CN115521883B - 一种具有缓解压力和改善睡眠功能的益生菌 - Google Patents
一种具有缓解压力和改善睡眠功能的益生菌 Download PDFInfo
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- CN115521883B CN115521883B CN202210142163.8A CN202210142163A CN115521883B CN 115521883 B CN115521883 B CN 115521883B CN 202210142163 A CN202210142163 A CN 202210142163A CN 115521883 B CN115521883 B CN 115521883B
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Abstract
本发明公开了一种具有缓解压力和改善睡眠功能的益生菌,本发明的具有缓解压力和改善睡眠功能的益生菌包括以下步骤:将大马士革玫瑰、藏红花和毛叶藜芦提取物加入到蛋白胨、葡萄糖、柠檬酸氢二铵、磷酸氢二钾、羧甲基淀粉钠、硫酸镁、麦芽糊精、甜味剂和水中混合制备成培养基,接种培养动物双歧杆菌、植物乳杆菌和嗜酸乳杆菌制备成一种具有缓解压力和改善睡眠功能的益生菌。与现有技术相比,本发明的缓解压力和改善睡眠功能的益生菌可以调节神经递质和γ‑氨基丁酸的含量水平,减少压力和失眠症状,而且副作用低,治疗效果明显。
Description
技术领域
本发明涉及微生物制剂技术领域,尤其涉及一种具有缓解压力和改善睡眠功能的益生菌及其制备方法。
背景技术
在日常生活中,人类暴露于各种暂时和长期的压力源,对心理健康产生负面影响。压力可以定义为一种产生生理或心理压力的刺激。我们的身体每天都会遭遇许多环境、生理或心理的压力源,而不会经历任何重大的变化,但长期的压力源可能会对我们产生负面影响,导致焦虑或失眠症状的发展。除了遗传因素外,当身体压力源与情绪压力源和不良的生理状态结合在一起时,会导致失眠症状的发生。失眠症状是一种常见的神经系统疾病,影响着全世界2.64亿人。由于缺乏了解,失眠仅仅被认为是精神不平衡的普通人群,人们对这种疾病的重视度远远不够,这也是大部分人不寻求治疗的原因。失眠会增加心血管疾病的风险,也会增加患上癌症、糖尿病、肥胖、哮喘、中风等一系列疾病的风险。失眠也会造成巨大的社会经济负担,因为它影响着个人工作和生活的状态。大多数失眠患者常出现白天功能障碍、认知能力下降、情绪问题等,给患者及其家人,甚至社会医疗和全球经济带来严重负担。
治疗失眠的方法有认知行为疗法、药物疗法和物理疗法。早期药物治疗主要以苯二氮卓类药物为主,但由于其导致认知能力下降、跌倒、精神和运动受损、睡眠结构改变、药物依赖、回弹性失眠等原因,逐渐被非苯二氮卓类药物所取代。目前临床较常用的非苯二氮卓类药物有佐匹克隆、埃佐匹克隆、唑吡坦和扎来普隆。其中唑吡坦使用较多。唑吡坦是一种短效非苯二氮卓类安眠药,属于咪唑吡啶类。用于睡眠不足、短暂性失眠、慢性失眠。虽然有报道称,批准剂量的唑吡坦(成人10mg,老年人5mg)在减少失眠患者的睡眠潜伏期和增加总睡眠时间方面有一定的效果,它也可以有效改善睡眠质量。但在临床上,其有效性和安全性仍受到使用者的担忧。
近年来,科学研究也表明部分益生菌对于身体的疾病和机能具有较好的治疗效果,对人类健康具有潜在的有益作用,例如调节菌群数量;提高宿主免疫防御和屏障功能;减轻氧化应激等,由于不同益生菌的菌株特异性机制,益生菌作为膳食补充剂来改善或预防能力有限。因此,开发具有特定作用的新型益生菌具有重要意义。
中国专利CN105769929A公开了一种熊果酸复合益生菌的制备方法,属于医学领域。该发明从苦丁茶中提取熊果酸并纯化,并制成含有熊果酸的发酵培养基,分别将乳酸菌菌株、双歧杆菌、嗜热链球菌接种到发酵培养基中,进行好养、厌氧培养,将培养完成的培养液分离后收集菌体,用磷酸盐缓冲液洗涤、超声波细胞粉碎机破碎处理,分离得上清液、回收溶剂后结晶,最后分离纯化即可,该发明制得的产物结合了熊果酸和多种益生菌的优点,不仅使熊果酸的活性更高,消炎、抗氧化、增强免疫等功效更明显,而且制备工艺简单、产品纯度高、应用广泛。但是该专利存在菌种复杂,制备过程繁杂,产品质量不稳定的缺点。
公开号为CN110432328A的发明专利公开一种益生菌,由以下重量百分比的原料制成:嗜酸乳杆菌冻干粉9%~11%、动物双歧杆菌冻干粉5%~7%、乳粉40%~42%、菊粉24%~26%、甘露醇16%~18%、二氧化硅0.9%~1.1%;制备方法包括以下步骤:1)原料的预处理:将检验合格的嗜酸乳杆菌冻干粉、动物双歧杆菌冻干粉、乳粉、菊粉、甘露醇及二氧化硅,通过筛网过滤后用;2)称量:将原料按重量百分比对原料进行称量;3)制备混合粉A:称量得到的嗜酸乳杆菌冻干粉和动物双歧杆菌冻干粉分别等量递增混合均匀后;4)制备混合粉B:将混合粉A与甘露醇按等量递增混合均匀后,得混合粉B;5)制备益生菌:混合粉B与乳粉、菊粉及二氧化硅加入三维混合机中,混合搅拌均后,得到益生菌。但是该方法制备的益生菌只是几种物质的混合料,功能性单一,并未充分发挥益生菌的有益效果。
公开号为CN110537704A的专利提供一种改善睡眠的益生菌发酵功能食品及其制备方法,是以大豆、茯苓、莲子的混合物作为固态发酵基质,采用植物乳杆菌、动物双歧杆菌、副干酪乳杆菌、青春双歧杆菌、凝结芽孢杆菌、枯草芽孢杆菌、罗伊氏乳杆菌、长双歧杆菌和乳酸片球菌等多种菌株进行混菌固态发酵后真空冷冻干燥制备而得。不仅能够提高其活性成分的含量,同时能够促进活性成分在人体内的吸收,对机体具有更好的保健作用。所述功能性食品主要用于改善入睡困难、睡眠质量下降和睡眠时间减少等症状。但是该方法采用的菌种较多,生产的产品质量不够稳定,而且茯苓和莲子具有一定的安神助眠作用,存在少量的毒性成份,影响使用效果和身体健康。
发明内容
有鉴于现有技术中制备存在功能单一、有效成份散失和毒性较大的缺点,本发明所要解决的技术问题是采用大马士革玫瑰、藏红花和毛叶藜芦培养益生菌的方式构造一种具有缓解压力和改善睡眠功能的益生菌。
为实现上述目的,本发明提供了一种具有缓解压力和改善睡眠功能的益生菌的制备方法,包括以下步骤,所述份数均为重量份:
步骤1、将10~18份蛋白胨、5~10份葡萄糖、3-7份柠檬酸氢二铵、6~10份磷酸氢二钾、3~6份羧甲基淀粉钠、1~3份硫酸镁、4~8 份麦芽糊精、1~3份甜味剂和70~100份水混合,混合均匀后采用 20~40wt%酸度调节剂调节pH值为6.2~6.8,配制成混合溶液,将混合溶液在800~900W超声功率下超声处理10~20min,向混合溶液中加入25~40份功能滤液,搅拌混合均匀,然后进行预处理,得到培养基;
步骤2、将5~8份动物双歧杆菌、10~15份植物乳杆菌和3~6份嗜酸乳杆菌,接种到70~90份步骤1制备的培养基中进行培养,先在 35~37℃下培养4~8h,再在37~40℃下厌氧培养10~15h,得到培养液;
优选的,所述步骤2中的动物双歧杆菌的浓度为 5×106~9×106CFU/g,植物乳杆菌的浓度为2×106~9×106CFU/g,嗜酸乳杆菌的浓度为4×106~8×106CFU/g。
步骤3、将40~60份步骤2制备的培养液在8000~12000r/min离心分离20~30min,分离后收集菌泥,将菌泥加入缓冲液中,控制缓冲液的量,调整菌数到4×109~6×109CFU/mL,制备成菌泥溶液;然后将菌泥溶液在10000~13000r/min离心分离20~40min,再次收集菌泥;在菌泥中添加保护剂,添加量为菌泥量重量的1~2倍;然后进行冷冻干燥,即得到缓解压力和改善睡眠功能的益生菌。
优选的,所述步骤1中的功能滤液的制备步骤如下,所述份数均为重量份:
S1、将15~25份大马士革玫瑰、10~20份藏红花、5~10份毛叶藜芦采用水进行清洗2~3遍,后置于50~70℃烘干3~4h;然后研磨成粉末,过50~200目筛,得到大马士革玫瑰粉、藏红花粉、毛叶藜芦粉,混合均匀,制备成混合粉料;
S2、将25~35份混合粉料加入200~400份85~95wt%的乙醇水溶液中混合,在80~90℃下回流提取2~3次,每次2~3h,回流结束后将提取液用200~400目滤布过滤,在40~50℃下蒸发除去溶剂,然后加入50~100份水,得功能滤液。
优选的,所述步骤1中的甜味剂为甜菜糖、白砂糖、麦芽糖、葡萄糖、果糖、乳糖和阿斯巴甜中的一种或两种及两种以上组合而成。
优选的,所述步骤1中的酸度调节剂为柠檬酸钠和三聚磷酸钠中的一种或两种。
优选的,所述步骤1的预处理是指均质、杀菌及冷却工艺;所述均质的温度为60~70℃,均质的压力为22~35MPa;所述杀菌的温度为90~97℃,杀菌的时间为5~120min;所述冷却工艺为在室温下冷却至20~28℃。
优选的,所述步骤3中缓冲液为1~3wt%醋酸钠水溶液和6~8wt%醋酸水溶液按照质量比1:1混合而成。
优选的,所述步骤3中冷冻干燥参数如下:在-42~-50℃的条件下预冻3~5h,冻结后进行抽真空干燥,干燥条件为:真空度为2~5Pa,干燥温度为-40℃逐步升温至26℃,干燥时间40~50h。
优选的,所述步骤3中保护剂为脱脂乳、蔗糖、海藻糖、麦芽糊精、蛋白胨、明胶中的一种或两种及两种以上组合而成。
失眠是一种多层面病理生理紊乱,通常被认为是一种与高度的生理、情感和认知活动有关的障碍。由一系列遗传、环境、分子、细胞和心理机制引起。从分子角度来看,主要是因为神经递质和激素(如 g-氨基丁酸、去甲肾上腺素、血清素、促肾上腺皮质激素释放激素、皮质醇和褪黑激素)受到干扰。因此,针对这些因素的治疗是治疗失眠的有效药物。由于物种的多样性,自然界存在植物性药物可以作为缓解压力进而增强睡眠的功能材料。
藏红花来自藏红花的柱头。它是一种广泛用于烹饪的天然添加剂,可增强食品的风味、香味和颜色。藏红花在传统医学中也有悠久的历史,可以治疗眼睛、皮肤、呼吸道和胃肠道疾病。
大马士革玫瑰是蔷薇科的芳香植物,主要种植在伊朗、保加利亚、土耳其、印度和摩洛哥。大马士革玫瑰的商业产品众多,包括玫瑰精油、玫瑰茶、玫瑰干花、玫瑰糖浆和果汁等,用于食品、香水、化妆品和医药行业。大马士革玫瑰的药用特性主要由于其中含有的丰富的苷类、萜类、黄酮类和花青素,被用于治疗民间各种疾病。
毛叶藜芦中的提取物白藜芦醇是一种天然多酚,可作为治疗和预防多种疾病的药物。可以有效缓解毒性成份对肾脏细胞的伤害,人体内存在一种内源性短链非编码,可通过靶向mRNA可以调控上游相关基因的表达参与细胞凋亡和坏死。具体通过靶向受体相互作用丝氨酸/苏氨酸激酶直接抑制其表达,进而抑制坏死和凋亡的细胞死亡。
压力和焦虑会加剧自身益生菌群的失调,而益生菌群现在被认为是对抗压力负面影响的治疗靶点。益生菌的使用可以影响大脑发育、功能和行为,当摄入足够的量时,会产生积极的健康益处。草药的治疗效果也存在一定的局限,而且草药中还含有部分毒性成份,对身体的其他机能存在不利的影响,益生菌的添加可以减少草药的毒性,增强治疗效果。大马士革玫瑰、藏红花和毛叶藜芦培育的益生菌群可以在脑肠轴中发挥作用,当使用时,通过调节免疫功能、皮质酮/皮质醇、神经递质和脑源性神经营养因子等对压力、失眠类症状有相当大的改善作用。上述草药培育的动物双歧杆菌和乳杆菌可以清除2,2- 二苯基-1-苦基肼(DPPH)自由基,使机体具有更高的抗氧化性能。服用本发明制备的益生菌可提高心肌梗死患者的抗氧化能力和抗炎能力,减少压力和失眠症状。作用机理主要是可通过提高丁酸盐水平,改善神经退行性变。食用含嗜酸乳杆菌、动物双歧杆菌和植物乳杆菌的益生菌可恢复神经突触传递和海马体的生长。通过产生丁酸盐、丙酸和醋酸等短链脂肪酸可以调节神经营养因子、γ-氨基丁酸、多巴胺和血清素水平,从而提高免疫系统和下丘脑-垂体-肾上腺轴的作用。而且丁酸盐产量的增加,防止多巴胺的耗竭,增加神经营养因子的水平来降低多巴胺能神经元退化,改善压力和焦虑的症状。
由于采用了以上的技术方案,与现有技术相比,本发明的一种具有缓解压力和改善睡眠功能的益生菌及其制备方法,其优点在于:1) 大马士革玫瑰、藏红花和毛叶藜芦培育的益生菌可以调节神经递质和γ-氨基丁酸的含量水平,减少压力和失眠症状。2)益生菌的培育可以减少草药中毒性物质的含量,增强草药的治疗效果,减轻副作用。
具体实施方式
实施例中采用的菌种的保藏信息:
植物乳杆菌FEED8:保藏编号为CGMCC No.15029,分类命名为:植物乳杆菌FEED8Lactobacillus plantarum,已于2017年12月7 日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮政编码:100101),保藏单位的缩写为CGMCC;
动物双歧杆菌乳亚种Y6:保藏编号为CGMCC No.15026,分类命名为:动物双歧杆菌乳亚种Y6乳亚种Bifidobacterium animalis subsp.lactis,已于2017年12月7日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮政编码:100101),保藏单位的缩写为 CGMCC;
嗜酸乳杆菌K43:保藏编号为CGMCC No.15705,分类命名为:嗜酸乳杆菌K43Lactobacillus acidophilus,已于2018年4月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心(地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮政编码:100101),保藏单位的缩写为CGMCC。
实施例中主要原料的来源:
大马士革玫瑰:产地陕西渭南。
藏红花:产地伊朗。
毛叶藜芦:产地浙江东阳。
柠檬酸氢二铵:江苏科伦多食品配料有限公司,CAS号: 3012-65-5。
羧甲基淀粉钠:四川华堂聚瑞生物科技有限公司,食品级,钠含量:4~6.5%。
醋酸铵:安徽巨达生物科技有限公司,CAS号:6131-90-4。
实施例1
一种具有缓解压力和改善睡眠功能的益生菌的制备方法,包括以下步骤,所述份数均为重量份:
步骤1、将15份蛋白胨、7份葡萄糖、5份柠檬酸氢二铵、8份磷酸氢二钾、4份羧甲基淀粉钠、2份硫酸镁、6份麦芽糊精、2份葡萄糖和80份水混合,混合均匀后采用30wt%柠檬酸钠水溶液调节pH 值为6.5,配制成混合溶液,将混合溶液在850W超声功率下超声处理12min,向混合溶液中加入30份功能滤液,搅拌混合均匀,然后进行均质、杀菌及冷却,均质的温度为65℃,均质的压力为26MPa,杀菌的温度为93℃,杀菌的时间为40min,室温下冷却至25℃,得到培养基;
步骤2、分别选取7份动物双歧杆菌乳亚种Y6、12份植物乳杆菌FEED8和4份嗜酸乳杆K43,接种到80份步骤1制备的培养基中进行培养,先在36℃下培养6h,再在39℃下厌氧培养12h,得到培养液;所述步骤2中的动物双歧杆菌乳亚种Y6的浓度为7×106CFU/g,植物乳杆菌FEED8的浓度为5×106CFU/g,嗜酸乳杆K43的浓度为 6×106CFU/g;
步骤3、将50份步骤2制备的培养液在10000r/min离心分离 25min,分离后收集菌泥,将菌泥加入2wt%醋酸钠水溶液和7wt%醋酸水溶液按照质量比1:1混合而成的缓冲液中,调整菌数到 5×109CFU/mL,制备成菌泥溶液;然后将菌泥溶液在11000r/min离心分离30min,再次收集菌泥;在菌泥中添加海藻糖,添加量为菌泥量重量的1.5倍,将菌泥置于冷冻干燥机中,在-45℃的条件下预冻3h,冻结后进行抽真空干燥,干燥条件为:真空度为3Pa,干燥温度为-40℃逐步升温至26℃,干燥时间45h,干燥结束后即可得到缓解压力和改善睡眠功能的益生菌。
所述步骤1中的功能滤液的制备步骤如下,所述份数均为重量份:
S1、将20份大马士革玫瑰、16份藏红花、7份毛叶藜芦采用水进行清洗2遍,后置于60℃烘箱烘干3.5h;然后采用研磨机磨成粉末,过100目筛,得到大马士革玫瑰粉、藏红花粉、毛叶藜芦粉,混合均匀,制备成混合粉料;
S2、将30份混合粉料加入300份90wt%的乙醇水溶液中混合,在80~90℃下回流提取3次,每次2.5h,回流结束后将提取液用300 目滤布过滤,在45℃下水浴蒸发除去溶剂,然后加入80份水,得功能滤液。
实施例2
一种具有缓解压力和改善睡眠功能的益生菌的制备方法,与实施例1基本相同,唯一区别仅仅在于:所述步骤1中的功能滤液的制备方法不一致。
所述步骤1中的功能滤液制备步骤如下,所述份数均为重量份:
S1、将16份藏红花、7份毛叶藜芦采用水进行清洗2遍,后置于60℃烘箱烘干3.5h;然后采用研磨机磨成粉末,过100目筛,得到藏红花粉、毛叶藜芦粉,混合均匀,制备成混合粉料;
S2、将30份混合粉料加入300份90wt%的乙醇水溶液中混合,在80~90℃下回流提取3次,每次2.5h,回流结束后将提取液用300 目滤布过滤,在45℃下水浴蒸发除去溶剂,然后加入80份水,得功能滤液。
实施例3
一种具有缓解压力和改善睡眠功能的益生菌的制备方法,与实施例1基本相同,唯一区别仅仅在于:所述步骤1中的功能滤液的制备方法不一致。
所述步骤1中的功能滤液制备步骤如下,所述份数均为重量份:
S1、将20份大马士革玫瑰、7份毛叶藜芦采用水进行清洗2遍,后置于60℃烘箱烘干3.5h;然后采用研磨机磨成粉末,过100目筛,得到大马士革玫瑰粉、毛叶藜芦粉,混合均匀,制备成混合粉料;
S2、将30份混合粉料加入300份90wt%的乙醇水溶液中混合,在80~90℃下回流提取3次,每次2.5h,回流结束后将提取液用300 目滤布过滤,在45℃下水浴蒸发除去溶剂,然后加入80份水,得功能滤液。
实施例4
一种具有缓解压力和改善睡眠功能的益生菌的制备方法,与实施例1基本相同,唯一区别仅仅在于:所述步骤1中的功能滤液的制备方法不一致。
所述步骤1中的功能滤液制备步骤如下,所述份数均为重量份:
S1、将20份大马士革玫瑰、16份藏红花采用水进行清洗2遍,后置于60℃烘箱烘干3.5h;然后采用研磨机磨成粉末,过100目筛,得到大马士革玫瑰粉、藏红花粉,混合均匀,制备成混合粉料;
S2、将30份混合粉料加入300份90wt%的乙醇水溶液中混合,在80~90℃下回流提取3次,每次2.5h,回流结束后将提取液用300 目滤布过滤,在45℃下水浴蒸发除去溶剂,然后加入80份水,得功能滤液。
对比例1
一种具有缓解压力和改善睡眠功能的益生菌的制备方法,与实施例1基本相同,唯一区别仅仅在于:所述步骤1中不添加功能滤液。
测试例1
改善睡眠功能测试
测试方法参考硕士论文(改善睡眠软胶囊的研究,作者:白金,吉林大学,2015),动物的品质与来源:BALB/c小鼠,清洁级,90 只,体重18~22g,雌雄各一半,由华中科技大学同济医学院实验动物学部提供。测试方法如下:
1、设置空白对照组,将小鼠随机分成6组,每组15只,空白对照组每天采用实验小鼠标准饲料早晚8点喂养两次,每次饲料为3g,同时喂入2mL蒸馏水;实施例和对比例小鼠采用相同方式喂养,每次喂养都另外添加1g上述制备的缓解压力和改善睡眠功能的益生菌。
2、在温度23±3℃、相对温度60±5%、人工光照12小时及通风的环境中,喂养30天,喂养结束后,收集小鼠的尿液,并加入 1mL1wt%NaN3溶液作为抑菌剂,收集到尿液置于-20℃冰箱中保存待用,供测试例2进行测试。在小鼠腹腔注射戊巴比妥钠,注射量为0.1mL/10gBW,记录30min内入睡的小鼠数量(翻正反射达1min以上者)和小鼠睡眠时间,以没有翻正反射为睡眠标准,计算小鼠睡眠时间平均值,测试结果见表1。
表1、小鼠睡眠功能测试
实验方案 | 入睡动物数(只) | 睡眠时间(min) |
实施例1 | 13 | 43.1±7.2 |
实施例2 | 10 | 34.0±6.1 |
实施例3 | 9 | 31.8±5.9 |
实施例4 | 9 | 30.3±5.2 |
对比例1 | 6 | 27.7±5.4 |
空白对照组 | 5 | 24.1±4.9 |
从表1测试结果可以看出,在小鼠睡眠功能测试中实施例1小鼠的入睡动物数量最多,平均睡眠时间更长。可能原因在于大马士革玫瑰、藏红花和毛叶藜芦培育的益生菌群可以在脑肠轴中发挥作用,通过调节免疫功能、皮质酮/皮质醇、神经递质和脑源性神经营养因子等对失眠类症状有相当大的改善作用。而且上述草药培育的动物双歧杆菌和乳杆菌可以清除2,2-二苯基-1-苦基肼(DPPH)自由基,使机体具有更高的抗氧化性能,通过提高丁酸盐水平,改善神经退行性变。食用含嗜酸乳杆K43、动物双歧杆菌乳亚种Y6和植物乳杆菌FEED8 的益生菌可恢复神经突触传递和海马体的生长。通过产生丁酸、丙酸和醋酸等短链脂肪酸可以调节神经营养因子、γ-氨基丁酸、多巴胺和血清素水平,既保留了草药的相关调理作用,又增强了益生菌的服用效果,两者协同增效,加强了改善睡眠的效果。
测试例2
试样毒性测试
将测试例1中采集的尿液常温解冻,将350μL尿液与350μL磷酸缓冲液(0.2M,PH=7.4)混合均匀后,在12000rpm离心10min,取上清液600μL,添加至已加入30μL TSP重水溶液(1mg/mL)的5mm核磁管中,搅拌混匀,对尿液中葡萄糖、肌酸、柠檬酸、TMAO(氧化三甲胺)、2-酮戊二酸和马尿酸等代谢产物的含量进行测试。
各空白对照组、实施例和对比例中小鼠尿液中的葡萄糖、肌酸、柠檬酸、TMAO、2-酮戊二酸和马尿酸均在正常范围内,而且实验过程中小鼠行为正常,小鼠体重呈逐渐上升趋势,未出现中毒迹象。说明大马士革玫瑰、藏红花和毛叶藜芦中的毒性成份被益生菌分解,培育出的功能化益生菌不含有毒性成份。
测试例3
缓解压力和改善睡眠测试
选择存在较大压力的200名人员,随机分成5组,每组各40名,每日睡前一次服用50mg(每克含活菌量2000亿),该实施例和对比例制备的缓解压力和改善睡眠功能的益生菌,服用后定期通过电话回访进行是否缓解压力和改善睡眠统计,统计结果如表2。
表2、缓解压力和改善睡眠测试结果
从表2中可以看出实施例1的缓解压力和改善睡眠的人数最多,对比例1的效果最差,可能原因在于大马士革玫瑰、藏红花和毛叶藜芦培育的益生菌群产生了协同增效作用,益生菌群通过提高丁酸盐水平,改善神经退行性变。食用含嗜酸乳杆K43、动物双歧杆菌乳亚种 Y6和植物乳杆菌FEED8的益生菌可恢复神经突触传递和海马体的生长。培养的益生菌群通过产生丁酸盐、丙酸和醋酸等短链脂肪酸可以调节神经营养因子、γ-氨基丁酸、多巴胺和血清素水平,其中实施例1益生菌群的γ-氨基丁酸含量相对于对比例1中有显著提升,γ- 氨基丁酸是公认的具有促进睡眠的作用。而且大马士革玫瑰、藏红花和毛叶藜芦培育的益生菌群可以在脑肠轴中发挥作用,提高免疫系统和下丘脑-垂体-肾上腺轴的作用,通过调节免疫功能、皮质酮/皮质醇、神经递质和脑源性神经营养因子等对压力、失眠类症状有相当大的改善作用。
Claims (2)
1.一种具有缓解压力和改善睡眠功能的益生菌的制备方法,其特征在于,采用以下步骤,以重量份计:
步骤1、将15份蛋白胨、7份葡萄糖、5份柠檬酸氢二铵、8份磷酸氢二钾、4份羧甲基淀粉钠、2份硫酸镁、6份麦芽糊精、2份甜味剂和80份水混合,混合均匀后采用30wt%酸度调节剂调节pH值为6.5,配制成混合溶液,将混合溶液在850W超声功率下超声处理12min,向混合溶液中加入30份功能滤液,搅拌混合均匀,然后进行预处理,得到培养基;
步骤2、将7份动物双歧杆菌、12份植物乳杆菌和4份嗜酸乳杆菌,接种到80份步骤1制备的培养基中进行混合培养,先在36℃下培养6h,再在39℃下厌氧培养12h,得到培养液;
步骤3、将50份步骤2制备的培养液在10000r/min离心分离25min,分离后收集菌泥,将菌泥加入缓冲液中,控制缓冲液的量,调整菌数,使得所述步骤2中的动物双歧杆菌的浓度为7×106CFU/g,植物乳杆菌的浓度为5×106CFU/g,嗜酸乳杆菌的浓度为6×106CFU/g;制备成菌泥溶液;
然后将菌泥溶液在10000r/min离心分离25min,再次收集菌泥;在菌泥中添加保护剂,添加量为菌泥量重量的1.5倍;然后进行冷冻干燥,即得到缓解压力和改善睡眠功能的益生菌;
所述步骤1中的功能滤液的制备步骤如下,以重量份计:
S1、将20份大马士革玫瑰、16份藏红花、7份毛叶藜芦采用水进行清洗2遍,后置于60℃烘干3.5h;然后研磨成粉末,过100目筛,得到大马士革玫瑰粉、藏红花粉、毛叶藜芦粉,混合均匀,制备成混合粉料;
S2、将30份混合粉料加入300份90wt%的乙醇水溶液中混合,在80~90℃下回流提取3次,每次回流时间2.5h,回流结束后将提取液用300目滤布过滤,在45℃下蒸发除去溶剂,然后加入80份水,得功能滤液;
所述步骤1中的甜味剂为葡萄糖;
所述步骤1中的酸度调节剂为柠檬酸钠;
所述步骤1的预处理是指均质、杀菌及冷却工艺;所述均质的温度为65℃,均质的压力为26MPa;所述杀菌的温度为93℃,杀菌的时间为40min;所述冷却工艺为在室温下冷却至25℃;
所述步骤2中动物双歧杆菌为动物双歧杆菌乳亚种Y6,其保藏编号为CGMCC No.15026;植物乳杆菌为植物乳杆菌FEED8,其保藏编号为CGMCC No.15029;嗜酸乳杆菌为嗜酸乳杆菌K43,其保藏编号为CGMCC No.15705;
所述步骤3中缓冲液为2wt%醋酸钠水溶液和7wt%醋酸水溶液按照质量比1:1混合而成;
所述步骤3中冷冻干燥参数如下:在-45℃的条件下预冻3h,冻结后进行抽真空干燥,干燥条件为:真空度为3Pa,干燥温度为-40℃逐步升温至26℃,干燥时间45h;
所述步骤3中保护剂为海藻糖。
2.一种具有缓解压力和改善睡眠功能的益生菌,其特征在于:采用如权利要求1所述的一种具有缓解压力和改善睡眠功能的益生菌的制备方法制备而成。
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