CN112877260A - Lactobacillus paracasei for relieving purgative colon and application thereof - Google Patents

Abstract

The invention discloses a strain of Lactobacillus paracasei for relieving laxative colon and its application, belonging to the technical field of microorganisms. The Lactobacillus paracasei CCFM1164 provided by the invention can shorten the time of the first black stool, increase the water content of the stool, increase the transcription level of mucin MUC-2 in colon tissue, reduce the transcription level of aquaporin AQP4 in colon tissue, and increase the Cajal level in colon tissue. The number of interstitial cells and glial cells, increase the level of glial cell-derived trophic factor GDNF and butyrate in colon tissue, improve intestinal health status, and fundamentally relieve laxative colon. The present invention can be applied to functional food and medicine for relieving the laxative colon, and has broad application prospect.

Description

Lactobacillus paracasei for relieving purgative colon and application thereof

Technical Field

The invention relates to lactobacillus paracasei for relieving purgative colon and application thereof, belonging to the technical field of microorganisms.

Background

Laxative colon is a condition that patients have dependence on laxatives due to the fact that laxatives are applied for a long time to damage the nervous system of the colon, so that the intestinal nerve is degenerated, gastrointestinal motility disorder occurs, and the responsiveness to the laxatives is reduced, and the laxatives are common in middle-aged and elderly people suffering from constipation.

In China, the incidence rate of chronic constipation reaches 20%, a series of irritant laxatives such as senna leaves, rheum officinale and aloe are favored by constipation patients due to quick effect and low cost, the patients need to use laxatives for a long time due to constipation, the dosage of the laxatives is inversely proportional to the effect along with the progress of the disease course, and finally the laxatives lose the catharsis effect to form 'laxative colon', and even serious patients can develop the melanosis of the colon. However, consumers have little knowledge of the side effects of the laxatives, and few reports of laxative colons are reported in China at present, which causes little attention to consumers, especially constipation patients, so that irritant laxatives are the primary choice for many families to have constipation symptoms, more and more laxative colons are available in the past, no effective means is available for treating the laxative colons at present, and serious patients need to perform colectomy to relieve the symptoms.

Probiotics have good constipation relieving effect as an effective adjuvant therapy means for relieving constipation, but the reasons for causing constipation are various, and the effect of the probiotics on different types of constipation is different. However, in current studies, there is no means to incorporate probiotics into the treatment of the laxative colon. Therefore, through the research on the colon of the laxative for the probiotics, the colon of the laxative is fundamentally relieved, and the constipation people are benefited.

Disclosure of Invention

Aiming at the technical defect that the prior purgative colon lacks an effective treatment means, the invention provides lactobacillus paracasei CCFM1164 which is easy to activate and short in growth period, the lactobacillus paracasei CCFM1164 can obviously improve the intestinal peristalsis capability, shorten the first-grain defecation blacking time, promote the healthy development of enteric nerves, provide a corresponding probiotic preparation or a functional food and effectively relieve the purgative colon.

The invention provides Lactobacillus paracasei (Lactobacillus paracasei) separated and screened from feces of 84-year-old people in southern region of Chongqing city, which is preserved in Guangdong province microorganism strain preservation center at 29 th 1 month in 2021, with the preservation number being GDMCC No: 61479, the preservation address is Guangzhou institute for microorganisms of Guangdong province, No. 59 building, No. 5 building, of Michelia Tokoro, Michelia Tourette.

The invention also provides a composition containing the lactobacillus paracasei CCFM 1164.

In one embodiment, the composition is a fermented food or dietary supplement.

In one embodiment, the fermented food is a fermented dairy product, a soy product, and a fruit and vegetable product; including but not limited to cow's milk, goat's milk, cheese, cream, milk-containing beverage or powdered milk; the bean product comprises soybean, soybean milk, bean curd, or soybean milk powder; the fruit and vegetable product is prepared from at least one of Chinese cabbage, radish, cucumber, beet, green bean, apple or waxberry product.

In one embodiment, the composition is a dietary supplement.

In one embodiment, the dietary supplement is in the form of a powder, capsule, fondant, or liquid comprising lactobacillus paracasei.

The invention also provides application of the lactobacillus paracasei CCFM1164 in preparing food or medicine for relieving slow transit constipation.

In one embodiment, the slow transit constipation includes, but is not limited to, laxative colon.

In one embodiment, the laxative colon is induced by senna leaves or extracts thereof.

In one embodiment, the relieving of slow transit constipation includes at least one of the following functions:

1) shortening the first grain black stool time of the cathartic colon mammal, and increasing the water content of the stool;

2) increasing mucin MUC-2 transcript levels in colonic tissue of a laxative colon mammal;

3) reducing the transcriptional level of aquaporin AQP4 in colonic tissue of a laxative colon mammal;

4) increasing the number of interstitial cells of Cajal in colon tissue of a laxative colon mammal;

5) increasing the number of glial cells in colonic tissue of a laxative colon mammal;

6) increasing the level of glial cell line-derived trophic factor (GDNF) in colonic tissue of a laxative colon mammal;

7) increasing butyric acid content in stool of laxative colon mammal.

In one embodiment, the mammal includes, but is not limited to, a human.

In one embodiment, the medicament further comprises a pharmaceutically acceptable carrier, including one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents, which are commonly used in medicine.

In one embodiment, the dosage form of the medicament is granules, capsules, tablets, pills or oral liquid.

The invention also provides a microbial inoculum containing the lactobacillus paracasei CCFM1164, and the microbial inoculum is powder obtained by drying a bacterial liquid containing the lactobacillus paracasei CCFM 1164.

In one embodiment, the drying is vacuum freeze drying or other bacterial liquid drying process.

In one embodiment, the cell number of Lactobacillus paracasei in the microbial inoculum is more than or equal to 1 × 10⁸CFU/g。

The invention has the beneficial effects

The lactobacillus paracasei CCFM1164 has excellent growth characteristics, and can obviously relieve the symptoms of purgative colon mice: the method has the advantages of shortening the first-grain black stool time, improving the water content of the stool, improving the transcription level of mucin MUC-2 in the colon tissue, reducing the transcription level of aquaporin AQP4 in the colon tissue, increasing the number of interstitial cells and glial cells of Cajal in the colon tissue, improving the level of glial cell-derived trophic factor GDNF in the colon tissue, increasing the content of butyric acid in the stool and improving the health state of the intestinal tract. Therefore, the bacterial strain can be used as a component for relieving or treating the purgative colon, is applied to medicines, health-care products or fermented foods for relieving the purgative colon, or is used as a medicine substitute of the existing medicines (such as mosapride), thereby playing the role of the bacterial strain and having wide application prospect.

Biological material preservation

Lactobacillus paracasei (Lactobacillus paracasei) CCFM1164 is classified and named as Lactobacillus paracasei and is preserved in Guangdong province microorganism strain preservation center 1-29 days 2021, with the preservation number being GDMCC No: 61479, the preservation address is Guangzhou institute for microorganisms of Guangdong province, No. 59 building, No. 5 building, of Michelia Tokoro, Michelia Tourette.

Drawings

FIG. 1 is a graph showing the first time of dark stool, water content of stool and small intestine propulsion rate of different groups of purgative colon mice.

FIG. 2 is a graph showing the variation of mucin MUC-2 transcript levels in colon tissues of laxative colon mice of different groups.

FIG. 3 is a graph showing the transcriptional level changes of aquaporin AQP4 in colon tissues of different groups of laxatives of colonic mice.

FIG. 4 is a graph showing the change in the expression level of the c-kit gene of stem cell factor receptor in colon tissues of different groups of purgative colon mice.

FIG. 5 is a graph showing the change in the number of glial cells in colon tissue of different groups of laxative colon mice.

FIG. 6 is a graphical representation of the change in expression of glial cell line-derived neurotrophic factor (GDNF) in colon tissue of laxative colon mice from different groups.

FIG. 7 is a graph showing butyric acid content in stool from colon mice for different groups.

Note: the upper bar indicates the level of data significance, p <0.05, p <0.01, # indicates p <0.001, # indicates p <0.0001 (compared to the model group), # indicates p <0.05, # indicates p <0.01, # indicates p <0.001, # indicates p <0.0001, and # indicates p <0.0001 (compared to the normal group).

Detailed Description

Example 1 screening, identification and culture of Lactobacillus paracasei CCFM1164

Separating and screening strains:

(1) collecting feces of 84-year-old people in Liangchang in Nanquan district of Chongqing city by using disposable sterile feces fetching device, spreading the feces sample in MRS culture medium, and placing in anaerobic incubator (N)₂:CO₂:H₂Enrichment in 80:10:10) for 12 h;

(2) coating the enriched sample on an LB solid plate after gradient dilution for anaerobic culture for 20 h;

(3) selecting single bacterial colony conforming to basic morphology of lactobacillus for plate streaking purification, and screening and separating out the selected bacterial strain;

(4) and culturing the single colony in a liquid MRS culture solution for 20h, performing gram staining, and selecting gram-positive bacteria for subsequent tests.

(II) preliminary identification of lactobacillus: caldolytic ring assay

(1) Culturing the lactic acid bacteria obtained by screening in the step (I) in a liquid sorbitol GM17 culture solution for 20h, and then centrifuging l mL of culture at 8000rpm for 2 min;

(2) with 0.05M KH₂PO₄Washing the solution twice;

(3) resuspending the resulting pellet and streaking on sorbitol GM 17-0.75% CaCO₃Culturing for 20 hours on the solid culture medium;

(4) selecting bacterial colonies which are obvious in calcium-dissolving ring, round in convex surface, fine, dense, white and sterile mycelia, and preliminarily determining lactobacillus by observing the bacteria in a rod shape through a microscope after gram staining.

(III) molecular biological identification of Lactobacillus

(1) Extracting a single-bacterium genome:

A. culturing the lactobacillus screened in the step (II) overnight, taking l mL of the overnight-cultured bacterial suspension into a 1.5mL centrifuge tube, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain thalli;

B. purging the thalli with l mL of sterile water, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain the thalli;

C. adding 200 μ LSDS lysate, and water-bathing at 80 deg.C for 30 min;

D. adding 200 μ L of phenol-chloroform solution into the thallus lysate, wherein the phenol-chloroform solution comprises Tris saturated phenol, chloroform and isoamylol at a volume ratio of 25:24:1, mixing, centrifuging at 12000rpm for 5-10min, and collecting 200 μ L of supernatant;

E. adding 400 μ L of glacial ethanol or glacial isopropanol into 200uL of supernatant, standing at-20 deg.C for 1h, centrifuging at 12000rpm for 5-10min, and discarding the supernatant;

F. adding 500 μ L70% (volume percentage) of glacial ethanol, resuspending the precipitate, centrifuging at 12000rpm for 1-3min, and discarding the supernatant;

drying in an oven at G.60 ℃ or naturally airing;

H.50μLddH₂re-dissolving the precipitate with O for PCR;

(2)16S rDNA PCR

A. bacterial 16S rDNA 50 μ LPCR reaction:

10 × Taq buffer, 5 μ L; dNTP, 5. mu.L; 27F, 0.5 μ L; 1492R, 0.5 μ L; taq enzyme, 0.5. mu.L; template, 0.5 μ L; ddH2O, 38 μ L.

PCR conditions:

95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；

(3) preparing 1% agarose gel, mixing the PCR product with 10000 × loading buffer, loading the sample by 5 μ L, running at 120V for 30min, and performing gel imaging;

(4) and (3) sequencing the PCR product of the 16S rDNA, searching and comparing the obtained sequence result in Gene Bank by using BLAST, selecting a sequencing result, identifying the sequencing result as a strain of lactobacillus paracasei, and preserving at-80 ℃ for later use.

(IV) preparation of lactobacillus suspension and freezing survival rate

Inoculating the activated 3-generation bacterial liquid into 1L of liquid MRS culture medium in an inoculation amount of 2%, shaking, mixing uniformly, and culturing in an anaerobic incubator at 37 ℃ for 20 h. Centrifuging at 8000g/min and 4 deg.C for 15min, removing supernatant, washing with sterile physiological saline for 2 times, centrifuging under the same conditions, removing supernatant, adding 10% (w/V) sterile skimmed milk solution (prepared by dissolving 10g skimmed milk powder in 100mL water) 2 times of the mass of bacterial sludge, and storing at-80 deg.C for one week. Before animal experiments, the viable bacteria quantity of the initial bacteria and the viable bacteria quantity of the frozen bacteria are measured by a flat plate pouring method after the bacteria liquid is uniformly vibrated. The formula of the MRS culture medium is as follows: 10g of beef extract; 10g of tryptone; 5g of yeast powder; 20g of glucose; 5g of anhydrous sodium acetate; MgSO (MgSO)₄·7H₂O 0.1g；MnSO₄·H₂0.05g of O; 2g of diammonium hydrogen citrate; k₂HPO₄·3H₂O2.6 g; tween 801 mL; the volume is up to 1L. Autoclaving at 115 deg.C for 20 min.

The experimental results are as follows: the initial viable count of the bacterial liquid is 9.6 multiplied by 10⁹CFU/mL, viable count after 1 week was 8.0X 10⁹CFU/mL, the magnitude of the order is not changed, which shows that the activity of the bacteria after the bacteria liquid is frozen is not changed greatly, the experiment is not influenced obviously, and the method can be used for animal experiments.

Example 2: relieving effect of lactobacillus paracasei CCFM1164 on senna diarrheagenic colon mice

Taking out the strain of Lactobacillus paracasei CCFM1164 in a refrigerator at minus 80 ℃, streaking the strain in an MRS plate, culturing for 20h at 37 ℃, selecting a single colony in an MRS liquid tube, culturing for 16h at 37 ℃, inoculating the single colony in a new MRS liquid culture medium according to the volume amount of 2%, culturing for 16h at 37 ℃, culturing for one generation again according to the same method, centrifuging the suspension of the Lactobacillus paracasei for 5min at 6000r/min and 4 ℃, then resuspending the suspension by 10% skim milk to obtain the strain with the concentration of 5 multiplied by 10⁹CFU/mL bacterial suspension, used for animal experiments.

24 healthy male C57BL/6J mice, 7 weeks old, were acclimatized for 1 week and randomized into 4 groups: control (normal), model (model), mosapride (mosapride) and lactobacillus paracasei CCFM164 intervention (CCFM1164), each containing 6 mice, each with a gavage starting at 9 am, each time at 0.2 mL. The grouping and treatment method of experimental animals is shown in Table 1

TABLE 1 grouping and treatment method of experimental animals

And after the gastric lavage is finished in the 16 th week, putting a single mouse into a cage box filled with absorbent paper, collecting excrement, weighing the excrement to obtain wet weight, freeze-drying the excrement to obtain dry weight, and calculating the water content of the excrement according to the following formula.

The water content of the excrement is (excrement wet weight-excrement dry weight)/excrement wet weight multiplied by 100%

Separately, each mouse was gavaged with 0.2mL of ink, and from the beginning of gavage, the time for each mouse to discharge the first black stool was recorded.

Before the mice are killed, each mouse is perfused with 0.2mL of ink, the mice are killed and dissected after 30min, the upper end of the mouse is cut from the lower end of a pylorus to a cecum, the total length of the small intestine is measured as the total length of the small intestine, the ink advancing length is measured from the pylorus to the front edge of the ink, and the small intestine advancing rate is calculated according to the following formula.

The small intestine propulsion rate ═ ink propulsion length (cm))/(total small intestine length (cm)) × 100%

The results of the water content of the feces, the first grain black stool time and the small intestine propulsion rate are shown in fig. 1, and it can be seen from the graph that compared with the control group, the water content of the feces of the mice after molding is reduced from 52.88% to 45.50%, the first grain black stool time is prolonged to 1.83 times of that of the normal group, and the small intestine propulsion rate has no significant change, namely the colon transport disorder exists in the model mice, and the colon molding of the purgative is successful. Compared with the purgative colon model group, the treatment of the lactobacillus paracasei CCFM1164 can shorten the first grain black stool time of the purgative colon model mouse to 66.48% of the model, improve the water content of the stool by 15.20% compared with the model group, enhance the colon peristalsis capability of the purgative colon mouse, and the effect is superior to that of the mosapride group. The lactobacillus paracasei CCFM1164 can obviously enhance the colon transport capacity of the purgative colon mice and relieve the symptoms of the purgative colon mice.

Example 3: lactobacillus paracasei CCFM1164 increases mucin MUC-2 levels in colonic tissue of purgative colonic mice

The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 2. The real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) is adopted to determine the expression quantity of the MUC-2 gene, firstly, the RNA is extracted from the fresh colon tissue, and the specific method is as follows:

0.2g of fresh colon tissue taken out after the mouse is dissected is repeatedly ground in a mortar (180 ℃, 4h high temperature enzyme deactivation) added with liquid nitrogen, 1mL of Trizol reagent is added into the mortar, the grinding is continued, after the liquid is basically clarified, the fresh colon tissue is collected into a 1.5mL enzyme-free centrifuge tube, the centrifuge tube is kept still at room temperature for 15min, 200 mu L of trichloromethane solution is added into the centrifuge tube, the centrifuge tube is gently shaken for 15s, the centrifuge tube is kept still at room temperature for 10min, the centrifuge tube is centrifuged for 15min at 4 ℃ and 12000r/min, 600 mu L of upper colorless aqueous phase is taken out to another enzyme-free centrifuge tube, and 500 mu L of isopropanol is added. And (2) reversing the upside down and mixing the mixture evenly, standing the mixture for 10min at room temperature, centrifuging the mixture for 10min at 12000r/min at 4 ℃ after the standing is finished, discarding the supernatant, leaving a white precipitate of RNA formed at the bottom of a centrifugal tube, adding 1mL of 75% ethanol solution prepared by DEPC water, oscillating and resuspending the mixture in a vortex, centrifuging the mixture for 5min at 4 ℃ at 7500r/min, discarding the supernatant, and naturally volatilizing and drying the mixture at room temperature. To the dried RNA, 30. mu.L of RNase free water was added, and after the RNA was dissolved, the concentration and purity of the RNA were determined by Nanodrop, and the quality of the RNA was checked by agarose gel electrophoresis. The extracted total RNA was used as a template, and cDNA was synthesized by reverse transcription according to the protocol of HiScript II Q Select RT Supermix for qPCR reverse transcription kit of Novozan Biotechnology Ltd, and was stored at-20 ℃.

Primers for the mouse MUC-2 gene and the reference gene mGAPDH are shown in Table 2.

TABLE 2 mouse MUC-2 protein Gene and mGAPDH Gene primer sequences

By using

The CFX96TM real-time fluorescent quantitative PCR instrument performs PCR amplification and reads the fluorescent signal.

The MUC-2 gene qRT-PCR reaction system is as follows:

the MUC-2 gene qRT-PCR reaction conditions are as follows:

30s at 95 ℃; 10s at 95 ℃ and 30s at 60 ℃ for 40 cycles. The results were analyzed by CFX96Manager software using mGAPDH gene as the reference gene. The results of the experiment are shown in FIG. 2.

Mucin MUC-2 is a main component constituting the mucus layer of the intestinal tract, and has the functions of lubricating the intestinal tract, retaining water, preventing inflammatory factors from invading and destroying normal cells, maintaining the integrity of the epithelium, maintaining the normal peristalsis of smooth muscles, and maintaining the immunological tolerance of nerves. As can be seen from FIG. 2, after modeling, the MUC-2 expression level of the purgative colon mice is significantly reduced to 47.45% (p <0.001), which indicates that the intestinal barrier of the purgative colon mice is damaged, and after gastric lavage of CCFM1164, the MUC-2 protein expression level of the purgative colon mice is increased to 2.56 times that of the model group (p < 0.0001). Therefore, lactobacillus paracasei CCFM1164 can obviously improve the expression quantity of MUC-2 in the colon of the purgative colon mouse, lubricate the intestinal tract, rebuild a healthy colon barrier and improve the colonic motility, thereby relieving the purgative colon.

Example 4: lactobacillus paracasei CCFM1164 reduces aquaporin AQP4 levels in colonic tissue of laxative colonic mice

The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 2. The real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression level of AQP4 gene in the same manner as in example 3.

The mouse AQP4 gene and the internal reference gene mGAPDH gene primers are shown in Table 3,

TABLE 3 mouse AQP4 protein Gene and mGAPDH Gene primer sequences

The results were analyzed by CFX96Manager software using mGAPDH gene as the reference gene. The results of the experiment are shown in FIG. 3.

Aquaporin is a key protein for regulating the water balance of the intestinal tract, and the expression of AQP4 in the colon is related to water absorption. As can be seen from FIG. 3, after modeling, the AQP4 expression level of the purgative colon mouse is significantly increased to 1.63 times (P <0.0001) of the normal group, which indicates that the purgative colon mouse has water solution regulation imbalance, excessive water loss in the intestinal lumen leads to dry and hard feces, and after gastric lavage of CCFM1164, the AQP4 protein expression level of the purgative colon mouse is significantly reduced to 51.69% (P <0.0001) of the model group, and the down-regulation effect of AQP4 is 16.14% (P <0.01) lower than that of the mosapride group. Therefore, the lactobacillus paracasei CCFM1164 can obviously reduce the expression quantity of AQP4 in the colon of the purgative colon mouse, restore the water balance of the colon, improve the water content of the excrement, and enable the excrement to be easier to be discharged through the colon, thereby relieving the purgative colon.

Example 5: lactobacillus paracasei CCFM1164 for improving c-kit gene expression level in colon tissues of purgative colon mice

The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 2. The expression level of c-kit gene was determined by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) in the same manner as in example 3.

The primers for the mouse c-kit gene and the reference gene mGAPDH gene are shown in Table 4,

TABLE 4 mouse c-kit protein Gene and mGAPDH Gene primer sequences

The results were analyzed by CFX96Manager software using mGAPDH gene as the reference gene. The results of the experiment are shown in FIG. 4.

The interstitial cells (ICC) of Cajal are intestinal pacemaking cells, and the ICC plays a role by combining with a receptor c-kit, opens a signal path, maintains the growth and development of the ICC, and the quantity of the ICC can be expressed by the expression quantity of the c-kit. As can be seen from FIG. 4, after modeling, the c-kit expression level of the purgative colon mice is significantly reduced to 47.34% (P <0.01) of the normal group, which indicates that the gastrointestinal motility of the purgative colon mice is insufficient, and after gastric lavage CCFM1164, the c-kit protein expression level of the purgative colon mice is significantly increased to 1.72 times (P <0.0001) compared with the model group, and the effect is far better than that of the drug mosapride group (increased by 36.83% compared with the normal group). Therefore, the lactobacillus paracasei CCFM1164 can obviously improve the c-kit expression quantity in the colon of the purgative colon mouse and increase the quantity of interstitial cells of Cajal, thereby improving the colonic motility and relieving the purgative colon.

Example 6: lactobacillus paracasei CCFM1164 increases the number of glial cells in colonic tissue of laxative colonic mice

The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 2. The quantity of glial cells in the enteric nerve was quantified using immunofluorescent-labeled glial cell-characteristic protein S100 beta. The specific method comprises the following steps:

when a mouse dies, fresh colon tissues are removed and washed clean by normal saline, the mouse is immediately put into neutral buffered paraformaldehyde fixing solution for fixation for 24 hours and washed overnight by running water, samples are dehydrated by 70 percent, 80 percent and 90 percent of ethanol solutions for 30 minutes respectively, and then are put into 95 percent and 100 percent of ethanol solutions for 2 times and 20 minutes each time. The sample is passed through 1/2 pure alcohol, 1/2 equal amount of mixed liquid of xylene for 15min, xylene I for 15min and xylene II for 15min to be transparent, mixed liquid of xylene and paraffin for 15min, paraffin I and paraffin II for 50-60 min respectively. And slicing the colon with the cross section of the colon on the bottom layer by using a come card handwheel type slicer. Putting the slices and the slice frame into a drying box at 60 ℃, soaking the slices for 10min by using dimethylbenzene until wax is melted, replacing the dimethylbenzene, then soaking for 10min for dewaxing, putting the samples into 100%, 95%, 85%, 75% ethanol and ddH2O for hydration, boiling the samples by using sodium citrate buffer solution (10mM sodium citrate, 0.05% Tween 20 and pH 6.0) for 20min for antigen retrieval, washing 3 times by TBS (TBS), dripping 50 mu l of BSA-PBST (bovine serum albumin-beta protein) into each slice, sealing the slices in a wet box at room temperature for 30min, discarding sealing solution, dripping 50 mu l of primary antibody (Anti-S100 beta antibody, Abcam) into each slice, incubating overnight in a wet box at 4 ℃, taking out the slices the next day, and re-warming the slices at room temperature for 60 min. TBS soaking for 5min, cleaning for 4 times, removing liquid on tissue, dripping fluorescence-labeled secondary antibody 50 μ l into each section, incubating in dark for 45-60min, soaking in TBS for 5min, cleaning for 4 times, dripping DAPI 50 μ l into each section, counterstaining cell nucleus, sealing with fluorescence-resistant quenching sealing liquid, and sealing with nail polish. The digital slice scanner scans and FJ analyses are performed, and the results are shown in fig. 5.

The glial cells of the mucous membrane are mainly involved in the epithelial barrier function, and the glial cells in the ganglia are mainly used for nerve repair, closely interact with neurons, support the differentiation of the cells and the glial cells, and participate in the generation and formation of nerves. Laxative colon is distinguished from common constipation by the most obvious characteristic that nerve is seriously damaged, the number of glial cells can well reflect the health condition of enteric nerve, and S100 beta is a characteristic protein of glial cells, and the number of glial cells can be reflected according to the quantity of S100 beta. As can be seen from FIG. 5, the positive expression area of the enterocyte in the purgative colon mice is reduced by 47.09% (p <0.001) compared with the normal group, which indicates that the enteric nerve is damaged, and after the gastric lavage of CCFM1164, the positive expression area of the enterocyte is increased to 2.00 times (p <0.001) compared with the model group, and is restored to the normal level, which indicates that the enteric nerve is rebuilt. It is known that the gavage CCFM1164 can restore the intestinal nerve health, thereby promoting the colon peristalsis, improving the colon motility and fundamentally relieving the purgative colon.

Example 7: lactobacillus paracasei CCFM1164 increases GDNF levels in colonic tissue of purgative colon mice

The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 2. The expression level of GDNF in colon tissue was quantified by ELISA. The specific method comprises the following steps:

the colon tissue was rinsed with pre-cooled PBS, residual blood removed, surrounding adipose tissue removed, weighed and minced. Crushing the sheared tissue and a PBS solution on a high-throughput tissue crusher according to the weight-volume ratio of 1:9, finally centrifuging the homogenate and 5000 Xg for 5-10 minutes, taking the supernatant for detection, carrying out an experiment according to the corresponding kit specification, and calculating the GDNF in the tissue according to a standard curve. The results are shown in FIG. 6.

GDNF is a polypeptide neurotrophic factor, has the functions of nourishing and protecting damaged nerve cells, and has reduced expression in degenerated neurons, thus indicating the damage of the neurons. As can be seen from FIG. 6, the GDNF level of purgative colon mice is reduced by 39.82% (p <0.0001) compared with normal group, suggesting that there is enteric nerve damage, the GDNF level after gastric perfusion CCFM1164 is obviously improved by 53.65% (p0.001) compared with model group, and can reach the GDNF level equivalent to the drug mosapride (54.64% is improved, p <0.001), and the effect of nourishing enteric nerve is achieved by improving the GDNF expression, the normal enteric nerve function is recovered, thereby relieving purgative colon.

Example 8: lactobacillus paracasei CCFM1164 for improving butyric acid content in feces of purgative colon mice

The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 2. The feces of mice collected before the end of the experiment, from which the moisture content of the feces was determined, were cryopreserved at-80 ℃. The specific method comprises weighing 20mg of lyophilized feces, resuspending with 500 μ L of saturated NaCl solution, and adding 20 μ L of 10% H2SO4 solution; adding 1000 μ L of anhydrous ether, shaking, extracting fatty acid, and centrifuging at 12000rpm at 4 deg.C for 15 min; getThe upper diethyl ether phase, 0.25g of anhydrous Na was added₂SO₄Drying is carried out; standing for 30min, centrifuging at 12000rpm at 4 deg.C for 5min, collecting the upper layer diethyl ether phase, and determining short chain fatty acid content in lyophilized feces of mouse by GC-MS. An Rtx-Wax column (column length 30m, inner diameter 25 μm) was used; the carrier gas is He, and the flow rate is 2 mL/min; the sample introduction volume is 1 mu L, the temperature is increased to 140 ℃ according to the speed of 7.5 ℃/min, then the temperature is increased to 200 ℃ according to the speed of 60 ℃/min, the temperature is kept for 3min, and the ionization temperature is 20 ℃; the analysis adopts a full scanning mode, and a standard curve is measured by an external standard method, so that the concentration of butyric acid is calculated. The results are shown in FIG. 7.

Butyric acid is an important substrate for maintaining the epithelium of the colon, can induce the proliferation of healthy colon cells, promote the secretion of mucin and the redistribution of tight junction protein, and maintain the normal barrier function of the colon, and can be used as an energy substance of the colon to provide power for the colon. As can be seen from FIG. 7, the content of butyric acid in feces of purgative colon mice is reduced by 79.90% (p <0.01) compared with normal group, after gastric lavage of CCFM1164, the content of butyric acid in feces is significantly increased by 5.65 times (p <0.01) compared with model group, and the content of butyric acid in feces can be restored to the level of control group, while the content of butyric acid in mosapride is only increased by 2.44 times although the content of butyric acid in feces of mosapride is restored, and the effect is inferior to that of CCFM 1164. Therefore, after the CCFM1164 is dried, the level of butyric acid which is a colon energy substance can be obviously improved, the power is provided for the colon, the colon peristalsis capability is enhanced, and the purgative colon is relieved. And, in combination with the results of example 2, the increased levels of butyric acid also restored a healthier intestinal barrier environment for laxative colon mice, thereby relieving the laxative colon.

Example 9: fermented food containing lactobacillus paracasei CCFM1164 prepared by the invention

Instantly sterilizing cow milk at high temperature, immediately cooling to 37 ℃, and then inoculating the lactobacillus paracasei CCFM1164 microbial inoculum starter prepared by the invention for fermentation to ensure that the concentration of the lactobacillus paracasei CCFM1164 microbial inoculum reaches 10⁶And (3) storing the mixture at a temperature of 4 ℃ for refrigeration at a concentration of CFU/mL or more to obtain fermented milk containing the viable bacteria of Lactobacillus paracasei CCFM1164 of the present invention.

Example 10: fermented food containing lactobacillus paracasei CCFM1164 prepared by the invention

The lactobacillus paracasei CCFM1164 can be used for fermenting and producing other fermented foods, wherein the fermented foods comprise solid foods, liquid foods and semi-solid foods. The fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, goat milk, cheese, cream, milk-containing beverage or milk powder; the bean product comprises soybean, soybean milk, bean curd, and soybean milk powder; the fruit and vegetable product comprises a fruit and vegetable product prepared from at least one of Chinese cabbage, radish, cucumber, beet, green bean, apple or waxberry product.

The fermented food prepared in example 9 and example 10 was applied to purgative colon mice, and the results show that the fermented food containing lactobacillus paracasei CCFM1164 also has the effects of shortening the first-grain black stool time, increasing the water content of feces, increasing the transcription level of mucin MUC-2 in the colon tissue, reducing the transcription level of aquaporin AQP4 in the colon tissue, increasing the number of interstitial cells and glial cells of Cajal in the colon tissue, increasing the level of glial cell-derived trophic factor GDNF in the colon tissue, increasing the content of butyric acid in the feces, and improving the intestinal health state of purgative colon mice.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> lactobacillus paracasei for relieving purgative colon and application thereof

<130> BAA2104349A

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<170> PatentIn version 3.3

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Claims (10)

1. A Lactobacillus paracasei (Lactobacillus paracasei) CCFM1164 is characterized by being preserved in Guangdong provincial academy of sciences microbiological research institute at 29 th 1 month in 2021, with the preservation number being GDMCC No: 61479.

2. a composition characterized by: contains the lactobacillus paracasei CCFM1164 as set forth in claim 1.

3. The composition of claim 2, wherein the cell number of Lactobacillus paracasei CCFM1164 is 1 x 10 or more⁸CFU/g or 1X 10⁸CFU/mL。

4. The composition according to claim 2 or 3, characterized in being a fermented food, a dietary supplement or a medicament.

5. The composition of claim 3, wherein the fermented food product is a fermented dairy product, a soy product, and a fruit and vegetable product prepared by fermentation using Lactobacillus paracasei CCFM 1164.

6. The composition as claimed in claim 3, wherein the dietary supplement comprises a powder, capsule, fondant or liquid formulation of Lactobacillus paracasei CCFM 1164.

7. Use of lactobacillus paracasei CCFM1164 as claimed in claim 1 for the preparation of a medicament for alleviating slow transit constipation or gastrointestinal motility disorders.

8. The use of claim 7, wherein the slow transit constipation includes, but is not limited to, laxative colon.

9. The use of claim 8, wherein the relief of slow transit constipation comprises at least one of:

1) shortening the first grain black stool time of the cathartic colon mammal, and increasing the water content of the stool;

2) increasing mucin MUC-2 transcript levels in colonic tissue of a laxative colon mammal;

3) reducing the transcriptional level of aquaporin AQP4 in colonic tissue of a laxative colon mammal;

4) increasing the number of interstitial cells of Cajal in colon tissue of a laxative colon mammal;

5) increasing the number of glial cells in colonic tissue of a laxative colon mammal;

6) increasing the level of glial cell line-derived trophic factor (GDNF) in colonic tissue of a laxative colon mammal;

7) increasing butyric acid content in stool of laxative colon mammal.

10. The medicament of claim 7, wherein the medicament is in the form of granules, capsules, tablets, pills or oral liquid.

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