WO2022016805A1 - 一种杜氏杆菌在延长寿命和抗衰老中的应用 - Google Patents

一种杜氏杆菌在延长寿命和抗衰老中的应用 Download PDF

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WO2022016805A1
WO2022016805A1 PCT/CN2020/139830 CN2020139830W WO2022016805A1 WO 2022016805 A1 WO2022016805 A1 WO 2022016805A1 CN 2020139830 W CN2020139830 W CN 2020139830W WO 2022016805 A1 WO2022016805 A1 WO 2022016805A1
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dubosiella
newyorkensis
lifespan
food
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朱升龙
陈永泉
姜旋
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Jiangnan University
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

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  • the invention relates to the application of Dunaliella in prolonging lifespan and anti-aging, and belongs to the technical fields of biomedicine and microorganisms.
  • Aging is a multifactorially triggered biological process common to all living organisms, characterized by progressive impairment of the host's response to stress and pervasive cellular degeneration of key metabolic pathways. Aging is associated with many detrimental physiological effects that affect the health and overall function of an organism. In humans and other mammals, aging can trigger a decline in immune function, increased susceptibility to disease (cancer, diabetes, cardiovascular disease, and neurodegenerative disease), increased chronic inflammation, decreased muscle mass, and increased Alzheimer's disease and the incidence of age-related degenerative diseases such as Huntington's disease.
  • the lifespan research model animal Caenorhabditis elegans N2 used in the present invention is a common small soil nematode. Because of its short life cycle and fast reproduction time, it is very similar to human biochemical and gene pathways. In addition, the genetic and pharmacological mechanisms of its regulation of lifespan have been clarified. The research on aging and anti-aging mechanisms, in fact, several major theories of aging mechanisms have been proved in nematodes. Therefore, drugs that have anti-aging effects on nematodes are generally considered to have the same effect on humans, and thus are used as a common model organism for the study of aging.
  • Dubosiella As a new genus isolated and identified from biological samples, Dubosiella has shown effects such as regulating metabolism in the body, improving intestinal immunity and promoting the body's resistance to inflammatory diseases, and can affect a variety of life activities of individuals.
  • the present invention first provides a strain of Dubosiella newyorkensis NYU-BL-A4, and the Dubosiella newyorkensis NYU-BL-A4 is published in the patent with the publication number of US2018125900A1.
  • the present invention provides the application of Dubosiella newyorkensis NYU-BL-A4 in the preparation of products for prolonging animal lifespan and/or anti-aging.
  • the product is used in at least one aspect of (a) to (b):
  • the product is used to enhance nematode reproduction.
  • the product includes, but is not limited to, food, health products, feed or additives thereof.
  • the food, health product, feed or additives thereof are used to improve the immunity of the body and strengthen the basal metabolic capacity.
  • the dosage forms of the food, health product, feed or its additives include tablets, granules, capsules, powders, liquids, jelly, and bars.
  • the food, health product, feed or its additives also contain common ingredients in the technical field that are appropriately selected by those skilled in the art according to the dosage form or purpose of use, and can be used together with other raw materials.
  • the prolongation of lifespan is an extension of the maximum length of an organism's lifespan or a delay in time to death.
  • the animal generally refers to an organism belonging to the kingdom Animalia in terms of biological classification, including but not limited to mammals.
  • the animal is a nematode.
  • the content of Dunaliella NYU-BL-A4 in the product is not less than 1.5 ⁇ 10 8 CFU/mL or 1.5 ⁇ 10 8 CFU/mg.
  • the Dubosiella newyorkensis strain (Dubosiella newyorkensis) NYU-BL-A4 of the invention can prolong the lifespan of Caenorhabditis elegans, does not affect the normal vital signs swallowing frequency of Caenorhabditis elegans, is safe and has no toxic side effects on nematodes, and proves that the Dubosiella newyorkensis strain (Dubosiella newyorkensis) NYU-BL-A4 has a good anti-aging effect and can be used to assist in the preparation of anti-aging products, so as to relieve aging and prolong life. It has good practical application value and is suitable for commercial production.
  • Figure 1 is a graph showing the relationship between Dubosiella newyorkensis NYU-BL-A4 and Caenorhabditis elegans lifespan.
  • Figure 2 is a graph showing the relationship between Dubosiella newyorkensis NYU-BL-A4 and C. elegans vital signs swallowing frequency.
  • Figure 3 is a graph showing the relationship between Dubosiella newyorkensis NYU-BL-A4 and Caenorhabditis elegans safety toxicity.
  • Figure 4 is a graph showing the relationship between the total amount of eggs laid by Dubosiella newyorkensis NYU-BL-A4 and Caenorhabditis elegans.
  • Figure 5 is a graph showing the relationship between the daily egg production of Dubosiella newyorkensis NYU-BL-A4 and Caenorhabditis elegans.
  • Figure 6 is a graph showing the relationship between Dubosiella newyorkensis NYU-BL-A4 and Caenorhabditis elegans dynamic swing frequency.
  • Example 1 Application of Dunaliella NYU-BL-A4 in prolonging the lifespan of Caenorhabditis elegans
  • NGM nematode growth medium
  • a single nematode is to be passaged, pick a nematode onto a new NGM medium with a self-made gold drill wire (3min in diameter) ignited by an alcohol lamp; Cut out the NGM medium in which the nematodes were cultured and paste it onto a new medium.
  • Synchronized nematodes were fed to the L4 stage on NGM plates coated with E. coli OP50 and then moved to E. coli OP50 coated with 1.0 x 10 8 CFU/mL and 3.0 x 10 8 CFU/mL of Dubosiella newyorkensis, respectively.
  • the NGM medium used in this experiment contained 50 mg/mL FUDR, and 20 nematodes were cultured in each petri dish, 3 parallels each time.
  • the lifespan experimental model counts the number of live nematodes every 2 days, and replaces the medium with a new medium. At the end of the experiment, the nematode lifespan curve is drawn.
  • the lifespan of the nematodes fed by Dubosiella newyorkensis NYU-BL-A4 was significantly longer than that of the control group.
  • the average lifespan of the control group was 16 days, and the average lifespan of the experimental group was 22 days.
  • the bacterial strain of the present invention has an extended lifespan. Effect.
  • Example 2 The effect of Dunaliella NYU-BL-A4 on the swallowing frequency of Caenorhabditis elegans vital signs
  • Nematode resuscitation, culture and synchronization methods are as described in Example 1.
  • Detection of nematode swallowing frequency Take the nematodes cultured to L2 to L3 stage after synchronization and move them to NGM coated with 1.0 ⁇ 10 8 CFU/mL E. coli OP50 and 3.0 ⁇ 10 8 CFU/mL Dunaliella NYU-BL-A4 respectively On the culture medium, the nematodes were observed under a stereo microscope and the number of phagocytosis within 1 min was counted, 10 times in each group. Statistical results are shown in Figure 2, the average number of times in the control group is 146 times, and the average number of times in the experimental bacteria group is 148 times.
  • M9 buffer was used to adjust Escherichia coli OP50 and Dunaliella NYU-BL-A4 to different concentrations (1.5, 3, 9, 12, unit: 1.0 ⁇ 10 8 CFU/mL) and spread on NGM on the plate medium. About 25 nematodes in the L4 phase after synchronization were taken per plate. After 10 days, the number of nematodes that survived was recorded and the survival rate was calculated.
  • Example 4 Dubosiella newyorkensis NYU-BL-A4 does not affect the reproductive capacity of C. elegans
  • the synchronized eggs were cultured to the L4 stage at 20°C, and one nematode in the L4 stage was picked and placed on an NGM culture plate (one plate) coated with 3.0 ⁇ 10 8 CFU/mL of Dunaliella or Escherichia coli OP50. One worm), transfer the egg-laying adults to a new plate every 24 hours until the nematode egg-laying is complete.
  • progeny nematodes When the progeny nematodes are cultured to the L2-L3 stage, counting and statistics are carried out. The number of progeny nematodes is the number of eggs laid by the mother nematodes. Finally, the number of eggs laid by the nematodes per day and the sum of the number of eggs laid during the spawning period are obtained. Three nematodes were selected for each group of experiments and the reproductive ability was tested at the same time. This experiment was repeated three times independently.
  • the level of nematode egg laying is the reference for the nematode's reproductive ability, as well as the standard for evaluating the nutritional status and physical condition of the nematode.
  • Research has shown that reproductive performance is closely related to longevity. Screening of anti-aging drugs or strains should be based on the premise of not impairing the reproductive ability of the body. For this reason, we evaluated the effect of Dubosiella newyorkensis on the egg-laying ability of nematodes.
  • Example 5 Dubosiella newyorkensis NYU-BL-A4 increases the dynamic swing frequency of Caenorhabditis elegans

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Abstract

杜氏杆菌在制备用于延长动物寿命和/或抗衰老的产品中的应用，所述产品用于提高肌肉能力和/或提高机体免疫能力。所述杜氏杆菌为NYU-BL-A4。所述产品包括但不限于食品、保健品、饲料或其添加剂。所述产品中杜氏杆菌的含量不低于1.0×10 8CFU/mL或1.0×10 8CFU/mg。

Description

一种杜氏杆菌在延长寿命和抗衰老中的应用 技术领域
本发明涉及一种杜氏杆菌在延长寿命和抗衰老中的应用，属于生物医药和微生物技术领域。
背景技术
衰老是所有活生物体共有的，由多因素引发的生物过程，其特征是宿主对应激反应的逐渐损害和关键代谢途径的普遍细胞退化。衰老与许多相关的有害生理效应，影响着生物体的健康和整体功能。在人类和其他哺乳动物中，衰老能引发免疫功能的下降，对疾病(癌症，糖尿病，心血管疾病和神经变性疾病)的易感性增加，慢性炎症增加，肌肉质量减少，增加阿尔茨海默病和亨廷顿氏病等与年龄相关的退行性疾病发病率。
近来研究表明，肠道微生物群的变化表现出与年龄相关联的表型，并与宿主的衰老过程有关，肠道细菌中，益生菌是人体的功能器官，因为它们对宿主健康有广泛的积极影响，包括对肠道微生物群调节，免疫调节，抗癌作用还有对宿主寿命的积极影响。益生菌的抗衰老作用早在1908年就已实现，当时研究人员报道保加利亚人食用大量发酵乳和乳杆菌的农民的寿命延长了。然而，关于益生菌的抗衰老作用的研究还很有限，影响宿主寿命的机制尚不清楚。很多研究大多局限于群体研究和宏基因组分析，对单一微生物的探索还比较缺乏。
本发明使用的寿命研究模式动物秀丽隐杆线虫(Caenorhabditis elegans)N2，是一种常见的小型土壤线虫，因其生命周期短，繁殖时间快，与人类的生化和基因通路上有很大的同源性，而且其调控寿命的遗传及药理学机制均已明确，研究衰老及抗衰老机制，实质上衰老机制的几大理论都是在线虫中被证明。因此，对线虫有抗衰老作用的药物通常也被认为对人类也具有同样功效，因而被作为一种研究衰老常见的模式生物。
发明内容
杜氏杆菌属(Dubosiella)作为从生物样本中被分离鉴定的新菌属，并表现出调节体内代谢，提高肠道免疫力和促进机体抵抗炎症疾病等功效，能影响个体多种生命活动。
为解决目前存在的问题，本发明首先提供了一株杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4，所述杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4公布于公开号为US2018125900A1的专利中。
本发明提供了杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4在制备用于延长动物寿命和/或抗衰老的产品中的应用。
在一种实施方式中，所述产品应用于(a)～(b)至少一方面：
(a)提高肌肉能力；
(b)提高机体免疫能力。
在一种实施方式中，所述产品应用于增强线虫繁殖能力。
在一种实施方式中，所述产品包括但不限于食品、保健品、饲料或其添加剂。
在一种实施方式中，所述食品、保健品、饲料或其添加剂用于提高机体免疫力，加强基础代谢能力。
在一种实施方式中，所述食品、保健品、饲料或其添加剂的剂型包括片剂、颗粒剂、胶囊、粉末剂、液体、果冻、棒状。
在一种实施方式中，所述食品、保健品、饲料或其添加剂中还含有本领域技术人员根据剂型或者使用目的适当选择配料的本技术领域的常用成分，可与其他原料一同使用。
在一种实施方式中，所述延长寿命是延长机体生命期的最大长度或延迟死亡时间。
在一种实施方式中，所述动物泛指生物学分类上归属于动物界的生物，包括但不限于哺乳动物。
在一种实施方式中，所述动物为线虫。
在一种实施方式中，所述产品中杜氏杆菌NYU-BL-A4的含量不低于1.5×10 8CFU/mL或1.5×10 8CFU/mg。
有益效果：
本发明杜氏杆菌属菌株(Dubosiella newyorkensis)NYU-BL-A4能够延长秀丽隐杆线虫的寿命，且不影响秀丽隐杆线虫正常生命体征吞咽频率，对线虫安全无毒性副作用，证明了杜氏杆菌属菌株(Dubosiella newyorkensis)NYU-BL-A4有较好抗衰老方面作用，可用于辅助制备抗衰老的产品，从而应用于缓解衰老、延长寿命，具备良好的实际应用价值，并适于商品化生产。
附图说明
图1是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4与秀丽隐杆线虫寿命关系图。
图2是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4与秀丽隐杆线虫生命特征吞咽频率关系图。
图3是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4与秀丽隐杆线虫安全毒性关系图。
图4是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4与秀丽隐杆线虫的产卵总量关系图。
图5是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4与秀丽隐杆线虫的每日产卵量关系图。
图6是杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4与秀丽隐杆线虫动态摆动频率关系图。
具体实施方式
实施例1：杜氏杆菌NYU-BL-A4在延长秀丽隐杆线虫寿命中的应用
(1)菌种活化：将冻存于-80℃环境下的杜氏杆菌NYU-BL-A4接种于改良MTGE肉汤培养基(MTGE肉汤培养基+0.05％半胱氨酸盐酸盐)中，温度37℃厌氧条件下培养48h，再以2％-4％的接种量液体传代培养2次，充分活化菌株。
(2)秀丽隐杆线虫的复苏、培养和传代
从-80℃冰箱中取出1管dauer时期(休眠期)线虫放在37℃水浴锅中1min快速融化后，涂布于含有尿嘧啶缺陷型大肠杆菌OP50的NGM(线虫生长培养基)培养基上，置于20℃生化培养箱里培养。若传代单只线虫，用酒精灯灼烧过的自制的钻金丝(直径化3min)挑取一只线虫到新的NGM培养基上；如需大量传代，用酒精灯火焰灼烧手术刀片，切下培养线虫的NGM培养基贴到新的培养基上。
(3)秀丽隐杆线虫的同步化
将生长状态良好的成虫收集到离心管里，加1mL M9缓冲液洗去多余的大肠杆菌OP50；离心管内保留上清液750μL，加入50μL 1M KOH溶液和200μL 5％NaClO溶液，在涡旋振荡器上振荡3min左右，一边振荡一边在体视显微镜下检测，若虫体断裂则尽快用M9缓冲液清洗3-5遍，清洗后用微型离心机离心，去掉多余的溶液并留下卵，再加500μL M9缓冲液放在20℃摇床，虫卵在16h孵化成同步化的L1幼虫。
(4)秀丽隐杆线虫寿命的检测
同步化后的线虫在涂有大肠杆菌OP50的NGM平板上喂养到L4阶段，然后移至分别涂有1.0×10 8CFU/mL的大肠杆菌OP50和3.0×10 8CFU/mL杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4的NGM培养基上，此实验用的NGM培养基含50mg/mL FUDR，每个培养皿培养线虫20只，每次3个平行。寿命实验模型每2天计数一次活线虫数，并更换新的培养基，实验结束绘制线虫寿命曲线。
如图1所示，杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4喂食的线虫寿命显著长于对照组，经计算，对照组的平均寿命16天，实验组平均寿命22天，本发明菌株具有延长寿命的效果。
实施例2：杜氏杆菌NYU-BL-A4对秀丽隐杆线虫生命体征吞咽频率的影响
线虫复苏、培养和同步化方法如同实施例1所述。
为验证杜氏杆菌的延寿作用是否是通过限制线虫食物摄入来实现的，当线虫吞咽节律下降，会导致食物摄入不足，从而使寿命大幅度延长。线虫吞咽频率的检测：取同步化后培养到L2到L3时期线虫移至分别涂有1.0×10 8CFU/mL的大肠杆菌OP50和3.0×10 8CFU/mL杜氏杆菌NYU-BL-A4的NGM培养基上，在体视显微镜下观察线虫并计数1min内吞咽的次数，每组计数10次。统计结果如图2所示，对照组平均次数146次，实验菌组平均次数148次，本发明菌株不影响秀丽隐杆线虫正常生命体征吞咽频率，不通过限制食物摄入发挥延长寿命作用。
实施例3：杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4对秀丽隐杆线虫安全性检测
对线虫毒性的检测：用M9缓冲液调大肠杆菌OP50、杜氏杆菌NYU-BL-A4菌液为不同浓度(1.5、3、9、12，单位：1.0×10 8CFU/mL)涂布在NGM平板培养基上。取同步化后L4期线虫约25只每平板。10天后记录线虫成活数并计算存活率。结果如图3所示，对比相同浓度条件下的喂有大肠杆菌OP50和杜氏杆菌的线虫存活率保持在同一水平，可认为杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4对线虫是安全的，并不影响其正常的生命活动。
实施例4：杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4不影响秀丽隐杆线虫的生殖能力
将同步化的虫卵在20℃条件下培养至L4期，挑取1条处于L4期的线虫到涂有3.0×10 8CFU/mL的杜氏杆菌或大肠杆菌OP50的NGM培养板上(一板一条虫)，每隔24小时将产卵的成虫转移至新的培养板上，直到线虫产卵完全结束。
将子代线虫培养至L2-L3期时进行计数统计，子代线虫的条数即为母代线虫产卵的枚数，最后得到线虫每天产卵的数量和产卵期内产卵数量的总和。每组实验分别选取3条线虫同时进行生殖能力检测。此实验独立重复三次。
线虫产卵实验结果：线虫产卵量的高低，是线虫繁殖能力的参考依据，也是线虫营养情况，身体状况评价的标准。研究表明，生殖能力与长寿密切相关。筛选抗衰老药物或菌株应以不损害机体生殖能力为前提条件，为此我们评估了杜氏杆菌(Dubosiella newyorkensis)对线虫产卵能力的影响。如图4和图5结果显示，饲喂杜氏杆菌(Dubosiella newyorkensis)后线虫生殖高峰期主要集中在前3天，并且在第2天其产卵量达到最大值。杜氏杆菌(Dubosiella newyorkensis)不改变线虫的产卵期，并且对线虫每天产卵的数量及产卵的总量均无显著的影响。因此我们得出结论，杜氏杆菌(Dubosiella newyorkensis)对线虫的生殖能力没有影响。
实施例5：杜氏杆菌(Dubosiella newyorkensis)NYU-BL-A4提高秀丽隐杆线虫的动态摆动频率
实验方法：同步化的L4期线虫在分别涂有3.0×10 8CFU/mL的杜氏杆菌或大肠杆菌OP50的NGM培养板上培养10天后，取一个12孔板，每个孔中均加入M9缓冲液2mL，每个小孔中放入15只线虫。放入后五分钟之内观察其身体摆动频率，即一分钟内线虫身体弯曲摆动次数(身体从一个方向弯曲到另一个方向，然后恢复原来的方向，记作一次摆动)，实验独立重复3次。
随着线虫的衰老，引起线虫肌肉退化其活动能力减慢，在生命后期线虫运动缓慢，对外界刺激不敏感。秀丽隐杆线虫在M9缓冲液中的身体动态摆动频率，在一定程度上可以说明其衰老的程度。结果如图6所示，与空白组相比，线虫饲喂杜氏杆菌后身体摆动频率明显增加，摆动频率提高了43.49％。说明杜氏杆菌提高了线虫的活动能力，对肌肉功能有正面的影响，在一定程度上减轻衰老对线虫肌肉的作用。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (9)

  1. 杜氏杆菌在制备用于延长动物寿命和/或抗衰老的产品中的应用，其特征在于，所述产品用于提高肌肉能力和/或提高机体免疫能力。
  2. 根据权利要求1所述的应用，其特征在于，所述杜氏杆菌为NYU-BL-A4。
  3. 根据权利要求2所述的应用，其特征在于，所述产品包括但不限于食品、保健品、饲料或其添加剂。
  4. 根据权利要求3所述的应用，其特征在于，所述食品、保健品、饲料或其添加剂用于提高机体免疫力，加强基础代谢能力。
  5. 根据权利要求4所述的应用，其特征在于，所述食品、保健品、饲料或其添加剂的形态包括片剂、颗粒剂、胶囊、粉末剂、液体、果冻、棒状。
  6. 根据权利要求5所述的应用，其特征在于，所述食品、保健品、饲料或其添加剂中还含有本领域技术人员根据剂型或者使用目的适当选择配料的本技术领域的常用成分，可与其他原料一同使用。
  7. 根据权利要求6所述的应用，其特征在于，所述延长寿命是延长机体生命期的最大长度或延迟死亡时间。
  8. 根据权利要求7所述的应用，其特征在于，所述动物泛指生物学分类上归属于动物界的生物，包括但不限于哺乳动物。
  9. 根据权利要求8所述的应用，其特征在于，所述产品中杜氏杆菌的含量不低于1.0×10 8CFU/mL或1.0×10 8CFU/mg。
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