CN116035069A - 用于调节睡眠的副干酪乳杆菌k56及其应用 - Google Patents
用于调节睡眠的副干酪乳杆菌k56及其应用 Download PDFInfo
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Abstract
本发明提供了用于调节睡眠的副干酪乳杆菌K56及其应用。本发明首先提供了副干酪乳杆菌(Lactobacillus paracasei subsp.paracasei)K56在制备具有调节睡眠功效的组合物中的应用,所述副干酪乳杆菌的保藏编号为CGMCC No.15139或DSM27447。本发明研究发现,该副干酪乳杆菌菌株发酵乳液具有调节睡眠等方向的应用,可广泛应用于食品、医药、饲料、化工等领域。
Description
本发明是申请号为202011348882.2、申请日为2020年11月26日、发明名称为“用于抗氧化、调节血压的副干酪乳杆菌K56及其应用”的分案申请。
技术领域
本发明涉及微生物发酵技术领域,尤其是涉及一种副干酪乳杆菌(Lactobacillusparacasei subsp.paracasei)K56(保藏编号CGMCC No.15139或DSM27447)发酵制备具有抗氧化、调节血压和/或调节睡眠功效的组合物中的相关应用。
背景技术
老化是人体器官最大的杀手,身体所产生的自由基(free radicals)则是造成器官老化的重要元凶。当身体调节自由基的机转丧失或失衡,过量的自由基产生则会破坏细胞DNA,改变细胞内蛋白质结构,攻击细胞膜,最后导致体细胞的死亡。皮肤老化也是因为长时间暴露于紫外线(UV exposure)或热源(heat exposure)下,诱发活性氧的量急剧增多,导致皮肤暗沉、皮肤癌等疾病。
高血压,与高血脂、高血糖统称为“三高”,是危害人类健康的一种心脑血管疾病。据专家推测,2025年全球范围的高血压患者人数将超过15亿。有效治疗或预防高血压疾病已成为一个全世界范围的健康问题。目前在体现降血压功效的发酵乳制品相关研究及产品主要集中在功能性成分物质的简单添加及应用。
从上世纪90年代开始,有关微生物尤其是乳酸菌的抗高血压研究受到高度重视,已有相关文献报道乳酸菌可降低血清胆固醇浓度、提高血清中SOD活性,降低过氧化脂质水平,具有辅助降血压血脂及抗氧化的功能,但是乳酸菌的这种辅助降血压血脂及抗氧化功能存在菌种甚至菌株的差异性,应用在具体的发酵乳制品中其功效有待进一步考证。
发明内容
本发明的一个目的在于提供副干酪乳杆菌K56的新用途。
副干酪乳杆菌(Lactobacillus paracasei subsp.paracasei)K56菌株已于2013年6月27日保存于德国微生物及细胞培养物收集中心(German Collection ofMicroorganisms and Cell Cultures),保藏编号DSM27447;另外,副干酪乳杆菌(Lactobacillus paracasei subsp.paracasei)K56菌株也已于2017年12月29日保存于中国微生物菌种保藏管理委员会普通微生物中心(China General MicrobiologicalCulture Collection Center,CGMCC),保藏编号CGMCC No.15139。
本发明发现副干酪乳杆菌(Lactobacillus paracasei subsp.paracasei)K56以主要含有乳成分的料液(乳基质)作为发酵底物,发酵产物中具有超氧化物歧化酶、血管紧张素转化酶抑制物、γ-氨基丁酸、共轭亚油酸、B族维生素等物质,具有优异的总抗氧化(T-AOC)能力,具有抗氧化、调节血压和/或调节睡眠功效。
从而,本发明提供了副干酪乳杆菌(Lactobacillus paracaseisubsp.paracasei)在制备具有抗氧化、调节血压和/或调节睡眠功效的组合物中的应用,所述副干酪乳杆菌的保藏编号为CGMCC No.15139或DSM27447。
根据本发明的具体实施方案,本发明的应用中,副干酪乳杆菌是通过发酵产生超氧化物歧化酶、血管紧张素转化酶抑制物、γ-氨基丁酸、共轭亚油酸和/或B族维生素而具有抗氧化、调节血压和/或调节睡眠功效。
根据本发明的具体实施方案,本发明的副干酪乳杆菌在制备具有抗氧化、调节血压和/或调节睡眠功效的组合物中的应用中,所述组合物可以为食品组合物、饲料组合物、化妆品组合物或药品组合物。
根据本发明的具体实施方案,本发明的应用中,利用副干酪乳杆菌CGMCCNo.15139或DSM27447发酵,制备得到发酵产物,得到的发酵产物作为或进一步用于制备所述具有抗氧化、调节血压和/或调节睡眠功效的组合物。
根据本发明的具体实施方案,本发明中,发酵时所用的发酵底物可以为乳液。优选地,所述乳液为可以为生鲜牛乳或是复原乳,可以是全脂乳、低脂乳或脱脂乳。所述乳液中,可以选择性地含有5%~10%的蔗糖。
根据本发明的具体实施方案,本发明中,所述发酵条件为:35℃~45℃,发酵1天~7天。
根据本发明的具体实施方案,本发明中,副干酪乳杆菌以种子液的形式接种于发酵底物中,接种量2%~5%。所述种子液的制备可以参照益生菌领域的常规技术进行。
根据本发明的具体实施方案,本发明中,作为副干酪乳杆菌K56的发酵底物的乳基质,可以是牛乳(全脂乳、低脂乳或脱脂乳)含量80%以上的乳液,其中可以根据需要选择性地添加甜味剂、稳定剂等辅料,例如可以是发酵乳或乳饮料制备过程中常用的发酵底物,本发明的副干酪乳杆菌K56发酵这些乳基质均可实现产生超氧化物歧化酶、血管紧张素转化酶抑制物、γ-氨基丁酸、共轭亚油酸和/或B族维生素等。
超氧化物歧化酶(Superoxide Dismutase,SOD)是生物体内存在的一种抗氧化金属酶,它能够催化超氧阴离子自由基歧化生成氧和过氧化氢,在机体氧化与抗氧化平衡中起到至关重要的作用。
血管紧张素转化酶(ACE)又称激肽酶Ⅱ或肽基-羧基肽酶,属血管内皮细胞膜结合酶。ACE主要功能有:催化血管紧张素Ⅰ转化为血管紧张素Ⅱ;使缓激肽失活。血管紧张素转化酶因这两种功能而成为治疗高血压等疾病的理想靶点。
γ-氨基丁酸(GABA)是一种四碳、非蛋白氨基酸,作为一种重要的抑制性神经递质,对哺乳动物具有安神、降低血压、改善睡眠等多种生理功能。有研究报道,GABA作为氧化代谢物的调控者发挥抗氧化作用,将拟南芥SSADH突变体暴露于高温下生长,发现其活性氧中间体(reactive oxygen intermediate,ROI)积累,使得植株死亡,而GABA可以减少ROI的积累使得生物免于高温带来的氧化损伤以及过氧化衰亡。
共轭亚油酸(CLA)是一系列双键亚油酸,具有清除自由基、增强人体的抗氧化能力和免疫能力、调节血液胆固醇和甘油三酸脂水平、防止动脉粥样硬化、促进脂肪氧化分解、对人体进行全面的良性调节等作用。有报道指出,CLA能有效的发挥“血管清道夫”的作用,可清除血管中的垃圾,有效调节血液黏稠度,扩张和松弛血管平滑肌、抑制血液运动中枢的作用,降低了血液循环的外周阻力,使血压下降,尤其是使舒张压下降更为明显,能够改善微循环、平稳血压。
B族维生素的抗氧化功效是众所周知的,B族维生素还可帮助清除肝脏脂肪,降低胆固醇,预防动脉硬化。此外,维生素B还能预防太阳晒伤及皮肤癌,保持皮肤光滑滋润,延迟皱纹的出现,还能促进毛发健康生长。有研究报道,B族维生素中的维生素B2、维生素B3、维生素B6等均不同程度参与机体氧化还原过程,在体内抗氧化防御***中起着重要作用。
根据本发明的具体实施方案,本发明测量了K56发酵乳液的上清中超氧化物歧化酶、血管紧张素转化酶抑制物、γ-氨基丁酸、共轭亚油酸和/或B族维生素,发酵产物的上清液作为或进一步用于制备所述具有抗氧化、调节血压和/或调节睡眠功效的组合物。
根据本发明的一些具体实施方案,本发明的副干酪乳杆菌K56发酵乳液,总抗氧化能力(T-AOC)达到1.0~3.0U/mL,且随着发酵过程的进行总抗氧化能力下降缓慢。
根据本发明的一些具体实施方案,本发明的副干酪乳杆菌K56发酵乳液,总超氧化物歧化酶(T-SOD)活力可达20~30U/mL。
根据本发明的一些具体实施方案,本发明的副干酪乳杆菌K56发酵乳液,血管紧张素转化酶抑制率(ACE)可达10~70%。
根据本发明的一些具体实施方案,本发明的副干酪乳杆菌K56发酵乳液中,γ-氨基丁酸含量(GABA)可达14~21μg/mL。
根据本发明的一些具体实施方案,本发明的副干酪乳杆菌K56发酵乳液中,共轭亚油酸(CLA)可达0.01~0.06mg/kg。
根据本发明的一些具体实施方案,本发明的副干酪乳杆菌K56发酵乳液中,维生素B1含量可达30~70μg/mL;维生素B2含量可达2.8~3μg/mL;维生素B3为50~80μg/mL;维生素B6含量为8~20μg/mL;维生素B9的含量为1.6~1.8μg/mL。
根据本发明的具体实施方案,本发明的副干酪乳杆菌K56发酵乳基质所得到的发酵产物可以直接作为食品(例如发酵乳、乳饮品、奶粉、固体饮料等)、日化品(化妆品等)、饲料(包括宠物食品或畜牧用益生菌产品)或药物等,或是进一步用于制备这些食品、日化品、饲料或药物产品。以发酵产物制备食品、日化品、饲料或药物产品的方法可以参照所属领域中的现有技术进行。
在本发明的一具体实施方案中,本发明的K56发酵产物是作为或是进一步用于制备食品,所述食品优选为发酵乳或发酵乳饮料。作为发酵底物的乳液中还可包括发酵乳或发酵乳饮料中常用的其他原料,例如适量的甜味剂、稳定剂、营养添加剂等。所述的发酵乳或发酵乳饮料可以是低温保存的活性菌的发酵乳或发酵乳饮料,也可以是常温保存的灭菌的发酵乳或发酵乳饮料。在本发明的一些更具体实施方案中,所述发酵乳或发酵乳饮料中副干酪乳杆菌CGMCC No.15139的含量为1×107cfu/mL~1×1011cfu/mL。
综上所述,本发明提供了副干酪乳杆菌K56的新用途,本发明发现该菌株单菌发酵乳基质,能够产生超氧化物歧化酶、血管紧张素转化酶抑制物、γ-氨基丁酸、共轭亚油酸和B族维生素等物质,具有抗氧化、调节血压与调节睡眠的潜能。本发明的副干酪乳杆菌K56菌株可用于具有抗氧化、调节血压、调节睡眠功效的发酵乳、乳饮品、奶粉、固体饮料、日化品等生产应用。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解,现结合具体实施例及对本发明的技术方案进行以下详细说明,应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中,各原始试剂材料均可商购获得,未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件,或按照仪器制造商所建议的条件。
实施例1:副干酪乳杆菌K56发酵乳生长动力学特征测定
1、实验菌株
本发明技术方案所用副干酪乳杆菌K56(CGMCC No.15139);
对照菌株1:商业副干酪乳杆菌菌株L.CASEI 431;
对照菌株2:商业副干酪乳杆菌菌株LPC-37。
2、菌株的活化与种子液培养
(1)菌株活化:以MRS培养基活化副干酪乳杆菌,取生长至对数期的菌株,甘油管保存备用。其中,MRS培养基(g/L):蛋白胨10.0g;酵母膏5.0g;葡萄糖20.0g;无水乙酸钠5.0g;柠檬酸二氢胺2.0g;吐温-80 1.0mL;磷酸氢二钾2.0g;七水硫酸镁0.2g;硫酸锰0.05g;牛肉膏10.0g;琼脂15g-20g。
(2)菌株生长动力特征测定
12%全脂乳粉+6%白砂糖常温溶解于水中,于95℃条件下灭菌5min,降温至36℃±1℃,作为乳基质接种菌株后发酵培养120h,于不同的发酵时间段取样,测定活菌数及pH值。
活菌数检测:25ml样品与75ml生理盐水混合均匀后,平板稀释法计数。
酸度检测:使用pH计测定样品pH并记录。取10g发酵乳制品,加入2滴酚酞溶液混匀,0.1M标准NaOH溶液滴定至粉红色。滴定酸度计算公式:
°T=C×V×100/(m×0.1)。
测定结果分别参见表1和表2。
表1副干酪乳杆菌在乳基质中不同发酵时间的菌落计数
由以上测试数据分析可知,K56发酵的pH值在28h活菌数增长迅速,之后降低达到平稳。对照菌株1、对照菌株2在36h活菌数增长迅速,之后降低达到平稳。K56与对照菌株1、对照菌株2分析p>0.05,无显著差异;对照菌株1与对照菌株2分析p>0.05,无显著差异。其中空白组为不加菌液的乳基质。
表2副干酪乳杆菌在乳基质中不同发酵时间的pH测定
由以上测试数据分析可知,K56、对照菌株1、对照菌株2在12h~36h内逐渐下降。K56与对照菌株2分析p<0.01,有极显著差异;K56与对照菌株1分析p>0.05,无显著差异;对照菌株1与对照菌株2分析p>0.05,无显著差异。由于温度原因,空白组中含有未杀灭的微生物导致奶腐败变质,pH值下降。其中空白组为不加菌液的乳基质。
(3)菌株种子液的制备:12%全脂乳粉+6%白砂糖常温溶解于水中,于95℃条件下灭菌5min,降温至36℃±1℃,接种菌株后36℃±1℃发酵培养48h±2h,可延长至72h±2h,作为种子液。
3、副干酪乳杆菌发酵乳抗氧化能力检测
发酵乳制备:生牛乳,加入占生牛乳总重量6.5%的蔗糖,搅拌,加热至95℃、5min杀菌,杀菌后的乳液冷却到37℃,分别加入各菌种子液(副干酪乳杆菌K56接种量3%,对照菌接种量根据活菌数折算成与K56等量),各样品分别于37℃发酵24、48、72、96、120h,发酵乳在4℃以8000r/min离心15min,收集发酵上清液,过0.22μm滤膜,滤液进行抗氧化能力的测定。
(1)总抗氧化(T-AOC)能力测定
本发明中,副干酪乳杆菌抗氧化能力主要通过测定总抗氧化(T-AOC)能力来评价。
总抗氧化(T-AOC)能力测定采用购于南京建成的总抗氧化能力(T-AOC)检测试剂盒,操作按照试剂盒说明书进行。抗氧化物质能把三价铁离子还原成二价铁离子,后者可以和菲啉类物质形成稳固的络合物,通过比色法可测定出其抗氧化能力的高低。测定过程中,待测样品与试剂盒提供的试剂混合时,漩涡混匀器充分混匀,37℃水浴30分钟放置10分钟,波长520nm,1cm光径,双蒸水调零,测定各管吸光度值。
本发明中,不同发酵时间的益生菌发酵上清的T-AOC能力测定结果参见表3。
表3总抗氧化能力
(2)K56发酵样品中其他抗氧化相关能力的检测
本发明中,还检测了K56发酵样品中其他抗氧化相关能力,包括不同发酵时间的上清液的总超氧化物歧化酶(T-SOD)活力、血管紧张素转化酶抑制率(ACE)、超氧阴离子自由基清除能力、OH自由基清除能力、DPPH自由基清除能力、γ-氨基丁酸含量(GABA)、共轭亚油酸(CLA)、B族维生素含量。
本发明中,总超氧化物歧化酶(T-SOD)活力测定采用购于南京建成的总超氧化物歧化酶(T-SOD)试剂盒,操作按照试剂盒说明书进行。通过黄嘌呤及黄嘌呤氧化酶反应***产生超氧阴离子自由基,后者氧化羟胺形成亚硝酸盐,在显色剂的作用下呈现***,用可见分光光度计测其吸光度。当被测样品种含有SOD时,则对超氧阴离子自由基有专一性的抑制作用,使形成的亚硝酸盐减少,比色时测定管的吸光度值低于对照管的吸光度值,通过计算可求出样品中SOD的活力。
血管紧张素转化酶(ACE)测定方法主要有比色法、酶偶联法等。本发明中,用紫外分光光度法测定血管紧张素转化酶(ACE)抑制活性:取2mL的离心管,加入底物为5mmol/LHippuryl-His-Leu(HHL)为底物的50mmol/L硼酸钠缓冲液50μL,再加入20μL待测液体,混合,在37℃水浴中预热3min,加入10μL ACE酶液,在37℃反应30min,再加入100μL 1mol/L盐酸终止反应。加入1.7mL乙酸乙酯,震荡15s,3000×g离心10min,分离上层1mL乙酸乙酯层(样品所在层)。加热挥发除去有机溶剂,再加入1mL去离子水,并震荡使马尿酸完全溶解,用微量紫外分光光度法在228nm测定样品吸光值。ACE抑制率(ACEI)计算公式如下:
其中,A表示ACE与HHL反应完全生成马尿酸的吸光值(对照);B表示待测液体与ACE和HHL反应后生成马尿酸的吸光值(样品);C表示先加入盐酸ACE和HHL反应后生成马尿酸的吸光值。
本发明中,超氧阴离子自由基清除能力测定采用邻苯三酚-分光光度计法。邻苯三酚-分光光度法被广泛应用于食品和药品行业的抗氧化剂初步筛选及各种活性物质清除超氧阴离子自由基的抗氧化功能评价。邻苯三酚自氧化过程为链式反应,可产生超氧阴离子自由基,其自身氧化产物的含量可用分光光度仪检测,通过此法间接评价抗氧化物质的抗氧化能力。具体步骤:取0.88mL 0.1mol/L Tris-HCl缓冲液(pH 8.2)于试管中,依次加入0.2mL 1.0mmol/L乙二胺四乙酸(EDTA)、0.2mL样品、0.3mL蒸馏水。于25℃水浴反应10min,再加入0.4mL 9.0mmol/L邻苯三酚,准确反应60min后,加100μL 12.0mol/L HCl终止反应。以上均平行测定3次取平均值,并且重复实验3次。计算公式:清除率=(Ac-As)/Ac×100%,其中,As:在波长325nm处测定吸光度;Ac:空白管以0.2mL蒸馏水代替样品,操作方法同样品管,测得吸光度。
本发明中,OH自由基清除能力采用邻二氮菲法测定。
本发明中,γ-氨基丁酸(GABA)含量测定方法根据“QB/T 4587-2013γ-氨基丁酸”进行。
本发明中,共轭亚油酸(CLA)含量测定方法根据“乳及乳制品中共轭亚油酸(CLA)含量测定气相色谱法NY/T 1671-2008”进行。
本发明中,B族维生素含量采用高效液相色谱仪测定方法,具体操作条件参照“HPLC检测黑皮鸡枞菌中的水溶性维生素(李艳,《食品研究与开发》,2018)”进行。
本发明中,K56不同发酵时间的益生菌发酵上清的总超氧化物歧化酶(T-SOD)活力、血管紧张素转化酶抑制率(ACE)、超氧阴离子自由基清除能力、OH自由基清除能力、DPPH自由基清除能力、γ-氨基丁酸含量(GABA)、共轭亚油酸(CLA)、B族维生素测定结果参见表4。
表4K56发酵上清相关抗氧化能力测定
由上述测定结果可知,K56发酵乳液具有优异的抗氧化能力,可用于具有抗氧化、调节血压、调节睡眠功效的发酵乳、乳饮品、奶粉、固体饮料、日化品等生产应用。
实施例2.副干酪乳杆菌K56发酵制备具有抗氧化、调节血压、调节睡眠潜力的乳饮料
本实施例提供了副干酪乳杆菌K56发酵制备的具有抗氧化、调节血压、调节睡眠潜力的乳饮料。该饮料的制作步骤如下:
(a)将副干酪乳杆菌K56接种于脱脂乳中进行发酵,发酵后得到发酵乳;
(b)利用甜味剂和/或酸味剂勾兑形成勾兑液,将勾兑液与步骤(a)制得的发酵乳混匀,均质,制备得到发酵乳饮料。
进一步地,所述步骤(a)中发酵的温度为37℃,发酵的时间为72h。
进一步地,步骤(b)中,所述发酵乳与勾兑液的混合体积比例为1:4。
进一步地,步骤(b)中均质的压力为30MPa。
进一步地,步骤(b)中可采用灭菌工艺,优选的灭菌的温度为120℃,灭菌的时间为15min。
本实施例的饮料在低温和常温两种产品品类中均具有良好的抗氧化、调节血压、调节睡眠的潜力。
Claims (10)
1.一种副干酪乳杆菌(Lactobacillus paracasei subsp.paracasei)在制备具有调节睡眠功效的组合物中的应用,所述副干酪乳杆菌的保藏编号为CGMCC No.15139或DSM27447。
2.根据权利要求1所述的应用,其中,副干酪乳杆菌通过发酵产生超氧化物歧化酶、血管紧张素转化酶抑制物、γ-氨基丁酸、共轭亚油酸和/或B族维生素而具有调节睡眠功效;
优选地,副干酪乳杆菌作为活性物质进一步具有抗氧化和/或调节血压功效。
3.根据权利要求1所述的应用,其中,所述组合物为食品组合物、饲料组合物、化妆品组合物或药品组合物。
4.根据权利要求1-3任一项所述的应用,其中,利用副干酪乳杆菌CGMCC No.15139或DSM27447发酵乳液,制备得到发酵产物,得到的发酵产物作为或进一步用于制备所述具有调节睡眠功效的组合物。
5.根据权利要求4所述的应用,其中,所述乳液为生鲜牛乳或复原乳,其中可选择性地含有5%~10%的蔗糖。
6.根据权利要求5所述的应用,其中,所述发酵条件为:35℃~45℃,发酵1天~7天。
7.根据权利要求5或6所述的应用,其中,副干酪乳杆菌以种子液的形式接种于乳液中,接种量2%~5%。
8.根据权利要求5所述的应用,其中,发酵产物的上清液作为或进一步用于制备所述具有调节睡眠功效的组合物。
9.根据权利要求4所述的应用,其中,所述具有调节睡眠功效的组合物为发酵乳、发酵乳饮料、奶粉或固体饮料。
10.根据权利要求9所述的应用,其中,所述发酵乳或发酵乳饮料中副干酪乳杆菌CGMCCNo.15139的含量为1×107cfu/mL~1×1011cfu/mL。
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