CN117603885A - Lactobacillus paracasei LP-116 and application thereof in preparation of products for regulating intestinal barrier damage - Google Patents
Lactobacillus paracasei LP-116 and application thereof in preparation of products for regulating intestinal barrier damage Download PDFInfo
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- CN117603885A CN117603885A CN202410069815.9A CN202410069815A CN117603885A CN 117603885 A CN117603885 A CN 117603885A CN 202410069815 A CN202410069815 A CN 202410069815A CN 117603885 A CN117603885 A CN 117603885A
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- lactobacillus paracasei
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- intestinal barrier
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses lactobacillus paracasei LP-116 and application thereof in preparation of products for regulating intestinal barrier damage, wherein the lactobacillus paracasei LP-116 is separated and screened from naturally fermented northeast pickled Chinese cabbage, the regulation influence of lactobacillus paracasei (live bacteria) and heat-inactivated lactobacillus paracasei (metazoan) bacteria powder on the intestinal barrier function is detected through in vitro experiments, an intestinal epithelial cell model is established by using Caco-2 cells and inflammation is induced by lipopolysaccharide LPS, and the applicant finds that the expression of compact proteins and inflammatory factors in Caco-2 cells induced by LPS can be effectively improved by adopting the freeze-dried powder of inactivated lactobacillus paracasei LP-116 and inactivated LGG probiotics for compounding.
Description
Technical Field
The invention relates to the technical field of microbial preparations, in particular to lactobacillus paracasei LP-116 and application thereof in preparation of products for regulating intestinal barrier damage.
Background
Consensus definition for probiotics is published in FAO/WHO (2001), and probiotics refer to "active microorganisms that produce health benefits to the host when ingested in sufficient quantities". This definition emphasizes that probiotics must meet their requirement of being "live". Probiotics have been used in the form of living bacteria mainly in the early years, but living bacteria preparations may have potential safety hazards, and cannot be mixed with antibiotics for use, and are easily unstable due to the influence of conditions such as processing, storage, and acidic environment of animal digestive tracts. With the expansion of the research scope and functions of probiotics in humans, researchers find that not only live bacteria can play a probiotic function, but also some components of non-live bacteria show obvious health promotion effects, such as inactivated bacterial cells, components released by dissolution after death of the bacterial cells, metabolites of bacteria and the like, wherein the bacterial components comprise lipoteichoic acid, cell surface proteins, peptidoglycan, capsular polysaccharide, pili, flagella and the like, and the metabolites comprise enzymes, polypeptides, short-chain fatty acids, extracellular polysaccharide and the like, and the inactivated bacteria and the metabolites with health promotion effects belong to the category of metazoan. To unify the definition of metagens, the international society for probiotics and prebiotics science (ISAPP) issued a consensus statement for metagens (Postbiotics) at month 5 of 2021: metazoan refers to a formulation of inanimate microorganisms and/or components thereof that are beneficial to the health of a host.
The human intestinal tract has a complex microenvironment, the existence of intestinal barrier is helpful for preservation of intestinal structures, and more than 1000 tens of thousands of people worldwide suffer from ulcerative colitis and crohn's disease. Studies have shown that intestinal dysbacteriosis and intestinal epithelial barrier dysfunction are directly related to the occurrence of UC and CD, when the intestinal barrier is compromised, local immunity is activated, the Tight Junction (TJ) protein structure is altered, resulting in cytokine imbalance, mediating reduced intestinal permeability, and the TJ protein formed by intercellular interactions is the primary defense barrier for intestinal epithelial cells; meanwhile, myosin light chain kinase (myosin light chain kinase, MLCK) interacts with TJ protein, regulates epithelial cell permeability, maintains the integrity of intestinal function; MLCK mediates myosin light chain (myosin light chain, MLC) phosphorylation (p-MLC), leading to destruction of TJ protein and an increase in intestinal epithelial cell permeability. Previous studies have shown that damage to the intestinal barrier can cause inflammation and high intestinal motility, which in turn can lead to the onset of inflammatory bowel disease, and therefore, maintaining the integrity of the intestinal barrier is critical.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide lactobacillus paracasei LP-116 and application thereof in preparing a product for regulating intestinal barrier damage.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the lactobacillus paracasei LP-116 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.26441 and a preservation address of North Chen Xili No. 1 and 3 in the Korean area of Beijing city, and has the name of LatinLactobacillus paracasei。
The invention also provides a microbial preparation for regulating intestinal barrier damage, comprising lactobacillus paracasei LP-116 and lactobacillus rhamnosus GG strain (LGG probiotics).
Preferably, the weight ratio of lactobacillus paracasei LP-116 to lactobacillus rhamnosus GG strain is 1-3:1.
Preferably, the active ingredient of the microbial preparation is an inactivated thallus of lactobacillus paracasei LP-116 and an inactivated thallus of lactobacillus rhamnosus GG strain.
Preferably, the microbial preparation is a probiotic freeze-dried powder, wherein the probiotics are lactobacillus paracasei LP-116 and lactobacillus rhamnosus GG strain; also included are prebiotics, wherein the prebiotics include maltodextrin, inulin, and galactooligosaccharides. .
Preferably, the mass ratio of the probiotics freeze-dried powder to the prebiotics is 4-6:4-6.
Preferably, the mass ratio of maltodextrin, inulin and galacto-oligosaccharides is 1-2:1-2:1.
Preferably, the microbial dosage form comprises a tablet, an oral liquid or a capsule.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, lactobacillus paracasei LP-116 is separated and screened from naturally fermented northeast pickled Chinese cabbage, the regulation influence of lactobacillus paracasei (viable bacteria) and heat-inactivated lactobacillus paracasei (metagen) bacterial powder on the intestinal barrier function is detected through in vitro experiments, an intestinal epithelial cell model is established by using Caco-2 cells, inflammation is induced by Lipopolysaccharide (LPS), and the applicant finds that the expression of compact proteins and inflammatory factors in Caco-2 cells induced by LPS can be effectively improved through compounding the inactivated lactobacillus paracasei LP-116 and the inactivated lactobacillus rhamnosus GG freeze-dried powder.
Drawings
FIG. 1 is a scanning electron microscope image of Lactobacillus paracasei LP-116;
FIG. 2 is a graph of 24-hour growth of Lactobacillus paracasei LP-116;
FIG. 3 is a graph showing the effect of a microbial agent on Caco-2 cell activity;
FIG. 4 is a graph showing the effects of microbial agents on intestinal epithelial barrier intercellular transmembrane resistance and permeability;
FIG. 5 is a graph showing the effect of a microbial agent on Caco-2 cell pro-inflammatory factor levels;
FIG. 6 is a graph showing the effect of a microbial agent on the expression level of a target protein in Caco-2 cells;
FIG. 7 is a graph showing the expression of inflammatory factors and ZO-1, claudin-1, occludin genes in a microbial preparation;
FIG. 8 is a graph showing the effect of microbial preparation 1 on improving intestinal damage;
FIG. 9 is a graph showing the effect of microbial preparation 1 on improving inflammatory reactions in the intestinal tract.
Detailed Description
The present invention will be described in further detail with reference to the following preferred examples, but the present invention is not limited to the following examples.
Unless otherwise specified, the chemical reagents involved in the present invention are all commercially available.
The invention provides a lactobacillus paracasei (LP-116), which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.26441, the preservation address of North Xielu No. 1, 3 in the Korean region of Beijing city and the Latin nameLactobacillus paracasei。
The invention also provides a method for separating and purifying lactobacillus paracasei (LP-116), which comprises the following steps:
sample collection
The lactobacillus paracasei (LP-116) is obtained by separating naturally fermented northeast pickled Chinese cabbage, and the sampling place is the Heilongjiang province of the Seilding city. Taking fresh fermented pickled Chinese cabbage juice 100 mL, adding 900. 900 mL sterile physiological saline (0.9%, w/v), and mixing thoroughly to obtain a collected sample.
(II) lactic acid bacteria enrichment
According to the inoculation amount of 1% (v/v), the culture medium is inoculated into 10mL of MRS broth culture medium for enrichment culture, and the culture medium is obtained after strict anaerobic culture for 48-72 hours at 37 ℃.
MRS broth formulation (1L): 10.0g of peptone, 5.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose and dipotassium hydrogen phosphate (K) 2 HPO 4 ) 2.0g of tri-ammonium citrate (C) 6 H 17 N 3 O 7 ) 2.0g, sodium acetate (CH) 3 COONa·3H 2 O) 5.0g, magnesium sulfate (MgSO) 4 ·7H 2 O) 0.2g, manganese sulfate (MnSO) 4 ·4H 2 O) 0.05g and Tween-80 1mL are added with deionized water 1L, stirred, heated and boiled until the mixture is completely dissolved, packaged and then autoclaved at 121 ℃ for 15 min, and cooled to normal temperature.
(III) isolation of strains
Taking culture solution 1mL, adding 9 mL sterile physiological saline (0.9%, w/v) for diluting 10 times, and sequentially diluting with physiological saline to 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 . Absorbing bacterial suspension with different dilution ratios of 0.1 and mL, and inoculating to CaCO by a coating method 3 MRS solid medium, strictly anaerobic at 37℃for 48 hours.
CaCO 3 -MRS solid medium formulation (1L): agar 20.0g, caCO, was added to MRS broth 3 10 And g, stirring, heating and boiling deionized water 1L until the deionized water is fully dissolved, sub-packaging, sterilizing at 121 ℃ for 15 min under high pressure, and pouring the mixture into a flat plate for standby after the mixture is cooled to about 60 ℃.
After the typical colony of the flat plate appears, the colony characteristics of the standard lactobacillus are selected, and single colony with the largest diameter of the calcium dissolving ring is generated for the next step of strain purification.
(IV) Strain purification
And continuously culturing the selected single colony for three times, wherein the colony morphology in the culture medium is consistent, the gram staining is purple, the cell morphology of the strain is single, namely, the pure culture strain is obtained, and the strain is inoculated with the MRS liquid culture medium and cultured for 24 hours at 37 ℃.
(fifth) Strain preservation
And (3) respectively taking 900 mu L of pure culture strain and 50% sterile glycerol, placing the pure culture strain and the sterile glycerol in a cell freezing tube, fully mixing, freezing and preserving at-80 ℃, inoculating the pure culture strain and the sterile glycerol into an MRS solid slant culture medium for preserving, and carrying out inspection and identification.
Bacteriological characterization of Lactobacillus paracasei (LP-116)
1. Basic features
The basic characteristics of Lactobacillus paracasei (LP-116) are shown in Table 1:
table 1: basic characteristics table of Lactobacillus paracasei LP-116
As can be seen from Table 1, LP-116 is a gram-positive, rod-shaped, sporeless, and contact and oxidase negative strain, and its scanning electron microscope image is shown in FIG. 1.
2. Growth curve
Activation of the LP-116 Strain: the strain is taken out from a refrigerator at the temperature of minus 80 ℃ and inoculated in an MRS liquid culture medium for activation for 24 hours, the activated strain is transferred to a corresponding MRS solid culture medium for culture for 48 hours in a constant temperature incubator at the temperature of 37 ℃, single bacterial colony is selected and inoculated in the MRS liquid culture medium, and the constant temperature culture at the temperature of 37 ℃ is continued for 18 hours to improve the activity of the strain for experiment standby.
Lactic acid bacteria were inoculated in an inoculum size of 1% (v/v) in MRS broth, cultured at 37℃for 24 hours, OD 600 values of the bacterial solutions were measured every 2 hours, and growth curves of the strains were established with time as abscissa, OD 600 values and pH as ordinate.
3. Molecular biology identification (16S rDNA)
(1) Template preparation
Bacterial liquid was dissolved in 50. Mu. l TaKaRa Lysis Buffer for Microorganism to Direct PCR (product number: D304), and the resultant was centrifuged to obtain a supernatant as a Template (Template DNA), and the reaction conditions were: 80 ℃ for 15 min.
(2) 16s rDNA PCR amplification
PCR amplification reagent 2X using TransGenTransTaqHigh Fidelity (HiFi) PCR Supermix I (cat# AS 131) for PCR amplification.
Table 2: PCR reaction System Table
The negative control used 1. Mu.l of 16S-free H 2 O replaces the template DNA; the positive control used 1. Mu.l of 16S rDNA of a known strain instead of the template DNA.
(3) 16s rDNA sequencing
Sequencing was performed using the ABI DNA sequencer (model: 3730 XL) using 1541R-new and 27F as primers.
Table 3:16s rDNA sequencing related primer information table
Molecular biological identification is carried out on the strain, the 16S rDNA sequence of the lactobacillus paracasei LP-116 is SEQ ID NO. 1, and the length of the 16S rDNA sequence obtained by amplification is 1454bp; the 16S rDNA sequence of the strain is found to have 100% homology with Lactobacillus paracasei after homology alignment in NCBI, and the result shows that the strain belongs to Lactobacillus paracasei.
Preparation of heat-inactivated lactobacillus paracasei (metazoan) fungus powder
Lactobacillus paracasei LP-116 was cultured with MRS broth, the inoculum size was 1% (v/v), the culture time was 24 hours, the cells were centrifuged at 8000 r/min for 10 min at 4℃after the completion of the culture, the cells were rinsed twice with sterile distilled water, and then resuspended, and sterilized at 100℃for 30 min.
Samples were taken before and after inactivation of the Lactobacillus paracasei LP-116 bacterial suspension, respectively, the MRS solid medium was diluted and spread with sterile physiological saline (0.9%), and plate counts were performed by stationary culture in a incubator at 37℃for 48 and h, and the inactivation effect was examined. The heat-treated plates showed no colony growth, indicating successful inactivation.
Freeze drying the inactivated thallus suspension in a sterile plate at-40deg.C, weighing, and heat inactivating lactobacillus paracasei (post-natal) powder to obtain lactobacillus paracasei number of 10 12 CFU/g。
Preparation of microbial preparations
Lactobacillus rhamnosus strain GG (LGG probiotic) was purchased from trade company, hansen (beijing) of the family;
bifidobacterium longum BL21 was purchased from micro Kang Yisheng (su zhou) inc;
the microbial preparation consists of 50% of probiotics freeze-dried powder and 50% of prebiotics, wherein the prebiotics comprise maltodextrin, inulin and galacto-oligosaccharides;
microbial preparation 1:25% inactivated Lactobacillus paracasei LP-116 lyophilized powder (bacterial count 10) 12 CFU/g), 25% inactivated lactobacillus rhamnosus GG strain freeze-dried powder (bacterial count 10) 12 CFU/g), 15% maltodextrin, 20% inulin and 15% galacto-oligosaccharides.
Microbial preparation 2:25% inactivated Lactobacillus paracasei LP-116 lyophilized powder (bacterial count 10) 12 CFU/g), 25% inactivated bifidobacterium longum BL21 lyophilized powder (bacterial count 10) 12 CFU/g), 15% maltodextrin, 20% inulin and 15% galacto-oligosaccharides.
Microbial preparation 3:50% inactivated Lactobacillus paracasei LP-116 lyophilized powder (bacterial count 10) 12 CFU/g), 15% maltodextrin, 20% inulin and 15% galacto-oligosaccharides.
Microbial preparation 4:50% non-inactivated Lactobacillus paracasei LP-116 lyophilized powder (bacterial count 10) 12 CFU/g), 15% maltodextrin, 20% inulin and 15% galacto-oligosaccharides.
The reagents and materials used in the invention are as follows: caco-2 human colorectal adenocarcinoma cell lines were derived from the Living technologies Co., ltd (China). Lipopolysaccharide (LPS) and Fluorescein Isothiocyanate (FITC) were purchased from Sigma-Aldrich Inc. (USA). Other cell culture materials were purchased from Shanghai Xiao peng Biotechnology Co., ltd (China), including Fetal Bovine Serum (FBS), DMEM high-sugar medium, penicillin streptomycin double antibody, trypsin, hanks' Balanced salt solution (HBSS) and phosphate buffered salt solution (PBS). Reference beta-actin (cat# 66009-1-Ig), myD88 (cat# 23230-1-AP), TLR4 (cat# 19811-1-AP), NF-. Kappa. B p65 (cat# 10745-1-AP), claudin-1 (cat# 13050-1-AP), occludin (cat# 27260-1-AP), ZO-1 (cat# 21773-1-AP), MLCK (cat# 21642-1-AP), MLC (cat# 10906-1-AP), HRP-conjugated Affinipure Goat Anti-Mouse IgG (cat# SA 00001-1), HRP-conjugated Affinipure Goat Anti-Rabbit (cat# SA 00001-2), coraLite488-conjugated Goat Anti-RabbIgG (cat# SA 00013-2), coraLite594-conjugated Goat Anti-R from proteintech Group biotechnology (cat# SA 00013-4), MLC (cat# 35-1-AP), MLC (cat# 35-6-Ser) and (cat# 35-35/Ser) (from U.S. No. 623-35) (see FIGS. 35-6).
Test 1: effect of microbial Agents on Caco-2 cell Activity
The Caco-2 cell culture procedure was as follows: placing the cells in DMEM complete culture solution (containing 10% fetal calf serum, 1% diabody, 1% glutamine, 1% nonessential amino acids), 37deg.C, 5% CO 2 Culturing in incubator, changing culture medium every 1-2 days, and when the cells are fused to 90%, digesting and passaging with pancreatin, and performing subsequent related experiments.
Taking Caco-2 cells in logarithmic growth phase according to 1×10 5 The density of each mL is inoculated in a 96-well plate, 100 mu L of each well is placed in a temperature of 37 ℃ and 5% CO 2 Culturing in an incubator until cell monolayer is formed, discarding the culture solution, adding 100 mu L of microbial preparation with a final concentration of 100 mu g/mL LPS and incubating 24 h with cells, discarding the culture solution, washing once with DMEM, measuring the absorbance value of each well by a CCK-8 method, and calculating the relative cell activity (%) of each well.
LPS group (only LPS at a concentration of 100. Mu.g/mL) and a blank group (Control group, no microbial agent or LPS was added) were simultaneously set.
As shown in the test result in FIG. 3, LPS with the concentration of 100 mug/mL can obviously reduce the activity of Caco-2 cells after 24 hours of stimulation, the activity is reduced to 68.24 percent, and the activity of the Caco-2 cells can be effectively improved by adding the inactivated lactobacillus paracasei LP-116. Meanwhile, experiments also find that the inactivated lactobacillus paracasei LP-116 and the inactivated lactobacillus rhamnosus GG strain are compounded (microbial preparation 1), compared with the inactivated lactobacillus paracasei LP-116 and the inactivated bifidobacterium longum BL21 (microbial preparation 2), the activity of Caco-2 cells can be obviously improved by approximately 20%. Comparing the capability of microbial preparation 1 with microbial preparation 3 for alleviating the apoptosis of Caco-2 induced by LPS, the 50% inactivated lactobacillus paracasei LP-116 freeze-dried powder is not 25% inactivated lactobacillus paracasei LP-116 freeze-dried powder and 25% inactivated lactobacillus rhamnosus GG strain freeze-dried powder has strong capability for alleviating cytotoxicity, which indicates that the synergistic effect of heat-inactivated LP-116 and heat-inactivated lactobacillus rhamnosus GG strain is superior to that of singly heat-inactivated LP-116 freeze-dried powder.
Test 2: effects of microbial agents on intestinal epithelial barrier intercellular transmembrane resistance and permeability
Culturing and digestion of Caco-2 cells reference the procedure in test 1 at 1X 10 4 cells/cm 2 Is inoculated into a 12-well transwell (Corning, lindfield, sydney, australia) cell culture chamber and cultured for 21 days until Caco-2 cells are fully differentiated and the resistance reaches 500 Ω/cm 2 In the above, LPS is an important component of gram-negative bacteria, and can change the permeability and resistance of intestinal epithelial cells, and Caco-2 cells cultured in a transwell culture plate can construct a cell monolayer, and can further establish an intestinal barrier dysfunction model through LPS induction, and the TEER value and FITC-glucan transport are evaluated to determine whether a microbial preparation can improve the intestinal barrier dysfunction caused by LPS.
The final TEER value for each well was determined by taking 100. Mu.L of the microbial preparation at a final concentration of 1. Mu.g/mL and adding LPS (final concentration of 100. Mu.g/mL) to the mucosal side of the cell monolayer together with 24 h. In addition, to evaluate the compactness and permeability of the Caco-2 cell monolayer model, 100. Mu.L of FITC-dextran (final concentration 1 mg/mL) was added to the mucosal side of the cell monolayer using FITC-dextran as a cell transport marker, and the mixture was placed at 37℃with 5% CO 2 Incubate in incubator for 24 hours.
Meanwhile, an LPS group (only 100 mug/mL of LPS is added) and a blank Control group (Control group, no microbial agent or LPS is added) are arranged, after incubation is completed, 100 mug of culture solution in the serosa side of each group of cell monolayers is respectively removed and placed in a black 96-well plate, and the culture solution is rapidly placed in an enzyme-labeled instrument to measure the fluorescence intensity of each group under the conditions that the excitation wavelength is 480 nm and the emission wavelength is 520 nm.
As shown in FIG. 4, the resistance and permeability of LPS group (100. Mu.g/mL) were significantly different from those of Control groupP<0.05 Indicating that LPS can significantly cause damage to the Caco-2 intestinal epithelial barrier cells. The addition of the lyophilized powder of Lactobacillus paracasei LP-116 in the group of microbial preparations resulted in a different degree of alleviation of intestinal barrier function impairment induced by LPS compared to the LPS group. Wherein the microbial preparation 1 has the best alleviating effect and has obvious difference with LPS groupP<0.05). The effect of the microbial preparation 1 is also better than that of other 3 preparations by comparing the capability of relieving the intestinal barrier function damage caused by LPS induction of 4 microbial preparations, which indicates that the probiotic effect after the combination of the inactivated lactobacillus paracasei LP-116 freeze-dried powder and the inactivated lactobacillus rhamnosus GG freeze-dried powder is better than that of the independent effect.
Test 3: effect of microbial preparation on Caco-2 cell proinflammatory factor levels
ELISA method is adopted to detect the secretion levels of TNF-alpha, IL-1 beta and IL-6 in Caco-2 cells, log-phase Caco-2 cells are inoculated into 6-hole plates, and each hole is 2 multiplied by 10 5 Individual cells, at 37 ℃,5% CO 2 After the cultured cells were fused to 90% in the incubator and the culture medium was discarded, 1000. Mu.L of the microbial preparation (final concentration 1. Mu.g/mL) was added to the 6-well plate, and LPS (final concentration 100. Mu.g/mL) was added to the plate to co-act 24 h.
Meanwhile, an LPS group (only 100 mug/mL of LPS is added) and a blank Control group (Control group, no microbial agent or LPS is added) are arranged, after the serosa side solution is taken and operated according to the flow of the instruction of the kit, an OD value is read at 450 nm by using an enzyme-labeled instrument, a standard curve is manufactured according to the instruction of the product and experimental data, ELISA data processing software is used for fitting the standard curve, and the contents of TNF-alpha, IL-1 beta and IL-6 in each group are calculated.
As shown in FIG. 5, the content of TNF-alpha, IL-1 beta and IL-6 in the induced Caco-2 cell monolayer is detected by ELISA kit, so that LPS can obviously induce Caco-2 cells to secrete inflammatory factorsP<0.01). The combination of LPS control group, microbial preparation group and blank control group shows that the pro-inflammatory factor secretion content in microbial preparation 1-4 groups is lowIn the LPS group, the microbial preparation can effectively relieve the secretion of inflammatory factors induced by LPS, thereby relieving inflammation. By analyzing and comparing the pro-inflammatory factor secretion conditions among microbial preparations, the pro-inflammatory factor secretion conditions in the group 1 of the microbial preparations are lower than those of other microbial preparations by 2-4, and the effect of relieving Caco-2 inflammation after the inactivated lactobacillus paracasei LP-116 lyophilized powder is compounded with the inactivated lactobacillus rhamnosus GG strain (microbial preparation 1) is superior to that of the inactivated lactobacillus paracasei LP-116 lyophilized powder and the inactivated BL21 lyophilized powder (microbial preparation 2).
Test 4: western blot detection of target protein expression level of microbial preparation in Caco-2 cells
The Western blot method is used for analyzing the expression of target proteins in Caco-2 cell whole proteins, and the method comprises the following steps: cell culture and experimental grouping were consistent with those in experiment 3, after stimulation was completed, 1.5 mL pre-chilled sterile PBS was added to scrape cells and collect them into 1.5 mL EP tubes, and after centrifugation (8000 r/min,4 ℃) for 5 min, cell pellet was obtained, RIPA (strong) cell lysate containing protease inhibitor was added to each EP tube, and after blowing was performed uniformly to allow the cells to be sufficiently lysed, the expression level of the target protein was detected.
Placing an EP tube containing cell lysate into a centrifuge to centrifuge (12000 r/min,4 ℃) for 15 min, removing supernatant of each cell protein, determining the concentration of each group of proteins by a BCA method, respectively removing supernatant of equal amount of proteins and a loading buffer solution, uniformly mixing according to a proportion, boiling water bath for 5 min to enable the proteins to be fully denatured, adding the mixed solution into each lane to carry out polyacrylamide gel electrophoresis separation and transfer membrane after a sample is cooled, placing a nitrocellulose membrane with target proteins into 5% skimmed milk, sealing 1 h at room temperature, incubating corresponding primary antibodies of each protein at 4 ℃) overnight, recovering the primary antibodies, washing protein strips for 3 times at room temperature, incubating the protein strips for 1 h at room temperature by corresponding secondary antibodies, washing the strips for 3 times by using TBST solution, placing the protein strips into a darkroom to carry out chemiluminescence, tabletting and X-ray film exposure and development.
The gel imaging system collects the Image result, and calculates the gray value of each protein band by Image J software, and the detection indexes comprise TLR-4, p-NFk B, NF kB and the tight junction proteins ZO-1, claudin-1, occludin and MLCK, MLC and p-MLC protein levels in the NF-kB signal path.
As shown in FIG. 6, the test results show that the Control group and the LPS group in FIG. 6 (A) can be used for obviously improving the expression of TLR4 and p-NFkB proteins in Caco-2 cells by the stimulation of LPS, which proves that the LPS can obviously induce the Caco-2 cells to improve the TLR4 and the p-NFkB proteinsP <0.05). The combination of LPS group, microbial preparation group and Control group shows that the microbial preparation 1 has the lowest TLR-4 and p-NFkB protein content, and the microbial preparation 1 has better effect of relieving inflammation caused by LPS compared with other preparations.
Referring to FIG. 6 (B), it is understood that the expression of p-MLC and MLCK proteins in Caco-2 cells stimulated by LPS is significantly improved by combining with Control group and LPS groupP <0.05). The combination of LPS group, microbial preparation group and Control group shows that the p-MLC and MLCK protein content in microbial preparation 1 group is the lowest, and the p-MLC protein content of microbial preparation 1 is obviously lower than that of other 3 groups of microbial preparations, which shows that the effect of the inactivated lactobacillus paracasei LP-116 after being compounded with the inactivated lactobacillus rhamnosus GG strain is the best, and the inflammatory pathway caused by LPS is reduced.
Referring to FIG. 6 (C), it was found that, in combination with the Control group and the LPS group, the expression level of TJ protein in Caco-2 cells was decreased by stimulation with LPS, and that LPS was able to inhibit the TJ protein expression ability of Caco-2 cellsP <0.05). The capability of improving the intestinal barrier function injury of the four microbial preparations is compared, and the results show that 1-3 groups of microbial preparations can improve TJ protein, and 4 groups of microbial preparations have the worst improvement effect, so that the intestinal barrier can be repaired at different degrees by adding the inactivated lactobacillus paracasei LP-116. In addition, the improvement effect after the LP-116 freeze-dried powder is compounded with the inactivated lactobacillus rhamnosus GG strain freeze-dried powder and the BL21 freeze-dried powder respectively is found by comparing the improvement effect after the inactivated lactobacillus casei LP-116 freeze-dried powder is compounded with the inactivated lactobacillus rhamnosus GG strain freeze-dried powder, and the compound lactobacillus rhamnosus GG strain freeze-dried powder has better improvement effect.
Test 5: real-time fluorescence quantitative PCR detection of inflammatory factor and ZO-1, claudin-1 and occludin gene expression
Detecting the expression of inflammatory factors and genes ZO-1, claudin-1 and occludin by adopting real-time fluorescence quantitative PCR, and the method comprises the following steps: cell culture and experimental grouping were consistent with those in experiment 3, after stimulation was completed, cells were scraped off and collected in an EP tube of 1.5 mL by adding 1.5 mL precooled sterile PBS, cell pellet was obtained after centrifugation (8000 r/min,4 ℃) for 5 min, 500. Mu.L Trizol was added for 10 min, and after vigorous shaking, the mixture was left at room temperature for 5 min to completely dissociate nucleoprotein, and supernatant was taken after centrifugation at 12,000 rpm for 10 min at 4 ℃; adding 0.2-mL chloroform according to each 1mL of Trizol, vigorously shaking for 15s, standing at room temperature for 2-3 min, freezing and centrifuging at 12000 rpm at 4 ℃ for 10-15 min, transferring an upper water phase into a clean EP tube after centrifuging, adding equal volume of isopropanol, standing at room temperature for 10 min after reversing and mixing uniformly, centrifuging to discard supernatant, washing precipitate with 75% ethanol with the same volume as that used by Trizol under the same centrifugation conditions, blowing until RNA precipitate is lightly suspended, but not blowing off, centrifuging at 12000 rpm at 4 ℃ for 3 min, discarding supernatant, taking care of not losing RNA precipitate, standing at room temperature for 2-3 min, airing, adding 50 mu L of non-enzyme water, fully dissolving RNA, and preserving the obtained RNA at-80 ℃ to prevent degradation.
The OD value was measured by a nucleic acid quantitative measuring instrument to quantify the concentration of RNA, and each sample RNA was diluted to the same concentration with enzyme-free water. Preparing a reverse transcription system according to the instruction book of Kangzhi reagent HiFiScript cDNA Synthesis Kit, incubating at 42 ℃ for 2 min, incubating at 42 ℃ for 15 min and incubating at 85 ℃ for 5 min for cDNA synthesis reaction, and centrifuging briefly after the reaction is finished and preserving at-20 ℃.
qPCR procedure fluorescence quantification was performed according to UltraSYBR Mixture instructions from Kao reagent Co., ltd under amplification conditions of pre-denaturation at 95℃for 10 min, followed by 40 cycles of denaturation at 95℃15s and annealing/extension at 60℃for 1 min, and thawing conditions at 95℃15s,60℃for 1 min,95℃15s,60℃15s. Firstly, normalizing target gene Ct values of an experimental group (test) and a control group (control) by using Ct values of reference genes; delta Ct test = test group target gene Ct value-test group reference gene Ct value, delta Ct control = control group target gene Ct value-control group reference gene Ct value, target gene expression level fold difference = 2 -（ΔCt test-ΔCt control） . Detection fingerTargets include pro-inflammatory factors (TNF- α, IL-1β, IL-6) and ZO-1, claudin-1, occludin gene expression.
Table 4: RT-qPCR primer sequence table
As shown in FIG. 7, the expression levels of mRNA of pro-inflammatory factors TNF-alpha, IL-1 beta and IL-6 in LPS group (100. Mu.g/mL) were significantly improved as compared with those in Control groupP<0.05). Compared with the LPS group, the microbial preparation group added with the inactivated lactobacillus paracasei LP-116 freeze-dried powder can relieve the expression quantity of the proinflammatory factor mRNA induced by LPS to different degrees. Wherein the microbial preparation 1 has the best alleviating effect and has obvious difference with LPS groupP<0.05). The effect of the microbial preparation 1 is also better than that of other 3 microbial preparations as compared with the capability of relieving intestinal barrier function injury (ZO-1, occludin, claudin-1) caused by LPS induction of 4 microbial preparations, which shows that the beneficial effect of the compound of the inactivated lactobacillus paracasei LP-116 freeze-dried powder and the inactivated lactobacillus rhamnosus GG strain freeze-dried powder is better than that of the independent effect.
Test 6: immunofluorescence observation of TJ protein expression
Taking Caco-2 cells in logarithmic phase, and adjusting cell concentration to 1.0X10 4 The cells/wells were inoculated in 24-well plate dishes at 37℃with 5% CO 2 Cells were cultured in an incubator to fuse to 80%. After co-stimulating Caco-2 cells 24 h with a final concentration of 1 μg/mL of microbial preparation 1 and LPS stimulation, the cell culture solution was discarded, washed three times with PBS, fixed with 4% paraformaldehyde for 20 min, then permeabilized with 0.5% Triton X-100 for 20 min, blocked with 5% FBS solution for 15 min at room temperature, and the method was performed according to 1: preparing ZO-1 primary antibody solution according to a proportion of 100, and standing overnight at 4 ℃; fluorescent secondary antibody is incubated for 1 hour at room temperature, DAPI is used for dying nuclei, the room temperature is 10 min, and glycerol PBS (phosphate buffered saline) sealing plates are used for observation and photographing.
As shown in FIG. 8, the LPS group (100. Mu.g/mL) can cause intestinal injury capability (ZO-1, occludin, claudin-1), and the effect of the microbial preparation 1 can be improved, which indicates that after the inactivated lactobacillus paracasei LP-116 freeze-dried powder and the inactivated lactobacillus rhamnosus GG freeze-dried powder are compounded, intestinal barrier improvement is facilitated.
Test 7: nuclear translocation observation of NF- κ B p65 in cells
The nuclear status of NF- κ B p65 was observed and recorded under an inverted fluorescence microscope. The method comprises the following steps: taking Caco-2 cells in logarithmic phase, and adjusting cell concentration to 1.0X10 3 The cells/wells were inoculated in 24-well plate dishes at 37℃with 5% CO 2 Cells were cultured in an incubator to 80%, incubated with 1. Mu.g/mL of microbial preparation 1, cell 6 was incubated h, and after stimulation of cells 24 h with LPS, cells were treated according to the NF- κB detection kit protocol, observed under an inverted fluorescence microscope and nuclear entry of NF- κ B p65 was recorded.
As shown in the test result in FIG. 9, the LPS group (100 mug/mL) can intensify inflammatory reaction, and the effect of the microbial preparation 1 can improve the phenomenon, so that the beneficial effect of the compound of the inactivated lactobacillus paracasei LP-116 freeze-dried powder and the inactivated lactobacillus rhamnosus GG strain freeze-dried powder is further demonstrated.
Finally, it should be noted that: the above examples are not intended to limit the present invention in any way. Modifications and improvements will readily occur to those skilled in the art upon the basis of the present invention. Accordingly, any modification or improvement made without departing from the spirit of the invention is within the scope of the invention as claimed.
Claims (8)
1. The lactobacillus paracasei LP-116 is characterized in that the strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.26441 and the preservation address of North Chenxi Lu No. 1 and 3 in the Korean area of Beijing city, and the Latin name isLactobacillus paracasei。
2. A microbial preparation for modulating impaired intestinal barrier comprising lactobacillus paracasei LP-116 and lactobacillus rhamnosus GG strain.
3. The microbial preparation for regulating intestinal barrier impairment according to claim 2, wherein the weight ratio of lactobacillus paracasei LP-116 and lactobacillus rhamnosus GG strain is 1-3:1.
4. The microbial preparation for regulating intestinal barrier impairment according to claim 2, wherein the active ingredients of the microbial preparation are inactivated cells of lactobacillus paracasei LP-116 and inactivated cells of lactobacillus rhamnosus GG strain.
5. The microbial preparation for modulating impaired intestinal barrier according to claim 2, wherein the microbial preparation is a probiotic lyophilized powder, wherein the probiotics are lactobacillus paracasei LP-116 and lactobacillus rhamnosus GG strain; also included are prebiotics, wherein the prebiotics include maltodextrin, inulin, and galactooligosaccharides.
6. A microbial preparation for modulating an impaired intestinal barrier according to claim 5, wherein the mass ratio of probiotic freeze-dried powder to prebiotic is 4-6:4-6.
7. The microbial preparation for regulating intestinal barrier impairment according to claim 5, wherein the mass ratio of maltodextrin, inulin and galacto-oligosaccharides is 1-2:1-2:1.
8. A microbial preparation for modulating an impaired intestinal barrier according to claim 2, wherein the dosage form of the microorganism comprises a tablet, an oral liquid or a capsule.
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