CN113493751A - Application of animal bifidobacterium in potential intervention of diabetes - Google Patents

Application of animal bifidobacterium in potential intervention of diabetes Download PDF

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CN113493751A
CN113493751A CN202110645457.8A CN202110645457A CN113493751A CN 113493751 A CN113493751 A CN 113493751A CN 202110645457 A CN202110645457 A CN 202110645457A CN 113493751 A CN113493751 A CN 113493751A
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bifidobacterium
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bifidobacterium animalis
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孟祥晨
朱宗涛
尚佳萃
赵乐
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Northeast Agricultural University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract

The invention discloses application of bifidobacterium animalis as a potential strain with the effect of intervening diabetes, and belongs to the technical field of food microorganisms and lactobacillus probiotics. Positive control strains of animal bifidobacterium BB12 and test strains of 20-9 and 15-2 are taken as research objects, and the antioxidant property, the alpha-glucosidase activity inhibition capacity, the resistance to acid, bile salt and simulated digestive tract environment, the adhesion capacity and the adhesion inhibition capacity of the positive control strains, the animal bifidobacterium BB12 and the test strains are analyzed. The results show that: the hydroxyl radical scavenging capacity and reducing capacity of the complete cells and cell extracts of the bifidobacterium animalis 15-2 are higher than those of the strain 20-9 and the strain BB12, the alpha-glucosidase activity inhibition capacity is higher than that of the strain 20-9, and the inhibition rates of the alpha-glucosidase activity and the strain BB12 are respectively 26.90% and 12.83%. The survival rate and the adhesion rate of the strain 15-2 to Caco-2 cells after simulation of the digestive tract are both higher than those of the strain 20-9 and the strain BB 12. Strain 15-2 is more acid tolerant than strains 20-9 and BB12, and is slightly inferior to strain BB12 in bile salt and adhesion inhibition, but still superior to strain 20-9. Therefore, the bifidobacterium animalis 15-2 can be further used for animal experiments as a potential diabetes intervention effect strain. The research result lays a foundation for further researching the potential intervention effect of the animal bifidobacterium on the diabetes.

Description

Application of animal bifidobacterium in potential intervention of diabetes
Technical Field
The invention belongs to the technical field of lactobacillus probiotic and food microorganism, and relates to application of bifidobacterium animalis in potential intervention of diabetes.
Background
Along with the development of urbanization and the change of life style, people gradually form bad life habits such as high-fat diet and reduced exercise amount, and the diabetic patients are increasing day by day. Diabetes is a metabolic disorder characterized by chronic hyperglycemia due to insufficient insulin production or insulin resistance. The long-term carbohydrate, fat and protein metabolism disorder can cause multi-system damage, and lead to chronic progressive lesion, hypofunction and failure of tissues and organs such as eyes, kidneys, nerves, hearts, blood vessels and the like. Acute severe metabolic disorders, such as diabetic ketoacidosis, hyperosmolar hyperglycemia complex, can occur when the condition is severe or under stress. Diabetes Mellitus (DM) has become one of four chronic diseases threatening human health, becomes a global and continuously-upgraded public health problem, and researches on effective measures for preventing and treating diabetes mellitus are urgent. The prevention and treatment of diabetes are hot problems, and more important, are civil problems. At present, except for diabetes mellitus which is treated by adopting medicines, more and more experiments prove that lactic acid bacteria have the function of resisting diabetes mellitus, and the blood sugar is reduced mainly by reducing oxidative stress, inhibiting the activity of alpha-glucosidase, balancing intestinal microorganisms, regulating immunity and the like. Oxidative stress refers to an elevated level of oxygen radicals that damage lipids, proteins, and DNA. Alpha-glucosidase is an enzyme from the brush border membrane of the small intestine, and has the functions of promoting the hydrolysis of carbohydrates such as starch, polysaccharide and the like into monosaccharides which are easy to absorb, inhibiting the activity of the monosaccharides, reducing the absorption of glucose and being beneficial to reducing the postprandial blood sugar.
Research shows that the animal bifidobacterium 01 has the function of antioxidation; bifidobacterium bifidum F-35 fermentation supernatant has the ability of inhibiting alpha-glucosidase activity; lactobacillus rhamnosus CCFM0528 can reduce postprandial blood glucose level of type II diabetic mice, which is probably related to the inhibition of alpha-glucosidase activity by Lactobacillus rhamnosus CCFM0528, and in vitro experiments have confirmed that Lactobacillus rhamnosus CCFM0528 has the ability to inhibit alpha-glucosidase activity. The study mainly compares the antioxidant capacity including DPPH scavenging capacity, hydroxyl free radical scavenging capacity and reducing capacity, the activity inhibition capacity of alpha-glucosidase, the resistance to simulated digestive tract environment and the capacity of adhering Caco-2 cells of two animal bifidobacteria, obtains the bifidobacteria strain with potential diabetes intervention effect by an in vitro screening method, and provides reference for the prevention and treatment of diabetes.
Disclosure of Invention
The purpose of the invention is as follows: provides the application of animal bifidobacterium in potential intervention of diabetes.
The technical problems solved by the invention are as follows: provides the application of animal bifidobacterium in potential intervention of diabetes, helps to solve the problem of metabolic disorder diseases characterized by chronic hyperglycemia, and provides a research basis for further research on prevention and treatment of diabetes for the technicians in the field.
The invention discloses the following technical effects: the invention obtains the bifidobacterium strain with potential effect of intervening diabetes through an in-vitro screening method. The result shows that the animal bifidobacterium 15-2 has the potential effect of intervening diabetes.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts;
FIG. 1 shows DPPH scavenging ability of intact cells and cell extracts of the strains (a) hydroxyl radical scavenging ability (b) and reducing ability (c);
fig. 2 is the survival of the strains when incubated at 37 ℃ under pH2 and pH3 conditions;
FIG. 3 shows the tolerance of the strains to bile salts at different concentrations;
FIG. 4 shows the ability of the strain to adhere to Caco-2 cells;
FIG. 5 is the competitive adhesion inhibition (a), exclusive adhesion inhibition (b) and alternative adhesion inhibition (c) capabilities of the strains;
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
Example 1
Application of animal bifidobacterium in potential intervention of diabetes
1. Materials and methods
1.1 Experimental materials
Bifidobacterium animalis (Bifidobacterium animalis) H15-2, wherein the Bifidobacterium animalis H15-2 is Bifidobacterium (Bifidobacterium), has been preserved in the northeast university of agriculture bacterial bank, and has a preservation date of 2017, 6-20 days and a preservation number of H15-2.
Alpha-glucosidase, 4-nitrophenol-alpha-D-glucopyranoside (PNPG), 1-diphenyl-2-trinitrophenylhydrazine (DPPH) Sigma USA; 1, 10-phenanthroline, trichloroacetic acid (TCA), 30% hydrogen peroxide (H)2O2) Ferrous sulfate (FeSO)4) Potassium ferricyanide, ferric chloride (FeCl)3) Domestic analysis is pure; the Shanghai cell bank of Caco-2 cells Chinese academy of sciences.
1.2 preparation of intact cells and cell-free extracts of the strains
The bacterial liquid cultured in mMRS medium for 18h is centrifuged for 10min at 8000r/min and 4 ℃. After washing with PBS 2 times, the cells were resuspended in PBS and adjusted to 1X 109CFU/mL, this is a suspension of whole cell bacteria. The strain suspension is subjected to ultrasonication under the ice bath condition, wherein the ultrasonication condition is as follows: the power is 200W, and the operation lasts for 8s every time for 10 min. Centrifuging the ultrasonic disrupted solution at 12000r/min for 20min, and filtering the supernatant with 0.22 μm filter membrane to obtain cell-free extract.
1.3 determination of antioxidant Capacity of Strain
1.3.1 determination of DPPH clearance of Lactobacillus cell-free extract
Adding 1mL of lactobacillus cell-free extract into a test tube, adding 1mL of 0.2mmol/L DPPH anhydrous ethanol solution, mixing well, reacting in a dark place for 30min, taking supernatant, measuring light absorption value at 517nm, and using PBS with equal volume instead of sample solution as a control group. Calculated according to the following formula: DPPH radical scavenging ratio (%) - (1-A)517sample/A517Control) × 100. In the formula: a. the517Sample and A517The control is the absorbance value measured at 517nm of the sample group and the control group respectively.
1.3.2 measurement of hydroxyl radical scavenging ability of lactic acid bacteria cell-free extract
Mixing 2.5 mmol/L1, 10-phenanthroline solution, PBS (pH7.4) and sampleThe solution was mixed with 1mL of each, and 1mL of 2.5mmol/L FeSO was added4The solution is fully mixed, 1mL of 20mmol/L H is added2O2And (3) solution. The mixed solution is put into a water bath with the temperature of 37 ℃ for constant temperature reaction for 1.5 h. The supernatant was collected by centrifugation at 6000r/min for 10min and the absorbance was measured at 536 nm. Meanwhile, according to the method, 1mL of sample solution to be detected in the mixed solution is replaced by 1mL of LPBS to be used as a blank control group, and the light absorption value at 536nm is measured. Calculated according to the following formula: hydroxy radical scavenging rate (%) - (1-A)536sample/A536Control) × 100. In the formula: a. the536Sample and A536The control is the absorbance of the sample and control at 536 nm.
1.3.3 measurement of the reducing ability of cell-free extract of lactic acid bacteria
0.5mL of the cell-free extract to be tested was added with 0.5mL of 1% potassium ferricyanide solution and 0.5mL of PBS (pH6.6), mixed well and placed in a 50 ℃ water bath for constant temperature reaction for 20 min. Taking out from the water bath, rapidly cooling in ice bath, adding 0.5mL of 10% trichloroacetic acid solution, mixing, centrifuging, and collecting supernatant (3000r/min, 10 min). Taking 1mL of supernatant, adding 1mL of 0.1% ferric chloride solution, and mixing uniformly. After 10min, the absorbance was measured at 700nm, using L-cysteine hydrochloride as the standard. Meanwhile, the sample in the mixed solution is replaced by distilled water as a control group, and the light absorption value is measured at the wavelength of 700 nm. The larger the light absorption value is, the stronger the reducing ability of the sample to be detected is.
1.4 measurement of alpha-glucosidase activity inhibition ability of Strain
Mixing 20 μ L of 0.1mol/L PBS (pH 7.0), 20 μ L of 2.5mmol/L PNPG, 20 μ L of 0.2U/mL alpha-glucosidase solution with 20 μ L of bacterial suspension or cell-free extract, adding 80 μ L of 0.2mol/L Na in water bath at 37 deg.C for 15min2CO3The reaction was terminated. The absorbance of the reaction solution was measured at 405 nm. The absorbance is proportional to the amount of free p-nitrophenol (PNP). In the reaction system, 0.1mol/L PBS with pH of 7.0 is used as a blank control of an alpha-glucosidase solution and a sample to be detected, and the inhibitory activity of the sample is calculated by adopting the following formula: enzyme activity inhibition (%) ([ 1- (C-D)/(A-B)] ×100. In the formula: a is a light absorption value measured by a sample containing alpha-glucosidase and not containing the alpha-glucosidase; b is a measured light absorption value without alpha-glucosidase and a sample; c is a determination light absorption value containing alpha-glucosidase and a sample; d is the measured absorbance of the sample without alpha-glucosidase.
1.5 Strain tolerance test
1.5.1 acid tolerance test
The strains were incubated at 37 ℃ and pH2, 3 and the survival rate, which is the percentage of the ratio of the log of viable bacteria at different incubation times to the log of viable bacteria incubated for 0h at the respective pH, was determined.
1.5.2 bile salt tolerance test
Experiments were performed at bile salt concentrations of 0.3%, 0.5% and 1% with reference to the method described at 1.5.1.
1.5.3 simulation of digestive tract tolerance test
The method described with reference to 1.5.1.
1.6 Strain adhesion Capacity test
The adhesion test is carried out in a well plate, and the plating process of a 12-well plate is as follows (the inoculation density is 2 multiplied by 10)5cells/well): adding 500 mu L of Caco-2 cell suspension which is washed for 2 times by PBS, digested by pancreatin, centrifuged and resuspended in DMEM culture medium into each hole of the hole plate, supplementing 500 mu L of cell culture medium to ensure that the culture system is 1mL/well, fully and uniformly mixing (which is very critical) in 8 or cross shape to prevent the cells from falling off due to uneven dispersion in the hole plate, sealing and placing in CO2Culturing in an incubator. The solution was changed every 2 days. After the cells grow to a monolayer (namely, a completely differentiated state is achieved, about 7 days), the adhesion test can be carried out (the cell culture medium needs to be changed into a double-antibody-free culture medium 24 hours before the test): the cultured overnight culture of Bifidobacterium is suspended in DMEM medium without dual-antibody after centrifugation and washing for 2 times, and the thallus concentration is adjusted to 108CFU/mL. The old medium in the well plate is discarded, the cell layer is washed for 2 times, the bacterial suspension is added according to 1mL/well, and the mixture is placed in an incubator for 2 hours. After the culture is finished, the cells and the adhered thalli are rinsed for 5 times, digested for 5min, and the eluted cells and the adhered thalli are collected into a centrifugal tube after the digestion is finished. Diluting and mixingAnd coating the mixture on a mMRS solid agar culture dish, and evaluating the adhesion capacity by calculating the ratio of the number of bacteria adhered to each hole to the number of bacteria added to each hole before adhesion.
1.7 statistical analysis
Data processing and significance analysis were performed using SPSS 20.0, with 3 replicates per experiment.
2 results
2.1DPPH scavenging Capacity
As shown in FIG. 1, intact cells and cell extracts of Bifidobacterium animalis BB12, H15-2 and H20-9 all had DPPH scavenging ability. Bifidobacterium animalis H15-2 and H20-9-intact cell DPPH clearance is significantly lower than BB12(p < 0.05), and there is no significant difference between strains H15-2 and H20-9 (p > 0.05). DPPH clearance of cell extracts of strains H15-2 and H20-9 is significantly higher than BB12(p < 0.05), wherein, the difference between the strains H20-9 and H20-2 is significant (p < 0.05).
2.2 hydroxyl radical scavenging Capacity
Bifidobacterium animalis BB12, 15-2 and 20-9 whole cells and cell extracts all have hydroxyl radical scavenging ability. The clearance rate of hydroxyl free radical of complete cells of the animal bifidobacterium 15-2 and 20-9 is obviously higher than BB12(p < 0.05), wherein the clearance rate of the animal bifidobacterium 15-2 is 24.59 percent at most and is obviously higher than 20-9(p < 0.05). The hydroxyl radical clearance rate of the 15-2 cell extract is obviously higher than that of BB12 and 20-9(p is less than 0.05). 20-9 was lower than BB12, with no significant difference (p > 0.05).
2.3 reducing ability
The whole cells and cell extracts of Bifidobacterium animalis BB12, 15-2 and 20-9 have certain reducing ability. Both the intact cells and the cell extract of Bifidobacterium animalis 15-2 were significantly higher in reducing power than strain BB 12. The reduction capability of the strain 20-9 for the intact cells is not obviously different from that of the strain BB12, but is obviously lower than that of the strain 15-2(p is less than 0.05). The cell extract reducing capacity of the strain 20-9 is obviously higher than that of the strain BB12(p is less than 0.05) and lower than that of the strain 15-2, and the two strains have no obvious difference.
2.4 alpha-glucosidase Activity inhibitory Capacity of Strain
The inhibition rate of bifidobacterium animalis 15-2 on alpha-glucosidase is obviously higher than that of the strain 20-9(p is less than 0.05), the inhibition rate of the bifidobacterium animalis on alpha-glucosidase is 26.90 percent and 12.83 percent respectively, and the strain BB12 has no inhibition effect on the activity of the alpha-glucosidase.
2.5 acid tolerance of the Strain
As shown in FIG. 2, the survival rates of Bifidobacterium animalis BB12, 15-2 and 20-9 were all decreased with the increase of the incubation time under acidic conditions. However, the survival rate of Bifidobacterium animalis 15-2 at pH2 was significantly higher than that of strain BB12(p < 0.05), whereas strain 20-9 was significantly lower than strain BB12(p < 0.05), with a significant difference between strain 15-2 and strain 20-9(p < 0.05). During the incubation for 0-3 h under the condition of pH3, the survival rate of the bifidobacterium animalis 15-2 is always higher than that of BB12, and the survival rate of the strain 20-9 is always lower than that of the strain BB 12. The survival rate of 15-2 was significantly higher than that of strain BB12(p < 0.05), that of strain 20-9 was significantly lower than that of strain BB12, and that of all the three was significantly different (p < 0.05) at 3h of incubation.
2.6 bile salt tolerance of the Strain
As shown in FIG. 3, the survival rates of Bifidobacterium animalis BB12, 15-2 and 20-9 were all decreased with the increase of the incubation time under the conditions containing 0.3%, 0.5% and 1% of bile salts. When the bifidobacterium animalis 15-2 is incubated for 8 hours under the condition of 0.3 percent of bile salt, the survival rate of the bifidobacterium animalis 15-2 is 72.41 percent, although the survival rate is higher than that of the strain BB12, the survival rate of the strain 20-9 is not obviously different from that of the strain BB12(p is less than 0.05). The survival rate difference between the strains 15-2 and 20-9 is significant (p < 0.05). There was no significant difference in survival between strains 15-2 and BB12, strains 20-9 and BB12, and strains 15-2 and 20-9 when incubated for 8h at a bile salt concentration of 0.5%. The survival rate of Bifidobacterium animalis 20-9 was significantly lower than that of strain BB12(p < 0.05) when incubated for 8h at 1% bile salt, the survival rate of strain 15-2 was not significantly different from that of strain BB12, nor was the difference between strain 20-9 and strain 15-2.
2.7 adhesion Capacity to Caco-2 cells
As shown in FIG. 4, the adhesion rates of Bifidobacterium animalis 15-2 and 20-9 to Caco2 cells were both higher than BB12, but both were not significantly different from BB12, and the adhesion rate of strain 15-2 was 27.59% at most, and was not significantly different from strain 20-9.
2.8 adhesion inhibition of the Strain
As shown in FIG. 5, Bifidobacterium animalis BB12, 15-2 and 20-9 had a competitive adhesion inhibition effect on Escherichia coli ATCC25922, the adhesion inhibition rate of Bifidobacterium animalis 20-9 was significantly lower than that of BB12(p < 0.05), there was no significant difference between 15-2 and BB12, and the adhesion inhibition rate of strain 15-2 was significantly higher than that of 20-9(p < 0.05). The three also have adhesion inhibition effect on salmonella typhimurium ATCC14028, bifidobacterium animalis 15-2 is lower than BB12, the adhesion inhibition rate of the strain 20-9 is lower than BB12, the adhesion inhibition rate of the strain 20-9 is not obviously different (p is more than 0.05), the strain 20-9 is higher than the strain 15-2, and the difference between the two is not obvious.
Bifidobacterium animalis BB12, 20-9 and 15-2 all have exclusive adhesion inhibition effect on Escherichia coli ATCC25922, and the adhesion inhibition rates of the Bifidobacterium animalis BB12, the Bifidobacterium animalis BB 899 and the Bifidobacterium animalis BB 25922 are 25.22%, 12.53% and 18.65%, wherein the adhesion inhibition rates of the strains 20-9 and 15-2 are respectively remarkably lower than BB12(p is less than 0.05), and the adhesion inhibition rate of the strain 15-2 is remarkably higher than 20-9(p is less than 0.05). The three strains also have adhesion inhibition effect on Salmonella typhimurium ATCC14028, the adhesion inhibition rate of the strain 20-9 is obviously lower than that of BB12, although the adhesion inhibition rate of 15-2 is higher than that of BB12, the difference is not obvious, and the bifidobacterium animalis 15-2 is obviously higher than 20-9(p is less than 0.05).
Bifidobacterium animalis BB12, 15-2 and 20-9 has an alternative adhesion effect on Escherichia coli ATCC25922, the adhesion inhibition rate of the strain BB12 is 24.36 percent at most, the adhesion inhibition rate of the strains 15-2 and 20-9 is obviously lower than that of BB12(p is less than 0.05), and the adhesion inhibition rate of the strain 20-9 is 6.41 percent at least and is obviously lower than that of the strain 15-2(p is less than 0.05). The three also have adhesion inhibition effect on Salmonella typhimurium ATCC14028, the adhesion inhibition rate of Bifidobacterium animalis 20-9 is significantly lower than BB12(p is less than 0.05), the adhesion inhibition rate of the Bifidobacterium animalis 15-2 is lower than BB12, no significant difference exists between the two, the adhesion inhibition rate of the Bifidobacterium animalis 15-2 is higher than 20-9, and the adhesion inhibition rate of the Bifidobacterium animalis 15-9 is significantly different between the two (p is less than 0.05).
3. Conclusion
In the experiment, the oxidation resistance (hydroxyl radical scavenging capacity and reducing capacity) of the bifidobacterium animalis 15-2 is higher than that of the strain 20-9 and the strain BB 12; the activity inhibition ability of the alpha-glucosidase is higher than that of the strain 20-9 and the strain BB 12; the survival rate and the adhesion rate of the strain 15-2 to Caco-2 cells after simulation of the digestive tract are higher than those of the strain 20-9 and the strain BB 12; the acid tolerance of the strain 15-2 is higher than that of the strains 20-9 and BB12, and the adhesion inhibition capability of the strain on Salmonella typhimurium ATCC14028 and Escherichia coli ATCC25922 is slightly inferior to that of the strain BB12, but still better than that of the strain 20-9. Therefore, the bifidobacterium animalis 15-2 can be used as a test strain for potential intervention in diabetes mellitus for the next animal experiment.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims (3)

1. The Bifidobacterium animalis (Bifidobacterium animalis) H15-2 is characterized in that the Bifidobacterium animalis H15-2 is Bifidobacterium (Bifidobacterium), is preserved in a northeast agriculture university strain bank, and has the preservation date of 2017, 6-20 days and the preservation number of H15-2.
2. Bifidobacterium animalis H15-2 as potential diabetes-intervening strain according to claim 1.
3. The bifidobacterium animalis H15-2 can enhance the antioxidant capacity of the body, the hydroxyl radical clearance rate of the intact cells is up to 24.59%, and the alpha-glucosidase inhibition rate is up to 26.90%.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
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CN107893044A (en) * 2017-12-27 2018-04-10 江南大学 One plant of bifidobacterium longum and its application
KR20180044245A (en) * 2018-04-06 2018-05-02 주식회사 종근당바이오 Novel Lactic Acid Bacteria Having Constipation Improvement Effect and Use Thereof

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