CN116790453A - Probiotic agent for preventing and/or treating gastrointestinal tract related diseases and application thereof - Google Patents
Probiotic agent for preventing and/or treating gastrointestinal tract related diseases and application thereof Download PDFInfo
- Publication number
- CN116790453A CN116790453A CN202311069571.6A CN202311069571A CN116790453A CN 116790453 A CN116790453 A CN 116790453A CN 202311069571 A CN202311069571 A CN 202311069571A CN 116790453 A CN116790453 A CN 116790453A
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- China
- Prior art keywords
- lactobacillus reuteri
- probiotic
- longum
- strain
- gastrointestinal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
The applicationDisclosed are a probiotic agent for preventing and/or treating gastrointestinal-related diseases and use thereof. The probiotics comprise lactobacillus reuteri LR08 strain and bifidobacterium longum BL11 strain. The application discovers lactobacillus reuteri for producing the antibacterial peptide for the first timeLactobacillus reuteriLR08 strain and bifidobacterium longum subspecies longumBifidobacterium longum subsp.Longum The BL11 strain combination can be effectively applied to the preparation of products for improving gastrointestinal related diseases, can obviously improve intestinal inflammation caused by bacterial infection, obviously improve intestinal barrier function damage caused by bacterial infection and effectively inhibit the growth of colorectal cancer cells of human, and has wide application prospect.
Description
Technical Field
The application belongs to the technical field of microorganisms, and relates to a probiotic for preventing and/or treating gastrointestinal tract related diseases and application thereof.
Background
The antibacterial peptide (Antimicrobial peptide, AMP) is a polypeptide micromolecule substance with microbial activity and immune regulation activity, generally contains 12-50 amino acid residues, has positive charges and amphiphilic molecular structure, has antibacterial, antifungal and antiviral effects, and can play important roles in regulating and controlling in organisms, and is also called host defense peptide. At present, a variety of different antimicrobial peptides have been identified from insects, plants, animals, microorganisms, and the like.
Lactic acid bacteria such as Streptococcus, bifidobacterium, and Lactobacillus reuteri produce a wide variety of AMPs. The bacteriocin and other antibacterial peptides produced by lactic acid bacteria have strong inhibition capability to pathogenic bacteria and food spoilage bacteria, and have relatively fixed antibacterial spectrum, and the AMP with small molecular weight and good thermal stability in the substances is recognized to have the most development and application prospect in the fields of foods, health-care products, medicines and the like.
The emergence of resistance to antibiotics, a single target, resulting from long-term widespread use, is becoming a major challenge in clinical infection management. In contrast, AMP shows advantages by acting on the plasma membrane and intracellular targets of pathogenic bacteria, and has a strong activity against drug-resistant bacteria, providing a new substitute for antibiotics, and development of APM has become one of the hot spots in the art. For example, CN112646743A discloses Lactobacillus reuteri CCFM1134 for preventing and relieving ulcerative colitis and application thereof, and discloses that Lactobacillus reuteri CCFM1134 can endure gastrointestinal environment of human body, remarkably reduce weight loss during the illness period of ulcerative colitis, improve fecal character and hematochezia condition, improve colonic mucosa injury, reduce MPO activity, reduce the content of pro-inflammatory factors TNF-alpha, IL-1 beta, IL-6, IFN-gamma in colon, up regulate the transcription level of colon tight junction related proteins Claudin-3, ZO-1, ZO-2 and Occludin, up regulate the transcription level of antimicrobial peptides Reg3g and Reg3b, up regulate the transcription level of mucin MUC2 in colon, improve intestinal flora diversity, and can be applied to preparing functional fermented foods for improving ulcerative colitis symptoms.
In summary, development of a microecological live bacteria preparation capable of producing AMP, which can effectively improve gastrointestinal diseases, is one of the technical problems to be solved currently.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the application provides a probiotic agent for preventing and/or treating gastrointestinal related diseases and application thereof, in particular to a probiotic agent for preventing and/or treating intestinal inflammation and colorectal cancer and application thereof.
In order to achieve the above purpose, the application adopts the following technical scheme:
in a first aspect, the present application provides a probiotic for the prevention and/or treatment of diseases associated with the gastrointestinal tract, the strains of said probiotic comprising lactobacillus reuteri and bifidobacterium longum; the Lactobacillus reuteri is Lactobacillus reuteriLactobacillus reuteriLR08 strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No. 1.12733 in the year 2020, month 07 and day 20; the Bifidobacterium longum is Bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum BL11 strain is preserved in China general microbiological culture Collection center (CGMCC) with preservation number of CGMCC No. 24412 and preservation address of 2022, 02 and 21The method comprises the following steps: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
In the present application, it was found thatLactobacillus reuteriLR08 strainBifidobacterium longum subsp. Longum BL11 strain, the two cooperate, it can inhibit the level of verification factors such as TNF-a, IL-6 and IL-10, relieve intestinal inflammation; regulating the expression level of the related genes of the antibacterial protein in intestinal tissues, including inhibiting the expression level of EG3 gamma and RemL beta genes, and up-regulating the expression level of TFF3 genes, thereby having an improvement effect on mucous layer functions and intestinal repair functions; can regulate and control the expression level of the intestinal canal compact junction protein ZO-1 and occludin to rise, and relieve the damage of intestinal canal barrier function of a mouse infected by bacteria; can obviously inhibit the growth of colorectal cancer cells; has wide application prospect.
Preferably, the lactobacillus reuteriLactobacillus reuteriLR08 strain and bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum The ratio of the viable count of BL11 strain is (1-3): (1-3), for example, 3:1, 2:1 or 1:1.
Preferably, the gastrointestinal related disorder comprises intestinal inflammation and/or colorectal cancer.
Preferably, the number of viable bacteria in the probiotic is not less than 1×10 9 CFU/mL。
Preferably, the formulation of the probiotic agent comprises freeze-dried powder, capsules, tablets or granules.
Preferably, the probiotic agent further comprises a lyoprotectant.
Preferably, the lyoprotectant comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.
Preferably, the probiotic agent further comprises a functional auxiliary agent.
Preferably, the functional auxiliary agent comprises any one or a combination of at least two of fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomalto-oligosaccharide, soy oligosaccharide, inulin, spirulina, arthrospira, coriolus versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.
In a second aspect, the present application provides a formulation for the prophylaxis and/or treatment of a gastrointestinal-related disorder, the formulation comprising lactobacillus reuteriLactobacillus reuteriAntibacterial peptide produced by LR08 strain and bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum An antibacterial peptide produced by BL11 strain.
Preferably, the lactobacillus reuteriLactobacillus reuteriAntibacterial peptide produced by LR08 strain and bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum The mass ratio of the antibacterial peptide produced by BL11 strain is (1-3): (1-3), for example, 3:1, 2:1 or 1:1, etc.
In a third aspect, the present application provides the use of a combination of lactobacillus reuteri and bifidobacterium longum for the manufacture of a product for the prevention and/or treatment of gastrointestinal related diseases, said lactobacillus reuteri being lactobacillus reuteriLactobacillus reuteriLR08 strain is preserved in China general microbiological culture Collection center (CGMCC) at 20/07/2020, and has a preservation number of CGMCC No. 1.12733, wherein the Bifidobacterium longum is Bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum BL11 strain is preserved in China general microbiological culture Collection center (CGMCC) at 2022, 02 and 21, and has a preservation number of CGMCC No. 24412 and a preservation address of: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
In the application, the lactobacillus reuteri is a probiotic which is included in a bacterial list for food, so that the lactobacillus reuteri LR08 obtained by screening is relatively safe for human bodies and is not easy to cause adverse reaction.
Preferably, the gastrointestinal related disorder comprises intestinal inflammation and/or colorectal cancer.
Preferably, the product comprises any one of a medicament, a functional fermented food or a functional bacterial agent.
Preferably, the viable count in the product is not less than 1X 10 9 CFU/mL。
Preferably, the product contains lyophilized powders of lactobacillus reuteri and bifidobacterium longum.
Preferably, the preparation method of the freeze-dried powder of lactobacillus reuteri and bifidobacterium longum comprises the following steps:
inoculating lactobacillus reuteri or bifidobacterium longum into a culture medium of liquid MRS for culture; centrifuging the culture solution to obtain thalli; re-suspending the thalli with a freeze-drying protective agent to obtain re-suspension; lyophilizing the heavy suspension, and mixing the two lyophilized powders; or alternatively, the process may be performed,
inoculating the lactobacillus reuteri and bifidobacterium longum into a culture medium of liquid MRS for culture; centrifuging the culture solution to obtain thalli; re-suspending the thalli with a freeze-drying protective agent to obtain re-suspension; the heavy suspension was lyophilized.
Preferably, the MRS medium includes, in concentration: peptone 0-15 g/L, beef extract 0-15 g/L, glucose 15-25 g/L, yeast powder 3-7 g/L, diammonium citrate 1-3 g/L, K 2 PO 4 ·3H 2 O 2-3 g/L、MgSO 4 ·7H 2 O 0.05-0.2 g/L、MnSO 4 0.01-0.1 g/L, tween 80 0.5-2 mL/L, cysteine amino acid salt 0.1-1 g/L.
Preferably, the method of lyophilization comprises vacuum freezing.
In a fourth aspect, the present application providesLactobacillus reuteriAntibacterial peptide produced by LR08 bacteriumBifidobacterium longum subsp. LongumUse of a bacterially produced antibacterial peptide combination for the preparation of a product for the prevention and/or treatment of diseases associated with the gastrointestinal tract, said product comprisingLactobacillus reuteriLR08 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No. 1.12733, and is preserved in the year 2020, month 07 and day 20Bifidobacterium longum subsp. Longum BL11 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 24412 in 2022 and 02 and 21.
Preferably, the gastrointestinal related disorder comprises intestinal inflammation and/or colorectal cancer.
Preferably, the method for preparing the antibacterial peptide comprises the following steps:
will beLactobacillus reuteriLR08 bacterium orBifidobacterium longum subsp. Longum Inoculating BL11 bacteria into culture medium, culturing, collecting fermentation liquid and supernatant, filtering supernatant, and collecting the supernatantLactobacillus reuteriAntibacterial peptide produced by LR08 bacteriumBifidobacterium longum subsp.Longum Mixing antibacterial peptides produced by BL11 bacteria; or alternatively, the first and second heat exchangers may be,
will beLactobacillus reuteriLR08 bacteriumBifidobacterium longum subsp. Longum BL11 bacteria are inoculated in a culture medium for culture, fermentation liquor is taken, supernatant fluid is collected, and the supernatant fluid is filtered.
Preferably, the saidLactobacillus reuteriAntibacterial peptide produced by LR08 bacteriumBifidobacterium longum subsp. Longum The mass ratio of the antibacterial peptide produced by BL11 bacteria is (1-3): 1-3, for example, the antibacterial peptide can be 3:1, 2:1 or 1:1, etc.
Preferably, the medium comprises, in concentration: peptone 0-15 g/L, beef extract 0-15 g/L, glucose 15-25 g/L, yeast powder 3-7 g/L, diammonium citrate 1-3 g/L, K 2 PO 4 ·3H 2 O 2-3 g/L、MgSO 4 ·7H 2 O 0.05-0.2 g/L、MnSO 4 0.01-0.1 g/L, tween 80 0.5-2 mL/L, cysteine amino acid salt 0.1-1 g/L.
Preferably, the filtration comprises filtration using a 0.22-0.45 μm pore size filter.
In a fifth aspect, the present application provides the use of a probiotic according to the first aspect for the prophylaxis and/or treatment of a gastrointestinal-related disorder or a formulation according to the second aspect for the prophylaxis and/or treatment of a gastrointestinal-related disorder in the manufacture of an inflammatory factor inhibitor.
In a sixth aspect, the present application provides the use of a probiotic agent for the prophylaxis and/or treatment of a gastrointestinal-related disease according to the first aspect or a formulation for the prophylaxis and/or treatment of a gastrointestinal-related disease according to the second aspect for the preparation of an inflammatory factor inhibitor for the purpose of non-disease diagnosis and/or treatment.
According to the research result of the present application, the probiotic agent for preventing and/or treating gastrointestinal-related diseases according to the first aspect or the agent for preventing and/or treating gastrointestinal-related diseases according to the second aspect has an effect of significantly inhibiting inflammatory factors, and thus the result shows that the agent can be used as an inflammatory factor inhibitor for non-diagnostic and/or therapeutic purposes in the scientific research field.
In a seventh aspect, the present application provides the use of a probiotic agent according to the first aspect for the prophylaxis and/or treatment of a gastrointestinal-related disease or a formulation according to the second aspect for the prophylaxis and/or treatment of a gastrointestinal-related disease in the preparation of a colorectal cancer cell proliferation inhibitor.
In an eighth aspect, the present application provides the use of a probiotic agent for the prophylaxis and/or treatment of a gastrointestinal-related disease according to the first aspect or a formulation for the prophylaxis and/or treatment of a gastrointestinal-related disease according to the second aspect for the preparation of a colorectal cancer cell proliferation inhibitor for non-diagnostic and/or therapeutic purposes.
According to the research results of the present application, the probiotic agent for preventing and/or treating gastrointestinal-related diseases according to the first aspect or the agent for preventing and/or treating gastrointestinal-related diseases according to the second aspect has a remarkable effect of inhibiting colorectal cancer cell proliferation, and thus the results indicate that the agent can be used in the scientific research field as an agent for inhibiting colorectal cancer cell proliferation for the purpose of non-diagnosis and/or treatment, for example, for researching more colorectal cancer cell growth and metabolic mechanisms or behaviors, for screening drugs for treating colorectal cancer, and the like.
Compared with the prior art, the application has the following beneficial effects:
the application creatively discovers lactobacillus reuteri producing the antibacterial peptideLactobacillus reuteriLR08 (LR)Bifidobacterium longum subsp. Longum BL11 fungus cooperatees, can show in showing that the relevant disease of gastrointestinal: (1) significantly ameliorating intestinal inflammation caused by bacterial infection; (2) The intestinal barrier function damage caused by bacterial infection is obviously improved; (3) can effectively inhibit the growth of human colorectal cancer cells. Therefore, the use of the modified starch in the preparation of products for preventing and/or treating gastrointestinal diseases has good prospect.
Drawings
FIG. 1 is a graph showing the results of the hemolysis rate of the antibacterial peptide in example 2;
FIG. 2 is a graph showing the results of cell viability in example 2;
FIG. 3 is a graph showing the survival rate of mice in example 3;
FIG. 4 is a graph showing the results of weight change in mice in example 3;
FIG. 5 is a graph showing the results of the serum cytokine levels of mice in example 3;
FIG. 6 is a graph showing the results of the expression level of the antimicrobial protein-related gene in colon tissue of the mouse of example 3;
FIG. 7 is a graph showing the results of the protein expression levels of mouse colon zonulin ZO-1, occludin in example 3;
FIG. 8 is a graph showing the results of colorectal cancer cell viability in example 4;
FIG. 9 is a graph showing the results of the serum cytokine levels of mice in example 5;
FIG. 10 is a graph showing the results of the protein expression levels of mouse colon zonulin ZO-1, occludin in example 5.
Detailed Description
The technical means adopted by the application and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the application and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The embodiment of the application relates to peptone, beef extract, glucose, yeast powder, diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O、MgSO 4 ·7H 2 O、MnSO 4 Tween 80 and cysteine amino acid salts were purchased from national pharmaceutical group chemical Co.
The culture medium involved in the embodiment of the application is as follows:
modified MRS Medium (g/L): peptone 10 g/L, beef extract 10 g/L, glucose 20 gL, yeast powder 5g/L, diammonium hydrogen citrate 2 g/L, K 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 O 0.1g/L、MnSO 4 0.05g/L, tween 80 1 mL/L, cysteine amino acid salt 0.5 g/L.
The Lactobacillus reuteri according to the embodiment of the application is named Lactobacillus reuteriLactobacillus reuteriThe LR08 strain has a preservation unit of China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation time of 2020, 07 and 20 months, and a preservation number of CGMCC No. 1.12733; the Bifidobacterium longum is Bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum BL11 is preserved in China general microbiological culture collection center (CGMCC) at 2022, 02 and 21, and has the preservation number of CGMCC No. 24412 and the address of: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
Example 1
Preparation of Lactobacillus reuteri LR08 antibacterial peptide (PLR 08) andBifidobacterium longumsubsp. Longum BL11 antibacterial peptide.
Inoculating preserved lactobacillus reuteri LR08 to MRS agar plate, culturing at 37deg.C for 24 h, inoculating the strain on MRS culture medium twice to recover strain activity, selecting single colony, inoculating on MRS liquid culture medium, culturing at 37deg.C for 24 h, centrifuging at 4deg.C for 20 min at 8000 r/min, discarding precipitate, filtering supernatant with 0.22 μm pore size filter membrane for sterilization to obtain crude extract antibacterial peptide, and concentrating by freeze-drying method for use to obtain antibacterial peptide PLR08.
Inoculating the preserved bifidobacterium longum BL11 to an MRS agar plate, culturing at 37 ℃ for 24 h, transferring the strain on the MRS culture medium for two times so as to recover the activity of the strain, selecting single bacterial colony, inoculating on the MRS liquid culture medium, culturing at 37 ℃ for 24 h, centrifuging at 4 ℃ for 10 min at 8000 r/min, discarding the precipitate, filtering the supernatant with a 0.22 mu m pore-size filter membrane for sterilization to obtain crude extract antibacterial peptide, and concentrating by a freeze-drying method for later use to obtain the antibacterial peptide PBL11.
Example 2
This example explores the evaluation of the safety of lactobacillus reuteri LR08 producing antimicrobial peptides.
Generally, insect-derived and frog-derived antimicrobial peptides have strong antimicrobial activity, such as melittin, which contains 26 amino acid residues, has a typical amphipathic alpha-helical structure, and is structurally stable. The low concentration of the melittin can achieve the sterilization effect, however, the melittin has higher side effects such as hemolysis, cytotoxicity and the like, and reduces the use value. The safety of the antibacterial peptide PLR08 takes melittin as a control peptide.
(1) Measurement of cell hemolytic Activity: referring to the prior art methods (Zweytick D, deutsch G, andr ä J, blondelle SE, vollmer E, jerala R, lohner K. Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides J Biol chem 2011 Jun 17;286 (24): 21266-76.), 1 mL human fresh blood was placed in a centrifuge and centrifuged (1000G, 10 min), the collected erythrocytes were washed with PBS buffer, the cells were resuspended in 10 mL PBS, 96-well plates, 50. Mu.L of each of the erythrocyte suspension was added to the first 10 wells, and 10 concentration gradients (256-0.5. Mu. Mol/L:0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256) of PBS were added, respectively, after dilution, to the antibacterial peptide PLR08; positive control: mu.L of 0.01% polyethylene glycol octylphenyl ether (Triton X-100) and red blood cell suspension were added, respectively; negative control: 50. Mu.L of PBS buffer and red blood cell suspension were added, respectively; after culturing at 37 ℃ for 1 h, centrifuging for 10 min to obtain supernatant, and detecting OD 570nm Absorbance values. Calculating the hemolysis rate: hemolysis ratio (%) = (a-A0)/(At-A0) ×100% [ formula, a: absorbance of the antimicrobial peptide sample test group; at: absorbance of positive control group; a0: negative control absorbance values ].
As shown in the figure 1, when the concentration of the peptide is 1-256 mu mol/L, the PLR08 hemolysis rate is obviously smaller than that of the melittin, the hemolysis rate of the melittin is 0-120%, and compared with the melittin, the hemolysis rate of the probiotic antibacterial peptide PLR08 is always smaller than 5%, which indicates that the hemolysis is very low.
(2) Cytotoxicity test: human kidney epithelial 293 cells (North Nanopsis) were resuscitated using MTT method and subjected to 5% CO at 37 ℃C 2 In the incubator of (2), the culture is carried out by DMEM until logarithmic phase, single cell suspension is prepared, and the antibacterial peptide is dilutedPLR08 with reference to (1), 50. Mu.L of the prepared single cell suspension was added. Positive control: only single cell suspensions were added. Negative control: neither single cell suspension nor antimicrobial peptide was added. At 5% CO 2 Culturing in incubator (37 ℃ C., 24 h) with 40. Mu.L MTT solution for 4h, adding 150. Mu.L DMSO solution, shaking for 10 min, and detecting OD 492nm The value, calculate the cell viability, the concrete method is: cell viability (%) = (OD Sample of - OD Background ）/（OD Control - OD Background ) X 100%, wherein: OD samples are absorbance values of the cell samples that received the treatment; OD background is background absorbance value containing medium or reagent alone; OD control is the absorbance value of the control sample without any treatment (untreated cells).
As shown in FIG. 2, when the peptide concentration is 1-64 mu mol/L, the toxicity of PLR08 to cells is obviously smaller than that of melittin, which indicates that the antibacterial peptide PLR08 produced by probiotics has little influence on cell growth, the cell survival rate is higher, and the result is similar when the antibacterial peptide PBL11 is tested by using the same method.
Example 3
This example explores the effect of the antimicrobial peptide PLR08 derived from lactobacillus reuteri LR08 on intestinal inflammation and intestinal barrier in bacterially infected mice.
(1) Animals were grouped and a bacterial infection mouse model was established:
preparation of E.coli bacterial suspension:
coli bacteriumE.coliPurchased from North Nanopsis (strain BNCC 336902), inoculated with a loop to pick BNCC336902 streaked on LB agar plates, cultured at 37℃for 18 h; inoculating single colony in 4 mL LB liquid medium, shaking table shaking 10 h at 37 ℃ and 250 rpm; 100. Mu.L of the bacterial suspension is sucked and transferred into 10 mL of LB liquid medium, the temperature is 37 ℃ and the rpm is 250, a shaking table is used for shaking 2 h, and the light absorption value OD is obtained 600nm About 0.5, a bacterial concentration of 3X 10 8 CFU/mL, and centrifuging (5000 rpm,3 min) to remove supernatant; adding 3 mL sterile PBS solution to resuspend bacteria to obtain BNCC336902 bacterial suspension with concentration of 1×10 9 CFU/mL。
And (3) establishing a model:
50C 57BL/6J mice weaned from 21 day old females were provided by Shanghai laboratory animal center. The mice are fed into the cages, the environment is clean and quiet, the temperature is 23-25 ℃, and the humidity is 50% -70%. All procedures involving mice were in compliance with guidelines provided by the Shanghai laboratory animal Care and animal Experimental center (license number 2022122007), with free feeding of drinking water, pre-feeding for 3 days. After 3 days of pre-feeding, 30 weaned mice were randomly divided into 5 groups of 10 mice each. The mouse feeds were purchased from Shanghai Laek laboratory animal Limited. Control group (CTL group): normal feed; model group (MC group): 1X 10 8 CFU BNCC336902 bacteria; antibacterial peptide PLR08 intervention group (P1): 1X 10 8 CFU BNCC336902 bacteria+plr08 (5.0 mg/kg antibacterial peptide PLR08 per mouse intraperitoneal injection); antibacterial peptide intervention group (P2): 1X 10 8 CFU BNCC336902 bacteria + bifidobacterium longum antibacterial peptide (5.0 mg/kg antibacterial peptide PBL11 per mouse intraperitoneal injection); antibacterial peptide combinatorial intervention group (P3): 1X 10 8 CFU BNCC336902 bacteria + antibacterial peptide PBL11 and antibacterial peptide PLR08 combination (antibacterial peptide PBL11 and antibacterial peptide PLR08 mass ratio of 1:3, 5.0 mg/kg antibacterial peptide combination per mouse intraperitoneal injection) 1 time per day for 3 consecutive days; the mental state and behavior of each group of mice were recorded daily during the test period, the infected mice and the uninfected mice were kept separately, weighed and survival rates were calculated.
The results are shown in figures 3 and 4, with the MC, control, P1 and P3 mice not dying during the entire trial, only one death in the P2 group, and the surviving mice behaved normally. UsingE.coliAfter infection, infected mice showed symptoms such as reaction retardation to different degrees and survival rate was obviously reduced to 60%, and 4 mice were dead. During the intervention period, the weight changes of the mice are different, wherein compared with the control group, the weight of the mice in the MC group is obviously reduced, the weight reduction of the mice is alleviated after the dry state of the antibacterial peptide PBL11, and the weight of the mice is returned after the dry state of the antibacterial peptide PLR08, and the normal level can be basically recovered after the dry state of the antibacterial peptide PBL11 and the antibacterial peptide PLR08.
(2) Sample collection:
the eyeballs of the mice are sampled, the blood is stored in a centrifuge tube, centrifuged for 15 min at 4 ℃ and 3000 and g, and serum is collected and stored at-20 ℃.
(3) Serum cytokine assay:
the cytokines TNF-a, IL-6 and IL-10 in serum are detected by adopting an ELISA kit, the kit is purchased from the Wuhan purity organism, and specific experimental steps are carried out according to the instruction of the kit.
The results are shown in FIG. 5, in which MC mice were treated as compared with control miceE.coliThe content of pro-inflammatory cytokines TNF-a, IL-6 and IL-10 in serum of mice after infection is obviously increased, and the content of the cytokines TNF-a, IL-6 and IL-10 in the serum of the mice is obviously reduced after the antibacterial peptide PLR08 or the antibacterial peptide PBL11 is dried, so that the normal level can be basically recovered, namely, the antibacterial peptide can relieve intestinal inflammation.
(4) Real-time fluorescent quantitative PCR detection:
extracting RNA of the colon tissue of the mouse by using a Trizol method, reversely transcribing the RNA into cDNA, and detecting the expression level of the related gene of the antibacterial protein in the colon tissue by using a real-time fluorescent quantitative PCR method. Specific methods and primer design are facilitated by Shanghai Biotechnology Inc.
As a result, as shown in FIG. 6, the gene expression of the intestinal antimicrobial-associated proteins REG3γ, remL β, TFF3 was detected by real-time fluorescent quantitative PCR. Compared with the control group, the expression level of REG3γ and RemL beta genes in the colon of the MC group mouse is obviously increased, the TFF3 level is reduced, the condition of the PLR08 group mouse is reversed under the intervention of the antibacterial peptide, and the expression level of REG3γ and RemL beta genes is reduced and is normal. Whereas TFF3 gene expression levels were significantly elevated. The REG family is a key antibacterial protein, REG3 is mainly expressed in small intestine tissues of mice and humans, meanwhile, the REG3 is also conditionally expressed when pathogen infection or inflammation occurs in large intestine tissues, and the experimental result shows that the relative expression quantity of REG3 gamma of MC mice is obviously increased, and the expression is obviously reduced after the antibacterial peptide PLR08 or the antibacterial peptide PBL11 is treated, and the combined use effect of the two is better. The intestinal tract is in direct contact with the outside environment and colonizes with a large number of microorganisms. Antibacterial proteins secreted by intestinal epithelial cells play an important role in maintaining intestinal epithelial and normal microbial flora homeostasis. RemL beta plays an important role in the innate immunity and host defense process, and the expression level of RemL beta gene of a infected mouse is abnormally increased, and after the antibacterial peptide treatment, the abnormal condition can be relieved and the normal level is trended. TFF3 is produced by mucous secreting cells, plays an important role in intestinal mucous layer function and mucous membrane repair function, and intervention of antibacterial peptide can effectively improve colon mucous layer thickness of a infected mouse, and mucous layer covers the surface of intestinal epithelium, so that the TFF3 is an important defense line for protecting intestinal epithelium integrity. The experimental results show that the intervention of the antibacterial peptide can have an improving effect on mucous layer functions and intestinal tract repairing functions.
(5) Western blot detection:
protein expression levels of mouse colon zonulin ZO-1, occludin were detected using an immunological assay Western blot.
As shown in fig. 7, the intestinal epithelial barrier is the first line of defense of the animal intestinal tract against bacterial invasion, and compared with the control group, the expression of the intestinal tract zonulin ZO-1 and occludin of the infected mice is reduced, so that the intestinal tract permeability is increased, and the intestinal epithelial barrier function is seriously damaged. After the effect of the probiotic antibacterial peptide, the expression quantity of the intestinal canal compact junction protein ZO-1 and the occludin is obviously improved, namely the antibacterial peptide PLR08 or the antibacterial peptide PBL11 can relieve the damage of the intestinal canal barrier function of E.coli infected mice, and the combined use effect of the two is better.
Example 4
This example demonstrates the effect of the antimicrobial peptide PLR08 on human colorectal cancer cell growth.
Cell culture: the human colon adenocarcinoma cell line SW480 (BNCC 100604) was purchased from northlasis. Cells were cultured in Leibovitz's L-15 (GIBCO) medium supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin.
MTT detection: cells treated with the antimicrobial peptide PLR08 were assayed for cell viability using the MTT method. The antibacterial peptide PLR08 with different concentration is added or not added with 20 mu M Z-VAD-FMK respectively, and poured into each carrier for 4×10 4 In a cell culture plate, 24 h is incubated at 37 ℃,the culture broth was removed, PBS buffer salt was added, and after incubation with MTT solution (0.5 mg/ml) (Sigma) for 4h, MTT solution was removed and incubation was continued for 10 min with addition of 100. Mu.L DMSO. OD was measured by ELISA using PBS as a control 565nm Values. Cell viability was determined as% cell viability = experimental OD value/control OD value.
The bifidobacterium longum antibacterial peptide and antibacterial peptide combination (PC group) treatment references the procedure described above.
As shown in fig. 8, the anti-diffusion capability of the probiotic antibacterial peptide PLR08 on the treated human colon adenocarcinoma cells SW480 was measured by the MTT assay, and the intervention of the antibacterial peptide PLR08 or the antibacterial peptide PBL11 can obviously inhibit the growth of cancer cells, and the combined use effect of the two is better.
Example 5
This example explores improvement of bacterial infection mice symptoms for BL11 derived from Lactobacillus reuteri LR08 and Bifidobacterium longum.
(1) Animals were grouped and bacterial infection mice were modeled as in example 3:
preparation of E.coli suspension was performed as in example 3;
preparation of lactobacillus reuteri LR08, bifidobacterium longum subspecies longum BL11, LR08 and BL11 combined group bacterial suspensions: inoculating each strain into skimmed milk, and culturing at 37deg.C for 18 h to activate to obtain activating solution; inoculating the activating solution into MRS liquid culture medium (the combination group is inoculated according to the ratio of LR08 to BL11 of 3:1) according to the inoculum size of 2% (v/v), and culturing at 37 ℃ for 18 h to obtain bacterial liquid; diluting to obtain the final product.
And (3) establishing a model: 50C 57BL/6J mice weaned from 21 day old females were provided by Shanghai laboratory animal center. The mice are fed into the cages, the environment is clean and quiet, the temperature is 23-25 ℃, and the humidity is 50% -70%. All procedures involving mice were in compliance with guidelines provided by the Shanghai laboratory animal Care and animal Experimental center (license number 2022122007), with free feeding of drinking water, pre-feeding for 3 days. After 3 days of pre-feeding, 30 weaned mice were randomly divided into 5 groups of 10 mice each. The mouse feeds were purchased from Shanghai Laek laboratory animal Limited. Control group (CTL group): normal feed; model group (MC group): 1X 10 8 CFU BNCC336902 bacteria; LR08 intervention group (LR 08): 1X 10 8 CFU BNCC336902 bacteria +1X10 8 CFU LR08; interference group (BL 11): 1X 10 8 CFU BNCC336902 bacteria +1X10 8 CFU BL11; combined interference group (LR 08+bl11): 1X 10 8 CFU BNCC336902 bacteria +1X10 8 CFU (LR 08+bl11) intervenes 1 time per day for 3 consecutive days; the mental state and behavior of each group of mice were observed daily during the test, and the infected mice and the uninfected mice were kept apart. The cytokines TNF-a, IL-6 and IL-10 in serum were detected using an ELISA kit purchased from Wohan purity organisms and specific experimental procedures were performed according to the kit instructions (see example 3). Protein expression levels of mouse colon zonulin ZO-1 and occludin were detected by Western blot using an immunological assay (see example 3).
The results are shown in FIG. 9, in which MC mice were treated as compared with control miceE.coliThe content of pro-inflammatory cytokines TNF-a, IL-6 and IL-10 in the serum of the mice after infection is obviously increased, and the content of the cytokines TNF-a, IL-6 and IL-10 in the serum of the mice is obviously reduced after the probiotics LR08 or BL11 are dried, so that the normal level can be basically recovered, namely the probiotics LR08 can relieve intestinal inflammation. In addition, when the LR08+BL11 combination acts, the effect of reversal is more pronounced.
As shown in fig. 10, the intestinal epithelial barrier is the first line of defense of the animal intestinal tract against bacterial invasion, and compared with the control group, the expression of the intestinal tract zonulin ZO-1 and occludin of the infected mice is reduced, so that the intestinal tract permeability is increased, and the intestinal epithelial barrier function is seriously damaged. After the probiotics act, the expression level of the intestinal canal compact junction protein ZO-1 and the occludin are obviously improved, namely LR08 and BL11 can be relievedE.coliIntestinal barrier function of infected mice is impaired. In addition, when the LR08+BL11 combination acts, the effect of repairing the intestinal barrier function damage is more prominent.
In conclusion, the application discovers lactobacillus reuteri producing the antibacterial peptide for the first timeLactobacillus reuteriLR08 strain and bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum BL11 strain can be effectively applied to preparation of drugs for improving gastrointestinal tract related diseasesThe product can obviously improve intestinal inflammation caused by bacterial infection, obviously improve the damage of intestinal barrier function caused by bacterial infection and effectively inhibit the growth of colorectal cancer cells of human, and has wide application prospect.
The applicant states that the detailed method of the present application is illustrated by the above examples, but the present application is not limited to the detailed method described above, i.e. it does not mean that the present application must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present application, equivalent substitution of raw materials for the product of the present application, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present application and the scope of disclosure.
Claims (10)
1. A probiotic for the prevention and/or treatment of gastrointestinal-related diseases, characterized in that the strains in the probiotic comprise lactobacillus reuteri and bifidobacterium longum;
the Lactobacillus reuteri is Lactobacillus reuteriLactobacillus reuteriLR08 strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No. 1.12733 in the year 2020, month 07 and day 20;
the Bifidobacterium longum is Bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum BL11 strain is preserved in China general microbiological culture Collection center (CGMCC) at 2022, 02 and 21, and the preservation number is CGMCC No. 24412.
2. A probiotic for the prevention and/or treatment of gastrointestinal-related diseases according to claim 1, characterized in that the lactobacillus reuteriLactobacillus reuteriLR08 strain and bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum The ratio of the viable count of BL11 strain is (1-3): 1-3.
3. The probiotic agent for preventing and/or treating gastrointestinal-related diseases according to claim 1, wherein the number of viable bacteria in the probiotic agent is not less than 1×10 9 CFU/mL。
4. The probiotic for the prevention and/or treatment of gastrointestinal-related diseases according to claim 1, wherein the formulation of the probiotic comprises a lyophilized powder, a capsule, a tablet or a granule.
5. The probiotic for preventing and/or treating gastrointestinal-related diseases according to claim 1, wherein the probiotic further comprises a lyoprotectant;
the freeze-drying protective agent comprises any one or a combination of at least two of skim milk, gelatin, dextrin, acacia, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.
6. The probiotic for preventing and/or treating gastrointestinal-related diseases according to claim 1, wherein the probiotic further comprises functional auxiliary agents;
the functional auxiliary agent comprises any one or a combination of at least two of fructo-oligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, isomalto-oligosaccharide, soybean oligosaccharide, inulin, spirulina, arthrospira, coriolus versicolor polysaccharide, stachyose, polydextrose, alpha-lactalbumin or lactoferrin.
7. A preparation for preventing and/or treating gastrointestinal-related diseases, characterized in that the preparation comprises lactobacillus reuteriLactobacillus reuteriAntibacterial peptide produced by LR08 strain and bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum An antibacterial peptide produced by BL11 strain.
8. The formulation for preventing and/or treating gastrointestinal-related diseases according to claim 7, wherein the lactobacillus reuteriLactobacillus reuteriAntibacterial peptide produced by LR08 strain and bifidobacterium longum subspecies longumBifidobacterium longum subsp. Longum BL11 strain production resistanceThe mass ratio of the mycopeptide is (1-3), namely (1-3).
9. Use of a probiotic according to any one of claims 1 to 6 for the prevention and/or treatment of diseases associated with the gastrointestinal tract or a formulation according to any one of claims 7 to 8 for the prevention and/or treatment of diseases associated with the gastrointestinal tract for the preparation of an inflammatory factor inhibitor.
10. Use of a probiotic agent according to any one of claims 1-6 for the prevention and/or treatment of a gastrointestinal related disease or a formulation according to any one of claims 7-8 for the prevention and/or treatment of a gastrointestinal related disease for the preparation of a colorectal cancer cell proliferation inhibitor.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607257A (en) * | 2019-09-06 | 2019-12-24 | 吉林恩泽生物技术开发有限公司 | Composite probiotics for preventing ulcerative colitis |
| CN113491706A (en) * | 2021-06-22 | 2021-10-12 | 中山大学 | Application of chickpea polysaccharide in preparation of medicine for treating and/or preventing ulcerative colitis |
| EP3909589A1 (en) * | 2020-05-11 | 2021-11-17 | "Jantar" Wody Mineralne Sp z o.o. | Liquid probiotic-herbal preparation |
| CN115305228A (en) * | 2022-09-16 | 2022-11-08 | 苏州益优生物科技有限公司 | Probiotic composition for improving colorectal cancer symptoms and preparation method and application thereof |
| CN116077536A (en) * | 2023-03-17 | 2023-05-09 | 微康益生菌(苏州)股份有限公司 | Microecological live bacteria preparation for improving obesity-related metabolic diseases and preparation method and application thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110607257A (en) * | 2019-09-06 | 2019-12-24 | 吉林恩泽生物技术开发有限公司 | Composite probiotics for preventing ulcerative colitis |
| EP3909589A1 (en) * | 2020-05-11 | 2021-11-17 | "Jantar" Wody Mineralne Sp z o.o. | Liquid probiotic-herbal preparation |
| CN113491706A (en) * | 2021-06-22 | 2021-10-12 | 中山大学 | Application of chickpea polysaccharide in preparation of medicine for treating and/or preventing ulcerative colitis |
| CN115305228A (en) * | 2022-09-16 | 2022-11-08 | 苏州益优生物科技有限公司 | Probiotic composition for improving colorectal cancer symptoms and preparation method and application thereof |
| CN116077536A (en) * | 2023-03-17 | 2023-05-09 | 微康益生菌(苏州)股份有限公司 | Microecological live bacteria preparation for improving obesity-related metabolic diseases and preparation method and application thereof |
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