Lactobacillus paracasei metaplasm with constipation relieving effect and application of lactobacillus paracasei metaplasm

Abstract

The invention discloses a Lactobacillus paracasei metagen with a constipation relieving effect and application thereof, and belongs to the field of microorganisms. The lactobacillus paracasei CCFM1451 provided by the invention is relatively good in activity, and has certain acid and alkali resistance and adhesion. According to the present invention, with the application of the lactobacillus paracasei CCFM1451, the inflammation level in the colon tissue can be significantly reduced, the pathological damage of the colon tissue can be improved, the levels of the proinflammatory factor and the anti-inflammatory factor can be adjusted, the aquaporin expression level can be reduced, and the motilin MTL content in the colon tissue can be increased; the content of vasoactive intestinal peptide VIP in the colon tissue is reduced; the compound has the advantages of enhancing intestinal tract movement, shortening intestinal tract transfer time and regulating intestinal tract steady state, and has a huge application prospect in preparation of medicines for relieving constipation.

Description

Technical Field

[0001] The invention relates to a Lactobacillus paracasei postbiotic with constipation relief effect and application thereof, belonging to the field of microorganisms. Background Art

[0002] Constipation is a common functional gastrointestinal disease, and its clinical symptoms mainly include: less bowel movements, low frequency, dry stools, and prolonged gastrointestinal emptying time. In recent years, with the aging of society, the accelerated pace of modern life, the improvement of people's living standards, changes in dietary structure, and the influence of social and psychological factors, the incidence of constipation in my country has been increasing year by year.

[0003] Many previous studies have shown that probiotics can be used to relieve constipation and treat a variety of gastrointestinal diseases. They can work through different mechanisms such as strengthening the intestinal barrier, improving the immune system, regulating metabolic processes and the brain-gut axis. However, there is also new scientific evidence that the beneficial effects of probiotics are not necessarily directly related to live bacteria. The metabolites and bacterial components of live bacteria may be important drivers of health promotion, making postbiotics (i.e., components and / or metabolites of probiotics) a new research direction as a potential probiotic substitute. These active ingredients can maintain the integrity of the intestinal barrier by regulating the intestinal flora, the nervous system, and promoting immune function, thereby relieving symptoms of gastrointestinal diseases such as constipation, diarrhea, irritable bowel syndrome, inflammatory bowel disease, and colorectal cancer. In addition, the drug resistance and safety of probiotics have gradually attracted people's attention. Scientific research shows that compared with probiotics, postbiotics have advantages such as good absorption, metabolism, clear chemical structure, and safe dosage parameters.

[0004] In summary, as people gain a deeper understanding of intestinal health, the role of postbiotics in the prevention and treatment of intestinal diseases has been recognized. The application of postbiotics has become a trend in the international market. Screening postbiotics that can relieve constipation and exploring the mechanism are of great significance for solving gastrointestinal health problems. Summary of the invention

[0005] [Technical issues]

[0006] The technical problem to be solved by the present invention is to provide a Lactobacillus paracasei postbiotic capable of relieving constipation and to provide application of the strain.

[0007] [Technical solution]

[0008] In view of the problem of exploring the mechanism of action of Lactobacillus paracasei postbiotics in relieving constipation at this stage, the present invention provides a strain of Lactobacillus paracasei, which can significantly reduce the level of inflammation in colon tissue, improve colon tissue pathological damage, and regulate the levels of pro-inflammatory and anti-inflammatory factors, reduce the expression level of aquaporins, and increase the content of motilin MTL in colon tissue; reduce the content of vasoactive intestinal peptide VIP in colon tissue, enhance intestinal peristalsis, shorten intestinal transit time, and regulate intestinal homeostasis. Provide corresponding postbiotic preparations, fermented foods and functional foods, so as to play the role of relieving constipation.

[0009] The present invention provides a strain of Lactobacillus paracasei, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, with a deposit number of GDMCC No: 65316.

[0010] In one embodiment of the present invention, the Lacticaseibacillusparacasei is from the feces of a 24-year-old woman in Fujian Province. The strain is sequenced and analyzed, and the sequence obtained by sequencing is compared with the nucleic acid sequence in NCBI Standard Nucleotide BLAST. The result shows that the similarity with the nucleic acid sequence of the genus Lacticaseibacillusparacasei is 100%; the result shows that the strain is Lacticaseibacillusparacasei, and it is named Lacticaseibacillusparacasei CCFM1451.

[0011] In one embodiment of the present invention, the Lactobacillus paracasei CCFM1451 has the following biological characteristics:

[0012] (1) Bacterial characteristics: Gram-positive, non-spore-forming, non-motile bacteria.

[0013] (2) Colony characteristics: round, smooth, and with neat edges.

[0014] (3) Growth characteristics: Under constant temperature of 37°C, the cells reached the late logarithmic phase after about 18 h of culture in MRS medium.

[0015] (4) It has strong tolerance to simulated gastrointestinal fluid.

[0016] (5) Significantly reduce the level of inflammation in colon tissue, improve colon tissue pathological damage, regulate the levels of pro-inflammatory and anti-inflammatory factors, reduce the expression level of aquaporin, increase the content of motilin (MTL) in colon tissue; reduce the content of vasoactive intestinal peptide (VIP) in colon tissue; enhance intestinal peristalsis, shorten intestinal transit time, and regulate intestinal homeostasis.

[0017] The invention also provides a microbial preparation containing the Lactobacillus paracasei CCFM1451.

[0018] The invention also provides postbiotics prepared by the Lactobacillus paracasei CCFM1451.

[0019] In one embodiment of the present invention, the postbiotics include inactivated bacteria, bacterial lysate and / or inactivated fermentation broth.

[0020] The present invention also provides a method for preparing the postbiotics, comprising (a) or (b):

[0021] (a) culturing the Lactobacillus paracasei CCFM1451 to a logarithmic growth phase, inactivating the cells by heat treatment, and centrifuging the cells to discard the supernatant to obtain postbiotics;

[0022] (b) culturing the Lactobacillus paracasei CCFM1451 to a logarithmic growth phase, inactivating it by heat treatment, and then crushing it by high-pressure homogenization to obtain postbiotics.

[0023] In one embodiment of the present invention, the postbiotics are processed into powder by a drying method and are used directly or dissolved in physiological saline for use.

[0024] In one embodiment of the present invention, the viable count of Lactobacillus paracasei CCFM1451 before inactivation is at least 8×10 9 CFU / mL or 10 9 CFU / g.

[0025] The invention also provides a product containing the Lactobacillus paracasei CCFM1451 and / or its postbiotics, and the product includes food, medicine or health care product.

[0026] In one embodiment of the present invention, the food is a fermented food, which is produced by fermentation with Lactobacillus paracasei CCFM1451 and then pasteurized. The pasteurized food includes solid food, liquid food, and semi-solid food.

[0027] In one embodiment of the present invention, the food includes dairy products, soy products or fruit and vegetable products.

[0028] In one embodiment of the present invention, the dairy product is a fermented dairy product, including fermented milk, fermented milk beverages, cream, cheese or milk powder; the soy product includes soy milk, soy milk beverages, soy milk powder; the fruit and vegetable products include fermented fruit and vegetable drinks or foods fermented with cabbage, white radish, cucumber, beet, yellow peach or bayberry as raw materials.

[0029] In one embodiment of the present invention, the medicine comprises Lactobacillus paracasei CCFM1451 and a pharmaceutically acceptable carrier.

[0030] In one embodiment of the present invention, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0031] In one embodiment of the present invention, the dosage form of the drug includes granules, capsules, tablets, pills or oral liquids.

[0032] The present invention also provides the use of the Lacticaseibacillus paracasei CCFM1451 and / or its postbiotics in preparing a medicine for relieving constipation.

[0033] In one embodiment of the present invention, the drug has at least one of the following functions:

[0034] (a) Improve colon tissue pathological damage;

[0035] (b) Reduce the expression of water channel protein AQP4 in colon tissue;

[0036] (c) reducing the expression of aquaporin AQP8 in colon tissue;

[0037] (d) reduce the expression of pro-inflammatory cytokine IL-1β in colon tissue;

[0038] (e) Increase the expression of anti-inflammatory factor IL-4 in colon tissue;

[0039] (f) Increase the content of motilin (MTL) in colon tissue;

[0040] (g) reducing the content of vasoactive intestinal polypeptide (VIP) in colon tissue;

[0041] (h) Enhance intestinal peristalsis, shorten intestinal transit time, and regulate intestinal homeostasis.

[0042] Beneficial Effects

[0043] (1) The Lactobacillus paracasei CCFM1451 of the present invention has good activity, can significantly reduce the level of inflammation in colon tissue, improve colon tissue pathological damage, regulate the levels of pro-inflammatory factors and anti-inflammatory factors, reduce the expression level of aquaporin, significantly increase the content of motilin MTL in colon tissue; reduce the content of vasoactive intestinal peptide VIP in colon tissue; enhance intestinal peristalsis, shorten intestinal transit time, and regulate intestinal homeostasis.

[0044] (2) The present invention can be regarded as a drug for relieving constipation, and can also be applied to medicines or some fermented foods and functional foods, so as to widely exert its effects and has very valuable application prospects.

[0045] Biomaterial Deposit

[0046] A strain of Lactobacillus paracasei CCFM1451, whose taxonomic name is: Lacticaseibacillus paracasei, was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, and the deposit number is GDMCC No: 65316. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : Schematic diagram of symptom-related indicators (time to first black stool, small intestinal propulsion rate, and number of fecal particles in 6 hours) in relieving constipation in mice with Lactobacillus paracasei CCFM1451.

[0048] Figure 2 : Schematic diagram of the gene expression of inflammatory factors in the colon tissue of constipated mice after intervention with Lacticaseibacillus paracasei CCFM1451. (A) Expression of inflammatory factor IL-4; (B) Expression of inflammatory factor IL-1β.

[0049] Figure 3 : Schematic diagram of H&E staining results of colon tissues of constipated mice after intervention with Lactobacillus paracasei CCFM1451 strain. (A) Blank group; (B) Model group; (C) CCMF1451; (D) FQHXN85L3.

[0050] Figure 4: Schematic diagram of the expression of aquaporin genes in the colon tissue of constipated mice after intervention with Lactobacillus paracasei CCFM1451. (A) Aquaporin AQP4 gene expression; (B) Aquaporin AQP8 gene expression.

[0051] Figure 5 : Schematic diagram of the changes in motilin (MTL) content (A) and vasoactive peptide (VIP) content (B) in colon tissue of constipated mice after intervention with Lacticaseibacillus paracasei CCFM1451.

[0052] Note: The symbols above the bar graph indicate the significance level of the data, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001 (compared with the M group), # indicates p<0.05, ## indicates p<0.01, ### indicates p<0.001, #### indicates p<0.0001 (compared with the NC group). DETAILED DESCRIPTION

[0053] The male BALB / C mice involved in the following examples were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.

[0054] The culture medium involved in the following examples is as follows:

[0055] MRS liquid medium: beef extract 10g; tryptone 10g; yeast powder 5g; glucose 20g; anhydrous sodium acetate 5g; MgSO 4 7H 2 O 0.1g; MnSO 4 ·H 2 O 0.05g; diammonium hydrogen citrate 2g; K 2 HPO 4 ·3H 2 O 2.6g; Tween 80 1mL; adjust pH to 6.8±0.2; make up to 1L. Autoclave at 115℃ for 20min.

[0056] MRS solid medium: Add 2% agar powder to MRS liquid medium.

[0057] Preparation of the Lactobacillus paracasei postbiotics involved in the following examples:

[0058] Inoculate Lactobacillus paracasei into MRS solid culture medium, culture at 37°C for 48 hours to obtain a single colony, inoculate the prepared single colony into MRS liquid culture medium, and culture at 37°C for 24 hours for activation;

[0059] The activated bacterial solution after 3 generations was inoculated into 1LMRS liquid culture medium at an inoculum volume of 2%, and then cultured in a constant temperature incubator at 37°C for 18 hours after oscillation and mixing. Centrifuge at 8000g / min, 4°C for 10 minutes, remove the supernatant, and wash twice with sterile saline. Centrifuge under the same conditions, remove the supernatant, and resuspend the bacteria with sterile saline to prepare a final concentration of 9×10 9 CFU / mL of Lactobacillus paracasei CCFM1451 suspension was heated in a 65°C water bath for one hour and then transferred to a high-throughput tissue grinder and ground 4-5 times at 65 Hz and 30 seconds. This obtained the postbiotic bacterial solution for use before intragastric administration. It was stored in a -80°C refrigerator.

[0060] Example 1: Acquisition of Lactobacillus paracasei CCFM1451

[0061] 1. Isolation and screening of Lactobacillus paracasei:

[0062] (l) A fecal sample from a woman in Ningde City, Fujian Province was collected using a disposable sterile fecal sampler and cultured at 37°C in an anaerobic environment for 72 hours; the colony morphology was observed and recorded, and the colonies were picked for streaking and purification; the samples were cultured in MRS liquid medium at 37°C for 48 hours, and the obtained colonies were Gram-stained and the colony morphology was recorded. Gram-negative bacterial strains and Gram-positive cocci in the colonies were discarded, and Gram-positive bacilli were selected.

[0063] (2) After catalase analysis, catalase-positive strains were discarded and catalase-negative strains were retained.

[0064] 2. Molecular biological identification of Lactobacillus paracasei:

[0065] (l) Single bacterial genome extraction: The catalase-negative strain screened in step 1 was cultured overnight, 1 mL of the overnight cultured bacterial suspension was taken into a 1.5 mL centrifuge tube, centrifuged at 10,000 rpm for 2 min, and the supernatant was discarded to obtain the bacterial cells; the bacterial cells were washed with 1 mL of sterile water, centrifuged at 10,000 rpm for 2 min, and the supernatant was discarded to obtain the bacterial cells; 200 μL SDS lysis solution, 80℃ water bath for 30min; add 200μL of phenol-chloroform solution to the bacterial lysis solution, wherein the composition and volume ratio of phenol-chloroform solution are Tris-saturated phenol: chloroform: isoamyl alcohol = 25:24:1, invert and mix, centrifuge at 12000rpm for 5-10min, and take 200μL of supernatant; add 400μL of ice ethanol or ice isopropanol to 200uL supernatant, let stand at -20℃ for 1h, centrifuge at 12000rpm for 5-10min, and discard the supernatant; add 500μL of 70% (volume percentage) ice ethanol to resuspend the precipitate, centrifuge at 12000rpm for 1-3min, and discard the supernatant; dry in a 60℃ oven, or dry naturally; 50μL ddH 2 O re-dissolve the pellet for PCR;

[0066] (2) 16S rDNA PCR:

[0067] A. Bacterial 16S rDNA 50μL PCR reaction system: 10×Taqbuffer, 5μL; dNTP, 5μL; 27F, 0.5μL; 1492R, 0.5μL; Taq enzyme, 0.5μL; template, 0.5μL; ddH 2 O, 38 μL.

[0068] B. PCR conditions: 95℃5min; 95℃10s; 55℃30s; 72℃30s; step2-430×; 72℃5min; 12℃2min;

[0069] C. Prepare 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging;

[0070] D. The PCR product was sent to a professional sequencing company. The sequencing results were searched and compared with similarity in GeneBank using BLAST. The product was identified as Lactobacillus paracasei and named Lactobacillus paracasei CCFM1451. It was stored at -80°C for future use.

[0071] Example 2: Lactobacillus paracasei CCFM1451 can significantly improve the number of fecal particles and intestinal transit time in male mice after 6 hours

[0072] 1. Take 32 healthy male SPF BALB / C mice aged 8 weeks, adapt to the environment for 1 week, and randomly divide them into 4 groups: normal group (blank control group), model group, Lactobacillus paracasei CCFM1451 postbiotic group, Lactobacillus paracasei FQHXN85L3 postbiotic group, with 8 mice in each group. The animal experimental model was constructed using loperamide hydrochloride. Loperamide hydrochloride is an opioid receptor agonist that antagonizes smooth muscle contraction by stimulating the μ-opioid receptors of the intestinal wall and blocking the release of acetylcholine and prostaglandins, thereby reducing intestinal peristalsis and secretion and prolonging the retention time of intestinal contents. It can significantly reduce the intestinal propulsion rate of mice, significantly reduce the amount of defecation, and can produce a relatively stable constipation mouse model. The experimental animal grouping and treatment methods are shown in Table 1.

[0073] Table 1 Experimental animal groups

[0074]

[0075] After the experiment, the mice were killed and their colon tissues were taken out for relevant tests or frozen at -80°C. Before killing, the following tests were performed:

[0076] (1) Number of fecal particles in 6 hours

[0077] The mice were placed in different cages, and the feces were collected after 6 hours. The number of feces in each cage was recorded as the number of feces particles in 6 hours.

[0078] (2) Time of first black stool

[0079] Mix gum arabic powder and water in a ratio of 1:10, heat on an induction cooker until transparent, stir constantly during heating, then add 10% (w / v) activated carbon powder, stir and boil until mixed evenly, and store in a refrigerator at 4°C after the solution cools. Measure the intestinal transit time of mice on the morning before the end of the experiment. To ensure the accuracy of the results, mice must fast overnight before measuring the intestinal transit time. During the measurement, each mouse is gavaged with 0.2 mL of the above ink, and the gavage time is recorded. Pay attention to the defecation status of the mouse at all times. When the first feces containing activated carbon is discharged, record the time. The time difference between the two is the time when the mouse excretes the first black stool.

[0080] (3) Small intestinal propulsion rate

[0081] The night before the mouse dissection, the mice were fasted but not watered. 12 hours later, the blank group was gavaged with sterile water, and the model group and the strain intervention group were gavaged with loperamide to establish the model. 1 hour later, each mouse was gavaged with 0.2 mL of ink, and the mice were anesthetized 30 minutes later and blood was taken immediately. During the dissection, the pylorus to cecum segment was laid flat, and the "total length of the small intestine" was the length from the pylorus to the front end of the cecum, and the "ink advancement distance" was the distance from the pylorus to the front end of the ink. The small intestine advancement rate was calculated according to the following formula:

[0082] Small intestine propulsion rate = (ink propulsion length (cm)) / (total small intestine length (cm)) × 100%

[0083] like Figure 1 As shown in the figure, after modeling, the number of fecal particles in the model group mice decreased significantly by 44.4% (P<0.01) compared with the normal group, indicating that the modeling was successful. Compared with the model group, the number of fecal particles in the constipated mice in the Lactobacillus paracasei CCFM1451 postbiotic group increased by 46.4% (P<0.05), while there was no significant difference between the Lactobacillus paracasei FQHXN85L3 postbiotic group and the model group. Small intestinal propulsion rate is another important indicator for evaluating fecal traits, such as Figure 1 As shown in the results, after modeling, compared with the normal group, the small intestinal propulsion rate of mice in the model group decreased significantly by 53.8% (P<0.05), and the time to discharge the first black stool increased by 92.8% (P<0.01). After one week of intervention, it can be seen that the postbiotics of Lactobacillus paracasei CCFM1451 can effectively improve the small intestinal propulsion rate and shorten the time to discharge the first black stool in mice with constipation. Compared with the model group, the postbiotics of Lactobacillus paracasei CCFM1451 in the intervention group increased the small intestinal propulsion rate of constipated mice by 41.6% (P<0.05) and shortened the time to discharge the first black stool in constipated mice by 23.6% (P<0.05), while there was no significant difference between the postbiotic group of Lactobacillus paracasei FQHXN85L3 and the model group. Comprehensive evaluation of these two indicators shows that Lactobacillus paracasei CCFM1451 has great potential in promoting intestinal peristalsis, improving stool characteristics and shortening intestinal transit time.

[0084] Example 3: Lactobacillus paracasei CCFM1451 reduces inflammation levels and colon tissue pathological damage caused by intestinal nerve damage in mice

[0085] The BALB / C mice were grouped, modeled and treated as in Example 2. The specific method is as follows:

[0086] Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression of IL-4 and IL-1β genes, and H&E staining of mouse colon tissue sections was used to characterize the pathological damage of mouse colon tissue. After the mouse was dissected, fresh tissue was taken and placed in polyformaldehyde fixative, soaked, and rinsed overnight. The samples were dehydrated in 70%, 80%, and 90% ethanol solutions for 30 minutes each, and then placed in 95% and 100% for 20 minutes each time. Put in a mixture of 1 / 2 pure alcohol and 1 / 2 xylene for 15 minutes, and wash in xylene for 15 minutes and wash in xylene for 15 minutes until transparent. Put in a mixture of half xylene and half paraffin for 15 minutes, and then put in paraffin I and paraffin II for 50-60 minutes each to remove the transparent agent. The processed samples were embedded, sliced, spread, baked, H&E stained and sealed.

[0087] IL-4 is an anti-inflammatory cytokine. It plays a key anti-inflammatory role in regulating immune responses and controlling inflammation. Figure 2 As shown in A, the anti-inflammatory factors in the model group decreased by 77.3% compared with the control group (p<0.001), indicating that the mice in the model group had intestinal inflammation. After gavage with Lactobacillus paracasei CCFM1451 postbiotics, the IL-4 gene expression level increased significantly by 156.4% compared with the model group (p<0.05). IL-1β has strong pro-inflammatory activity, can induce a variety of pro-inflammatory mediators, and ultimately lead to extensive inflammatory events. The results are shown in Figure 2 As shown in B, the pro-inflammatory factors in the model group increased significantly by 54.5% compared with the control group (p<0.01), indicating that constipated mice had intestinal inflammation. After gavage with Lactobacillus paracasei CCFM1451 postbiotics, the IL-1β gene expression level was significantly reduced by 33.8% compared with the model group (p<0.01).

[0088] Observe the pathological sections of mouse colon tissue, such as Figure 3 As shown in the figure, compared with the control group, the model group mice had thinner mucus layer, shorter crypt structure, fewer goblet cells, and inflammatory infiltration in the colon tissue. However, after oral administration of Lactobacillus paracasei CCFM1451 postbiotics, the mucus layer in the colon tissue of mice became thicker, the boundaries of goblet cells became clearer, and the smooth muscle structure was relatively complete. For the Lactobacillus paracasei FQHXN85L3 postbiotic group, the mucus layer in the colon tissue did not thicken, and the inflammation phenomenon still existed.

[0089] Example 4: Lactobacillus paracasei CCFM1451 significantly increases the expression of aquaporins AQP4 and AQP8 in colon tissue of constipated mice

[0090] The experimental animal grouping and treatment method are the same as in Example 2. After the experiment, about 20 mg of mouse colon stored at -80°C was taken, and the total RNA of mouse colon was extracted using the TRIzol method: the mouse colon tissue was cut into pieces and placed in an enzyme-free centrifuge tube together with zirconium oxide beads that had been inactivated by high temperature, and 1 mL of TRIzol lysis solution was added and then fully broken using a high-throughput crusher, 200 μL of chloroform was added and vortexed for 30 seconds to fully emulsify it, and it was allowed to stand at 4°C for 5 minutes, and then centrifuged at 4°C 12000g for 15 minutes. Carefully aspirate 400 μL of supernatant and transfer it to a new enzyme-free centrifuge tube, add an equal volume of pre-cooled isopropanol, flip several times to mix, stand at 4°C for 10 minutes, and centrifuge again at 4°C 12000g for 15 minutes. After discarding the supernatant, 1 mL of pre-cooled DEPC water-prepared 75% ethanol was slowly added along the tube wall to wash the extracted RNA twice, and the bottom of the tube was flicked to suspend the RNA precipitate. After standing for 3-5 minutes, DEPC water was added to dissolve the precipitate. 1 μL RNA sample was taken and the concentration, purity and integrity of RNA were tested by ultra-micro spectrophotometer. According to the instructions provided by the Novozyme kit, the extracted total RNA was used as a template to synthesize cDNA, and real-time fluorescence quantitative PCR was performed. The system was prepared according to the instructions of HiScript III RTSuperMix for qPCR and the RT-qPCR program was run. Reaction system: 2×ChamQ Universal SYBR qPCR Master Mix 5 μL; 1 μL each of forward and reverse primers (10 μM); 1 μL cDNA; ddH 2 O 2μL. Reaction procedure: pre-denaturation at 95℃ for 10s; amplification at 95℃ for 10s, 57℃ for 30s, 40 cycles; melting curve at 95℃ for 15s, 60℃ for 60s, and 95℃ for 15s.

[0091] Real-time fluorescence quantitative PCR (RT-qPCR) was used to determine the gene expression levels of AQP4 and AQP8 in mouse colon tissue.

[0092] AQPs can control the transport of water and some small molecules and maintain the homeostasis of water molecules. Aqp4 and Aqp8 are a type of AQPs that have been studied more. Studies have shown that AQP4 and AQP8 can increase the intestinal cavity's absorption of water to feces, making the feces dry and hard and difficult to excrete. Previous studies in our laboratory have found that there is a correlation between fecal water content and Aqps gene transcription levels. Figure 4As shown, compared with blank mice, the transcription levels of Aqp4 and Aqp8 genes in the colon of constipated mice were significantly increased by 218.1% (P<0.01) and 84.9% (P<0.01). There was no significant difference in the transcription levels of Aqp4 and Aqp8 genes in the colon of mice in the postbiotic group of Lactobacillus paracasei FQHXN85L3 compared with the model group. However, the transcription levels of Aqp4 and Aqp8 genes in the colon of mice in the postbiotic intervention group of Lactobacillus paracasei CCFM1451 decreased by 35.3% (P<0.05) and 50.8% (P<0.01) compared with the model group. Therefore, the postbiotic Lactobacillus paracasei CCFM1451 has the ability to significantly downregulate the transcription levels of Aqp4 and Aqp8 genes. This indicates that the postbiotics of Lactobacillus paracasei CCFM1451 can reduce the intestinal water absorption capacity of feces by downregulating the transcription levels of Aqp4 and Aqp8 genes, thereby upregulating the water content of feces and relieving constipation.

[0093] Example 5: Lactobacillus paracasei CCFM1451 postbiotics significantly altered gastrointestinal active peptides in constipated mice

[0094] Specific implementation method Referring to Example 2, after the experiment, about 20 mg of mouse colon was taken and the expression of motilin MTL protein was detected by enzyme-linked immunosorbent assay. The determination method was referred to the instructions of the kit. The concentration of MTL substance contained in the sample was calculated according to the absorbance.

[0095] Gastrointestinal active peptides can be roughly divided into excitatory and inhibitory types. The motilin (MTL) involved in this study is an excitatory type, and vasoactive intestinal peptide (VIP) is an inhibitory type. Figure 5 As shown in the results, compared with the blank group, the MTL level of constipated mice in the model group decreased by 15.3% (P<0.01), and the VIP level increased significantly by 123.5% (P<0.01), indicating that constipation can lead to changes in gastrointestinal active peptides, inhibit the movement of intestinal muscles, and thus weaken the intestinal motility. Compared with the mice in the model group, the level of motilin (MTL) in the colon of mice in the postbiotic intervention group of Lactobacillus paracasei CCFM1451 increased by 21.8% (P<0.01), and the level of vasoactive peptide (VIP) decreased by 54.5% (P<0.01), while the content of motilin and vasoactive peptide in the colon tissue of mice in the postbiotic FQHXN85L3 bacteria intervention group was not significantly different from that in the model group. This result shows that postbiotics may promote intestinal motility and restore transport capacity by increasing excitatory gastrointestinal active peptides (MTL) and reducing inhibitory active peptides (VIP).

[0096] Therefore, it can be seen from the above examples that the postbiotics of Lactobacillus paracasei CCFM1451 can significantly reduce the level of inflammation in colon tissue, improve pathological damage of colon tissue, regulate the levels of pro-inflammatory and anti-inflammatory factors, reduce the expression level of aquaporin, promote neurotransmitter secretion, and significantly increase the content of motilin MTL in colon tissue; reduce the content of vasoactive intestinal peptide VIP in colon tissue; enhance intestinal peristalsis, shorten intestinal transit time, and regulate intestinal homeostasis.

[0097] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Lactobacillus paracasei CCFM1451, characterized in that: The Lactobacillus paracasei was deposited in the Guangdong Provincial Microbial Culture Collection Center on October 23, 2024, with the storage address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC No: 65316.

2. a microbial agent containing the Lactobacillus paracasei CCFM1451 of claim 1.

3. the postbiotic prepared by Lactobacillus paracasei CCFM1451 according to claim 1.

4. The postbiotic according to claim 3, characterized in that The postbiotics include inactivated bacterial cells, bacterial lysates and / or inactivated fermentation broth.

5. A method for preparing the postbiotics according to claim 3 or 4, characterized in that: Include (a) or (b): (a) culturing the Lactobacillus paracasei CCFM1451 of claim 1 to a logarithmic growth phase, inactivating the mixture by heat treatment, and centrifuging the mixture, discarding the supernatant, to obtain postbiotics; (b) culturing the Lactobacillus paracasei CCFM1451 described in claim 1 to the logarithmic growth phase, inactivating it by heat treatment, and then crushing it by high-pressure homogenization to obtain postbiotics.

6. The method according to claim 5, characterized in that The postbiotics are processed into powder by a drying method and are used directly or dissolved in physiological saline for use.

7. The method according to claim 5 or 6, characterized in that: The viable count of Lactobacillus paracasei CCFM1451 before inactivation is at least 8×10 9 CFU / mL or 10 9 CFU / g.

8. A product containing Lactobacillus paracasei CCFM1451 and / or its postbiotics according to claim 1, characterized in that: The products include food, medicine or health products.

9. Use of the Lactobacillus paracasei CCFM1451 and / or its postbiotics according to claim 1 in preparing a medicine for alleviating constipation.

10. The use according to claim 9, characterized in that: The drug has at least one of the following functions: (a) Improve colon tissue pathological damage; (b) Reduce the expression of water channel protein AQP4 in colon tissue; (c) reducing the expression of aquaporin AQP8 in colon tissue; (d) reduce the expression of pro-inflammatory cytokine IL-1β in colon tissue; (e) Increase the expression of anti-inflammatory factor IL-4 in colon tissue; (f) Increase the content of motilin (MTL) in colon tissue; (g) reducing the content of vasoactive intestinal polypeptide (VIP) in colon tissue; (h) Enhance intestinal peristalsis, shorten intestinal transit time, and regulate intestinal homeostasis.

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