Kluyveromyces marxianus KUST3801 and application thereof in relieving constipation

Abstract

The invention discloses Kluyveromyces marxianus KUST3801 and application thereof in relieving constipation, and the preservation number of the Kluyveromyces marxianus KUST3801 is CGMCC (China General Microbiological Culture Collection Center) NO.32767. The Kluyveromyces marxianus strain is classified and named as Kluyveromyces marxianus, and the Kluyveromyces marxianus strain is named as Kluyveromyces marxianus. The sequence of the 16SrRNA is as shown in SEQ ID NO. 1; the composition is used for improving constipation symptoms. After the kluyveromyces marxianus KUST3801 is applied, the excrement water content and the small intestine propulsion rate of a mouse with ICR constipation induced by loperamide hydrochloride are remarkably improved, and the defecation frequency and the defecation number within the same time are remarkably increased; intestinal mucosa tissues of constipation mice can be protected, and intestinal mucosa injury is relieved; the concentration of SP and MTL is obviously improved, and the concentration of VIP is reduced; iL-6 concentration is reversed, and inflammatory response is relieved; the concentrations of acetic acid, butyric acid, isobutyric acid and isovaleric acid in the short-chain fatty acid are obviously higher than those of mice with constipation, and the composition has a relatively strong effect of relieving constipation symptoms.

Description

Technical Field

[0001] This application relates to the technical field of constipation improvement, particularly a Kluyveromyces marxianus KUST3801 and its application in relieving constipation. Background Art

[0002] Constipation is a common intestinal disease related to intestinal microbiota dysbiosis. Constipation can reduce the quality of life of patients. In severe cases, it can threaten the health of patients, cause inflammatory reactions, damage the intestinal mucosa, leading to abdominal pain, abdominal distension, anal fissure, hemorrhoids, and intestinal perforation. It can even induce hypertension, cardiovascular diseases, and colon cancer, etc. At the same time, due to the defecation pressure, some people will suffer from mental illnesses to varying degrees. Currently, the treatment methods for constipation include: traditional Chinese medicine treatment, Western medicine treatment, surgical treatment, and intestinal microecological adjustment. However, currently, 60% of constipation patients mainly relieve symptoms through stimulant laxatives and stool softeners. These drugs have problems such as large side effects and easy drug dependence, resulting in about 1 / 3 of the patients being unable to obtain ideal treatment effects.

[0003] Intestinal microecological dysregulation is one of the causes of constipation. Intestinal flora disorders can lead to bile acid metabolism disorders, causing proximal colon motility disorders and resulting in the occurrence of constipation. Probiotics are defined as "live microorganisms that, when administered in sufficient amounts, confer health benefits to the host".

[0004] Probiotics can be used to regulate the composition of the host intestinal microbiota, improve the intestinal environment, and are widely used in the research of relieving constipation. Because of its advantages such as no side effects, relieving intestinal inflammation, and promoting intestinal peristalsis, it has received extensive attention. Existing probiotics for relieving constipation symptoms include Bifidobacterium bifidum, Bifidobacterium lactis, etc. The existing types of probiotics need to be urgently broadened.

[0005] Some strains of Kluyveromyces marxianus show good lactic acid tolerance and low pH tolerance (CN202310540115.9), and there is no report on its application in constipation-related symptoms.

[0006] The information disclosed in the background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0007] This application provides a Kluyveromyces marxianus KUST3801 and its application in relieving constipation for the above technical problems.

[0008] The present application provides a Kluyveromyces marxianus KUST3801 and its application in relieving constipation, with the preservation number CGMCC NO. 32767; the taxonomic name is Kluyveromyces marxianus; the 16S rRNA sequence is as shown in SEQ ID NO. 1; it is used for the improvement of constipation symptoms.

[0009] Preferably, improving constipation symptoms includes: increasing the defecation volume, fecal weight, water content, and small intestine propulsion rate after constipation; restoring the overall morphology of the colon tissue and protecting the intestinal mucosa tissue after constipation;

[0010] Preferably, increasing the defecation volume to 3.2 times that of the model group; increasing the fecal weight to 4.3 times that of the model group; increasing the small intestine propulsion rate to 1.5 times that of the model group.

[0011] Preferably, improving constipation symptoms includes: increasing the SP level, increasing the MTL content, decreasing the VIP content, and decreasing the IL-6 concentration in the blood after constipation;

[0012] Preferably, increasing the SP level to 45.52 pg / mL; increasing the MTL content to 82.87 pg / mL; decreasing the VIP content to 50.40 pg / mL; decreasing the IL-6 concentration to 5.11 pg / mL.

[0013] Preferably, improving constipation symptoms includes: increasing the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid in the blood after constipation;

[0014] Preferably, increasing the acetic acid content in the blood after constipation to 1.42 times that of the model group; increasing the propionic acid content in the blood after constipation to 1.49 times that of the model group; increasing the isobutyric acid content in the blood after constipation to 3.09 times that of the model group; increasing the butyric acid content in the blood after constipation to 2.05 times that of the model group; increasing the isovaleric acid content in the blood after constipation to 1.81 times that of the model group.

[0015] On the other hand, the present application also provides an application of the Kluyveromyces marxianus as described above in the preparation of a preparation for preventing or treating constipation.

[0016] Preferably, the preparation includes: the Kluyveromyces marxianus KUST3801 strain or the fermentation product of the Kluyveromyces marxianus KUST3801 strain;

[0017] Preferably, the viable count of the Kluyveromyces marxianus KUST3801 strain in the preparation is greater than or equal to 1×10 8 CFU / g;

[0018] Preferably, the viable count of the Kluyveromyces marxianus KUST3801 strain in the preparation is 1×10 8~1×10 10 CFU / g.

[0019] Preferably, the method for preparing the fermentation product of Kluyveromyces marxianus KUST3801 strain comprises the following steps: picking a single colony of activated Kluyveromyces marxianus KUST3801 and inoculating it into a test tube containing YEPD liquid medium, culturing it with shaking at 25 - 30 °C for 20 - 30 h to obtain the fermentation product of Kluyveromyces marxianus KUST3801 strain;

[0020] Preferably, the inoculation amount of a single colony of Kluyveromyces marxianus KUST3801 in YEPD liquid medium is 1 - 3% of the volume of YEPD liquid medium.

[0021] Preferably, the formula of YEPD liquid medium: is prepared according to the ratio of 20 - 25 g / L glucose, 15 - 20 g / L peptone, 5 - 10 g / L yeast extract powder, and 1 L deionized water.

[0022] Preferably, the preparation includes: solid dosage form or liquid dosage form.

[0023] Preferably, the solid dosage form includes: any one of powder, liquid dosage form, tablet, capsule, powder, pill, and film patch;

[0024] The liquid dosage form includes: any one of injection, oral liquid, syrup dosage form, and suspension.

[0025] The beneficial effects that this application can produce include:

[0026] 1) For the Kluyveromyces marxianus KUST3801 provided by this application and its application in relieving constipation, after using Kluyveromyces marxianus KUST3801, the water content of feces and the small intestine propulsion rate of ICR constipated mice induced by Loperamide hydrochloride are significantly increased, the defecation frequency and the number of defecation grains within the same time are significantly increased; and it has a certain protective effect on the intestinal mucosal tissue of constipated mice, and can reduce intestinal mucosal damage; in addition, the concentrations of gastrointestinal regulatory peptides substance P (SP) and motilin (MTL) are significantly increased, and the concentration of vasoactive intestinal peptide (VIP) is decreased; effectively reverse the concentration of inflammatory cytokine interleukin - 6 (IL - 6), and reduce the inflammatory response; in addition, the concentrations of acetic acid, butyric acid, isobutyric acid, and isovaleric acid in short - chain fatty acids are significantly higher than those of constipated mice, and it has a strong effect of relieving constipation symptoms.

[0027] 2) The Kluyveromyces marxianus KUST3801 provided by this application and its application in relieving constipation. The Kluyveromyces marxianus KUST3801 can increase the defecation volume to 3.2 times that of the model group; increase the defecation weight to 4.3 times that of the model group; increase the water content of feces to protect the intestinal mucosa; increase the small intestine propulsion rate to 1.5 times that of the model group relative to the model group; the overall morphology of the colon tissue in the KUST3801 group is closer to that of the blank group, and only slight damage appears in the villus structure, which has a certain protective effect on the intestinal mucosa tissue of constipated mice and can also reduce and repair intestinal mucosa damage.

[0028] 3) The Kluyveromyces marxianus KUST3801 provided by this application and its application in relieving constipation. The Kluyveromyces marxianus KUST3801 can significantly increase the SP level of constipated mice to 45.52 pg / mL; significantly increase the MTL content to 82.87 pg / mL, which is higher than the level of normal mice, indicating that the Kluyveromyces marxianus KUST3801 can significantly improve the motilin content in the blood of mice; both significantly reduce the VIP content to 50.40 pg / mL; significantly reduce the concentration of IL-6 to 5.11 pg / mL. The results show that the Kluyveromyces marxianus KUST3801 can effectively reverse the abnormal production of inflammatory cytokines and relieve the inflammatory response of constipated mice.

[0029] 4) The Kluyveromyces marxianus KUST3801 provided by this application and its application in relieving constipation. The Kluyveromyces marxianus KUST3801 can increase the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid in model constipated mice to 1.42, 1.49, 3.09, 2.05, and 1.81 times that of the model group. In particular, it can increase the content of butyric acid, the main energy source of colon cells, and can maintain the integrity of the colon mucosa, prevent colorectal cancer and inflammation. It shows that the intervention of Kluyveromyces marxianus KUST3801 can be used as an effective way to relieve constipation.

[0030] The Kluyveromyces marxianus KUST3801 was deposited on November 25, 2024, at the China General Microbiological Culture Collection Center (CGMCC), No. 3, Building 1, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101; the deposit number is CGMCC NO. 32767; the taxonomic name is Kluyveromyces marxianus. Description of the Drawings

[0031] Figure 1 It is a photograph of the colony morphology of the Kluyveromyces marxianus KUST3801 obtained in Example 2 of this application;

[0032] Figure 2This is a growth curve of Kluyveromyces marxii KUST3801 obtained in Example 3 of the present application at inoculation amounts of 1%, 2%, and 3%;

[0033] Figure 3 This is a bar graph of the number of stool particles in mice after treatment with the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxensis KUST3801 group (KUST3801 group) on days 7 and 14 in Example 4 of the present application;

[0034] Figure 4 This is a bar graph of the feces weight results of mice treated with the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxensis KUST3801 group (KUST3801 group) on days 7 and 14 in Example 4 of the present application;

[0035] Figure 5 This is a bar graph of the fecal water content results of mice treated with the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxensis KUST3801 group (KUST3801 group) on days 7 and 14 in Example 4 of the present application;

[0036] Figure 6 This is a bar graph of the small intestinal propulsion rate results of mice in the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxense KUST3801 group (KUST3801 group) treated on the 15th day in Example 4 of the present application;

[0037] Figure 7 The figure is the H&E staining result of the colon of mice in the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxist KUST3801 group (KUST3801 group) in Example 4 of the present application after 15 days of treatment; the figure at the intersection of 40× and the blank group is the H&E staining result of the colon of mice obtained in the blank group, and the meanings of the remaining figures are expressed by analogy.

[0038] Figure 8 This is a bar graph showing the results of the substance P (SP) content in the blood of mice treated with the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxense KUST3801 group (KUST3801 group) for 15 days in Example 4 of the present application;

[0039] Figure 9Bar chart of the motilin (MTL) content in the blood of mice obtained after 15 days of treatment with the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxianus KUST3801 group (KUST3801 group) in Example 4 of this application;

[0040] Figure 10 Bar chart of the vasoactive intestinal peptide (VIP) content in the mice obtained after 15 days of treatment with the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxianus KUST3801 group (KUST3801 group) in Example 4 of this application;

[0041] Figure 11 Bar chart of the interleukin-6 (IL-6) content of inflammatory factors in the mice obtained after 15 days of treatment with the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxianus KUST3801 group (KUST3801 group) in Example 4 of this application;

[0042] Figure 12 Bar chart of the short-chain fatty acid (SCFAs) content in the mice obtained after 15 days of treatment with the blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxianus KUST3801 group (KUST3801 group) in Example 4 of this application; Detailed implementation mode

[0043] The technical solutions provided by this application will be explained and described in detail below in combination with specific embodiments and drawings.

[0044] Embodiment

[0045] Unless otherwise specified in the following embodiments, the detection methods used are all existing methods, and the materials used are all commercially available products.

[0046] The preparation method of the culture medium used in the following embodiments is as follows:

[0047] Embodiment 1: Strain isolation and culture

[0048] The Kluyveromyces marxianus KUST3801 provided by the present invention was isolated from the handmade natural fermented yak yogurt made by herdsmen in Shangri-La City, Diqing Tibetan Autonomous Prefecture, Yunnan on June 10, 2024. The specific steps are as follows:

[0049] 1. Strain isolation and culture: After shaking the collected yak yogurt sample evenly, weigh 0.5 mL and perform ten-fold serial dilution with 0.9% sterile physiological saline (1×10 -1 ~1×10 -9) Next, spread it on YEPD agar plate medium, and anaerobically culture it at 30 °C for 48 h. After picking a single colony, continue to streak and purify it on YEPD agar medium. Repeat the purification step three times to obtain a pure culture. Use 50% glycerol as a cryoprotectant for the purified strain and store it in a -80 °C refrigerator for later use.

[0050] Preparation of the YEPD medium used: Prepare YEPD agar medium according to the ratio of 22 g / L glucose, 17 g / L peptone, 8 g / L yeast extract powder, 17 g / L agar powder, and 1 L deionized water; sterilize it at 160 °C for 17 min to obtain YEPD agar medium.

[0051] 2. Strain morphological identification: After activating the strain obtained in step 1, inoculate it on YEPD agar plate medium and anaerobically culture it at 30 °C for 48 h to obtain a colony plate as shown in the appendix Figure 1 As can be seen from observing this plate, the colony morphology of this strain is milky white, round and convex, with a neat, transparent or translucent edge and a gelatinous and viscous texture.

[0052] Example 2: Molecular biological identification of the strain

[0053] Inoculate the strain isolated in Example 1 in YEPD liquid medium, culture it at 30 °C for 48 h, and then perform 16S rRNA sequencing identification. Compare the measured 16S rRNA sequence with the NCBI BLAST database. The identification result is Kluyveromyces marxianus, and it is named Kluyveromyces marxianus KUST3801.

[0054] The 16S rRNA sequence is as shown in SEQ ID NO.1:

[0055]

[0056] Kluyveromyces marxianus KUST3801 was deposited on November 25, 2024, at the China General Microbiological Culture Collection Center (CGMCC), No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101; the deposit number is CGMCC NO. 32767; the taxonomic name is Kluyveromyces marxianus.

[0057] Example 3: Growth ability test of Kluyveromyces marxianus KUST3801

[0058] 1. Preparation of YEPD liquid medium: Prepare YEPD liquid medium according to the ratio of 20 - 25 g / L glucose, 15 - 20 g / L peptone, 5 - 10 g / L yeast extract powder, and 1 L deionized water; sterilize it at 115 - 121 °C for 15 - 20 min to obtain YEPD liquid medium.

[0059] 2. Inoculation and culture: Pick a single colony of Kluyveromyces marxianus KUST3801 from the YEPD agar plate medium and inoculate it into a test tube filled with YEPD liquid medium, and culture it with shaking at 30 °C for 24 h to obtain a Kluyveromyces marxianus KUST3801 bacterial suspension.

[0060] Measure the absorbance (OD) of the bacterial suspension at 600 nm with an enzyme - linked immunosorbent assay (ELISA) reader, and adjust the OD600nm to 1 with the uninoculated YEPD liquid medium.

[0061] 3. Measurement of growth amount: Inoculate the adjusted - concentration Kluyveromyces marxianus KUST3801 bacterial suspension into YEPD liquid medium at inoculation amounts of 1%, 2%, and 3% of the volume of the liquid medium, and culture it with constant - temperature shaking at 30 °C. Use the uninoculated YEPD liquid medium as a blank control, and start measuring the OD600nm value of the culture solution at 0 h. The measurement time interval is 1 h or 2 h.

[0062] 4. Drawing of growth curve: Use time as the abscissa and OD600nm value as the ordinate to draw the growth curve of Kluyveromyces marxianus KUST3801. As shown in the appendix Figure 2 shown, Kluyveromyces marxianus KUST3801 with three different inoculation amounts all reached the stationary phase at 25 - 30 h, at which time the viable cell count remained relatively stable and reached the highest level.

[0063] Example 4: Alleviating effect of Kluyveromyces marxianus KUST3801 on constipated mice

[0064] 1. Animal grouping and model establishment

[0065] After acclimating 40 six-week-old male ICR mice for one week, they were randomly divided into 4 groups: blank control group (blank group), constipation model group (model group), drug treatment group (drug group), and Kluyveromyces marxianus KUST3801 group (KUST3801 group). The blank control group was intragastrically administered 0.2 mL of normal saline twice a day; the constipation model group was intragastrically administered 0.2 mL of loperamide hydrochloride solution (10 mg / kg b.w) once a day, and 0.2 mL of normal saline was intragastrically administered 1 hour later; the drug treatment group was intragastrically administered 0.2 mL of loperamide hydrochloride solution (10 mg / kg b.w) once a day, and an equal volume of bisacodyl solution (100 mg / Kg b.w) was intragastrically administered 1 hour later; the Kluyveromyces marxianus KUST3801 group was intragastrically administered 0.2 mL of loperamide hydrochloride solution (10 mg / kg b.w) once a day, and 1×10 9 CFU / mL of Kluyveromyces marxianus KUST3801 bacterial suspension was intragastrically administered continuously.

[0066] 2. Effects of Kluyveromyces marxianus KUST3801 on the number, weight, and water content of feces in constipated mice

[0067] Mice feces were collected at 8 - 10 am on the 7th and 14th days. Each mouse was placed separately in a clean cage box, and feces were collected in a timely manner. The wet weight and the number of fecal pellets were recorded, and they were stored in an ice box. After freeze-drying, the dry weight of the feces was recorded, and the fecal moisture content was calculated.

[0068] The results of the number of fecal pellets of each group of mice are shown in the appendix Figure 3 As shown, there were significant differences between the KUST3801 group and the model group. On the 7th and 14th days, the model group had the least amount of feces discharged, which were 1.9 pellets and 1.3 pellets respectively, while the KUST3801 group had 4.8 pellets and 5.3 pellets respectively, which were 3.2 times that of the model group. In addition, compared with the number of fecal pellets on the 7th day, the number of fecal pellets in the model group decreased by 0.6 pellets on the 14th day, while the number of fecal pellets in the other groups increased on the 14th day.

[0069] The results of the fecal weight of each group of mice are shown in the appendix Figure 4 As shown, on the 7th and 14th days, the fecal weight of the model group at 2 h was the lowest, which were 0.52 g and 0.66 g respectively, while the fecal weight of the KUST3801 group was 2.1 g and 2.88 g respectively, which were 4.3 times that of the model group.

[0070] Feces of each treatment group of mice were collected respectively after treatment, and the water content of the obtained feces was calculated according to the following formula: fecal water content (%) = (fecal wet weight - fecal dry weight) ÷ fecal wet weight × 100%; the fecal dry weight was obtained by weighing the collected feces after freeze-drying in a vacuum environment.

[0071] The results of fecal water content in each group of mice are shown in the appendix Figure 5 As shown, the fecal water content in the KUST3801 group was higher than that in the model group, indicating that the KUST3801 strain can effectively improve the problem of overly dry feces in the constipation model mice, which is beneficial to alleviating the problem of intestinal mucosal damage caused by overly dry feces due to constipation.

[0072] As can be seen from the appendix Figures 3 to 5 Compared with the model group, Kluyveromyces marxianus KUST3801 can significantly improve the defecation quantity, weight and water content of the modeled mice, and show outstanding effects on increasing the number of fecal pellets, fecal weight and improving fecal water content and other indicators.

[0073] 3. Effect of Kluyveromyces marxianus KUST3801 on the small intestine propulsion rate of constipated mice;

[0074] Each treatment group of mice was normally fed until the 15th day, and the small intestine propulsion rate of the mice was measured. After 14 days of gavage, the mice were fasted for 12 h before being sacrificed. All groups except the blank control group were gavaged with 0.2 mL of loperamide hydrochloride solution (10 mg / kg b.w). After 1 h, each group was gavaged with 0.25 mL of ink containing the corresponding test substance; after 0.5 h, the mice were sacrificed by cervical dislocation after taking blood from the eyeball, the small intestine was taken out, the total length of the small intestine from the pyloric end to the cecal segment was measured and recorded as the total length of the small intestine; the distance from the pylorus to the most forward end of the ink propulsion in the intestinal tube was measured and recorded as the ink propulsion length, and then the ink propulsion rate was calculated according to the following formula:

[0075] Small intestine propulsion rate (%) = (ink propulsion length ÷ total length of small intestine) × 100%

[0076] The results obtained in each treatment group are shown in the appendix Figure 6 As shown, there were significant differences in the small intestine propulsion rate of each group of mice compared with the model group. Compared with the model group, the small intestine propulsion rate in the KUST3801 group increased by 28.11%, which was 1.5 times that of the model group. The results showed that Kluyveromyces marxianus KUST3801 can significantly increase the small intestine propulsion rate of constipated mice, which is beneficial to improving constipation symptoms.

[0077] The preparation method of the above-mentioned ink used: Take 50 g of gum arabic powder and mix it with 400 mL of water, heat and boil until the solution becomes transparent, add 25 g of activated carbon powder, boil it three times repeatedly, filter out the insoluble substances, dilute it with water to 500 mL, store it at 4 °C, and shake it well before use.

[0078] 4. Effect of Kluyveromyces marxianus KUST3801 on the histopathology of the colon in constipated mice

[0079] The dissected mouse colons in each group were immersed in 4% paraformaldehyde fixative overnight, rinsed to obtain the samples to be tested. The obtained samples to be tested were dehydrated with gradient alcohol, washed with xylene, embedded in paraffin, sectioned, and stained with HE.

[0080] The sections of mouse colon tissue were observed under a microscope, and the results were as Figure 7 shown. The pathological morphology of the colon in the blank group was normal, the mucosa was intact, and there was no inflammatory infiltration. In the model group, the intestinal villi and crypts of the mouse colon were damaged. After magnifying 400 times, it could be seen that the number of goblet cells in the colon of the model group mice increased and the cell volume enlarged, with obvious inflammatory infiltration, erosion of the mucosa, and submucosal edema;

[0081] In the drug group, submucosal edema of the colon was visible;

[0082] The overall morphology of the colon tissue in the Kluyveromyces marxianus KUST3801 group was closer to that of the blank group, and only slight damage occurred to the villus structure, indicating that Kluyveromyces marxianus KUST3801 had a certain protective effect on the intestinal mucosal tissue of constipated mice and could also reduce and repair intestinal mucosal damage.

[0083] 5. Effect of Kluyveromyces marxianus KUST3801 on gastrointestinal regulatory peptides in constipated mice

[0084] After the extracted mouse blood was placed at room temperature for 2 h, it was centrifuged for 15 min (3000×g, 4 °C), and the obtained supernatant was the serum sample to be tested. The serum was aliquoted into 200 μL centrifuge tubes, and the determination methods for gastrointestinal regulatory peptides (substance P (SP), motilin (MTL), vasoactive intestinal peptide (VIP)) were carried out according to the instructions of the ELISA kit.

[0085] The change in the content of substance P (SP) in mouse serum was as shown in the appendix Figure 8 shown. Kluyveromyces marxianus KUST3801 and the drug group significantly increased the SP level in constipated mice (the KUST3801 group was 45.52 pg / mL, the drug group was 41.43 pg / mL, and the model group was 29.45 pg / mL).

[0086] The content of motilin (MTL) in the blood of mice in each group after 15 days of treatment was as shown in the appendix Figure 9As shown, the MTL content in constipated mice (54.46 pg / mL in the model group) was significantly lower than that in normal mice (77.10 pg / mL in the blank group). Kluyveromyces marxianus KUST3801 significantly increased the MTL content to 82.87 pg / mL, higher than the level in normal mice, indicating that Kluyveromyces marxianus KUST3801 can significantly improve the motilin content in the blood of mice; while the MTL level in the bisacodyl drug treatment group of mice (70.84 pg / mL in the drug group) was comparable to that in the blank group of mice.

[0087] The vasoactive intestinal peptide (VIP) content of the mice obtained after 15 days of treatment in each group is as shown in the appendix Figure 10 As shown, compared with healthy mice, the VIP level in constipated mice was significantly increased (72.17 pg / mL in the model group and 49.55 pg / mL in the blank group). Intervention with Kluyveromyces marxianus KUST3801 (50.40 pg / mL) and bisacodyl treatment (55.05 pg / mL) both significantly reduced the VIP level.

[0088] 6. Effect of Kluyveromyces marxianus KUST3801 on the concentration of inflammatory factors in constipated mice

[0089] The blood samples collected from the constipated mouse experiment were placed at room temperature for 1 h, and then centrifuged at 3500 rpm for 15 min at 4 °C to obtain serum samples. According to the manufacturer's instructions, the concentration of the inflammatory cytokine IL-6 was measured using an Elisa kit.

[0090] The results obtained in each treatment group are as shown in Figure 11 As shown, compared with the blank group of mice, the concentration of the inflammatory cytokine IL-6 in the model group of mice (13.23 pg / mL total protein in the model group concentration) was significantly increased. Both the Kluyveromyces marxianus KUST3801 group and the drug group could significantly reduce the concentration of IL-6. The KUST3801 group had the best effect (5.11 pg / mL total protein in concentration), followed by the drug group (7.76 pg / mL total protein in concentration). The results indicate that Kluyveromyces marxianus KUST3801 can effectively reverse the abnormal production of inflammatory cytokines and alleviate the inflammatory response in constipated mice.

[0091] 7. Effect of Kluyveromyces marxianus KUST3801 on the content of short-chain fatty acids (SCFAs) in constipated mice

[0092] On the 14th day of gavage, fecal samples of each group were collected and freeze-dried to obtain samples to be measured. The samples to be measured (20 - 50 mg) were dissolved in saturated sodium chloride solution and acidified with sulfuric acid (10%). Total SCFAs were extracted with ether. The concentration of SCFA was determined by gas chromatography-mass spectrometry.

[0093] The results obtained by each treatment group are shown in the appendix Figure 12 As shown, from the results of the model group, it can be seen that constipation led to a significant decrease in the contents of acetic acid (481.67 μmol / g), isobutyric acid (9.20 μmol / g), butyric acid (58.13 μmol / g), and isovaleric acid (17.27 μmol / g) in the feces of mice, while no change in valeric acid was observed.

[0094] The intervention of Kluyveromyces marxianus KUST3801 significantly increased the contents of acetic acid (681.73 μmol / g), isobutyric acid (28.46 μmol / g), butyric acid (119.20 μmol / g), and isovaleric acid (31.21 μmol / g). Compared with the model group, the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid in the KUST3801 group increased by 1.42, 1.49, 3.09, 2.05, and 1.81 times, respectively. Among SCFAs, butyric acid is the main energy source for colon cells, which can maintain the integrity of the colonic mucosa, prevent colorectal cancer and inflammation. The recovery effect of the butyric acid content in the Kluyveromyces marxianus KUST3801 group was obvious, and it was basically close to the level of mice in the blank group, indicating that the intervention of Kluyveromyces marxianus KUST3801 can be used as an effective way to relieve constipation.

[0095] Example 5

[0096] The preparation method of the fermentation product of Kluyveromyces marxianus KUST3801 strain includes the following steps: Pick a single colony of activated Kluyveromyces marxianus KUST3801 and inoculate it into a test tube containing YEPD liquid medium, and shake culture at 25 °C for 200 h to obtain the fermentation product of Kluyveromyces marxianus KUST3801 strain.

[0097] The formula of the used YEPD liquid medium: Prepare according to the ratio of 20 g / L glucose, 15 g / L peptone, 5 g / L yeast extract powder, and 1 L deionized water.

[0098] The obtained fermentation product is mixed with pure water at a volume ratio of 1:1 to make a liquid dosage form oral liquid, and the viable count of the strain in the oral liquid is 1×10 8 .

[0099] Example 6

[0100] The preparation method of the fermentation product of Kluyveromyces marxianus KUST3801 strain includes the following steps: Pick a single colony of activated Kluyveromyces marxianus KUST3801 and inoculate it into a test tube containing YEPD liquid medium, and shake culture at 30 °C for 30 h to obtain the fermentation product of Kluyveromyces marxianus KUST3801 strain.

[0101] The formulation of the YEPD liquid medium used: Prepared according to the ratio of 25 g / L glucose, 20 g / L peptone, 10 g / L yeast extract powder, and 1 L deionized water.

[0102] The obtained fermentation product is made into tablets, and the viable count of the strain is 1×10 10 CFU / g.

[0103] The tablet preparation process is as follows: After spray-drying the fermentation product of Kluyveromyces marxianus strain KUST3801 to obtain drug powder, it is mixed evenly with excipients, and then 2% of the total mass of binder (such as water, ethanol, etc.) is added to make soft material; the soft material is made into wet granules through a sieve or granulator, and granulated after drying; after adding lubricant and disintegrant, it is mixed evenly, and finally pressed into tablets by a tablet press.

[0104] Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Kluyveromyces marxii strain KUST3801, characterized in that: The deposit number is CGMCC NO.32767; the classification name is Kluyveromyces marxianus; the 16SrRNA sequence is shown in SEQ ID NO.1; and it is used for improving constipation symptoms.

2. The Kluyveromyces marxii KUST3801 according to claim 1, characterized in that Improvement of constipation symptoms includes: increasing stool volume, stool weight, water content and small intestinal propulsion rate after constipation; restoring the overall morphology of colon tissue after constipation and protecting intestinal mucosal tissue; Preferably, the defecation volume is increased to 3.2 times that of the model group; the defecation weight is increased to 4.3 times that of the model group; and the small intestinal propulsion rate is increased to 1.5 times that of the model group.

3. The Kluyveromyces marxii KUST3801 according to claim 1, characterized in that Improvement of constipation symptoms includes: increasing the level of SP in the blood after constipation, increasing the content of MTL, decreasing the content of VIP, and decreasing the concentration of IL-6; Preferably, the SP level is increased to 45.52 pg / mL; the MTL content is increased to 82.87 pg / mL; the VIP content is reduced to 50.40 pg / mL; and the IL-6 concentration is reduced to 5.11 pg / mL.

4. The Kluyveromyces marxii KUST3801 according to claim 1, characterized in that Improvement of constipation symptoms includes: increasing the levels of acetic acid, propionic acid, isobutyric acid, butyric acid and isovaleric acid in the blood after constipation; Preferably, the acetic acid content in the blood after constipation is increased to 1.42 times that of the model group; the propionic acid content in the blood after constipation is increased to 1.49 times that of the model group; the isobutyric acid content in the blood after constipation is increased to 3.09 times that of the model group; the butyric acid content in the blood after constipation is increased to 2.05 times that of the model group; and the isovaleric acid content in the blood after constipation is increased to 1.81 times that of the model group.

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

6. The use according to claim 5, characterized in that: Preparations include: Kluyveromyces marxianus strain KUST3801 or a fermentation product of Kluyveromyces marxianus strain KUST3801; Preferably, the number of viable bacteria of Kluyveromyces marxii KUST3801 strain in the preparation is greater than or equal to 1×10 8 CFU / g; Preferably, the viable count of Kluyveromyces marxii KUST3801 strain in the preparation is 1×10 8 ~1×10 10 CFU / g.

7. The use according to claim 6, characterized in that: The method for preparing the fermentation product of Kluyveromyces marxense KUST3801 strain comprises the following steps: picking a single colony of Kluyveromyces marxense KUST3801 after activation and inoculating it into a test tube filled with YEPD liquid culture medium, and shaking and culturing it at 25-30° C. for 20-30 hours to obtain the fermentation product of Kluyveromyces marxense KUST3801 strain; Preferably, the inoculation amount of a single colony of Kluyveromyces marxii KUST3801 in the YEPD liquid culture medium is 1-3% of the volume of the YEPD liquid culture medium.

8. The use according to claim 7, characterized in that: The formula of YEPD liquid culture medium: prepared according to the ratio of 20-25g / L glucose, 15-20g / L peptone, 5-10g / L yeast extract powder, and 1L deionized water.

9. The use according to claim 5, characterized in that: Preparations include: Solid dosage form or liquid dosage form.

10. The use according to claim 9, characterized in that: Solid dosage forms include: any one of powder, liquid dosage form, tablet, capsule, powder, pill, and film-forming agent; Liquid dosage forms include: any one of injection, oral solution, syrup, and suspension.

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