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 CCFM1452 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 CCFM1452, 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 gastrointestinal disease characterized by difficult, infrequent or incomplete defecation. It has a great impact on the patient's quality of life. About 12-14% of people worldwide suffer from constipation. In Asia, with changes in the environment, increased mental stress and prolonged sitting time, the prevalence of constipation in some populations is as high as 20%. Since there are many and complex causes of constipation, once constipation occurs, especially severe and long-lasting constipation, the right solution should be found.

[0003] Probiotics are living microorganisms that can have beneficial effects on host health when given in sufficient quantities. With the continuous deepening of research on the relationship between probiotics and humans and animals, and the continuous revelation of the physiological significance of probiotics to humans and animals, the application of probiotics in the field of therapeutic medicine has been continuously expanded. Its role in relieving constipation has been widely studied. However, many previous studies have shown that probiotics can be used to maintain intestinal homeostasis and treat a variety of gastrointestinal diseases. They can play a role through different mechanisms such as enhancing the intestinal barrier, improving the immune system, regulating metabolic processes and the brain-gut axis. However, the safety of probiotics remains controversial. In order to overcome the problems related to the clinical, technical and economic application of live bacterial cells, new probiotic-related concepts have been proposed. The International Association of Probiotics and Prebiotics published a consensus on postbiotics in 2021, officially defining "postbiotics" as "preparation of inanimate microorganisms and / or their components that are beneficial to host health." The active ingredients of postbiotics are mainly inactivated bacteria, bacterial lysates, cell wall components and metabolites. 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. Recently, more and more evidence supports that the protective effect of postbiotics on gastrointestinal function is similar to or even better than that of live probiotics. Compared with the controversial safety issues of probiotics, postbiotics avoid the spread of virulence factors and drug-resistant genes, and the survival ability of probiotics in the intestine is no longer a problem. Its stability and safety are more conducive to storage and transportation at room temperature. Therefore, compared with probiotics, postbiotics have more obvious advantages. However, probiotics that can use postbiotics to relieve constipation have not yet been found.

[0004] In summary, the application of postbiotics has become a trend in the international market, and there is an urgent need to screen strains of postbiotics that can be used to relieve constipation. 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 the mechanism of action of Lactobacillus paracasei postbiotics in relieving constipation at this stage, the present invention provides a strain of Lactobacillus paracasei CCFM1452, the postbiotics of which can significantly reduce the level of inflammation in colon tissue, improve colon tissue pathological damage, and regulate the reduction of pro-inflammatory factors and the increase of 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. Corresponding postbiotic preparations, fermented foods, and functional foods are provided to play the role of relieving constipation.

[0009] The present invention provides a strain of Lactobacillus paracasei CCFM1452, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, with a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a deposit number of GDMCC No: 65317.

[0010] In one embodiment of the present invention, the Lacticaseibacillus paracasei CCFM1452 is derived from 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 Lacticaseibacillus paracasei is 100%; the result shows that the strain is Lacticaseibacillus paracasei, and it is named Lacticaseibacillus paracasei CCFM1452.

[0011] In one embodiment of the present invention, the Lactobacillus paracasei CCFM1452 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] (6) 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, 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.

[0017] The invention also provides a microbial preparation containing the Lactobacillus paracasei CCFM1452.

[0018] The invention also provides postbiotics prepared by the Lactobacillus paracasei CCFM1452.

[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 CCFM1452 to a logarithmic growth phase, inactivating the mixture by heat treatment, and centrifuging the mixture, discarding the supernatant, and obtaining postbiotics;

[0022] (b) Lactobacillus paracasei CCFM1452 is cultured to the logarithmic growth phase, inactivated by heat treatment, and then crushed 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 CCFM1452 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 CCFM1452 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 CCFM1452 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 comprises 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 contains the above-mentioned Lactobacillus paracasei CCFM1452 or its postbiotics or the above-mentioned microbial agent.

[0030] In one embodiment of the present invention, the medicine comprises Lactobacillus paracasei CCFM1452 or its postbiotics, and a pharmaceutically acceptable carrier.

[0031] 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.

[0032] In one embodiment of the present invention, the dosage form of the drug includes granules, capsules, tablets, pills or oral liquids.

[0033] The present invention also provides the use of the Lacticaseibacillus paracasei CCFM1452 or its postbiotics, or the microbial agent in preparing a medicine for relieving constipation.

[0034] In one embodiment of the present invention, the drug has at least one of the following functions:

[0035] (a) Improve colon tissue pathological damage;

[0036] (b) Reduce the expression of water channel protein AQP4 in colon tissue;

[0037] (c) reducing the expression of aquaporin AQP8 in colon tissue;

[0038] (d) reduce the expression of pro-inflammatory cytokine IL-1β in colon tissue;

[0039] (e) Increase the expression of anti-inflammatory factor IL-4 in colon tissue;

[0040] (f) Increase the content of motilin (MTL) in colon tissue;

[0041] (g) reducing the content of vasoactive intestinal polypeptide (VIP) in colon tissue;

[0042] (h) Enhance intestinal peristalsis, shorten intestinal transit time, and regulate intestinal homeostasis.

[0043] Beneficial Effects

[0044] (1) The activity of the Lactobacillus paracasei CCFM1452 of the present invention is good. The postbiotics prepared by the Lactobacillus paracasei CCFM1452 can significantly reduce the level of inflammation in colon tissue, improve pathological damage of colon tissue, regulate the levels of pro-inflammatory factors 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. After only one week of action, the effect of significantly relieving constipation can be achieved, and various indicators of constipated mice can reach levels comparable to those of the control group.

[0045] (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.

[0046] Biomaterial Deposit

[0047] A strain of Lactobacillus paracasei CCFM1452, whose taxonomic name is: Lacticaseibacillus paracasei, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024. The collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the collection number is GDMCC No: 65317. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] 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) of Lactobacillus paracasei CCFM1452 in alleviating constipation in mice.

[0049] Figure 2: Schematic diagram of the gene expression of inflammatory factors in the colon tissue of constipated mice after intervention with Lacticaseibacillus paracasei CCFM1452. (A) Expression of inflammatory factor IL-4; (B) Expression of inflammatory factor IL-1β.

[0050] Figure 3 : Schematic diagram of H&E staining results of colon tissues of constipated mice after intervention with Lactobacillus paracasei CCFM1452 strain. (A) Blank group; (B) Model group; (C) CCMF1452; (D) CCFM1451.

[0051] Figure 4 : Schematic diagram of the expression of aquaporin genes in the colon tissue of constipated mice after intervention with Lactobacillus paracasei CCFM1452. (A) Aquaporin AQP4 gene expression; (B) Aquaporin AQP8 gene expression.

[0052] 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 CCFM1452.

[0053] 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

[0054] The male BALB / C mice involved in the following examples were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.

[0055] The culture medium involved in the following examples is as follows:

[0056] MRS liquid culture medium: beef extract 10g; tryptone 10g; yeast powder 5g; glucose 20g; anhydrous sodium acetate 5g; MgSO4·7H2O 0.1g; MnSO4·H2O 0.05g; diammonium hydrogen citrate 2g; K2HPO4·3H2O 2.6g; Tween 80 1mL; adjust pH to 6.8±0.2; make up to 1L. Autoclave at 115℃ for 20min.

[0057] MRS solid medium: Add 2% agar powder to MRS liquid medium.

[0058] Preparation of the Lactobacillus paracasei postbiotics involved in the following examples:

[0059] 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;

[0060] 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 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 gavage. Store in a -80°C refrigerator.

[0061] Example 1: Acquisition of Lactobacillus paracasei CCFM1452

[0062] 1. Isolation and screening of Lactobacillus paracasei:

[0063] (l) A fecal sample from a female student 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.

[0064] (2) After catalase analysis, catalase-positive strains were discarded and catalase-negative strains were retained.

[0065] 2. Molecular biological identification of Lactobacillus paracasei:

[0066] (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 buffer, 80℃ water bath for 30min; add 200μL of phenol-chloroform solution to the bacterial lysis buffer, 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; re-dissolve the precipitate with 50μL ddH2O for PCR;

[0067] (2) 16S rDNA PCR:

[0068] 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; ddH2O, 38μL.

[0069] B. PCR conditions: 95℃5min; 95℃10s; 55℃30s; 72℃30s; step2-430×; 72℃5min; 12℃2min;

[0070] 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;

[0071] 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 CCFM1452. It was stored at -80°C for future use.

[0072] Example 2: Lactobacillus paracasei CCFM1452 can significantly improve the number of fecal particles and intestinal transit time in male mice after 6 hours

[0073] 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 CCFM1452 postbiotic group, and Lactobacillus paracasei CCFM1451 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 preventing 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.

[0074] Table 1

[0075]

[0076]

[0077] After the experiment, the mice were killed and their colon tissues were taken out for relevant tests, or frozen at -80°C. The following tests were performed before killing:

[0078] (1) Number of fecal particles in 6 hours

[0079] 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.

[0080] (2) Time of first black stool

[0081] 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.

[0082] (3) Small intestinal propulsion rate

[0083] 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:

[0084] Small intestine propulsion rate = (ink propulsion length (cm)) / (total small intestine length (cm)) × 100%

[0085] like Figure 1 As shown in the figure, after modeling, the number of fecal particles of mice in the model group decreased significantly by 40.0% (P<0.01) compared with the normal group, proving that the modeling was successful. Compared with the model group, the number of fecal particles of constipated mice in the Lactobacillus paracasei CCFM1452 postbiotic group increased by 43.1% (P<0.01), while the number of fecal particles of constipated mice in the Lactobacillus paracasei CCFM1451 postbiotic group increased by 29.6% (P<0.05). Therefore, judging from the number of fecal particles, the effect of CCFM1452 postbiotics in relieving constipation is better than that of CCFM1451 postbiotics. Small intestinal propulsion rate is another important indicator for evaluating stool characteristics, 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 66.1% (P<0.01), and the time to the first black stool increased by 99.8% (P<0.01). After one week of intervention, compared with the model group, the postbiotics of Lactobacillus paracasei CCFM1452 in the intervention group increased the small intestinal propulsion rate of mice with constipation by 165.4% (P<0.01) and shortened the time to the first black stool of mice with constipation by 26.2% (P<0.01), while the postbiotics of Lactobacillus paracasei CCFM1451 in the intervention group increased the small intestinal propulsion rate of mice with constipation by 124.3% (P<0.01) and shortened the time to the first black stool of mice with constipation by 18.6% (P<0.01). It can be seen that Lactobacillus paracasei CCFM1452 postbiotics can more effectively improve the small intestinal propulsion rate and shorten the first black stool discharge time of constipated mice than Lactobacillus paracasei CCFM1451 postbiotics. Comprehensive evaluation indicators show that Lactobacillus paracasei CCFM1452 postbiotics are superior to CCFM1451 postbiotics, and CCFM1452 postbiotics have great potential in promoting intestinal peristalsis, improving stool characteristics and shortening intestinal transit time.

[0086] Example 3: Lactobacillus paracasei CCFM1452 reduces inflammation levels and colon tissue pathological damage caused by intestinal nerve damage in mice

[0087] The BALB / C mice were grouped, modeled and treated as in Example 2. The specific method is as follows:

[0088] Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression of IL-1β gene, 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.

[0089] IL-4 is an anti-inflammatory cytokine. It plays a key anti-inflammatory role in regulating immune responses and controlling inflammation. Figure 2 -A shows that the anti-inflammatory factors in the model group decreased significantly by 73.9% compared with the control group (p<0.001), indicating that the mice in the model group had intestinal inflammation. After gavage with Lactobacillus paracasei CCFM1452 postbiotics, the IL-4 gene expression level increased significantly by 170.9% compared with the model group (p<0.05), while after gavage with Lactobacillus paracasei CCFM1451 postbiotics, the IL-4 gene expression level increased by 132.2% 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 widespread inflammatory events. The results are shown in Figure 2 -B shows that the model group had a significant increase in pro-inflammatory factors by 54.7% (p<0.01) compared with the control group, indicating that constipated mice had intestinal inflammation. After oral administration of Lactobacillus paracasei CCFM1452 postbiotics, the IL-1β gene expression level was significantly reduced by 70.7% compared with the model group (p<0.05), while after oral administration of Lactobacillus paracasei CCFM1451 postbiotics, the IL-1β gene expression level was reduced by 58.5% (p<0.05) compared with the model group. The comprehensive evaluation indicators show that Lactobacillus paracasei CCFM1452 postbiotics are superior to CCFM1451 postbiotics in regulating the levels of pro-inflammatory and anti-inflammatory factors.

[0090] In addition, the pathological sections of mouse colon tissue were observed, such as Figure 3As 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 CCFM1452 postbiotics, the mucus layer in the colon tissue of mice became thicker, the boundaries of goblet cells became clearer, the smooth muscle structure was more complete, and the effect of eliminating inflammation was better than CCFM1451.

[0091] Example 4: Lactobacillus paracasei CCFM1452 significantly increases the expression of aquaporins AQP4 and AQP8 in colon tissue of constipated mice

[0092] The grouping, modeling and treatment methods of BALB / C mice were the same as those 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. 400 μL of supernatant was carefully pipetted into a new enzyme-free centrifuge tube, an equal volume of pre-cooled isopropanol was added, and it was mixed by inverting several times, allowed to stand at 4°C for 10 minutes, and centrifuged again at 4°C 12000g for 15 minutes. After discarding the supernatant, slowly add 1 mL of pre-cooled 75% ethanol prepared with DEPC water along the tube wall to wash the extracted RNA twice, flick the bottom of the tube to suspend the RNA precipitate, let it stand for 3-5 minutes, and then add DEPC water to dissolve the precipitate. Take 1 μL of RNA sample and test the concentration, purity and integrity of RNA by ultra-micro spectrophotometer. According to the instructions provided by the Novozyme kit, cDNA was synthesized using the extracted total RNA as a template, 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 of cDNA; 2 μL of ddH2O. Reaction procedure: pre-denaturation at 95°C for 10 s; amplification at 95°C for 10 s, 57°C for 30 s, and 40 cycles; melting curve: 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s.

[0093] Real-time fluorescence quantitative PCR (RT-qPCR) was used to determine the expression levels of AQP4 and AQP8 genes in mouse colon tissue.

[0094] 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 4 As shown, compared with blank mice, the transcription levels of Aqp4 and Aqp8 genes in the colon of constipated mice were significantly increased by 257.6% (P<0.01) and 85.2% (P<0.01). The transcription levels of Aqp4 and Aqp8 genes in the colon of constipated mice in the postbiotic intervention group of Lactobacillus paracasei CCFM1452 were significantly decreased by 50.7% (P<0.01) and 47.7% (P<0.05) compared with the model group. The transcription levels of Aqp4 and Aqp8 genes in the colon of constipated mice in the postbiotic intervention group of Lactobacillus paracasei CCFM1451 were significantly decreased by 53.0% (P<0.01) and 61.0% (P<0.05) compared with the model group. Therefore, the postbiotic Lactobacillus paracasei CCFM1452 has the ability to significantly downregulate the transcription levels of Aqp4 and Aqp8 genes. This indicates that both Lactobacillus paracasei CCFM1452 and CCFM1451 postbiotics can reduce the intestinal tract's ability to absorb water from feces by downregulating the transcription levels of Aqp4 and Aqp8 genes, thereby increasing the water content of feces and alleviating constipation.

[0095] Example 5: Lactobacillus paracasei CCFM1452 postbiotics significantly altered gastrointestinal active peptides in constipated mice

[0096] Specific implementation method Referring to Example 2, after the experiment, the 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.

[0097] 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 5As shown, compared with the blank group, the MTL level of the constipated mice in the model group decreased by 15.3% (P<0.01), and the VIP level increased significantly by 123.8% (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 mice in the postbiotic intervention group of Lactobacillus paracasei CCFM1452 increased significantly by 20.8% (P<0.01), and the level of vasoactive peptide (VIP) decreased by 43.4% (P<0.01). The level of motilin (MTL) in the mice in the postbiotic intervention group of Lactobacillus paracasei CCFM1451 increased significantly by 21.8% (P<0.01), and the level of vasoactive peptide (VIP) decreased by 55.3% (P<0.01). The results indicate that postbiotics may promote intestinal motility and restore transport capacity by increasing excitatory gastrointestinal peptide (MTL) and reducing inhibitory peptide (VIP), and the effect is equivalent to that of CCFM1451.

[0098] Therefore, it can be seen from the above examples that the postbiotics of Lactobacillus paracasei CCFM1452 can significantly reduce the level of inflammation in colon tissue, improve pathological damage of colon tissue, regulate the expression levels of pro-inflammatory factors 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.

[0099] 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 CCFM1452, 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: 65317.

2. a microbial agent containing the Lactobacillus paracasei CCFM1452 of claim 1.

3. the postbiotic prepared by Lactobacillus paracasei CCFM1452 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 CCFM1452 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 CCFM1452 according to claim 1 to a 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 CCFM1452 before inactivation is at least 8×10 9 CFU / mL or 10 9 CFU / g.

8. A product containing Lactobacillus paracasei CCFM1452 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 and / or its postbiotics according to claim 1 in preparing a medicine for relieving 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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