CN115029260A - Lactobacillus gasseri with anti-inflammatory and antioxidant properties and application thereof - Google Patents
Lactobacillus gasseri with anti-inflammatory and antioxidant properties and application thereof Download PDFInfo
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- CN115029260A CN115029260A CN202210532248.7A CN202210532248A CN115029260A CN 115029260 A CN115029260 A CN 115029260A CN 202210532248 A CN202210532248 A CN 202210532248A CN 115029260 A CN115029260 A CN 115029260A
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- lactobacillus gasseri
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Abstract
The invention discloses a lactobacillus gasseri with anti-inflammatory and antioxidant properties and application thereof, belonging to the technical field of microorganisms. The strain disclosed by the invention can better tolerate the gastrointestinal tract environment, has stronger anti-inflammatory and antioxidant capabilities, and can remarkably relieve the abnormal increase of the expression levels of proinflammatory cytokines TNF alpha, IL-6 and IL-1 beta caused by LPS. Compared with other lactobacilli, the Lactobacillus gasseri FWJL-4 has advantages in the aspects of cellular immune response and maintenance and antioxidant stress, and the live bacteria culture supernatant can be directly used as an oral vaccine and cause stronger cellular immune response, can be used as a microbial drug or product with good industrial prospect, plays a positive role in reducing organism inflammation, and has important practical significance in promoting the healthy development of intestinal tracts.
Description
Technical Field
The invention relates to a Lactobacillus gasseri with anti-inflammatory and antioxidant properties and application thereof, belonging to the technical field of microorganisms.
Background
Recent studies have found that the intestinal flora plays a significant role in human health and disease. The gut flora may play a role by affecting host functions such as metabolism, digestion, and gut mucosal integrity and immune response. The disturbance of intestinal flora refers to the persistent imbalance of intestinal microbial composition, which results in relatively increased pathogenic microorganisms and abnormal metabolite content of intestinal flora. Numerous studies have shown that disturbances of the intestinal flora are involved in the pathogenesis of various diseases. Therefore, the use of probiotics or metabolites of probiotics, or the use of prebiotics, diet improvement and the like to regulate the intestinal flora or promote anti-inflammatory and antioxidant environments, and at the same time, positively regulate the immune system and energy balance, thereby delaying or preventing the occurrence and development of certain diseases.
Lactobacillus belongs to the main genus in the human digestive tract and is one of the earliest discovered probiotics. Lactobacillus is closely related to human life, and is one of beneficial microorganisms widely applied to the fields of food fermentation, industrial lactic acid fermentation and medical care. The lactobacillus gasseri is one of lactobacilli, is a main dominant bacterium in the intestinal tracts of mothers and babies, can produce substances such as adhesins, bacteriocins, bile salt hydrolase and the like, and can resist inflammation and inhibit cancers due to extracellular polysaccharide, so that the lactobacillus gasseri has the characteristic of being capable of tolerating extreme environments (such as gastric acid, bile salt, digestive enzyme and the like) of a human body, and has the function of adhering to the epithelium of the intestinal tracts of the human body so as to regulate the immunity of the organism.
Disclosure of Invention
The invention provides a strain of Lactobacillus gasseri FWJL-4 with probiotic characteristics and anti-inflammatory and antioxidant characteristics.
The invention provides Lactobacillus gasseri FWJL-4 which is preserved in Guangdong province microorganism strain preservation center at 11 days 4 months 2022, wherein the preservation number is GDMCC No: 62365, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city. For the sake of convenience of description, Lactobacillus gasseri FWJL-4 is hereinafter referred to simply as "Lactobacillus gasseri FWJL-4".
The invention also provides application of the Lactobacillus gasseri FWJL-4 in preparation of medicaments with anti-inflammatory effects.
In one embodiment, the Lactobacillus gasseri FWJL-4 in the medicament is more than or equal to 1 x 10 8 CFU/mL or 1X 10 8 CFU/g。
In one embodiment, the medicament further comprises a pharmaceutically acceptable carrier.
In one embodiment, the anti-inflammatory drug is suitable for diseases/conditions such as inflammatory bowel disease/gastritis.
In one embodiment, the medicament is an oral vaccine comprising the culture of lactobacillus gasseri FWJL-4 and/or lactobacillus gasseri FWJL-4.
The invention also provides application of the Lactobacillus gasseri FWJL-4 and/or the culture thereof in improving the activity of antioxidant enzymes and/or relieving oxidative stress, and is suitable for gastrointestinal diseases/symptoms such as inflammatory bowel diseases.
In one embodiment, the culture is a fermentation supernatant obtained by culturing the Lactobacillus gasseri FWJL-4 in a medium for a certain period of time and then centrifuging to remove the cells.
The invention also provides application of the Lactobacillus gasseri FWJL-4 in preparation of food, functional food, formula food for special medical application or health care products which are helpful for oxidation resistance and/or inflammation resistance.
Has the advantages that: compared with the existing lactobacillus gasseri, the lactobacillus gasseri FWJL-4 separated by the invention has the following advantages: the strain has strong acid resistance and bile salt resistance, and can better resist gastrointestinal tract environment; has better antibiosis (broad-spectrum antibiosis capability) and antibiotic sensibility, which shows that the strain is sensitive to antibiotics and is safer to human bodies. The strain of the invention also has strong anti-inflammatory and antioxidant capacity in vivo, and the culture solution has no antibiotic resistance and inhibits harmful pathogenic bacteria in intestines, thereby proving that the strain has good probiotic property. Compared with other lactobacilli, the screened Lactobacillus gasseri FWJL-4 has advantages in terms of cellular immune response and maintenance and antioxidant stress, and the live bacteria culture supernatant can be directly used as an oral vaccine and cause stronger cellular immune response, can be used as a microbial drug or product with good industrial prospect, plays a positive role in reducing organism inflammation, and has important practical significance in promoting the healthy development of intestinal tracts.
Biological material preservation
Lactobacillus gasseri FWJL-4, taxonomically named Lactobacillus gasseri, has been deposited at the Guangdong province collection of microorganisms at 11.4.2022 with the deposit number GDMCC No: 62365, the preservation address is No. 59 building 5 of No. 100 Dazhong Jie-Lu-100 Guangzhou city.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a colony morphology of Lactobacillus gasseri FWJL-4.
FIG. 2 is a morphological diagram of gram-stained cells of Lactobacillus gasseri FWJL-4.
FIG. 3 is the electrophoretic identification chart of Lactobacillus gasseri FWJL-4 amplified with Lactobacillus specific primers.
FIG. 4 shows the survival rate of Lactobacillus gasseri FWJL-4 in simulated digestive juices.
FIG. 5 shows the effect of Lactobacillus gasseri FWJL-4 on macrophage cytokine expression.
FIG. 6 shows the effect of Lactobacillus gasseri FWJL-4 on macrophage antioxidase and hydrogen peroxide production.
FIG. 7 is a graph of the effect of Lactobacillus gasseri FWJL-4 on the oxidative stress-mediated Nrf2/HO-1 signaling pathway. P < 0.01, p < 0.05.
Detailed Description
The invention is further illustrated by the following figures and examples in conjunction with the description. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The experimental procedures, for which specific conditions are not noted in the examples below, are generally performed according to conditions conventional in the art or according to conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art.
Lactobacillus rhamnosus GG (ATCC 7469) was obtained from american type culture collection bank (ATCC); lactobacillus gasseri FWJL-4 was isolated from feces of pure breast-fed healthy infants in Wuxi city, Jiangsu province.
Lactobacillus rhamnosus FSJ-13 and Lactobacillus plantarum Fjias-5 were isolated from feces of pure breast-fed healthy infants in Wuxi city, Jiangsu province in the same lot.
Example 1 screening and characterization of Lactobacillus gasseri FWJL-4
1. Screening of Lactobacillus gasseri FWJL-4
1.1 sample sources
The bacterial strain used in the invention is separated from the excrement of pure breast-feeding healthy infants in Wuxi city, Jiangsu province.
1.2 isolation and purification of the Strain
Approximately 5g of fresh sample was collected in a sterile tube and immediately sent to the laboratory for strain isolation. Putting 1g of sample into 9mL of MRS broth culture medium, mixing uniformly by vortex, and carrying out enrichment culture in an anaerobic incubator at 37 ℃ for 48 h; then sucking 1mL of enrichment liquid in a super clean bench, performing tenfold gradient dilution by using sterile physiological saline, and selecting 10 -6 、10 -7 、10 -8 And (3) three dilution gradients, wherein 100 mu L of bacterial liquid of each gradient is taken and smeared on an MRS agar culture medium, and the bacterial liquid is subjected to anaerobic culture at 37 ℃ for 48 hours. After the culture is finished, selecting a plate with 50-150 single colonies growing in the agar medium, selecting colonies with different shapes, sizes and colors, streaking and purifying the colonies on an MRS agar plate for multiple times until the colony shapes on the whole plate are consistent, and selecting single colonies to be cultured in an MRS broth medium for enrichment. The obtained strains were all stored frozen at-80 ℃ in MRS broth containing 40% glycerol.
Wherein, the MRS culture medium can be composed of: 10g of soybean peptone, 5g of beef extract, 5g of yeast powder, 20 g of glucose, 801 mL of tween-801, 2g of sodium dihydrogen phosphate, 5g of anhydrous sodium acetate, 2g of triammonium citrate, 0.02g of manganese sulfate, 0.1g of magnesium sulfate and 1L of distilled water, adjusting the pH to be about 6.2, 15g of agar, sterilizing at 121 ℃ for 15 min.
2. Identification of Lactobacillus gasseri FWJL-4
2.1 characteristics of the colonies
After culturing the Lactobacillus gasseri FWJL-4 in MRS agar medium for 48h, the diameter is between 0.3 mm and 1.5mm, the colony is round, the edge is neat, the color is white, and the surface is moist and smooth, which is shown in figure 1.
2.2 microscopic morphology:
smear of colonies of Lactobacillus gasseri FWJL-4: gram staining is positive, no spore is produced, short rod shape is distributed singly, in pairs or in clusters, and two ends are in round shape, as shown in figure 2.
2.316S rDNA identification
Extracting the target strain genome DNA by using an Ezup column type bacterial genome DNA extraction kit, taking the extracted lactobacillus genome DNA as a template for PCR amplification, carrying out a PCR experiment by using a lactobacillus specific primer, and after the PCR reaction amplification is finished, taking a PCR product to carry out agarose gel detection and photograph, wherein the length of an amplified fragment is about 550bp, which is shown in figure 3. And then, carrying out 16S rDNA PCR experiments by using bacterial universal primers 27F and 1492R, carrying out agarose gel detection and photographing on a PCR product after PCR amplification is finished, wherein the length of an amplified fragment is about 1.2Kbp, and the PCR product is sent to Shanghai biological engineering Co., Ltd by using the primers for sequencing, and the result is shown as SEQ ID No. 1. BLAST sequence alignment on the NCBI website showed that the sequence has over 99% homology with the 16S rDNA sequence of Lactobacillus gasseri.
Combining the sequence comparison result of the strain FWJL-4 with the physiological and biochemical results to determine that the screened Lactobacillus fwJL-4 is Lactobacillus gasseri FWJL-4(Lactobacillus gasseri FWJL-4).
The strain FWJL-4 of the invention has a preservation name of Lactobacillus gasseri, and the preservation unit comprises: the preservation address of Guangdong province microbial strain preservation center is No. 59 building No. 5 building of Michelia media Dazhou No. 100, Guangzhou city, and the preservation number is GDMCC No: 62365, preservation time 2022, 4 months and 11 days.
Example 2 confirmation of tolerance of Lactobacillus gasseri FWJL-4 digestive juices
1. Preparation of simulated digestive juice
1.1 preparation method of simulated saliva: after PBS was sterilized, 3g/L of alpha-amylase (purchased from Sigma, USA, having a product number of 9000-90-2, enzyme activity of 300- 2 And 1.2g/L NaHCO 3 The simulated saliva is prepared by filtering and sterilizing with a 0.22 μm microporous filter membrane.
1.2 preparation method of simulated gastric juice: sterilizing PBS, adding pepsin 3.0g/L (purchased from Sigma, USA, with a product number of 9001-75-6, and enzyme activity of 2000U/mg protein), NaCl 3g/L, KCl 1.1g/L, and CaCl 0.15g/L 2 And 0.6g/L NaHCO 3 Adjusting pH to 2.5 with 1mol/L HCL, and filtering with 0.22 μm microporous membrane for sterilization to obtain simulated gastric fluid.
1.3 preparation method of simulated intestinal juice: sterilizing PBS, adding 3g/L of ox bile salt, 0.1g/L of lipase (purchased from Sigma, USA; product number: 9001-62-1; enzyme activity is not less than 20,000U/mg protein), 1g/L of trypsin (purchased from Sigma, USA; product number: 9002-07-7; enzyme activity is 1,000- 2 And 0.6g/L NaHCO 3 Adjusting pH to 8.0 with 0.1mol/L NaOH, and filtering with 0.22 μm microporous membrane for sterilization to obtain simulated intestinal juice.
2. Digestive juice tolerance test method:
the Lactobacillus gasseri FWJL-4 strain was activated and cultured in MRS medium for two generations, centrifuged twice, the thallus was collected, resuspended in 1mL of simulated saliva for 5min, then the cells were centrifuged (4 ℃, 12000rpm, 2min) and resuspended in 2mL of simulated gastric juice, and incubated at 37 ℃ for 2 h. And simultaneously, counting and determining the viable bacteria rate by using an MRS agar culture medium pouring method. Each sample was repeated 3 times and averaged.
Subsequently, the digested 2h artificial bacteriological gastric fluid was re-centrifuged (4 ℃, 12000rpm, 2min) and resuspended in 2mL simulated intestinal fluid and incubated at 37 ℃ for 2 h. And finally, diluting the bacterial suspension, inoculating the bacterial suspension on MRS agar, and culturing for 36-48h at 37 ℃ under an anaerobic condition. And counting and determining the viable bacteria rate by using an MRS agar culture medium pouring method. The survival rates of the strains in simulated gastric fluid and simulated intestinal fluid were calculated according to the following formula, respectively.
Percent survival (LogN) 1 /LogN 0 ×100
Wherein N is 1 Representing the number of viable bacteria in a strain system treated by simulated gastric fluid or simulated intestinal fluid; n is a radical of 0 Representing the initial viable count in the strain system.
As shown in fig. 4, the number of viable bacteria of lactobacillus gasseri FWJL-4 survived for 2 hours under simulated gastric juice (pH 2.5) tended to decrease, the survival rate at 2 hours was 96.33%, and after maintaining at 37 ℃ for 2 hours in artificial gastric juice at pH 2.5, the cells were transferred to artificial intestinal juice at pH 8.0 for 2 hours, and the survival rate was as high as 91.32%.
From the above experimental results, it can be seen that the lactobacillus gasseri FWJL-4 provided by the present invention has excellent gastrointestinal fluid tolerance, can enter human intestinal tract in a living state, and live in gastrointestinal organs of animals including human to exert health efficacy, and the above characteristics are the basis of the strain as probiotic.
Example 3 use of Lactobacillus gasseri FWJL-4 for anti-inflammatory applications
1. Preparation of Strain fermentation broth
The strain preserved in glycerol tube is first streaked and activated 2-3 times on MRS agar plate, then single colony is selected and enlarged cultured in MRS broth culture medium for 18h (cultured under 37 deg.C anaerobic condition), and the culture solution is adjusted to thallus concentration of 1 × 10 with distilled water 9 Centrifuging at 4 deg.C and 8000r/min for 20min under CFU/mL, collecting supernatant as fermentation supernatant, and filtering with 0.22 μm filter membrane to remove thallus.
Each strain is subjected to 3 parallels, each group of experiments are repeated for 3 times, meanwhile, MRS broth culture medium without strain inoculation is used as a negative control, Lactobacillus rhamnosus GG is used as a positive control, and the other 2 strains of Lactobacillus plantarum Fjias-5 and Lactobacillus rhamnosus FSJ-13 screened in the process of the embodiment 1 are used as comparison; the 2 strains of lactobacillus were also prepared as described above to obtain the corresponding supernatants.
2. Determination of anti-inflammatory Capacity of Strain fermentation broth
2.1 culture of RAW264.7 mouse macrophages
Macrophages are a key effector cell in inflammatory bowel disease, and show a causal relationship between macrophage infiltration, intestinal inflammation regression deficiency and mononuclear-macrophage differentiation change in inflammatory bowel disease patients, so the macrophages are considered as potential new targets for developing new treatment methods for inflammatory bowel disease. (see, documents "Mucosal profiling of clinical-once clinical and IBD temporal common pathology and therapeutic Pathways", "Macrophages in intracellular information and resolution: a potential therapeutic target in IBD", etc.)
Resuscitating mouse macrophage (RAW264.7) in DMEM medium containing 10% (v/v) fetal calf serum and 1% penicillin/streptomycin, and placing in 5% CO at 37 deg.C 2 Culturing in a humidifying incubator. After passage twice, the stable cells were plated at 1X 10 5 The cells were seeded at a density of one cell/mL in six-well cell culture plates and cultured for 24 h.
2.2 confirmation of anti-inflammatory ability of fermentation broth of Strain on macrophage of RAW264.7 mouse
Adding a fermentation supernatant prepared according to the method described in step 1 to a six-well culture plate containing RAW264.7 cells, the cell number of the fermentation supernatant before centrifugation: macrophage number 100: 1; adding the cell after strain fermentation supernatant at 37 deg.C and 5% CO 2 The cells were co-cultured for 1 hour, washed 2 times with sterile PBS, and then cultured for 20 hours with the addition of LPS (1. mu.g/mL).
2.2.1 qPCR determination of the Gene expression level of cytokines
RNA was extracted from cells by Trizol method according to the instructions. The extracted RNA was reverse transcribed into cDNA using RNase Free ddH with 500ng RNA, 2. mu.L 5 XPrimeScript RT Master Mix 2 Make up to 10. mu.L of O. The reverse transcription conditions were 37 ℃ for 15min, 85 ℃ for 5s, and 4 ℃ for 10 min. Using cDNA as a PCR template, a qPCR reaction mix (50. mu.L) contained 50ng cDNA, 1. mu.L 10. mu.M forward primer, 1. mu.L 10. mu.M reverse primer, and 25. mu.L
Green PCR Master Mix and ddH 2 And (O). qPCR reactions were amplified using the following parameters: initial denaturation at 95 ℃ for 5min, 40 cycles of 94 ℃ for 30s, 95 ℃ for 10s and 55 ℃ for 20s, followed by extension at 72 ℃ for 2 min. mRNA levels of beta-actin (housekeeping genes) were used to normalize relative mRNA expression levels in samples and comparative cycle thresholding was used (2) -ΔΔCt ) Fold change of mRNA in each sample was calculated. Each sample was repeated 3 times and averaged. The expression of the different genes was calculated according to the following formula.
The results are shown in figure 5, and LPS-treated cells showed significantly enhanced expression of pro-inflammatory cytokines relative to cells without any treatment. Macrophages can differentiate into two classes of macrophages, namely: proinflammatory macrophages (M1) and suppressive macrophages (M2). M1-type macrophages promote inflammation by secreting TNF α, IL-6 and IL-1 β, while M2-type macrophages inhibit inflammation by secreting IL-10. In diseases associated with damage to the gastrointestinal mucosa such as peptic ulcer, gastrointestinal cancer and inflammatory bowel disease, macrophages are polarized to M1 type macrophages, and abnormal increases in TNF α, IL-6 and IL-1 β are observed (see the documents "ECM 1 is an addressing factor for the determination of M1 pathological localization in IBD in stress to LPS stimulation" and "Oxidative stress: an addressing factor in the pathogenesis of intestinal mucosal diseases of intestinal mucosa of inflammatory diseases of intestinal tract). After macrophages are treated by LPS, TNF alpha, IL-6 and IL-1 beta are respectively improved by 14 times, 72 times and 21 times, and the expression level of common anti-inflammatory cytokine IL-10 is reduced by 10 times. Compared with LPS treated cells, the cells treated by the Lactobacillus gasseri FWJL-4 can reduce the expression of proinflammatory cytokines TNF alpha, IL-6 and IL-1 beta by 64%, 94% and 75%, respectively, while the expression of the anti-inflammatory cytokine IL-10 is obviously increased by 270%.
From the experimental results, the lactobacillus gasseri FWJL-4 provided by the invention has good anti-inflammatory properties, can inhibit the polarization of macrophages, and is beneficial to playing a key role in the fields of preventing and/or treating inflammatory bowel diseases and the like.
Example 4 use of Lactobacillus gasseri FWJL-4 for antioxidation
Inflammatory bowel disease is a chronic gastrointestinal disease and has an increasing global incidence in recent years. There is increasing evidence that oxidative stress plays a crucial role in the pathogenesis and progression of inflammatory bowel disease. In addition, the relevant clinical test results also show that the oxidative stress state of patients with inflammatory bowel diseases can be obviously improved by taking the probiotics, and the treatment potential of the Lactobacillus gasseri FWJL-4 in the disease is suggested. (see the literature, "Pathology of oxidative stress in fluidic bow area and spatial antioxidant therapeutics," "biological impact on oxidative stress values in fluidic bow area: random double-blocked plasma-controlled pit velocity"). In this example, Lactobacillus gasseri FWJL-4 was used to treat mouse macrophage RAW264.7 to verify the antioxidant capacity of the strain.
1. Preparation of Strain fermentation broth
The strain preserved in glycerol tube is first streaked and activated for 2-3 times on MRS agar plate, then single colony is picked up and enlarged cultured in MRS broth culture medium for 18h (cultured under 37 deg.C anaerobic condition), and the culture solution is adjusted to lactobacillus concentration of 10 with distilled water 9 Centrifuging at 4 deg.C and 8000r/min for 20min under CFU/mL, collecting supernatant as fermentation supernatant, and filtering with 0.22 μm filter membrane to remove thallus.
Each strain is subjected to 3 parallels, each group of experiments are repeated for 3 times, meanwhile, MRS broth culture medium without strain inoculation is used as a negative control, Lactobacillus rhamnosus GG is used as a positive control, and the other 2 strains of Lactobacillus plantarum FSJ-13 and Lactobacillus rhamnosus Fjias-5 are screened in the same sample in the same batch according to the method of the embodiment 1 and are used for comparison; the 2 strains of lactobacillus were also prepared as described above to obtain the corresponding supernatants.
2. Determination of antioxidant capacity of strain fermentation liquor
2.1 culture of RAW264.7 mouse macrophages
Resuscitating mouse macrophage (RAW264.7) in DMEM medium containing 10% (v/v) fetal calf serum and 1% penicillin/streptomycin, and placing in 5% CO at 37 deg.C 2 Culturing in a humidifying incubator. After passage twice, the stable cells were plated at 1X 10 5 The cells were seeded at a density of one cell/mL in six-well cell culture plates and cultured for 24 h.
2.2 confirmation of antioxidant capacity of fermentation broth of Strain on macrophages of RAW264.7 mice
Adding a fermentation supernatant prepared according to the method described in step 1 to a six-well culture plate containing RAW264.7 cells, the cell number of the fermentation supernatant before centrifugation: macrophage number 100: 1; adding the cell after strain fermentation supernatant at 37 deg.C and 5% CO 2 The cells were co-cultured for 1h, washed 2 times with sterile PBS, and cultured for 20h with the addition of LPS (1. mu.g/mL).
Cells were harvested and the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), Glutathione (GSH) and glutathione disulfide (GSSG) in the cells was determined using the test kit according to the instructions. Each sample was repeated 3 times and averaged.
For H 2 O 2 For measurement, cells were first treated with culture supernatant of the strain for 1h, and then with 1. mu.g/mL of LPS for 2h, 4h, 6h, 8h, 10h and 12h, respectively. After LPS treatment, cells were washed with PBS and incubated with Amplex Red (50. mu.M) for 30min at 37 ℃ in the dark. Subsequently, the cells were washed twice with PBS and the corresponding values were measured at OD571 nm with a fluorescent microplate reader. Each sample was repeated 3 times and averaged.
As shown in FIG. 6, the ratio of antioxidase (SOD, GPx) and GSH/GSSG produced by the cells of the Lactobacillus gasseri FWJL-4 treated group was significantly higher than that of the other Lactobacillus treated groups. Furthermore, treatment with Lactobacillus gasseri FWJL-4 significantly reduced the amount of hydrogen peroxide in the cells. The results show that the fermentation supernatant of the Lactobacillus gasseri FWJL-4 provided by the invention has better antioxidant capacity, and is helpful for playing a key role in the fields of preventing and/or treating inflammatory bowel diseases and the like.
Example 5 molecular mechanisms of Lactobacillus gasseri FWJL-4 against oxidative stress injury to macrophages
The Nrf-2/HO-1 signaling pathway is not only considered to be a cytoprotective factor that can also regulate the expression of genes encoding antioxidant, anti-inflammatory and detoxification proteins, but it is also a powerful regulator of species longevity. Nrf2 is a cytoprotective factor that regulates anti-inflammatory and antioxidant proteins. HO-1 is one of the major effectors of Nrf 2-dependent cellular antioxidant responses, and it also exerts beneficial effects by protecting against oxidative damage, modulating inflammation, and modulating apoptosis. Researches show that probiotics playing an anti-oxidation effect can improve diseases such as inflammatory bowel diseases and the like by regulating the Nrf-2/HO-1 signal pathway, and suggest that the mechanism of the Lactobacillus gasseri FWJL-4 playing the anti-oxidation effect is possibly related to the Nrf-2/HO-1 signal pathway. (see The documents of roll of Nrf2/HO-1system in maintenance, oxidative stress and diseases: an experimental structural contained mechanism, The protective effect of Lactobacillus veras 5-aminosalicylic acid in enzymatic stability model of gut microbial and Nrf2/Ho-1 pathway) this example uses Lactobacillus grignard FWJL-4 for treating mouse macrophage RAW264.7 to verify The antioxidant ability of The strain.
1. Preparation of Strain fermentation broth
The strain preserved in glycerol tube is first streaked and activated 2-3 times on MRS agar plate, then single colony is selected and enlarged cultured in MRS broth culture medium for 18h (cultured under 37 deg.C anaerobic condition), and the culture solution is adjusted to lactobacillus concentration of 1 × 10 with distilled water 9 Centrifuging at 4 deg.C and 8000r/min for 20min under CFU/mL, collecting supernatant as fermentation supernatant, and filtering with 0.22 μm filter membrane to remove thallus.
Each strain was done in 3 replicates, each experiment was repeated 3 times, and the remaining 2 strains of lactobacillus (lactobacillus plantarum Fjias-5 and lactobacillus rhamnosus FSJ-13) screened in the process of example 1 were compared; the 2 strains of lactobacillus were also prepared as described above to obtain the corresponding supernatants.
2. Determination of molecular mechanism for alleviating oxidative stress damage of macrophages by strain fermentation liquor
2.1 culture of RAW264.7 mouse macrophages
Mouse macrophage (RAW264.7) was recovered in DMEM medium containing 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin and then incubated at 37 ℃ in 5% CO 2 Culturing in a humidifying incubator. After two passages, the stable cells were plated at 1X 10 5 The cells were seeded at a density of one cell/mL in six-well cell culture plates and cultured for 24 h.
2.2 Effect of Strain fermentation broth on macrophage Nrf2/HO-1 Signal pathway
Adding a fermentation supernatant prepared according to the method described in step 1 to a six-well culture plate containing RAW264.7 cells, the cell number of the fermentation supernatant before centrifugation: macrophage number 100: 1; adding the cell after strain fermentation supernatant at 37 deg.C and 5% CO 2 The cells were co-cultured for 1 hour, washed 2 times with sterile PBS, and then cultured for 6 hours with the addition of LPS (1. mu.g/mL).
2.2.1 extraction of cellular proteins and detection of protein expression
Nuclear/cytoplasmic proteins were extracted separately using the nuclear and cytoplasmic protein extraction kits according to the instructions. An equal amount of protein was separated by SDS-PAGE and then transferred to nitrocellulose membrane. Membranes were blocked with 5% skim milk at room temperature and then incubated overnight at 4 ℃ with the corresponding primary antibody containing 5% BSA. The membrane was then incubated with horseradish peroxidase-conjugated rabbit secondary antibody (1:5000) for 2h at room temperature. A chemically amplified luminescent solution was then added and the bands visualized by ChemiDoc Imager software. The bands were densitometrically analyzed using AlphaView software, with β -actin or histone H3 as internal controls.
As shown in FIG. 7, the Lactobacillus gasseri FWJL-4 screened by the invention can remarkably improve the expression level of Nrf2 protein in macrophage nucleoprotein and antioxidant enzyme HO-1 at the downstream of the macrophage nucleoprotein. And the treatment effect of the Lactobacillus gasseri FWJL-4 group is obviously better than that of other Lactobacillus treatment groups.
The results show that the fermentation supernatant of the Lactobacillus gasseri FWJL-4 provided by the invention has better antioxidant capacity, and the molecular mechanism of the fermentation supernatant is as follows: the protein expression level related to the Nrf2/HO-1 signal pathway mediated by oxidative stress is obviously improved, and the lactobacillus gasseri FWJL-4 plays a key role in the fields of preventing and/or treating inflammatory bowel diseases and the like.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> Lactobacillus gasseri with anti-inflammatory and antioxidant properties and application thereof
<130> BAA220387A
<160> 1
<170> PatentIn version 3.3
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<211> 1408
<212> DNA
<213> Lactobacillus gasseri
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aagtctgatg tgaaagcctt cggctcaacc ggagaattgc atcagaaact gttgaacttg 600
agtgcagaag aggagagtgg aactccatgt gtagcggtgg aatgcgtaga tatatggaag 660
aacaccagtg gcgaaggcgg ctctctggtc tgcaactgac gctgaggctc gaaagcatgg 720
gtagcgaaca ggattagata ccctggtagt ccatgccgta aacgatgagt gctaagtgtt 780
gggaggtttc cgcctctcag tgctgcagct aacgcattaa gcactccgcc tggggagtac 840
gaccgcaagg ttgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 900
gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatccagtg caaacctaag 960
agattaggag ttcccttcgg ggacgctgag acaggtggtg catggctgtc gtcagctcgt 1020
gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgtcatta gttgccatca 1080
ttaagttggg cactctaatg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 1140
caagtcatca tgccccttat gacctgggct acacacgtgc tacaatggac ggtacaacga 1200
gaagcgaacc tgcgaaggca agcggatctc tgaaagccgt tctcagttcg gactgtaggc 1260
tgcaactcgc ctacacgaag ctggaatcgc tagtaatcgc ggatcagcac gccgcggtga 1320
atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtctgt aacacccaaa 1380
gccggtggga taacctttat aggagtca 1408
Claims (10)
1. A strain of Lactobacillus gasseri FWJL-4, which has been deposited at 11.4.2022 in Guangdong province of microorganism culture Collection with the deposit number GDMCC No: 62365.
2. use of lactobacillus gasseri FWJL-4 according to claim 1 for the preparation of a medicament with anti-inflammatory and/or antioxidant action.
3. The use according to claim 2, wherein the anti-inflammatory includes, but is not limited to, the prevention and/or treatment of inflammatory bowel disease, gastritis.
4. The use according to claim 2, wherein the anti-oxidation includes, but is not limited to, increasing the activity of anti-oxidative enzymes and/or reducing oxidative stress.
5. The use of claims 2-4, wherein the medicament comprises Lactobacillus gasseri FWJL-4 ≥ 1 x 10 8 CFU/mL or 1X 10 8 CFU/g。
6. Use according to claims 2 to 4, wherein the medicament comprises a culture of said Lactobacillus gasseri FWJL-4 and/or Lactobacillus gasseri FWJL-4.
7. The use according to claim 6, wherein the culture is a fermentation supernatant obtained by culturing the Lactobacillus gasseri FWJL-4 in a culture medium for a period of time and then centrifuging the culture to remove the bacterial cells.
8. The use according to any one of claims 2 to 7, wherein the medicament further comprises a pharmaceutically acceptable carrier.
9. The use according to claim 5, wherein the medicament is an oral vaccine comprising the Lactobacillus gasseri FWJL-4 and a culture.
10. Use of lactobacillus gasseri FWJL-4 according to claim 1 for the preparation of food products, functional food products, formulated food products for special medical use or health products that are helpful for anti-oxidation and/or anti-inflammation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116622584A (en) * | 2023-06-16 | 2023-08-22 | 广东南芯医疗科技有限公司 | Application of lactobacillus gasseri LS03 in preparation of antioxidant and anti-aging products |
| CN117025479A (en) * | 2023-08-30 | 2023-11-10 | 上海市儿童医院 | Lactobacillus formatus SHMB0001 and application thereof in preventing and relieving acute colitis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105733983A (en) * | 2011-02-25 | 2016-07-06 | 保健乳制品生物免疫中心 | Isolated microorganism strains lactobacillus plantarum mcc1 dsm 23881 and lactobacillus gasseri mcc2 dsm 23882 and their use |
| CN112469812A (en) * | 2018-05-23 | 2021-03-09 | Ko生物技术有限公司 | Lactobacillus gasseri KBL697 strain and application thereof |
-
2022
- 2022-05-09 CN CN202210532248.7A patent/CN115029260B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105733983A (en) * | 2011-02-25 | 2016-07-06 | 保健乳制品生物免疫中心 | Isolated microorganism strains lactobacillus plantarum mcc1 dsm 23881 and lactobacillus gasseri mcc2 dsm 23882 and their use |
| CN112469812A (en) * | 2018-05-23 | 2021-03-09 | Ko生物技术有限公司 | Lactobacillus gasseri KBL697 strain and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116622584A (en) * | 2023-06-16 | 2023-08-22 | 广东南芯医疗科技有限公司 | Application of lactobacillus gasseri LS03 in preparation of antioxidant and anti-aging products |
| CN117025479A (en) * | 2023-08-30 | 2023-11-10 | 上海市儿童医院 | Lactobacillus formatus SHMB0001 and application thereof in preventing and relieving acute colitis |
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| CN115029260B (en) | 2023-08-25 |
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