CN113308421B - Lactobacillus plantarum BUFX and application thereof in metabolic syndrome - Google Patents
Lactobacillus plantarum BUFX and application thereof in metabolic syndrome Download PDFInfo
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- CN113308421B CN113308421B CN202110867435.6A CN202110867435A CN113308421B CN 113308421 B CN113308421 B CN 113308421B CN 202110867435 A CN202110867435 A CN 202110867435A CN 113308421 B CN113308421 B CN 113308421B
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Abstract
The invention provides a lactobacillus plantarum (A)Lactobacillus plantarum) BUFX and application thereof in metabolic syndrome, relating to the technical field of microorganisms and application thereof. The strain is preserved in the China general microbiological culture Collection center, and the preservation numbers are as follows: CGMCC No. 22172. The lactobacillus plantarum is obtained by separating and screening adult excrement, can promote glucose consumption of HepG2 liver cancer cells, inhibit alpha-amylase activity, remarkably reduce the weight and the blood fat content of high-fat diet mice, improve glucose tolerance and have important application significance for preventing or treating metabolic diseases such as diabetes, obesity and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms and application thereof, in particular to lactobacillus plantarum (A)Lactobacillus plantarum) BUFX and its use in metabolic syndrome.
Background
With the improvement of living standard of people and the change of dietary structure, people can eat and drink food without avoiding food, eat more food and move less food, and the diabetes is also called as 'riches and honor disease'. This modern lifestyle is prone to excess glucose in the body, resulting in elevated blood glucose, which often leads to the development of diabetes mellitus as a result of prolonged hyperglycemia or elevation of blood glucose. Over time, diabetes can damage the cardiovascular, renal and nervous systems, and is currently one of the major causes of blindness, renal failure, heart attack, stroke and lower limb amputation, severely harming human health. According to international world health organization statistics, the number of diabetic patients rises from 1.08 billion in 1980 to 4.22 billion in 2014, and the number of deaths due to hyperglycemia or diabetes is as high as several million each year.
The relationship between intestinal microorganisms and human health is increasingly recognized, and healthy intestinal flora contributes to maintaining the health of the organism to a great extent. The quantity and diversity of intestinal probiotic flora are closely related to the glucose tolerance of human body. Dysbacteriosis is one of the important factors for the development of metabolic diseases such as diabetes and obesity. Numerous studies have shown that blood glucose levels show different decreases after ingestion of certain probiotics or probiotic fermented foods, whether in animal models in animal experiments or in diabetic patients in clinical trials.
Traditional hypoglycemic treatments mainly include drug and diet control of blood glucose and exercise. The long-term use of hypoglycemic drugs can easily improve the drug resistance of pancreatic islets and cause organ function decline, the other drug is insulin injection, but is easy to cause allergy, has short effective period, can cause rebound of blood sugar level when the drugs are stopped, and can cause hypoglycemia and is more dangerous when the dosage is excessive. Because the probiotics have edibility, the probiotics have high safety in reducing blood sugar, and can not generate additional side effect on organisms. At present, 35 edible probiotics or subspecies probiotics are published in China, but because the pathogenesis of diabetes is complex, and the functions of the probiotics have strain specificity, the influence of the probiotics on intestinal flora and host metabolism is different. Therefore, the search and discovery of probiotic strains with excellent hypoglycemic effects have important market significance for preventing and treating hyperglycemia and diabetes.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum with the efficacy of reducing hyperglycemia and application thereof; the lactobacillus plantarum can promote glucose consumption of HepG2 liver cancer cells, inhibit alpha-amylase activity, remarkably reduce the weight, blood fat and high blood sugar content of high-fat diet mice, improve glucose tolerance and insulin resistance and relieve liver steatosis.
The invention provides lactobacillus plantarum (with preservation number of CGMCC number 22172) in the first aspectLactobacillus plantarum) Or a culture supernatant thereof or a dead bacterium thereof.
Wherein, the lactobacillus plantarum (A)Lactobacillus plantarum) Named BUFX, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC 22172 at 12.4.2021 and the preservation address of China academy of sciences microbial research institute No. 3, Xilu No. 1, North Cheng, the Korean district, Beijing.
The lactobacillus plantarum BUFX with the efficacy of reducing the hyperglycemia is screened, and the strain is identified to be lactobacillus plantarum (BUFX) through mass spectrometryLactobacillus plantarum）。
The strain can also be prepared into a microbial agent containing the bacteria (live bacteria or dead bacteria) of lactobacillus plantarum BUFX or a culture (such as culture supernatant) of lactobacillus plantarum BUFX. In a specific scheme, the microbial agent can be a liquid microbial agent or a solid microbial agent. Further, the total number of the colonies of Lactobacillus paracasei contained in the microbial agent is 0.5 × 107-2.5×1012 cfu·mL-1Or 0.5X 107-2.5×1012 cfu·g-1。
In a second aspect, the invention provides the use of the lactobacillus plantarum described above, or a culture supernatant thereof, or a dead bacterium thereof, in the preparation of a medicament for ameliorating and/or preventing metabolic syndrome.
In a third aspect, the present invention provides the use of the lactobacillus plantarum described above, or a culture supernatant thereof, or a dead bacterium thereof, for the preparation of a medicament for ameliorating and/or preventing hyperglycemia. The lactobacillus plantarum has the effects of promoting glucose consumption of HepG2 liver cancer cells and inhibiting alpha-amylase activity, and therefore, the lactobacillus plantarum also has the effects of promoting glucose consumption of HepG2 liver cancer cells and inhibiting alpha-amylase activity.
In a fourth aspect, the present invention provides the use of the lactobacillus plantarum described above, or a culture supernatant thereof, or a dead bacterium thereof, in the preparation of a medicament for improving lipid metabolism.
In a fifth aspect, the present invention provides the use of the lactobacillus plantarum described above, or a culture supernatant thereof, or a dead bacterium thereof, for the preparation of a medicament for improving and/or preventing lipid accumulation.
The sixth aspect of the present invention provides the use of the lactobacillus plantarum described above, or a culture supernatant thereof, or a dead bacterium thereof, for the preparation of a medicament for ameliorating and/or preventing hyperlipidemia.
The seventh aspect of the present invention provides the use of the lactobacillus plantarum described above, or a culture supernatant thereof, or a dead bacterium thereof, in the preparation of a medicament for improving and/or preventing fatty liver.
The tenth aspect of the present invention provides a pharmaceutical composition comprising the lactobacillus plantarum described above or a culture supernatant thereof or a dead bacterium thereof.
In an eleventh aspect, the present invention provides a method of treating metabolic syndrome in a patient, comprising administering to said patient lactobacillus plantarum or its culture supernatant or dead bacteria thereof according to the first aspect, or a pharmaceutical composition according to the tenth aspect.
The metabolic syndrome referred to in the present invention includes, but is not limited to, obesity, hyperglycemia, hypertension, dyslipidemia, high blood viscosity, hyperuricemia, high fatty liver incidence, insulin resistance, and hyperinsulinemia.
It is noted that the improvement referred to in the present invention includes, but is not limited to, lowering blood glucose levels, reducing body weight, lowering blood lipid levels, inhibiting alpha-amylase activity, reducing insulin resistance, increasing glucose tolerance, and alleviating hepatic steatosis.
It is to be noted that the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, such as a solid carrier or a liquid carrier, specifically, for example, bentonite, calcium carbonate, zeolite, starch; or vegetable oil, mineral oil, water, and the like. The pharmaceutical composition may be prepared in various dosage forms such as lyophilized powder, tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions or dosage forms well known to those of ordinary skill in the art of pharmaceutical formulation.
Where the dosage form or pharmaceutically acceptable carrier is a solid pharmaceutical composition (e.g., capsules, tablets, and powders), suitable binders including starches, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like, lubricants, disintegrants, colorants, flavoring agents, glidants, melting agents, and the like, may be included. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to: starch, methyl cellulose, agar, bentonite, xanthan gum, etc.
In addition, capsules (e.g., gelatin capsules) may contain the active ingredient and powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be made into immediate release products or sustained release products. Compressed tablets may be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated to allow selective disintegration in the gastrointestinal tract.
Wherein, when the dosage form or pharmaceutically acceptable carrier is a liquid pharmaceutical composition, the liquid pharmaceutical composition comprises a solution or suspension in water, pharmaceutically acceptable fats and oils, alcohols including esters or other organic solvents, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may include, for example, suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, thickeners, and melting agents.
Wherein the liquid pharmaceutical composition for oral administration may contain coloring and flavoring agents to improve patient acceptance. Generally, water, suitable oils, saline, aqueous dextrose (glucose) and corresponding sugar solutions, and glycols (e.g., propylene glycol or polyethylene glycol) are suitable carriers for parenteral solutions. For example, in the form of tablets or capsules for oral use, the active ingredient may be mixed with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.
Compared with the prior art, the invention has the beneficial effects that:
(1) the lactobacillus plantarum BUFX provided by the invention can promote glucose consumption of HepG2 liver cancer cells, inhibit alpha-amylase activity, remarkably reduce the body weight and blood fat content of high-fat diet mice, improve glucose tolerance and insulin resistance, and relieve liver steatosis, and has important application significance for preventing or treating metabolic diseases such as diabetes, obesity and the like.
(2) The Lactobacillus plantarum BUFX strain disclosed by the invention is obtained by screening through a large amount of work, and has more excellent blood sugar reducing capability than other Lactobacillus plantarum strains. The discovery of the strain provides a new microbial resource for hypoglycemic drugs.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a diagram showing the mass spectrometric identification of Lactobacillus plantarum BUFX according to the invention;
FIG. 2 shows the resistance of Lactobacillus plantarum BUFX in the present invention to artificial gastric juice (A), artificial intestinal juice (B) and bile salts (C);
FIG. 3 shows the inhibition of the activity of alpha-amylase (A) and alpha-glucosidase (B) by the live Lactobacillus plantarum BUFX bacteria and fermentation supernatant according to the invention; wherein, acarbose is used as a positive control;
FIG. 4 shows the effect of Lactobacillus plantarum BUFX fermentation supernatant in the present invention on glucose consumption by HepG2 hepatoma cells, with insulin as positive control and 3 concentrations of supernatant set for 10%, 20% and 30% (v/v cell culture medium volume), respectively;
FIG. 5 shows the effect of live and dead Lactobacillus plantarum BUFX solution in the present invention on the body weight of mice fed with high fat;
FIG. 6 shows that Lactobacillus plantarum BUFX in the present invention improves glucose and insulin resistance in high fat fed mice; (A) fasting blood glucose value of mice fed with high fat, (B) OGTT oral glucose tolerance test of mice;
FIG. 7 shows that Lactobacillus plantarum BUFX in the present invention reduces the serum triglyceride TG (A), total cholesterol TC (B) and low-density lipoprotein cholesterol LDL-c (C) levels in high-fat-fed mice.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Unless otherwise defined, all terms (including 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. In case of conflict between the prior art and the present disclosure, the present disclosure should control.
Example 1: sample collection and strain isolation
The method comprises the steps of collecting excrement of healthy adults living in Beijing urban areas for a long time as a screening sample, and collecting the history that the target people do not take antibiotic medicines before collection, and have no probiotic taking history and no gastrointestinal disease history. Diluting the collected excrement sample, coating the diluted excrement sample on a YCFA culture medium, carrying out anaerobic culture at 37 ℃ for 24-48h, and separating to obtain different single colonies on the plate. And selecting different single colonies by using the sterilized inoculating loops, streaking and purifying the single colonies on a new YCFA solid culture medium plate, and carrying out anaerobic culture at 37 ℃ for 48h to obtain 29 purified colonies which are respectively marked as L1-L29.
It should be noted that the dilution of the stool sample is a conventional method in the art, and the present invention is not limited thereto, and in one embodiment of the present invention, the stool sample is diluted with clear water.
Example 2: preparation of live bacterial liquid, metabolite and inactivated thallus
2.1 cultivation of the Strain
Coating the frozen bacteria liquid at-80 ℃ on a YCFA solid plate, performing inverted culture at 37 ℃ for 24-48h, inoculating a single colony in a liquid YCFA culture medium, and performing culture at 37 ℃ for 18-24h to obtain a first generation bacteria liquid; inoculating 10% of a first-generation bacterial liquid to a fresh YCFA liquid culture medium, and culturing at 37 ℃ for 18-24h to obtain a second-generation bacterial liquid; inoculating 10% of the second-generation bacterial liquid into a fresh YCFA liquid culture medium, and culturing at 37 ℃ for 18-24h to obtain a working bacterial liquid.
2.2 obtaining of viable bacteria liquid
In the embodiment of the invention, the working bacterial liquid obtained in the step 1 is placed at 13000 rpm and centrifuged at 4 ℃ for 15 min, then the supernatant is discarded, the precipitate is collected and resuspended by normal saline, and the viable bacterial liquid with viable bacteria is obtained.
The viable cell liquid can also be obtained by other means in the technical field as long as the cells can be enriched from the culture solution. This can be achieved, for example, by means of centrifugation and/or filtration.
2.3 obtaining of metabolites
Since metabolites of bacterial cells generally exist in a culture solution of bacterial cells, the metabolites can be obtained by subjecting the culture solution of bacterial cells to solid-liquid separation to obtain a supernatant. Of course, the preparation of the bacterial culture supernatant can also be carried out in an anaerobic environment. In one embodiment of the invention, specifically, the working bacteria liquid is centrifuged at 13000 rpm and 4 ℃ for 15 min, the supernatant is left and transferred to a sterile centrifuge tube, and then the bacterial culture supernatant is obtained and stored at 4 ℃ for later use, and the metabolite is obtained.
2.4 obtaining of dead bacteria solution
The dead bacteria may be prepared by means conventional in the art, for example, heating, irradiation, and the like. In one embodiment of the invention, the dead bacteria liquid of dead bacteria is obtained by heating live bacteria for 1h at the temperature of 65-85 ℃ for killing.
Example 3 screening and identification of Strain having cell glucose cell promoting action
3.1 identification of the Strain
And (3) carrying out streak culture on the separated single colony, then selecting the single colony to be coated on a mass spectrum plate, adding lysis solution and matrix respectively, drying, and then identifying by using a MALDI-TOF MS 1000 mass spectrometer (Autobio) to obtain the information of each strain.
3.2 cell glucose consumption detection experiment
The human liver cancer HepG2 cells used by the invention are purchased from national biomedical experimental cell resource libraries. The cell culture is performed by a culture method known in the art, and the present invention is not limited thereto.
HepG for human liver cancer2 cell culture at 37 ℃ and 5% CO2The double antibody (100 mug/ml penicillin and 100 mug/ml streptomycin) was added to the culture medium at a ratio of 1:100 in a high glucose DMEM medium containing 10% Fetal Bovine Serum (FBS) under the conditions, and the fresh culture medium was replaced every 1-2 d.
And observing and inoculating the human liver cancer HepG2 cells with good growth state and logarithmic growth phase into a 96-well plate at proper concentration, adding 100 mu l of cell suspension into each well, reserving a row of non-added cells as a blank group, and culturing for 24h until the cells are completely attached to the wall. The original culture medium is discarded during the experiment, and the 96-well plate is lightly tapped to ensure that the culture medium is completely poured. And changing a high-sugar phenol red-free incomplete culture medium into one group of 8 holes, and then carrying out corresponding treatment, wherein 30% of fermentation supernatant (L1-L29) of the working strain prepared in the above volume is added into each hole of the sample group, YCFA of the culture medium used by the culture strain with the same volume is added into the control group, and insulin solution (the final concentration is 10 mu mol/L) prepared by the incomplete culture medium is added into the positive group. Putting the cells into an incubator for incubation and culture for 24h, taking 2 mul of culture medium supernatant from each well, putting the culture medium supernatant into a new 96-well plate, adding 250 mul of reaction liquid according to the operational instruction of building a glucose oxidase kit from Nanjing, and putting the reaction liquid into an oven at 37 ℃ for reaction for 10 min. After the reaction solution is completely developed, measuring the absorbance at 505 nm by using an enzyme-labeling instrument, and then calculating the promoting effect of the strain fermentation liquid on the glucose consumption of HepG2 cells according to the kit specification, wherein the experimental results are shown in the following table 1.
As can be seen from Table 1, the 29 isolated strains all belong to the genus Lactobacillus, including 5 species of Lactobacillus mucosae, Lactobacillus salivarius, Lactobacillus pentosus, Lactobacillus plantarum and Lactobacillus sake. The 29 strains all show different degrees of promotion effects on the glucose consumption of HepG2 cells, wherein the L14 lactobacillus plantarum has the strongest promotion effect on the consumption, which suggests that the strains can reduce the storage of glucose and achieve the purpose of reducing blood sugar by promoting the glucose consumption of the cell cells. The mass spectrometric identification result of FIG. 1 shows number L14The bacteria belong to the species Lactobacillus plantarum, from which we have named this strain Lactobacillus plantarum (A)Lactobacillus plantarum）BUFX。
Example 4 evaluation of gastrointestinal tolerance of Lactobacillus plantarum BUFX
4.1 tolerance assay for Lactobacillus plantarum BUFX to Artificial gastric and intestinal juices
4.1.1 preparation of Artificial gastrointestinal fluids
The preparation of the artificial gastrointestinal fluid used in the invention refers to Chinese pharmacopoeia.
Artificial gastric juice: firstly, preparing dilute hydrochloric acid, measuring 234 ml of concentrated hydrochloric acid, adding water to dilute the concentrated hydrochloric acid to 1000 ml to obtain 9.5-10.5% dilute hydrochloric acid, then taking 16.4 ml of dilute hydrochloric acid, adding 800 ml of water and 10g of pepsin, shaking up, and adding water to dilute the dilute hydrochloric acid to 1000 ml to obtain the artificial gastric juice.
Artificial small intestine liquid: taking 6.8 g of monopotassium phosphate, adding 500 ml of water to fully dissolve the monopotassium phosphate, adjusting the pH value to 6.8 by using 0.1mol/L sodium hydroxide solution, weighing 10g of pancreatin, adding water to dissolve the pancreatin, mixing the two solutions, and adding water to dilute the solution to 1000 ml to obtain the artificial intestinal juice.
4.1.2 tolerance testing of strains
Collecting cultured viable bacteria, centrifuging at 13000 rpm at 4 deg.C for 15 min, discarding liquid, collecting thallus precipitate, resuspending with normal saline, and counting viable bacteria number 109CFU/ml is respectively inoculated into the artificial stomach/intestinal juice, samples are taken after 0h, 1h, 2h, 3h and 4h of culture at the temperature of 37 ℃ for viable count detection, the viable count of 0h is taken as a control, the survival rate is calculated, the survival rate (%) = (viable count of 1h/2h/3h/4 h/viable count of 0 h) × 100%, and the experimental result is shown as A, B in figure 2.
The results in fig. 2 a show that the strain of lactobacillus plantarum BUFX according to the present invention exhibits better acid resistance to artificial gastric juice, and after 1h incubation, the survival rate of lactobacillus plantarum BUFX was 92.3%, and after 2h survival rate was greater than 82.5%, and after 4h incubation time, the survival rate of BUFX strain was as high as 62.4%, which indicates that lactobacillus plantarum BUFX can maintain a higher survival rate in a strong acid environment, can withstand the examination of the strong acid environment in the stomach, and is favorable for its full probiotic effectiveness.
The results in fig. 2B show that the lactobacillus plantarum BUFX strain has better intestinal fluid tolerance, and the survival rate of the lactobacillus plantarum BUFX of the present invention is as high as 81.4% after 1h in an environment with pH 6.8, more than 70.3% after 2h, and 53.7% after 4h, indicating that lactobacillus plantarum BUFX has better intestinal fluid tolerance and can retain a higher bacterial load in the intestinal tract, thereby contributing to the probiotic effect.
4.2 Lactobacillus plantarum BUFX bile salt tolerance assay
Pressing the collected live bacteria 109CFU/ml was inoculated into solutions of bile salt concentration 0, 0.25%, 0.5% and 1%, respectively, cultured at 37 ℃ for 4 hours, and then viable cell count was measured and calculated, with viable cell count of 0 hours as a control, and the survival (%) = (0.25%/0.5%/viable cell count in 1% bile salt solution/viable cell count of 0 hours after 4 hours) × 100%, with the results of the measurement shown in C in fig. 2.
According to the results shown in C in FIG. 2, it can be seen that the Lactobacillus plantarum BUFX strain of the invention has better tolerance to bile salts, the survival rate of the Lactobacillus plantarum BUFX of the invention can reach 65.2% when 0.25% of bile salts is added compared with the group without bile salts, and the survival rate of the Lactobacillus plantarum BUFX strain can still be 48.4% when the content of bile salts is increased to 1%, therefore, the above results show that the Lactobacillus plantarum BUFX of the invention can well tolerate bile salts and meet the survival requirement of probiotics in the human gastrointestinal digestive tract.
Example 5: inhibition of amylases and glycosidases by Lactobacillus plantarum BUFX
5.1 inhibition of Amylase inhibition assay by Lactobacillus plantarum BUFX
Taking 2 ml of alpha-amylase solution (prepared by PBS buffer solution with the concentration of 50 mM and the pH value of 7.0) with the mass concentration of 2 mg/ml, respectively adding 2 ml of sample solution (BuFX viable bacteria suspension and fermentation supernatant), reacting at 37 ℃ for 30 min, adding 2 ml of 1% soluble starch, reacting at 37 ℃ for 15 min, adding iodine solution for color development, and measuring the light absorption value OD (optical density) at 660 nm by using an enzyme-labeling instrument660. In which YCFA medium was used as a negative control and acarbose was used as a positive control, and the results of the experiment are shown in FIG. 3A.
The results a in fig. 3 show that: compared with the positive drug acarbose, the average value of the amylase inhibition rates of the BuFX viable bacteria suspension and the fermentation supernatant is about 47.5 percent and 42.1 percent, which shows that the bacterial strain and the metabolite thereof have higher amylase inhibition activity, and the bacterial strain provided by the invention has the potential of effectively inhibiting the activities of salivary amylase and pancreatic amylase.
5.2 Effect of Lactobacillus plantarum BUFX on alpha-glucosidase
Taking 30 mul of Lactobacillus plantarum BUFX sample (comprising viable bacteria suspension and fermentation supernatant) to mix with 30 mul of alpha-glucosidase enzyme solution (0.1U/ml), incubating for 10 min at 37 ℃, then adding 60 mul of substrate PNPG (0.5 mM), reacting for 20 min at 37 ℃, and adding 100 mul of 2M sodium carbonate solution to terminate the reaction. Then, the absorbance OD at 405nm was measured by a microplate reader405. In which YCFA medium was used as a negative control and acarbose was used as a positive control, and the results of the experiment are shown in FIG. 3B.
The B results in fig. 3 show: the inhibition rates of alpha-glucosidase of the BuFX viable bacteria suspension and the fermentation supernatant are respectively 24.1% and 26.8%, and the inhibition rate of acarbose can reach 33.2%. The strain and the metabolite secreted to the extracellular have strong alpha-glucosidase inhibitory activity, and the substance with the alpha-glucosidase inhibitory activity can effectively prevent and treat postprandial hyperglycemia and relieve hyperinsulinemia, so that the strain provided by the invention has high possibility of exerting the effects of preventing and treating the postprandial hyperglycemia and relieving the hyperinsulinemia in vivo.
Example 6: quantitative effect analysis of Lactobacillus plantarum BUFX fermentation supernatant for promoting hepatocyte glucose consumption
The human liver cancer HepG2 cells used by the invention are purchased from national biomedical experimental cell resource libraries. The cell culture is performed by a culture method known in the art, and the present invention is not limited thereto.
The cell culture and operation methods in this example were substantially the same as those in example 3. The human liver cancer HepG2 cells with good growth state and logarithmic growth phase are inoculated into a 96-well plate at proper concentration, 100 mu l of cell suspension is added into each well, a row of cells which are not added is reserved as a blank group, and the cells are cultured for 24h until the cells are completely attached to the wall. The original culture medium is discarded during the experiment, and the 96-well plate is lightly tapped to ensure that the culture medium is completely poured. Changing a high-sugar phenol red-free incomplete culture medium for every 8 holes, and then carrying out corresponding treatment, wherein the fermentation supernatant of BUFX strain with the volume of 10%/20%/30% is added into each hole of the sample group, the culture medium YCFA used by the culture strain with the same volume is added into the control group, and the insulin solution (the final concentration is 10 mu mol/L) prepared by the incomplete culture medium is added into the positive group. And putting the cells into an incubator for incubation and culture for 24h, and then determining the glucose content in the supernatant of the culture medium. 2 mul of culture medium supernatant is taken from each well and placed in a new 96-well plate, 250 mul of reaction liquid is added according to the operational instruction of building a glucose oxidase kit from Nanjing, and the reaction liquid is placed in a 37 ℃ oven for reaction for 10 min. After the reaction solution is completely developed, the absorbance at 505 nm is measured by using a microplate reader, then the glucose consumption of each group is calculated according to the kit specification, and the experimental result is shown in figure 4.
As can be seen from the results of the experiment in FIG. 4, the glucose content in the original cell culture medium was 22.5 mmol/L, the glucose content in the cell culture medium was significantly reduced after the addition of the Lactobacillus plantarum BUFX fermentation supernatant of the invention (10%, 20% and 30%), the average values were about 16.6 mmol/L and 15.8 mmol/L, respectively, and the glucose content in the culture medium was reduced to an average value of about 13.1 mmol/L after the positive control group of insulin-treated cells. Therefore, the fermentation supernatant of the Lactobacillus plantarum BUFX strain provided by the invention can obviously promote the HepG2 liver cancer cell to consume glucose and reduce the accumulation of glucose in the cell.
Example 7: application of lactobacillus plantarum BUFX in improving glycolipid metabolic disorder of high-fat mice
This example illustrates the efficacy of Lactobacillus plantarum BUFX of the invention in lowering blood glucose and improving glucose tolerance.
Animal experiments mice were purchased from Beijing Wittiaxle laboratory animals, and 8 week-old male C57BL/6 mice were selected and randomly divided into 4 groups (normal group Chow, model group HFD, Lactobacillus plantarum BUFX live bacterial liquid group, BUFX dead bacterial group), and 8 animals per group were randomly allocated. The normal group was given standard mouse diet, and the remaining groups were given high fat diet with 60% fat function for 8 weeks. Thereafter, a gavage experiment was performed in which live BuFX groups were used eachDaily administration is 1X 109 Live Lactobacillus plantarum BUFX from CFU; BuFX dead bacteria group was given daily to a killed bacteria heavy suspension, and model group was given an equal volume of physiological saline. The mice were weighed weekly for 4 weeks and the body weight data recorded, and the weight gain was recorded as the pre-anatomical weight minus the pre-gavage weight. And (3) performing an OGTT experiment, performing overnight fast on the day before, preparing a 20% glucose solution, performing glucose gavage according to a common dosage of 1 g/kg, timing, and detecting blood glucose values of glucose gavage at time points of 0, 30, 60, 90 and 120 min by using a Roche glucometer. Orbital bleeds were performed after the entire animal experiment was completed and supernatants were centrifuged at 4 ℃ as serum samples for measuring blood glucose and serum triglyceride TG, total cholesterol TC and low density lipoprotein cholesterol LDL-c levels.
The results in fig. 5 show that the mice gained significantly higher weight after high fat diet feeding than the normal feeding group, whereas the mice gained less weight after gavage of lactobacillus plantarum BUFX of the present invention (including live and dead bacterial groups), indicating that the BUFX strain was able to inhibit weight gain. As shown in FIG. 6, after analyzing serum samples of various groups of mice, it is found that the lactobacillus plantarum BUFX (including live bacterial group and dead bacterial group) in the invention can reduce the hyperglycemic value (238 mg/dL vs 157 mg/dL, 173 mg/dL) of mice in a high-fat model group in a fasting state, and the oral glucose tolerance OGTT experiment shows that both live bacteria and dead bacteria of BUFX can effectively improve the glucose tolerance caused by high-fat diet and enhance the sensitivity of organisms to glucose. Further serum biochemical indicator test results (fig. 7) show that: high-fat diet can lead to significant increase in the levels of triglyceride (B), total cholesterol (C) and low-density lipoprotein cholesterol (D) in mice, presenting a pronounced hyperlipidemia phenotype, suggesting a dysfunction in glycolipid metabolism in mice. The live lactobacillus plantarum BUFX bacteria and the inactivated bacteria can also obviously reduce the blood fat (TG, TC and LDL-c) level of animals, and the results comprehensively show that the lactobacillus plantarum BUFX has good blood sugar and blood fat reducing activity and can improve glycolipid metabolic disorder caused by high-fat diet.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. Lactobacillus plantarum (CGMCC number 22172 in preservation numberLactobacillus plantarum）BUFX。
2. Use of a lactobacillus plantarum as claimed in claim 1 for the preparation of a medicament for the amelioration and/or prevention of hyperglycemia.
3. Use of a lactobacillus plantarum as claimed in claim 1 for the preparation of a medicament for improving and/or preventing lipid accumulation.
4. Use of lactobacillus plantarum as claimed in claim 1 for the preparation of a medicament for the improvement and/or prevention of hyperlipidemia.
5. A pharmaceutical composition comprising the lactobacillus plantarum strain defined in claim 1.
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