CN112167346B - 用以抗氧化的含乳酸菌菌株的食品组合物以及医药组合物 - Google Patents
用以抗氧化的含乳酸菌菌株的食品组合物以及医药组合物 Download PDFInfo
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- CN112167346B CN112167346B CN201910604927.9A CN201910604927A CN112167346B CN 112167346 B CN112167346 B CN 112167346B CN 201910604927 A CN201910604927 A CN 201910604927A CN 112167346 B CN112167346 B CN 112167346B
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
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- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G4/00—Chewing gum
- A23G4/06—Chewing gum characterised by the composition containing organic or inorganic compounds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
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Abstract
本发明提供了一种用以抗氧化的含乳酸菌菌株的食品组合物以及医药组合物。具体地，本发明提供一种长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)GB‑1496菌株，此乳酸菌菌株具有抗氧化及清除自由基的活性效果，且以食品组合物或医药组合物的形式存在。
Description
技术领域
本发明是有关一种食品组合物以及医药组合物，特别是一种包含具有抗氧化活性效果的乳酸菌菌株的食品组合物以及医药组合物。
背景技术
老化是人体器官最大的杀手，器官老化会导致慢性肾病、失智症、心血管疾病、糖尿病、癌症或其他慢性疾病，严重时可导致个体死亡。而身体所产生的自由基(freeradicals)则是造成器官老化的重要元凶，身体所产生的自由基主要分为活性氧类(reactive oxygen species)以及活性氮类(reactive nitrogen species)。活性氧类是生物正常代谢中会产生的副产品，包含一些氧离子、过氧化氢等等，这些活性氧物质在细胞信号传导、对抗微生物感染和保持机体恒常性中扮演着很重要的角色。而活性氮类则是在病原菌入侵时，大量被免疫细胞产生，主要用于杀死入侵的病原菌。血管内皮细胞也会分泌少量的活性氮物质，来促进血管扩张及讯息传导。因此，调节并维持良好的自由基平衡对人体非常重要。
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。慢性肾病(chronic kidneydisease)的其中一个原因则是因为慢性发炎而肾脏累积了过多的活性氧物质，造成肾细胞的死亡，使得肾器官逐渐丧失其生理功能，到了末期患者只能洗肾度日。阿兹海默症则是脑内类淀粉的堆积造成慢性发炎，而发炎所产生的自由基诱发神经元细胞死亡，最后造成失智症。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。
综上所述，发展出一种安全且可长期使用的具有抗氧化活性效果的营养补充品有其迫切性。而乳酸菌一般来说是安全的，因此，找出具有抗氧化活性效果的乳酸菌菌株便是目前极需努力的目标。
发明内容
本发明提供一种含乳酸菌菌株的食品组合物及医药组合物，其具有抗氧化的活性效果，因而能够减少自由基浓度，以抑制器官老化。
本发明一实施例的含乳酸菌菌株的食品组合物包含具有抗氧化的活性效果的乳酸菌菌株，此乳酸菌株为长双歧杆菌婴儿亚种(Bifidobacterium longumsubsp.infantis)GB-1496菌株，保藏编号为CCTCC NO：M2011122，上述乳酸菌菌株保藏于中国典型培养物保藏中心(保藏日期2011年04月10日；保藏单位地址：中国武汉武汉大学，430072；分类命名：长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis))；以及生理上可接受的赋形剂、稀释剂或载体。
本发明另一实施例的含乳酸菌菌株的医药组合物包含具有抗氧化活性效果的乳酸菌菌株，此乳酸菌株为长双歧杆菌婴儿亚种(Bifidobacterium longumsubsp.infantis)GB-1496菌株，保藏编号为CCTCC NO：M2011122，上述乳酸菌菌株保藏于中国典型培养物保藏中心；以及医药上可接受的赋形剂、稀释剂或载体。
以下藉由具体实施例配合所附的附图详加说明，当更容易了解本发明的目的、技术内容、特点及其所达成的功效。
附图说明
图1为本发明的乳酸菌菌株的清除自由基能力分析的试验结果。
图2为本发明的乳酸菌菌株的还原能力分析的试验结果。
图3为本发明的乳酸菌菌株和人类肠上皮细胞株(Caco-2)共同培养后，肠上皮细胞所产生的超氧化物歧化酶活性分析的试验结果。
图4为本发明的乳酸菌菌株和人类肠上皮细胞株(Caco-2)共同培养后，肠上皮细胞所产生的过氧化氢酶活性分析的试验结果。
用于专利程序的微生物保藏：
本发明的GB-1496菌株
保藏日期：2011年04月10日
保藏单位：中国典型培养物保藏中心
保藏单位地址：中国武汉武汉大学，430072
保藏编号：CCTCC NO：M2011122
分类命名：长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)
具体实施方式
以下将详述本发明的各实施例，并配合附图作为例示。除了这些详细说明之外，本发明亦可广泛地施行于其它的实施例中，任何所述实施例的轻易替代、修改、等效变化都包含在本发明的范围内，并以请求保护范围为准。在说明书的描述中，为了使读者对本发明有较完整的了解，提供了许多特定细节；然而，本发明可能在省略部分或全部特定细节的前提下，仍可实施。此外，众所周知的步骤或组件并未描述于细节中，以避免对本发明形成不必要的限制。附图中相同或类似的组件将以相同或类似符号来表示。特别注意的是，附图仅为示意之用，并非代表组件实际的尺寸或数量，有些细节可能未完全绘出，以求附图的简洁。
本发明所述的乳酸菌菌株的冷冻干燥培养物已保藏于中国典型培养物保藏中心(CCTCC，中国武汉武汉大学，430072)。保藏的详细资料如表1所示：
表1乳酸菌菌株的保藏资料
如表1所列已保藏的长双歧杆菌婴儿亚种(Bifidobacterium longumsubsp.infantis)GB-1496菌株被发现具有抗氧化(高还原力)及清除自由基的活性效果。
本发明包含具有抗氧化及清除自由基活性效果的乳酸菌菌株，该乳酸菌菌株为长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)GB-1496菌株，保藏编号为CCTCC NO：M2011122，上述乳酸菌菌株保藏于中国典型培养物保藏中心；以及生理上可接受的赋形剂、稀释剂或载体所组成的食品组合物，或医药上可接受的赋形剂、稀释剂或载体所组成的医药组合物，其中乳酸菌菌株为具有活性的菌株。
于食品组合物的实施例中，生理上可接受的赋形剂、稀释剂或载体可为一食品。举例而言，食品可包含但不限于乳制饮品、茶、咖啡、口香糖、洁牙糖(例如***片、咀嚼锭、软糖等)或以上的组合，其中乳制饮品可包含发酵乳、优格、奶酪或乳制饮品乳粉等。医药组合物可为口服剂型。举例而言，口服剂型可为锭剂、胶囊、溶液剂及粉剂等。
于食品组合物或医药组合物的实施例中，乳酸菌菌株的数量为106CFU以上；较佳者，乳酸菌菌株的数量为1010CFU以上。
实施例1：本发明的乳酸菌菌株的形态学以及一般性质
根据16S rDNA序列分析以及API细菌鉴定***分析结果来确认菌株在分类学上的特征。本发明的乳酸菌菌株在形态学及一般性质上的特征详细列于表2：
表2本发明的乳酸菌菌株的形态学及一般性质特征
实施例2：本发明的乳酸菌菌株的收集、培养与保存
本发明的乳酸菌菌株是以20％甘油保存于-80℃。使用前，以含有0.05％cysteine的MRS broth(DIFCO)，37℃下活化(24小时)二次后使用。使用于研究的本发明乳酸菌菌株：长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)GB-1496菌株，来源为人类母乳。
实施例3：本发明的乳酸菌菌株GB-1496菌株的自由基清除活性分析
DPPH(di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium)是稳定自由基分子，DDPH自由基在甲醇溶液中在波长517nm下有最高的吸收值。当DPPH自由基与抗氧化物质作用后，抗氧化物质提供氢质子而清除自由基，而DPPH自由基就会失去本身蓝紫色的特性而造成吸光值的下降。利用测定OD517值的下降来测定受测乳酸菌菌株的自由基清除能力。
乳酸菌菌株的自由基清除能力的检测方法如下。将本发明的长双歧杆菌婴儿亚种GB-1496菌株的含菌株菌液(约为2×109CFU)、10μg/ml维生素C(阳性对照组，positivecontrol)、不具抗氧化活性效果的嗜热链球菌(Streptococcus thermophiles)SY-66菌株、鼠李糖乳酸杆菌(Lactobacillus rhamnosus)gL-35菌株、副干酪乳酸杆菌(Lactobacillusparacasei)gL-30菌株及gL-180菌株的含菌株菌液(约为2×109CFU，阴性对照组，negativecontrol)及二次水(空白组，blank)分别与0.2mM DPPH在甲醇溶液中1：1混合。混和均匀后，在黑暗室温下反应30分钟。接着于4℃离心(12000rpm,2min)后，取200μl到96孔盘中并测定OD517值。自由基清除能力的计算公式如下：
自由基清除能力＝ODblank-ODsample/ODblank×100％
其中ODsample为受测样品的吸光值，ODBlank为空白组的吸光值。
请参照图1，其为本发明的乳酸菌菌株清除自由基能力分析(DPPH assay)的试验结果，其中符号**表示p值<0.01，亦即统计学上具有显著差异。由图1的试验结果可知，相较于SY-66菌株(Streptococcus thermophiles)、gL-35菌株(Lactobacillus rhamnosus)、gL-30菌株(Lactobacillus paracasei)及gL-180菌株(Lactobacillus paracasei)，本发明的长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)GB-1496菌株具有强清除自由基的能力。
实施例4：本发明的乳酸菌菌株GB-1496菌株的抗氧化还原力活性分析
抗氧化还原力活性分析(Ferric-reducing ability of power，FRAP assay)是一种普遍用来测试抗氧化剂还原力活性的一种方法，其是以样品整体的还原能力作为抗氧化力。于酸性环境(pH 3.6以下)下，FRAP试剂中的三价铁(Fe3+)会被抗氧化物如维生素C还原成二价铁(Fe2+)，而造成颜色的改变。利用TPTZ(2,4,6-Tri-(2-pyridyl)-5-triazine)的呈色特性可测得样品的还原能力。当Fe3+-TPTZ复合物被还原成Fe2+-TPTZ时，会由黄色转为蓝色，蓝色越深表示抗氧化力越强。因此，检测OD593值即可计算出抗氧化剂的还原能力也就是抗氧化能力。
本次实验是将本发明的长双歧杆菌婴儿亚种GB-1496菌株的含菌株菌液(约为6×109CFU)、5μg/ml维生素C(阳性对照组，positive control)、不具抗氧化特性的SY-66菌株(Streptococcus thermophiles)、gL-35菌株(Lactobacillus rhamnosus)、gL-30菌株(Lactobacillus paracasei)及gL-180菌株(Lactobacillus paracasei)菌株菌液(约为6×109CFU)，阴性对照组，negative control)和Fe3+-TPTZ做反应，并检测OD593值。所测的值和所制备的已知浓度的FeSO4标准液混合FRAP试剂所得到的标准检量线做比对。利用检量线公式计算出含本发明的长双歧杆菌婴儿亚种GB-1496菌株的菌液的还原力(μg/ml,Fe2+)。
请参照图2，其为本发明的乳酸菌菌株还原力分析试验(FRAP assay)的试验结果，其中符号***表示p值<0.005，亦即统计学上具有非常显著的差异。由图2的试验结果可知，相较于SY-66菌株(Streptococcus thermophiles)、gL-35菌株(Lactobacillusrhamnosus)、gL-30菌株(Lactobacillus paracasei)及gL-180菌株(Lactobacillusparacasei)，本发明的长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)GB-1496菌株具有很强的还原力，也就是抗氧化力。
实施例5：本发明的乳酸菌菌株诱发肠上皮细胞表达抗氧化酶分析
身体内对于太多的自由基(oxidative stress)会有所谓的调控机制，体细胞会产生抗氧化剂(antioxidant)来因应，如合成谷胱甘肽(glutathione)、泛醇(ubiquinol)和尿酸(uric acid)等物质来吸收游离电子，另一方面从食物中摄取抗氧化剂如维生素C、维生素E等，也能抑制自由基的产生。身体细胞的另一个抗氧化***为抗氧化酶(antioxidantenzymes)网络。超氧化物歧化酶(superoxide dismutase，SOD)是一种重要的抗氧化酶，可以将超氧化物通过歧化反应转化为氧气和过氧化氢，人体内有三种超氧化物歧化酶，分别存在于胞外、细胞质(cytoplasm)及粒线体(mitochondria)中。另一种酶为过氧化氢酶(catalase)是可以将超氧化物歧化酶所产生的过氧化氢转化为氧气和水。在饱和的状态下，一个过氧化氢酶分子每秒能将四千万个过氧化氢分子转化为水和氧气。
Caco-2细胞为人类结肠腺癌细胞上皮细胞，其细胞结构和功能相似于分化的小肠上皮细胞。Caco-2细胞具有微绒毛等结构并含有与小肠刷状缘上皮相关的酶系，因而被广泛应用在模拟体内肠细胞生理活动模式。在细胞培养***中，Caco-2细胞可以长成单一层细胞，细胞彼此排列紧密，不仅在形态学上和小肠上皮细胞相似，且具有同样的胞饮作用、Tight junction等结构。
本实验在Caco-2细胞的培养***中加入本发明的长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)GB-1496菌株的活菌(实验组)、Streptococcus thermophiles SY-66菌株的活菌(对照组)及不加入任何活菌(空白对照组，Control)，以比例1：100(cells：probiotics)共同培养16小时后，洗掉菌株，再将Caco-2细胞打破萃取蛋白质，以检测细胞内的超氧化物歧化酶(superoxide dismutase，SOD)活性(其试验结果如图3所示)及过氧化氢酶(catalase)活性(其试验结果如图4所示)。SOD活性分析是使用SOD Assay Kit(Cayman Cat.706002)，而catalase活性分析是使用CatalaseAssay Kit(Cayman Cat.707002)。所有实验流程皆依照kit的说明书建议进行分析。
请参照图3及图4，以说明本发明的乳酸菌菌株诱发肠上皮细胞表达氧化酶的效果，其中符号*表示p值<0.05，亦即统计学上具有显著差异；符号***表示p值<0.005，亦即统计学上具有非常显著的差异。相较于对照组Streptococcus thermophiles SY-66菌株，本发明的长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)GB-1496菌株可以诱发Caco-2细胞增加抗氧化酶的表达，以分解体内过多的自由基。
需注意的是，乳酸菌对身体健康的功能在于菌株(strain)的特异性，而非菌种(species)，此种对于人身体健康有特殊功效的菌株称为功能性益生菌(Guidelines forthe evaluation of probiotics in food；Report of joint FAO/WHO working group ondrafting guidelines for the evaluation of probiotics in food；London Ontario,Canada April 30 and May 1,2002：1-7)。举例而言，根据2018年发表在ScientificReports的一篇论文，野生种Bifidobacterium longum subsp.infantis在有氧的环境下，由于烟碱酰胺腺嘌呤二核甘酸磷酯(nicotinamide adenine dinucleotide phosphate，NADPH)氧化酶的作用，会在菌体内产生过多的活性氧自由基(H2O2)。因此，野生种Bifidobacterium longum subsp.infantis似乎无法有效清除自由基，并造成菌体的死亡或抑制其生长。而本发明的长双歧杆菌婴儿亚种(Bifidobacterium longumsubsp.infantis)GB-1496菌株具有清除自由基的功效，相较于野生种Bifidobacteriumlongum subsp.infantis具有其独特性。
综合上述，相较于其它受测的乳酸菌菌株，本发明的长双歧杆菌婴儿亚种GB-1496菌株具有较佳的自由基清除能力以及还原能力，且能诱发Caco-2细胞增加抗氧化酶的表达，因此，本发明的长双歧杆菌婴儿亚种GB-1496菌株具有抗氧化的活性效果，且能够减少自由基浓度，以抑制器官老化。
以上所述的实施例仅是为说明本发明的技术思想及特点，其目的在使熟习此项技艺的人士能够了解本发明的内容并据以实施，当不能以之限定本发明的专利范围，即大凡依本发明所揭示的精神所作的均等变化或修饰，仍应涵盖在本发明的专利范围内。
Claims (9)
1. 一种含乳酸菌菌株的食品组合物，其包含：
具有抗氧化的活性效果的乳酸菌菌株，该乳酸菌菌株为长双歧杆菌婴儿亚种(Bifidobacterium longum subsp. infantis) GB-1496 菌株，保藏编号为CCTCC NO：M2011122，上述乳酸菌菌株保藏于中国典型培养物保藏中心；所述乳酸菌菌株为具有活性的菌株；以及
生理上可接受的载体。
2.如权利要求1所述的含乳酸菌菌株的食品组合物，其中所述载体为一食品。
3.如权利要求2所述的含乳酸菌菌株的食品组合物，其中所述食品包含乳制饮品、茶、咖啡、口香糖、洁牙糖或以上的组合。
4.如权利要求2所述的含乳酸菌菌株的食品组合物，其中所述食品包含发酵乳。
5.如权利要求2所述的含乳酸菌菌株的食品组合物，其中所述食品包含优格。
6.如权利要求2所述的含乳酸菌菌株的食品组合物，其中所述食品包含奶酪。
7.如权利要求2所述的含乳酸菌菌株的食品组合物，其中所述食品包含乳粉。
8. 一种含乳酸菌菌株的医药组合物，其包含：
具有抗氧化的活性效果的乳酸菌菌株，该乳酸菌菌株为长双歧杆菌婴儿亚种GB-1496菌株，保藏编号为CCTCC NO：M2011122，上述乳酸菌菌株保藏于中国典型培养物保藏中心；所述乳酸菌菌株为具有活性的菌株；以及
生理上可接受的载体。
9.如权利要求8所述的含乳酸菌菌株的医药组合物，其为口服剂型。
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