0038] Strain survival rate (%) = N1 / N0×100% Among them, N1 represents the number of viable bacteria in the strain system after treatment, CFU / mL; N0 represents the initial number of viable bacteria in the strain system, that is, the number of viable bacteria measured at 0 h, CFU / mL.

[0039] Test Example 2 This test case explores the effects of the C-1 strain on various indicators of mice with intestinal environmental imbalance (constipation). The specific steps are as follows: (1) Experimental animals: 6-week-old SPF male BALB / c mice, weighing 19±1g, housed in a temperature of 25°C, humidity of 50%, and lighting for 12 h. They were fed with a basic mouse diet for one week (free access to food and water).

[0040] (2) Intervention methods: During the experiment, mice in each group had free access to food and water.

[0041] After one week of adaptive feeding, 64 mice were randomly selected and given loperamide hydrochloride by gavage at 20 mg / kg for one week to establish a constipation model. The mice were randomly divided into 8 groups: S1 group (C-1 bacterial suspension), S2 group (CKCC 1913 bacterial suspension), S3 group (C-1 bacterial suspension + CKCC 1913 bacterial suspension, the ratio of live bacteria was 10:1), S4 group (C-1 bacterial suspension + CKCC 1913 bacterial suspension, the ratio of live bacteria was 1:1), S5 group (C-1 bacterial suspension + CKCC 1913 bacterial suspension, the ratio of live bacteria was 1:10), S6 group (CICC 24210 bacterial suspension + CKCC1913 bacterial suspension, the ratio of live bacteria was 10:1), and S7 group was the positive control group (1 g / ml lactulose oral solution), S8 group was the model group (normal saline), S9 group was the blank group (normal mice without modeling, normal saline), each group had 8 mice, and the total viable bacteria in each group of S1-S6 was 1×10 9 CFU / mL. Each group of mice was gavaged once a day, and the gavage volume of each bacterial suspension / lactulose oral solution / normal saline was 0.2 mL. The experiment lasted for 4 weeks, with the first week being adaptive feeding and the following three weeks being gavage treatment.

[0042] (3) Detection and analysis of relevant indicators (a) Fecal water content On the 14th and 28th days of the experiment, the mice were transferred to clean empty cages and fecal samples were collected for 3 hours. 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, and the fecal water content was determined. The fecal water content was calculated using the following formula. The results are shown in the figure. Figure 1 shown.

[0043] Fecal moisture content (%) = (fecal wet weight - fecal dry weight) / fecal wet weight × 100% Fecal water content is one of the important indicators reflecting the degree of constipation. It is also determined by measuring the fecal water content to determine whether the model is successful. Therefore, this experiment measured the fecal water content on the 14th day of the mouse experiment (the 7th day of gavage). As shown in the figure, the water content of the model group mice was significantly reduced compared with the normal unmodeled mice in the blank group. It shows that after gavage with loperamide hydrochloride, the water content of the mouse feces decreased, indicating that the model group mice had dry and hard feces, and the constipation model was successfully established. Both C-1 and CKCC 1913 strains can increase the water content of mouse feces and improve constipation symptoms, and the C-1 bacterial suspension has the best effect. In addition, it was unexpectedly found that when the C-1 and CKCC 1913 strains were used in combination, the effect of improving the constipation symptoms of mice was better.

[0044] (b) Time of first black stool excretion, small intestinal transit rate and number of fecal particles in constipated mice Determination of the time of first black stool discharge: After the fourth week of the experiment, each group of mice was orally infused with 0.2 mL of 20% ink suspension and placed in a clean cage. This time was recorded as the start time. After the mouse discharged the first black stool, the corresponding time was recorded as the end time. The interval between the two times was the time of first black stool discharge. Determination of small intestinal transit rate: After the fourth week of the experiment, the mice were fasted overnight but not watered. They were gavaged with 20% ink for 30 minutes and immediately killed by cervical dislocation. The killed mice were placed supinely on a wooden board and fixed with nails. The stomach was found in the upper left abdominal cavity of the mouse, the gastroesophageal connection was cut at the pyloric opening, the intestines were 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 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 the "small intestinal transit rate". The ink transit rate was calculated according to the following formula.

[0045] Small intestinal transit rate (%) = ink propulsion length (cm) / total small intestinal length (cm) × 100% The above test and calculation results are as follows Figure 2 As shown; Determination of fecal pellet number: After the fourth week of the experiment, each group of mice was placed in a cage, and feces were collected for 5 hours. The number of fecal pellets of each mouse was recorded, which was the 5-hour fecal pellet number. The results are shown in Table 1.

[0046] Table 1

[0047] Through Table 1 and Figure 2 The relevant test results show that the time of first black stool discharge of S8 (model group) mice was significantly longer than that of S9 (control group), and the small intestinal transit rate was significantly reduced. The number of feces, that is, the number of defecation, is an important indicator of the health of an individual's digestive system. Less feces discharge is an important manifestation of constipation. The feces discharge of S8 (model group) mice was significantly lower than that of S9 (control group), indicating that the constipation mouse model was successfully constructed.

[0048] Both C-1 and CKCC 1913 strains can shorten the time of first black stool excretion and increase small intestinal transit rate and stool volume after oral gavage, and C-1 bacterial suspension has the best effect. In addition, it was unexpectedly found that the combined use of C-1 and CKCC 1913 strains can promote intestinal peristalsis and improve constipation symptoms in mice.

[0049] (c) Analysis of gastrointestinal regulatory peptide levels After the fourth week of the experiment, the mice were killed, blood was collected from the eyeballs, and the blood samples were allowed to stand for 30 min before centrifugation to collect serum samples.

[0050] ELISA kits were used to determine the levels of gastrointestinal regulatory peptides in serum, including excitatory gastrointestinal regulatory peptides motilin (MTL) and gastrin (GAS) and inhibitory gastrointestinal regulatory peptides vasoactive intestinal peptide (VIP) and somatostatin (SS). The test method was referred to the instructions of the kit. The results were as follows: Figure 3 shown.

[0051] From the statistical results in the figure, we can see that the levels of excitatory neurotransmitters (MTL and GAS) in the serum of S8 (model group) mice were significantly decreased compared with those of S9 (control group), and the levels of inhibitory neurotransmitters (VIP and SS) were significantly increased, indicating that loperamide hydrochloride has an inhibitory effect on MTL and GAS of 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.

[0052] In the groups treated with C-1 and CKCC 1913 bacterial suspensions, the levels of various gastrointestinal regulation-related peptides in mouse serum were effectively improved, and the effect of C-1 bacterial suspension was the best. In addition, it was unexpectedly found that when C-1 and CKCC 1913 strains were used in combination, the improvement effect of related peptide index levels was better.

[0053] (d) Analysis of short-chain fatty acid content In the fourth week of the experiment, the feces of mice in each group were collected, and the content of short-chain fatty acids (SCFAs) in the feces of mice was quantitatively analyzed by gas chromatography (GC). 20 mg of fecal samples from each group of mice were accurately weighed into a centrifuge tube, immersed in a saturated NaCl solution, and mashed until there were no obvious lumps. 20 μL of 10% H2SO4 solution was added for acidification, and then 500 μL of anhydrous ether was added for extraction. The extracted mixture was centrifuged at 18000 g for 10 min, and the supernatant was transferred to a centrifuge tube containing 0.25 g of anhydrous Na2SO4 for drying; after standing for 30 min, it was centrifuged at 12000 g for 5 min, and the upper ether phase was transferred to a sample bottle for quantitative analysis. The results are as follows: Figure 4 shown.

[0054] From the quantitative statistical results in the figure, we can see 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 C-1 bacteria and CKCC 1913 strain bacterial suspensions, the contents of various short-chain fatty acids in the feces of mice were increased to varying degrees, and the C-1 bacterial suspension had the best improvement effect. In addition, it was unexpectedly found that when C-1 and CKCC 1913 strains were used in combination, the improvement effect of relevant indicators was better.

[0055] (e) Histopathological observation of colon sections At the end of the fourth week of the experiment, the mice were killed, and the colon tissues of the mice in the S1, S7, S8 and S9 groups were collected, rinsed with normal saline, and fixed overnight in a 10% paraformaldehyde solution. Paraffin sections were made using paraffin embedding technology, and hematoxylin-eosin staining was performed after xylene dewaxing. The pathological changes of colon sections were observed under an optical microscope. The results are as follows: Figure 5 shown.

[0056] The staining results in the figure show that the colon tissue of mice in S9 (control group) is normal, the colon wall is intact, the crypts are normally differentiated, and the goblet cells are neatly arranged without damage or wrinkles; the goblet cells in S8 (model group) are incomplete, the colon wall is damaged, and the thickness of the mucosal muscular layer is thinner; intervention with C-1 bacterial suspension can effectively alleviate constipation-induced damage and increase the thickness of the colon mucosa.

[0057] (f) Analysis of protein expression levels in colon tissue At the end of the fourth week of the experiment, the mice were killed, and the colon tissues of each group of mice were collected. The RNA in the mouse colon tissues was extracted according to the instructions of the RNA Easy Fast Animal Tissue / Cell RNA Extraction Kit. After reverse transcription into cDNA using a reverse transcription (ABScriptIII RT Master Mix for qPCR) kit, real-time fluorescence quantitative PCR was performed using a kit (2X Universal SYBRGreen Fast qPCR Mix) to analyze the expression levels of mucin-2 (MUC-2), zonula occludens-1 (ZO-1), tight junction protein-1 (Claudin-1), and occudin. The results are shown in Figure 2. Figure 6 shown.

[0058] From the statistical results of the tests in the figure, we can see that the expression levels of mucin, zonula occludens, tight junction proteins and occludens in the colon of S8 (model group) mice were significantly reduced. The intestinal mucosal layer was destroyed due to constipation, which led to an inflammatory response. The inflammatory response affected the related proteins between epithelial cells, thereby changing the permeability of the intestine. In the groups treated with C-1 bacteria and CKCC 1913 strain 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 C-1 and CKCC 1913 strains were used in combination, the improvement effect of related indicators was better.

[0059] (g) Analysis of inflammatory marker levels Real-time fluorescence quantitative PCR analysis was used to detect inflammatory factors in colon tissue, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10); ELISA kits were used to detect the levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-10 (IL-10) in mouse serum; The above test results are as follows Figure 7 shown.

[0060] IL-1β and TNF-α are two highly effective inflammatory factors that play an important role in inflammatory responses. IL-1β can guide inflammatory cells into the lesion site, promote the expression of vascular leukocyte adhesion molecules, and cause inflammatory responses; TNF-α mainly induces multiple cells to produce other inflammatory factors in the early stages of inflammation, producing important synergistic effects; IL-10 is a multi-cell-derived, multifunctional 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.

[0061] The statistical results in the figure show that the relative expression of IL-1β and TNF-α in the colon tissue and serum of S8 (model group) mice increased significantly compared with S9 (control group), while the relative expression level of the anti-inflammatory factor IL-10 decreased significantly, indicating that the intestinal environment of constipated mice was unbalanced and produced an inflammatory response. After oral administration of C-1 bacteria and CKCC 1913 strains, the relative expression of IL-1β and TNF-α in the colon can be reduced to varying degrees, and the relative expression of IL-10 can be increased, reducing the damage of inflammatory cells to intestinal tissue, and the C-1 bacterial suspension has the best effect. In addition, it was unexpectedly found that the combined use of C-1 and CKCC 1913 strains can reduce the release of inflammatory factors in the intestine and have a better effect in alleviating intestinal damage.

[0062] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0063] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of 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.

[0064] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A probiotic agent for regulating the intestinal environment, characterized in that The strains in the probiotics for regulating the intestinal environment include Bifidobacterium animalis subsp. lactis 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 ratio of the live bacteria count of Bifidobacterium animalis subspecies lactis C-1 to Lactobacillus gasseri CKCC 1913 is 1:10-10:

1.

4. 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.

5. 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.

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

7. Use of the probiotic according to any one of claims 1 to 5 in balancing gastrointestinal regulating peptides.

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