CN116694528B - Application of bifidobacterium longum LF04 in preparation of antioxidant and anti-aging products - Google Patents

Application of bifidobacterium longum LF04 in preparation of antioxidant and anti-aging products Download PDF

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CN116694528B
CN116694528B CN202310731195.6A CN202310731195A CN116694528B CN 116694528 B CN116694528 B CN 116694528B CN 202310731195 A CN202310731195 A CN 202310731195A CN 116694528 B CN116694528 B CN 116694528B
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bifidobacterium longum
zebra fish
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antioxidant
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CN116694528A (en
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陈涛
郑康帝
刘彦
张召
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Guangdong Longsee Medical Technology Co ltd
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Abstract

The invention discloses application of bifidobacterium longum LF04 in preparation of antioxidant and anti-aging products, and belongs to the technical field of microorganisms. The bifidobacterium longum LF04 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 using the bifidobacterium longum LF 04.

Description

Application of bifidobacterium longum LF04 in preparation of antioxidant and anti-aging products
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of bifidobacterium longum LF04 in preparation of antioxidant and anti-aging products.
Background
Oxidation is an essential metabolic process of the body, but excessive oxidation causes damage to biological macromolecules. Oxidative stress is the root cause of aging and aging-related diseases in the body. The free radical has strong oxidation effect, and can trigger lipid peroxidation to damage cell membrane, thereby leading to cell death. When the human body has excessive free radical generation, the free radical can combine with biomacromolecules such as protein, nucleic acid and the like in cells of the human body and destroy the structure and the function of the biomacromolecules. The probiotics can regulate intestinal flora balance, improve metabolism of protein and vitamins, generate antibiotics to inhibit growth of harmful flora, resist tumor, enhance immunity, reduce serum cholesterol, resist oxidation, delay aging, etc.
Therefore, providing the use of bifidobacterium longum LF04 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 bifidobacterium longum LF04 in the preparation of antioxidant and anti-ageing 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:
The application of bifidobacterium longum (Bifidobacterium longum) LF04 in preparing antioxidant and anti-aging products is characterized in that the preservation number of the bifidobacterium longum LF04 is CGMCC No.23316, the bifidobacterium longum LF04 is preserved in China general microbiological culture Collection center (CGMCC), the institute of microbiology, national academy of sciences 3, the Xiya 1, north Star, the Beijing city, and the preservation date is 2021, 08, 30, and the bifidobacterium longum Bifidobacterium longum.
Further, the bifidobacterium longum LF04 is applied to the preparation of products for reducing the ROS level in vivo and improving the SOD activity in vivo.
Further, the bifidobacterium longum LF04 is a bacterial suspension.
The bifidobacterium longum LF04 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, enhances the capability of the organism for scavenging free radicals, and has good antioxidant and anti-aging effects.
Compared with the prior art, the application of the bifidobacterium longum LF04 in preparing antioxidant and anti-aging products is disclosed, the bifidobacterium longum LF04 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 theoretical reference and guiding basis are provided for developing antioxidant probiotic preparations by using the bifidobacterium longum LF 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 drawing showing the colony morphology of bifidobacterium longum LF04 on BS agar plates;
FIG. 2 is a visual graph showing the effect of bifidobacterium longum LF04 on ROS levels in a menaquinone-induced zebra fish oxidative stress model;
Wherein A: normal group; b: a model group; c: a positive control group; d: 1X 10 6 CFU/mL Bifidobacterium longum 15708; e: 1X 10 6 CFU/mL Bifidobacterium longum LF04;
FIG. 3 is a graph showing the statistical effect of bifidobacterium longum LF04 on ROS levels in a menaquinone-induced zebra fish oxidative stress model;
FIG. 4 is a graph showing the effect of bifidobacterium longum LF04 on SOD activity in menaquinone-induced zebra fish oxidative stress models.
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; bifidobacterium longum 15708 (ATCC 15708) was purchased from Beijing Bai Oubo Biotechnology Co.
EXAMPLE 1 isolation, identification and preservation of bifidobacterium longum LF04
(1) Separating:
1) The feces (about 0.1 g) of the elder with long life are dissolved in a 1.5mL centrifuge tube filled with 1mL sterile physiological saline, and are fully blown and evenly mixed by 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) From the 1 st centrifuge tube containing 10 -1 sample dilutions, 100 μl of the liquid was pipetted into the 2 nd centrifuge tube (10 -2) and diluted to 10 -2;
4) From the 2 nd centrifuge tube containing 10 -2 sample dilutions, 100 μl of liquid was pipetted into the 3 rd centrifuge tube (10 -3) and diluted to 10 -3;
5) The above steps were repeated until the dilution was 10 -4、10-5、10-6、10-7.
6) 100 Mu L of sample diluent is sucked from a centrifuge tube filled with 10 -4 sample diluent and respectively inoculated to MRS solid culture medium, BS solid culture medium and BHI solid culture medium, 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.
7) The above steps were repeated to complete the dilution coating at a dilution gradient of 10 -5、10-6、10-7.
8) After the coating, the dishes were cultured at 37℃under anaerobic conditions for 48 hours, and then were subjected to observation and recording.
9) Single colony on the plate is picked up by an inoculating loop and streaked into a BS solid culture medium, anaerobic culture is carried out for 48 hours at 37 ℃, and pure colony is obtained by separation.
10 Inoculating pure bacterial colony on the flat plate into BS liquid culture medium, anaerobic culturing at 37 ℃ for 12-16 h, adding 20% glycerol, and storing in a refrigerator at-80 ℃.
(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 feces of the elderly in Jiangling county of plum state, guangdong province is identified by morphological observation and 16S rDNA, wherein the strain LF04 is identified as bifidobacterium longum, and the 16S rDNA sequence is shown as SEQ ID NO. 3.
TGCAAGTCGAACGGGATCCATCAGGCTTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCAGTTGATCGCATGGTCTTCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAGCGAGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACGGTCGTAGAGATACGGCTTCCCTTCGGGGCGGGTTCACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTATGCCGGGAACTCACGGGGGACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCTTACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACGCGGCGACGCGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGCGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAAGCCGGTGGCCTAACCCCTTGTGGGATGAGCCGTCTAA;SEQ ID NO.3.
The strain LF04 single colony is inoculated on a BS solid culture medium, grows well under anaerobic conditions at 37 ℃, and has milky white, spherical, neat-edged and smooth surface (figure 1). The strain BS is preserved in China general microbiological culture Collection center (CGMCC), and has the preservation date of 2021, 08 and 30 days, the classification name of bifidobacterium longum Bifidobacterium longum and the preservation number of CGMCC No.23316, wherein the CGMCC is called No. 1 and No.3 of West Hirudo in the Korean region North Star of Beijing.
EXAMPLE 2 preparation of Bifidobacterium longum LF04 bacterial suspension (thallus)
Inoculating bifidobacterium longum LF04 into a BS 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 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 preparation of Bifidobacterium longum 15708 suspension (thallus)
Inoculating bifidobacterium longum 15708 after activation culture in a BS 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 4 Effect of Bifidobacterium longum LF04 on ROS levels in the zebra fish oxidative stress model
Healthy wild-type AB zebra fish that developed to 4dpf (days post fertilization) 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 bifidobacterium longum 15708 intervention group, and a bifidobacterium longum LF04 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 bifidobacterium longum 15708 was added to the intervention group (1X 10 6 CFU/mL) and 1X 10 6 CFU/M bifidobacterium longum 15708; bifidobacterium longum LF04 intervention group (1×10 6 CFU/mL) 1×10 6 CFU/mL Bifidobacterium longum LF04 was added, incubated at 28℃2.5mL per well, and 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, bifidobacterium longum 15708 intervention group, bifidobacterium longum LF04 intervention group, respectively (menaquinone was first formulated with DMSO as 600 μm stock solution and then diluted with PBS to 6 μm), 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 adopted Data 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. 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+/-7.21%), the ROS level (211.80 +/-10.19%) 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 114.22 +/-6.37%, and the difference is obvious (P < 0.005) compared with the model group (211.80 +/-10.19 percent); thus, GSH has a pronounced antioxidant effect, consistent with clinical results. Bifidobacterium longum 15708 intervention group (1X 10 6 CFU/mL) red fluorescence intensity in zebra fish was similar to model group; the ROS levels in zebra fish of the same duration bifidobacterium 15708 intervention group (1 x 10 6 CFU/mL) were 205.60 ±9.92%, with no significant differences (P > 0.05) compared to the model group (211.80 ±10.19%). Compared with the model group, the green fluorescence intensity of the bifidobacterium longum LF04 in the zebra fish body is weakened, which shows that the bifidobacterium longum LF04 can reduce the ROS level in the zebra fish body in a menaquinone-induced zebra fish oxidative stress model; the ROS level in zebra fish of the same duration bifidobacterium LF04 intervention group (1 x 10 6 CFU/mL) was 126.78 ±9.72%, with significant worse variability (P < 0.005) compared to the model group (211.80 ±10.19%). Therefore, the results show that at the same concentration, the bifidobacterium longum LF04 has stronger effect of reducing the ROS level in the zebra fish body than the bifidobacterium longum 15708 in the body oxidative stress model, and has good anti-oxidation and anti-aging effects.
Example 5 Effect of Bifidobacterium longum LF04 on SOD Activity in a zebra fish oxidative stress model
Healthy wild-type AB zebra fish that developed to 4dpf (days post fertilization) 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 bifidobacterium longum 15708-interfered group, and a bifidobacterium longum LF 04-interfered 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 bifidobacterium longum 15708 was added to the intervention group (1X 10 6 CFU/mL) and 1X 10 6 CFU/mL bifidobacterium longum 15708; bifidobacterium longum LF04 intervention group (1×10 6 CFU/mL) 1×10 6 CFU/mL Bifidobacterium longum LF04 was added, incubated at 28℃2.5mL per well, and 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, bifidobacterium longum 15708 intervention group, bifidobacterium longum LF04 intervention group, respectively (menaquinone was first formulated with DMSO as 600 μm stock solution and then diluted with PBS to 6 μm), 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 (2.79+ -0.17U/mg), the SOD activity (0.82+ -0.05U/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 2.17+/-0.13U/mg, and the difference is obvious (P < 0.005) compared with the model group (0.82+/-0.05U/mg), which shows that GSH has obvious antioxidation effect and is consistent with clinical results. The SOD activity in zebra fish of the bifidobacterium longum 15708 intervention group (1X 10 6 CFU/mL) is 0.98+/-0.11U/mg, and compared with the model group (0.82+/-0.05U/mg), the SOD activity in zebra fish has no significant difference (P > 0.05). Whereas the activity of SOD in zebra fish in the interference group of bifidobacterium longum LF04 (1X 10 6 CFU/mL) is 1.98+/-0.13U/mg, the dissimilarity is obvious (P < 0.005) compared with the model group (0.82+/-0.05U/mg). Therefore, the result shows that at the same concentration, the bifidobacterium longum LF04 has stronger effect of improving the SOD activity in the zebra fish body than the bifidobacterium longum 15708 in the in-vivo oxidative stress model, namely the capability of enhancing the body to remove free radicals, and has good effects of resisting oxidation and aging.
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 (2)

1. The application of bifidobacterium longum (Bifidobacterium longum) LF04 in preparing antioxidant and anti-aging products is characterized in that the preservation number of the bifidobacterium longum LF04 is CGMCC No.23316.
2. The application of bifidobacterium longum LF04 in preparing a product for reducing the ROS level and/or improving the SOD activity in vivo is characterized in that the preservation number of the bifidobacterium longum LF04 is CGMCC No.23316.
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CN115279385A (en) * 2020-01-15 2022-11-01 佰欧波利斯有限公司 Probiotic compositions as antioxidants

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EP4091673A4 (en) * 2020-01-17 2024-01-31 Morinaga Milk Industry Co., Ltd. Anti-aging composition

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