CN115992075A - Lactobacillus rhamnosus capable of inhibiting pathogenic bacteria and promoting defecation and application thereof - Google Patents
Lactobacillus rhamnosus capable of inhibiting pathogenic bacteria and promoting defecation and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses lactobacillus rhamnosus LRJ-1 capable of inhibiting pathogenic bacteria and promoting defecation and application thereof, wherein the lactobacillus rhamnosus is lactobacillus rhamnosus (Lactobacillus rhamnosus), the strain number of the lactobacillus rhamnosus is LRJ-1, and the registration number of the lactobacillus rhamnosus in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No.25104. The rhamnose milk lever and the product containing the same can have the following effects: 1) Improving defecation promoting capability; 2) Increasing motilin levels; 3) Reducing somatostatin levels; 4) Reducing inflammatory cell infiltration of colon.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus rhamnosus LRJ-1 capable of inhibiting pathogenic bacteria and promoting defecation and application thereof.
Background
The functional pathological changes of the gastrointestinal tract are caused by the control disorder of the nervous and muscle tissues of the gastrointestinal tract, and are mainly represented by gastrointestinal symptoms such as pantothenic acid, abdominal distension, constipation, diarrhea and the like, and the common diseases such as irritable bowel syndrome, functional dyspepsia and the like which are clinically common diseases are not structurally or biochemically changed in routine examination. The gastrointestinal motility decrease is one of the main manifestations of functional lesions of the gastrointestinal tract, and symptoms such as abdominal distension, constipation and the like can occur clinically.
Constipation refers to normal changes in bowel movement morphology, reduced number of times of bowel movement, difficulty in bowel movement, etc., and is a clinically common complex symptom, and it is known that constipation has a prevalence of up to 27% in the population. Chronic constipation is overlooked by people in the past quite long period, and in recent years, the prevalence of constipation is increased year by year due to unreasonable diet structure, sedentary sitting, reduced physical labor and the like, and patients often have a lot of trouble due to complex disease causes and repeated lingering disease courses, which seriously affect the life quality of people. More and more studies have shown that healthy intestinal flora helps promote intestinal motility, and that the lack of beneficial bacteria can lead to intestinal dysfunction and even constipation. Lactobacillus is a beneficial symbiotic bacteria which can be planted in human intestinal tracts, and can grow and be planted in human bodies by utilizing nutrient components in the intestinal tracts, and the beneficial effects on organisms are achieved by inhibiting pathogenic bacteria or conditional pathogenic bacteria to regulate intestinal flora, promote intestinal microecological balance. Intestinal flora can affect the enteroendocrine cells of the body, and secreted peptide substances such as motilin play an important role in regulating intestinal motility, absorption and secretion. Somatostatin is a kind of brain-intestine peptide, and is widely distributed in central nervous system and gastrointestinal tract, and can strongly inhibit exocrine of digestive tract glands such as gastrointestinal pancreas, etc., and inhibit gastrointestinal peristalsis. Gamma-aminobutyric acid is an important neurotransmitter, and researches show that the gamma-aminobutyric acid has the effects of aiding sleep, stabilizing, resisting convulsion, improving senile dementia, enhancing brain activity nerve cells and the like. The multifunctional probiotics which can inhibit pathogenic bacteria, promote gastrointestinal peristalsis, promote sleep and the like are provided to relieve or treat constipation and promote human health.
Disclosure of Invention
The invention aims to provide lactobacillus rhamnosus LRJ-1 capable of inhibiting pathogenic bacteria and promoting defecation and application thereof. The lactobacillus rhamnosus is lactobacillus rhamnosus (Lactobacillus rhamnosus), the strain number of the lactobacillus rhamnosus is LRJ-1, and the registration number of the lactobacillus rhamnosus in the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No.25104.
The application of the lactobacillus rhamnosus in any of the following should also be within the scope of the present invention:
1) Preparing gamma-aminobutyric acid;
2) Inhibiting salmonella and escherichia coli;
3) Improving gastrointestinal motility.
Wherein the improving gastrointestinal motility is at least one of:
1) Improving defecation promoting capability;
2) Increasing motilin levels;
3) Reducing somatostatin levels;
4) Reducing inflammatory cell infiltration of colon.
The invention provides a microbial inoculum, which is characterized in that: the microbial inoculum contains the lactobacillus rhamnosus and/or metabolites of the lactobacillus rhamnosus, wherein the metabolites refer to a culture solution mixture of the lactobacillus rhamnosus cultured in the culture medium or other similar culture media.
The application of the microbial inoculum in any one of the following should also be within the scope of the invention:
1) Preparing gamma-aminobutyric acid;
2) Inhibiting salmonella and escherichia coli;
3) Improving gastrointestinal motility.
Wherein the improving gastrointestinal motility is at least one of:
1) Improving defecation promoting capability;
2) Increasing motilin levels;
3) Reducing somatostatin levels;
4) Reducing inflammatory cell infiltration of colon.
The invention provides a composition for preparing gamma-aminobutyric acid, which is characterized in that the product comprises lactobacillus rhamnosus and a culture medium.
The invention provides a medicine for improving gastrointestinal motility, which comprises the lactobacillus rhamnosus or the microbial inoculum.
The improving gastrointestinal motility is at least one of the following:
1) Improving defecation promoting capability;
2) Increasing motilin levels;
3) Reducing somatostatin levels;
4) Reducing inflammatory cell infiltration of colon.
The rhamnose milk lever and the product containing the same can have the following effects: 1) Improving defecation promoting capability; 2) Increasing motilin levels; 3) Reducing somatostatin levels; 4) Reducing inflammatory cell infiltration of colon.
Preservation description
Strain name: lactobacillus rhamnosus
Latin name: lactobacillus rhamnosus
Strain number: LRJ-1
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Preservation date: 2022, 7, 27
Accession numbers of the preservation center: CGMCC No.25104.
Drawings
FIG. 1 shows the culture of Lactobacillus rhamnosus LRJ-1 and Bacillus cereus on blood plates.
FIG. 2 shows gastrointestinal tolerance of Lactobacillus rhamnosus LRJ-1.
FIG. 3 shows the production of gamma-aminobutyric acid by Lactobacillus rhamnosus LRJ-1 and Lactobacillus rhamnosus LGG.
FIG. 4 shows the antibacterial effect of Lactobacillus rhamnosus LRJ-1 against Salmonella and Escherichia coli.
FIG. 5 is the effect of Lactobacillus rhamnosus LRJ-1 on the grain number, weight and humidity of mouse faeces.
FIG. 6 is the effect of Lactobacillus rhamnosus LRJ-1 on blood parameters of mice.
FIG. 7 is the effect of Lactobacillus rhamnosus LRJ-1 on constipation in mice colon tissue morphology.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
1. Isolation and identification of lactobacillus rhamnosus Lactobacillus rhamnosus LRJ-1
Lactobacillus rhamnosus LRJ-1 is separated from healthy adult feces, grows well in MRS culture medium, is identified by 16SrRNA complete sequence sequencing, and is finally identified and separated to obtain lactobacillus rhamnosus, which is named lactobacillus rhamnosus (Lactobacillus rhamnosus) LRJ-1. The 16S rRNA sequence of lactobacillus rhamnosus LRJ-1 is shown as a sequence 1 in a sequence table.
2. Preservation of lactobacillus rhamnosus Lactobacillus rhamnosus LRJ-1
Lactobacillus rhamnosus (Lactobacillus rhamnosus) LRJ-1 has been deposited in the general microbiological center of the China Committee for culture Collection of microorganisms, with a deposit number of cgmccno.25104, at 7.27 of 2022.
Example 2
1. Haemolytic Activity of lactobacillus rhamnosus LRJ-1
Lactobacillus rhamnosus LRJ-1 and the positive control bacteria of example 1 were inoculated as streaks on MRS plates containing 4% defibrinated sheep blood (TXWYX 100, beijing RunZuokang Biotechnology Co., ltd.) with Bacillus cereus having hemolysis as positive control bacteria, and cultured at 37℃for 24 hours, and the results are shown in FIG. 1, and as can be seen from FIG. 1, only the positive control strain has hemolysis and lactobacillus rhamnosus LRJ-1 has no hemolysis.
2. Sugar utilization characteristics of lactobacillus rhamnosus LRJ-1
The sugar utilization characteristics of lactobacillus rhamnosus LRJ-1 were tested using the API 50CHL kit (cat No. 50410, biomeriuex), and the results are shown in table 1, and it can be seen from table 1 that lactobacillus rhamnosus LRJ-1 can be metabolized using 30 kinds of sugars such as ribose, galactose, glucose, fructose, etc.
TABLE 1 sugar utilization Properties of Lactobacillus rhamnosus LRJ-1
+: has activity; -: inactive; w: weak activity
3. Gastrointestinal fluid tolerance of lactobacillus rhamnosus LRJ-1 after incubating lactobacillus rhamnosus LRJ-1 with an initial viable count of 8.76±0.02 (lg CFU/mL) in artificial gastric fluid, intestinal fluid and intestinal fluid, respectively, for 2 hours, the results are shown in fig. 2, and as can be seen from fig. 2, the viable count of lactobacillus rhamnosus LRJ-1 after incubating in artificial gastric fluid (cat No. CZ0211, league), intestinal fluid (cat No. CZ0201, league) and colonic fluid (cat No. CZ0206, league) for 2 hours, respectively, is maintained at 6.52±0.02 (lg CFU/mL), 7.87±0.04 (lg CFU/mL), and 7.81±0.09 (lg CFU/mL). Indicating that lactobacillus rhamnosus LRJ-1 has certain gastrointestinal fluid tolerance.
4. Lactobacillus rhamnosus LRJ-1 has the ability to produce gamma-aminobutyric acid (GABA)
And (3) quantitatively determining gamma-aminobutyric acid by adopting a colorimetric method. Preparing gamma-aminobutyric acid standard solution with the concentration of 0.1-0.6 g/L, and respectively measuring the light absorption values of gamma-aminobutyric acid standard solutions with different concentrations at 640nm wavelength by using a spectrophotometer. And (3) preparing a GABA standard curve by taking an OD640 nm absorbance value as an ordinate and the concentration of gamma-aminobutyric acid standard solution as an abscissa. Lactobacillus rhamnosus LRJ-1 monoclonal is cultured in 3mL MRS culture medium, after fermentation for 24 hours at 37 ℃, bacterial liquid 10000r/min is centrifugated for 5min, 1mL of supernatant is taken, 0.4mL of dilution is taken after dilution for 10 times, OD640 nm absorbance value is measured, and the content of gamma-aminobutyric acid produced by LRJ-1 is calculated by using a standard curve.
As a result, as shown in FIG. 3, it was revealed from FIG. 3 that Lactobacillus rhamnosus LRJ-1 produced gamma-aminobutyric acid, and that under laboratory conditions (monoclonal in MRS medium, cultured at 37℃for 24 hours), the yield of gamma-aminobutyric acid was 1.78.+ -. 0.59mg/mL, and that of the control strain Lactobacillus rhamnosus LGG (Thirty years of Lactobacillus Rhamnosus GG: areview, J Clin gateway, lucio Capurso,2019Mar;53Suppl 1:S1-S41) was 0.88.+ -. 0.11mg/mL.
5. Antibacterial activity and antibacterial spectrum of lactobacillus rhamnosus LRJ-1
1) Lactobacillus rhamnosus LRJ-1 was activated and inoculated onto MRS medium at an inoculum size of 1%, and after 24 hours of culture at 37 ℃ the supernatant of the culture broth was taken.
2) Salmonella typhimurium 14028 (Yuehua Jiao, han Yang, nditange Shigwedha, shuang Zhang, fei Liu, lanwei Zhang. Probiological Effects and Metabolic Products of Enterococcus faecalis LD with resolution capability, food. 2022Feb20, respectively; 11 (4) 606) and toxin-producing E.coli K88 (Wang T) # ,Teng K # Liu G, liu Y, zhang J, zhang X, zhang M, tao Y, zhong J, lactobacillus reuteri HCM, 2, protects mice against Enterotoxigenic Escherichia coli through modulation of gut, microbiota 2018, scientific reports, nov 30;8 (1) 17485.doi:10.1038/s 41598-018-35702-y.) into LB medium (1X 10) 6 CFU/mL), an indicator fungus plate was prepared, and the plate was perforated, 50ul of the supernatant of LRJ-1 was added to the well, and after incubation at 37℃for 24 hours, the presence or absence of a zone of inhibition was observed. Sterile medium served as negative control. The results are shown in FIG. 4.
As can be seen from FIG. 4, the culture supernatant of Lactobacillus rhamnosus LRJ-1 has a certain inhibition ability on the growth of pathogenic Salmonella typhimurium 14028 and toxin-producing E.coli K88.
Example 3
Grouping treatment of 1 mice
1) 27 SPF mice (Beijing Vitrendy laboratory animal technologies Co., female, 8 weeks old, 20-22 g) were divided into 3 groups of 9.
2) 9 mice of the Control group were perfused daily with phosphate buffered saline (PBS, 100. Mu.L) in addition to normal culture on days 1 to 21.
3) 9 mice of the LOP group were subjected to gastric lavage treatment with 100. Mu.L of Loperamide (LOP) per day at a dose of 10mg/kg/d, in addition to normal culture for the first to seven days. On days 8-21, each mouse was perfused daily with PBS (100. Mu.L) in addition to normal culture.
4) 9 mice of LRJ-1 group were treated with Loperamide (LOP) at 10mg/kg/d per mouse, except for the first to seven days of normal culture; on days 8-21, each mouse was used for 5X 10 in addition to normal culture 8 The CFU/L/D lactobacillus rhamnosus LRJ-1 was subjected to gastric lavage.
2. Defecation promoting ability of lactobacillus rhamnosus LRJ-1
On days 14 and 21, the faeces of three groups of mice were collected and the particle count, weight, humidity of the faeces of the mice and the feed intake of the mice were counted. The results are shown in fig. 5, and on day 14, the LOP group significantly reduced fecal pellet count, weight and moisture compared to Control, indicating that LOP induced constipation in mice. LOP does not affect the feed intake of mice. While intervention with lactobacillus rhamnosus LRJ-1 significantly increased fecal pellet number (P < 0.05), weight (P < 0.01) and humidity (P < 0.05) in constipation mice. On day 21, intervention by lactobacillus rhamnosus LRJ-1 significantly increased the feed intake of constipation mice (P < 0.001). These results demonstrate that LRJ-1 can promote defecation and ingestion in mice, and has a certain effect of relieving constipation in mice.
3. Effect of Lactobacillus rhamnosus LRJ-1 on blood parameters of mice
On day 21, serum was collected from three groups of mice, and the motilin and somatostatin contents in the blood were analyzed, and the results are shown in fig. 6. Motilin (MTL) is a short peptide secreted by the small intestine and is responsible for improving gastrointestinal motility or peristalsis, normal filling and emptying of the gastrointestinal system, and stimulation and response to food intake and hunger. Somatostatin (SS) is a brain-intestinal peptide that strongly inhibits gastric acid secretion. The results in figure 6 show that lactobacillus rhamnosus LRJ-1 significantly increases the blood MTL level (P < 0.05) and decreases the SS content (P < 0.01) in constipation mice. This suggests that LRJ-1 relief of constipation in mice is associated with its increase in MTL and decrease in SS.
4. Effect of Lactobacillus rhamnosus LRJ-1 on constipation mouse colon tissue morphology
On day 21, colon tissues of three groups of mice were collected, and the structure of the colon tissues of the mice was examined by slicing, and the results are shown in fig. 7. As can be seen from FIG. 7, the colon tissue structure of the Control group is basically normal, the visual field can see that the epithelial cells do not necrosed and fall off, and the tissues do not see inflammatory cell infiltration. Whereas more inflammatory cell infiltration was seen in the LOP group, indicating that inflammation of the intestinal tract may be caused when LOP induces constipation. After LRJ-1 intervention, colon tissues are normal, necrosis and shedding are not found, so that LRJ-1 reduces inflammatory cell infiltration of colon tissues of constipation mice.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (9)
1. Lactobacillus rhamnosus, its characterized in that: the lactobacillus rhamnosus is lactobacillus rhamnosus (Lactobacillus rhamnosus), the strain number of the lactobacillus rhamnosus is LRJ-1, and the registration number of the lactobacillus rhamnosus in the common microorganism center of the China Committee for culture Collection of microorganisms is CGMCC No.25104.
2. Use of lactobacillus rhamnosus as claimed in claim 1 in any of the following:
1) Preparing gamma-aminobutyric acid;
2) Inhibiting salmonella and escherichia coli;
3) Improving gastrointestinal motility.
3. The use according to claim 2, wherein: the improving gastrointestinal motility is at least one of the following:
1) Improving defecation promoting capability;
2) Increasing motilin levels;
3) Reducing somatostatin levels;
4) Reducing inflammatory cell infiltration of colon.
4. The microbial inoculum is characterized in that: the microbial inoculum contains the lactobacillus rhamnosus of claim 1 and/or a metabolite of the lactobacillus rhamnosus.
5. The use of the microbial agent of claim 4 in any of the following:
1) Preparing gamma-aminobutyric acid;
2) Inhibiting salmonella and escherichia coli;
3) Improving gastrointestinal motility.
6. The use according to claim 5, wherein: the improving gastrointestinal motility is at least one of the following:
1) Improving defecation promoting capability;
2) Increasing motilin levels;
3) Reducing somatostatin levels;
4) Reducing inflammatory cell infiltration of colon.
7. A composition for preparing gamma-aminobutyric acid, characterized in that the product comprises lactobacillus rhamnosus as claimed in claim 1 and a medium.
8. A medicament for improving gastrointestinal motility, the medicament comprising the lactobacillus rhamnosus of claim 1 or the microbial agent of claim 4.
9. The pharmaceutical product of claim 8, wherein the gastrointestinal motility improvement is at least one of:
1) Improving defecation promoting capability;
2) Increasing motilin levels;
3) Reducing somatostatin levels;
4) Reducing inflammatory cell infiltration of colon.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116676225A (en) * | 2023-05-23 | 2023-09-01 | 河北源民生物科技有限公司 | Lactobacillus rhamnosus LR-28 strain with nerve soothing and sleep aiding effects, fermentation product, hypnotic fungus group mixture and application |
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| CN117586927A (en) * | 2024-01-18 | 2024-02-23 | 山东中科嘉亿生物工程有限公司 | Lactobacillus rhamnosus JYLR-127 for improving difficult defecation after fracture, microbial inoculum and application thereof |
| CN117586927B (en) * | 2024-01-18 | 2024-04-23 | 山东中科嘉亿生物工程有限公司 | Lactobacillus rhamnosus JYLR-127 for improving difficult defecation after fracture, microbial inoculum and application thereof |
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