CN117089480A - Bacillus marinus BH03 and application thereof in preparation of antioxidant and anti-aging products - Google Patents
Bacillus marinus BH03 and application thereof in preparation of antioxidant and anti-aging products Download PDFInfo
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- 241000006384 Jeotgalibacillus marinus Species 0.000 title claims abstract description 61
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 18
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 18
- 230000003712 anti-aging effect Effects 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 230000000694 effects Effects 0.000 claims abstract description 19
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 238000001727 in vivo Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 5
- 241001344239 Bacillus haynesii Species 0.000 claims description 4
- 241000252212 Danio rerio Species 0.000 abstract description 45
- 230000036542 oxidative stress Effects 0.000 abstract description 15
- 244000005700 microbiome Species 0.000 abstract description 3
- 239000006041 probiotic Substances 0.000 abstract description 2
- 230000000529 probiotic effect Effects 0.000 abstract description 2
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- 102000019197 Superoxide Dismutase Human genes 0.000 description 16
- 108010012715 Superoxide dismutase Proteins 0.000 description 16
- HYPYXGZDOYTYDR-HAJWAVTHSA-N 2-methyl-3-[(2e,6e,10e,14e)-3,7,11,15,19-pentamethylicosa-2,6,10,14,18-pentaenyl]naphthalene-1,4-dione Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 HYPYXGZDOYTYDR-HAJWAVTHSA-N 0.000 description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 235000019143 vitamin K2 Nutrition 0.000 description 12
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- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
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- 239000007788 liquid Substances 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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- 230000001105 regulatory effect Effects 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
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- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 108700024526 zebrafish sox32 Proteins 0.000 description 1
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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Abstract
The invention discloses bacillus marinus BH03 and application thereof in preparation of antioxidant and anti-aging products, and belongs to the technical field of microorganisms. The bacillus marinus BH03 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 bacillus marinus BH 03.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus marinus BH03 and application thereof in preparing antioxidant and anti-aging products.
Background
Radicals are also called radicals, which are atoms containing unpaired electrons, radicals, molecules, ions or groups thereof. Oxygen containing unpaired electrons, called Oxygen Radical (ORF), accounts for more than 95% of the free radicals in the body, and is an active harmful substance released during oxidation in the human body. It is a device which is used to destroy the structure of cells, DNA, RNA and protein by using its electrons to prey on other molecules in body, so that the functions of cell tissue and organ in body are reduced, and can not be repaired again, so that the function of immune system in body is reduced, and various diseases are resulted, even death is resulted. The aging process involves a number of internal and external factors, the most common endogenous biochemical factors involved in the aging process being free radicals.
Antioxidant substances (antioxidants) are substances that are capable of scavenging oxygen radicals, inhibiting or eliminating and slowing down oxidation reactions. At present, natural antioxidant substances are mainly extracted from animals and plants, such as vitamins, enzymes, natural medicines and the like. However, the natural antioxidant substances in animals and plants have low content, the extraction process is complex, and the extraction from the animal and plant resources is liable to seriously destroy the animal and plant resources, so that the loss of biodiversity and ecological environment is caused.
Accordingly, providing bacillus marinus BH03 and its use in preparing antioxidant and anti-aging products is a problem that one skilled in the art would need to solve.
Disclosure of Invention
In view of this, the present invention provides bacillus marinus BH03 and its use in preparing 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:
bacillus marinus (Bacillus haynesii) BH03 with the preservation number of CGMCC No.22984 is preserved in the China general microbiological center of the Committee for culture Collection of microorganisms, CGMCC for short, and the national institute of microbiology, national academy of sciences of China, no.3, north Chen West Lu, 1, beijing, chaoyang, with the preservation date of 2021, and classified and named as Bacillus marinus Bacillus haynesii.
Further, the application of the bacillus marinus BH03 in preparing antioxidant and anti-aging products.
Further, the bacillus marinus BH03 is applied to the preparation of products for reducing the ROS level in vivo and improving SOD activity in vivo.
Further, the bacillus marinus BH03 is a bacterial suspension.
Bacillus marinus BH03 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 to remove free radicals, thereby having good antioxidation and anti-aging effects.
Compared with the prior art, the invention discloses bacillus marinus BH03 and application thereof in preparing antioxidant and anti-aging products, the bacillus marinus BH03 has the potential of obviously reducing the ROS level in zebra fish and obviously improving the SOD activity in zebra fish in a zebra fish oxidative stress model, and theoretical reference and guiding basis are provided for developing antioxidant probiotic preparations by utilizing bacillus marinus BH 03.
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 drawing showing colony morphology of Bacillus marinus BH03 of the present invention on a PYG agar plate;
FIG. 2 is a visual graph showing the effect of Bacillus marinus BH03 on ROS levels in a menaquinone-induced zebra fish oxidative stress model in accordance with the present invention;
wherein A: normal group; b: a model group; c: a positive control group; d: 1X 10 4 CFU/mL Bacillus marinus BH03; e: 1X 10 5 CFU/mL Bacillus marinus BH03; f: 1X 10 6 CFU/mL Bacillus marinus BH03;
FIG. 3 is a graph showing the statistical effect of Bacillus marinus BH03 on ROS levels in a menaquinone-induced zebra fish oxidative stress model in accordance with the present invention;
FIG. 4 is a graph showing the effect of Bacillus marinus BH03 on SOD activity in a menaquinone-induced zebra fish oxidative stress model in accordance with the present invention.
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-fluorescein diacetate (DCFH-DA) and superoxide dismutase (SOD) detection kits were purchased from Sigma-Aldrich.
EXAMPLE 1 isolation, identification and preservation of Bacillus marinus BH03
(1) Separating:
1) Kimchi (about 0.5 g) is dissolved in a 1.5mL centrifuge tube filled with 1mL sterile physiological saline, and is fully blown and evenly mixed with a 1mL sterile gun head for standby.
2) Into each of 6 sterile 1.5mL centrifuge tubes, 900. Mu.L of sterile physiological saline was added.
3) Diluting the sample to 10 by adopting a gradient dilution method -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 。
4) From the package 10 -4 100 mu L of sample diluent is sucked into a centrifuge tube of the sample diluent and coated on MRS solid culture medium, BS solid culture medium and PYG solid culture medium respectively, and 100 mu L of bacterial liquid is spread and dried, and the coating method is mild, fast in action and needs to be operated near the flame of an alcohol lamp. After coating, the side of the dish was marked, including information on name, sample number, medium name, incubation time, dilution gradient, incubation conditions (anaerobic/aerobic), etc.
5) Repeating the above steps to obtain 10 -5 、10 -6 、10 -7 Dilution coating of dilution gradient.
6) After the coating, the dishes were cultured at 37℃under anaerobic conditions for 48 hours, and then were subjected to observation and recording.
7) Single colony on the plate is picked by an inoculating loop and streaked into PYG solid culture medium, anaerobic culture is carried out for 48 hours at 37 ℃, and pure colony is obtained by separation.
8) Pure bacterial colony on the flat plate is inoculated into PYG liquid culture medium, anaerobic culture is carried out for 12-16 h at 37 ℃, 20 percent of glycerol is added, and the flat plate is placed in a refrigerator at-80 ℃ for preservation. The bacterial strain BH03 single colony is inoculated on a PYG solid culture medium, grows well under anaerobic conditions at 37 ℃, and has milky white colony, neat edge and smooth surface (figure 1).
(2) Molecular biological identification of strains: genomic DNA was extracted from the obtained strain, and a full-length fragment of 16S rDNA was amplified by PCR technique using the universal primers 27F and 1492R of 16S rDNA, followed by sequencing to identify the species of the strain.
The primer sequences of the universal primers 27F and 1492R are as follows:
27F：5’-AGAGTTTGATCCTGGCTCAG-3’；SEQ ID NO.1；
1492R：5’-GGTTACCTTGTTACGACTT-3’；SEQ ID NO.2。
experimental results: the strain screened from Guangzhou pickle in Guangdong province is identified by morphological observation and 16S rDNA, wherein the strain BH03 is identified as bacillus marinus, and the 16S rDNA sequence is shown as SEQ ID NO. 3.
GACCGGGAGTGCTATACTGCAGTCGAGCGCACTGGCCCAACTGGTATGACGTGGTTGGACTGATTTGACGATGGATTACCAGGGGGCGGGGGACGGGTGAGTAACACGTGGGCAACCTGGCCTGGAGCGGGGGATAACATTTGGAAACAGATGGTAATACCGCATAATAATCAAAACCACATGGTTTTGATTTCAAAGATGGCTTCGGCTATTACTCCAGGATGGGCCCGCGGTGGATTAACTAGTTGGTAAGGGAACGGCTTACCAAGGGGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAAACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGGTGGTGAAAAAAAACGTGCGTGAGAATAACTGGTCATGGAGTGACGGTATTCAACCAGAAAGGCACGGCTAACTACCTGCCAGCAGCCGCGGTAATACGTAAGTGGC；SEQ ID NO.3。
And combining colony characteristics of the BH03 strain and a 16srDNA amplification result, and finally identifying the bacillus marinus. The strain BH03 is preserved in China general microbiological culture Collection center (CGMCC), the national institute of microbiological culture Collection center (China) of national academy of sciences of China, no.3, north Chen West Lu, korea, beijing, the preservation date is 2021, 07, 30 days, and the strain is classified and named as Bacillus marinus Bacillus haynesii, and the preservation number is CGMCC No.22984.
EXAMPLE 2 preparation of Bacillus marinus BH03 suspension (thallus)
Inoculating bacillus marinus BH03 into a PYG liquid culture medium after activating and culturing for 24 hours at 37 ℃, and centrifuging for 10 minutes at 4 ℃ and 6000r/min to obtain bacterial precipitate; after the bacterial cell precipitate is washed twice by PBS, the bacterial cell is resuspended by PBS, and the cell concentration is regulated to be 1 multiplied by 10 4 CFU/mL、1×10 5 CFU/mL、1×10 6 CFU/mL gave a bacterial suspension (cell).
Example 3 Effect of Bacillus marinus BH03 on ROS levels in the zebra fish oxidative stress model
Select development to 4dpf (days post fertiliza)the healthy wild-type AB-line zebra fish of (j) were placed in 6-well cell culture plates with 20 fish per well. Experimental setting Normal group, model group, positive control Group (GSH), bacillus marinus BH03 (1×10) 4 CFU/mL、1×10 5 CFU/mL、1×10 6 CFU/mL) intervention group. PBS was added to both the normal and model groups, GSH solution (100. Mu.M) was added to the positive control group, and 1X 10 was added 4 CFU/mL 1X 10 intervention group addition of Bacillus marinus BH03 4 CFU/mL Bacillus marinus BH03, 1X 10 5 CFU/mL 1X 10 intervention group addition of Bacillus marinus BH03 5 CFU/mL Bacillus marinus BH03, 1X 10 6 CFU/mL 1X 10 intervention group addition of Bacillus marinus BH03 6 CFU/mL Bacillus marinus BH03, 2.5mL per well incubated at 28℃and replaced with new solution after every 24 h; after 48h incubation, the normal group was added with 2.5mL PBS (1% DMSO), and the model group, positive control group, B.marinus BH03 (1X 10) 4 CFU/mL、1×10 5 CFU/mL、1×10 6 CFU/mL) intervention 6 μm menaquinone (menaquinone was first formulated with DMSO as a 600 μm stock solution, then diluted with PBS to 6 μm) was added separately, 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:
SPSS 19.0 software is adopted for statistical data processing, and experimental data are all adoptedData represent, analyzed by T-test, compared to normal group: ### P<0.005, compared to model group: ** P<0.01， *** P<0.005。
the results are shown in fig. 2 and 3; as can be seen from fig. 2 and 3, the intensity of green fluorescence in the zebra fish body 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+/-5.91%), the ROS level (201.47 +/-8.36%) 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 109.89 +/-8.01%, and the difference is obvious (P) compared with the model group (201.47 +/-8.36%)<0.005 A) is provided; thus, GSH has a pronounced antioxidant effect, consistent with clinical results. Compared with the model group, bacillus marinus BH03 (1×10) 4 CFU/mL、1×10 5 CFU/mL、1×10 6 CFU/mL) group of zebra fish has reduced green fluorescence intensity, which indicates that bacillus marinus BH03 can reduce ROS level in the zebra fish in a menaquinone induced zebra fish oxidative stress model; at the same time, the concentration of Bacillus marinus BH03 is 1 multiplied by 10 4 CFU/mL、1×10 5 CFU/mL、1×10 6 At CFU/mL, the ROS levels in zebra fish were 163.90 + -8.70%, 142.51 + -7.37%, 120.72 + -8.82%, respectively, and the variability was significant (P) compared with the model group (201.47 + -8.36%)<0.01). Therefore, the results show that the bacillus marinus BH03 can obviously reduce the ROS level in the zebra fish body in an in-vivo oxidative stress model, and has good antioxidant and anti-aging effects.
Example 4 Effect of Bacillus marinus BH03 on SOD Activity in a zebra fish oxidative stress model
Healthy wild-type AB-line zebra fish developed to 4dpf (days post fertilization) were selected and placed in 6-well cell culture plates with 20 fish per well. Experimental setting Normal group, model group, positive control Group (GSH), bacillus marinus BH03 (1×10) 4 CFU/mL、1×10 5 CFU/mL、1×10 6 CFU/mL) intervention groups, 3 duplicate wells per group. PBS was added to both the normal and model groups,positive control group was added with GSH solution (100. Mu.M), 1X 10 4 CFU/mL 1X 10 intervention group addition of Bacillus marinus BH03 4 CFU/mL Bacillus marinus BH03, 1X 10 5 CFU/mL 1X 10 intervention group addition of Bacillus marinus BH03 5 CFU/mL Bacillus marinus BH03, 1X 10 6 CFU/mL 1X 10 intervention group addition of Bacillus marinus BH03 6 CFU/mL Bacillus marinus BH03, 2.5mL per well incubated at 28℃and replaced with new solution after every 24 h; after 48h incubation, the normal group was added with 2.5mL PBS (1% DMSO), and the model group, positive control group, B.marinus BH03 (1X 10) 4 CFU/mL、1×10 5 CFU/mL、1×10 6 CFU/mL) intervention 6 μm menaquinone (menaquinone was first formulated with DMSO as a 600 μm stock solution, then diluted with PBS to 6 μm) was added separately, 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).
SPSS 19.0 software is adopted for statistical data processing, and experimental data are all adoptedData represent, analyzed by T-test, compared to normal group: ### P<0.005, compared to model group: *** P<0.005。
the results are shown in FIG. 4; as can be seen from FIG. 4, compared with the normal group (3.03+ -0.18U/mg), the SOD activity (0.96+ -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 2.60+/-0.14U/mg, and the difference is obvious (P) compared with the model group (0.96+/-0.09U/mg)<0.005 Indicated that GSH has obvious antioxidant effectThe effect of the drug is consistent with clinical results. Bacillus marinus BH03 at a concentration of 1X 10 4 CFU/mL、1×10 5 CFU/mL、1×10 6 When CFU/mL, the SOD activities in the zebra fish body are respectively 1.72+/-0.12U/mg, 1.89+/-0.11U/mg and 2.21+/-0.14U/mg, and the difference is obvious (P) compared with the model group (0.96+/-0.09U/mg)<0.005). Therefore, the results show that the bacillus marinus BH03 can obviously improve the SOD activity in the zebra fish in an in-vivo oxidative stress model, enhance the capability of the organism for scavenging 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 (5)
1. Bacillus marinus (Bacillus haynesii) BH03, which is characterized by having a preservation number of CGMCC No.22984.
2. Use of bacillus marinus BH03 according to claim 1 for the preparation of antioxidant and anti-ageing products.
3. The use of bacillus marinus BH03 according to claim 2, for the preparation of antioxidant and anti-ageing products, wherein said bacillus marinus BH03 is a bacterial suspension.
4. Use of bacillus marinus BH03 according to claim 1 for the preparation of a product for reducing ROS levels and increasing SOD activity in vivo.
5. The use of bacillus marinus BH03 according to claim 4 for the preparation of a product for reducing ROS levels and increasing SOD activity in vivo, wherein said bacillus marinus BH03 is a bacterial suspension.
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