CN117264814A - Lactobacillus rhamnosus with effects of preventing and treating digestive tract diseases - Google Patents
Lactobacillus rhamnosus with effects of preventing and treating digestive tract diseases Download PDFInfo
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- CN117264814A CN117264814A CN202311155674.4A CN202311155674A CN117264814A CN 117264814 A CN117264814 A CN 117264814A CN 202311155674 A CN202311155674 A CN 202311155674A CN 117264814 A CN117264814 A CN 117264814A
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Abstract
The invention provides lactobacillus rhamnosus with a preventive and therapeutic effect on digestive tract diseases, and the preservation number of the lactobacillus rhamnosus is CGMCC No.27365. The strain has tolerance to gastric acid and bile salts, and can relieve diarrhea of mice induced by antibiotics. The strain has a preventive effect on mice with DSS induced colonitis. The strain can also relieve AOM/DSS induced colon cancer mice.
Description
Technical Field
The invention belongs to the technical field of microbial screening, and particularly relates to lactobacillus rhamnosus with a preventive and therapeutic effect on digestive tract diseases.
Background
The digestive system absorbs nutrients such as protein and amino acid in the food after the food is taken, maintains the health of the body, and has the functions of promoting digestion and absorption and running the food. Digestive tract diseases usually show symptoms such as abdominal pain, diarrhea and the like, and severe diseases can cause colonitis and further cause colon cancer.
At present, antibiotics are mainly used for clinically treating diarrhea, but abuse of antibiotics can lead to removal of beneficial flora, cause dysbacteriosis in intestinal tracts and threaten human health. The traditional clinical treatment medicine for colonitis mainly comprises 5-aminosalicylic acid, corticosteroid, immunosuppressant, biological agent and the like, and can cause side effects such as anaphylactic reaction, liver injury and the like after long-term use. The operation treatment is a main treatment means of colon cancer, but chemical and radiation therapy causes great harm to human bodies, anticancer drugs used clinically have certain toxicity and drug resistance, and have great toxic and side effects, thus being easy to cause discomfort to patients.
The probiotics are beneficial microorganisms and have the functions of regulating intestinal flora balance, promoting digestion and absorption, resisting fatigue, resisting diabetes, resisting cancer, relieving anxiety and the like.
The invention researches the functions of the lactobacillus rhamnosus and the digestive tract diseases such as diarrhea, colonitis and colon cancer through a mouse model by separating the lactobacillus rhamnosus AFY05 from the Xinjiang traditional fermented yogurt.
Disclosure of Invention
The invention aims to provide lactobacillus rhamnosus with a preventive and therapeutic effect on digestive tract diseases, which can be used for preparing products for preventing and relieving digestive tract diseases.
The preservation number of the strain of the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) AFY05 provided by the invention is CGMCC No.27365, and the preservation date is 2023, 05 and 17; the preservation unit is China general microbiological culture Collection center, and the address is North Chen Xili No. 1 and 3 in the Chaoyang area of Beijing city.
The lactobacillus rhamnosus AFY05 strain provided by the invention can be used for preventing and treating digestive tract diseases.
The digestive tract diseases, as one of the examples, mainly include diarrhea, colitis and colon cancer.
In a further aspect, the present invention provides a bacterial preparation for preventing and treating digestive tract diseases;
the bacterial preparation contains the live bacteria of the Lactobacillus rhamnosus AFY05 strain or the fermentation product thereof.
The screened lactobacillus rhamnosus AFY05 strain is proved by a mouse model test to be capable of relieving diarrhea caused by antibiotics, mouse colonitis induced by DSS and colon cancer induced by AOM/DSS, and is a beneficial bacterium for treating digestive tract diseases; the strain can obviously reduce the incidence rate of digestive tract diseases.
Drawings
Fig. 1: figure of AFY05 tolerance to artificial gastric juice and bile salts;
fig. 2: a water content diagram of the mouse feces;
fig. 3: average feeding/water intake profile for mice, panel a for food intake profile, panel B for water intake profile;
fig. 4: intestinal tissue section;
fig. 5: an effect diagram of AFY05 on the level of cell inflammatory factors IL-6 and IL-1 beta in mouse serum;
fig. 6: an effect map of AFY05 on diarrhea mice 5-HT, MDA;
fig. 7: mRNA expression levels in the colon of mice;
fig. 8: body weight trend graph of mice;
fig. 9: mouse DAI score change profile;
fig. 10: colonic length measurement map of colitis mice;
fig. 11: a plot of H & E pathology of the mouse colon;
fig. 12: colon cancer mice colon length map;
fig. 13: colon tumor count and observation diagram of mice;
fig. 14: 4 x pathology observation diagram of colon H & E staining of mice;
fig. 15: mouse colon H & E staining 10 x pathology view.
Detailed Description
The present invention will be described in detail with reference to specific embodiments and drawings.
Example 1: isolation and purification of strains
The method comprises the steps of taking natural fermented yoghourt from Altai area of Xinjiang, sucking 40mL of natural fermented yoghourt, placing the natural fermented yoghourt into a sterile centrifuge tube, placing the sterile centrifuge tube into a food sampling box, and placing the food sampling box into a refrigerator at the temperature of 4 ℃ in a laboratory for later use.
1. Isolation and identification of lactic acid bacteria
1.1 isolation and purification of lactic acid bacteria
Taking 1mL of natural fermentation yoghurt sample respectively, and carrying out 10-time gradient dilution to 10 by using sterile normal saline -6 Then take 10 -4 、10 -5 、10 -6 The bacterial solutions of 3 gradients were plated at 100. Mu.L, incubated at 37℃for 24-48h, and colony morphology was observed and recorded. And (3) picking colonies with different forms on the flat plate for streak separation, culturing at 37 ℃ for 48 hours, and then picking single colonies with different forms on the flat plate again for streak separation, and repeating the steps for 2 to 3 times until pure single colonies with consistent forms are obtained.
1.2 lactic acid bacteria DNA extraction
The purified suspected target strain is inoculated into MRS broth, and after being cultured for 18-24 hours at 37 ℃, the DNA extraction is carried out by adopting a bacterial genome DNA extraction kit. Numbering the extracted DNA, and preserving in a freezer at-20 deg.C.
1.4 PCR amplification of genomic DNA and detection by agarose gel electrophoresis
The extracted DNA was subjected to PCR amplification in which 1. Mu.L of the upstream primer 27F (5'-AGA GTT TGA TCC TGGCTC AG-3'), 1. Mu.L of the downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3'), 12.5. Mu.L of the 2 XTaq plus Buffer, and 1. Mu.L of the template DNA were used, and the system was made up to 25. Mu.L with sterile dd H2O. And sterile ultrapure water was used as a negative control instead of the template DNA. The amplification conditions were: 94 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 1min, 29 cycles in total, and finally 72℃for 5min.
Then, 5. Mu.L of the amplified product was subjected to agarose gel electrophoresis under conditions of 110V for 45min at an agarose concentration of 1.5%. The PCR products that were successfully detected were sent to beijing department biotechnology limited for sequencing, and the sequences that were successfully sequenced were aligned using the BLAST (Basic Local Alignment Search Tool) program in NCBI.
The screened strain is determined to be the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus), named AFY05 strain, and is preserved in China center for culture Collection of microorganisms (CGMCC) with the preservation number of 27365 in the year 023, month 05 and month 17.
Example 2: tolerance of AFY05 to gastric acid and bile salts
1mL of the bacterial suspension was inoculated into 9mL of artificial gastric juice, and then cultured at 37 ℃, the viable count at 0h and 3h was recorded, and the survival rate was calculated using the formula.
The liquid culture medium containing 0.0% and 0.3% of bile salt is inoculated with clean bacterial suspension according to the inoculum size of 2%, and then is cultured for 24 hours at 37 ℃, the OD value of the culture medium at 600nm is measured by an enzyme-labeling instrument (blank culture medium is used as a control), and the bile salt tolerance of the Lactobacillus rhamnosus AFY05 is calculated by using a formula.
As can be seen from FIG. 1, AFY05 has a survival rate as high as 91.85% at pH 3.0, and is well-tolerated by gastric acid. The survival rate of AFY05 in 0.3% of bile salt solution is also higher and reaches 82.25%, so that the AFY05 has good activity in bile salt, namely the AFY05 has good bile salt tolerance.
Example 3: alleviation of antibiotic-induced diarrhea in mice by Lactobacillus rhamnosus AFY05
Treatment of test animals: KM male mouse (seven weeks old)The weight is 20+/-5 g, the feeding temperature is controlled at 25+/-2 ℃, the mice are adaptively fed for one week under the condition of meeting 12 hours light/dark circulation, the KM mice are randomly divided into 5 groups according to the average weight value, and 10 of the KM mice in each group are respectively a normal group, a model group, a treatment group, a high-concentration bacteria group and a low-concentration bacteria group. From the first day, the normal group was removed, and the remaining groups were filled with lincomycin hydrochloride injection (15 mg/kg) daily in the morning and continuously with distilled water for 7 days. Live bifidobacterium tablet (21.06 mg/kg) with equal dose for stomach infusion in afternoon treatment group, and fungus height (1 multiplied by 10 9 CFU/mL), low bacterial count (1X 10) 8 CFU/mL) concentration groups were filled with bacteria of different concentrations for 14 days. The body weight, food intake and water intake of the mice were measured daily, and the feces of the mice were collected, the water content of the feces was measured, and the state of the mice was observed. On day 15, mice were fasted without water, and after 12 hours the orbital veins were bled and dissected to collect serum, small intestine and colon tissue.
3.1 Effect of Lactobacillus rhamnosus AFY05 on moisture content of mouse faeces
FIG. 2 is the fecal content results for each group of mice. The fecal moisture content was significantly higher in the diarrhea group compared to the normal group. The fecal water content of the treatment group, the high bacteria concentration group and the low bacteria concentration group is higher than that of the normal group, and the fecal water content of the three groups is obviously reduced (p < 0.05) compared with that of the diarrhea group. AFY05 was demonstrated to be effective in reducing fecal moisture in mice.
3.2 Effect of Lactobacillus rhamnosus AFY05 on the feeding intake of mice
The results in figure 3 show that the feeding water intake was significantly reduced in the diarrhea group mice compared to the normal group. The differences between the treated group and the normal group were not significant. The average feed and water intake of mice were increased after AFY05 interference compared to diarrhea group. AFY05 was demonstrated to help promote feeding and drinking in mice.
3.3 Effect of Lactobacillus rhamnosus AFY05 on mouse intestinal tissue
During observation of small intestine tissue sections (fig. 4), it was found that diarrhea group mice had edema of inner wall of intestinal tract, which was loosely arranged and disturbed, compared with normal group; intestinal mucosa epithelial cells fall off, dissolve, and cavitation phenomenon occurs in part of cells. Compared with diarrhea groups, the mice with high AFY05 and low AFY05 have relatively regular and complete intestinal epithelial cell arrangement, and the intestinal dysbacteriosis degree of the mice is reduced to different degrees.
3.4 Effect of Lactobacillus rhamnosus AFY05 on mouse serum
Arterial blood of mice was collected, centrifuged at 4000r/min at 4℃for 10min using a bench-top high-speed cryocentrifuge, and the supernatant was collected and assayed for levels of propylene glycol (MDA), 5-hydroxytryptamine (5-HT), mouse interleukin 1 beta (IL-1 beta), and mouse interleukin 6 (IL-6) in the serum of mice according to the protocol described in the kit.
From FIG. 5, it can be seen that the IL-1. Beta. And IL-6 inflammatory factor levels in the serum of mice from diarrhea group were significantly higher than those from normal group. After gastric lavage with AFY05, the serum IL-1 beta and IL-6 levels were significantly lower in mice than in diarrhea groups, and the inflammatory factor level change of IL-6 was not apparent compared to normal groups.
As shown in FIG. 6, the mice in the diarrhea group had a small increase in both 5-HT and MDA relative to the normal group, and were treated with the treatment group to have a different degree of decrease. Compared with diarrhea groups, the expression level of 5-HT and MDA in the serum of mice in the high AFY05 group and the low AFY05 group is obviously reduced, and the AFY05 has a certain treatment effect on the peroxidation damage degree of intestinal tissues.
3.5 Effect of Lactobacillus rhamnosus AFY05 on mouse mRNA expression
Methods for detecting relative expression of mRNA using fluorescent quantitative PCR. Taking 50mg of intestinal tissue, placing the intestinal tissue into a homogenizing tube with homogenizing beads, adding 1mLTRIzol, placing into a homogenizer for homogenizing to obtain liquid, separating nucleic acid from protein complex, adding 200 mu L of chloroform, shaking, standing for 30min, placing into a 4 ℃ high-speed refrigerated centrifuge, centrifuging for 15min at 14000r/min, carefully sucking supernatant, adding equal volume of isopropanol, shaking, standing for 10min, placing into a 4 ℃ high-speed refrigerated centrifuge, centrifuging for 20min at 14000r/min, carefully discarding supernatant, adding 1mL of precooled ethanol for washing, placing into a 4 ℃ high-speed refrigerated centrifuge, centrifuging for 15min at 14000r/min, naturally volatilizing, adding 20 mu L of DEPC water for dissolving, detecting by a micro-spectrophotometer, and calculating the concentration. RNA was transcribed into cDNA according to the cDNA reverse transcription reagent instructions.
Eight-piece tubes were added in a proportion of 2. Mu.L of template (cDNA), 4. Mu.L of primer (primer sequences see Table 1), 10. Mu.L of LSYBR and 4. Mu.L of sterile ultra-pure water, and PCR reactions were performed in a 20. Mu.L reaction volume in a fluorescent PCR instrument.
Table 1: gene primer sequence table
The effect of lincomycin hydrochloride on the mRNA expression of CFTR, EGFR, NHE1 and TGF-. Beta.1 after induction was shown in FIG. 7. The expression levels of CFTR, EGFE, NHE and TGF- β1 mRNA were significantly reduced in the colon in the treatment and AFY05 groups compared to the diarrhea group, and the AFY05 group significantly inhibited the increase in the expression levels of CFTR, EGFR, NHE and TGF- β1 mRNA in the colon. It is proved that AFY05 can reduce the expression content of primer factors mRNA such as CFTR, EGFR, NHE1, TGF-beta 1 and the like in colon and relieve the dysbacteriosis of intestinal tract.
Example 4: prevention effect of lactobacillus rhamnosus AFY05 on DSS-induced colitis mice
Treatment of test animals: 40C 57BL/6 mice, body weight (20+ -3 g), all mice were fed in an environment with a temperature of 25+ -2deg.C and a light dark time of 12/12 h. Mice were fed adaptively for 1 week and divided equally into 4 groups (n=10): normal group, DSS induced colitis model group, lactobacillus rhamnosus AFY05 group, sulfasalazine (SASP) drug control group. After the adaptive feeding of the mice, 2.5% DSS water was continuously and freely drunk for 7 days, and the mice of the lactobacillus rhamnosus AFY05 group were daily gavaged with AFY05 bacterial suspension (10 9 CFU/kg), SASP group mice were perfused daily with sulfasalazine (100 mg/kg); normal groups of mice drink drinking water throughout 0-28d, and the remaining groups are free to drink water from 15-28 days. On day 28, all mice were fasted without water for 12h, sacrificed by cervical dislocation after orbital vein blood collection, and dissected promptly.
4.1 influence of Lactobacillus rhamnosus AFY05 on the weight of mice with colon inflammation
In the experimental process, the weight change of the mice is shown in fig. 8, and the weight of the normal group mice is in an ascending trend; during the molding period, the weight of mice in the model group, SASP group and Lactobacillus rhamnosus AFY05 group is reduced; the SASP group, the Lactobacillus rhamnosus AFY05 group mice began to return to body weight on day seven and recovered essentially to their original weight by day 14. The results showed that DSS induced weight loss in colitis mice, which decreased by about 17.8% compared to the initial weight by day 11; compared with the model group, SASP and Lactobacillus rhamnosus AFY05 both have remarkable (p < 0.05) slowing effect on weight loss of mice with enteritis, and the effect is basically similar, but the weight loss rate of mice in the Lactobacillus rhamnosus AFY05 group is lower than that of the SASP group.
4.2 Effect of Lactobacillus rhamnosus AFY05 on the index of the murine DAI against colon inflammation
As shown in fig. 9, the DSS group mice showed a significant increase in DAI index (p < 0.05) at the beginning of the fifth day of modeling, and the DAI index increased to a different extent in both the SASP group and the lactobacillus rhamnosus AFY05 group, but the rising trend was slower in the lactobacillus rhamnosus AFY05 group than in the SASP group. Experimental results show that the score of the DSS induced colonitis mouse DAI is obviously increased, SASP and lactobacillus rhamnosus AFY05 can slow down the rising trend of the mouse DAI, and the experimental data show that the inhibiting effect of the lactobacillus rhamnosus AFY05 is more obvious than SASP. (p < 0.05)
4.3 Effect of Lactobacillus rhamnosus AFY05 on colon length of mice with colon inflammation
After dissection of the mice, the colorectal length of the mice was measured and photographed. As shown in fig. 10, the colon of the model group mice was significantly shortened (p < 0.05) compared to the normal group, and slight hyperemia was observed in the colon mucosa of the model group. The colon shortening is caused by inflammation, congestion, edema and other phenomena of the colon, the colon shortening phenomenon of the SASP group and the Lactobacillus rhamnosus AFY05 group is lightened, compared with the model group, the colon shortening phenomenon is obviously reduced (p is less than 0.05), and the phenomenon shows that the Lactobacillus rhamnosus AFY05 has obvious inhibition effect on colon shortening of colon of mice with enteritis.
4.4 influence of Lactobacillus rhamnosus AFY05 on colonic tissue of colonic mice on colonic inflammation
After the colon tissue is fixed by 4% paraformaldehyde solution, a paraffin block is formed by paraffin embedding, the paraffin block is sliced by a slicing machine and then stained by hematoxylin and eosin (H & E), and the colon histopathology is observed under an optical microscope.
DSS induces a marked damage to colonic tissue with the phenomena of intestinal epithelial destruction, goblet cell destruction, strong inflammatory infiltrate and crypt disappearance. As shown in fig. 11, normal group mice have complete colon structure, obvious crypt, abundant goblet cells and no obvious damage; the colon mucous membrane of the mice in the model group is damaged, crypt epithelial cells are distorted, infiltration of a large number of inflammatory cells occurs, and intestinal barriers are damaged; the SASP group has complete colon structure, but has the phenomena of partial inflammatory cell infiltration and intestinal wall edema; the crypt of the group AFY05 of the Lactobacillus rhamnosus is clearly visible, and almost no edema or inflammatory cell infiltration phenomenon exists. These results indicate that lactobacillus rhamnosus AFY05 alleviates tissue damage of DSS to the colon of mice with a more pronounced effect than SASP.
Example 5: prevention effect of AFY05 on AOM/DSS induced colon cancer mice
Treatment of test animals: 40 male C57BL/6 mice of 6 weeks of age, body weight (20.0.+ -. 5.0 g). All mice were randomly and evenly divided into a normal group, an AOM/DSS induced group, an aspirin group, an AFY05 group after one week of adaptive feeding, 1mg/mL AOM reagent was intraperitoneally injected except for the normal group, whereas 2.5% DSS water was used for one week in the third week, followed by two weeks of pure water feeding and three cycles, during which period the aspirin group mice were gavaged with 10mg/mL aspirin solution daily, and the AFY05 group mice were gavaged with 109CFU/kg of Lactobacillus rhamnosus AFY05 bacterial suspension daily. Body weight measurements and recordings were made on all mice. All mice are fasted for 16 hours after the experimental period is finished, then blood is taken from the orbital veins, the mice are centrifuged at 4000r/min and 4 ℃ for 15min, serum is obtained after separation, and the mice are frozen at-80 ℃ for later use. After the mice were sacrificed, colon and liver tissues were collected and recorded by weighing.
5.1 influence of Lactobacillus rhamnosus AFY05 on weight and liver index of colon cancer mice
From the data in Table 2, it can be seen that the body weight of the normal group mice is at the highest level, while the liver quality and liver index are at the lowest level. The mice in the AOM/DSS-induced group had significantly lower body weight than the other groups, while the liver weight and liver index exhibited significant advantages, indicating that the AOM/DSS-induced group mice had significantly impaired body. Aspirin group significantly reduced liver weight, liver index (p < 0.05) in AOM/DSS-induced group. The liver weight and liver index reduction effect of the AFY05 group are equivalent to that of the aspirin group, compared with the AOM/DSS induction group, the reduction trend is obvious (p < 0.05), the body weight of the AOM/DSS induction group is the lowest, and the ingestion of the AFY05 significantly slows down the body weight reduction of mice (p < 0.05).
Table 2: index table of body weight and liver of mice
5.2 Effect of Lactobacillus rhamnosus AFY05 on colon length and tumor count in colon cancer mice
Table 3: colon length and tumor number table of mice
As can be seen from Table 3 and FIG. 12, the colon length of the normal group mice is longest and is 7.30.+ -. 0.41cm, and the colon length of the AOM/DSS induced group is significantly shortened compared with that of the normal group, and is 6.29.+ -. 0.24cm. The AFY05 group had a colon length of 6.67.+ -. 0.20cm, which was longer (p < 0.05) relative to the AOM/DSS induced group.
From table 3 and fig. 13, it can be observed that the colon of the normal group of mice exhibited a perfect flesh color and the surface was smooth and flawless; in contrast, a number of larger tumor tissues were observed in the mid-posterior segment of the colon in AOM/DSS-induced mice, distributed in the area near the anus, and the colon tissue of the mice exhibited a pronounced red color. These results indicate that the colon of AOM/DSS induced mice has been compromised, while the colon of normal mice remains healthy. In addition, mice from the aspirin and AFY05 groups had fewer colon tumors and were small compared to the AOM/DSS-induced groups, and the inflammatory phenomenon was also reduced.
5.3 influence of Lactobacillus rhamnosus AFY05 on colon tissue of colon cancer mice
The colon tissue of the mice fixed with 4% paraformaldehyde solution was dehydrated by soaking in ethanol for 24 hours, followed by paraffin embedding, slicing and H & E staining, and finally observed and photographed under an optical microscope.
According to fig. 14 and 15, the colonic myolayer and mucosal layer of the normal mice exhibited a highly intact morphology, and the crypt arrangement was well ordered without any lesions. In contrast, the inner colon wall of AOM/DSS-induced mice exhibited a cauliflower-like bulge while the tumor had infiltrated the colonic mucosal layer, resulting in back-to-back and coanda of the glands, thus obscuring the local structure, while the myometrium at the tumor site had developed fibrosis, indicating that the malignancy of the tumor was quite high. The colonic myolayer and mucosal layer of the aspirin group exhibited better integrity and clarity, but some inflammatory cells had also infiltrated, which means that the area had undergone an inflammatory response. Similarly, the colonic tissue mucosa of AFY05 mice was substantially intact, accompanied by partial inflammatory cell infiltration, and the crypt was slightly distended, but the presence of intact crypt cavities was still seen, and in addition, the colonic muscle layer was slightly thickened, all of which demonstrated that the malignancy of the mice tumors was significantly lower than that of AOM/DSS-induced groups.
Claims (8)
1. The lactobacillus rhamnosus is characterized in that the preservation number of the lactobacillus rhamnosus is CGMCC No.27365.
2. Use of the lactobacillus rhamnosus of claim 1 for the prevention and treatment of digestive tract diseases.
3. Use of the lactobacillus rhamnosus of claim 1 in the manufacture of a product for the prevention and treatment of digestive tract disorders.
4. The use according to claim 2, wherein the product is a functional food, pharmaceutical or health product.
5. The use of claim 4 wherein the product is a probiotic.
6. The use according to claim 2, wherein the digestive tract disease is diarrhea, colitis or colon cancer.
7. A bacterial preparation for preventing and treating digestive tract diseases, which is characterized by comprising the living lactobacillus rhamnosus of claim 1.
8. The bacterial preparation according to claim 7, wherein the bacterial preparation comprises the fermentation product of lactobacillus rhamnosus according to claim 1.
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