CN116606776B - Application of lactobacillus johnsonii LS04 in preparation of antioxidant and anti-aging products - Google Patents
Application of lactobacillus johnsonii LS04 in preparation of antioxidant and anti-aging products Download PDFInfo
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Abstract
The invention discloses application of lactobacillus johnsonii LS04 in preparation of an antioxidant and anti-aging product, and belongs to the technical field of microorganisms. The lactobacillus johnsonii LS04 disclosed by the invention has the potential of obviously reducing the ROS level in the zebra fish body and obviously improving the SOD activity in the zebra fish body in a zebra fish oxidative stress model, and provides theoretical reference and guiding basis for developing an antioxidant probiotic preparation by utilizing the lactobacillus johnsonii LS 04.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of lactobacillus johnsonii LS04 in preparation of antioxidant and anti-aging products.
Background
Reactive Oxygen Species (ROS) are byproducts of cellular aerobic metabolism and play an important role in the cell life cycle. The low concentration of ROS can act as a key signaling molecule within cells to regulate cell growth, proliferation, and differentiation. However, accumulation of ROS can severely damage cellular biological macromolecules such as proteins, lipids, and DNA, etc., resulting in a variety of chronic diseases including atherosclerosis, arthritis, diabetes, neurodegenerative diseases, aging, inflammatory bowel disease, etc. A large number of experimental researches show that the antioxidant or the free radical scavenger is helpful for avoiding the accumulation of ROS, has a protective effect on oxidative damage, can effectively control the occurrence of the diseases, and plays a good role in preventing. However, most of the chemically synthesized or plant-extracted antioxidative drugs are not recommended for long-term use due to potential adverse reactions. Compared with the traditional antioxidant drugs, probiotics with antioxidant effect are paid attention to because of the characteristics of small side effect, other probiotic effects and the like.
Thus, providing the use of lactobacillus johnsonii LS04 in the preparation of antioxidant and anti-aging products is a problem to be solved by the person skilled in the art.
Disclosure of Invention
In view of this, the present invention provides the use of lactobacillus johnsonii LS04 for the preparation of antioxidant and anti-aging products.
Menaquinone is an oxidizing agent that produces unstable semiquinones through the intracellular reductase system (microsomal P450 reductase and mitochondrial respiratory chain reductase), which enter the redox cycle, producing reactive oxygen species. Menaquinone can induce zebra fish to establish an oxidative stress model.
Through specific fluorescent staining (green, mainly located in cell nuclei and mitochondria), the whole body of the zebra fish subjected to oxidative stress reaction is obviously much more green than that of normal zebra fish, and the active oxygen content in the zebra fish can be observed under a fluorescent microscope.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
Use of lactobacillus johnsonii (Lactobacillusjohnsonii) LS04 having a preservation number of CGMCC No.22094 (see patent No. 202211558460.7) for the preparation of an antioxidant and anti-aging medicament.
Further, the lactobacillus johnsonii LS04 is applied to the preparation of medicines for reducing the ROS level in the body and improving the SOD activity in the body.
Further, the lactobacillus johnsonii LS04 is a bacterial suspension.
The lactobacillus johnsonii LS04 can obviously reduce the ROS level in the zebra fish body and obviously improve the SOD activity in the zebra fish body in an in-vivo oxidative stress model, and can strengthen the capability of the organism for scavenging free radicals, thereby having good antioxidation and anti-aging effects.
Compared with the prior art, the application of lactobacillus johnsonii LS04 in preparing antioxidant and anti-aging medicines is disclosed, and the lactobacillus johnsonii LS04 has the potential of remarkably reducing the ROS level in the zebra fish body and remarkably improving the SOD activity in the zebra fish body in a zebra fish oxidative stress model, so that theoretical reference and guiding basis are provided for developing antioxidant probiotic preparations by using the lactobacillus johnsonii LS 04.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a visual graph showing the effect of Lactobacillus johnsonii LS04 on ROS levels in a menaquinone-induced zebra fish oxidative stress model according to the present invention;
Wherein A: normal group; b: a model group; c: a positive control group; d: 1X 10 6 CFU/mL Lactobacillus johnsonii 33200; e: 1X 10 6 CFU/mL Lactobacillus johnsonii LS04;
FIG. 2 is a graph showing the statistical effect of Lactobacillus johnsonii LS04 on ROS levels in a menaquinone-induced zebra fish oxidative stress model according to the present invention;
FIG. 3 is a graph showing the effect of Lactobacillus johnsonii LS04 of the present invention on SOD activity in a menaquinone-induced zebra fish oxidative stress model.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Reduced Glutathione (GSH), menaquinone, dimethyl sulfoxide (DMSO) were all purchased from shanghai source leaf biotechnology limited; 2',7' -dichloro-dihydro-fluorescein diacetate (DCFH-DA) and superoxide dismutase (SOD) detection kits were purchased from Sigma-Aldrich company; lactobacillus johnsonii 33200 (ATCC 33200) was purchased from bio-technology limited of beijing Bai-o-borg.
EXAMPLE 1 preparation of Lactobacillus johnsonii LS04 suspension (thallus)
Inoculating lactobacillus johnsonii LS04 after activation culture in MRS liquid culture medium, culturing at 37 ℃ for 24 hours, and centrifuging at 4 ℃ for 10 minutes at 6000r/min to obtain bacterial precipitate; after the cell pellet was washed twice with PBS, the cell was resuspended in PBS, and the cell concentration was adjusted to 1X 10 6 CFU/mL to obtain a cell suspension (cell).
EXAMPLE 2 preparation of Lactobacillus johnsonii 33200 bacterial suspension (thallus)
Inoculating lactobacillus johnsonii 33200 after activation culture in MRS liquid culture medium, culturing at 37deg.C for 24 hr, and centrifuging at 4deg.C for 10min at 6000r/min to obtain bacterial precipitate; after the cell pellet was washed twice with PBS, the cell was resuspended in PBS, and the cell concentration was adjusted to 1X 10 6 CFU/mL to obtain a cell suspension (cell).
Example 3 Effect of Lactobacillus johnsonii LS04 on ROS levels in the zebra fish oxidative stress model
Healthy wild-type AB zebra fish that developed to 4dpf (dayspostfertilization) were selected and placed in 6-well cell culture plates with 20 fish per well. The experiments set up normal, model, positive control (GSH), lactobacillus johnsonii 33200, and lactobacillus johnsonii LS 04. PBS was added to both the normal and model groups, GSH solution (100. Mu.M) was added to the positive control group, and Lactobacillus johnsonii 33200 was added to the intervention group (1X 10 6 CFU/mL) and 1X 10 6 CFU/mL Lactobacillus johnsonii 33200; lactobacillus johnsonii LS04 intervention group (1X 10 6 CFU/mL) 1X 10 6 CFU/mL of Lactobacillus johnsonii LS04 was added, incubated at 28℃at 2.5mL per well, and the new solution was changed after every 24 h; after 48h incubation, 2.5mL of PBS (1% DMSO) was added to the normal group, and 6 μm menaquinone was added to the model group, positive control group, lactobacillus johnsonii 33200 intervention group, lactobacillus johnsonii LS04 intervention group (menaquinone was first made up with DMSO as 600 μm stock solution and then diluted with PBS to 6 μm) and 2.5mL per well; after 24h incubation at 28 ℃, the solution is discarded, the zebra fish is washed 3 times by PBS, 20 mug/mL DCFH-DA solution is added, 3mL of each hole is incubated for 1h at 28 ℃ in a dark place, the zebra fish is washed 3 times by PBS, and the fluorescence intensity in the zebra fish is observed under a fluorescence microscope and recorded by photographing. Quantitative statistical analysis of fluorescence intensity (S) in zebra fish was performed using Image J software. ROS levels in zebra fish were calculated as follows:
SPSS19.0 software was used to statistically process the data, experimental data were all expressed as x+ -SEM data, analyzed by T-test, compared to normal group: ### P <0.005, compared to model group: *** P <0.005.
The results are shown in figures 1 and 2; as can be seen from fig. 1 and 2, the intensity of green fluorescence in zebra fish reflects the level of ROS; compared with the normal group, the green fluorescence intensity in the zebra fish body of the model group is enhanced, which indicates that the ROS level in the zebra fish body of the model group is increased; meanwhile, compared with a normal group (100.00+/-7.48%), the ROS level (205.66 +/-8.57%) in the zebra fish body of the model group is obviously increased (p < 0.005), which indicates that the current zebra fish oxidative stress model is successfully established.
Compared with the model group, the positive control Group (GSH) has reduced green fluorescence intensity in the zebra fish body, which indicates that GSH can reduce the ROS level in the zebra fish body in a menaquinone induced zebra fish oxidative stress model; meanwhile, the ROS level in the zebra fish of the positive control group is 116.85 +/-7.43%, and the difference is obvious (P < 0.005) compared with the model group (205.66 +/-8.57 percent); thus, GSH has a pronounced antioxidant effect, consistent with clinical results. Lactobacillus johnsonii 33200 intervention group (1X 10 6 CFU/mL) red fluorescence intensity in zebra fish was similar to model group; meanwhile, the level of ROS in zebra fish in the interference group (1X 10 6 CFU/mL) of lactobacillus johnsonii 33200 is 195.42 +/-10.12 percent, and no significant difference (P > 0.05) is generated compared with the model group (205.66 +/-8.57 percent). Compared with the model group, the green fluorescence intensity of the lactobacillus johnsonii LS04 zebra fish is reduced, which shows that the lactobacillus johnsonii LS04 can reduce the ROS level in the zebra fish in a menaquinone induced zebra fish oxidative stress model; meanwhile, the ROS level in zebra fish of the interference group (1X 10 6 CFU/mL) of lactobacillus johnsonii LS04 is 123.32+/-6.00%, and the difference is obvious (P < 0.005) compared with the model group (205.66 +/-8.57%). Therefore, the results show that at the same concentration, the lactobacillus johnsonii LS04 has stronger effect of reducing the ROS level in zebra fish bodies than the lactobacillus johnsonii 33200 in the in-vivo oxidative stress model, and has good anti-oxidation and anti-aging effects.
Example 4 Effect of Lactobacillus johnsonii LS04 on SOD Activity in a zebra fish oxidative stress model
Healthy wild-type AB zebra fish that developed to 4dpf (dayspostfertilization) were selected and placed in 6-well cell culture plates with 20 fish per well. The experiments set up a normal group, a model group, a positive control Group (GSH), a lactobacillus johnsonii 33200 intervention group, a lactobacillus johnsonii LS04 intervention group, 3 duplicate wells per group. PBS was added to both the normal and model groups, GSH solution (100. Mu.M) was added to the positive control group, and Lactobacillus johnsonii 33200 was added to the intervention group (1X 10 6 CFU/mL) and 1X 10 6 CFU/mL Lactobacillus johnsonii 33200; lactobacillus johnsonii LS04 intervention group (1X 10 6 CFU/mL) 1X 10 6 CFU/mL of Lactobacillus johnsonii LS04 was added, incubated at 28℃at 2.5mL per well, and the new solution was changed after every 24 h; after 48h incubation, 2.5mL of PBS (1% DMSO) was added to the normal group, and 6 μm menaquinone was added to the model group, positive control group, lactobacillus johnsonii 33200 intervention group, lactobacillus johnsonii LS04 intervention group (menaquinone was first made up with DMSO as 600 μm stock solution and then diluted with PBS to 6 μm) and 2.5mL per well; after 24h incubation at 28 ℃, the solution was discarded, the zebra fish was washed 3 times with PBS, and the zebra fish was collected into 1.5mL centrifuge tubes, 50mg zebra fish per tube, 6 tubes per experimental group; after the water in the centrifuge tube was sucked dry, 250. Mu.L of a buffer solution (a buffer solution of a superoxide dismutase (SOD) detection kit) was added. The zebra fish homogenate was broken up by holding a micro-electric tissue homogenizer with S-18KS until no distinct tissue fragments were present, centrifuged at 15000 Xg at 4℃for 15min and the supernatant was collected. The SOD activity of each group was measured using a superoxide dismutase (SOD) detection kit (Sigma-Aldrich).
SPSS19.0 software was used to statistically process the data, experimental data were all expressed as x+ -SEM data, analyzed by T-test, compared to normal group: ### P <0.005, compared to model group: *** P <0.005.
The results are shown in FIG. 3; as can be seen from FIG. 3, compared with the normal group (2.94+ -0.09U/mg), the SOD activity (0.88+ -0.09U/mg) in the zebra fish of the model group is significantly reduced (p < 0.005), which indicates that the current zebra fish oxidative stress model is successfully established.
The SOD activity in the zebra fish of the positive control group is 2.33+/-0.13U/mg, and the difference is obvious (P < 0.005) compared with the model group (0.88+/-0.09U/mg), which shows that GSH has obvious antioxidation effect and is consistent with clinical results. The SOD activity in zebra fish of the interference group (1X 10 6 CFU/mL) of Lactobacillus johnsonii 33200 was 1.01+ -0.09U/mg, and there was no significant difference (P > 0.05) compared with the model group (0.88+ -0.09U/mg). Whereas the activity of SOD in zebra fish of the interference group (1X 10 6 CFU/mL) of Lactobacillus johnsonii LS04 was 2.08+ -0.13U/mg, the dissimilarity was significant (P < 0.005) compared with the model group (0.88+ -0.09U/mg). Therefore, the results show that at the same concentration, the lactobacillus johnsonii LS04 has stronger effect of improving the SOD activity in zebra fish bodies than lactobacillus johnsonii 33200 in an in-vivo oxidative stress model, namely the capability of enhancing the body to remove free radicals, and has good anti-oxidation and anti-aging effects.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (4)
1. The application of lactobacillus johnsonii (Lactobacillus johnsonii) LS04 in preparing antioxidant and anti-aging medicines is characterized in that the preservation number of the lactobacillus johnsonii LS04 is CGMCC No.22094.
2. Use of lactobacillus johnsonii LS04 according to claim 1 for the preparation of an antioxidant and anti-ageing medicament, wherein said lactobacillus johnsonii LS04 is used in the form of a bacterial suspension.
3. Use of lactobacillus johnsonii LS04 as claimed in claim 1 for the manufacture of a medicament for reducing ROS levels and increasing SOD activity in vivo.
4. Use of lactobacillus johnsonii LS04 according to claim 3 for the manufacture of a medicament for reducing ROS levels and increasing SOD activity in vivo, wherein said lactobacillus johnsonii LS04 is used in the form of a bacterial suspension.
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CN115992059A (en) * | 2022-07-06 | 2023-04-21 | 南京财经大学 | Lactobacillus johnsonii for producing feruloyl esterase and application thereof in relieving ulcerative colitis |
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