CN115485362A - 抑制肌肉萎缩基因的表达的双歧杆菌 - Google Patents
抑制肌肉萎缩基因的表达的双歧杆菌 Download PDFInfo
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Abstract
本发明提供一种来源于婴儿肠道的双歧杆菌,其抑制肌肉萎缩基因的表达,并且可以适用于食品和饮料中,以及含有该双歧杆菌的食品和饮料。提供了抑制肌肉萎缩基因表达的短双歧杆菌菌株N106(NITE BP‑03231)。肌肉萎缩基因是一个MuRF1基因。还提供了含有该双歧杆菌的食品和饮料。即使是运动有困难的人,也可以通过饮食来预防肌肉萎缩,提高生活质量。
Description
技术领域
本发明涉及抑制肌肉萎缩基因表达的来自婴儿肠道的双歧杆菌(Bifidobacterium)菌株,以及含有该菌株的食品和饮料产品。特别地,本发明涉及短双歧杆菌(Bifidobacterium breve)。
背景技术
世界卫生组织(WHO)将总人口中65岁及以上占7%至14%的社会定义为老龄化社会,占14%至21%的社会定义为老龄社会,占21%或以上的社会定义为超老龄社会。根据总务省(日本)2019年公布的数据,日本65岁及以上的老年人口的数量估计为3588万,占总人口的28.4%。因此,现在的日本属于超老龄社会的范畴。人口老龄化的原因被认为是由于医学进步和其他因素导致的平均预期寿命增加。
即使平均预期寿命增加,身体也会随着年龄的增长而衰老。老化过程的一个例子是肌肉力量(肌肉质量)的损失。肌肉是通过合成和降解之间的平衡形成的。已知PGC-1α和MuRF1基因分别参与肌肉合成和肌肉降解。MuRF1已显示可诱导构成骨骼肌组织的“肌球蛋白重链”降解。如果肌肉降解超过肌肉合成,肌肉降解就会进展,肌肉质量会减少。随着肌肉降解的进一步进展,肌肉萎缩(肌萎缩)。
一定程度的肌肉质量损失被诊断为肌肉减少症(sarcopenia)。特别是,如果沿着骨骼分布并支持身体活动的骨骼肌质量减少,人们将无法正常移动身体,从而增加因跌倒而导致骨折、住院和卧床不起的风险。因此,人们需要防止肌肉质量的损失,以过上健康和文雅的生活。
建议进行习惯性锻炼以防止肌肉质量下降。但是,对于因衰老而肌肉萎缩的人、卧床不起或不能拉紧运动器官(骨骼和关节)和循环器官的人,不限于老年人和每天忙碌的人,很难继续持续锻炼。结果,即使他或她希望这样做,也难以充分恢复不能锻炼的人的肌肉。此外,当一个人由于受伤或其他原因被固定在石膏中时,肌肉收缩并加速肌肉萎缩。
引文列表
专利文献
[PTL 1]JP-2005-154387-A
发明概述
技术问题
鉴于上述问题做出了本发明。本发明的目的在于提供一种通过运动以外的手段来预防肌肉萎缩的方法。
问题的解决方案
本发明人着眼于已知的肌肉萎缩基因MuRF1,并研究了饮食对MuRF1表达的抑制。结果,本发明人发现在从婴儿粪便分离的双歧杆菌中存在抑制MuRF1表达的双歧杆菌,从而完成了本发明。
为了解决上述问题,本发明的特征在于属于短双歧杆菌属种的双歧杆菌,其抑制肌肉萎缩基因的表达。本发明中的肌肉萎缩基因是包括编码直接或间接参与肌肉萎缩的蛋白质的基因序列的概念。本发明的特征还在于肌肉萎缩基因是MuRF1。
为了解决上述问题,本发明的特征还在于双歧杆菌为短双歧杆菌菌株N106(NITEBP-03231)。
为了解决上述问题,本发明的特征还在于含有双歧杆菌的食品和饮料。
发明的有益效果
本发明的双歧杆菌抑制肌肉萎缩基因的表达。这使得即使是那些运动有困难的人也可以通过饮食来预防肌肉萎缩并提高他们的生活质量。此外,通过摄入双歧杆菌可以改善肠道环境。
附图说明
图1是比较本发明和比较菌株中MuRF1表达的抑制的图。
图2是比较本发明菌株和模式菌株中MuRF1表达的抑制的图。
具体实施方式
现在将详细描述本发明。
1.短双歧杆菌菌株N106(NITEBP-03231)
本发明中的双歧杆菌为短双歧杆菌。本发明中的符号N106是日清食品控股株式会社(Nissin Foods Holdings Co.Ltd.)独立分配给菌株的编号。该菌株是发明人首次从婴儿粪便中分离得到的。
短双歧杆菌菌株N106在以下条件下保藏:
(1)保藏机构名称:国家技术评价研究院专利微生物保藏中心(NITE)
(2)联系方式:日本千叶县木更津市上总镰足2-5-8号122室,292-0818
(3)受托人编号:NITE BP-03231
(4)识别标识:N106
(5)交付日期:2020年6月18日
短双歧杆菌菌株N106的细菌学特征如下表1和表2所示。细菌学特征是根据泊杰氏的系统细菌学手册第2卷(1986年)中描述的方法确定的。表1显示了与该菌株有关的形状和其他特性,表2显示了使用Api20A(Biomérieux)进行的生理生化表征结果。在表2中,符号“+”表示阳性,符号“-”表示阴性。
[表1]
+:阳性,-;阴性
[表2]
反应/酶 | 结果 | 反应/酶 | 结果 | ||
1 | 吲哚的产生 | - | 11 | 明胶的水解 | - |
2 | 脲酶 | - | 12 | 七叶苷的水解 | - |
3 | 葡萄糖发酵 | + | 13 | 甘油发酵 | - |
4 | 甘露醇发酵 | + | 14 | 纤维二糖发酵 | + |
5 | 乳糖发酵 | + | 15 | 甘露糖发酵 | + |
6 | 蔗糖发酵 | + | 16 | 松三糖(Meletitose)发酵 | + |
7 | 麦芽糖发酵 | + | 17 | 棉子糖发酵 | + |
8 | 水杨苷发酵 | + | 18 | 山梨糖醇发酵 | + |
9 | 木糖发酵 | + | 19 | 鼠李糖发酵 | + |
10 | 阿拉伯糖发酵 | - | 20 | 海藻糖发酵 | + |
2.抑制MuRF1表达的评价
如以下实验例所示,本发明的短双歧杆菌菌株N106在抑制肌肉萎缩基因MuRF1的表达方面具有高度活性。通过以下测试方法评价MuRF1基因表达的抑制。
<双歧杆菌悬液的制备>
在MuRF1表达抑制评价中使用的样品(双歧杆菌悬液)在表3所示的GAM培养基(商品名“GAM Broth”:Nippon Suisan Kaisha,Ltd.)中于37℃培养24小时。Anaeropak(Mitsubishi Gas Chemical Company,Inc.)用于在厌氧条件下培养。然后通过离心收集增殖的细菌。收集的细菌用无菌水洗涤3次,加热灭菌,冷冻。然后使用冷冻干燥机将细菌冷冻干燥以产生干燥的细菌粉末。将干燥的细菌粉末作为双歧杆菌悬浮液悬浮在PBS中。
[表3]
<MuRF1表达抑制的评价>
在体外研究中评估了对MuRF1表达的抑制。人骨骼肌成肌细胞(LonzaK.K.)用于该研究。根据制造商的方案,将冷冻细胞添加到生长培养基(商品名“SkBM2”:Lonza K.K.)中进行培养和生长。然后在96孔板的每个孔中接种2.0×104个细胞。将细胞在CO2培养箱(5%CO2,37℃)中培养3天,使其在板上贴壁生长,然后将生长培养基更换为分化培养基(商品名“DMEM:F12培养基”):由Lonza K.K.用2%马血清制备),然后培养5天以分化成肌管细胞。将上述双歧杆菌悬液加入分化培养基中至终浓度为10μg/mL。向培养基中添加已知上调骨骼肌中MuRF1表达的地塞米松至0.1μM。温育24小时后,使用Rneasy迷你试剂盒(QIAGEN)提取RNA。使用ReverTra Ace qPCR RT Master Mix(Toyobo Co.,Ltd.)对提取的RNA进行cDNA合成。使用THUNDERBIRD SYBR qPCR mix(Toyobo Co.,Ltd.)和LightCycler 480 System II(Roche Diagnostics K.K.)通过RT-PCR测量基因表达。RNA提取、cDNA合成和RT-PCR根据各自试剂盒和仪器的方案进行。用于测定的引物序列列于表4。
[表4]
引物名称 | 序列 |
MuRF1_F | TGGGGGAGCCACCTTCCTCT |
MuRF1_R | ATGTTCTCAAAGCCCTGCTCTGTCT |
RPLP0_F | GGAAACTCTGCATTCTCGCTTCCT |
RPLP0_R | CCAGGACTCGTTTGTACCCGTTG |
3.食品和饮料
本发明的双歧杆菌可以复合在食品和饮料中使用。本发明的双歧杆菌特别适用于饮料,例如发酵乳和乳酸菌饮料。根据现行的《牛奶和乳制品成分标准部令》(MinisterialOrdinance on Standard of Ingredients for Milk and Dairy Products),成分标准要求发酵乳(脱脂乳的固体含量为8.0%或更高)和乳制品乳酸菌饮料(脱脂牛奶的固体含量为3.0%或更高)最低为为1.0×107cfu/mL和乳酸菌饮料(脱脂牛奶的固体含量小于3.0%)最低为1.0×106cfu/mL,但上述细菌计数可以通过在发酵溶液(例如牛奶)中生长或以最终产品的形式生长来实现。除了含有双歧杆菌的发酵乳和乳酸菌饮料之外,双歧杆菌还可以用于黄油等乳制品、蛋黄酱等加工蛋制品和奶油蛋糕等糕点。双歧杆菌也可以适当地用于加工食品,例如方便面和饼干。除了这些应用之外,本发明的食品产品可以与适当的载体和必要的添加剂一起制成双歧杆菌的制剂形式(例如粉末、颗粒、胶囊和片剂)。
本发明的双歧杆菌除了一般的饮料、食品以外,还可以复合到特定保健用食品和营养补充剂中。
本发明的双歧杆菌还可以应用于化妆品领域,如乳液,医药领域,如肠道调节剂,日用品领域,如牙膏,以及动物饲料和植物肥料领域,如青贮饲料、动物饲料,以及植物液体肥料,除了食品以外。
工业实用性
双歧杆菌(短双歧杆菌N106)具有抑制肌肉萎缩基因MuRF1表达的高活性。
[实施例]
现在将描述本发明的实施例,但本发明不限于以下实施例。
<实施例1>MuRF1表达抑制的评价
评价了本发明的短双歧杆菌菌株N106和日清食品控股株式会社拥有的11个比较菌株的抑制MuRF1表达的活性。
如表3所示,本发明菌株、模式菌株和比较菌株各自在GAM培养基(商品名“GAMBroth”:Nippon Suisan Kaisha,Ltd.)中于37℃温育24小时。Anaeropac(Mitsubishi GasChemical Company,Inc.)用于在厌氧条件下培养细菌。然后通过离心收集细菌。收集的细菌用无菌水洗涤3次,加热灭菌,冷冻。然后使用冷冻干燥机将细菌冷冻干燥以产生干燥的细菌粉末。将干燥的粉末作为双歧杆菌悬浮液悬浮在PBS中。
然后将人骨骼肌成肌细胞以2.0×104个细胞/孔接种在96孔板中并培养三天。将培养基更换为分化培养基后,培养5天,分化为肌管细胞。然后将每个样品组添加到相应的培养基中,最终浓度为10μg/mL。地塞米松也添加到培养基中至0.1μM。温育24小时后,从细胞中提取RNA,并通过RT-PCR测量基因表达。制备仅含有地塞米松但不含双歧杆菌悬浮液的对照。
图1显示了每个样品组的添加结果。结果表明MuRF1/RPLP0比率,对照设置为“1”以进行比较。
图1表明一些双歧杆菌促进了MuRF1的表达,而另一些则抑制了表达。其中,菌株N106和N185在抑制MuRF1表达方面表现出高活性。
<实施例2>短双歧杆菌菌株N106的优越性评价
然后将短双歧杆菌菌株N106与模式菌株短双歧杆菌JCM1192进行比较,以确认短双歧杆菌菌株N106的优越性。与实施例1一样进行3次试验,取平均值进行比较。结果如图2所示。
图2明显表明,与对照组相比,含有短双歧杆菌菌株N106的组中MuRF1的表达被显著抑制。
如上所述,短双歧杆菌菌株N106具有有效抑制肌肉萎缩基因MuRF1表达的功能。
序列表
<110> 日清食品控股株式会社
<120> 抑制肌肉萎缩基因的表达的双歧杆菌
<130> JP-5641
<150> JP 2020-147207
<151> 2020-09-02
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> RT-PCR检测基因表达的引物
<400> 1
tgggggagcc accttcctct 20
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> RT-PCR检测基因表达的引物
<400> 2
atgttctcaa agccctgctc tgtct 25
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> RT-PCR检测基因表达的引物
<400> 3
ggaaactctg cattctcgct tcct 24
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence
<220>
<223> RT-PCR检测基因表达的引物
<400> 4
ccaggactcg tttgtacccg ttg 23
Claims (4)
1.属于短双歧杆菌种的双歧杆菌,其抑制肌肉萎缩基因的表达。
2.根据权利要求1所述的双歧杆菌,其中,所述肌肉萎缩基因是MuRF1基因。
3.根据权利要求1或2所述的双歧杆菌,其中,所述双歧杆菌为短双歧杆菌菌株N106(NITE BP-03231)。
4.含有权利要求1至3中任一项所述的双歧杆菌的食品或饮品。
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