Application of dietary fiber probiotic composition

Abstract

The invention relates to application of a dietary fiber probiotic composition, in particular to application of a composition of ganoderma lucidum dietary fibers and lactobacillus fermentum CEC-T5716 in preparation of drugs or health care products for preventing or relieving constipation. The constipation symptom can be effectively improved by influencing the weight of mice, the first black stool time, the excrement water content, the small intestine propulsion rate, the colon tissue state, colon tissue aquaporin, the intestinal barrier, inflammatory factor related genes and excrement intestinal flora expression and adjusting the short-chain fatty acid content in cecum contents; the symptoms of dry and hard excrement, prolonged defecation time and the like caused by constipation are relieved, the intestinal peristalsis capability of mice with constipation can be promoted, and the damage of constipation to intestinal tracts is relieved.

Description

Technical Field

[0001] The invention relates to the technical field of health food, in particular to the application of a dietary fiber probiotic composition. Background Art

[0002] Constipation is a widespread health problem. According to the "Chinese Expert Consensus on Chronic Constipation (2019)", the prevalence of chronic constipation in adults is 4.0~10.0%, which means that about 50 million adults are suffering from constipation, which seriously affects the quality of life and physical and mental health of patients. Long-term constipation may cause intestinal nerve dysfunction, affect the normal peristalsis and defecation function of the intestine, and further aggravate the symptoms of constipation. Traditional methods for treating constipation mainly include drug therapy, dietary adjustment and improvement of lifestyle habits. However, these methods have certain limitations in terms of treatment effect and safety. For example, long-term use of laxatives may lead to decreased intestinal function and form drug dependence; while dietary adjustment and improvement of lifestyle habits require patients to persist for a long time, and the effect varies from person to person. It is understood that probiotics and dietary fiber can promote each other in the intestine and play a role in moisturizing the intestine and laxative. Probiotics can increase the number of beneficial bacteria in the intestine, while dietary fiber can provide an ideal breeding ground for these beneficial bacteria and promote their growth and reproduction. At the same time, dietary fiber can also increase the volume and weight of feces, promote intestinal peristalsis, and make stool easier to excrete. Therefore, the combined use of probiotics and dietary fiber has a good application prospect in the preparation of health products and clinical treatment of constipation.

[0003] Lactobacillus fermentum CEC-T5716 is a probiotic belonging to the genus Lactobacillus. It can produce lactic acid and other metabolites through fermentation, which is beneficial to intestinal health, can improve digestive function, enhance immunity, increase stool volume and moisture, and improve the intestinal environment, effectively relieving constipation symptoms. Appropriate intake of fermented foods containing Lactobacillus fermentum CEC-T5716, such as yogurt, cheese, etc., can help increase the number of Lactobacillus fermentum CEC-T5716 in the body, thereby exerting its probiotic effect.

[0004] Lingzhi dietary fiber is a unique nutrient found in Lingzhi. It is effective in relieving constipation caused by changes in stool frequency, consistency, hardness and transit time. Lingzhi is a precious Chinese herbal medicine and health food. Its dietary fiber is not only rich in content but also of high quality. With its unique physical properties, it can significantly increase the volume of stool. This change helps stimulate the intestinal wall and promotes the natural peristalsis of the intestine, thereby accelerating the digestion process and allowing waste to be excreted more smoothly. This process not only relieves the discomfort caused by constipation, but also promotes intestinal health.

[0005] The Chinese patent with the publication number CN118104812A recently applied by the applicant discloses a method for extracting low molecular weight water-soluble dietary fiber from Ganoderma lucidum using a composite enzyme-hot water, which comprises the following steps: S1 drying and crushing the Ganoderma lucidum fruiting body to obtain Ganoderma lucidum powder; S2 mixing the Ganoderma lucidum powder obtained in S1 with phosphate buffer, first adding papain for primary enzymolysis, boiling and killing the enzyme after the enzymolysis is completed, then adding cellulase and hemicellulase for secondary enzymolysis, boiling and killing the enzyme after the enzymolysis is completed to obtain an enzymolysis solution; S3 heating the enzymolysis solution obtained in S2 for leaching, and after the leaching is completed, post-treating to obtain an extract; S4 mixing the extract obtained in S3 with an ethanol solution, first precipitating it with alcohol overnight, and then post-treating it to obtain low molecular weight water-soluble dietary fiber from Ganoderma lucidum. Soluble dietary fiber (SDF) refers to low molecular weight polysaccharides with a degree of polymerization of more than 3 that are soluble and cannot be digested and absorbed by the human body. This type of substance can stimulate intestinal peristalsis, facilitate the excretion of feces, and prevent constipation, rectal cancer, hemorrhoids, etc.; it can prevent the occurrence of cardiovascular diseases such as atherosclerosis and coronary heart disease; prevent the formation of gallstones; produce a sense of fullness, which is beneficial for obese patients to eat, and can be used as a weight loss food; improve glucose tolerance, can regulate the blood sugar level of diabetics, and can be used as a food for diabetics; improve intestinal flora, appendicitis, etc.

[0006] The combination of probiotics and dietary fiber is a golden pair for intestinal health. The two complement each other, not only improving constipation, but also promoting the balance of intestinal microecology, enhancing the body's immune defenses and optimizing nutrient absorption. As a commercial industrialized probiotic, Lactobacillus fermentum CEC-T5716 can effectively relieve constipation by regulating the structure of intestinal flora. At the same time, its metabolites can enhance the intestinal barrier function, resist the invasion of harmful bacteria, and build a solid defense line for intestinal health. Lingzhi dietary fiber, derived from the natural treasure Lingzhi, is rich in water-soluble and insoluble fiber, which can increase the water content of feces and promote intestinal peristalsis, thereby solving constipation problems for constipation patients. Combining these two ingredients to develop innovative drugs for constipation patients is not only highly feasible, but also expected to become an innovative product in the field of constipation treatment. Such drugs can not only quickly relieve constipation symptoms, but also gradually improve the intestinal environment and improve the overall health level in long-term use, bringing good news to constipation patients and reducing their physical and mental burden. Summary of the invention

[0007] The problem to be solved by the present invention is to provide an application of a dietary fiber probiotic composition in view of the above-mentioned deficiencies existing in the prior art. The dietary fiber probiotic composition can effectively improve the symptoms of constipation, relieve the symptoms such as dry and hard stool, prolonged defecation time, etc. caused by constipation, promote the intestinal peristalsis ability of constipated mice, and relieve the damage of constipation to the intestine by affecting the weight of mice, the time of the first black stool, the water content of stool, the propulsion rate of small intestine, the state of colon tissue, the water channel protein of colon tissue, the intestinal barrier, the genes related to inflammatory factors, the expression of fecal intestinal flora, and the content of short-chain fatty acids in the contents of the cecum.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The invention discloses an application of a dietary fiber-probiotic composition, and an application of a composition of ganoderma dietary fiber and fermented lactobacillus CEC-T5716 in the preparation of medicines or health products for preventing or relieving constipation.

[0010] Furthermore, the composition comprises 900-110 mg / mL of Ganoderma lucidum dietary fiber solution and 2.5×10 9 ~3.5×10 9 CFU / mL fermented Lactobacillus CEC-T5716 bacterial liquid composition.

[0011] Furthermore, the volume ratio of the Ganoderma lucidum dietary fiber solution to the fermented Lactobacillus CEC-T5716 bacterial solution is 1:0.8-1.2.

[0012] Furthermore, the medicine or health product is administered orally, and the daily oral dose of the composition is 0.2 mL / 18-20 g.

[0013] Furthermore, the medicine or health product for preventing or relieving constipation is a medicine or health product that promotes recovery of intestinal function and water retention in the body.

[0014] And / or, the medicine or health product for preventing or alleviating constipation is a medicine or health product that restores stool consistency by reducing the expression of aquaporins AQP3, AQP4 and AQP8.

[0015] And / or, the medicine or health product for preventing or alleviating constipation is a medicine or health product that protects the integrity of the intestinal barrier by increasing the expression of MUC-2 and ZO-1.

[0016] And / or, the medicine or health product for preventing or alleviating constipation is a medicine or health product that improves intestinal immune response disorders by reducing the expression of inflammatory factors IL-6, IL-10 and IL-1β.

[0017] And / or, the medicine or health product for preventing or alleviating constipation is a medicine or health product that improves intestinal flora disorder by increasing the expression of Firmicutes and decreasing the expression of Bacteroidetes and Proteobacteria.

[0018] And / or, the medicine or health product for preventing or alleviating constipation is a medicine or health product that increases the content of one or more short-chain fatty acids (SCFAs) among acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.

[0019] In summary, the beneficial technical effects of the present invention are:

[0020] 1. Effectively improve constipation symptoms: Application principle: By combining the use of Ganoderma lucidum dietary fiber and fermented Lactobacillus CEC-T5716, the present invention can significantly increase the intestinal motility of constipated mice, promote feces excretion, shorten the time of the first black stool, and increase the water content of feces, thereby effectively improving constipation symptoms; compared with the use of dietary fiber or probiotics alone, this combination can more comprehensively promote intestinal health and improve the therapeutic effect of constipation;

[0021] 2. Alleviate the damage to the intestine caused by constipation: The combination of Ganoderma lucidum dietary fiber and fermented Lactobacillus CEC-T5716 can reduce the expression of water channel proteins AQP3, AQP4 and AQP8, thereby restoring the normal consistency of feces and reducing excessive loss of intestinal water. At the same time, they can also improve the integrity of the intestinal barrier and protect the intestine from damage by increasing the expression of MUC-2 and ZO-1; this combination not only solves the symptoms of constipation, but also fundamentally alleviates the potential damage to the intestine caused by constipation, which helps maintain the long-term health of the intestine.

[0022] 3. Improve intestinal flora disorder and intestinal immune response: By regulating intestinal flora, the present invention can significantly increase the expression of Firmicutes and reduce the expression of Bacteroidetes and Proteobacteria, thereby improving intestinal flora disorder. In addition, it can also reduce the expression of inflammatory factors IL-6, IL-10 and IL-1β, and improve intestinal immune response disorder; this regulation helps to restore the balance of intestinal flora, reduce the growth of harmful bacteria, and reduce intestinal inflammatory response, providing a healthier environment for the intestine.

[0023] 4. Increase the content of short-chain fatty acids and promote intestinal health: Application principle: The combination of Ganoderma lucidum dietary fiber and fermented Lactobacillus CEC-T5716 can significantly increase the content of short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. These short-chain fatty acids are important markers of intestinal health and can promote intestinal peristalsis, maintain intestinal barrier function, regulate immune response, etc. By increasing the content of short-chain fatty acids, the present invention can further enhance intestinal health and provide comprehensive treatment and support for patients with constipation.

[0024] 5. In summary, the key point of the present invention is to achieve effective improvement of constipation symptoms, alleviation of intestinal damage, improvement of intestinal flora disorder and intestinal immune response, and increase of short-chain fatty acid content by combining Ganoderma lucidum dietary fiber and fermented Lactobacillus CEC-T5716. These effects together constitute the technical points to be protected by the present invention, providing a comprehensive and effective treatment plan for constipation patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a growth curve diagram of Lactobacillus fermentum CEC-T5716 of Preparation Example 1 of the present invention;

[0026] Figure 2 This is a colony growth diagram of Lactobacillus fermentum CEC-T5716 of Preparation Example 1 of the present invention;

[0027] Figure 3 This is a graph showing the weight changes of mice in Example 1 and Comparative Examples 1 to 3 of the present invention;

[0028] Figure 4 It is a graph showing the time of the first black stool of mice in Example 1 and Comparative Examples 1 to 3 of the present invention;

[0029] Figure 5 It is a graph of the 4-hour feces water content of mice in Example 1 and Comparative Examples 1 to 3 of the present invention;

[0030] Figure 6 It is a graph of the small intestine propulsion rate of mice in Example 1 of the present invention and Comparative Examples 1 to 3;

[0031] Figure 7 The results of HE staining and AB-PAS staining of the mouse colon and ileum in Example 1 and Comparative Examples 1 to 3 of the present invention are shown;

[0032] Figure 8 Graph showing the effects of the expression of the mouse colon tissue water channel protein genes AQP3, AQP4, and AQP8 in Example 1 and Comparative Examples 1 to 3 of the present invention;

[0033] Fig. 9 1 is an effect diagram of the expression of the mouse colon tissue water channel protein genes MUC-2 and ZO-1 in Example 1 of the present invention and Comparative Examples 1 to 3;

[0034] Fig.10 This is an effect diagram of the expression of IL-6, IL-10, and IL-1β genes in the mouse colon tissue of Example 1 and Comparative Examples 1 to 3 of the present invention;

[0035] Fig.11 It is an influence diagram of the expression of genes Firmicutes, Bacteroidetes, and Proteobacteria in the intestinal flora of mice feces in Example 1 and Comparative Examples 1 to 3 of the present invention;

[0036] Fig.12 It is a graph showing the content of short-chain fatty acids in the cecal contents of mice in Example 1 and Comparative Examples 1 to 3 of the present invention. DETAILED DESCRIPTION

[0037] In order to make the technical means, creative features, objectives and functions achieved by the present invention clearer and easier to understand, the present invention is further explained below in conjunction with the accompanying drawings and specific implementation methods.

[0038] Preparation Example

[0039] Preparation Example 1: The preparation process of Lactobacillus fermentum CEC-T5716 bacterial liquid includes:

[0040] S1 prepares the culture medium,

[0041] MRS liquid 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 801mL, L-cysteine ​​hydrochloride 0.8g, adjust pH to 6.8±0.2, make up to 1L, and sterilize at 115℃ for 20min;

[0042] MRS solid medium: add 2wt% agar powder to MRS liquid medium;

[0043] cMRS liquid medium: MRS liquid medium plus 0.08wt% cysteine ​​hydrochloride;

[0044] cMRS solid medium: MRS solid medium + 0.05~0.1%wt cMRS liquid medium;

[0045] S2 inoculated fermented Lactobacillus CEC-T5716 into cMRS solid culture medium, cultured at 37°C for 24h, streaked to activate single colonies, inoculated the activated fermented Lactobacillus CEC-T5716 into cMRS liquid culture medium at a 3% inoculation rate, cultured anaerobically at 37°C, with the inoculation time as 0h, sampled every 2h to measure the OD600 value, and set three parallels at the same time, then plotted a growth curve with the culture time as the horizontal axis and the corresponding absorbance value as the vertical axis. The results are as follows: Figure 1 As shown;

[0046] S3 Take 50 μl of the bacterial solution at each inoculation time point, apply it to cMRS solid medium, and culture it at 37°C for 24 h to obtain colonies. Count the number of colonies CFU. Set up three parallel sets at the same time to obtain the relationship between CFU and OD value. The results are as follows: Figure 2 As shown;

[0047] S4 Inoculate the activated bacterial solution into 10 mL cMRS liquid medium at a 3% (v / v) inoculation volume, shake and mix, and culture in an anaerobic incubator at 37°C for 24 h. Centrifuge at 8000 g / min, 4°C for 15 min, remove the supernatant, wash twice with sterile saline containing 0.05%-0.1% L-cysteine ​​hydrochloride, centrifuge under the same conditions to obtain bacterial sludge, resuspend with saline, and measure OD 600 The viable count is 3.0×10 9 CFU / mL, and obtain the fermented Lactobacillus CEC-T5716 bacterial liquid.

[0048] Preparation Example 2: The preparation process of the Ganoderma lucidum dietary fiber solution includes obtaining low molecular weight Ganoderma lucidum water-soluble dietary fiber, i.e., Ganoderma lucidum dietary fiber, according to the method of Example 1 of CN118104812A, and then dissolving it with physiological saline to obtain a 100 mg / mL Ganoderma lucidum dietary fiber solution.

[0049] Preparation Example 3: The preparation process of the composition of Ganoderma dietary fiber and fermented Lactobacillus CEC-T5716 includes mixing the fermented Lactobacillus CEC-T5716 bacterial liquid prepared in Preparation Example 1 and the Ganoderma dietary fiber solution prepared in Preparation Example 2 in a volume ratio of 1:1 to obtain the composition of Ganoderma dietary fiber and fermented Lactobacillus CEC-T5716.

[0050] Example

[0051] Example 1: Application of a dietary fiber probiotic composition disclosed in the present invention, application of the composition of Ganoderma lucidum dietary fiber and fermented Lactobacillus CEC-T5716 prepared in Preparation Example 3 in the preparation of a medicine or health product for preventing or relieving constipation. The medicine or health product is administered orally, and the daily oral dose of the composition is 0.2mL / 18~20g.

[0052] Example 2: Application of a dietary fiber probiotic composition disclosed in the present invention. The difference from Example 1 is that the composition is composed of 90 mg / mL Ganoderma lucidum dietary fiber solution and 2.5×10 9 CFU / mL fermented Lactobacillus CEC-T5716 bacterial liquid composition.

[0053] Example 3: Application of a dietary fiber probiotic composition disclosed in the present invention. The difference from Example 1 is that the composition is composed of 110 mg / mL Ganoderma lucidum dietary fiber solution and 3.5×10 9 CFU / mL fermented Lactobacillus CEC-T5716 bacterial liquid composition.

[0054] Example 4: An application of a dietary fiber probiotic composition disclosed in the present invention, which is different from Example 1 in that the volume ratio of the Ganoderma lucidum dietary fiber solution to the fermented Lactobacillus CEC-T5716 bacterial solution is 1:0.8.

[0055] Example 5: An application of a dietary fiber probiotic composition disclosed in the present invention, which is different from Example 1 in that the volume ratio of the Ganoderma lucidum dietary fiber solution to the fermented Lactobacillus CEC-T5716 bacterial solution is 1:1.2.

[0056] Example 6: This is an application of a dietary fiber probiotic composition disclosed in the present invention. The difference from Example 1 is that the medicine or health product for preventing or relieving constipation is a medicine or health product that promotes the recovery of intestinal function and water retention in the body.

[0057] Example 7: An application of a dietary fiber probiotic composition disclosed in the present invention. The difference from Example 1 is that the medicine or health product for preventing or relieving constipation is a medicine or health product that restores stool consistency by reducing the expression of water channel proteins AQP3, AQP4 and AQP8.

[0058] Example 8: This is an application of a dietary fiber probiotic composition disclosed in the present invention. The difference from Example 1 is that the medicine or health product for preventing or relieving constipation is a medicine or health product that protects the integrity of the intestinal barrier by increasing the expression of MUC-2 and ZO-1.

[0059] Example 9: An application of a dietary fiber probiotic composition disclosed in the present invention. The difference from Example 1 is that the medicine or health product for preventing or relieving constipation is a medicine or health product that improves intestinal immune response disorders by reducing the expression of inflammatory factors IL-6, IL-10 and IL-1β.

[0060] Example 10: An application of a dietary fiber probiotic composition disclosed in the present invention. The difference from Example 1 is that the medicine or health product for preventing or relieving constipation is a medicine or health product that improves intestinal flora disorder by increasing the expression of Firmicutes and reducing the expression of Bacteroidetes and Proteobacteria.

[0061] Example 11: This is an application of a dietary fiber probiotic composition disclosed in the present invention. It is different from Example 1 in that the medicine or health product for preventing or relieving constipation is a medicine or health product that increases the content of one or more short-chain fatty acids (SCFAs) among acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.

[0062] Comparative Example

[0063] Comparative Example 1: is an application of a dietary fiber probiotic composition disclosed in the present invention, which is different from Example 1 in that the composition is a polyethylene glycol 4000 solution.

[0064] Comparative Example 2 is an application of a dietary fiber probiotic composition disclosed in the present invention. The difference from Example 1 is that the composition is the fermented Lactobacillus CEC-T5716 bacterial liquid prepared in Preparation Example 1.

[0065] Comparative Example 3 is an application of a dietary fiber probiotic composition disclosed in the present invention. The difference from Example 1 is that the composition is the Ganoderma lucidum dietary fiber solution prepared in Preparation Example 2.

[0066] Performance testing

[0067] The experimental animals were 6-week-old male BALB / c mice weighing 18-20 g. They were kept in a mouse house with a controlled environment of (23±2)°C, relative humidity of 45-55%, 12 hours of lighting and 12 hours of darkness per day, in line with the day and night pattern. Five mice were placed in each cage, fed with standard feed and purified water, with free access to food and water, and the bedding was changed every 3 days.

[0068] After a one-week adaptation period, 60 BALB / c mice were randomly divided into 6 groups, including a normal control group (CON), a constipation model group (LOP), a positive drug group (P, comparative example 1), a probiotic group (B, comparative example 2), a dietary fiber group (F, comparative example 3) and a combination group (B+F, Example 1). During the one-week adaptation period, each group was free to eat and drink, and then entered a 7-day modeling period, with the 1st to 7th day being the modeling period and the 8th to 28th day being the treatment period, and gavage was performed at 9 am every day.

[0069] Modeling period: The CON group was gavaged with 0.2 mL of normal saline, and the other experimental groups were gavaged with 0.2 mL of 5 mg / kg.bw loperamide hydrochloride solution for one week.

[0070] During the treatment period, the CON group continued to be gavaged with 0.2 mL of normal saline, and the other groups were gavaged with 0.2 mL of 5 mg / kg.bw loperamide hydrochloride solution. After 1 hour, the treatment drugs were gavaged: the P group was gavaged with 0.2 mL of 3000 mg / kg.bw polyethylene glycol 4000 solution, and the B group was gavaged with 3.0×10 9 The patients in group F were given 0.2 mL of CFU / mL fermented Lactobacillus CEC-T5716 bacterial solution, group F was given 0.2 mL of 1000 mg / kg.bw Ganoderma lucidum dietary fiber solution by gavage, and group B+F was given 0.2 mL of mixed solution of bacteria and dietary fiber by gavage, for three weeks.

[0071] On day 29, mice were weighed, eyeballs were removed to collect blood, and mice were killed by cervical dislocation. The liver and spleen were removed and weighed, the small intestine segment was measured, the liver, ileum, and colon were collected, and the blood was centrifuged to obtain the supernatant, which was temporarily frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0072] The mice were weighed with an electronic scale at 9 a.m. every day. Figure 3 As shown, the status of the mice and their feces were observed. During the modeling period, the mouse feces were collected every day and the water content of the feces was measured to determine whether the modeling was successful. After verification, the modeling was successful.

[0073] Experimental Example 1: On the 27th day, the feces of mice were collected, and the time of the first black stool and the water content of the feces at 4 hours were measured. Except for the CON group, which was gavaged with normal saline, the mice in the other experimental groups were gavaged with 0.2 mL of 5 mg / kg.bw loperamide hydrochloride solution. One hour later, the CON group was gavaged with 0.2 mL of cuttlefish juice, and the other experimental groups were gavaged with 0.2 mL of cuttlefish juice containing the corresponding therapeutic drug. After the gavage, the mice were placed in a cage with an absorbent pad and were allowed to eat and drink freely. The time from the completion of gavage to the production of the first black stool was recorded. The results are as follows Figure 4 As shown, it is the time of the first black stool. At the same time, all feces discharged within 4 hours are collected in a sample bag, and the wet weight of feces and the number of feces are weighed and recorded. The sample bag is pierced with holes and placed in a 55℃ drying oven for 24 hours, which is recorded as the dry weight of feces. After collecting the mouse feces, they are placed in the original cages according to the groups. The water content of feces is calculated according to the following formula. The results are as follows: Figure 5 As shown:

[0074] ×100%

[0075] Preparation of cuttlefish ink: 1g cuttlefish powder is dissolved in 1mL of normal saline.

[0076] Test Example 2: At 9:00 p.m. on the 28th day, the mouse food was removed, the mice were fasted for 12 hours, and were allowed to drink water freely. On the next morning, 0.2 mL of cuttlefish juice was gavaged into the mice, and 30 minutes later, the mice were killed by cervical dislocation. The abdominal cavity was opened, the stomach was found, the small intestine segment was quickly separated, placed on filter paper, excess blood was sucked away, the small intestine was gently straightened, and carefully laid on white paper, the total length of the small intestine and the distance the cuttlefish juice advanced forward were measured, and the small intestine advancement rate was calculated according to the following formula. The results are as follows: Figure 6 As shown:

[0077] ×100%

[0078] Experimental Example 2: After the mice were killed, 1 cm of the middle section of the colon was cut off, immersed in the fixative, stored in a refrigerator at 4°C, and stained with hematoxylin-eosin (HE) according to the following steps: the fixed sample was rinsed under tap water for 30 minutes, then dehydrated and embedded in paraffin. The embedded wax block was chilled for 2 hours, spread, baked, baked and sliced, dewaxed and hydrated, placed in hematoxylin for staining for 10 minutes, washed twice with double distilled water for a total of 5 minutes, then stained with eosin for 30 seconds, dehydrated and transparent, 50 μL of neutral gum was added to each slide, and the slide was sealed to remove bubbles and observed under a microscope. The results are as follows: Figure 7 shown.

[0079] Test Example 3: Dewaxing of paraffin-coated sections: successively put the sections into xylene for 120 minutes, then put them into xylene for 120 minutes, anhydrous ethanol for 15 minutes, 75% alcohol for 5 minutes, and finally wash them with tap water; Alcian blue staining: put the sections in step A into Alcian blue staining solution for 5 minutes, then wash them with tap water for 2 minutes; periodic acid staining: use periodic acid to stain the sections for 15 minutes, then wash them with tap water, and then wash them twice with distilled water; Shepherd's staining: Shepherd's staining The sections were stained with hematoxylin for 30 minutes in a dark environment, and then rinsed with running water for 5 minutes; hematoxylin staining: the sections were stained with hematoxylin for 3-5 minutes, and then washed with tap water, differentiated with differentiation solution, washed twice with tap water, blued with blueing solution, and rinsed with running water; dehydration and sealing: the sections were placed in anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol, xylene I and xylene II in turn, and placed in each liquid for 5 minutes until dehydrated and transparent, and finally sealed with Zhongxing gum. The results are shown in the figure. Figure 7 shown.

[0080] Experimental Example 4: To extract colon RNA, weigh about 30 mg of mouse colon tissue, put it into a 1.5 mL centrifuge tube, add 500 μL of Trizol reagent, and grind it thoroughly with a fast grinder. Each group has 8 samples;

[0081] After the sample is fully ground and lysed, add 120 μL of chloroform to the Trizol mixture, vortex for 15 seconds, let it stand on ice for 5 minutes, and then centrifuge at 4°C, 12000 rpm for 10 minutes. After the centrifugation, three layers of liquid can be seen. Use a pipette to draw 200 μL of the top clear liquid into a new 1.5 mL centrifuge tube; add an equal volume (200 μL) of isopropanol, mix gently, and let it stand on ice for 5 minutes; centrifuge at 4°C, 12000 rpm for 10 minutes, and discard the top clear liquid. Remove the supernatant, use a pipette to absorb as much liquid as possible, then add 1mL of prepared 75% ethanol, at this time, RNA can be seen as white blocks at the bottom of the centrifuge tube, gently flick the RNA off the tube wall to make it float in 75% ethanol; centrifuge at 4℃, 12000rpm for 10min, discard the supernatant, use a pipette to absorb as much liquid as possible, open the centrifuge tube cover to dry, after the liquid in the tube is dried, add 30μL of ultrapure water to dissolve the RNA, centrifuge and mix, obtain mouse colon total RNA solution, and store at -80℃ for use. All steps were performed on ice.

[0082] Test Example 5: Colon RNA reverse transcription: Thaw the previously extracted RNA solution on ice, denature at 65°C in a dry thermostat for 5 minutes to open the RNA secondary structure; measure the purity and concentration of each RNA sample, and the 260 / 280 ratio of the pure RNA sample is between 1.8 and 2.0. If the concentration is greater than 800ng / μL, dilute with ultrapure water to ensure that the RNA concentration is between 400 and 500ng / μL; configure the reaction system according to the instructions of the reverse transcription kit: RNA 1μg, 4μL reverse transcription reagent, and make up the system to 20μL with ultrapure water. The reaction conditions are: 50°C water bath reaction for 15min, 85°C water bath for 5s to inactivate the enzyme to terminate the reaction, quickly insert the centrifuge tube into ice to cool, briefly centrifuge to obtain the colon cDNA solution, and store it at -80°C for use.

[0083] Experimental Example 6: Extraction of intestinal flora genomic DNA, weigh about 50 mg of mouse colon contents and place them in a 1.5 mL sterile centrifuge tube, with 8 parallels per group. A magnetic bead DNA extraction kit was used to extract total DNA from mouse colon contents, and all operations were performed strictly in accordance with the instructions. The specific steps are as follows: add 200μL PBS and 10μL proteinase K to the centrifuge tube containing the sample, grind it thoroughly with a grinder, and let it stand for 3 minutes; add 400μL Lysis / Binding Buffer, mix it thoroughly (use a vortex oscillator to shake it 5-10 times), place it in a 68℃ water bath for 1.5-2 hours, and gently shake the centrifuge tube every 15 minutes to mix it; centrifuge it at 4℃, 13000rpm for 10 minutes, take the supernatant to a new 1.5μL centrifuge tube, add 1.5μL RNase, mix it well and let it stand at room temperature for 5 minutes; add 30μL Foreal Beads that have been shaken and mixed beforehand, then add 400μL anhydrous ethanol, mix it thoroughly with a vortex oscillator, place the centrifuge tube on a magnetic stand, and aspirate the liquid; add 500μL Washing Buffer I, mix it well and place it on a magnetic stand for magnetic separation, and aspirate the liquid; add 500μL Washing Buffer Ⅱ. After mixing, place on a magnetic stand for magnetic separation, discard the liquid, and repeat this step once; open the lid at room temperature for 5 minutes to dry, add 50μL Elution Buffer, gently oscillate for 5-10 seconds, mix well, place in a 63℃ water bath for 10 minutes, shake the centrifuge tube gently every 2-3 minutes, then separate the magnetic beads on the magnetic stand, carefully pipette the supernatant into a new 1.5mL centrifuge tube, and store at -80℃ for later use.

[0084] Test Example 7: Fluorescence quantitative PCR reaction: The concentration of the extracted colon content DNA was measured by an ultra-micro UV-visible spectrophotometer, diluted to 10 ng / μL with ultrapure water, and the reaction system was prepared on ice according to the instructions of the fluorescence quantitative PCR kit for detection of gene expression detection of bacterial abundance changes at the intestinal phylum level. The cDNA samples obtained by reverse transcription of the colon samples were used to detect genes related to the intestinal barrier and mucus barrier. 18s was used as the housekeeping gene, and the reaction procedure was: 95℃+3min, 95℃+10s and 60℃+30s, and the latter two steps were cycled 40 times; 16s was used as the housekeeping gene for the bacterial flora determination, and the reaction procedure was: 50℃+2min; 95℃+10min; 95℃+15s, 56℃+30s, 72℃+1min, 72℃+10min, and the latter three steps were cycled 40 times; 72℃ was fully extended for 10min; the relevant genes and primer sequences used are shown in Table 1, and the test results are as follows Figures 8 to 11 shown.

[0085] Table 1

[0086] Gene name Primer sequences 18s F:5′-GGCCGTTCTTAGTTGGTGGAGCG~3′R:5′-CTGAACGCCACTTGTCCCTC~3′ AQP3 F:5′-GCTTTTGGCTTCGCTGTCAC~3′R:5′-TAGATGGGCAGCTTGATCCAG~3′ AQP4 F:5′-CTTTCTGGAAGGCAGTCTCAG~3′R:5′-CCACACCGAGCAAAAAAAGAT~3′ AQP8 F:5′-TGTGTAGTATGGACCTACCTGAG~3′R:5′-ACCGATAGACATCCGATGAAGAT~3′ MUC-2 F:5′-TACGCTCTCCACCAGTTCCT~3′R:5′-CAGCTCTCGATGTGTGTGTAGG~3′ ZO-1 F:5′-ATCCCAAATAAGAACAGAGC~3′R:5′-GGCGTTACATCTAATAAAGC~3′ IL-6 F:5′-CCACTTCACAAGTCGGAGGCT~3′R:5′-GCAAGTGCATCATCGTTGTTCA~3′ IL-10 F:5′-GCTCTTACTGACTGGCATGAG~3′R:5′-CAATACATTGACCTGCCGAT~3′ IL-1β F:5′-CCAACAAGTGATATTCTCCATG~3′R:5′-ACTCTGCAGACTCAAACTCCA~3′ 16s F:5′-GGGTTGCGCTCGTTGC~3′R:5′-ATGGYTGTCGTCAGCTCGTG~3′ Firmicutes F: 5′-GGAGYATGTGGTTTAATTCGAAGCA~3'R: 5′-AGCTGACGACAACCATGCAC~3' Bacteroidetes F:5′-CCGCACAGTTGGCGAGATGA~3′R:5′-CGACAGTTATGACGCCCTCC~3′ Proteobacteria F:5′-GAGTTTGATCATGGCTCA~3′R:5′-GTATTACCGCGGCTGCTG~3′

[0087] Test Example 8: Before analyzing the sample with a gas chromatography-mass spectrometer (GC-MS), a standard curve should be drawn. The specific operation is as follows: Preparation of standard solution 1: Take 10 μL of each SCFAs (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid) and add it to 1 mL with anhydrous ether; Preparation of standard solution 2: Take 100 μL of the mixture of standard solution 1 and add it to 1 mL with anhydrous ether; Preparation of standard curves of different concentrations: Take 200 μL, 150 μL, 100 μL, 75 μL, 50 μL, 25 μL, 15 μL of the mixture of standard solution 2, add it to 1 mL with anhydrous ether, and detect it on the machine; Use nitrogen as the carrier gas and control the gas flow rate to 1 mL / min. Set the column temperature to 100°C and maintain it for 0.5 min. Then, increase the temperature to 180°C at 8°C / min and maintain it for 1 min. Then the temperature was raised to 200°C at 20°C / min and maintained for 5 min. The detector temperature was set to 240°C, the injection port temperature was set to 200°C, and the injection volume was 1 μL. The detection time for each sample was about 17.5 min.

[0088] Sample pretreatment: Weigh 20~50mg of fecal sample and place it in a 2mL EP tube. Add 500μL of saturated NaCl to the EP tube (weigh 20g of sodium chloride, place it in a beaker, and add 40mL of water). Let it stand for 30 minutes, homogenize, and add 20μL of sulfuric acid to acidify (the configuration of sulfuric acid is 0.5mL concentrated sulfuric acid + 5mL water). After adding sulfuric acid, vortex on a vortex shaker for 30s to mix. Add 800μL of ether in the fume hood to extract short-chain fatty acids. After adding ether, vortex on a vortex shaker for 30s and let it stand for 30 minutes. After oscillation, centrifuge at 14000rpm for 15min. After centrifugation, take the supernatant, add the supernatant to a 2mL EP tube containing 0.25g of anhydrous sodium sulfate, and centrifuge again under the same conditions to remove residual water. After centrifugation, transfer the supernatant to a gas phase injection bottle for injection testing. The content of short-chain fatty acids in the sample is calculated according to the standard curve, and the results are as follows. Fig.12 shown.

[0089] Note x (μmol / g), the formula is as follows:

[0090] X=

[0091] Where x: content in sample, residual amount of the measured component in the sample, μmol / g;

[0092] c: average value, concentration of the measured component solution obtained from the standard working curve, μmol / mL;

[0093] v: fixed volume of sample solution, mL;

[0094] m: sample weight in g.

[0095] from Figures 3 to 12 It can be seen that

[0096] 1. The health status of mice was as follows: the weight of mice in each group increased rapidly during the adaptation period. After oral administration of loperamide hydrochloride during the modeling period, the weight of each group increased, but the growth rate of the LOP group was slower;

[0097] 2. The time of the first black stool in mice in the LOP group was significantly prolonged, while that in the B+F group was almost the same as that in the CON group. The time from longest to shortest was LOP, P, B, F, B+F, and CON groups. The fecal water content in the LOP group was significantly reduced, while that in the B+F group was the highest, with the B group being similar to the F group, and the P group being the second. This indicates that the B+F compound treatment effectively promoted the recovery of intestinal function and water retention.

[0098] 3. The propulsion rate of the small intestine of mice was as follows: the propulsion rate of the small intestine of the LOP group was significantly reduced; compared with the LOP group, the propulsion rate of the small intestine of the other experimental groups was significantly increased, which played a role in improving the propulsion force of the small intestine, among which the effect of the B+F group was the most obvious;

[0099] 4. The changes in colon and ileum tissues were as follows: compared with the CON group, local inflammatory cell infiltration, reduced goblet cells, and damaged colon mucosa were observed in the colon tissues of the LOP group mice; in contrast, lymphokines accumulated in the colon tissues of the B and F groups; the P and B+F groups were similar to the CON group, with clear goblet cell structures and a large number, and little inflammatory infiltration; the tissue layer of the ileum became thinner, and the thickness of the tissue layer of the other groups was almost the same as that of the CON group;

[0100] 5. The results of AB-PAS staining of colon tissue showed that the mucus secretion of mice in the LOP group was significantly reduced compared with the CON group; while the mucus secretion in each experimental group increased, especially in the B+F group, which significantly improved the mucus secretion level of constipated mice;

[0101] 6. The q-PCR results of mouse colon tissue showed that compared with the CON group, the expression levels of water channel proteins AQP3, AQP4, and AQP8 in the LOP group increased significantly. However, after treatment, the expression levels of these water channel proteins decreased, especially in the B+F group, which had the most significant effect, indicating that this group could more effectively regulate the expression of water channel proteins, thereby helping to restore the normal consistency of feces. Further detection of the expression of genes related to intestinal barrier function showed that the expression levels of MUC-2 and ZO-1 in the LOP group were significantly lower than those in the CON group, reflecting the damage of intestinal barrier function. However, the expression levels of MUC-2 and ZO-1 in the B+F group increased significantly after treatment, indicating that it can effectively enhance the intestinal barrier function. At the same time, the detection results of inflammatory factors showed that the levels of IL-6 and IL-1β in the LOP group were significantly higher than those in the CON group, indicating that there was an inflammatory response in the intestine. While improving the intestinal barrier function, the B+F group may also have a positive effect on alleviating this inflammatory response.

[0102] 7. The results of q-PCR of intestinal flora in feces showed that at the phylum level, the expression of Firmicutes in the LOP group mice was significantly decreased compared with that in the CON group, while the expression of Proteobacteria and Bacteroidetes was significantly increased; this change indicated that the LOP group had intestinal flora disorder; however, after constipation was relieved, the intestinal flora expression trend of each experimental group was opposite to that of the LOP group, especially the B+F group, which was able to significantly regulate the intestinal flora disorder of constipated mice, thereby effectively relieving constipation symptoms;

[0103] 8. The test results of short-chain fatty acids (SCFAs) in feces showed that compared with the CON group, the contents of SCFAs such as acetate, propionate, butyrate, isobutyrate, valeric acid, and isovaleric acid in the LOP group were decreased; these SCFAs are essential for intestinal health, and their reduced content may further aggravate the constipation in the LOP group; and after treatment, the contents of these SCFAs in the B+F group increased significantly, approaching or exceeding the levels of the CON group, which further confirmed the effectiveness of the B+F group in improving intestinal flora and relieving constipation.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An application of a dietary fiber probiotic composition, characterized in that: Application of a composition of ganoderma lucidum dietary fiber and fermented lactobacillus CEC-T5716 in the preparation of medicines or health products for preventing or relieving constipation.

2. The use of a dietary fiber probiotic composition according to claim 1, characterized in that: The composition comprises 900-110 mg / mL of ganoderma lucidum dietary fiber solution and 2.5×10 9 ~3.5×10 9 CFU / mL fermented Lactobacillus CEC-T5716 bacterial liquid composition.

3. The use of a dietary fiber probiotic composition according to claim 2, characterized in that: The volume ratio of the ganoderma dietary fiber solution to the fermented lactobacillus CEC-T5716 bacterial solution is 1:0.8-1.

2.

4. The use of a dietary fiber probiotic composition according to claim 2, characterized in that: The medicine or health product is administered orally, and the daily oral dosage of the composition is 0.2 mL / 18-20 g.

5. The use of a dietary fiber probiotic composition according to claim 1, characterized in that: The medicine or health product for preventing or relieving constipation is a medicine or health product that promotes the recovery of intestinal function and water retention in the body.

6. The use of a dietary fiber probiotic composition according to claim 1, characterized in that: The medicine or health product for preventing or alleviating constipation is a medicine or health product that restores stool consistency by reducing the expression of aquaporins AQP3, AQP4 and AQP8.

7. The use of a dietary fiber probiotic composition according to claim 1, characterized in that: The medicine or health product for preventing or alleviating constipation is a medicine or health product that protects the integrity of the intestinal barrier by increasing the expression of MUC-2 and ZO-1.

8. The use of a dietary fiber probiotic composition according to claim 1, characterized in that: The medicine or health product for preventing or alleviating constipation is a medicine or health product that improves intestinal immune response disorders by reducing the expression of inflammatory factors IL-6, IL-10 and IL-1β.

9. The use of a dietary fiber probiotic composition according to claim 1, characterized in that: The medicine or health product for preventing or alleviating constipation is a medicine or health product that improves intestinal flora disorder by increasing the expression of Firmicutes and reducing the expression of Bacteroidetes and Proteus.

10. The use of a dietary fiber probiotic composition according to claim 1, characterized in that: The medicine or health product for preventing or alleviating constipation is a medicine or health product that increases the content of one or more short-chain fatty acids among acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid.

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