CN118165896A - Lactobacillus rhamnosus JYLR-985 for relieving constipation of medicine, product and application - Google Patents
Lactobacillus rhamnosus JYLR-985 for relieving constipation of medicine, product and application Download PDFInfo
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- CN118165896A CN118165896A CN202410586781.0A CN202410586781A CN118165896A CN 118165896 A CN118165896 A CN 118165896A CN 202410586781 A CN202410586781 A CN 202410586781A CN 118165896 A CN118165896 A CN 118165896A
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Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to a preparation method of post-generated metaflour of lactobacillus rhamnosus JYLR-985 for relieving constipation of a medicine, a product and application, and the post-generated metaflour of lactobacillus rhamnosus JYLR-985 for relieving constipation of the medicine, which comprises the following steps: preparing a fermentation broth of lactobacillus rhamnosus JYLR-985; step two: centrifuging the fermentation liquor to obtain bacterial mud; step three: dissolving the bacterial mud in distilled water for sterilization to obtain an inactivated bacterial liquid; step four: freeze-drying the inactivated bacterial liquid to obtain inactivated bacterial powder; step five: and diluting the inactivated bacterial powder by using maltodextrin to obtain the metapowder of the lactobacillus rhamnosus JYLR-985. The invention adopts the post-metagen preparation of the rhamnose cheese bacillus JYLR-985 to prevent and/or treat the constipation induced by the antipsychotic drug, has obvious effect and higher safety, and is beneficial to relieving the pain of the psychotic in the antipsychotic treatment process.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus JYLR-985 for relieving constipation of a medicine, a product and application.
Background
Constipation refers to abnormal defecation caused by abnormal large intestine transport, and is mainly manifested by the following symptoms: 1. constipation and long defecation period; 2. the defecation period is not long, but the excrement is dry and hard; 3. the feces are not hard and are intentional, but are unsmooth. Constipation has an average prevalence of 16% in general adults and 14% in children, with female prevalence being higher than male.
In recent years, the incidence of constipation induced by drugs is increasing, and the drugs which may induce constipation mainly comprise anticholinergic drugs, antipsychotic and antidepressant drugs, narcotic analgesic drugs, cation-containing preparation drugs, antihypertensive drugs, antitumor drugs, diuretics, stimulant laxatives, nonsteroidal anti-inflammatory drugs and the like. Clozapine belongs to a diphenyl diazapine antipsychotic drug, and the drug can be combined with dopamine D2 and D3 receptors in the central nervous system to play a role in inhibiting dopamine; can also bind with 5-hydroxytryptamine receptor of central nervous system to affect metabolism of 5-hydroxytryptamine, raise 5-hydroxytryptamine level in brain, and improve delusional impulse and sedative hypnotic effects. Clozapine is commonly used clinically for the treatment of schizophrenia, sustained suicide or self-wound behavioural schizophrenia, and other diseases that are refractory to other antipsychotic therapies.
Studies have shown that clozapine has a strong blocking effect on the 5-hydroxytryptamine receptor and the dopamine D1, D4 receptors, which reduces intestinal motility and causes adverse effects on the digestive system, and that 30% -60% of every 1000 patients treated with clozapine have constipation, at least four have serious gastrointestinal complications (e.g. ileus) and at least one die. In the prior art, adverse reactions of digestive systems such as constipation and the like are generally relieved by taking bisacodyl or a probiotic preparation, and the bisacodyl promotes intestinal peristalsis by directly stimulating intestinal wall nerves, so that drug dependence and organism injury can be caused after long-term administration; the probiotic preparation does not cause obvious side effects, but the activity of probiotics is greatly influenced by the outside, few viable bacteria which can enter and adhere to the intestinal tract are provided, the effect of regulating the intestinal tract function is difficult to ensure, and even if the probiotics are successfully adhered to the intestinal tract, the probiotics can exert effect after the intestinal tract is colonised for a period of time.
Disclosure of Invention
Aiming at the technical problems of drug dependence and organism injury caused by using bisacodyl to relieve constipation in the prior art, the invention provides the lactobacillus rhamnosus JYLR-985 for relieving drug constipation, the product and the application, and the metagen preparation of the lactobacillus rhamnosus JYLR-985 is used for preventing and/or treating the constipation induced by the antipsychotic drug, so that the effect is remarkable, the safety is higher, and the pain of a psychotic in the antipsychotic treatment process is relieved.
In a first aspect, the invention provides a strain of lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) JYLR-985 for relieving constipation of the medicine, wherein the strain of lactobacillus rhamnosus JYLR-985 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the 18 th month of 2024, and the preservation address is North Star Xiyu No.1, 3 in the Korean region of Beijing and the preservation number is CGMCC No.29658.
In a second aspect, the present invention provides a prebiotic formulation of Lactobacillus rhamnosus JYLR-985 for alleviating constipation of a medicament as described above, the prebiotic formulation comprising a prebiotic powder of Lactobacillus rhamnosus JYLR-985.
Further, the preparation method of the post-metapowder of the lactobacillus rhamnosus JYLR-985 comprises the following steps:
step one: preparing a fermentation broth of lactobacillus rhamnosus JYLR-985;
step two: centrifuging the fermentation liquor to obtain bacterial mud;
Step three: dissolving the bacterial mud in distilled water for sterilization to obtain an inactivated bacterial liquid;
step four: freeze-drying the inactivated bacterial liquid to obtain inactivated bacterial powder;
Step five: and diluting the inactivated bacterial powder by using maltodextrin to obtain the metapowder of the lactobacillus rhamnosus JYLR-985.
Further, in the first step, the fermentation broth of Lactobacillus rhamnosus JYLR-985 is prepared as follows: single colonies of Lactobacillus rhamnosus JYLR-985 were inoculated into liquid medium and fermented at 37℃for 24h.
Further, the raw materials of the liquid medium comprise 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of tri-ammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate and 1000mL of distilled water. After the raw materials of the liquid culture medium are uniformly mixed, the liquid culture medium is sterilized at high temperature under the low vacuum condition of 0-0.1MPa, the temperature of the high temperature sterilization is 120-122 ℃, and the time of the high temperature sterilization is not less than 20min.
Further, in the second step, the viable count of the bacterial sludge is 1.0X10 10 cfu/g.
In the third step, the mass ratio of the bacterial sludge to the distilled water is 1:3. The sterilization temperature is 115 ℃ and the sterilization time is 30min. Cooling the sterilized bacterial liquid to room temperature to obtain an inactivated bacterial liquid.
In the fifth step, the number of the bacteria of the metapowder is 1.0X10 9 cfu/g.
In a third aspect, the invention also provides the use of a metagen preparation of Lactobacillus rhamnosus JYLR-985 as described above for the preparation of a medicament for the prevention and/or treatment of antipsychotic drug-induced constipation.
Further, the antipsychotic agent is clozapine.
The invention has the beneficial effects that:
The post-metapreparation of the rhamnose cheese bacillus JYLR-985 provided by the invention can improve the levels of substances P, motilin and 5-hydroxytryptamine in serum and colon, stimulate the contraction of colon smooth muscle, increase the peristaltic frequency of intestinal tracts, promote the peristaltic frequency of the intestinal tracts, shorten the defecation period and relieve constipation caused by long defecation period; the expression level of aquaporins closely related to intestinal functions such as VIP, AQP2, AQP3 and the like is obviously reduced, the colonic water metabolism is quickened, and constipation caused by dry feces is relieved; reducing the release of high-efficiency inflammatory factors such as Interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (Tumor necrosis factor alpha, TNF-alpha) in colon tissues, and relieving intestinal damage caused by antipsychotics; improving the level of superoxide dismutase (SOD) and reduced Glutathione (GSH), improving the antioxidant capacity of organism, and treating intestinal tract oxidative damage caused by constipation.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Example 1
Separation and identification of strains
1. Strain screening and purification
(1-1) Strain Source
Pickled garlic was collected in WiHai City, shandong, 7.19 days 2023.
(1-2) Preparation of samples
S1: 1g of the sample is added into a conical flask containing 9ml of sterile physiological saline with mass concentration of 0.9%, and the mixture is stirred and oscillated for 30min at 4 ℃ to obtain a sample raw solution, wherein the mark of the sample raw solution is 1#;
S2: and (3) respectively carrying out gradient dilution on the sample raw solution in the step (S1) to 10 -2、10-3、10-4、10-5、10-6、10-7 times by using sterile normal saline, and sequentially marking the sample raw solution as No. 2, no. 3, no. 4, no. 5, no. 6 and No. 7 for later use.
(1-3) Preparation of MRS plate Medium containing 0.5% CaCO 3
10G of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 5gCaCO 3 g of agar, 15g of agar and 1000mL of distilled water are mixed, the pH is adjusted to 6.8, the mixture is heated to 95 ℃ and uniformly mixed, sterilization is carried out for 20min at 121 ℃ and 0.1MPa, and then the sterilized culture medium is poured into a plate and is cooled for standby.
(1-4) Cultivation
Coating 1# -7# sample on the MRS flat plate culture medium of 0.5% CaCO 3 prepared in the step (1-3) respectively by using a coating rod, and culturing at 37 ℃ under anaerobic condition for 48h.
(1-5) Selection of colonies
After the colony is formed, thirty-two single colonies with diameters of 1-2mm, roundness, neat edges, micro white with bulges in the middle and larger calcium dissolving ring are selected.
(1-6) Separation and purification
And (3) inoculating thirty-two single colonies selected in the step (1-5) to the MRS flat-plate culture medium in the step (1-3) respectively by adopting a streaking method, culturing for 48h at 37 ℃ under anaerobic conditions, repeating the operation for 2-3 times, selecting single colonies, placing the single colonies into a glycerol tube for preservation at the temperature of-70 ℃, and sequentially marking the single colonies as the candidate strain 1-candidate strain 32.
2. Preparation of metapowder of alternative strains
(2-1) Preparation of liquid culture Medium
10G peptone, 5g beef powder, 5g sodium acetate trihydrate, 2g dipotassium phosphate heptahydrate, 1mL Tween 80, 0.05g manganese sulfate tetrahydrate, 2g tri-ammonium citrate, 20g glucose, 0.2g magnesium sulfate heptahydrate and 1000mL distilled water were mixed, the pH was adjusted to 6.8, and the mixture was sterilized at 121℃under 0.1MPa for 20 minutes.
(2-2) Preparation of fermentation broth
Culturing the alternative strain 1-alternative strain 32 by adopting the streaking method of (1-6), respectively picking a single colony of an inoculating loop, inoculating the single colony into a liquid culture medium, and culturing for 24 hours at 37 ℃ to obtain fermentation liquor of the alternative strain 1-alternative strain 32 for later use.
(2-3) Preparation of metapowder
Centrifuging the fermentation liquor of the alternative strain 1-32 to obtain bacterial sludge, and taking 1g of bacterial sludge for plate counting, wherein the viable count of the bacterial sludge is 1.0X10 10 cfu/g; 1g of bacterial mud is taken and dissolved in 3g of distillation Shui Xilin bottle to obtain bacterial liquid. Sterilizing the bacterial liquid at 115 ℃ for 30min, cooling to obtain an inactivated bacterial liquid, freeze-drying the inactivated bacterial liquid to prepare an inactivated bacterial powder, and weighing and refrigerating. And (3) diluting the inactivated bacterial powder into metapowder with the bacterial cell number of 1.0X10 9 cfu/g by using maltodextrin by means of detecting the bacterial count of bacterial mud. Thirty-two candidate strains are respectively used for preparing respective metazoan powder. Maltodextrin was purchased from bowling organism stock.
3. Facilitating selection of diamine oxidase (DAO) active strains
(3-1) Preparation of sample to be tested
And (3) mixing 125 mu L of inactivated bacterial solutions of the alternative strain 1-the alternative strain 32 with 100 mu L of diamine oxidase solution, and placing the mixture on ice to obtain a sample to be tested. Wherein the concentration of the diamine oxidase solution is 1U, and the solvent is phosphate buffer solution.
(3-2) Detection and screening of strains facilitating diamine oxidase Activity
And (3) detecting the sample to be detected in the step (3-1) according to the instruction of the diamine oxidase kit, wherein each candidate strain is provided with three parallel samples to be detected, and the average value of the relative enzyme activities is taken, and the initial enzyme activity of the diamine oxidase solution is 100%. Among the alternative strains 1 to 32, the alternative strains 1,2 and 3 had a remarkable effect of promoting diamine oxidase activity, and the relative enzyme activity detection results of the alternative strains 1,2 and 3 are shown in table 1. Diamine oxidase and diammine oxidase kits were purchased from beijing solebao corporation.
TABLE 1 relative enzyme Activity detection results for alternative Strain 1-alternative Strain 3
As can be seen from Table 1, the relative enzyme activity of the alternative strain 1 was 75.3%, which is significantly higher than that of the alternative strain 2 and the alternative strain 3, and the diamine oxidase was abundant in the intestinal villus epithelial cells, and was able to play a role in metabolism of histamine and various polyamines. By promoting the activity of diamine oxidase, the metabolism of polyamine substances can be accelerated, the intestinal peristalsis can be regulated, and the excretion can be promoted. The candidate strain 1 having the highest relative enzyme activity was sent for identification, identification unit: the primers used in the identification process of the biological engineering (Shanghai) Co., ltd were as follows:
Primer sequence:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'
1492R：5'- GGTTACCTTGTTACGACTT-3'
The gene sequence of the alternative strain 1 is as follows:
TCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTCAAAATTAAATCAAGATGCAAGCACCTTTCAATAATCAGAACTCGTTCGACTTGCATGTATAGCACGACG
Alternative strain 1 was identified as Lacticaseibacillus rhamnosus (lactobacillus rhamnosus).
The identified candidate strain 1 is named as lactobacillus rhamnosus JYLR-985, and the lactobacillus rhamnosus JYLR-985 is sent to the China general microbiological culture Collection center for preservation of microorganisms, and the preservation information is as follows;
the classification is named: lactobacillus rhamnosus (Lacticaseibacillus rhamnosus), date of preservation: 2024 1 month 18, deposit address: games 3, gao Yu 1, north Star, qing dynasty, beijing city, post code: 100101, accession number: CGMCC No.29658.
Example 2 relief of constipation by means of the metapowder of Lactobacillus rhamnosus JYLR-985 against psychotropic drugs
1. Mouse molding and administration
Six-week-old male SPE mice are selected, the mice are purchased from Tian Du biotechnology Co Ltd in Changsha, weight is 20 g+/-2 g, after the mice are fed for 2 weeks in an adaptive mode, the mice are randomly divided into six groups, twelve mice in each group are respectively marked as a control group, a constipation model group, a bisacodyl positive drug group, JYLR-985 metatuples, an alternative strain 2 metatuple and an alternative strain 3 metatuple. The six groups of mice were continuously dosed for 15 days by the following method: control group: each mouse is irrigated with 0.2 mL sterile physiological saline every day; constipation model group, bisacodyl positive drug group, JYLR-985 metagroup, alternative strain 2 metagroup and alternative strain 3 metagroup: each mouse was perfused with 0.2 mL sterile normal saline +10mg/kg mouse body weight per day of clozapine and a drug-induced constipation model was established. After 15 days, the mice were successfully modelled.
Mice with successful modeling were dosed continuously for four weeks as follows, with free water during dosing:
control group: each mouse is irrigated with 0.2 mL sterile physiological saline every day;
constipation model group: each mouse is irrigated with 0.2 mL sterile physiological saline every day;
bisacodyl positive drug group: each mouse is irrigated with 0.2 mL sterile normal saline plus 100mg/kg of bisacodyl per day;
JYLR-985 metatuple: each mouse is irrigated with 0.2 mL sterile normal saline +100mg/kg of post-natal powder of lactobacillus rhamnosus JYLR-985 of mouse body weight per day;
Alternative strain 2 metatuple: each mouse is irrigated with 0.2 mL sterile normal saline plus 100mg/kg of candidate strain 2 metapowder of mouse body weight every day;
Alternative strain 3 metatuple: each mouse was perfused daily with 0.2 mL sterile normal saline+100 mg/kg of strain 3 candidate metapowder of mouse body weight.
2. Detection of basic index of constipation
Mice of the control group, constipation model group, bisacodyl positive drug group, JYLR-985 metagroup, alternative strain 2 metagroup and alternative strain 3 metagroup are fasted overnight on the same day after last administration, and are free to drink water. Each group of mice was perfused with 0.2mL of magenta solution after 12h of fasting, and each mice was fed in a single cage, and the first time of defecation, the fecal water content, and the number of fecal particles in 6h were counted, and the statistical results are shown in table 2.
Table 2 statistics of the first time to discharge red stool, the stool moisture content, and the stool grain number in 6h for each group of mice (n=12, x±s)
Note that: # P <0.05 compared to control; * P <0.05 compared to constipation model group; NC Compared with the bisacodyl positive drug group, the bisacodyl positive drug has no significant difference
As can be seen from table 2, the first time of the red stool discharge of the mice in the constipation model group was significantly prolonged compared with the control group, which proves that the modeling of the mice in the constipation model group was successful. The mice of JYLR-985 metagroups had significantly shorter time to first-time bowel movement than the constipation model group, while the mice of the alternative strain 2 metagroups and the alternative strain 3 metagroups had no significant difference in time to first-time bowel movement than the constipation model group. The result shows that the post-generated metaflour of the lactobacillus rhamnosus JYLR-985 can obviously shorten the first defecation time of the constipation mice.
The number of fecal particles in 6h of mice in the constipation model group was significantly reduced compared to the control group. The number of fecal particles in mice of JYLR-985 metazoan groups is obviously increased compared with a constipation model group, which shows that JYLR-985 metazoan powder has the effect of promoting defecation of constipation mice. The fecal moisture content of mice in the constipation model group was significantly reduced compared to the control group. The water content of the feces of mice with JYLR-985 metatuple is obviously improved compared with a constipation model group, and the metapowder of the surface lactobacillus rhamnosus JYLR-985 can effectively improve the water content of the feces.
Cutting off the abdominal cavity of each group of mice to dissect, taking out the gastrointestinal tract completely, placing on a photographic background plate, and measuring the advancing distance of the front end of the red solution and the total length of the small intestine by using a metric ruler, wherein the total length of the small intestine is the total length from the pylorus to the ileocecum. The small intestine thrust rate was calculated according to the following formula:
Small intestine propulsion = distance forward of magenta solution/pylorus to ileocecum total length x 100%;
the small intestine propulsion rates of the mice in each group are shown in Table 3.
Table 3 small intestine propulsive rate (n=12, x±s) for each group of mice
Note that: # P <0.05 compared to control; * P <0.05 compared to constipation model group; NC Compared with the bisacodyl positive drug group, the bisacodyl positive drug has no significant difference
As can be seen from table 3, the mice in the constipation model group had significantly lower intestinal thrust than the control group; the small intestine propulsion rate of mice with JYLR-985 metatuple and bisacodyl positive drug group is obviously higher than that of a constipation model group, and the result shows that the metapowder of the lactobacillus rhamnosus JYLR-985 can effectively promote intestinal peristalsis and is beneficial to defecation.
3. Detection of neurotransmitters and neuropeptides in serum and colon tissues
The serum samples and colon samples of each group of mice were tested for the amounts of substance P, motilin and 5-hydroxytryptamine by ELISA, and the test results are shown in tables 4 and 5.
Table 4 content of substance P, motilin and 5-hydroxytryptamine in serum samples of mice of each group (n=12, x±s)
Note that: # P <0.05 compared to control; * P <0.05 compared to constipation model group;
Table 5 content of substance P, motilin and 5-hydroxytryptamine in colon samples of mice of each group (n=12, x±s)
Note that: # P <0.05 compared to control; * P <0.05 compared to constipation model group;
From tables 4 and 5, it can be seen that the content of substance P, motilin and 5-hydroxytryptamine in serum samples and colon samples of mice of JYLR-985 metatuple is significantly higher than that of constipation model group, alternative strain 2 metatuple and alternative strain 3 metatuple, indicating that the metapowder of lactobacillus rhamnosus JYLR-985 can increase the level of substance P, motilin and 5-hydroxytryptamine in serum and colon tissues of mice, stimulate contraction of colon smooth muscle, increase intestinal motility frequency, and thus relieve constipation.
4. Detection of colonic water metabolism
The expression levels of mRNA of Vasoactive Intestinal Peptide (VIP), AQP2 and AQP3 in the colon of each group of mice were tested by RT-qPCR and WB methods, and the test results are shown in Table 6.
Table 6 expression levels of VIP, AQP2 and AQP3 mRNA in the colon of each group of mice (n=12, x±s)
Note that: # P <0.05 compared to control; * P <0.05 compared to constipation model group;
As can be seen from table 6, the expression levels of VIP, AQP2 and AQP3 mRNA in the colon of mice in the constipation model group were significantly higher than that in the control group, indicating that clozapine induced an increase in the expression levels of VIP, AQP2 and AQP3 mRNA in the colon of mice. And the expression level of the mRNA of VIP, AQP2 and AQP3 in the colon of the mice of JYLR-985 metagroup is obviously lower than that of constipation model group, candidate strain 2 metagroup and candidate strain 3 metagroup, and is close to the control group and bisacodyl positive medicine group, which shows that the metapowder of the lactobacillus rhamnosus JYLR-985 can regulate the expression level of the mouse aquaporin, thereby relieving constipation.
Example 3 prevention of Constipation by means of an anti-psychotic drug from the metapowder of Lactobacillus rhamnosus JYLR-985
1. Mouse molding and administration
Six week old male SPF grade mice were selected and purchased from Gekko Biotechnology Co., ltd, weight 19+ -3 g, and after 2 weeks of adaptive feeding, the mice were randomly divided into four groups of twelve mice, each group, and the six groups of mice were labeled as a second control group, a second constipation model group, a second JYLR-985 metagroup, and a JYLR-985 metagroup+clozapine group, respectively. The four groups of mice were continuously dosed for 15 days by the following method: second control group: each mouse is irrigated with 0.2mL sterile physiological saline every day; a second constipation model group: each mouse is irrigated with 0.2mL of sterile physiological saline plus 10mg/kg of clozapine per day per mouse body weight; second JYLR-985 metatuple: each mouse is irrigated with 0.2mL sterile normal saline +100mg/kg of post-natal powder of lactobacillus rhamnosus JYLR-985 of mouse body weight per day; JYLR-985 metagen+clozapine group: each mouse is irrigated with 0.1mL sterile normal saline +100mg/kg mouse body weight of post-natal powder of Lactobacillus rhamnosus JYLR-985 and 0.1mL sterile normal saline +10mg/kg mouse body weight of clozapine daily,
Mice were free to drink water during dosing.
2. Detection of basic index of constipation
Mice of the second control group, the second constipation model group, the second JYLR-985 metazoan group and the JYLR-985 metazoan+clozapine group were fasted overnight on the day after last dose, and were free to drink water. Each group of mice was perfused with 0.2mL of magenta solution after 12h of fasting, and each mice was fed in a single cage, and the first time of defecation, the fecal water content, the number of fecal particles in 6h and the small intestine propulsion rate of the mice were counted, and the statistical results are shown in table 7.
TABLE 7 influence of metazoan on the defecation time, the number of faeces particles and the water content of faeces in mice (n=12, x.+ -.s)
Note that: # P <0.05 compared to the second control group; * P <0.05 compared to the second constipation model group;
As can be seen from Table 7, the first time to flush the red feces was the shortest in mice of the second JYLR-985 metazoan group, significantly lower than in the second constipation model group. Compared with a second control group, the mice small intestine propulsion rate of JYLR-985 metagen and clozapine group has no obvious difference, but is obviously higher than that of a second constipation model group, which shows that the metagen powder of the lactobacillus rhamnosus JYLR-985 can promote intestinal peristalsis and prevent constipation.
3. Detection of neurotransmitters and neuropeptides in serum and colon tissues
The serum samples and colon samples of each group of mice were tested for the amounts of substance P, motilin and 5-hydroxytryptamine by ELISA, and the test results are shown in tables 8 and 9.
Table 8 content of substance P, motilin and 5-hydroxytryptamine in serum samples of mice of each group (n=12, x.+ -.s)
Note that: # P <0.05 compared to the second control group; * P <0.05 compared to the second constipation model group;
Table 9 content of substance P, motilin and 5-hydroxytryptamine in colon samples of mice of each group (n=12, x±s)
Note that: # P <0.05 compared to the second control group; * P <0.05 compared to the second constipation model group;
As can be seen from tables 8 and 9, the second JYLR-985 metazoan group of mice had the highest levels of substance P, motilin and 5-hydroxytryptamine in the serum samples and colon samples, while the second constipation model group of mice had the lowest levels of substance P, motilin and 5-hydroxytryptamine in the serum samples and colon samples. Compared with a serum sample and a colon sample of a mouse of a second constipation model group, the substances P of the mice of the JYLR-985 metagroup and the clozapine group are respectively improved by 58.5 percent and 14.0 percent; motilin levels were increased by 69.5% and 37.1%, respectively; the 5-hydroxytryptamine levels were increased by 75.2% and 49.4%, respectively. The results show that the post-metapowder of Lactobacillus rhamnosus JYLR-985 can promote the level of excitatory neurotransmitter, increase gastrointestinal motility, promote defecation, and prevent constipation.
Example 4 improving Effect of post-metaflour of Lactobacillus rhamnosus JYLR-985 on psychotropic drug-induced damage to the body
1. Mouse molding and administration
Six-week-old male SPF-grade mice are selected, purchased from Gekko Biotechnology Co., ltd, and fed with 20 g.+ -.2 g weight, and after 2 weeks of adaptive feeding, the mice were randomly divided into five groups of twelve mice, each group of five mice being labeled as a third control group, a third constipation model group, a JYLR-985 metazoan low dose group, a JYLR-985 metazoan medium dose group, and a JYLR-985 metazoan high dose group, respectively. The five groups of mice were continuously dosed for two weeks by the method of gavage as follows: third control group: each mouse is irrigated with 0.2mL sterile physiological saline every day; a third constipation model group, JYLR-985 metazoan low dose group, JYLR-985 metazoan medium dose group, and JYLR-985 metazoan high dose group: each mouse was perfused with 0.2mL of sterile physiological saline +10mg/kg of mouse body weight per day of clozapine, and a drug-induced constipation model was established. After two weeks the mice were successfully modelled.
Mice with successful modeling were dosed continuously for 30 days as follows, with free water during dosing:
Third control group: each mouse is irrigated with 0.2 mL sterile physiological saline every day;
third constipation model group: each mouse is irrigated with 0.2 mL sterile physiological saline every day;
JYLR-985 metasew low dose group: each mouse is irrigated with 0.2 mL sterile normal saline +100mg/kg of post-natal powder of lactobacillus rhamnosus JYLR-985 of mouse body weight per day;
JYLR-985 metazoan dose group: each mouse is irrigated with 0.2 mL sterile normal saline +200mg/kg of post-natal powder of lactobacillus rhamnosus JYLR-985 of mouse body weight per day;
JYLR-985 metasequoyins high dose group: each mouse was perfused daily with 0.2 mL sterile normal saline+300 mg/kg mouse body weight of the post-natal powder of Lactobacillus rhamnosus JYLR-985.
2. Effect of post-metaflour of Lactobacillus rhamnosus JYLR-985 on constipation mice inflammation level
The content of IL-1. Beta. And TNF-alpha. In colon tissue of mice was measured by ELISA, and the measurement results are shown in Table 10.
TABLE 10 IL-1 beta and TNF-alpha content in colon tissue of mice
# P <0.05 compared to the third control group; * P <0.05 compared to the third constipation model group; * P <0.01 compared to the third constipation model group.
As can be seen from table 10, the contents of 1L-1 β and TNF- α in the colon tissue of the mice of the third constipation model group were significantly increased compared to the third control group, indicating that there was a significant change in the inflammatory factor level in the colon tissue of the mice after the post-baking powder lavage of lactobacillus rhamnosus JYLR-985, and the inflammatory factor decrease level was more significant as the post-baking powder lavage dose of lactobacillus rhamnosus JYLR-985 was increased. This suggests that the post-metapowder of Lactobacillus rhamnosus JYLR-985 can reduce the release of inflammatory factors and alleviate the mechanism of intestinal injury.
3. Effect of post-metaflour of Lactobacillus rhamnosus JYLR-985 on antioxidant capacity in colon tissue of constipation mice
The amounts of superoxide dismutase (SOD), reduced glutathione (GSH-Px), and Malondialdehyde (MDA) in the serum of each group of mice were measured using corresponding kits, all purchased from Beijing Soy Corp, and the measurement results are shown in Table 11.
TABLE 11 content of SOD, GSH-Px, MDA in serum of mice of each group
# P <0.05 compared to the third control group, p <0.05 compared to the third constipation model group; * P <0.01 compared to the third constipation model group
As can be seen from table 11, the serum content of SOD and GSH in the mice of the third constipation model group was significantly reduced, and the MAD content was significantly increased, compared to the third control group; compared with the third constipation model group, the levels of SOD and GSH-Px in the serum of mice in the JYLR-985 metazoan low-dose group and the JYLR-985 metazoan medium-dose group are obviously increased, and the levels of SOD and GSH-Px in the serum of mice in the JYLR-985 metazoan high-dose group are extremely obviously increased. The result shows that the metapowder of the lactobacillus rhamnosus JYLR-985 has good treatment effect on oxidative damage caused by constipation.
Although the present invention has been described in detail in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (10)
1. The lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) JYLR-985 for relieving constipation of the medicine is characterized in that the lactobacillus rhamnosus JYLR-985 is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 1 month and 18 days of 2024, and the preservation address is North Star Xili No. 1, 3 in the Chaoyang district of Beijing city, and the preservation number is CGMCC No.29658.
2. The post-cursor preparation of lactobacillus rhamnosus JYLR-985 for relieving constipation of a medicament according to claim 1, wherein the post-cursor preparation comprises post-cursor powder of lactobacillus rhamnosus JYLR-985.
3. The post-cursor preparation of lactobacillus rhamnosus JYLR-985 as claimed in claim 2, wherein the preparation method of the post-cursor powder of lactobacillus rhamnosus JYLR-985 comprises the following steps:
step one: preparing a fermentation broth of lactobacillus rhamnosus JYLR-985;
step two: centrifuging the fermentation liquor to obtain bacterial mud;
Step three: dissolving the bacterial mud in distilled water for sterilization to obtain an inactivated bacterial liquid;
step four: freeze-drying the inactivated bacterial liquid to obtain inactivated bacterial powder;
Step five: and diluting the inactivated bacterial powder by using maltodextrin to obtain the metapowder of the lactobacillus rhamnosus JYLR-985.
4. The post-cursor preparation of lactobacillus rhamnosus JYLR-985 of claim 3, wherein in step one, the fermentation broth of lactobacillus rhamnosus JYLR-985 is prepared as follows: single colonies of Lactobacillus rhamnosus JYLR-985 were inoculated into liquid medium and fermented at 37℃for 24h.
5. The metagen preparation of JYLR-985 of lactobacillus rhamnosus as claimed in claim 4 wherein the liquid medium comprises 10g peptone, 5g beef powder, 5g sodium acetate trihydrate, 2g dipotassium phosphate heptahydrate, 1mL tween 80, 0.05g manganese sulfate tetrahydrate, 2g tri-ammonium citrate, 20g glucose, 0.2g magnesium sulfate heptahydrate and 1000mL distilled water as raw materials.
6. The post-cursor preparation of lactobacillus rhamnosus JYLR-985 of claim 3, wherein in step two, the viable count of the bacterial sludge is 1.0x10 10 cfu/g.
7. The post-natal preparation of lactobacillus rhamnosus JYLR-985 of claim 3, wherein in the third step, the mass ratio of the bacterial sludge to distilled water is 1:3, the sterilization temperature is 115 ℃, and the sterilization time is 30min.
8. The post-cursor preparation of lactobacillus rhamnosus JYLR-985 of claim 3, wherein in step five, the number of cells of the post-cursor powder is 1.0x10 9 cfu/g.
9. Use of the metagen preparation of lactobacillus rhamnosus JYLR-985 according to any one of claims 2-8 for the preparation of a medicament for the prevention and/or treatment of antipsychotic drug-induced constipation.
10. The use according to claim 9, wherein the antipsychotic agent is clozapine.
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