CN116875516B - Bifidobacterium adolescentis BA-3 and its application in resisting aging, oxidation and inflammation - Google Patents
Bifidobacterium adolescentis BA-3 and its application in resisting aging, oxidation and inflammation Download PDFInfo
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- CN116875516B CN116875516B CN202311148046.3A CN202311148046A CN116875516B CN 116875516 B CN116875516 B CN 116875516B CN 202311148046 A CN202311148046 A CN 202311148046A CN 116875516 B CN116875516 B CN 116875516B
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Abstract
The application belongs to the field of microorganisms, and particularly relates to bifidobacterium adolescentis BA-3 and application thereof in resisting aging, oxidization and inflammation. The bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 can break the vicious circle of free radicals-oxidative stress-inflammation-oxidative stress-free radicals and can realize comprehensive improvement in multiple aspects, so that the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 has performance advantages remarkably superior to other strains of the same kind when being used for resisting aging, resisting oxidation and resisting inflammation.
Description
Technical Field
The application belongs to the field of microorganisms, and particularly relates to bifidobacterium adolescentis BA-3 and application thereof in resisting aging, oxidization and inflammation.
Background
Aging is a physiological process which naturally occurs in organisms but cannot be avoided, and it is currently widely recognized that the primary purpose of aging is to prolong the life as much as possible and on the basis of this, to improve the vitality. However, no known and effective intervention measures for delaying senescence are available at present, so that the deep research on the mechanism of senescence and the development of safe and effective anti-aging products are of great significance.
Aging is often accompanied by a chronic, low-grade, systemic inflammation involving activation of the inflammatory network and release of related cytokines, including key pro-inflammatory mediators such as TNF- α, IL-6, and IL-1β, which are important driving forces for the induction of aging and aging-related diseases. At the same time, aging is also related to oxidative stress. In 1956, harman in the United states has proposed a well-known theory of free radicals. The theory suggests that under normal conditions, the formation of oxygen radicals in the body is balanced with the scavenging. However, with age, the body's ability to scavenge free radicals decreases, the balance between free radical generation and scavenging is broken, and oxygen free radicals are generated in excess of the antioxidant capacity of cells, which can put the body in oxidative stress and cause aging. In fact, the inflammatory state is also closely related to the oxidative stress state, and oxidative stress and inflammation caused by free radicals are mutually influenced by regulating the transcription level, the inflammatory reaction is aggravated by the oxidative stress, and the inflammation promotes the oxidative state through inflammatory mediators, so that the vicious circle of free radicals-oxidative stress-inflammation-oxidative stress-free radicals is formed.
Intestinal flora is always in the process of dynamic changes from birth to death. Queue studies of different people at home and abroad prove that the intestinal flora compositions and the abundance of different age groups are obviously different. Research shows that after the organism is gradually aged and enters the aged, the quantity and abundance of bifidobacteria, bacteroides and the like are obviously reduced, the quantity of facultative anaerobes such as fusobacterium and the like is obviously increased, the diversity of intestinal microbiota is reduced, and the variation of the difference between individuals is larger than that of young people. On the strain level, more researches show that the abundance of bifidobacterium adolescentis in the intestinal tract can be obviously reduced with the increase of age.
The research results published in 2021, nature Aging, indicate that the dietary supplement mode strain bifidobacterium adolescentis ATCC15703 may increase the health life and life of various animal models by up-regulating the expression and activity of host Catalase (CAT).
In addition, "Gut Microbes" reports that Bifidobacterium adolescentis can alleviate DSS-induced chronic colitis by inducing a protective Th2/Treg response and remodelling intestinal microbiota, suggesting Bifidobacterium adolescentis or improving the clinical therapeutic effect of inflammatory bowel disease.
Disclosure of Invention
Three dominant candidate bifidobacterium adolescentis BA-3, BA-4 and BA-11 are obtained from healthy young female human bodies through screening, an inflammation model of a mouse is induced by LPS (lipopolysaccharide), and the bifidobacterium adolescentis BA-3 has remarkably better anti-inflammatory and antioxidant effects through checking pro-inflammatory factors TNF-alpha, IL-6, IL-1 beta level, SOD activity and MDA level. Then, an in vivo anti-aging experiment of drosophila is carried out, and compared with the known model strain ATCC15703 with anti-aging function, the effect of bifidobacterium adolescentis BA-3 on the aspect of deferring organism aging is proved to be obviously superior to ATCC15703.
Based on the above findings, the present application provides Bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3, which is deposited in China general microbiological culture Collection center (CGMCC, address: north Chen West Lu No. 1, 3, china academy of sciences of microorganisms, post code 100101) at the day of 14, 6, 2023, and classified and named Bifidobacterium adolescentisBifidobacterium adolescentisThe preservation number is CGMCC NO.27625.
Furthermore, the application also provides a microbial inoculum which contains the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3.
In some embodiments, the bacterial agent may be a liquid bacterial agent or a solid bacterial agent.
In some embodiments, the microbial inoculum also contains other functional bacteria.
In some embodiments, the microbial agent further comprises auxiliary materials allowed in the field of microbial preparations.
In a specific embodiment, the microbial inoculum can be prepared by adopting a conventional technical means.
Furthermore, the application also provides a fermented product or a fermented extract, which is obtained by fermenting the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3.
In the application, the fermentation broth supernatant and thalli obtained after the fermentation of the bifidobacterium adolescentis BA-3 have positive effects in resisting aging, resisting oxidization and resisting inflammation.
In the present application, the fermented product is a fermentation broth supernatant and/or a cell obtained by fermenting the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3.
In the present application, the fermentation extract is an organic solvent extract of a fermentation broth supernatant and/or a cell obtained by fermenting the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3.
Furthermore, the application also provides the application of the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the microbial inoculum or the ferment extract in at least one aspect of the following: 1) Anti-aging; 2) Oxidation resistance; 3) Anti-inflammatory.
References to "anti-aging" in the present application are also to be understood as meaning "delaying aging", "preventing or slowing signs of aging", "prolonging life" and the like. Likewise, references to "antioxidant" in the present application are also to be understood as meaning "preventing or reducing peroxidic damage", "eliminating excessive oxidative free radicals", and the like. Reference herein to "anti-inflammatory" is also to be understood as meaning "preventing or reducing inflammation" and the like. The use of the strains, inoculants or ferments or fermentation extracts of the present application in the above-described aspects is also within the scope of the present application.
Further, the application also provides the use of said bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or said bacterial agent or said ferment or fermented extract for the preparation of a composition for use in at least one of the following: 1) Anti-aging; 2) Oxidation resistance; 3) Anti-inflammatory.
In some embodiments, the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the bacterial agent or the ferment or fermentation extract is used to reduce the level of pro-inflammatory factors.
Preferably, the pro-inflammatory factor comprises one or more of TNF-alpha (tumor necrosis factor-alpha; tumor necrosis factor-alpha), IL-1β (Interleukin-1β; interleukin-1β) and IL-6 (Interleukin-6; interleukin-6). TNF-alpha, IL-1 beta, IL-6, etc. are pro-inflammatory factors that play an important role in inflammation in vivo, and changes in levels may manifest in vivo inflammation.
In some embodiments, the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the bacterial agent or the ferment or fermented extract is used for at least one of the following purposes: a) Improving the activity of antioxidant enzyme; b) Reducing the level of peroxidation products.
Preferably, the antioxidant enzyme comprises SOD (superoxide dismutase; superoxide dismutase). SOD is a type of catalyst capable of catalyzing superoxide anion free radical (O 2 - ) Disambiguation to H 2 O 2 And O 2 Has antioxidant and antiaging effects, and has the action mechanism of scavenging superoxide anion free radical (O) 2 - ）。
Preferably, the peroxidation product comprises MDA (Malondialdehyde). MDA is a product of intracellular lipid peroxidation and is also an important index of oxidative stress.
In some embodiments, the subject of the use of the application includes humans and other animals other than humans. Other animals than humans mentioned herein may be primates such as monkeys, gorillas, gibbons, gorillas, etc.; livestock animals such as pigs, cattle, sheep, horses, camels, cats, dogs, etc.; poultry animals such as chicken, duck, goose, quail, etc.; etc.
In some embodiments, the composition is a food composition, a feed composition, a topical skin composition, or a pharmaceutical composition.
The application also provides a food composition, which contains the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the microbial inoculum or the fermentation product or the fermentation extract.
In some embodiments, the food composition is a health functional food.
The type of the food composition of the present application is not particularly limited, and the food composition may be in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., or may be added to ordinary foods such as sugar, biscuits, chewing gum, ice cream, noodles, bread, beverages, etc.
The food composition of the present application can be prepared by using one or more of a filler, an extender, a binder, a wetting agent, a disintegrant, a sweetener, a flavoring agent, a preservative, a surfactant, a lubricant, an excipient, etc. according to the form, according to the conventional method.
The application also provides a feed composition, which contains the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the microbial inoculum or the fermentation product or the fermentation extract.
In some embodiments, the feed composition may be a carbohydrate feed, a protein feed, a green feed, a mineral feed, or the like. Meanwhile, the specific components of the above feeds may be confirmed by those skilled in the art in combination with common general knowledge, for example, corn, wheat bran, wheat, barley, sorghum, rice bran, etc. may be contained in the carbohydrate feed.
The application also provides a skin external composition which contains the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the microbial inoculum or the ferment extract.
The external composition for skin according to the present application means any substance that can be externally applied to the skin and can include various pharmaceutical dosage forms. For example, the composition may be in the form of an ointment, lotion, gel, cream, spray, suspension, emulsion, patch, etc., but is not limited thereto.
In some embodiments, the skin external composition may further comprise functional additives and ingredients contained in general skin external compositions. Wherein the functional additive may comprise a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, sphingolipids and seaweed extracts. The ingredients contained in the general skin external composition include oil components, moisturizers, emollients, surfactants, organic or inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, colorants, flavors, blood circulation promoters, coolants, antiperspirants, and purified water.
The application also provides a pharmaceutical composition, which contains the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the microbial inoculum or the fermentation product or the fermentation extract.
The pharmaceutical compositions of the application may be administered orally (e.g., by administration or inhalation) or parenterally (e.g., by injection, transdermal absorption, rectal administration). The injection may be, for example, intravenous, subcutaneous, intramuscular, or intraperitoneal.
The pharmaceutical composition of the present application may be formulated into tablets, capsules, granules, fine granules (fine granules), powders, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, sprays and the like depending on the route of administration. The pharmaceutical compositions according to the present application in the above-described various forms can be manufactured by known techniques using a pharmaceutically acceptable carrier (carrier) commonly used in each dosage form. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrants (disintegrating agent), lubricants, preservatives, antioxidants, isotonic agents, buffers, coating agents, sweeteners, dissolving agents, base agents, dispersing agents, wetting agents, suspending agents, stabilizers, colorants, and the like.
The term "and/or", "and/or" as used herein includes a selection of any one of two or more of the listed items and also includes any and all combinations of the listed items, including any two or more of the listed items, or all combinations of the listed items. It should be noted that, when at least three items are connected by a combination of at least two conjunctions selected from the group consisting of "and/or", "and/or", it should be understood that, in the present application, the technical solutions include technical solutions that all use "logical and" connection, and also include technical solutions that all use "logical or" connection. For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, and/or B, and/or C, and/or D" include any one of A, B, C, D (i.e., the technical scheme of "logical or" connection), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and also include four combinations of A, B, C, D (i.e., the technical scheme of "logical and" connection).
The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
In the present application, the terms "plurality", and the like refer to, unless otherwise specified, 2 or more in number.
In the application, the technical characteristics described in an open mode comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme comprising the listed characteristics.
In the present application, "preferred", "better", "preferred" are merely embodiments or examples which are better described, and it should be understood that they do not limit the scope of the present application. In the present application, "optional" means optional or not, that is, means any one selected from two parallel schemes of "with" or "without". If multiple "alternatives" occur in a technical solution, if no particular description exists and there is no contradiction or mutual constraint, then each "alternative" is independent.
The bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 can break the vicious circle of free radicals-oxidative stress-inflammation-oxidative stress-free radicals and can realize comprehensive improvement in multiple aspects, so that the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 has performance advantages remarkably superior to other strains of the same kind when being used for resisting aging, resisting oxidation and resisting inflammation.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the effect of different Bifidobacterium adolescentis interventions on body weight, food intake and water intake of mice in an example of the application; in the figure, a: the weight of the mice with different bifidobacteria adolescents continuously perfused for 14 days changes; b: feeding of mice; c: water intake of mice.
FIG. 2 shows the effect of different Bifidobacterium adolescentis interventions on inflammatory factors in serum of mice with LPS inflammatory models in an embodiment of the application; in the figure, a: TNF- α levels in serum; IL-1 beta levels in serum; IL-6 levels in serum; * P <0.05, < P <0.01, < P < 0.001 vs LPS group; #P <0.05, #P <0.01, #P < 0.001 vs Control Group.
FIG. 3 shows the effect of different Bifidobacterium adolescentis interventions on oxidative stress factors in serum of mice with LPS inflammation model in examples of the present application; in the figure, a: SOD activity in serum; b: MDA levels in serum; * P <0.05, < P <0.01, < P < 0.001 vs LPS group; #P <0.05, #P <0.01, #P < 0.001 vs Control Group.
FIG. 4 shows exogenous addition of BA-3 to wild-type Drosophila w in an embodiment of the present application 1118 Influence of lifetime. In the figure, a and B are respectively: wild female and male Drosophila w 1118 Survival curves of (2); c and D are respectively: wild female and male Drosophila w 1118 Is a comparison of the average life, median life and maximum life; * P (P)<0.05, **P<0.01，***P＜0.001 vs Placebo group；#P<0.05, ##P<0.01，###P＜0.001 vs ATCC15703 group。
FIG. 5 shows exogenous addition of BA-3 to wild-type Drosophila w in an embodiment of the present application 1118 Influence of the anti-gravity climbing ability; in the figure, a and B are respectively: wild female and male Drosophila w 1118 Comparison of the countergravity crawling ability on day 30; * P (P)<0.05, **P<0.01，***P＜0.001 vs Placebo group；#P<0.05, ##P<0.01，###P＜0.001 vs ATCC15703 group。
Detailed Description
The following describes specific embodiments of the present application in detail. It will be understood that the embodiments described herein are for the purpose of illustration and explanation only and are not intended to limit the present application, as many modifications and variations of the present application may be made by those skilled in the art without departing from the scope or spirit thereof. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise defined, all terms (including technical and scientific terms) used to describe the application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present application. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.
Embodiments of the present application will be described in detail below with reference to examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not noted, are preferably referred to in the guidelines given in the present application, and may be according to the experimental manuals or conventional conditions in the art, and may be referred to other experimental methods known in the art, or according to the conditions suggested by the manufacturer.
The bifidobacterium adolescentis BA-4 and bifidobacterium adolescentis BA-11 used in the following examples are other bifidobacterium adolescentis dominant strains obtained when the dominant strains are selected according to the present application. The bifidobacterium adolescentis ATCC15703 used in the following examples is obtained by a commercially available route.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
EXAMPLE 1 isolation screening and identification of Bifidobacterium adolescentis Bifidobacterium adolescentis BA-3
1. Separation and screening of bifidobacterium adolescentis Bifidobacterium adolescentis BA-3
A number of healthy young women without inflammation or any intestinal disease were enrolled in providing samples, and participants passed the health examination of the physical examination center and provided information about their age (20-25), menstrual cycle, and other health activities, etc. by questionnaires. No antibiotics were taken by the population within 6 months prior to sample collection, no history of probiotic administration, and no history of inflammation.
And diluting the collected fecal sample, then coating the diluted fecal sample on a GAM blood agar culture medium, carrying out anaerobic culture at 36-38 ℃ for 48-72 h, picking up single bacteria, streaking and culturing on a new GAM blood agar culture medium plate to obtain purified bacterial colonies, and obtaining the bifidobacterium adolescentis BA-3 after separation, activity screening and identification.
2. Physiological characteristics of Bifidobacterium adolescentis BA-3
After bifidobacterium adolescentis BA-3 is subjected to anaerobic culture on a GAM blood agar medium for 48 hours, bacterial colonies are round and convex, edges are neat, and surfaces are moist and smooth; gram-positive staining, no spores, no capsules and no flagella, and the thalli are in a straight shape or a curved shape.
3. Molecular characterization of Bifidobacterium adolescentis BA-3
Inoculating bifidobacterium adolescentis BA-3 into a GAM broth culture medium, performing anaerobic culture at 36-38 ℃ for 24 hours, centrifuging to collect thalli, extracting DNA and performing whole genome sequencing.
The molecular identification results showed that the ANI (Average Nucleotide Identity ) value of bifidobacterium adolescentis BA-3 and GCF_003467335.1 (Bifidobacterium adolescentis) was 98.7072%.
The bifidobacterium adolescentis BA-3 is preserved in the China general microbiological culture collection center (CGMCC) at the 14 th month of 2023, and is classified and named as bifidobacterium adolescentis by the classification of the 3 rd national institute of microbiology, the institute of China academy of sciences, the 1 st part of North Chen West Lu, the 3 rd part of the Korean area of BeijingBifidobacterium adolescentisThe preservation number is CGMCC NO.27625.
4. Whole genome data analysis
Full genome sequencing of bifidobacterium adolescentis BA-3, construction of libraries of different inserts using full genome shotgun (Whole Genome Shotgun, WGS) strategy, sequencing using a second generation sequencing technology (Next-Generation Sequencing, NGS), based on Illumina NovaSeq sequencing platform, and sequencing using a third generation single molecule sequencing technology, based on PacBio sequence sequencing platform.
Then, the drug resistance gene, virulence factor and biogenic amine potential of the bifidobacterium adolescentis BA-3 are analyzed by combining the sequencing result, and the specific analysis process and the specific analysis result are as follows:
1) Bifidobacterium adolescentis BA-3 drug resistance gene analysis
Analysis method
Uploading the whole genome sequence of BA-3 to https:// card.mcmaster.ca/analysis/RGI by using the CARD self-contained software RGI (v6.0.2), comparing the CARD 3.2.7 database, and selecting default parameters for analysis, particularly Select Data Type: DNA sequence; select criterion Perfect and Strict hits only; nudge is more than or equal to 95 percent identity Loose hits to Strict, namely Exclude Nudge; sequence Quality: high Quality/coverage.
Conclusion of analysis
By comparing the drug-resistant gene database, the ultra-broad spectrum beta lactamase, vancomycin drug-resistant gene, carbapenem drug-resistant gene, methicillin drug-resistant gene and multiple drug-resistant genes are not detected in the bifidobacterium adolescentis BA-3 genome.
2) Bifidobacterium adolescentis BA-3 virulence gene analysis
Analysis method
The virulence factor database (VFDB,http://www.mgc.ac.cn/VFs/) The virulence factor gene database VFDB (version 2023.04.11) was downloaded, parameters were chosen to be identity > 80%, coverage > 70% based on the blastx algorithm using diamondv2.0.15.153 software (command line: -id 80-query-cover 70), and the amino acid sequences in the BA-3 genome nucleotide sequences and databases.
Analysis results
By comparing the virulence gene database, no gene annotation in the bifidobacterium adolescentis BA-3 genome is the virulence gene.
3) Bifidobacterium adolescentis BA-3 producer amine potential analysis
Analysis method
The amino acid sequence of the bifidobacterium adolescentis BA-3 whole genome gene was aligned with the eggNOG (evolutionary gene genealogy Nonsupervised Orthologous Groups) database using the emermer-2.1.9 software.
Analysis results
By aligning the egNOG database and searching the genes related to the biogenic amine, genes related to the biogenic amine such as histidine decarboxylase, tyrosine decarboxylase, ornithine decarboxylase, lysine decarboxylase and the like are not detected in the bifidobacterium adolescentis BA-3 genome.
Example 2 LPS-induced mouse inflammation model
Experimental method
1. Grouping and modeling method
Healthy ICR mice are all male, 30 animals are all male, the weight range is 22+/-2 g, the ambient temperature is 20-25 ℃, the healthy ICR mice are fed with standard feed and drink water freely, and the healthy ICR mice circulate every twelve hours. Mice were adaptively maintained for 1 week, and randomly divided into a Control group (Control), a model Control group (LPS), a Bifidobacterium adolescentis BA-3-intervention group (LPS+BA-3), a Bifidobacterium adolescentis BA-4-intervention group (LPS+BA-4), and a Bifidobacterium adolescentis BA-11-intervention group (LPS+BA-11), each group being 6. Bifidobacterium adolescentis (sterile PBS was resuspended to a final concentration of 10) was administered daily intragastrically to the intervention group 9 CFU/mL) 200 μl, and the blank and model control groups were filled with equal volumes of sterile PBS solution daily for 14 days of continuous intervention. After the last administration, the model control group and the interference group mice of bifidobacterium adolescentis BA-3, bifidobacterium adolescentis BA-4 and bifidobacterium adolescentis BA-11 are subjected to intraperitoneal injection of LPS 5 mg/kg (prepared by normal saline), an acute inflammation model of the LPS mice is built, the blank control group is subjected to injection of an equal volume of normal saline, and the mice are sacrificed after 2 hours and blood is taken.
2. Body weight change, food intake and water intake change
During the intervention period, the body weight, 24-hour intake and water intake of each group of mice were recorded every two days. Data were analyzed for significance using GraphPad Prism 9.
3. Determination of IL-1 beta, IL-6 and TNF-alpha levels in serum
After blood is taken from each group of mice, the mice are placed at room temperature and centrifuged for 20 min at 3000 g, serum is carefully sucked and used for subsequent index determination, and the concentration of IL-6, TNF-alpha and IL-1 beta in each group of serum is detected by adopting a double antibody sandwich method according to the description method of ELISA detection kit. Data were analyzed for significance using GraphPad Prism 9.
4. Determination of SOD and MDA levels in mouse serum
The detection of SOD and MDA levels in the serum of the mice is carried out according to the instruction of the kit, and the absorbance value is measured by an enzyme-labeled instrument under the condition of 450 and nm. Data were analyzed for significance using GraphPad Prism 9.
Experimental results
1. Body weight change, food intake and water intake change
The growth was normal during each group of mice intervention, and there was no significant difference in mouse body weight between the different groups (fig. 1, A). Compared with the blank group and the model group, the 24-hour ingestion and water intake of mice in the intervention group of the bifidobacterium adolescentis BA-3, BA-4 and BA-11 do not show significant difference (figures 1B and C), and the preliminary explanation that the bifidobacterium adolescentis BA-3, BA-4 and BA-11 strains do not have adverse effect on the health condition of the mice.
2. Changes in inflammatory factor levels in serum
Lipopolysaccharide LPS can elicit an inflammatory response by activating inflammatory signaling pathways and promoting secretion of inflammatory cytokines. Among them, TNF- α, IL-1β, IL-6, etc. are all pro-inflammatory factors that play an important role in the LBP-induced acute inflammation model. Thus, the inflammatory status of mice can be assessed by detecting changes in the levels of pro-inflammatory factors in the serum of the mice. The levels of inflammatory factors in the serum of each group of mice after intraperitoneal injection of LPS are shown in FIG. 2. Compared with the blank control group, the levels of TNF-alpha, IL-6 and IL-1 beta of the model control group are obviously increased, which proves that the LPS-induced acute inflammation model is successfully constructed. The bifidobacterium adolescentis BA-3, BA-4 and BA-11 were able to reduce the levels of pro-inflammatory factors in serum to a different extent compared to the model control group. Wherein, the BA-3 has the best effect of reducing the proinflammatory factors in 3 strains, and compared with a model control group, the levels of TNF-alpha, IL-1 beta and IL-6 in serum of LPS mice in a BA-3 intervention group are obviously reduced. Whereas the levels of TNF- α and IL-1β were significantly reduced in the serum of LBP mice in BA-11 intervention group compared to model control group, no significant difference was observed in IL-6. In the BA-4 intervention group, there was a significant decrease in IL-1β levels, but there was no significant difference in TNF- α and IL-6 levels compared to the model control group. Experimental results show that bifidobacterium adolescentis BA-3, BA-4 and BA-11 show a certain anti-inflammatory effect in improving LPS-induced mouse inflammation, wherein the anti-inflammatory effect of the BA-3 strain is optimal.
3. Variation of SOD and MDA levels in serum
Oxidative stress is closely related to inflammation and is also one of the bases of inflammation caused by LPS. SOD is the main antioxidant enzyme for scavenging free radicals in the organism, and has the function of blocking the primary reaction of the free radicals caused by superoxide anion free radicals, thereby reducing the generation of other active oxygen; MDA is the product of lipid peroxidation, and the level of MDA indirectly reflects the severity of free radical attack on body cells. To evaluate the remission of different bifidobacteria adolescents on oxidative stress injury in LPS model mice, SOD activity and MDA levels in serum were measured to evaluate the antioxidant capacity of the strain. As shown in fig. 3, the model group showed significantly decreased SOD activity and significantly increased MDA levels after 2 hours of LPS injection compared to the blank group. The dry prognosis of the bifidobacterium adolescentis BA-3 and BA-4 and the dry prognosis of the bifidobacterium adolescentis BA-11 are all different in degree, so that the SOD activity is improved, and the MDA level is reduced. Wherein, compared with the model control group, BA-3 significantly improves the activity of serum SOD, while BA-4 and BA-11 have no significant difference compared with the model control group. While BA-3, BA-4 and BA-11 all significantly reduced serum MDA levels compared to the model control. Experimental results show that bifidobacterium adolescentis BA-3, BA-4 and BA-11 show a certain antioxidation effect in improving LPS-induced oxidative stress of mice.
Conclusion(s)
In the embodiment, bifidobacterium adolescentis BA-3, BA-4 and BA-11 are continuously fed into healthy male ICR mice for 14 days, then an LPS acute inflammation model is established, and the condition of the mice is not adversely affected by bifidobacterium adolescentis BA-3, BA-4 and BA-11 through monitoring of body weight, food intake and water intake. And the determination of the levels of the pro-inflammatory factors shows that the levels of TNF-alpha, IL-6 and IL-1 beta in serum of mice with LPS models can be reduced to a certain extent by all BA-3, BA-4 and BA-11, wherein the BA-3 can obviously reduce the levels of 3 pro-inflammatory factors at the same time. The detection results of the oxidative stress related factors show that BA-3, BA-4 and BA-11 all show a certain effect of resisting oxidative stress in LPS inflammatory mice. In the whole, among three strains of bifidobacterium adolescentis BA-3, BA-4 and BA-11, BA-3 has no adverse effect on the health of mice, and exhibits more excellent anti-inflammatory and antioxidant effects.
Example 3 Drosophila experiments
Experimental method
The drosophila as a model organism widely applied to the aging research field has the advantages of short growth period, strong fertility, easy distinction of male and female, and the like, and meanwhile, the drosophila has high gene conservation, has about 75 percent of genes homologous to human diseases, and has very similar metabolic pathways to human beings.
1. Cultivation of Drosophila
The wild type Drosophila strain used in this example is w 1118 . All were kept in Drosophila incubators at 25℃constant temperature, 65% constant humidity, 12 h light/12 h darkness. The medium formulation is shown in Table 1. In the intervention group, bifidobacterium adolescentis BA-3 and ATCC15703 were resuspended to 10 using sterile PBS 10 CFU/mL, and mixing the bifidobacterium adolescentis BA-3 suspension, ATCC15703 suspension and basal medium to a final concentration of 10 9 CFU/mL, control group using the same volume of sterile PBS mixed with basal medium.
Table 1 culture medium formulation
2. Life test
After free mating of the new born drosophila, 48 and h, 300 female drosophila and male drosophila are randomly selected, transferred into empty culture tubes, fasted for 2 hours, randomly divided into 3 groups, and 100 female drosophila and male drosophila in each group are respectively transferred into culture tubes containing bifidobacterium adolescentis BA-3-containing culture medium, ATCC 15703-containing culture medium or basal culture medium, and 10 female drosophila and male drosophila in each group are respectively transferred into each tube. Every 3 days transfer to a new culture tube and observe the number of drosophila that died recorded until all drosophila died. The survival time per tube of Drosophila was recorded and a survival curve was drawn and statistically analyzed for mean, median and longest (maximum) life (the longest life is the average life of the last 10% of Drosophila per group). Data were analyzed for significance using GraphPad Prism 9.
3. Reverse gravity crawling capability test
The female and male drosophila were cultured in a culture tube containing a medium containing bifidobacterium adolescentis BA-3, a medium containing ATCC15703 or a basal medium for 30 days, and 20 female and male drosophila were randomly selected from each group and transferred to a measuring tube, respectively. After the drosophila is adapted for 3min, the test tube is gently beaten to enable the drosophila to fall at the bottom, timing is started, the number of drosophila at the marker position with the crawling distance exceeding 8cm within 10s is measured, the experiment is repeated for 3 times, more than 10min each time, and the test tube is horizontally placed during the interval. And calculating the climbing index of the drosophila according to a formula, wherein the higher the climbing index is, the stronger the climbing ability of the drosophila against gravity is represented. Data were analyzed for significance using GraphPad Prism 9.
Climbing index= (number of drosophila/total number of drosophila successfully climbing to 8 cm) ×100%.
Experimental results
1. Life test
Bifidobacterium adolescentis BA-3 had a longevity-extending effect on male and female wild-type drosophila compared with ATCC 15703-interfered and control groups (fig. 4A, B). Comparing the bifidobacterium adolescentis BA-3 intervention group with the control group, the average life span, median life span and maximum life span of the bifidobacterium adolescentis BA-3 intervention group in the female and male drosophila are all significantly higher than those of the control group (fig. 4C, D). Comparing the bifidobacterium adolescentis BA-3-interfered group with ATCC 15703-interfered group, the mean life span of the bifidobacterium adolescentis BA-3-interfered group in female wild-type drosophila was significantly higher than that of ATCC 15703-interfered group, whereas in male wild-type drosophila, the median life span of the bifidobacterium adolescentis BA-3-interfered group was significantly different from that of ATCC 15703-interfered group (fig. 4C, D).
2. Reverse gravity crawling experiment
The anti-gravity crawling ability is a sport ability and is an important index for representing the health life of the drosophila melanogaster. Compared with a control group, the bifidobacterium adolescentis BA-3 group has obviously improved anti-gravity climbing capacity of female and male wild type drosophila melanogaster on day 30. There was a significant difference in the ability of male wild-type Drosophila to climb against gravity on day 30 in the bifidobacterium adolescentis BA-3 group compared to the ATCC15703 group.
Conclusion(s)
In the embodiment, the bifidobacterium adolescentis BA-3 is added into a basic culture medium of the drosophila, and the delaying effect of the bifidobacterium adolescentis BA-3 on the aging of the drosophila is investigated by detecting the life index of the drosophila and detecting the reverse gravity crawling ability of the drosophila at a specific time node. Wild type Drosophila w of different sexes 1 1 1 8 In the bifidobacterium adolescentis BA-3 intervention group, the median survival time, the mean life span and the mean maximum life span were significantly higher than those of ATCC15703 group and control group, as shown in fig. 4A, 4B, 4C, 4D. Furthermore, on day 30, the reverse gravity crawling ability of the bifidobacterium adolescentis BA-3 intervention group of different sexes was also significantly better than that of ATCC15703 group and control group, as shown in fig. 5A and 5B.
Taken together, this example demonstrates that exogenous bifidobacterium adolescentis BA-3 supplementation can increase the longevity of wild-type drosophila melanogaster of different natures and improve the healthy longevity index of wild-type drosophila melanogaster, and that the effect is superior to that of model strain ATCC15703, which has been reported to have anti-aging function.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application.
Claims (13)
1. Bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.27625.
2. A microbial agent comprising the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 as claimed in claim 1.
3. Use of bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 as claimed in claim 1 or a microbial agent as claimed in claim 2 for non-disease therapeutic use in at least one of the following: 1) Anti-aging; 2) Oxidation resistance; 3) Anti-inflammatory.
4. Use of bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 as claimed in claim 1 or a bacterial agent as claimed in claim 2 in the manufacture of a composition for use in at least one of the following: 1) Anti-aging; 2) Oxidation resistance; 3) Anti-inflammatory.
5. The use according to claim 3 or 4, wherein the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the bacterial agent is used to reduce the level of pro-inflammatory factors.
6. The use of claim 5, wherein the pro-inflammatory factor comprises one or more of TNF- α, IL-1β, and IL-6.
7. The use according to claim 3 or 4, wherein the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the bacterial agent is used for at least one of the following purposes: a) Improving the activity of antioxidant enzyme; the antioxidant enzyme comprises SOD; b) Reducing the level of peroxidation products; the peroxidation product comprises MDA.
8. The use according to claim 3 or 4, wherein the subject of administration of the use comprises humans and other animals than humans.
9. The use according to claim 4, wherein the composition is a feed composition, a composition for external use on the skin or a pharmaceutical composition.
10. A food composition comprising the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the microbial agent of claim 2 of claim 1.
11. A feed composition comprising the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the microbial agent of claim 2 of claim 1.
12. A composition for external use for skin comprising the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the microbial agent of claim 2 as claimed in claim 1.
13. A pharmaceutical composition comprising the bifidobacterium adolescentis (Bifidobacterium adolescentis) BA-3 or the microbial agent of claim 2 of claim 1.
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