Strong-affinity lactobacillus plantarum for improving intestinal constipation from saussurea involucrata and application of lactobacillus plantarum

Abstract

The invention discloses a plant lactobacillus plantarum, which is a plant lactobacillus plantarum TSXueLian-3, is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on September 20, 2024, and has the preservation number of GDMCC No.65165. The plant lactobacillus plantarum is a plant lactobacillus plantarum strain, and the plant lactobacillus plantarum is a plant lactobacillus plantarum strain, and is preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on September 20, 2024, and the preservation number is GDMCC No.65165. The invention further discloses a preparation method of the plant lactobacillus plantarum strain. The invention also provides an application of the plant lactobacillus and a fermented product prepared from the plant lactobacillus and used for improving constipation. The plant lactobacillus provided by the invention not only can promote intestinal tract movement, but also can reduce the quantity of probiotics adhered to excrement and avoid the loss of viable bacteria.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to a plant lactobacillus derived from Saussurea involucrata, which has strong affinity and can improve intestinal constipation, and an application thereof. Background Art

[0002] As people age, the incidence of constipation gradually increases. The prevalence rate in people over 60 years old is as high as 33.5%, and the prevalence rate in women is higher than that in men (1.5:1). In addition to common symptoms such as hardened stools, long defecation cycles, frequent bowel movements, and a feeling of incomplete defecation, long-term or frequent constipation can cause serious harm to the human body, such as: inducing / aggravating cardiovascular and cerebrovascular diseases; causing / aggravating rectal and anal diseases; causing gastrointestinal nerve dysfunction; accumulation of toxins in the human body and sub-healthy skin; increasing the risk of colorectal cancer; leading to the formation of "fecal ulcers", etc. There are many reasons for constipation, such as: insufficient intake of cellulose and water; reduced intestinal smooth muscle tension and weakened peristalsis; mechanical obstruction of intestinal peristalsis; and activity disorders of the defecation muscle group. However, studies in recent years have found that intestinal microecology is involved in the occurrence and development of constipation.

[0003] The traditional view is that if constipated patients take more vegetables, perhaps constipation can be improved, but the result is that many people who take a lot of vegetables, even sweet potatoes, experience abdominal distension and even harder stools. The reason is that the intestinal flora of constipated patients is insufficient and there is no way to ferment. If too much food residue or food containing dietary fiber is added, the intestinal flora will be overloaded and unable to ferment normally, resulting in harder stools. Another view is that by taking some laxatives to expel the hard stool, it will not be difficult to defecate later, but the result is that when constipated patients take laxatives, in addition to the stool being excreted, the normal flora is also excreted, resulting in insufficient flora. Once the patient stops using these laxatives, the stool will become harder after defecation, forming a vicious cycle.

[0004] Therefore, how to effectively improve constipation and make defecation form a virtuous cycle has become a problem that needs to be solved urgently. Summary of the invention

[0005] The first aspect of the present invention aims to provide a plant lactobacillus derived from Saussurea involucrata, which can not only promote intestinal peristalsis, but also reduce the number of probiotics adhering to feces and avoid the loss of live bacteria.

[0006] The second aspect of the present invention aims to provide a fermented product prepared by fermenting a composition with Lactobacillus plantarum derived from Saussurea involucrata, which has the effect of improving constipation.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention provides a plant lactobacillus, which is Lactobacillus plantarum (Lactiplantibacillus plantarum) TS XueLian-3, which was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on September 20, 2024, with a deposit number of GDMCC No.65165, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The inventors used Tianshan Snow Lotus as a sample, placed it in a specific culture medium A and cultured it for a period of time, then aspirated the culture solution and applied it to culture medium B. After continuing to culture for a period of time, they selected colonies and further purified and separated them to obtain a new strain, which was identified as Lactiplantibacillus plantarum by molecular biology and named TSXueLian-3.

[0010] In some embodiments of the present invention, the culture medium A is: 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 0.58 g of magnesium sulfate, and 0.25 g of manganese sulfate, dissolved in 1000 mL of distilled water, adjusted to pH 6.5, sterilized at 121° C. for 20 min, and cooled to 37° C.

[0011] In some embodiments of the present invention, the culture medium B is: 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, and 15 g of agar, dissolved in 1000 mL of distilled water, adjusted to pH 6.5, sterilized at 121° C. for 20 min, and cooled to 37° C.

[0012] A second aspect of the present invention provides a bacterial agent, wherein the bacterial agent comprises the above-mentioned Lactobacillus plantarum.

[0013] Furthermore, the bacterial agent may be various preparations containing the above-mentioned Lactobacillus plantarum, including but not limited to bacterial suspension, fermentation broth, etc.

[0014] The third aspect of the present invention provides the use of the Lactobacillus plantarum in the preparation of a preparation for improving constipation.

[0015] The fourth aspect of the present invention provides the use of the Lactobacillus plantarum in the preparation of a preparation for promoting intestinal motility.

[0016] The fifth aspect of the present invention provides the use of the Lactobacillus plantarum in the preparation of a preparation for reducing the excretion of probiotics in the intestine.

[0017] The sixth aspect of the present invention provides the use of the bacterial agent in the preparation of a preparation for improving constipation.

[0018] The seventh aspect of the present invention provides the use of the bacterial agent in the preparation of a preparation for promoting intestinal motility.

[0019] The eighth aspect of the present invention provides the use of the bacterial agent in the preparation of a preparation for reducing the excretion of probiotics in the intestine.

[0020] Furthermore, the preparations include but are not limited to medicines, foods and health products.

[0021] The ninth aspect of the present invention provides a fermented product for improving constipation, wherein the fermented product is obtained by inoculating the plant lactobacillus into a composition and fermenting the composition; the composition comprises the following components by weight: 25-45 parts of young barley shoots, 20-40 parts of plums, 10-30 parts of mangosteens, 5-25 parts of pitayas, 1-10 parts of monohydrate glucose, and 100-150 parts of water.

[0022] Barley seedlings are the seedlings of barley, a plant of the Poaceae family. They are generally considered to be barley seedlings that have grown to 20 to 30 cm. They were recorded as early as in the "Compendium of Materia Medica", which mentioned that barley seedlings have "spicy, cold, and non-toxic taste; can sober up and treat poisonous sores; can be taken after crushing and filtering to detoxify; can be taken after boiling to eliminate boredom, treat seasonal epidemics, and is beneficial to intestinal health." Modern pharmacological research has found that barley seedlings are rich in protein and enzymes, amino acids, vitamins, minerals, chlorophyll, flavonoids and other nutrients. They are also low in carbohydrates, low in fat, high in vitamins, high in dietary fiber, and high in protein. They help to increase stool volume and lower intestinal pH, thereby accelerating intestinal peristalsis and stool excretion, shortening the transit time of stool in the gastrointestinal tract, and reducing the risk of colorectal cancer.

[0023] Plums are rich in nutrients, including organic acids, niacin, vitamin C, flavonoids, anthocyanins, polyphenols and other nutrients. They can promote the digestion and metabolism of food in the human body, that is, by promoting gastric acid secretion, they can promote gastrointestinal motility and promote food digestion. In addition, plums still retain a high antioxidant activity after digestion, which is beneficial to repair oxidative damage to the intestinal mucosa of patients with constipation and reduce intestinal inflammation.

[0024] Mangosteen, also known as mangosteen, formerly known as phoenix fruit, is rich in citric acid, vitamin C, vitamin B, lipids, sugars, minerals (Ca, K, Mg) and amino acids (alanine, glutamic acid) and other nutrients. It is cool in nature and has the effects of cooling and reducing fire, appetizing, regulating qi, resolving phlegm and stopping vomiting. It can also treat constipation, abdominal pain, diarrhea, suppuration, chronic ulcers and other diseases.

[0025] Pitaya, also known as red dragon fruit, fairy fruit, auspicious fruit, etc., is rich in dietary fiber, anthocyanins, flavonoids, plant albumin, carbohydrates, protein, minerals, vitamins and other nutrients. Among them, the high content of dietary fiber can promote intestinal peristalsis and help prevent / improve constipation; plant albumin plays the role of a scavenger in the human body. It can remove acidic substances produced by cell metabolism (ie, garbage in the body) from the body, while avoiding poisoning caused by the absorption of heavy metal ions, and has a protective effect on the stomach wall.

[0026] The composition provided by the present invention selects fresh herbs and fruits of specific types, and is rich in chlorophyll, organic acid, dietary fiber, albumin and other functional ingredients that have a promoting effect on improving constipation. The types and proportions of the composition are very innovative. In addition, after fermentation with Lactobacillus plantarum, the obtained fermentation product has strong affinity for the human body and high bioavailability. While promoting intestinal peristalsis, it can reduce the adhesion of probiotics in feces and avoid the occurrence of flora disorder. It can fundamentally and better improve constipation and protect the intestinal health of the human body.

[0027] In a preferred embodiment of the present invention, the composition comprises the following components by weight: 35 parts of young barley shoots, 25 parts of plums, 20 parts of mangosteens, 15 parts of pitayas, 5 parts of glucose monohydrate, and 130 parts of water.

[0028] The tenth aspect of the present invention also provides a method for preparing the fermented product, comprising the following steps:

[0029] S1. Wash, drain, and chop the young barley seedlings, place them in a wall-breaking machine, add water to break the wall, and filter to obtain filtrate A; wash and drain the plums, mangosteens, and pitaya, take the pulp, place it in a wall-breaking machine, add glucose monohydrate and the remainder of water, break the wall, and obtain base material B;

[0030] S2. After the filtrate A and the base material B are mixed evenly, sterilized to obtain base material C;

[0031] S3. Inoculating Lactobacillus plantarum into the base material C, and obtaining the fermented product after anaerobic fermentation.

[0032] In the above step S1, in terms of raw material selection, fresh, non-corrupted, disease- and insect-free barley seedlings are preferably selected; fresh, ripe, and complete plums, mangosteens, and pitayas are selected; and non-hygroscopic, high-quality glucose monohydrate is selected.

[0033] In a preferred embodiment of the present invention, the above step S1 is: wash the young barley seedlings with clean water, drain, and chop, weigh 35 parts, put them in a wall breaking machine, add 70 parts of purified water, break the wall at 22000r / min for 1min, filter with a 300-mesh filter, and take filtrate A; wash the plums, mangosteens, and pitaya with clean water, drain, and take the pulp, weigh 25 parts of plums, 20 parts of mangosteens, and 15 parts of pitayas, put them in a wall breaking machine, add 5 parts of monohydrate glucose and 60 parts of purified water, break the wall at 28000r / min for 1min, and obtain base material B.

[0034] In the above step S2, the sterilization method is: heating the mixed solution to 50-70°C and maintaining it for 20-40 minutes. Preferably, the heating temperature is 60°C and maintained for 30 minutes.

[0035] In the above step S3, the plant lactobacillus is first activated and prepared into a bacterial suspension, and then inoculated into the base material C. In some embodiments of the present invention, the activation treatment method is: the plant lactobacillus frozen in the cryopreservation tube is inoculated into 10mL of culture medium, and cultured at 37°C under constant temperature and anaerobically for 24h as the first generation of activated bacteria; 2% (v / v) of the inoculum volume ratio is taken from the first generation of bacterial solution, inoculated into 40mL of culture medium, and cultured at 37°C under constant temperature and anaerobically for 24h as the second generation of activated bacteria; 2% (v / v) of the inoculum volume ratio is taken from the second generation of bacterial solution and inoculated into 2L of culture medium, and cultured at 37°C under constant temperature and anaerobically for 24h to obtain the activated bacterial solution D.

[0036] In some embodiments of the present invention, the preparation method of the culture medium is as follows: 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 0.58 g of magnesium sulfate, and 0.25 g of manganese sulfate are dissolved in 1000 mL of distilled water, the pH is adjusted to 6.5, sterilized at 121° C. for 20 min, and cooled to 37° C.

[0037] In some embodiments of the present invention, the preparation method of the bacterial suspension is as follows: the activated bacterial solution D is placed in a high-speed centrifuge, centrifuged at 4°C and 4000 r / min for 15 min, the supernatant is poured out, the bacterial mud is washed twice with sterile saline, and the number of live bacteria is diluted with sterile saline to 1×10 9 CFU / g, and obtain bacterial suspension E.

[0038] In some embodiments of the present invention, in the above step S3, the inoculation amount of the bacterial suspension E is 3 portions, and the fermentation conditions are: constant temperature anaerobic fermentation at 37° C. for 8 hours to obtain a fermentation product.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The present invention provides a plant lactobacillus derived from Saussurea involucrata, which can not only promote intestinal peristalsis, but also reduce the amount of probiotics adhering to feces and avoid the loss of live bacteria.

[0041] 2. The fermented product for improving constipation provided by the present invention has strong affinity to the human body and high bioavailability after being fermented by Lactobacillus plantarum. While promoting intestinal peristalsis, it can reduce the adhesion of probiotics in feces and avoid the occurrence of flora disorder. It can fundamentally and better improve constipation and protect the intestinal health of the human body. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0044] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are all conventional methods, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0045] Isolation of Lactobacillus plantarum from Saussurea involucrata

[0046] 1. Sample processing: remove the outer leaves of the Tianshan Saussurea involucrata, rinse with sterile water for 3 times, drain the water, and obtain sample A;

[0047] 2. Culture medium preparation

[0048] (1) Culture medium A: 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 0.58 g of magnesium sulfate, and 0.25 g of manganese sulfate were dissolved in 1000 mL of distilled water, the pH was adjusted to 6.5, sterilized at 121°C for 20 min, and cooled to 37°C.

[0049] (2) Culture medium B: 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate, and 15 g of agar, dissolved in 1000 mL of distilled water, adjusted to pH 6.5, sterilized at 121°C for 20 min, and cooled to 37°C.

[0050] 3. Strain culture and isolation: Take 1g of sample A and place it in culture medium A, and culture it at 37℃ for 72h; take 0.1mL and spread it on culture medium B, and culture it at 37℃ for 48h, select single colonies, further purify and isolate them, and obtain strains, and store them in a freezer at -80℃.

[0051] 4. Strain identification: The strain was identified by 16sRNA and was confirmed to be Lactobacillus plantarum.

[0052] The specific gene sequence identification results are as follows:

[0053]

[0054] Test Example 1

[0055] 1. Experimental strains: Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus salivarius thermophilus, Pediococcus pentosaceus. All the above strains are standard strains.

[0056] 2. Sample preparation

[0057] (1) Preparation of culture medium: Dissolve 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 0.58 g of magnesium sulfate, and 0.25 g of manganese sulfate in 1000 mL of distilled water, adjust the pH to 6.5, sterilize at 121°C for 20 min, and cool to 37°C.

[0058] (2) Activation of bacterial strains: The experimental bacterial strains stored in the cryopreservation tubes were inoculated into 10 mL of culture medium, and cultured at 37° C. under constant temperature and anaerobism for 24 h to obtain the first generation of activated bacterial strains; 2% (v / v) of the inoculum volume ratio was taken from the first generation bacterial solution and inoculated into 40 mL of culture medium, and cultured at 37° C. under constant temperature and anaerobism for 24 h to obtain the second generation of activated bacterial strains; 2% (v / v) of the inoculum volume ratio was taken from the second generation bacterial solution and inoculated into 2 L of culture medium, and cultured at 37° C. under constant temperature and anaerobism for 24 h to obtain the activated bacterial solution.

[0059] (3) Preparation of bacterial suspension: Place the activated bacterial suspension in a high-speed centrifuge and centrifuge at 4°C and 4000 r / min for 15 min. Pour off the supernatant and wash the bacterial sludge twice with sterile saline. Then dilute the suspension with sterile saline until the number of viable bacteria is 1×10 9 CFU / g, and obtain bacterial suspension.

[0060] (4) Preparation of fermentation liquid: Weigh 10 parts of soybeans and place them in a wall-breaking machine, add 8 parts of white sugar and 82 parts of purified water, break the wall at 18000 r / min for 2 min, boil, sterilize at 121°C for 20 min, and cool to 37°C to obtain stock solution A. Add 3 parts of bacterial suspension to the stock solution, stir evenly, and culture at 37°C under constant temperature and anaerobic conditions for 8 h to obtain fermentation liquid.

[0061] (5) The fermentation broths prepared by fermenting five bacterial species, namely, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus salivarius subsp. thermophilus, and Pediococcus pentosaceus, were numbered as fermentation broth A to fermentation broth E.

[0062] 3. Test methods:

[0063] (1) 140 wild-type AB zebrafish were selected 5 days after hatching and treated with Nile red for 16 hours. The zebrafish were kept in 28°C fish water (water quality: 200 mg of instant sea salt was added to 1 L of reverse osmosis water, conductivity was 480-510 μs / cm; pH was 6.9-7.2; hardness was 53.7-71.6 mg / L).

[0064] (2) After Nile red was removed, zebrafish were randomly divided into 7 groups and distributed in six-well plates. Each well was treated with 20 zebrafish and treated with water-soluble drugs for 24 h. The drug administration conditions are shown in Table 1. Group A was used as a blank group, and groups B-G were used as experimental groups.

[0065] Table 1 Zebrafish grouping and drug administration

[0066] Group Dosage Group A 0.8mg stock solution A + 3mL fish water Group B 0.09mg domperidone + 3mL fish water Group C 0.8mg fermentation liquid A + 3mL fish water Group D 0.8mg fermentation liquid B + 3mL fish water Group E 0.8mg fermentation liquid C + 3mL fish water Group F 0.8mg fermentation liquid D + 3mL fish water Group G 0.8mg fermentation liquid E + 3mL fish water

[0067] (2) Ten zebrafish were randomly selected from each group to detect the Nile red fluorescence intensity (S) in the intestine and calculate the intestinal motility promotion rate. The results are shown in Table 2.

[0068]

[0069] 4. Results and Analysis

[0070] Table 2 Promotion rate of intestinal motility in zebrafish

[0071]

[0072]

[0073] As shown in Table 2, compared with the blank group A, the intestinal motility promotion rate of group B was greater than 0, indicating that domperidone has a promoting effect on intestinal motility. Due to the difference in strain species, the intestinal motility promotion rates of groups C to G were different and all greater than 0, indicating that the five strains of Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Streptococcus salivarius thermophilus subsp. and Pediococcus pentosaceus all have the effect of promoting intestinal motility.

[0074] In addition, compared with other groups, the intestinal motility promotion rate of group D was the highest, indicating that the sample of group D (fermentation liquid B) had the best effect on promoting intestinal motility of zebrafish. This shows that compared with Lactobacillus acidophilus, Lactobacillus rhamnosus, Streptococcus salivarius thermophilus subsp. and Pediococcus pentosaceus, the fermentation liquid made by Lactobacillus plantarum can better promote intestinal motility.

[0075] Test Example 2

[0076] 1. Experimental strains: Lactobacillus plantarum CICC 25282 (preservation number: CICC 25282), Lactobacillus plantarum CICC25283 (preservation number: CICC 25283), Lactobacillus plantarum TSXueLian-3, Lactobacillus plantarum GDMCC 1.4957 (preservation number: GDMCC 1.4957).

[0077] 2. Sample preparation

[0078] (1) Preparation of culture medium: Dissolve 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 0.58 g of magnesium sulfate, and 0.25 g of manganese sulfate in 1000 mL of distilled water, adjust the pH to 6.5, sterilize at 121°C for 20 min, and cool to 37°C.

[0079] (2) Activation of bacterial strains: The experimental bacterial strains stored in the cryopreservation tubes were inoculated into 10 mL of culture medium, and cultured at 37° C. under constant temperature and anaerobism for 24 h to obtain the first generation of activated bacterial strains; 2% (v / v) of the inoculum volume ratio was taken from the first generation bacterial solution and inoculated into 40 mL of culture medium, and cultured at 37° C. under constant temperature and anaerobism for 24 h to obtain the second generation of activated bacterial strains; 2% (v / v) of the inoculum volume ratio was taken from the second generation bacterial solution and inoculated into 2 L of culture medium, and cultured at 37° C. under constant temperature and anaerobism for 24 h to obtain the activated bacterial solution.

[0080] (3) Preparation of bacterial suspension: Place the activated bacterial solution in a high-speed centrifuge and centrifuge at 4°C and 4000 r / min for 15 min. Pour away the supernatant, wash the bacterial sludge twice with sterile saline, and dilute with sterile saline until the number of live bacteria is 1×109 CFU / g to obtain a bacterial suspension.

[0081] 3. Test methods

[0082] (1) Experimental animals: Kunming mice, male, SPF grade, weight 20±2g. The breeding environment was clean, with a temperature of 22°C and a relative humidity of 50%; the light intensity was 12h / 12h alternating light and dark; the cages were changed once a day.

[0083] (2) After 5 days of adaptive feeding, the experimental mice were randomly divided into 11 groups according to their body weight, with 8 mice in each group, numbered as Group A, Group B, Group C, Group D, Group E, Group F, Group G, Group H, Group I, Group J, and Group K. The mice in Groups B to K were gavaged with loperamide hydrochloride solution (0.26 mg / mL of loperamide hydrochloride) once a day, 0.6 mL each time, for 14 consecutive days; the mice in Group A were gavaged with normal saline once a day, 0.6 mL each time, for 14 consecutive days. 30 minutes after the last administration, Indian ink was gavaged, and the timing started immediately after gavage. Each mouse was placed in a separate cage with free access to water. The feces of the mice were collected within 6 hours, observed every 15 minutes, and the time required for the first black feces to be discharged was recorded (min). The results are shown in Table 4.

[0084] (3) On the 15th day, the mice were given free access to food and water and were given drugs once a day at 8:00 a.m. for 7 consecutive days. The groups and drug administration are shown in Table 3.

[0085] Table 3 Grouping and administration of mice

[0086] Group Dosage Group A 0.6 mL normal saline Group B 0.6 mL normal saline Group C 0.3mL domperidone solution (100mg / mL) + 0.3mL normal saline Group D 0.3mL domperidone solution (100mg / mL) + 0.3mL Lactobacillus plantarum CICC25282 bacterial suspension Group E 0.3mL domperidone solution (100mg / mL) + 0.3mL Lactobacillus plantarum CICC25283 bacterial suspension Group F 0.3mL domperidone solution (100mg / mL) + 0.3mL Lactobacillus plantarum TSXueLian-3 bacterial suspension Group G 0.3mL domperidone solution (100mg / mL) + 0.3mL Lactobacillus plantarum GDMCC1.4957 bacterial suspension Group H 0.6mL Lactobacillus plantarum CICC25282 bacterial suspension Group I 0.6mL Lactobacillus plantarum CICC25283 bacterial suspension Group J 0.6mL Lactobacillus plantarum TSXueLian-3 bacterial suspension Group K 0.6mL Lactobacillus plantarum GDMCC1.4957 bacterial suspension

[0087] (4) On day 22, all mice were no longer given medication and were kept normally for 7 days.

[0088] (5) During the period from 8:00 a.m. to 18:00 p.m. on the 21st and 28th days, the feces of the mice were collected, and 3 fresh feces were taken for intestinal flora detection, and the number of Lactobacillus and Bifidobacterium was calculated. The results are shown in Table 4. The weight of the remaining feces before and after drying was weighed, and the drying condition was 105°C for 30 min. The water content of the feces was calculated, and the results are shown in Table 4.

[0089]

[0090] (6) Intestinal flora detection: Weigh 0.1 g of fresh mouse feces, place in a sterilized and dried triangular flask with glass beads, add 10 mL of sterile water, shake and mix to obtain a fecal suspension, and then dilute it 10 times in sequence to 10 -9 Times. Take 1mL of bacterial suspension and inoculate it on the culture medium, and repeat 3 times. Lactobacillus and Bifidobacterium use MRS and BSM culture medium respectively; MRS and BSM culture medium plates are placed at 37℃ under anaerobic culture for 48h and then counted; according to the colony morphology, the number of colonies formed by each bacterial group per gram of feces is calculated by smear analysis, and the results are expressed as the logarithm of colonies lg (CFU / g).

[0091] 4. Results and Analysis

[0092] Table 4 Various indicators of mice

[0093]

[0094] As shown in Table 4, compared with Group A, the first defecation time of mice in Groups B to K was significantly increased, indicating that the constipation mouse model was successfully established. Compared with Group B, the fecal water content of mice in Group C increased significantly on the 21st day, and the lactobacillus and bifidobacteria in the feces increased significantly, indicating that domperidone can promote intestinal peristalsis and improve constipation. At the same time as defecation, more lactobacillus and bifidobacteria in the intestine are discharged. On the 28th day, the fecal water content of mice in Group C decreased significantly, indicating that the mice were constipated. Although the consumption of domperidone during constipation can improve constipation in constipated mice, due to excessive discharge of intestinal probiotics, the constipation of mice recurred again after stopping eating domperidone for a period of time.

[0095] Compared with group B, the fecal water content of mice in groups D to G increased significantly on the 21st day, and compared with group C, the fecal lactobacillus and bifidobacteria in mice in groups D to G decreased significantly. This shows that when domperidone promotes intestinal peristalsis, bacterial suspensions of different strains can reduce the excretion of lactobacillus and bifidobacteria in the intestine; the fecal water content on the 28th day was still significantly higher than that of group B, indicating that constipation in mice did not recur. Among them, the fecal water content of mice in group F was the highest on the 21st day, and the number of lactobacillus and bifidobacterium colonies was the lowest, indicating that plant lactobacillus TSXueLian-3 had the best effect in reducing the excretion of lactobacillus and bifidobacteria in the intestine during intestinal peristalsis.

[0096] Compared with group B, the fecal water content of mice in groups H to K increased significantly on the 21st day, and compared with group C, the lactobacillus and bifidobacteria in the feces of mice in groups H to K decreased significantly. This shows that when the bacterial suspension is used alone, even if the strains are different, it can promote intestinal peristalsis and improve constipation, and at the same time, it can reduce the excretion of lactobacillus and bifidobacteria in the intestine; on the 28th day, the fecal water content of mice in groups H to K was still significantly higher than that in group B, indicating that constipation in mice did not recur. Among them, the fecal water content of mice in group J was the highest on the 21st day, and the number of lactobacillus and bifidobacterium colonies was the lowest, indicating that plant lactobacillus TSXueLian-3 has the best effect in promoting intestinal peristalsis, improving constipation, and reducing the excretion of lactobacillus and bifidobacteria in the intestine. On the 28th day, the fecal water content of mice in group J was still the highest and close to that of group A, and the number of fecal Lactobacillus and Bifidobacterium colonies was lower than that of group A, indicating that Lactobacillus plantarum TSXueLian-3 can not only improve constipation, but also reduce the adhesion of probiotics in feces, reduce the loss of intestinal probiotics, reduce the recurrence of constipation, fundamentally improve constipation, and protect intestinal health.

[0097] Therefore, compared with other strains, Lactobacillus plantarum TSXueLian-3 can better reduce the adhesion of probiotics in feces.

[0098] Example 1

[0099] This embodiment 1 provides a fermented product for improving constipation, which is obtained by inoculating Lactobacillus plantarum into a composition and fermenting it. The composition includes the following components by weight: 35 parts of young barley shoots, 25 parts of plums, 20 parts of mangosteens, 15 parts of pitaya, 5 parts of monohydrate glucose, and 130 parts of water.

[0100] The preparation method of the fermentation product of this embodiment is as follows:

[0101] S1. Raw material selection: Select fresh, non-corrupted, disease- and insect-free barley seedlings; select fresh, ripe, and complete plums, mangosteens, and dragon fruits; select non-hygroscopic, high-quality glucose monohydrate.

[0102] S2. Pretreatment: Wash, drain and chop the young barley seedlings with clean water, weigh several portions, put them in a wall-breaking machine, add 70 portions of purified water, break the wall at 22000r / min for 1 min, filter with a 300-mesh filter, and take filtrate A; wash and drain the plum, mangosteen and pitaya with clean water, take the pulp, weigh several portions of plum, mangosteen and pitaya, put them in a wall-breaking machine, add monohydrate glucose and 60 portions of purified water, break the wall at 28000r / min for 1 min, and obtain base material B.

[0103] S3. Sterilization: Mix filtrate A and base material B evenly, heat to 60°C, maintain for 30 minutes, cool to 37°C, and obtain base material C.

[0104] S4. Preparation of culture medium: dissolve 10.0 g of peptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 2.0 g of diammonium citrate, 0.58 g of magnesium sulfate, and 0.25 g of manganese sulfate in 1000 mL of distilled water, adjust the pH to 6.5, sterilize at 121°C for 20 min, and cool to 37°C.

[0105] S5. Activation of bacterial strains: inoculate the plant lactobacillus TSXueLian-3 frozen in a cryopreservation tube into 10 mL of culture medium, and culture at 37°C under constant temperature and anaerobically for 24 h as the first-generation activated bacterial strain; take 2% (v / v) of the inoculum by volume from the first-generation bacterial solution, inoculate it into 40 mL of culture medium, and culture it at 37°C under constant temperature and anaerobically for 24 h as the second-generation activated bacterial strain; take 2% (v / v) of the inoculum by volume from the second-generation bacterial solution, inoculate it into 2 L of culture medium, and culture it at 37°C under constant temperature and anaerobically for 24 h to obtain the activated bacterial solution D.

[0106] S6. Preparation of bacterial suspension: Place the activated bacterial suspension D in a high-speed centrifuge and centrifuge at 4°C and 4000 r / min for 15 min. Pour off the supernatant and wash the bacterial sludge twice with sterile saline. Then dilute the suspension with sterile saline until the number of viable bacteria is 1×10 9 CFU / g, and obtain bacterial suspension E.

[0107] S7. Fermentation: Take 3 portions of bacterial suspension E and place them in base material C, stir evenly, and culture at 37°C, constant temperature, and anaerobic conditions for 8 hours to obtain a fermentation product.

[0108] Comparative Examples 1 to 6

[0109] The fermented products provided in Comparative Examples 1 to 6 are prepared in the same manner as in Example 1, except that the ratios of the compositions are different.

[0110] The specific formulas of the compositions in Example 1 and Comparative Examples 1 to 6 are shown in Table 5.

[0111] Table 5 Weight parts of each component in the fermentation product

[0112]

[0113] Comparative Example 7

[0114] The proportions of the composition in Comparative Example 7 are the same as those in Example 1, except that bacterial suspension E is not added for fermentation in Comparative Example 7, and bacterial suspension E is replaced by an equal amount of sterile physiological saline.

[0115] Test Example 3

[0116] 1. Crowd Testing

[0117] 1. Subject recruitment: People aged 5 to 65 years old, who had constipation symptoms 6 months ago and had 2 or more symptoms as described in the inclusion criteria in the past 3 months.

[0118] (1) Inclusion criteria

[0119] 1) Straining during bowel movements at least 25% of the time;

[0120] 2) having lumpy or hard stools at least 25% of the time;

[0121] 3) Feeling of incomplete bowel movements at least 25% of the time;

[0122] 4) Anorectal obstruction during bowel movements at least 25% of the time;

[0123] 5) Defecation requires manual assistance (such as finger assistance, pelvic floor support) at least 25% of the time;

[0124] 6) Spontaneous bowel movements less than 3 times per week;

[0125] (2) Exclusion criteria

[0126] (1) Those with unclear chief complaint; those who are physically weak and unable to undergo the test; and women who are pregnant, lactating, or menstruating.

[0127] (2) Those who have undergone surgical operations within 30 days and have constipation symptoms. Those who have had acute gastrointestinal diseases within 30 days. Those who have recent difficulty in defecation due to severe organic diseases (colon cancer, severe enteritis, intestinal obstruction, inflammatory bowel disease, etc.).

[0128] (3) Patients with serious systemic diseases such as cardiovascular, liver, kidney and hematopoietic system diseases, those with constipation and pain, or those who are undergoing treatment for other concomitant diseases.

[0129] (4) Those who cannot take food orally or cannot take the test sample as required. Those who take items related to the function of the test sample in a short period of time, which may affect the judgment of the results.

[0130] (5) Those who have taken antibiotics for a long time or within 15 days before the start of the trial.

[0131] (6) Those who took probiotics or fermented products within 7 days before the start of the trial.

[0132] 2. The subjects were randomly divided into 8 groups, 6 people in each group, and the fermented products of Examples 1 to 8 were consumed respectively. The groups and consumption conditions are shown in Table 6. The consumption method was to consume the fermented products half an hour after meals every day, 3 times a day, 5g each time, for 14 consecutive days.

[0133] Table 6 Types of fermented products consumed by the subjects

[0134]

[0135]

[0136] 3. The subjects recorded the number of their own completely autonomous bowel movements every day and scored the stool characteristics. The scoring criteria are shown in Table 7 for 21 consecutive days. On the 1st day, 14th day, and 21st day, the number of completely autonomous bowel movements per week in each group was calculated and the average was calculated. The number of people with completely autonomous bowel movements ≥ 3 times per week was counted, and the average score of stool characteristics was calculated. The results are shown in Table 8.

[0137] Table 7 Scoring criteria

[0138]

[0139] 2. Results and Analysis

[0140] Table 8 Defecation status of subjects

[0141]

[0142]

[0143] As shown in Table 8, compared with the first day, the number of subjects with completely autonomous bowel movements per week and the number of people with bowel movements ≥ 3 times on the 14th day increased to varying degrees, and the scores of stool characteristics decreased to varying degrees, indicating that consuming fermented products fermented by Lactobacillus plantarum TSXueLian-3 and fermented products not fermented by Lactobacillus plantarum TSXueLian-3 are beneficial to increase the number of completely autonomous bowel movements, reduce stool hardness, further improve bowel comfort, and improve constipation symptoms. Compared with group H, the number of subjects in groups A to G increased to varying degrees on the 14th day, and the scores of stool characteristics decreased to varying degrees, indicating that the fermented products fermented by Lactobacillus plantarum TSXueLian-3 have strong affinity for the human body and high bioavailability, and their effect on improving constipation is better than that of fermented products not fermented by Lactobacillus plantarum TSXueLian-3. At the same time, even if the weight of each component in the fermented product is different, the fermented product obtained after fermentation by Lactobacillus plantarum TSXueLian-3 can better improve constipation. Among them, Group G had the highest number of completely autonomous bowel movements per week, the largest number of people with bowel movements ≥ 3 times, and the lowest score for stool characteristics, indicating that the fermented product of Group G was most effective in improving constipation during the consumption period, and could increase the number of completely autonomous bowel movements per week in people with constipation and reduce the hardness of stool.

[0144] Compared with the 14th day, the subjects' weekly completely autonomous bowel movements on the 21st day decreased to varying degrees, and the scores of stool characteristics increased to varying degrees, indicating that the fermented products fermented by Lactobacillus plantarum TSXueLian-3 and the fermented products not fermented by Lactobacillus plantarum TSXueLian-3 have different sustained abilities to improve constipation. Compared with the H group, the subjects in groups A to G had different degrees of increase in the weekly completely autonomous bowel movements on the 21st day, and the scores of stool characteristics decreased to varying degrees, indicating that the fermented products fermented by Lactobacillus plantarum TSXueLian-3 have better sustained effects on improving constipation than the fermented products not fermented by Lactobacillus plantarum TSXueLian-3. At the same time, even if the weight of each component in the fermented products is different, after fermentation by Lactobacillus plantarum TSXueLian-3, the fermented products obtained can still play a role in improving constipation after stopping consumption. Among them, group G had the highest number of completely autonomous bowel movements per week, the largest number of people with bowel movements ≥ 3 times, and the lowest score for stool characteristics, indicating that the fermented product of group G can continue to protect intestinal health after stopping consumption, and has the best effect in improving constipation. It can increase the number of completely autonomous bowel movements per week in people with constipation and reduce the hardness of stool.

[0145] Therefore, the fermented product of Example 1 of the present invention has a better effect of improving constipation.

[0146] In summary, the plant lactobacillus TSXueLian-3 from the Saussurea involucrata provided by the present invention has strong affinity for the human body, and after colonization in the intestinal tract, it can not only promote intestinal peristalsis and promote completely autonomous defecation, but also reduce probiotic adhesion in feces and reduce the lack of intestinal probiotic species, thereby improving constipation symptoms and blocking the development of constipation. The fermented product made of the plant lactobacillus TSXueLian-3 can not only improve constipation during consumption, but also after stopping consumption for a period of time, its effect of improving constipation will not rebound, and can continuously prevent and improve constipation, protecting the health of the human intestinal tract.

[0147] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A plant lactobacillus, characterized in that The plant lactobacillus is Lactobacillus plantarum TS XueLian-3, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on September 20, 2024, with a deposit number of GDMCC No.65165.

2. A bacterial agent, characterized in that The bacterial agent comprises the Lactobacillus plantarum described in claim 1.

3. A bacterial agent according to claim 2, characterized in that: The bacterial agent includes bacterial suspension and fermentation liquid.

4. Use of the plant lactobacillus according to claim 1 or the bacterial agent according to claim 2 in the preparation of a preparation for improving constipation.

5. Use of the plant lactobacillus according to claim 1 or the bacterial agent according to claim 2 in the preparation of a preparation for promoting intestinal peristalsis.

6. Use of the plant lactobacillus according to claim 1 or the bacterial agent according to claim 2 in the preparation of a preparation for reducing the excretion of probiotics in the intestine.

7. The use according to any one of claims 4 to 6, characterized in that: The preparations include medicines, foods and health products.

8. A fermented product for improving constipation, characterized in that: The fermented product is obtained by inoculating the plant lactobacillus described in claim 1 into a composition and fermenting it; the composition comprises the following components by weight: 25-45 parts of young barley shoots, 20-40 parts of plums, 10-30 parts of mangosteens, 5-25 parts of pitayas, 1-10 parts of monohydrate glucose, and 100-150 parts of water.

9. The fermented product for improving constipation according to claim 8, characterized in that: The composition comprises the following components by weight: 35 parts of young barley shoots, 25 parts of plums, 20 parts of mangosteens, 15 parts of pitayas, 5 parts of glucose monohydrate, and 130 parts of water.

10. The method for preparing a fermented product for improving constipation according to claim 8 or 9, characterized in that: The following steps are involved: S1. Wash, drain, and chop the young barley seedlings, place them in a wall-breaking machine, add water to break the wall, and filter to obtain filtrate A; wash and drain the plums, mangosteens, and pitaya, take the pulp, place it in a wall-breaking machine, add glucose monohydrate and the remainder of water, break the wall, and obtain base material B; S2. After the filtrate A and the base material B are mixed evenly, sterilized to obtain base material C; S3. Inoculating Lactobacillus plantarum into the base material C, and obtaining the fermented product after anaerobic fermentation.

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