CN116574648A - Lactobacillus plantarum and application thereof in relieving constipation - Google Patents
Lactobacillus plantarum and application thereof in relieving constipation Download PDFInfo
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- CN116574648A CN116574648A CN202310536685.0A CN202310536685A CN116574648A CN 116574648 A CN116574648 A CN 116574648A CN 202310536685 A CN202310536685 A CN 202310536685A CN 116574648 A CN116574648 A CN 116574648A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a lactobacillus plantarum (Lactobacilli splantarum) HM-22, with preservation number: CCTCCNO: m2023282; the separation method comprises the following steps: mixing the obtained sample of Xinjiang traditional fermented acid cow milk, performing gradient dilution, coating on an agar medium, performing anaerobic culture at 37 ℃ for 48 hours, and streaking and purifying the single strain on the agar medium to obtain a pure culture. The invention can obviously improve the water content of the feces of the constipation mice and the small intestine propulsion rate, and shortens the first granule blacking time by about 30 percent compared with the constipation mice; the concentrations of the excitatory gastrointestinal regulating peptide and the 5-HT are obviously increased, and the concentrations of propionic acid, butyric acid, acetic acid and total acid in the short chain fatty acid are obviously higher than those of a constipation mouse, so that the constipation symptom is relieved strongly.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum with a probiotic function and application thereof in relieving constipation symptoms.
Background
Constipation is one of the most common gastrointestinal disorders, occurring in people of all ages worldwide. Constipation is characterized by dysuria, which is mainly characterized by dry stool, difficult or irregular defecation, with symptoms of abdominal pain, abdominal distension, etc. The reasons for the formation are mainly the change of the dietary structure; psychological or mental stress; other diseases cause; drug stimulation: abuse of laxatives results. Current treatments for constipation include general diet adjustment treatments, medication treatments, biofeedback treatments, surgical treatments, and the like. At present, drug therapy is still the most important therapeutic means, but long-term use and abuse of stimulant laxatives can also produce side effects on the body.
Probiotics are beneficial bacteria that naturally grow in the intestinal tract and are viable microorganisms that promote human health. Study data confirm that constipation patients have intestinal microecological imbalance. The probiotics can regulate intestinal microecological balance, increase the abundance of beneficial bacteria in intestinal tracts and improve the diversity of intestinal microorganisms. The increase of beneficial bacteria in the intestinal tract promotes the metabolism of short chain fatty acids, thereby increasing the concentration of excitatory gastrointestinal regulatory peptides to improve intestinal motility, increasing fecal water content, and thus improving constipation symptoms. The most common genera currently used in relieving constipation are bifidobacteria and enterococci.
Disclosure of Invention
The invention aims to provide a novel lactobacillus plantarum with a probiotic function and application thereof in relieving constipation symptoms, so as to solve the problem of insufficient strain for relieving constipation symptoms in the aspect of regulating intestinal flora structure in the prior art.
To achieve the above object, the present invention provides a Lactobacillus plantarum (Lactobacilli splantarum) HM-22, accession number: CCTCCNO: m2023282.
The invention provides a method for separating lactobacillus plantarum HM-22, which comprises the following steps:
mixing the obtained sample of Xinjiang traditional fermented acid cow milk, performing gradient dilution, coating on an agar medium, performing anaerobic culture at 37 ℃ for 48 hours, and streaking and purifying the single strain on the agar medium to obtain a pure culture.
Preferably, the agar medium is one of MRS agar medium or M17 agar plate medium.
Preferably, the MRS agar medium is prepared by mixing glucose, peptone, yeast extract powder, beef extract, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, mgSO 4 ·7H 2 O、MnSO 4 ·5H 2 O and Tween-80 are uniformly mixed and dissolved in deionized water, an MRS culture medium is obtained through sterilization, agar powder is added, and the MRS agar culture medium is obtained after sterilization.
Preferably, the feed is prepared according to 20-25g/L glucose, 10-15g/L peptone, 5-10g/L yeast extract powder, 10-15g/L beef extract, 2-7g/L dipotassium hydrogen phosphate, 1.5-2g/L hydrogen diamine citrate, 5-8g/L sodium acetate, 0.58-1g/L MgSO 4 ·7H 2 O, 0.25-0.5g/L MnSO 4 ·5H 2 The MRS agar medium is prepared according to the ratio of O to Tween-80 of 1 mL/L.
Preferably, the method of sterilization comprises sterilizing at a temperature of 120-121 ℃ for 15-20min.
Preferably, the dosage of the agar powder is 15-20g/L.
Preferably, the gradient dilution method comprises ten times gradient dilution with 0.85% sterile physiological saline to 10 -4 ，10 -5 ，10 -6 。
The invention provides application of lactobacillus plantarum HM-22 for relieving constipation symptoms.
Preferably, the use in any one of the following:
A. is used for adjusting the water content of the excrement and the gastrointestinal creep capacity;
B. for alleviating lesions of colon tissue;
C. for increasing the content of excitatory gastrointestinal modulator peptides and 5-HT;
D. for promoting short chain fatty acid metabolism;
E. for increasing intestinal flora diversity.
Compared with the prior art, the invention has the beneficial effects that:
under the action of lactobacillus plantarum HM-22, the water content and the small intestine propulsion rate of BALB/c constipation mice induced by Loperamide (Loperamide) are obviously improved, and the first granule black discharge time is shortened by about 30 percent compared with the constipation mice; the concentrations of the excitatory gastrointestinal regulating peptide and the 5-HT are obviously increased, and the concentrations of propionic acid, butyric acid, acetic acid and total acid in the short chain fatty acid are obviously higher than those of a constipation mouse, so that the short chain fatty acid can be applied to a probiotic product for relieving constipation symptoms, and has a stronger effect of relieving constipation symptoms.
Compared with the traditional Chinese medicine preparation for relieving constipation, the probiotic preparation prepared from the lactobacillus HM-22 is safer in treatment and wider in applicable crowd range.
Preservation description
Preservation information of biological material samples according to the present invention: the microorganism (strain) of the reference is HM-22, is classified and named as lactobacillus plantarum (Lactplantibilum) and is preserved by China Center for Type Culture Collection (CCTCC) for 3 months and 9 days in 2023, with the preservation number: cctccc NO: m2023282. The CCTCC address is No. 299 of the Wuchang district of Wuhan, hubei province.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum HM-22 of the present invention;
FIG. 2 is a microscopic image of the Lactobacillus plantarum HM-22 cell of the present invention after 1000 times magnification;
FIG. 3 is a phylogenetic dendrogram of Lactobacillus plantarum HM-22 according to the invention;
FIG. 4 is a graph showing comparison of the effect of the haemolytic activity of Lactobacillus plantarum HM-22 according to the invention;
FIG. 5 is a statistical graph showing the effect of Lactobacillus plantarum HM-22 of the present invention on the moisture content of constipation mice feces;
FIG. 6 is a graph showing the statistics of the black time of the HM-22 strain of Lactobacillus plantarum of the present invention on the gastrointestinal first granule discharge of constipation mice;
FIG. 7 is a graph showing the statistics of the rate of intestinal motility of Lactobacillus plantarum HM-22 according to the present invention on constipation mice;
FIG. 8 is a statistical graph of the effect of Lactobacillus plantarum HM-22 of the present invention on constipation mice intestinal histopathology;
FIG. 9 is a statistical graph showing the effect of Lactobacillus plantarum HM-22 of the present invention on concentration of substance P in the gastrointestinal tract of constipation mice;
FIG. 10 is a statistical plot of the effect of Lactobacillus plantarum HM-22 of the present invention on gastrin concentration in the gastrointestinal tract of constipation mice;
FIG. 11 is a statistical graph showing the effect of Lactobacillus plantarum HM-22 of the present invention on motilin concentration in the gastrointestinal tract of constipation mice;
FIG. 12 is a statistical graph showing the effect of Lactobacillus plantarum HM-22 of the present invention on somatostatin concentration in the gastrointestinal tract of constipation mice;
FIG. 13 is a statistical graph showing the effect of Lactobacillus plantarum HM-22 of the present invention on vasoactive intestinal peptide concentration in the gastrointestinal tract of constipation mice;
FIG. 14 is a statistical plot of the effect of Lactobacillus plantarum HM-22 of the present invention on endothelin-1 concentration in the gastrointestinal tract of constipation mice;
FIG. 15 is a statistical plot of the effect of Lactobacillus plantarum HM-22 of the present invention on constipation mouse 5-hydroxytryptamine concentration;
FIG. 16 is a statistical plot of the effect of Lactobacillus plantarum HM-22 of the present invention on the content of SCFAs in constipation mice faeces;
FIG. 17 is a statistical graph showing the effect of Lactobacillus plantarum HM-22 of the present invention on the alpha diversity of intestinal microorganisms in constipation mice;
FIG. 18 is a statistical graph showing the effect of Lactobacillus plantarum HM-22 of the present invention on beta diversity of intestinal microorganisms in constipation mice;
FIG. 19 is a statistical plot of the effect of Lactobacillus plantarum HM-22 of the present invention on constipation mice intestinal flora.
Detailed Description
The invention is further illustrated below in connection with specific examples, but is not limited in any way.
The instruments, reagents, materials, etc. used in the examples described below are conventional instruments, reagents, materials, etc. known in the art, and are commercially available. The experimental methods, detection methods, and the like in the examples described below are conventional experimental methods, detection methods, and the like that are known in the prior art unless otherwise specified.
Example 1 Strain culture
The lactobacillus plantarum HM-22 is obtained by separating from Xinjiang traditional fermented yogurt, and comprises the following specific steps:
MRS medium preparation: according to 20g/L glucose, 10g/L peptone, 5g/L yeast extract powder, 10g/L beef extract, 2g/L dipotassium hydrogen phosphate, 2g/L diamine hydrogen citrate, 5g/L sodium acetate and 0.58g/L MgSO 4 ·7H 2 O, 0.25g/L MnSO 4 ·5H 2 Weighing raw materials of O and 1mL/L Tween-80, uniformly mixing and dissolving in deionized water, and sterilizing at 120-121 ℃ for 15-20min to obtain the MRS culture medium; adding 15-20g/L agar powder into the culture medium, and sterilizing at 120-121deg.C for 15-20min to obtain MRS agar culture medium.
II, separating strains: as shown in FIG. 1, the sample of Xinjiang traditional fermented acid milk was weighed uniformly into 0.5mL, and subjected to ten-fold gradient dilution (10 -4 ，10 -5 ，10 -6 ) Next, the culture was spread on MRS agar medium or M17 agar plate medium, anaerobic cultured at 37℃for 48 hours, single colony was picked, and streaking purification was continued on MRS agar medium to obtain a pure culture.
III, strain morphology identification: as shown in figure 2, bacteria positive in gram staining and negative in catalase experiment are temporarily designated as lactic acid bacteria, bacterial colonies of the strain are milky round raised bacterial colonies, edges are neat and opaque, and cells are in a long rod shape, single or chain shape after gram staining.
IV. Molecular biology identification: the strain is inoculated in MRS liquid culture medium, and is cultured for 24 hours at 37 ℃ for 16SrRNA sequencing identification, and the identification result is lactobacillus plantarum (Lactplatinum sp.) which is named as lactobacillus plantarum HM-22, and a phylogenetic dendrogram is shown in figure 3.
Preservation information of biological material samples according to the present invention: the microorganism (strain) of the reference is HM-22, is classified and named as lactobacillus plantarum (Lactplantibilum) and is preserved by China Center for Type Culture Collection (CCTCC) for 3 months and 9 days in 2023, with the preservation number: cctccc NO: m2023282. The CCTCC address is No. 299 of the Wuchang district of Wuhan, hubei province.
Example 2: bacterial strain probiotics
I. Gastrointestinal fluid tolerance test activated Lactobacillus plantarum HM-22 bacterial solution was centrifuged (4 ℃ C., 3500r/min, 10 min), washed 3 times with 0.1mol/L PBS and resuspended as initial bacterial solution. The sample was inoculated into simulated gastric fluid containing pepsin at an inoculum size of 1% (v/v), and poured into a water bath at 37℃for 3 hours, and the plate was poured out after the completion of the inoculation. Then, the bacterial liquid treated with simulated gastric fluid was inoculated into simulated intestinal fluid containing trypsin in an inoculum size of 1% (v/v), and the bacterial liquid was subjected to a water bath at 37℃for 8 hours, and after the completion of the water bath, the bacterial liquid was poured into a plate. And strain survival was calculated according to the formula. The results are shown in Table 1, and Lactobacillus plantarum HM-22 shows a strong tolerance to gastrointestinal fluids, with survival rates in the gastrointestinal fluids reaching 88.28% and 89.05%, respectively.
Wherein: n (N) 1 Refers to the number of viable bacteria (CFU/mL) after treatment with simulated artificial gastric fluid or intestinal fluid;
N 0 refers to the viable count (CFU/mL) of the strain before treatment;
II, adhesion experiment: centrifuging activated Lactobacillus plantarum HM-22 bacterial solution (4deg.C, 3500r/min, 5 min), washing bacterial precipitate with 0.1mol/L PBS buffer 3 times, and re-suspending in RPMI-1640 culture solution (without inactivated fetal bovine serum and double antibody) to adjust bacterial concentration to 5×10 8 CFU/mL is ready for use. Human colon cancer gland cell line HT-29 cells were cultured in RPMI-1640 medium (containing 1% diabody and 10% fetal bovine serum) and grown to 2X 10 cells when the flask area was about 90% 5 Inoculating the mixture to 12-well plate, and placing in a 37 ℃ constant temperature and humidity incubator (90% humidity, 5% CO) 2 ) Medium culture for adhesionAnd (5) carrying out an experiment. The cultured waste solution of the 12-well plate was discarded, the solution was washed with the above PBS buffer, then the bacterial suspension (1 mL) was added, after 2 hours of culture, the solution was washed 5 times with PBS buffer, 0.5% Triton-X100 was added, and after 10 minutes of ice bath, the plate was poured. The adhesion index is calculated according to the following formula:
wherein: the number of adherent colonies refers to the number of colonies (CFU/mL) of the strain adhering to HT-29 cells as determined by plate decantation;
cell number refers to the average cell number per well (cell/mL);
adhesion of probiotics to the intestinal surface is important for the human intestinal tract, and it prevents the removal of probiotics by intestinal peristalsis. As shown in Table 1, the adhesion index reaches 19.11cfu/cell, which shows that Lactobacillus plantarum HM-22 has stronger adhesion capability.
Table 1, survival rate of Lactobacillus plantarum HM-22 according to the invention in simulated gastrointestinal fluids and adhesion index to HT-29 cells example 3: strain safety test
I. Evaluation of haemolysis of Lactobacillus plantarum HM-22: activated Lactobacillus plantarum HM-22 and Listeria monocytogenes (L.unicytogenes) were streaked onto Columbia solid medium supplemented with 7% (v/v) goat blood, and incubated at 37℃for 48h. If no transparent ring appears, it is safe to indicate that the strain has gamma-hemolysis. Beta-hemolysis is usually pathogenic if transparent rings appear around the growing colonies. As shown in FIG. 4, L.Monocytogenes were used as positive control in the experiment, and hemolysis rings appeared around the colony of L.Monocytogenes, whereas the colony of Lactobacillus plantarum HM-22 of the present invention did not have hemolysis rings, indicating that Lactobacillus plantarum HM-22 is a safer strain.
II, evaluating the sensitivity of the Lactobacillus plantarum HM-22 antibiotics: lactobacillus plantarum HM-22 was tested for susceptibility to antibiotics according to standard methods published by the American clinical laboratory standards institute (Clinical and Laboratory Standards Institute, CLSI) and selected from the following antibiotic classes (kanamycin, erythromycin, ampicillin, chloramphenicol, tetracycline, clindamycin, gentamicin, vancomycin). The Minimum Inhibitory Concentration (MIC) of Lactobacillus plantarum HM-22 for an antibiotic is compared to a threshold value, and when the MIC value is less than or equal to the threshold value, the Lactobacillus is indicated to be susceptible to the antibiotic. As shown in Table 2, lactobacillus plantarum HM-22 was sensitive to all seven antibiotics and safe.
S sensitive to antibiotics
TABLE 2 sensitivity of Lactobacillus plantarum HM-22 of the invention to antibiotics
III, evaluating the capacity of lactobacillus plantarum HM-22 to produce biogenic amine: lactobacillus plantarum HM-22 was cultured in MRS medium (seven precursor amino acids 0.1% and 0.005% pyridoxal added) at 37℃for 18h. The resulting culture was centrifuged (8000 r/min,4 ℃) and the supernatant (750. Mu.L) was taken and added with an equivalent amount of dansyl chloride derivative reagent (750. Mu.g dansyl chloride, 100mL acetone) and 150. Mu.L of saturated sodium carbonate solution, and after 30min at 45℃in a water bath, the biogenic amine content was quantitatively determined using a high performance liquid phase.
As shown in Table 3, lactobacillus plantarum HM-22 produces trace amounts of biogenic amine and has safety.
ND: i.e. no biogenic amine is detected
TABLE 3 capability Table of Lactobacillus plantarum HM-22 to produce biogenic amines according to the invention
Example 4: effect on fecal moisture content in constipation mice
I. Grouping animals: six week old male BALB/c mice were randomly divided into 5 groups, normal (Nor) groups, respectively,Constipation model group (Lop), bacteria control group (Lop+JCM 1132), bacteria control group (Lop+LGG), HM-22 group (Lop+HM-22) were 10 per group. Lactobacillus acidophilus (Lactobacillus acidophilus) JCM1132 and Lactobacillus rhamnosus (Lactobacillus rhamnosus) GG were used as bacterial controls. All groups of mice except the Nor group were perfused with loperamide (10 mg/kg. BW,0.2 mL) once daily for 7d to establish a constipation model. At the same time, nor mice were infused with the same volume of 0.9% sterile saline. After successful establishment of constipation model, nor mice were given sterile saline, lop group, lop+JCM1132 group, lop+LGG group, lop+HM-22 group mice were filled with loperamide (10 mg/kg. BW,0.2 mL), and after 1h, were filled with saline and each bacterial suspension (10) 9 CFU/mL), 14d in succession, as shown in table 4.
Table 4, animal group and daily lavage content
II effects on Constipation mouse faeces moisture content
Fecal samples were collected at experiment 7d and 21d, wet weights were weighed and dried at 105 c for 5 hours to constant weight, weights were recorded as dry weights after constant weight, and fecal moisture content was calculated according to the following formula. As a result, as shown in FIG. 5, at 7d, the fecal moisture content of the Lop group, lop+JCM1132 group, lop+LGG group, and Lop+HM-22 group was significantly lower than that of the Nor group, indicating that the constipation model was successfully established at this time. At 21d, the fecal moisture content of constipation group mice, which were subjected to the intervention of Lactobacillus plantarum HM-22 of the present invention, was significantly increased to 63.96+ -4.47% and higher than that of the two-group bacteria control group mice. The lactobacillus plantarum HM-22 can effectively relieve the drop of the water content of the excrement caused by constipation.
Example 5: effect of Lactobacillus plantarum HM-22 on gastrointestinal motility in constipation mice
At 20d, mice of all groups except Nor group were givenAfter 1h of gastric lavage loperamide, nor group and Lop group were lavaged with active carbon solution, and the other group was lavaged with each bacterial suspension (10 9 CFU/mL) activated carbon solution (10% activated carbon and 0.5% carboxymethyl cellulose suspension, 0.2 mL). The discharge time of the first black stool (first black stool discharge time, min) was recorded.
All but the Nor group of mice were given loperamide at 21d, and after 1h, the Nor and Lop groups were given gavage with activated carbon solutions, the remaining groups were given gavage with respective bacterial suspensions (10 9 CFU/mL). Mice were sacrificed after 30min and the small intestine (upper end from pylorus, lower end to cecum) was removed to calculate the activated carbon passage distance.
The first grain black discharge time and the small intestine propulsion rate are important indexes for evaluating the gastrointestinal vermicular ability. As shown in FIGS. 6 and 7, the first graining black time and the small intestine propulsion rate of the Lop group were 147.67.+ -. 19.64min and 42.35.+ -. 3.74%, respectively. After the lactobacillus plantarum HM-22 is dried, the first granule black discharge time is obviously shortened, and the small intestine propulsion rate is obviously increased, namely 97.5+/-4.98 min and 79.65 +/-0.49 percent respectively. And the gastrointestinal vermicular ability of the Lop+HM-22 mice is superior to that of the Lop+JCM1132 mice (first granule black time: 101+ -3.46 min, small intestine propulsion rate: 76.81+ -1.32%) and the Lop+LGG mice (first granule black time: 106.33 + -1.67 min, small intestine propulsion rate: 76.29 + -5.68%), which further proves the advantage of the Lactobacillus plantarum HM-2 in improving gastrointestinal motility of constipation mice.
Example 6: effect of Lactobacillus plantarum HM-22 on colon histopathology in constipation mice
Colon tissues of each group of mice were taken out and fixed in 4% paraformaldehyde, stored at room temperature and sent to Shanghai, sanghai Biotechnology Co., ltd. For H & E staining. As a result, as shown in FIG. 8, lactobacillus plantarum HM-22 effectively restored colonic tissue integrity in constipation mice, intestinal epithelial folds were relatively intact and regular, crypt structures were restored, and goblet cells increased, as compared to Lop group mice, and colonic characteristics were similar to that of Nor group. The lactobacillus plantarum HM-22 can effectively improve colon tissue structure damage caused by constipation.
Example 7: effect of Lactobacillus plantarum HM-22 on Constipation mouse gastrointestinal Conditioning peptide and 5-HT
Mice were bled from the orbit prior to sacrifice and serum concentrations of gastrointestinal regulatory peptide and 5-HT were determined using ELISA kits and each measurement was repeated three times. Gastrointestinal regulatory peptides play a key role in regulating gastrointestinal motility. Excitatory gastrointestinal modulator peptides (substance P, motilin and gastrin) and 5-hydroxytryptamine alleviate constipation symptoms by modulating contractile intestinal smooth muscle and aqueous electrolyte transport. Inhibitory gastrointestinal modulatory peptides (somatostatin, endothelin-1 and vasoactive intestinal peptide) extend intestinal transit time by inhibiting the release of motilin and gastrin. As shown in FIGS. 9-15, lactobacillus plantarum HM-22 significantly increased the concentration of excitatory gastrointestinal regulatory peptides (substance P, motilin and gastrin) and 5-HT, 70.32 + -6.44 ng/mL, 936.71 + -28.07 ng/L, 721.23 + -49.34 ng/L, 157.44+ -10.22 ng/mL, respectively, compared to constipation mice. And significantly reduces the concentration of inhibitory gastrointestinal regulatory peptides (somatostatin, vasoactive intestinal peptide and endothelin-1) at 34.65+ -1.05 ng/L, 102.49 + -8.82 ng/L, 412.44 + -18.45 ng/L, respectively. Wherein the concentration of somatostatin and endothelin-1 in the Lop+HM-22 group is significantly lower than that in the Lop+JCM1132 group (81.11 + -8.43 ng/L) and the Lop+LGG group (116.19 + -3.84 ng/L); the 5-hydroxytryptamine concentration was significantly higher than that of the Lop+JCM1132 group (131.74 + -12.14 ng/mL) and Lop+LGG group (129.40 + -9.70 ng/mL). Illustrating that lactobacillus plantarum HM-22 of the present invention can alleviate constipation by improving the gastrointestinal transit peptide and 5-HT content.
Example 8: effect of Lactobacillus plantarum HM-22 on the content of SCFAs in constipation mice faeces
The mouse feces obtained on day 21d are placed in a 2mL sterile cryopreservation tube, quickly frozen by liquid nitrogen and then stored at-80 ℃. Under the condition of dry ice, the mixture is sent to Shanghai Meiji biological medicine technology Co.Ltd for measuring the content of SCFAs. LGG was not analyzed for short chain fatty acids and intestinal flora, as it had poor constipation relieving effect compared to l.acidophilus JCM 1132. The results are shown in FIG. 16, in which FIG. 16 (a) is an acetic acid concentration versus graph, FIG. 16 (b) is a butyric acid concentration versus graph, FIG. 16 (c) is a propionic acid concentration versus graph, and FIG. 16 (d) is a total short chain fatty acid concentration versus graph, the Lop+HM-22 group significantly increased acetic acid (3405.51 + -301.07 μg/g), butyric acid (1160.13 + -50.80 μg/g), propionic acid (760.84 + -133.56 μg/g), and total SCFAs (4785.66 + -561.83) concentrations compared to the Lop group. And the content of all organic acids in the Lop+HM-22 group is obviously higher than that in the Lop+JCM1132 group, which shows that the Lactobacillus plantarum HM-22 can relieve constipation by increasing the concentration of SCFAs.
Example 9: effect of Lactobacillus plantarum HM-22 on Constipation mice intestinal flora content
The mouse feces obtained in 21d are placed in a 2mL sterile cryopreservation tube, quickly frozen by liquid nitrogen and then stored at-80 ℃. Intestinal flora sequencing was performed by Miseq PE300 platform, carried out by Shanghai Meiji Biotechnology Co.Ltd under dry ice conditions. As shown in FIG. 17, the Lop+HM-22 group had significantly higher chao1 index than the Nor, lop and Lop+JCM1132 groups, indicating increased intestinal flora diversity. The non-metric dimensional scores shown in FIG. 18 demonstrate some degree of separation from the Lop group after the HM-22 dry prognosis of Lactobacillus plantarum, indicating some modulation of intestinal flora structure. As shown in FIG. 19, lactobacillus plantarum HM-22 treatment down-regulates the abundance of Moraxella (Moraxellaceae) and Aerococcaceae (Aerococcaceae) at the family level, up-regulates the abundance of Lactobacillus (Lactobacillus), the intestinal flora is balanced, and the Lactobacillus abundance is increased. It was demonstrated that Lactobacillus plantarum HM-22 can alleviate constipation symptoms by modulating the intestinal flora.
In conclusion, the lactobacillus plantarum HM-22 can effectively regulate the water content of excrement and the gastrointestinal vermicular ability, relieve colon tissue lesions, increase the content of excitatory gastrointestinal regulatory peptide and 5-HT, promote the metabolism of short-chain fatty acid, increase the diversity of intestinal flora, and have the effect of relieving constipation symptoms, and can effectively relieve the symptoms of constipation patients when being applied to relieving constipation, so that the constipation patients can be treated more safely.
Many possible variations and modifications of the disclosed technology can be made by anyone skilled in the art without departing from the scope of the technology, or the technology can be modified to be equivalent. Therefore, any simple modification, equivalent variation and modification of the above embodiments according to the technical substance of the present invention shall still fall within the scope of the technical solution of the present invention.
Claims (10)
1. Lactobacillus plantarum (Lactobacilli) HM-22, characterized by a deposit number: CCTCCNO: m2023282.
2. A method for separating lactobacillus plantarum HM-22, which is characterized by comprising the following steps:
mixing the obtained Xinjiang fermented acid cow milk sample, performing gradient dilution, coating on an agar medium, performing anaerobic culture at 37 ℃ for 48 hours, and streaking and purifying the single strain on the agar medium to obtain a pure culture.
3. The method for isolating lactobacillus plantarum HM-22 according to claim 2, wherein the agar medium is one of MRS agar medium or M17 agar plate medium.
4. The method for separating Lactobacillus plantarum HM-22 according to claim 3, wherein the MRS agar medium is prepared by mixing glucose, peptone, yeast extract, beef extract, dipotassium hydrogen phosphate, diamine hydrogen citrate, sodium acetate, mgSO 4 ·7H 2 O、MnSO 4 ·5H 2 O and Tween-80 are uniformly mixed and dissolved in deionized water, an MRS culture medium is obtained through sterilization, agar powder is added, and the MRS agar culture medium is obtained after sterilization.
5. The method for separating Lactobacillus plantarum HM-22 according to claim 4, wherein the glucose is 20-25g/L, peptone is 10-15g/L, yeast extract is 5-10g/L, beef extract is 10-15g/L, dipotassium hydrogen phosphate is 2-7g/L, diamine hydrogen citrate is 1.5-2g/L, sodium acetate is 5-8g/L, mgSO is 0.58-1g/L 4 ·7H 2 O, 0.25-0.5g/L MnSO 4 ·5H 2 O and Twe of 1mL/LThe ratio of en-80 is used for preparing MRS agar culture medium.
6. The method for separating lactobacillus plantarum HM-22 according to claim 4, wherein the sterilization method comprises sterilization at a temperature of 120-121 ℃ for 15-20min.
7. The method for separating Lactobacillus plantarum HM-22 according to claim 4, wherein the amount of the agar powder is 15-20g/L.
8. The method for separating lactobacillus plantarum HM-22 according to claim 2, wherein the gradient dilution method comprises ten times of gradient dilution with 0.85% sterile physiological saline to 10 -4 ，10 -5 ，10 -6 。
9. Use of lactobacillus plantarum HM-22 for alleviating symptoms of constipation.
10. Use of lactobacillus plantarum HM-22 according to claim 9, characterised by the use in any one of the following:
A. is used for adjusting the water content of the excrement and the gastrointestinal creep capacity;
B. for alleviating lesions of colon tissue;
C. for increasing the content of excitatory gastrointestinal modulator peptides and 5-HT;
D. for promoting short chain fatty acid metabolism;
E. for increasing intestinal flora diversity.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116803300A (en) * | 2023-05-12 | 2023-09-26 | 黑龙江飞鹤乳业有限公司 | Synbiotic composition capable of improving chronic constipation symptom and preparation method and application thereof |
| CN117165497A (en) * | 2023-11-02 | 2023-12-05 | 微康益生菌(苏州)股份有限公司 | Lactobacillus plantarum Lp18 for improving constipation, application and product thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116803300A (en) * | 2023-05-12 | 2023-09-26 | 黑龙江飞鹤乳业有限公司 | Synbiotic composition capable of improving chronic constipation symptom and preparation method and application thereof |
| CN117165497A (en) * | 2023-11-02 | 2023-12-05 | 微康益生菌(苏州)股份有限公司 | Lactobacillus plantarum Lp18 for improving constipation, application and product thereof |
| CN117165497B (en) * | 2023-11-02 | 2024-01-02 | 微康益生菌(苏州)股份有限公司 | Lactobacillus plantarum Lp18 for improving constipation, application and product thereof |
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