CN103596579A - 含有利用泡菜乳酸菌发酵的稻米糖化液作为活性成分并具有抗菌和抗病毒效果的稻米发酵食品组合物 - Google Patents
含有利用泡菜乳酸菌发酵的稻米糖化液作为活性成分并具有抗菌和抗病毒效果的稻米发酵食品组合物 Download PDFInfo
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Abstract
本发明涉及一种稻米发酵食品组合物，所述组合物含有利用泡菜乳酸菌发酵的稻米糖化液作为活性成分，并具有抗菌和抗病毒效果。更具体而言，本发明涉及一种稻米发酵食品组合物和一种包含其的功能健康食品，所述组合物通过将泡菜乳酸菌添加至稻米糖化液作为主要成分并使该混合物发酵而制备，并具有针对特应性皮炎和禽流感病毒等的抗菌和抗病毒效果。
Description
技术领域
本发明涉及一种稻米发酵食品组合物，所述组合物含有利用泡菜乳酸菌(kimchi lactobacillus)发酵的稻米糖化液作为活性成分，并具有抗菌和抗病毒效果。更具体而言，本发明涉及一种稻米乳酸菌发酵食品组合物和一种包含其的功能健康食品，所述组合物通过将泡菜乳酸菌添加至稻米糖化液作为主要成分并使该混合物发酵而制备，并具有针对特应性皮炎和禽流感病毒的抗菌和抗病毒效果。
背景技术
韩国将乳酸菌用于食品中历史悠久，最具代表性的利用乳酸菌的食品是泡菜。作为传统韩国发酵食品的泡菜，其优越性已为全世界所知，因此在2001年7月5日召开的食品法典委员会(Codex Alimentarius Commission)第24届大会中，韩国食品泡菜的Codex标准率先被采纳为韩国食品。
发现构成泡菜的乳酸菌为乳杆菌属(Lactobacillus)、明串珠菌属(Leuconostoc)和魏斯氏菌属(Weissella)这三个属，并且代表性乳酸菌种为清酒乳杆菌清酒亚种(Lactobacillus sakei subsp.sakei)、柠檬明串珠菌(Leuconostoc citreum)和韩国魏斯氏菌(Weissella koreensis)。
这些菌种的构成可以根据泡菜的内外部环境而改变，并且乳酸菌的种类会根据泡菜而变化。因此，泡菜的味道也极大地依赖于获自泡菜乳酸菌新陈代谢的发酵副产物。随着发酵的进行，泡菜中存在的有害好氧菌被杀死，厌氧有益菌繁殖，乳酸浓度升高，酸性降低，并且更多的乳酸逐渐增加。这些泡菜乳酸菌可增强体内免疫功能，并具有通过产生细菌素和有机酸等而抑制有害细菌的抗菌活性，由此有助于肠道微生物群落的产生和维护。此外，也已知道，泡菜乳酸菌具有血液胆固醇降低和抗癌效果。
在从泡菜中分离的乳酸菌中，清酒乳杆菌(Lactobacillus sakei)属于产生有机酸的乳酸菌(LAB)群。通常，乳杆菌主要发现于肉类和织物发酵产物和发酵的鱼类中。该 细菌是商业上必不可少的细菌，被用作制造发酵香肠产品用酵母，及用作肉类和鱼类食品的生物防腐培养物。清酒乳杆菌产生有机酸作为初级代谢产物，具有作为起子(starter)和防腐培养物的重要作用，基于有机酸的酸化对食品防腐，并有助于改善产品品质。另外，清酒乳杆菌通过产生细菌素而具有抗菌活性。该抗菌活性是针对鱼类致病菌的抗菌活性，其有助于预防鱼类的细菌性疾病并抑制肉类发酵过程中的李斯特氏杆菌(listeria)。
因此，本发明人成功地发现了清酒乳杆菌的菌株，所述菌株在作为泡菜中所富含的乳酸菌的清酒乳杆菌中，具有有力的预防过敏功效，并通常用于香肠和肉类发酵。本发明人成功地从泡菜中分离了作为清酒乳杆菌的亚种的清酒乳杆菌ProBio-65，所述清酒乳杆菌ProBio-65具有耐酸性、耐胆汁性、抗菌活性和耐抗生素性。
即，所开发的菌株通过以下方式获得：逐步稀释泡菜汁，在乳酸菌选择培养基中培养泡菜汁，从培养基中仅分离乳酸菌，选择所分离的细菌，在MRS培养基中培养该细菌并最终选择有力地抑制作为特应性皮炎中加重因子的金黄色葡萄球菌(Staphylococcus aureus)增殖的微生物，该微生物被称作“清酒乳杆菌ProBio-65”(保藏号：KCTC10755BP)并作为韩国专利0479719被授予专利。
源自泡菜的乳酸菌的清酒乳杆菌ProBio-65具有抑制称做特应性皮炎的疾病加重因子的金黄色葡萄球菌生长的优越功能，以及针对各种病原菌的抗菌活性，对于IgE产生的优越的抑制活性和对于免疫系统的优异的活化能力。另外，清酒乳杆菌可有效地到达肠，并因优越的耐酸性和耐胆汁酸性，通过分泌抗菌物质而有助于对有害菌的生长抑制和肠道微生物群落的正常化。
同时，特应性皮炎是一种炎性皮肤病，引起严重的瘙痒和特异性湿疹样皮损(pathognomonic eczematous skin lesion)，并且主要在婴幼儿时期发作。特应性皮炎是特别常发于冬季的慢性发作的特应性疾病。特应性皮炎的发作经常导致支气管哮喘或过敏性鼻炎，甚至在成年期也经常复发。特应性皮炎患者所遭受的严重瘙痒会导致对环境的适应性、活力和工作功效降低、失眠症和情绪失调等，并且伴随色素沉着的湿疹样皮损会导致皮肤畸形并扰乱正常的人际关系或社交活动。另外，干而敏感的皮肤会经常导致刺激性接触性皮炎，限制职业选择。
近年来，特应性皮炎在全世界范围内有所增加，在韩国随着过敏性疾病的急剧增加而增加。基于2000年韩国过敏和呼吸系统疾病学会(Korean academy of allergy and respiratory Disease)在全国范围内对整个韩国小学生和中学生中的43,045名学生的调研结果，可以看出有24.9%的小学生和12.8%的中学生被诊断有特应性皮炎。10年来特应性皮炎增长了50%，并且其增加量与年龄成正比。即，特应性皮炎的发作率很高，对于成年人来说高达约17%。
特应性皮炎的引发原因已知与遗传、免疫和环境因子有关。遗传因子为特应性疾病相关基因，而免疫基因是特应性疾病相关基因的重要部分。迄今被发现的特应性疾病相关基因包括人类白细胞抗原(HLA)和染色体6p，T-细胞受体(TCR)和染色体7p，IgE高亲合力受体(FcεRI-β)和染色体11q，以及IL-4和染色体5q等。环境因子包括扁虱、屋尘、真菌、有害物质(如甲醛水和甲苯，源自用于建筑房屋的材料或涂料，并暴露于空气)和包含于食品中的化学品或食品。
另外，特应性皮炎的另一加重因子为金黄色葡萄球菌。在80%～100%的特应性皮炎患者的湿疹样病变中发现了金黄色葡萄球菌蔟。该值远高于健康皮肤中发现的金黄色葡萄球菌蔟的量，即，约5%～约30%。金黄色葡萄球菌蔟加重皮炎部位，例如增加脓疱病，感染他人并使其易感特应性皮炎。由金双色葡萄球菌产生的外毒素对于加重特应性皮炎的超抗原作用，其相关证据已被提出。外毒素的种类包括葡萄球菌肠毒素A-D(SEA-D)和中毒性休克综合征毒素-1(TSST-1)，这些外毒素使IGE的产生增加至8倍以上。
常用于特应性皮炎患者的治疗的实例包括抗生素治疗、抗组胺治疗、类固醇治疗和免疫治疗等。抗生素治疗用于如上述金黄色葡萄球菌等微生物导致的继发性感染，但具有与抗生素的常见不正确施用相关的许多问题，并且会使耐抗生素金黄色葡萄球菌增加。世界上重要的耐抗生素菌株包括耐甲氧西林金黄色葡萄球菌(MRSA)和耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)等。
因此，对于下述替代物存在不断提高的需求，所述替代物可预防抗生素的滥用和副作用以及耐抗生素细菌产生，并且可预防特应性皮炎的根本病因并可治疗特应性皮炎。
另一方面，禽流感是适于鸟类的急性病毒感染，自从1983年首次发现于欧洲(如比利时和法国)以来，至今已成为全世界范围内的问题。禽流感对于鸡和火鸡等家禽造成巨大破坏。感染禽流感的动物在大多数国家被屠杀，感染禽流感的国家的家禽产品出口受到限制。
感染通常因直接接触被感染的鸟类的粪便或者通过液滴、水、人脚、饲料运输车辆、设备、装置和蛋壳中污染的排泄物等而导致。症状随所感染的病毒的病原性而不同，但常见症状包括呼吸道症状、腹泻和产卵率急剧下降。在一些情形中，如鸡冠等头部区域中会产生紫绀，发生面部水肿，羽毛集中在一处，并且死亡率也视病原性而在0%～100%内变动。
一般而言，已知禽流感病毒不直接感染人类，其通过作为中间宿主的猪感染人类，但根据病原性分为三类，即，高致病性禽流感、低致病性禽流感和非致病性禽流感。在这些禽流感中，高致病性禽流感也感染人类。
最近受到关注的关于高致病性禽流感(HPAI)病毒H5N1对于人类的感染所提出的禽流感病毒人类感染的病理机制证实，由于感染HAPI病毒时T细胞的显著降低和干扰素-γ的降低，在早期阶段病毒的体内增殖未得到抑制，病毒感染在整个身体内转移，结果，发现病毒在器官(如肺等呼吸器官和血液以及直肠)中增殖，并且发生这些病毒繁殖部位中的细胞凋亡所导致的细胞的死亡，和大量释放的炎性细胞因子所导致的非化脓性肺炎和多器官功能障碍，由此造成死亡。
因此，感染禽流感对于人类造成的破坏变得愈发严重，例如，在东南亚(如越南和泰国)禽流感造成了灾难。为减少此破坏，已经付出了许多努力来开发禽流感疫苗和药物，但仅开发和报道了一些不能完全预防和治疗禽流感的疫苗和药物。韩国专利10-2007-0035203号公报提出一种使用鱼腥草(Houttuynia cordata)提取物作为抗禽流感病毒试剂的东方药物组合物，并且韩国专利第0485056号提出一种含有酵母的水溶性葡聚糖低聚物的组合物，所述组合物通过促进细胞内NO和TNF-α的产生而被用作间接预防和治疗禽流感的药物。
同时，稻米是韩国以及全世界一半以上的人口用作主食的谷类。基于品种改良和农耕技术的开发，稻米在韩国的生产得到迅速增加，但稻米消费因西方饮食文化的影响而逐年显著降低。这种稻米消费的降低归因于食品消耗方式的多样化、健康文化的传播以及饮食习惯的改变。稻米消费降低的另一重要原因在于利用稻米的各种食品不够充足。即，稻米大多在初步加工后被食用，或者有少量被面包房使用及用于面包中等。由于国民收入提高和不充分加工食品所导致的饮食习惯的改变，稻米消费降低并且库存增加。
另外，稻米含有改善视力的胡萝卜素、强化骨骼的钙和铁，和抑制胆固醇的生物 合成的红曲霉组分，非常适于韩国人体质。特别是，当将稻米与乳、作为稻米中所缺乏的氨基酸的牛奶补充剂赖氨酸组合使用时，会显著地改善发酵牛奶的饮食和营养价值，并有助于促进近来消费降低的稻米的消费。
迄今已报道了一些含有稻米作为主要成分的食品。韩国专利公开2002-0016109号公报公开了一种具有极佳味道的稻米饮料组合物及其制备方法，其中将精米与糙米分开加工，在精米加工过程中省略烘烤，温度、时间和产生淀粉糖浆(糖稀)时的酶含量得到控制以提高热稳定性，进行烘烤，但在糙米加工过程中省略碾磨和糖稀生产，以防止热量导致的褐化而不管是否为充分微生物杀菌而进行加热杀菌，由此保持精米固有的白色并使营养物破坏最小化。另外，韩国专利公开2000-0006601号公报公开了一种制备稻米饮料的方法，所述方法通过酶分解烘烤的糙米和精米而获得澄清的液体，其中糙米和精米的营养物破坏被最小化，味道得到良好的平衡，并且该稻米饮料是非酸性的，适于低温灭菌并适于填充PET容器。
但是，传统的专利仅公开了包括制备稻米糖化液并将糖化液与辅助添加剂等简单混合的制备稻米食品的方法，而未报道赋予人类有益组分的利用乳酸菌的发酵食品，并且通过利用乳酸菌使稻米发酵而获得的食品在得到开发。
利用稻米的乳酸菌发酵食品预防肥胖和成人病，促进利用难消化的碳水化合物作为营养物的肠道微生物的繁殖，并降低血液胆固醇，从而有助于预防成人病。另外，由于稻米含有二十八烷醇作为精力(耐力)增强剂和助于特应性改善的神经酰胺，因此稻米加工食品得到积极开发。
韩国专利公开1998-075079号公报公开了一种制备液体稻米酸奶的方法，所述方法包括将68重量份～76重量份水与5重量份～18重量份阿尔法稻米粉、10重量份～15重量份脱脂奶粉、2重量份葡萄糖和2重量份蔗糖混合，然后均化、热分解并冷却以获得样品，对该样品接种混合乳酸菌种子培养物，静止培养该样品，并将所获得的发酵原液与糖液混合，随后均化。
另外，韩国专利公开第0527419号公开了一种包含稻米发酵液的稻米发酵食品组合物，所述稻米发酵液通过以下方式制备：将双歧杆菌和乳酸菌分别添加至稻米糖化液作为主要成分并使所获得的混合物发酵，或者添加双歧杆菌和乳酸菌的混合物并使该混合物发酵。
另外，韩国专利公开10-2010-0074389号公报公开了一种通过以下方法制备的稻 米酸奶，所述方法包括(1)第一步，通过下述方式制备稻米浓缩物，所述方式为通过初步酶处理使稻米液化，通过二次酶处理将稻米糖化，并浓缩稻米直至浓缩物的白利糖度为50°Bx～75°Bx，以提高稻米成分的含量并改善味道；(2)第二步，通过下述方式制备稻米糖浆，所述方式为将稻米浓缩物与糖、膳食纤维和添加剂混合以遮掩稻米气味并改善味道；(3)第三步，通过下述方式制备乳酸菌发酵液，所述方式为将脱脂奶粉溶于原料乳或复原乳中，使该溶液接种冷冻干燥的混合粉末状乳酸菌，使所获得的溶液发酵，并在滴定酸度达到0.75%时冷却该溶液；(4)第四步，将第二步的稻米糖浆与第三步的乳酸菌发酵液混合，随后均化以平衡甜度和酸度；和(5)第五步，使用自动填充包装机将该混合物填充至塑料瓶中并包装塑料瓶。
但是，未发现证实以下内容的测试结果：通过泡菜乳酸菌的发酵获得的稻米发酵食品组合物，除了乳酸菌的一般效果(如免疫功能的改善、有害细菌的抑制、正常肠道菌群的产生和维护、血液胆固醇的降低和抗癌效果)之外，具有针对特应性皮炎和流感病毒感染的抑制活性，特别是针对禽流感病毒繁殖的抑制活性。在此情况下，本发明人发现，通过使作为韩国主食的稻米接种本发明人开发的作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(KCTC10755BP)并使所获得的稻米发酵而制备的乳酸菌发酵稻米食品组合物，表现出对于特应性皮炎和禽流感病毒等的抗菌和抗病毒效果，由此完成了本发明。
发明内容
技术问题
因此，鉴于以上问题进行了本发明，本发明的一个目的是提供一种稻米乳酸菌发酵食品组合物，所述组合物通过使作为主要组分的稻米糖化液接种泡菜乳酸菌并随后发酵而制备，并且具有针对特应性皮炎和禽流感病毒等的抗菌和抗病毒效果，不具有安全性问题并可以每日食用。
技术方案
根据本发明的一个方面，以上和其他目的可以通过提供抗病毒组合物来实现，所述组合物包含作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)或其培养物，并表现出针对禽流感病毒的抗病毒效果。
根据本发明的另一方面，提供一种稻米乳酸菌发酵食品组合物，所述组合物包含 稻米乳酸菌发酵液，并表现出针对特应性皮炎的抗菌效果，所述稻米乳酸菌发酵液通过将作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)添加至稻米糖化液并随后发酵而制备。
根据本发明的另一方面，提供一种稻米乳酸菌发酵食品组合物，所述组合物包含稻米乳酸菌发酵液，并表现出针对禽流感病毒的抗病毒效果，所述稻米乳酸菌发酵液通过将作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)添加至稻米糖化液并随后发酵而制备。
发明效果
本发明提供一种稻米乳酸菌发酵食品组合物，所述组合物通过使作为韩国主食的稻米接种清酒乳杆菌ProBio-65(KCTC10755BP)并随后发酵而制备。该稻米乳酸菌发酵食品组合物能够制备具有增强免疫功能的较高附加价值的稻米发酵食品，并能提高过多生产的稻米的可用性。当每日食用时，该稻米发酵食品组合物表现出显著地效果，如针对特应性皮炎和禽流感病毒等的抗菌和抗病毒效果。
附图说明
通过与附图一同提供的以下详细描述，本发明的上述和其它目的、特征及其它优点将会得到更加清楚的理解，附图中：
图1是说明制备稻米乳酸菌发酵食品组合物的方法的流程图；
图2是显示根据本发明通过细胞系测试鉴定抗病毒活性的照片；
图3是显示根据本发明的鸡胚蛋培养和鉴定抗病毒活性的照片；和
图4是显示根据本发明鉴定抗菌活性的照片。
具体实施方式
本发明的特征在于，提供一种抗病毒组合物，所述组合物包含作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)或其培养物，并表现出针对禽流感病毒的抗病毒效果。
另外，本发明的特征在于，提供一种稻米乳酸菌发酵食品组合物，所述组合物包含稻米乳酸菌发酵液，并表现出针对特应性皮炎的抗菌效果，所述稻米乳酸菌发酵液通过将作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP) 添加至稻米糖化液并随后发酵而制备。
另外，本发明的特征在于，提供一种稻米乳酸菌发酵食品组合物，所述组合物包含稻米乳酸菌发酵液，并表现出针对禽流感病毒的抗病毒效果，所述稻米乳酸菌发酵液通过将作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)添加至稻米糖化液并随后发酵而制备。
下文中，将详细提供本发明的优选实施方式，本发明所属技术领域的本领域技术人员可以容易地执行实施这些实施方式。本发明可以以各种方式实施，并不限于本文所述的实施方式。
首先，如图1所示，稻米糖化液的制备可以通过以下方式实施：称重稻米、将稻米浸入纯化水(purified water)中，将稻米与水共混并将该共混物糊化。糊化可以通过在约100℃保持20分钟～40分钟的一步法实施，或者通过两步法实施，所述两步法包括在约60℃保持约20分钟～40分钟的初步糊化和之后在约100℃保持约20分钟～40分钟的主糊化。
接下来，在约80℃～100℃使用α-淀粉酶将该糊化液初步糖化约40分钟～2小时。在初步糖化之后，将所获得的液体利用葡糖淀粉酶在约60℃～约75℃二次糖化约40分钟～2小时，随后过滤以制备稻米糖化液。
同时，稀释家中所制备的泡菜液体，涂在BCP固体培养基上，并在37℃培养48小时。在使用显微镜观察在预定时间后产生的菌落之后，选择480个球菌和杆菌，在MRS培养基中培养并在零下80℃储藏，鉴定抑制作为特应性疾病的加重因子的金黄色葡萄球菌KCTC1621增殖的微生物，分离清酒乳杆菌ProBio-65(KCTC10755BP)菌株，并将分离的ProBio65菌株在37℃于MRS培养基中培养以制备清酒乳杆菌ProBio-65(KCTC10755BP)。
即，ProBio-65菌株的培养通过以下方式进行：在37℃于MRS培养基(含有葡萄糖2％、蛋白胨2％、酵母提取物0.5％、乙酸钠0.5％、磷酸氢二钾0.2％、硫酸铵0.2％、硫酸镁0.1％、硫酸锰0.005％和吐温80(Tween80)0.1％)中培养该菌株12小时，以13,000rpm离心该培养液3小时，将所获得的微生物细胞与水解脱脂奶粉等量混合，均化该混合物，在-40℃初步冷冻该混合物，如下冷冻干燥该混合物，即在-40℃至-20℃的升温下保持7小时然后在-20℃保持5小时，在-20℃至-5℃的升温下保持9小时然后在-5℃保持10小时，在-5℃至10℃的升温下保持10小时然后在10℃保持 10小时，并且在10℃至20℃的升温下超过8小时，最终在20℃完成冷冻干燥，并研磨该冷冻干燥的微生物细胞以制备清酒乳杆菌ProBio-65(KCTC10755BP)。
接下来，将经过滤的稻米糖化液在100℃杀菌20分钟，将1.5重量%的所制备的清酒乳杆菌ProBio-65(KCTC10755BP)添加至稻米糖化液，在37℃进行发酵12小时，直至最终pH达到4.1，并向其添加5重量%的淀粉糖浆，以制备稻米乳酸菌发酵食品组合物。
当将清酒乳杆菌ProBio-65作为发酵起子添加至所制备的稻米糖化液时，所添加的清酒乳杆菌ProBio-65的量相对于稻米糖化液重量优选为0.1重量%～5重量%，并且稻米糖化液可以还包含选自全脂奶粉、牛奶、脱脂大豆蛋白和各种低聚糖、pH调节剂和缓冲剂、各种糖和甜味剂((sweeteners))以及其他添加剂等中的至少一种。
适当的低聚糖的实例包括异麦芽低聚糖、低聚半乳糖、麦芽低聚糖、环糊精、低聚果糖、低聚乳果糖、葡萄糖基蔗糖、龙胆低聚糖、帕拉金低聚糖、大豆低聚糖和低聚木糖等。
对于液体产品通常需要pH调节剂和缓冲剂，并将pH由约4.0调整至约6.5。pH调节剂或缓冲剂的实例包括弱酸，如柠檬酸、酒石酸、苹果酸、乳酸和碳酸及其盐，如柠檬酸钠、柠檬酸铵、酒石酸钠、苹果酸钠、乳酸钠、乳酸钙、碳酸钠和碳酸氢钠等。磷酸氢二钠也可以用作pH调节剂或缓冲剂。这些酸及其盐可以单独使用，或以其两种以上组合使用。混合比例可以在获得适当pH的同时确定。通常，相对于组合物的重量，混合比例优选为2重量%以下，更优选为0.05重量%～0.3重量%。
另外，可以将各种糖或甜味剂添加至饮料形式的食品组合物并与其共混。糖的实例包括：单糖，如葡萄糖和果糖；二糖，如麦芽糖和蔗糖；多糖，如糊精和环糊精；糖醇，如木糖醇、赤藓糖醇、山梨糖醇；和糖酯。甜味剂的实例包括：天然甜味剂，如甜叶菊提取物，例如莱鲍迪甙A、甜叶菊苷元(sormatin)和甘草甜素；和合成甜味剂，如糖精和阿斯巴甜。相对于所获得的饮料的重量，这些糖和甜味剂的混合比例通常为约15重量%以下，优选为约13重量%以下。
如果需要，根据本发明的食品组合物还包含一种或两种以上以下添加剂。添加剂的实例包括：各种水果汁(或浓缩汁及其粉状汁)，如柚子、苹果、橙子、柠檬、菠萝、香蕉和梨；维生素和维生素原(水溶性和脂溶性维生素，如视黄醇棕榈酸酯、双苯酰硫胺、核黄素、盐酸吡多辛、氰钴维生素、抗坏血酸钠、烟酰胺、泛酸钙、叶酸、生 物素、胆钙化甾醇、胆碱酒石酸氢盐、维生素E和β-胡萝卜素；香精(如柠檬香精、橙子香精、柚子香精和香草香精)；氨基酸、核酸及其盐(如谷氨酸、谷氨酸钠、甘氨酸、丙氨酸、天门冬氨酸、天冬氨酸钠、肌苷酸)；植物纤维(如聚右旋糖、果胶、黄原胶、阿拉伯胶和藻酸)；矿物质和微量元素(如氯化钠、乙酸钠、硫酸镁、氯化钾、氯化镁、碳酸镁、氯化钙、磷酸氢二钾、磷酸钠、甘油磷酸钙、柠檬酸亚铁钠、柠檬酸亚铁铵、柠檬酸铁、硫酸锰、硫酸铜、碘化钠、山梨酸钾、锌、锰、铜、碘和钴)。
此外，添加剂可以包括谷类(如糙米、黑米、糯米、黄米、高粱、大麦、绿豆、小米、薏仁米(Coixseed)、α-玉米、玉米、小麦、红豆和芝麻)、豆类(大豆、花生、黄豆、豌豆和青豆)、坚果(如松子、核桃、开心果、杏仁、葵花子、山核桃、板栗和葡萄等)、水果类（苹果、柠檬、温柏(quince)、柿子、桃、香蕉、草莓和柠檬)、蔬菜类(如西葫芦、萝卜、芜菁(Wuqing)、白菜、胡萝卜、牛蒡、莲藕、当归、松针、paselri、myeongilyeop、水芹(dolminari)和菠菜)、gugeunryu类(如土豆、雪莲果、胡萝卜、党参、牛蒡、红萝卜(radish)、莲藕、麻、羽衣甘蓝和桔梗)、叶菜类蔬菜(如芥兰、当归和花椰菜)、蘑菇类(如桦褐菌、灵芝、桑黄、猴头菇、伞菌、香菇(shiitake mushrooms)、指菇(fingering mushrooms)、蘑菇、平菇、岩石蘑菇、冬菇和冬虫夏草)、微生物类(酵母、乳酸菌、芽孢杆菌和红麴)、微藻类(如小球藻和螺旋藻)、藻类(如紫菜、海带、海藻、紫菜和莱茵衣藻(Chop))、植物药(如绿茶、香草、甘草、hwangchil、刺五加、人参、红参、枸杞子和芦荟)、动物药(如鹿茸和桑蚕)。
<实施例1>
稻米乳酸菌发酵食品组合物的制备
称重稻米，将其浸入纯化水中，共混，在60℃初步糊化40分钟，然后在100℃糊化40分钟，以制备糊化液。将糊化液在100℃使用α-淀粉酶初步糖化40分钟。在完成初步糖化之后，将所获得的液体在75℃使用葡糖淀粉酶二次糖化40分钟以制备糖化液，并将该糖化液过滤，以制备稻米糖化液。
独立于稻米糖化液，将陈泡菜液稀释，涂在BCP固体培养基(由Eiken Chemical Co.,Ltd.制造，日本)上，在37℃培养48小时，在使用显微镜观察在预定时间后产生的菌落之后，选择480个球菌和杆菌，在MRS培养基(由Difco Inc.制造)中培养并在零下80℃储藏，鉴定抑制作为特应性疾病的加重因子的金黄色葡萄球菌KCTC1621增殖的微生物，分离清酒乳杆菌ProBio-65(KCTC10755BP)菌株，并将分离的 ProBio-65菌株在37℃于MRS培养基中培养12小时，以13,000rpm离心该培养液3小时，将所获得的微生物细胞与水解脱脂奶粉混合，将该混合物均化，在-40℃预冷冻，如下冷冻干燥，即在-40℃至-20℃的升温下保持7小时然后在-20℃保持5小时，在-20℃至-5℃的升温下保持9小时然后在-5℃保持10小时，在-5℃至10℃的升温下保持10小时然后在10℃保持10小时，并且在10℃至20℃的升温下超过8小时，所述冷冻干燥最终在20℃完成，并研磨该冷冻干燥的微生物细胞以制备清酒乳杆菌ProBio-65菌。
将全脂奶粉以相对于稻米糖化液重量为6重量%的量添加至稻米糖化液，并在100℃的升温下进行杀菌20分钟。将1.5重量%的所制备的清酒乳杆菌ProBio-65(KCTC10755BP)添加至该液体，将所获得的混合物在37℃发酵12小时，直至最终pH达到约4.1，并向其添加5重量%的淀粉糖浆，以制备最终的稻米乳酸菌发酵食品组合物。
<实施例2>
稻米乳酸菌发酵食品组合物的特应性皮炎SCORAD的测量与测试
在为期12周的人体试验中，将根据本发明的稻米乳酸菌发酵食品组合物和对照食品施用于54名中度过敏的特应性的患者。结果，对特应性皮炎的严重指数(SCORAD)进行分析，结果显示在下表1。
表1
<SCORAD的变化(平均值±标准差)>
如从上表1中可以看出的，施用稻米乳酸菌发酵食品组合物的组(也简称为“稻米乳酸菌发酵食品组合物组”)和对照食品组的作为特应性皮炎的临床指标的SCORAD(特应性皮炎的打分)均降低，但稻米乳酸菌发酵食品组合物组的SCORAD变化高于对照食品组，特别是，施用稻米乳酸菌发酵食品组合物组的SCORAD降低20%以上，并且施用稻米乳酸菌发酵食品组合物组中症状改善的患者的比例为70%， 是对照食品组的两倍以上，对照食品组中该值为29.6%。
<实施例3>
关于稻米乳酸菌发酵食品组合物的CCL17、CCL27和CCL18变化的测试
除实施例2之外，CCL17(趋化因子(c-c模体)配体17)、CCL27(趋化因子(c-c基序)配体27)和CCL18(趋化因子(c-c基序)配体18)也参与了特应性皮炎的炎症中动员T淋巴细胞的机制，测量了作为特应性皮炎的免疫学和客观指标(生物标志物)的CCL17、CCL27和CCL18的浓度，结果显示在表2中。
表2
<趋化因子和细胞因子的变化>
如从上表2中可看出的，稻米乳酸菌发酵食品组合物组的血液中CCL17、CCL27和CCL18的浓度在统计学上显著降低。该结果在临床上证实了，根据本发明的稻米乳酸菌发酵食品组合物在改善特应性皮炎症状方面是有效的。
<实施例4>
清酒乳杆菌Probio-65(KCTC10755BP)的抗病毒活性测试
[细胞系培养]
将MDCK(马-达二氏犬肾(MDCK)细胞系；韩国细胞系库，KCLB No.10034)细胞在35℃于含有牛血清和抗生素(100U/ml青霉素和100ug/ml链霉素)的伊格尔最低必需培养基(MEM)中培养48小时～72小时。
[病毒培养]
将H9N2病毒(禽流感H9N2病毒；国家兽医研究检疫局(National Veterinary Research and Quarantine Service))在35℃于MDCK细胞系中培养48小时～72小时。使用磷酸盐缓冲液洗涤MDCK单层细胞两次，并接种H9N2病毒，使该病毒吸附在细胞上约30分钟，对其添加不含牛血清的伊格尔最低必需培养基，并在温育器中于37℃培养该细胞的同时观察细胞病变效应(CPE)。当在单层细胞的约70%中观察到细 胞病变效应时，在-70℃反复冷冻和解冻含有MEM培养基的受感染的细胞三次并离心，并在将不含细胞组分的上清液于-70℃储藏时获得病毒。
[清酒乳杆菌Probio-65(KCTC10755BP)的培养]
在实施例1中制备清酒乳杆菌ProBio-65(KCTC10755BP)的过程中将微生物细胞与脱脂奶粉混合并且冷冻干燥该混合物之前，将培养液离心，获得上清液并将其用于测试。
[抗病毒活性的鉴定]
将MDCK单层细胞接种在96孔细胞培养板上并使用磷酸盐缓冲液洗涤两次，将在-70℃储藏的病毒通过10倍连续稀释法稀释，并在各稀释步骤中接种在10个孔中，并在含有5%CO2的37℃温育器中培养72小时。此时，观察细胞病变效应。基于这些结果，检验培养液中病毒的含量。
在抗病毒活性测试中，使用不含牛血清的最低必需培养基稀释病毒培养基，使得病毒滴度达到1.0TCID50/0.1mL、10.0TCID50/0.1mL、100TCID50/0.1mL和1000TCID50/0.1mL。将MDCK单层细胞接种在96孔细胞培养板上，并使用磷酸盐缓冲溶液洗涤两次。对于每个清酒乳杆菌ProBio-65培养基样品使用4个孔。将90μl的1.0TCID50/0.1mL、10.0TCID50/0.1mL、100TCID50/0.1mL和1000TCID50/0.1mL分别添加至第一、第二、第三和第四孔，并将10μl(10%)清酒乳杆菌ProBio-65培养基立即添加至各孔，并在含有5%CO2的37℃细胞温育器中培养。在测试后24小时、48小时和72小时观察细胞病变效应。在观察到细胞病变效应的情形中，确定培养液不具有抗病毒活性。如图2中所示，使用清酒乳杆菌ProBio-65培养液处理的组未表现出细胞病变效应，这意味着培养液具有抗病毒活性。
[鸡胚蛋的培养和抗病毒活性的鉴定]
将鸡胚蛋在温育器中于37℃孵化9～11天。确定蛋壳的接种点，在鸡蛋的不存在血管的位置接种0.2ml病毒和清酒乳杆菌ProBio-65的混合物。在接种病毒和乳杆菌之后，在培养鸡蛋4～5天时观察到鸡胚的血管和壳风化(shell weathering)的存在。由鸡胚蛋收获尿囊液，并对尿囊液进行血凝反应，以鉴定病毒的增殖。血凝反应如下进行。通过2倍连续稀释法，使用25μl尿囊液和25μl磷酸盐缓冲液稀释各96孔培养板。将25μl分离自鸡的1%红细胞接种在稀释的96孔细胞培养板上，并在室温观察血凝反应40分钟。在鸡胚蛋测试过程中观察到血凝并且血细胞沉积在96孔细胞培 养板上的情形中，确定培养液不具有抗病毒活性。如图3所示，确定使用清酒乳杆菌ProBio-65培养基处理的组不具有抗病毒活性，因为血凝的物质未沉积在96孔细胞培养板上。
<实施例5>
稻米乳酸菌发酵食品组合物的抗病毒活性测试
以与实施例4中清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)的抗病毒活性测试相同的方式测试实施例1中制备的稻米乳酸菌发酵食品组合物。结果，获得了与实施例4中相同的结果。
<实施例6>
稻米乳酸菌发酵食品组合物的抗菌活性测试
以与实施例3中清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)的抗菌活性测试相同的方式测试实施例1中制备的稻米乳酸菌发酵食品组合物。获得的结果显示在表2中。如从表3和表4可以看出的，抗菌活性得到确认。
表3
虽然出于说明性目的公开了本发明的优选实施方式，但是本领域技术人员会意识到，可以进行各种修改、添加和取代，而不偏离所附权利要求中公开的本发明的范围和精神。
工业实用性
本发明提供一种稻米乳酸菌发酵食品组合物和一种包含它的功能健康食品，所述组合物通过将泡菜乳杆菌添加至稻米糖化液作为主要成分并使该混合物发酵而制备，并具有针对特应性皮炎和禽流感病毒的抗菌和抗病毒效果，由此具有工业实用性。
国际承认用于专利程序的微生物保藏布达佩斯条约
国际表格
原始保藏的接受
根据细则7.1
致：Probionic Corp
Bio Venture Center304,Korea Research Institute of Bioscience Biotechnology
#52.Qun-Dong.Yuseong-gu,Daejeon305-333
韩国
Form BP/4(KCTC From17) 单页
Claims (6)
1.一种抗病毒组合物，所述抗病毒组合物包含作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)或它的培养物，所述抗病毒组合物具有针对禽流感病毒的抗病毒效果。
2.一种稻米乳酸菌发酵食品组合物，所述稻米乳酸菌发酵食品组合物包含稻米乳酸菌发酵液，所述稻米乳酸菌发酵液通过将作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)添加至稻米糖化液并随后发酵而制备，所述稻米乳酸菌发酵食品组合物具有针对特应性皮炎的抗菌效果。
3.一种稻米乳酸菌发酵食品组合物，所述稻米乳酸菌发酵食品组合物包含稻米乳杆菌发酵液，所述稻米乳酸菌发酵液通过将作为提取自泡菜的乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)添加至稻米糖化液并随后发酵而制备，所述稻米乳酸菌发酵食品组合物具有针对禽流感病毒的抗病毒效果。
4.根据权利要求2或3所述的稻米乳酸菌发酵食品组合物，其中，所述稻米糖化液通过以下方式制备：称重稻米，将所述稻米浸入纯化水中，将所述稻米共混，并将所述共混物在100℃糊化20分钟～40分钟或者将所述共混物在60℃初步糊化20分钟～40分钟，然后将所述共混物在100℃主糊化20分钟～40分钟以制备糊化液，使用α-淀粉酶在80℃～100℃初步糖化所述糊化液40分钟～2小时并使用葡糖淀粉酶在60℃～75℃二次糖化所获得的液体40分钟～2小时，随后过滤。
5.一种制备如权利要求1或2所述的稻米乳酸菌发酵食品组合物的方法，所述方法包括：
糊化，所述糊化包括称重稻米，将所述稻米浸入纯化水中，将所述稻米共混，并将所述共混物在100℃糊化20分钟～40分钟或者将所述共混物在60℃初步糊化20分钟～40分钟，然后将所述共混物在100℃主糊化20分钟～40分钟以制备糊化液；
稻米糖化，所述稻米糖化包括使用α-淀粉酶在80℃～100℃初步糖化所述糊化液40分钟～2小时并使用葡糖淀粉酶在60℃～75℃二次糖化所获得的液体40分钟～2小时，随后过滤以制备稻米糖化液；
混合和杀菌，所述混合和杀菌包括将选自全脂奶粉、牛奶、脱脂大豆蛋白、低聚糖、pH调节剂、缓冲剂、糖、甜化剂(sweetenings)、果汁、维生素、甜味剂、氨基酸、核酸及其盐、植物纤维和矿物质中的至少一种添加剂添加至所述稻米糖化液，随后在100℃杀菌20分钟；
制备作为乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)，所述制备包括将陈泡菜液稀释，将所述泡菜稀释液涂在BCP固体培养基上，将所述泡菜稀释液在37℃培养48小时，将选定的球菌和杆菌在MRS培养基中培养，将所述球菌和杆菌在零下80℃储藏，分离清酒乳杆菌ProBio-65(KCTC10755BP)菌株并将所分离的ProBio-65菌株在37℃于MRS培养基中培养，以制备清酒乳杆菌ProBio-65菌；和
将所述乳酸菌发酵，所述发酵包括将作为所述乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)以相对于所述稻米糖化液的重量为0.1重量%～5重量%的量，添加至在所述混合和杀菌步骤中制备的所述混合杀菌稻米糖化液，随后在37℃发酵12小时，直至最终pH达到4.1。
6.如权利要求5所述的方法，其中，在作为所述乳酸菌的清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)的制备步骤中，所述清酒乳杆菌ProBio-65(保藏号：KCTC10755BP)菌株的培养和所述乳酸菌的制备通过以下方式进行：将所述清酒乳杆菌ProBio-65菌株在37℃于MRS培养基(含有葡萄糖2％、蛋白胨2％、酵母提取物0.5％、乙酸钠0.5％、磷酸氢二钾0.2％、硫酸铵0.2％、硫酸镁0.1％、硫酸锰0.005％和吐温80(Tween80)0.1％)中培养12小时，以13,000rpm将所述培养液离心3小时，将所获得的微生物细胞与水解脱脂奶粉等量混合，均化所述混合物，将所述混合物在-40℃初步冷冻，如下冷冻干燥所述混合物，即在-40℃至-20℃的升温下保持7小时然后在-20℃保持5小时，在-20℃至-5℃的升温下保持9小时然后在-5℃保持10小时，在-5℃至10℃的升温下保持10小时然后在10℃保持10小时，并且在10℃至20℃的升温下超过8小时，在20℃完成所述冷冻干燥，并研磨所述冷冻干燥的微生物细胞以制备清酒乳杆菌ProBio-65(KCTC10755BP)。
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- 2011-05-12 KR KR1020110044557A patent/KR101241385B1/ko active IP Right Grant
- 2011-05-12 JP JP2014510227A patent/JP2014516524A/ja active Pending
- 2011-05-12 WO PCT/KR2011/003522 patent/WO2012153885A1/ko active Application Filing
- 2011-05-12 CN CN201180070795.8A patent/CN103596579A/zh active Pending
- 2011-05-12 US US14/115,458 patent/US20140356338A1/en not_active Abandoned
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104351698A (zh) * | 2014-10-17 | 2015-02-18 | 北华大学 | 一种天女木兰香型党参泡菜及其制作方法 |
| CN104351698B (zh) * | 2014-10-17 | 2020-01-17 | 北华大学 | 一种天女木兰香型党参泡菜及其制作方法 |
| CN109310720A (zh) * | 2016-06-13 | 2019-02-05 | 株式会社村田制作所 | 抗菌抗病毒药物、抗菌抗病毒部件以及抗菌抗病毒药物的制造方法 |
| CN107969661A (zh) * | 2017-11-28 | 2018-05-01 | 海南鸿大海实业有限公司 | 一种酸芥菜食品及其制备方法 |
| CN107969661B (zh) * | 2017-11-28 | 2021-03-05 | 海南鸿大海实业有限公司 | 一种酸芥菜食品及其制备方法 |
| CN108324586A (zh) * | 2018-04-08 | 2018-07-27 | 北京皓尔生物科技有限公司 | 一种抗菌消炎组合物及其应用 |
| CN112858364A (zh) * | 2020-07-27 | 2021-05-28 | 苏州泰纽测试服务有限公司 | 一种利用核磁共振测量岩心物性的方法 |
| CN113750000A (zh) * | 2020-12-12 | 2021-12-07 | 广州元基生物科技有限公司 | 一种封层修护护肤品配方及其制备方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2012153885A1 (ko) | 2012-11-15 |
| WO2012153885A9 (ko) | 2013-03-21 |
| US20140356338A1 (en) | 2014-12-04 |
| JP2014516524A (ja) | 2014-07-17 |
| KR101241385B1 (ko) | 2013-03-11 |
| KR20120126619A (ko) | 2012-11-21 |
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