WO2018218694A1 - 具有头孢抗性且高表达Sir2蛋白的长双歧杆菌及其应用 - Google Patents

具有头孢抗性且高表达Sir2蛋白的长双歧杆菌及其应用 Download PDF

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WO2018218694A1
WO2018218694A1 PCT/CN2017/087285 CN2017087285W WO2018218694A1 WO 2018218694 A1 WO2018218694 A1 WO 2018218694A1 CN 2017087285 W CN2017087285 W CN 2017087285W WO 2018218694 A1 WO2018218694 A1 WO 2018218694A1
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bifidobacterium longum
high expression
sir2
aging
sir2 protein
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English (en)
French (fr)
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孙晗笑
郭晴
利时雨
刘梦鸽
陈露平
黄金群
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广州弘宝元生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/533—Longum

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  • the invention belongs to the field of screening and application of new strains, and more particularly to a Bifidobacterium longum BL16 having cephalosporin resistance and high expression of Sir2 protein and its application in related foods or medicines.
  • Bifidobacteria are obligate anaerobic bacteria that have important regulatory functions in the digestive tract of animals and are considered to be a Gram-positive probiotic, which is dominant in the large intestine, followed by the small intestine. There is a small amount of oral distribution.
  • the Tissier researcher from the Pasteur Institute in France separated for the first time in the feces of breast-fed healthy infants.
  • the fungus belongs to the genus Bifidobacterium of the actinomycetes and has various forms and manifestations due to different growth environments.
  • Bending rod shape, V shape, L or Y shape, etc. in addition to the discovery of Bifidobacterium globularis, when cultured on solid medium, the colonies are convex round, milky white, smooth and complete surface, soft texture; Suitable growth temperature is 37 ° C ⁇ 41 ° C, the pH can be grown 5.0 ⁇ 8.0, the most suitable is 6.5 ⁇ 7.0; according to DNA homology and sugar fermentation characteristics, a total of 24 species, and There are only 12 species of human origin, and among the 12 species, only 5 species, including Bifido.bifidum and Bifidobacterium adolescentis, are used in the human intestinal health and can be colonized in the human intestine. (Bifido.adolescentis), Bifido.infantis, Bifido.breve, and Bifido.1ongum.
  • Bifidobacteria does not produce endotoxin in the body, nor does it produce pathogenic substances and harmful gases. It is a recognized intestinal bacterium that is harmless to the human body and exerts many beneficial physiological functions on the human body.
  • the Sir2 (silence information regulator) gene is a class of NAD+-dependent histone/non-histone deacetylases that are highly conserved from bacteria to higher eukaryotes.
  • the Sir2-related protein encoded by the Sir2 family gene is collectively named Sirtuin.
  • Sir2 protein plays an important role in the regulation of physiological activities such as cell survival, apoptosis and senescence. There have been no reports of bifidobacteria that highly express the Sir2 protein.
  • the present invention further studies Sir2 protein and bifidobacteria by screening for bifidobacteria having cephalosporin resistance, and screening for long bifidobacteria expressing Sir2 protein by immunoblotting to make it in disease treatment or related foods and health care. Play a role in the product.
  • the present invention screens a Bifidobacterium longum BL16 with cephalosporin resistance and high expression of Sir2 protein, and preserves it in the Guangdong Provincial Collection of Microorganisms and Cultures, with the preservation number GDMCC No. 60191, the preservation address. It is the 5th floor of Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong province. The deposit date is May 24, 2017.
  • the Bifidobacterium longum BL16 having the cephalosporin resistance and high expression of the Sir2 protein of the present invention can be used for preparing an anti-aging, immune-enhancing, antibacterial food or medicine.
  • the present invention also provides an anti-aging food comprising an effective amount of the cephalosporin-resistant and high-expressing Sir2 protein-producing Bifidobacterium longum BL16 as an active ingredient.
  • the present invention also provides an anti-aging medicine comprising an effective amount of the cephalosporin-resistant and high-expressing Sir2 protein-producing Bifidobacterium longum BL16 as an active ingredient, and pharmaceutically acceptable Accepted carrier.
  • the present invention also provides an immunostimulating food comprising an effective amount of the cephalosporin-resistant and high-expressing Sir2 protein-producing Bifidobacterium longum BL16 as an active ingredient.
  • the present invention also provides a medicine for enhancing an immune function comprising an effective amount of the cephalosporin-resistant and high-expressing Sir2 protein-producing Bifidobacterium longum BL16 as an active ingredient, and a pharmacy An acceptable carrier.
  • the present invention also provides an antibacterial agent comprising an effective amount of the cephalosporin-resistant and high-expressing Sir2 protein-producing Bifidobacterium longum BL16 as an active ingredient, and pharmaceutically acceptable a.
  • the present invention also provides a food for regulating intestinal flora, which comprises an effective amount of the Bifidobacterium bifidobacterium Bifidobacterium having cephalosporin resistance and high expression of Sir2 protein according to claim 1 as an active ingredient.
  • a food for regulating intestinal flora which comprises an effective amount of the Bifidobacterium bifidobacterium Bifidobacterium having cephalosporin resistance and high expression of Sir2 protein according to claim 1 as an active ingredient.
  • the food is particularly suitable for the regulation of intestinal flora disturbances such as diarrhea caused by the use of antibiotics, and restores the balance of the intestinal flora.
  • the present invention also provides a pharmaceutical preparation for regulating intestinal flora, which comprises an effective amount of the Bifidobacterium bifidobacterium Bifidobacterium having cephalosporin resistance and high expression of Sir2 protein according to claim 1 as an active ingredient.
  • the drug is especially suitable for the regulation of intestinal flora disorders such as diarrhea caused by the use of antibiotics. Restore the balance of the intestinal flora.
  • Bifidobacterium longum BL16 having cephalosporin resistance and high expression of Sir2 protein has certain antibacterial activity, and has strong cephalosporin resistance, which can significantly improve organs.
  • the MAD content reduces the activity of SOD, GSH-Px, NO and NOS. Therefore, the Bifidobacterium longum BL16 having cephalosporin resistance and high expression of Sir2 protein can improve the body's antioxidant capacity and remove the aging body. Excessive free radicals inhibit the peroxidation process of the body, tissues and cells, and ultimately play a role in delaying aging and enhancing immunity. They can be used to prepare related foods or medicines and have good application value.
  • Figure 1 shows the results of identification of the sir2 gene of Bifidobacterium longum in the present invention.
  • FIG. 2 is a diagram showing the electrophoresis of Bifidobacterium longum proteins.
  • Figure 3 is a result of Western blot analysis of the Sir2 protein of Bifidobacterium longum.
  • the pure culture of the isolated and purified strain was inoculated into the enrichment liquid medium, incubated at 37 ° C for 24 h, and after Gram staining, the cell morphology was observed under a light microscope, and a microscope with a digital imaging system was used. Select a suitable field of view for photographic recording.
  • PCR reaction system 25 ⁇ L: primer 1/1.0 ⁇ L; primer 2/1.0 ⁇ L; 10 ⁇ PCR Buffer/2.5 ⁇ L; dNTP mix/2.0 ⁇ L; Taq enzyme/0.3 ⁇ L; DNA template/1.0 ⁇ L; ultrapure water/25 ⁇ L.
  • the PCR reaction conditions were: 94 ° C - 4 min; then 94 ° C - 30 s, 55 ° C - 40 s, 72 ° C - 90 s cycle 30 times, and finally 72 ° C - 10 min.
  • the template was replaced with sterile deionized water as a negative control.
  • 3.0 ⁇ L of the amplified product was subjected to agarose gel electrophoresis. After the electrophoresis is finished, the tape to be tested is sent to Shanghai Biotech for sequence determination.
  • the sequence results of the assay strains were compared with the known 16S rDNA sequences in NCBI by Blast to obtain the closest strain to determine the species of the bacteria isolated from the experiment.
  • test bacteria were the above-obtained bacteria, and the control strain was Lactobacillus rhamnosus, and 100 ⁇ L of each strain was subjected to activation and resuscitation in 100 mL of lactic acid bacteria MRS enrichment broth medium.
  • the indicator bacteria were Escherichia coli ATCC11105, Clostridium difficile NY-5, Staphylococcus aureus NT-12.
  • Susceptibility testing was performed according to the KB method promulgated by the American Association of Clinical Laboratory Standards. First, use a sterile forceps to separately take the drug-sensitive papers containing different antibiotics, and paste them on each plate that has been inoculated with the test bacteria (200 ⁇ L of the suspension of the bacteria to be tested is 3.0 ⁇ 10 8 CFU/mL, respectively.
  • 200 ⁇ L of the suspension of the bacteria to be tested is 3.0 ⁇ 10 8 CFU/mL, respectively.
  • a lactic acid bacteria MRS enrichment broth agar medium with a temperature of about 50 ° C, mix well and pour the plate. Approximately 5 sheets of susceptibility paper are attached to each plate, and the distance between the sheets is approximately equal and marked.
  • the anaerobic culture was carried out at 37 ° C overnight, and the inhibition of the plate was observed. The size of the inhibition zone was measured by a vernier caliper and recorded.
  • the Bifidobacterium longum sir2 primer the upstream primer is shown in SEQ ID NO: 3, and the downstream primer is shown in SEQ ID NO: 4.
  • PCR reaction system template - 1 ⁇ L; upstream primer - 0.5 ⁇ L; downstream primer - 0.5 ⁇ L; dNTP (10 mM) - 0.5 ⁇ L; LA Taq (5 U / ⁇ L) - 0.5 ⁇ L; 10 ⁇ PCR buffer - 2.5 ⁇ L; - 19.5 ⁇ L.
  • Glue Dispose 12% of the separation gel and inject it into the gap of the glass plate to 1.5cm from the upper edge, add the appropriate amount of 75% ethanol; to solidify, pour off the upper liquid, inject 5% concentrated glue, insert the comb, naturally Dry.
  • Luminescence detection TBST liquid washing membrane, incubated with luminescent liquid for 3 min, blotting the luminescent liquid with filter paper, placing the film and PVDF film into the tableting box for 1 min, taking out the film fixing for 30 s, washing with water, drying, scanning.
  • the three strains of Bifidobacterium longum Sir2 protein have a bright band at 2.7 KD.
  • B. longum BL16 which has a cephalosporin resistance and high expression of the Sir2 protein, was successfully screened.
  • sub-acute aging mice induced by D-galactose are used as aging animal models by Bifidobacterium longum BL16 (hereinafter referred to as B16), and the index and biochemical indexes of various organs of the mouse are measured and compared. Explore the role of B16.
  • mice half male and half female, weighing (20 ⁇ 2) g, were provided by the Experimental Animal Center of Jinan University. After adaptive breeding of 1W, they were randomly divided into four groups of 10 each. The first group was a blank control group, the second group was a model control group, and the third group was a bifidobacterium group.
  • Example 2 D-galactose was purchased from Beijing Dingguo Biological Co., Ltd., and SOD, MDA, GSH-Px, NO, and NOS kits were purchased from Nanjing Jiancheng Bioengineering Research Institute.
  • the model control group and the administration group received daily subcutaneous injection of D-galactose 0.15 mg/g body weight (prepared in physiological saline), and the control group was injected with the same amount of normal saline.
  • Gastric B16 solution Boifidobacterium concentration to 10 10 , daily intragastric administration of 1 mL
  • blank control group and model control mice were given the same amount of normal saline for 40 days.
  • the animals in each group were weighed, sacrificed by cervical dislocation, and the brain, thymus, spleen, liver and kidney were taken out.
  • the residual blood was washed with 0.9% physiological saline and accurately weighed to calculate the organ index.
  • the brain tissue used for sectioning was fixed in 10% formaldehyde, and the remaining organ tissues were wrapped with tin foil paper and stored in an ultra-low temperature refrigerator at -80 °C for use.
  • the frozen brain tissue was placed in a glass tube for use in a tissue homogenizer, and 9 volumes of pre-cooled physiological saline (distilled water) was added thereto, and after thorough grinding, a 10% brain tissue homogenate was prepared.
  • the content of total protein in brain homogenate was determined by Coomassie brilliant blue method; the content of MDA was determined by thiobarbituric acid reaction method; the activity of GSH-Px was detected by enzymatic method; the specific detection method of NO by nitrate reduction method was referred to the kit manual.
  • the weight of tissues and organs, especially the weight changes of vital organs such as brain, thymus, spleen, liver and kidney are important indicators reflecting the degree of aging of organisms.
  • the most obvious immune system decay is the thymus and spleen.
  • the reduction of thymus and spleen weight, atrophy, and functional decline can reduce immune function, leading to an increase in the incidence of malignant diseases in elderly animals (human) and accelerate the aging process.
  • the results showed that D-galactose-induced subacute aging cerebellum, thymus, spleen, liver and kidney index were significantly decreased.
  • BL16 treatment significantly increased the brain, thymus, spleen, liver and kidney weight of aging mice, indicating that BL16 can effectively fight against The aging of the mouse brain, maintaining the body's immune function, and improving disease resistance, has a positive significance for delaying aging and enhancing immunity.
  • the organism has an antioxidant system in which SOD is one of the most critical enzymes. This enzyme can scavenge superoxide anion free radicals and protect cells from damage. Its activity reflects the antioxidant capacity of the body. Studies by the American Bardimo Medical Research Center have shown that there is a significant positive correlation between SOD content and life span in mammals. The results of this experiment showed that BL16 can significantly enhance the SOD activity in brain tissue of D-galactose-induced aging mice, improve the ability of brain tissue to scavenge free radicals, and block free radical response.
  • Free radicals are produced by normal metabolism of the body, but excessive free radicals can invade cells, especially unsaturated fatty acids in brain tissue, causing lipid peroxidation, forming metabolite MDA, which in turn forms lipofuscin, through proteins and nucleic acids. Cross-linking of biological macromolecules, destroying the function of normal cells. Therefore, the MDA content often reflects the degree of lipid peroxidation in the body, indirectly reflecting the degree of cell damage, and is an important indicator of aging.
  • NO is endogenously synthesized by NOS using L-arginine. It is a small molecular substance with a wide range of biological functions. It is soluble in water and lipids, and can rapidly diffuse to adjacent cells through paracrine, and intracellular ornithine ring. The heme gene on the enzyme activates the enzyme, and ornithine cyclase catalyzes the production of ornithine (cGMP). cGMP acts as a second messenger, causing a series of biological effects through signal cascade amplification should.
  • the main role of NO in the nervous system is to mediate the response of neurons to excitatory amino acids and enhance the body's learning and memory ability.
  • NOS is a rate-limiting enzyme for NO synthesis.
  • Neurons containing NOS are present in many parts of the central nervous system, including the cerebral cortex, hippocampus, and hypothalamus. NO plays an important role in the regulation of neuronal activity in these sites. Physiological aging is closely related to the decrease of NOS activity in brain tissue.
  • the decrease of NOS activity leads to a decrease in NO content in the brain, which can lead to a decrease in the phagocytic ability of macrophages; a decrease in the content of active guanine cyclase, a decrease in the ability to regulate various metabolisms, and an accelerated aging.
  • the results of this experiment showed that the NOS activity and NO content of the model aging mice decreased significantly, and BL16 could significantly increase the NOS activity and NO content of aging model mice.
  • the above results suggest that BL16 can increase the production of NO by increasing the activity of NOS, and its biological effects can be sustained, which can delay the aging of the body.
  • GSH-Px is an important catalytic peroxidation enzyme widely distributed in the body. It specifically catalyzes the reduction of hydrogen peroxide by GSH and inhibits the formation of OH by nicotinamide adenine dinucleotide (NADH). - Impairing the damage to lipid peroxidation, thereby protecting the structure and function of the cell membrane.
  • NADH nicotinamide adenine dinucleotide
  • this experiment shows that bifidobacteria can increase the levels of SOD, GSH-Px, NO and NOS, and reduce the content of MDA, suggesting that bifidobacteria may increase the body's antioxidant capacity and remove excessive free radicals from the aging body. It inhibits the peroxidation process of the body, tissues and cells, and plays a role in delaying aging.

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Abstract

提供了具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16，及其在制备抗衰老、增强免疫功能或调节肠道菌群的食品或药品中的应用。具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16可以通过改善肠道微生态环境，清除体内自由基，延缓动物的衰老过程，还可显著提高衰老器官的重量，保持机体的免疫功能，提高抗病能力，因此具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16可用于制备抗衰老、增强免疫功能或调节肠道菌群的食品或药品。

Description

具有头孢抗性且高表达Sir2蛋白的长双歧杆菌及其应用 技术领域
本发明属新菌种筛选和应用领域，更具体地说，本发明涉及一种具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16及其在相关食品或药品中的应用。
背景技术
双歧杆菌是一类存在于动物消化道中对宿主具有重要调节功能的专性厌氧菌，被认为是一种革兰氏阳性益生菌，该菌在大肠中占绝对优势，次之在小肠，口腔分布有少量。在1899年，由法国巴斯德研究院的Tissier研究员第一次在母乳喂养的健康婴儿粪便中分离出来，该菌类属于放线菌科双歧杆菌属，形态多样，因生长环境不同而表现出弯曲杆形、V形、L或Y形等，除此外还发现有球形双歧杆菌，当固体培养基上培养时，其菌落为凸状圆形，乳白色，表面光滑完整，质地柔软；最适合的生长温度为37℃～41℃，可以生长的pH值为5.0～8.0，最适为6.5～7.0；根据DNA的同源性和糖发酵特性来分，总共分为24个种属，而人类来源的只有12个种属，在这12个种属中，用人类健康事业并能在人体肠道内定植的仅有5种，包括两歧双歧杆菌(Bifido.bifidum)、青春双歧杆菌(Bifido.adolescentis)、婴儿双歧杆菌(Bifido.infantis)、短双歧杆菌(Bifido.breve)以及长双歧杆菌(Bifido.1ongum)。
双歧杆菌在体内不产生内毒素，也不产生致病物质和有害气体，是一种公认的对人体无害的肠道细菌，对人体发挥许多有益生理功能。
Sir2(silence information regulator)基因是一类从细菌到高等真核生物都高度保守的NAD+依赖的组蛋白/非组蛋白去乙酰化酶。Sir2家族基因所编码的Sir2相关蛋白统一命名为Sirtuin。Sir2蛋白对细胞的存活、凋亡、衰老等生理活动的调节具有重要作用。目前尚未有高表达Sir2蛋白的双歧杆菌的相关报道。
发明内容
本发明进一步对Sir2蛋白和双歧杆菌进行研究，通过筛选具有头孢抗性的双歧杆菌，再通过免疫印迹筛选出高表达Sir2蛋白的长双歧杆菌，使其在疾病治疗或相关食品、保健品中发挥作用。
为了实现上述发明目的，本发明筛选出一种具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16，保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60191，保藏地址为广东省广州市越秀区先烈中路100号大院59号楼5楼，保藏日期为2017年5月24日。
本发明具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16可用于制备抗衰老、增强免疫功能、抗菌的食品或药品。
为了实现上述发明目的，本发明还提供了一种抗衰老食品，其包括有效剂量的作为活性成分的所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16。
为了实现上述发明目的，本发明还提供了一种抗衰老药品，其包括有效剂量的作为活性成分的所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16，以及药学上可接受的载体。
为了实现上述发明目的，本发明还提供了一种增强免疫功能的食品，其包括有效剂量的作为活性成分的所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16。
为了实现上述发明目的，本发明还提供了一种增强免疫功能的药品，其包括有效剂量的作为活性成分的所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16，以及药学上可接受的载体。
为了实现上述发明目的，本发明还提供了一种抗菌药物，其包括有效剂量的作为活性成分的所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16，以及药学上可接受的载体。
为了实现上述发明目的，本发明还提供了一种调节肠道菌群的食品，其包括有效剂量的作为活性成分的权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16和/或其菌体蛋白成分。该食品特别适用于因使用抗生素导致的腹泻等肠道菌群紊乱的调节，使肠道菌群恢复平衡。
为了实现上述发明目的，本发明还提供了一种调节肠道菌群的药品，其包括有效剂量的作为活性成分的权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16和/或其菌体蛋白成分，以及药学上可接受的载体。该药品特别适用于因使用抗生素导致的腹泻等肠道菌群紊乱的调节， 使肠道菌群恢复平衡。
相对于现有技术，本发明经实验发现，具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16具有一定的抑菌活性，还具有很强的头孢抗性，可显著提高器官中的MAD含量，降低SOD、GSH-Px、NO和NOS的活性，因此本发明具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16可通过提高机体的抗氧化能力，清除衰老机体产生的过多自由基，抑制机体、组织、细胞的过氧化过程，最终起到延缓衰老和增强免疫的作用，可用于制备相关的食品或药品，具有良好的应用价值。
附图说明
图1为本发明长双歧杆菌sir2基因鉴定结果。
图2为本发明长双歧杆菌蛋白电泳图。
图3为本发明长双歧杆菌Sir2蛋白Western blot结果。
具体实施方式
以下结合实施例，对本发明进行进一步详细说明。
实施例1
1.益生菌初筛
收集3个正常人粪便样品各1.0g，用LB培养基扩增培养，蒸馏水稀释至10-8共9个稀释度，取1mL，涂布到含有8～12g/L碳酸钙的MRS分离固体培养基和改良MRS分离固体培养基上，置于厌氧培养箱中，37℃厌氧培养48h。培养结束后，取出平板挑选出具有可见溶钙圈或蓝色、浅蓝色的菌落，并转接至MRS增菌肉汤培养基中在37℃厌氧培养48h，并放入4℃冰箱保存备用。
2.菌株的形态学观察。
将分离纯化的菌株纯培养物接种到增菌液体培养基中，37℃培养24h复活后，经革兰氏染色操作后，在光学显微镜下观察其细胞形态特征，并用带有数字成像系统的显微镜选取合适视野进行照相记录。
3.分子生物学-16S rDNA鉴定：
16S rDNA全长通用引物：引物1如SEQ ID NO:1所示；引物2如SEQ ID NO:2所示。
PCR反应体系(25μL)：引物1/1.0μL；引物2/1.0μL；10×PCR Buffer/2.5μL； dNTP mix/2.0μL；Taq酶/0.3μL；DNA模板/1.0μL；超纯水/25μL。
PCR反应条件为：先94℃—4min；再94℃—30s，55℃—40s，72℃—90s下循环30次，最后72℃—10min。
以无菌去离子水替代模板作为阴性对照。扩增结束后取3.0μL扩增后的产物进行琼脂糖凝胶电泳。电泳结束后，切割待测胶带送上海生工进行序列测定。将测定菌株的序列结果与NCBI中已知的16S rDNA序列进行Blast对比分析得到最相近的菌株，以确定实验所分离细菌的种属。
4.抑菌活性测定
待测菌为上述获得的菌，对照菌株为鼠李糖乳杆菌，分别取各菌100μL于100mL的乳酸菌MRS增菌肉汤培养基中进行活化复苏。指示菌为Escherichia coli ATCC11105、Clostridium difficile NY-5、Staphylococcus aureus NT-12。
5.药敏试验
选择10种抗菌药物药敏纸片：头孢吡肟(FEP,20μg)，头孢噻肟(CTX,20μg)，利福平(Rf,10μg)，氨苄西林(AM,10μg)，四环素(TE,20μg)，氯霉素(CHL,20μg)，环丙沙星(CIP,10μg)，阿米卡星(AK,20μg)，庆大霉素(GM,10μg)，甲氧苄氨嘧啶(TMP,10μg)，均购自广州市宜康生物科技有限公司(Oxoid)。
根据美国临床实验室标准化协会颁布的KB法进行药敏试验。首先用无菌镊子分别夹取含有不同抗生素的药敏纸片，分别贴在已接种待测菌的各平板(200μL待测菌菌悬液含菌数为3.0×108CFU/mL，分别加入到温度为50℃左右的乳酸菌MRS增菌肉汤琼脂培养基)中，迅速混合均匀，倒平板。每个平板贴大约5张药敏纸片，各纸片间距离大致相等，并做好标记。在37℃厌氧过夜培养，观察平板抑菌情况，游标卡尺测定抑菌圈的大小并记录。
6.sir2基因的鉴定
以待测菌的基因组DNA为模板，长双歧杆菌sir2引物：上游引物如SEQ ID NO:3所示，下游引物如SEQ ID NO:4所示
PCR反应体系：模板—1μL；上游引物—0.5μL；下游引物—0.5μL；dNTP(10mM)—0.5μL；LA Taq(5U/μL)—0.5μL；10×PCR buffer—2.5μL；灭菌水—19.5μL。
7.Tricine-SDS-PAGE
A.蛋白多肽粗提
(a)收取DMEM缓冲培养基培养过夜之后的菌液100ml，10000rpm离心10min，分别收集菌体沉淀及上清液；
(b)菌体沉淀转移至破壁管中，机械破壁，系数为4.0，时间为1min，重复6次，加入适量去离子水，10000rpm离心10min，除去沉淀，收集上清；
(c)用无菌蒸馏水清洗过滤膜组件3次，洗干净保护液，流尽清洗液，将10kDa中空纤维滤膜装上，安装好膜组件，再用PBS缓冲液冲洗组件3次；
(d)将离心后收集的上清液通过过滤膜组件，压力为0.15MPa，温度为37℃，时间为2h，得到的残渣，用适量的无菌水溶解，重复过滤二次；
(e)取过滤液，加入固体硫酸铵至饱和度为40％，静置30min，离心除去沉淀，离心后得到的上清液继续加入固体硫酸铵至饱和度为60％，于0℃下静放3h，12000rpm离心10min，弃去上清，收集沉淀物，
(f)使用0.1M的醋酸铵甲醇溶液清洗沉淀两次，再用80％已预冷的的丙酮溶液润洗三次，加入100％冷冻丙酮洗一次，低温干燥30min，加入适量蒸馏水溶解干燥物，备用。
B.电泳
(a)先注入分离胶，待分离胶凝固45min后，再注入夹层胶，约45min待夹层胶凝固，最后加入浓缩胶，凝固45min；
(b)在电泳槽底部加入正极缓冲液，把制好的胶板同其制胶装置一起放入电泳槽内，在胶层间加入负极缓冲液，将整个电泳装置置于冰上，用60v的电压先电泳30min；
(c)取少量分离得到的蛋白样品，用样品缓冲液稀释至5ml，开始加样电泳，当染料从夹层胶进入分离胶后，电压加到120v，继续电泳，直至染料到达分离胶顶端，停止电泳；
(d)取出电泳槽中的胶板，即刻置于固定液中浸泡固定1.5h，再将胶板取出，于45℃的染色液中浸泡1.5h，然后用无菌蒸馏水将胶板洗涤两次，最后用变换脱色液，清洗多次，当看到明显的条带和清晰的背景则停止冲洗；
(e)洗脱后，用凝胶成像仪成像，观察目的蛋白的表达，并拍照保存。
8.Western blotting
(1)制胶：配置12％的分离胶并注入玻璃板间隙至离上边缘1.5cm处，上层加适量75％乙醇；待凝固，倒掉上层液体，注入5％浓缩胶，插入梳子，自然晾干。
(2)上样：将蛋白质样品100℃水浴3min，在电泳槽中倒入新配置的电泳液，分别在两边泳道加入8μl和5μl蛋白maker，用1×蛋白上样缓冲液补足体积到10μl，其余泳道各加10μl样品，50V恒压跑电泳。
(3)转膜：电泳结束后，剪取11cm×8cm滤纸和合适大小的0.22μm的PVDF膜，用甲醇活化5min，按照“海绵-6层滤纸-凝胶-PVDF膜-6层滤纸-海绵”组装转印夹层，夹板组装后转移至转膜槽，200mA恒流转移60min。
(4)孵育抗体：转膜结束后，TBS液洗膜5min，5％的脱脂奶粉封闭1h，TBST液洗膜3次，每次5min，加一抗兔抗人SIRT3多克隆抗体稀释液4℃过夜孵育；TBST液洗膜3次，每次5min，加二抗兔抗稀释液室温孵育1h。
(5)发光检测：TBST液洗膜，加发光液孵育3min，用滤纸吸干发光液，将胶片和PVDF膜放入压片盒中压片1min，取出胶片定影30s，用水清洗，烘干，扫描。
实验结果：
1.菌体的生长情况的检查结果见表1。
表1菌体生长情况检查结果
Figure PCTCN2017087285-appb-000001
Figure PCTCN2017087285-appb-000002
2. 16S rDNA鉴定结果
进入NCBI核酸数据库http://blast.ncbi.nlm.nih.gov/Blast.cgi，输入待测菌株的16S rDNA序列，点击Blast进入比对。
比对结果显示，有三种菌株的16S rDNA序列与其它多株长双歧杆菌的16S rDNA序列有99％的相似性，初步判断为长双歧杆菌(Bifidobacterium longum)，将其命名为Bifidobacterium longum BL12，Bifidobacterium longum BL14，Bifidobacterium longum BL16。
3.抑菌活性测定
抑菌活性的试验结果，见表2，三株长双歧杆菌都有一定的抑菌能力。
表2 B.longum抑菌活性的试验结果
Figure PCTCN2017087285-appb-000003
4.药敏试验，结果见表3所示。筛选到的三株长双歧杆菌都具有很强的头孢抗性。
表3 B.longum的药敏试验结果
Figure PCTCN2017087285-appb-000004
注：S-敏感；R-耐药；I-中度耐药。
5.Sir2基因PCR鉴定结果
如图1所示，在651bp处分别扩增出三条长双歧杆菌sir2基因条带。
6.Tricine-SDS-PAGE结果
如图2所示，三株长双歧杆菌Sir2蛋白分别在2.7KD处有一条明亮的条带。
7.Western blotting
如图3 Western blot结果显示，长双歧杆菌中B.longum BL16的Sir2蛋白表达量最高。因此，成功筛选到带有头孢抗性且高表达Sir2蛋白的长双歧杆菌B.longum BL16。
实施例2
本实施例通过长双歧杆菌Bifidobacterium longum BL16(下称B16)喂饲用D-半乳糖所致亚急性衰老小鼠为衰老动物模型，对小鼠各器官的指数和生化指标进行测定和比较，探讨B16的作用。
1.动物分组
昆明种小鼠40只，雌雄各半，体重(20±2)g，暨南大学实验动物中心提供。适应性饲养1W后，随机分为四组，每组10只。第一组为空白对照组、第二组为模型对照组，第三组为双歧杆菌组。
2.药品及试剂
B16为实施例1所得；D-半乳糖购至北京鼎国生物有限公司，SOD、MDA、GSH-Px、NO、NOS试剂盒购自南京建成生物工程研究所。
3.模型建立及给药方案
模型对照组和给药组小鼠每日接受颈背部皮下注射D-半乳糖0.15mg/g体重 (生理盐水配制)，对照组注射等量的生理盐水；同时给药组小鼠每日分别灌胃B16液(双歧杆菌浓度到1010，每日灌胃给药1mL)，空白对照组和模型对照组小鼠灌胃等量的生理盐水，共40d。
4.检测指标
各组动物经连续处理40d后称重，断颈处死，取出脑，胸腺、脾、肝、肾，用0.9％生理盐水洗净残留血液后精确称重，计算器官指数。用于切片的脑组织置于10％甲醛中固定，其余脏器组织用锡箔纸包好，置-80℃超低温冰箱保存待用。
5.各脏器指数的测定
准确称取小鼠全脑、胸腺、脾脏湿重后，计算各脏器指数(mg/g)〔胸腺重量(mg)/体重(g)〕。
6.脑组织中生化指标的测定
将冻存的脑组织放入组织匀浆机配用的玻璃管中，加入9倍体积预冷的生理盐水(蒸馏水)，充分研磨后，制成10％脑组织匀浆。采用考马斯亮蓝法测定脑组织匀浆中总蛋白含量；硫代巴比妥酸反应物法测定MDA含量；酶法检测GSH-Px的活性；硝酸还原法检测NO具体检测方法参照试剂盒说明书。
7.统计学处理
所有数据均以x±s表示,多组间比较采用SPSS13.0统计学分析软件进行单因素方差分析。
表4实验组对衰老小鼠脏器指数的影响
Figure PCTCN2017087285-appb-000005
表5实验组对脑组织中生化指标的影响
Figure PCTCN2017087285-appb-000006
结果：
1.BL16对衰老小鼠脏器指数的影响(见表4)。与空白对照组比较，模型组小鼠脑、胸腺、脾，肝、肾指数均减小(P<0.05)，实验组小鼠各器官指数明显升高。
组织器官的重量，特别是脑、胸腺、脾、肝、肾等重要器官的重量变化是反映生物体衰老程度的重要指标。免疫器官衰变最明显的是胸腺和脾，胸腺和脾重量的降低、萎缩、功能衰退可使免疫功能降低，导致老年动物(人)恶性疾病发生率增高，加速老化进程。本结果显示D-半乳糖诱导的亚急性衰老小脑、胸腺、脾脏、肝、肾指数均显著下降，BL16治疗明显提高了衰老小鼠脑、胸腺、脾脏、肝、肾重量，说明BL16能够有效对抗小鼠脑的衰老，保持机体的免疫功能，提高抗病能力，从而对延缓衰老和增强免疫力具有积极的意义。
2.BL16对衰老小鼠组织SOD、MAD、GSH-Px、NO、NOS的影响(见表5)。与空白对照组比较，模型组小鼠脑组织MAD含量显著提高，差异有统计学意(P<0.01)，SOD、GSH-Px、NO、NOS活性明显降低(P<0.01)，差异在统计学意义(P<0.01)。
生物体存在抗氧化系统，其中SOD是最关键的酶之一。此酶能清除超氧阴离子自由基、保护细胞免受损伤，其活性高低间接反映了机体的抗氧化能力。美国巴乐的摩老年医学研究中心研究证明，哺乳动物体内SOD含量与其寿命之间呈明显正相关关系。本实验结果发现:BL16能明显增强D-半乳糖诱导衰老小鼠的脑组织SOD活性，提高脑组织对自由基的清除能力，阻断自由基应答。
自由基是机体正常代谢产生，但过多的自由基可侵犯细胞，特别是脑组织中的不饱和脂肪酸，引起脂质过氧化，形成代谢产物MDA，继而形成脂褐素，通过与蛋白质、核酸等生物大分子交联，破坏正常细胞的功能。因此，MDA含量常常可反映机体内脂质过氧化的程度，间接反映细胞的损伤程度，是老化的重要指标。
NO由NOS利用左旋精氨酸内源性合成，是具有广泛的生物学作用的小分子物质，能溶于水和脂质，可通过旁分泌快速扩散至邻近细胞，与细胞内鸟氨酸环化酶上的血红素基因结合激活该酶，由鸟氨酸环化酶催化鸟氨酸生成环鸟营(cGMP)。cGMP作为第二信使，通过信号级联放大作用，引起一系列生物学效 应。NO在神经系统中的主要作用为介导神经元对兴奋性氨基酸的反应和增强机体的学习记忆能力等，其含量降低与衰老及许多老年性疾病如老年性痴呆、高血压、动脉粥样硬化等的发生有关。NOS是NO合成的限速酶，含有NOS的神经元在中枢神经系统许多部位存在，包括大脑皮层、海马、下丘脑等，NO在这些部位的神经元活动调控中起重要作用。生理性衰老与脑组织内NOS活性降低密切相关。NOS活性降低，使脑中NO含量下降，从而可导致巨噬细胞吞噬能力下降；使有活性的鸟昔酸环化酶含量降低，调节多种代谢的能力减弱，可进一走加速衰老。本实验结果显示:模型衰老小鼠的NOS活性和NO含量显著下降，BL16能明显提高衰老模型小鼠的NOS活性和NO含量。上述结果提示BL16能通过增强NOS活性，使NO生成量增多，其生物学效应能持久发挥，从而可延缓机体衰老。
GSH-Px是广泛存在于机体内的一种重要的催化过氧化分解的酶，它特异性地催化GSH对过氧化氢的还原反应，抑制烟酸胺腺嘌呤二核苷酸(NADH)形成OH-，削弱对脂质过氧化作用的损伤，从而起到保护细胞膜结构和功能完整的作用。本实验结果表明BL16可显著提高D-半乳糖致衰老小鼠脑组GSH-Px活性，提高体内抗氧化酶活性，减轻自由基对生物大分子的伤害，而起到延缓衰老的作用。
综上，本实验表明双歧杆菌能提高SOD、GSH-Px、NO和NOS的水平，降低MDA的含量，提示双歧杆菌可能是通过提高机体抗氧化能力，清除衰老机体产生过多的自由基，抑制机体、组织、细胞的过氧化过程，而起到延缓衰老作用。
Figure PCTCN2017087285-appb-000007

Claims (10)

  1. 一种具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16，其特征在于，所述长双歧杆菌Bifidobacterium longum BL16保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.60191，保藏地址为广东省广州市越秀区先烈中路100号大院59号楼5楼，保藏日期为2017年5月24日。
  2. 权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16在制备抗衰老的食品或药品中的应用。
  3. 权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16在制备增强免疫功能的食品或药品中的应用。
  4. 权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16在制备调节肠道菌群的食品或药品中的应用。
  5. 一种抗衰老食品，其特征在于，包括有效剂量的作为活性成分的权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16和/或其菌体蛋白成分。
  6. 一种抗衰老药品，其特征在于，包括有效剂量的作为活性成分的权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16和/或其菌体蛋白成分，以及药学上可接受的载体。
  7. 一种增强免疫功能的食品，其特征在于，包括有效剂量的作为活性成分的权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16和/或其菌体蛋白成分。
  8. 一种增强免疫功能的药品，其特征在于，包括有效剂量的作为活性成分的权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16和/或其菌体蛋白成分，以及药学上可接受的载体。
  9. 一种调节肠道菌群的食品，其特征在于，包括有效剂量的作为活性成分的权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16和/或其菌体蛋白成分。
  10. 一种调节肠道菌群的药品，其特征在于，包括有效剂量的作为活性成分 的权利要求1所述具有头孢抗性且高表达Sir2蛋白的长双歧杆菌Bifidobacterium longum BL16和/或其菌体蛋白成分，以及药学上可接受的载体。
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