CN110249055A - 发酵物及其制造方法 - Google Patents
发酵物及其制造方法 Download PDFInfo
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- CN110249055A CN110249055A CN201780079994.2A CN201780079994A CN110249055A CN 110249055 A CN110249055 A CN 110249055A CN 201780079994 A CN201780079994 A CN 201780079994A CN 110249055 A CN110249055 A CN 110249055A
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- skin
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- fermentation
- honey
- improvement
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Abstract
本发明提供利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到的发酵物。
Description
技术领域
本发明涉及发酵物及其制造方法。
背景技术
皮肤、特别是表皮具有屏障功能是已知的。屏障功能是指防止从生物体内蒸散过量的水分、防止细菌等异物从体外侵入的功能。认为存在于表皮颗粒层的紧密连接(tightjuction)在皮肤的屏障功能中担负着重要作用。紧密连接不仅使相邻的细胞彼此密合，还将细胞与细胞的间隙密封而对物质透过进行控制。构成紧密连接的是作为细胞膜蛋白质的密封蛋白(claudin)、闭合蛋白(occludin)及紧密连接蛋白。认为这些蛋白质构成紧密连接束(tight junction strand)的骨架，控制紧密连接的屏障功能。专利文献1记载了含有植物提取物的密封蛋白生成促进剂及闭合蛋白生成促进剂。专利文献2记载了包含仙人掌果果实提取物(其是由属于仙人掌科仙人掌属的植物的果实而得到的)的皮肤屏障功能改善剂。
现有技术文献
专利文献
专利文献1：日本专利第5860577号公报
专利文献2：日本特开2012-121871号公报
发明内容
发明要解决的课题
专利文献1中记载了使用德国洋甘菊(Matricaria chamomilla L.)提取物来研究闭合蛋白的生成促进作用。然而，专利文献1公开了德国洋甘菊提取物几乎不具有闭合蛋白生成促进作用。
本发明的目的在于提供具有优异的皮肤改善作用的新型蜂蜜发酵物。
用于解决课题的手段
本申请的发明人新发现了使用昆氏乳杆菌(Lactobacillus kunkeei)将蜂蜜和德国洋甘菊或其处理物发酵而得到的发酵物具有优异的皮肤改善作用。
本发明提供利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到的发酵物。该发酵物具有优异的皮肤改善作用。
上述发酵物中，上述德国洋甘菊或其处理物优选为德国洋甘菊花的干燥粉末。通过使用德国洋甘菊花的干燥粉末，能够更为简便地得到具有更好的皮肤改善作用的发酵物。
上述发酵物中，上述蜂蜜与上述德国洋甘菊或其处理物的质量比优选为26:1～4:1。若质量比在上述范围，则可得到更好的皮肤改善作用。
上述发酵物适合用于选自由皮肤屏障功能改善用途、皮肤保湿用途、皮肤的弹力改善用途、皮肤的暗沉改善用途、皮肤的皱纹改善用途、肌肤粗糙改善用途、皮肤的抗老化用途、肌肤的肌理改善用途及敏感肌改善用途组成的组中的至少一者。
本发明还涵盖利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到的发酵物，所述发酵物用于选自由皮肤屏障功能改善、皮肤保湿、皮肤的弹力改善、皮肤的暗沉改善、皮肤的皱纹改善、肌肤粗糙改善、皮肤的抗老化、肌肤的肌理改善及敏感肌改善组成的组中的至少一者。
本发明涵盖选自由皮肤屏障功能改善方法、皮肤保湿方法、皮肤的弹力改善方法、皮肤的暗沉改善方法、皮肤的皱纹改善方法、肌肤粗糙改善方法、皮肤的抗老化方法、肌肤的肌理改善方法及敏感肌改善方法组成的组中的至少一者，上述方法包括将利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到的发酵物经口施予至需要所述发酵物的对象的步骤、或者将所述发酵物应用于该对象的皮肤的步骤。
本发明还涵盖利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到的发酵物在下述制剂的制造中的用途，所述制剂为选自由皮肤屏障功能改善剂、皮肤保湿剂、皮肤的弹力改善剂、皮肤的暗沉改善剂、皮肤的皱纹改善剂、肌肤粗糙改善剂、皮肤的抗老化剂、肌肤的肌理改善剂及敏感肌改善剂组成的组中的至少一者。
本发明提供包含上述发酵物的食品、化妆品、医药品或准医药品。
本发明还提供发酵物的制造方法，所述制造方法包括利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到发酵物的工序。利用该制造方法而得到的发酵物具有优异的皮肤改善作用。
上述制造方法中，德国洋甘菊或其处理物优选为德国洋甘菊花的干燥粉末。
上述制造方法中，上述蜂蜜与上述德国洋甘菊或其处理物的质量比优选为26:1～4:1。
利用上述制造方法而得到的发酵物适合用于选自由皮肤屏障功能改善用途、皮肤保湿用途、皮肤的弹力改善用途、皮肤的暗沉改善用途、皮肤的皱纹改善用途、肌肤粗糙改善用途、皮肤的抗老化用途、肌肤的肌理改善用途及敏感肌改善用途组成的组中的至少一者。
本发明还提供包括添加利用上述制造方法而得到的发酵物的工序的食品、化妆品、医药品或准医药品的制造方法。
发明效果
本发明的发酵物具有优异的皮肤改善作用。
具体实施方式
以下，对用于实施本发明的方式进行详细说明。但是，本发明不限于以下的实施方式。本说明书中，只要没有特别记载，则“％”是指“质量％”。
本实施方式涉及的发酵物是利用昆氏乳杆菌(Lactobacillus kunkeei)将蜂蜜、和德国洋甘菊或其处理物(以下，也称为“德国洋甘菊原料”)发酵而得到的。
蜂蜜是将蜜蜂从植物的花蜜、树液、寄生于植物的虫的分泌液等中收集的蜜作为主原料而制成的。本实施方式中，供于发酵的原料蜂蜜的种类没有特别限制，可使用例如麦卢卡蜂蜜(Manuka Honey)、洋槐蜜、甘露蜜、三叶草蜂蜜、橙蜜、紫云英蜜、迷迭香蜂蜜、向日葵蜂蜜、油菜花蜂蜜、咖啡蜂蜜等。蜂蜜可单独使用1种，也可合用2种以上。用于采集蜂蜜所利用的蜜蜂的种类、及蜂蜜的产地没有特别限定。
德国洋甘菊为菊科(Compositae)的植物，学名为Matricaria chamomilla L.。作为使用的德国洋甘菊的部位，没有特别限制，可进行适当选择，可举出例如叶、茎、根、花、花蕾、种子、果实、果皮、果核、地上部、全株、它们的混合物等。使用的德国洋甘菊的部位优选为花。
供于发酵的德国洋甘菊原料可为采集而得到的德国洋甘菊自身，也可为德国洋甘菊的处理物。作为德国洋甘菊处理物，可举出例如对德国洋甘菊进行了干燥、粉碎、提取等处理而得到的处理物。上述处理可单独实施1种，也可合用2种以上。
德国洋甘菊处理物可为例如德国洋甘菊的干燥物、粉碎物、破碎物、粉末、提取物等。就德国洋甘菊的干燥而言，可通过例如日晒干燥、热风干燥、冷冻干燥、喷雾干燥等进行。粉碎及破碎可分别使用例如粉碎机、破碎机等进行。德国洋甘菊原料优选为德国洋甘菊的干燥粉末。德国洋甘菊的干燥粉末可通过例如将德国洋甘菊干燥、然后进行粉碎而得到。德国洋甘菊优选在采集后立即进行干燥、粉碎。作为德国洋甘菊处理物，可使用市售品。
德国洋甘菊的提取物可利用在植物的提取中通常使用的方法从德国洋甘菊提取而得到。例如，向装满提取溶剂的处理槽中投入作为提取原料的德国洋甘菊的提取部位，一边根据需要进行适当搅拌、一边使可溶性成分溶出，然后进行过滤来除去提取残渣，从而得到提取液。
作为德国洋甘菊的提取物，可以直接使用从德国洋甘菊提取而得到的提取液，还可使用例如进一步进行了稀释、浓缩、干燥、过滤、纯化等处理的物质。
作为提取溶剂，例如可为水性溶剂、亲水性有机溶剂等。作为水性溶剂，可举出例如纯水、纯化水、自来水、井水、矿泉水、含矿物质水、温泉水、泉水、淡水、热水、离子交换水、生理盐水、磷酸缓冲液、磷酸缓冲生理盐水等。作为亲水性有机溶剂，可举出例如甲醇、乙醇、丙醇、异丙醇等碳原子数为1～5的低级醇；丙酮、甲基乙基酮等低级脂肪族酮；1,3-丁二醇、丙二醇、甘油等碳原子数为2～5的多元醇等。提取溶剂可单独使用1种，也可合用2种以上。
发酵可使用昆氏乳杆菌来进行。用于得到本实施方式涉及的发酵物的昆氏乳杆菌优选为针对葡萄糖及果糖显示同化性的昆氏乳杆菌，优选为针对葡萄糖、果糖、蔗糖、海藻糖及葡萄糖酸盐显示同化性的昆氏乳杆菌。昆氏乳杆菌可为昆氏乳杆菌BPS402。昆氏乳杆菌BPS402于2011年10月3日以保藏号FERM P-22177保藏于独立行政法人产业技术综合研究所专利生物保藏中心(日本国茨城县筑波市东1丁目1番地1中央第6(邮政编码305-8566))，可以获得。另外，该菌株目前已移交至国际保藏，保藏号为FERM BP-11439。
用于发酵的昆氏乳杆菌的培养可按照常规方法进行。培养基只要能够培养该菌即可，没有特别限制，可使用天然培养基、合成培养基、半合成培养基等。作为培养基，可使用含有氮源及碳源的培养基，作为氮源，可举出肉提取物、蛋白胨、酪蛋白、酵母提取物、谷蛋白(gluten)、大豆粉、大豆水解物、氨基酸等，作为碳源，可举出葡萄糖、乳糖、果糖、肌醇、山梨糖醇、糖浆、淀粉、曲汁、麦麸、甘蔗渣、糖蜜等。另外，可添加无机质(例如，硫酸铵、磷酸钾、氯化镁、食盐、碳酸钙、铁、锰、钼)、各种维生素类等。
培养温度例如可为4～45℃、优选为25～40℃、更优选为28～33℃。培养时间例如可为8～72小时。培养可进行通气振荡或通气搅拌而实施。培养基的pH例如可为4.0～9.0、优选为6.0～8.0。作为培养方法，可举出例如将菌体接种于MRS培养基、于30℃培养48小时。
培养昆氏乳杆菌而得到的菌液可直接接种于包含蜂蜜及德国洋甘菊原料的发酵培养基而用于发酵，也可用培养基等稀释而使用。
由昆氏乳杆菌进行的蜂蜜及德国洋甘菊原料的发酵例如可于4～45℃实施、优选于25～40℃实施。发酵时间可为例如12～60小时，优选为24～48小时。发酵开始时的含有蜂蜜的物质的pH例如可为3～8、优选为4～6。发酵还可进行通气振荡或通气搅拌而实施。
蜂蜜及德国洋甘菊原料的发酵可通过在包含蜂蜜及德国洋甘菊原料的发酵培养基中培养昆氏乳杆菌来进行。包含蜂蜜及德国洋甘菊原料的发酵培养基只要是昆氏乳杆菌能够进行发酵的形态即可，例如可为液体、膏(paste)、凝胶等形态。发酵培养基例如还可为使蜂蜜及德国洋甘菊原料溶解或悬浮于例如水等溶剂而得的培养基。
发酵开始时的发酵培养基中的蜂蜜浓度相对于发酵培养基总量而言例如可为0.1～40％，可为1～30％，也可为8～13％。另外，就发酵开始时的发酵培养基中的蜂蜜浓度而言，作为固态成分，相对于发酵培养基总量而言例如可为0.08～32％，可为0.8～24％，也可为6.4～10.4％。
就发酵开始时的发酵培养基中的德国洋甘菊原料浓度而言，作为固态成分，相对于发酵培养基总量而言，例如可为0.01～20％、可为0.1～5％、也可为0.5～2％。发酵开始时的发酵培养基中的蜂蜜(总量)与德国洋甘菊原料(干燥质量)的质量比例如可为4000:1～1:200、也可为26:1～4:1。发酵培养基还可进一步含有例如维生素类、氨基酸类、矿物质类、盐类、表面活性剂、脂肪酸、金属等对于乳酸菌的培养而言必需的已知营养素。用于发酵的蜂蜜还可通过加热等进行杀菌处理。
本实施方式涉及的发酵物可为利用发酵而得到的发酵物自身，也可为施以加热、过滤、纯化、浓缩、蒸发干燥、冷冻干燥、喷雾干燥等处理而得到的发酵物。本实施方式涉及的发酵物可包含昆氏乳杆菌的活菌体及/或死菌体，也可利用过滤等来分离除去菌体。就本实施方式涉及的发酵物而言，由于在发酵培养基中包含蜂蜜，因此能够期待与原料蜂蜜相比，维生素、氨基酸等成分增加，生成原料蜂蜜所不含的有机酸。
本实施方式涉及的发酵物具有促进选自由闭合蛋白、密封蛋白-1、密封蛋白-4、紧密连接蛋白-1、紧密连接蛋白-2、紧密连接蛋白-3、III型胶原蛋白α1链、IV型胶原蛋白、水孔蛋白(aquaporin)3、丝氨酸棕榈酰转移酶2及TIMP(组织金属蛋白酶抑制剂)2组成的组中的至少1种蛋白质的生成的作用。因此，本实施方式涉及的发酵物也适合用于闭合蛋白生成促进用途、密封蛋白-1生成促进用途、密封蛋白-4生成促进用途、紧密连接蛋白-1生成促进用途、紧密连接蛋白-2生成促进用途、紧密连接蛋白-3生成促进用途、III型胶原蛋白α1链生成促进用途、IV型胶原蛋白生成促进用途、水孔蛋白3生成促进用途、丝氨酸棕榈酰转移酶2生成促进用途及/或TIMP2生成促进用途。另外，上述发酵物还适合用于编码上述蛋白质的各基因的表达促进用途。
本实施方式涉及的发酵物具有抑制选自由白细胞介素-1α、白细胞介素-1β、环氧合酶2及基质金属蛋白酶-9组成的组中的至少1种蛋白质的生成的作用。因此，本实施方式涉及的发酵物也适合用于白细胞介素-1α生成抑制用途、白细胞介素-1β生成抑制用途、环氧合酶2生成抑制用途及/或基质金属蛋白酶-9生成抑制用途。另外，上述发酵物还适合用于编码上述蛋白质的各基因的表达抑制用途。
关于上述各种蛋白质，以下情况是已知的。闭合蛋白、密封蛋白-1、密封蛋白-4、紧密连接蛋白-1、紧密连接蛋白-2及紧密连接蛋白-3为紧密连接构成蛋白质，参与屏障功能。当紧密连接变得脆弱，会引起水分蒸散亢进、保湿成分及钙离子的流出、与角质层水分量有关的聚丝蛋白(filaggrin)及细胞间脂质的合成不良、角质肥厚等，从而导致干燥肌、敏感肌、肌肤粗糙、肌肤缺乏透明感、肌肤暗沉等。通过强化紧密连接，能够期待预防干燥肌、敏感肌、肌肤粗糙、及暗沉、提高透明感等效果。
III型胶原蛋白富含于胎儿的皮肤，担负着保持肌肤的弹性的作用。IV型胶原蛋白为存在于表皮和真皮之间的基底膜的主要构成成分。丝氨酸棕榈酰转移酶2为合成神经酰胺时的初始限速酶。TIMP2为MMP(基质金属蛋白酶)的抑制剂。
已知白细胞介素-1α及白细胞介素-1β促进免疫细胞的活化、引起炎症反应。特别是白细胞介素-1α大量存在于角质形成细胞内。已知环氧合酶2为参与炎症的前列腺素的合成酶，其表达因紫外线等而上升。环氧合酶2的生成降低会抑制作用于黑色素细胞的前列腺素的生成，从而对由紫外线引起的色斑的产生进行抑制。
报道了MMP-9分解基底膜构成蛋白质，其表达因紫外线而上升。通过防止MMP-9的增加，可抑制基底膜的破坏，从而可抑制松弛及皱纹的形成。另外，通过防止基底膜的破坏，可防止在表皮中生成的黑色素向真皮侧的移动，从而对不易消失的色斑的产生进行抑制。
因此，本实施方式涉及的发酵物的皮肤改善作用优异，具体而言，例如皮肤屏障功能改善作用、皮肤保湿作用、皮肤的弹力改善作用、皮肤的暗沉改善作用、皮肤的皱纹改善作用、肌肤粗糙改善作用、皮肤的抗老化作用、肌肤的肌理改善作用及敏感肌改善作用优异。因此，本实施方式涉及的发酵物适合用于例如选自由皮肤屏障功能改善用途、皮肤保湿用途、皮肤的弹力改善用途、皮肤的暗沉改善用途、皮肤的皱纹改善用途、肌肤粗糙改善用途、皮肤的抗老化用途、肌肤的肌理改善用途及敏感肌改善用途组成的组中的至少一者。皮肤的抗老化例如包括皮肤的皱纹、色斑、暗沉、松弛、粗糙等的预防或改善、肌理的改善等。
本发明的一个实施方式为利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到的发酵物，所述发酵物用于选自由皮肤屏障功能改善、皮肤保湿、皮肤的弹力改善、皮肤的暗沉改善、皮肤的皱纹改善、肌肤粗糙改善、皮肤的抗老化、肌肤的肌理改善及敏感肌改善组成的组中的至少一者。
本发明的一个实施方式为选自由皮肤屏障功能改善方法、皮肤保湿方法、皮肤的弹力改善方法、皮肤的暗沉改善方法、皮肤的皱纹改善方法、肌肤粗糙改善方法、皮肤的抗老化方法、肌肤的肌理改善方法及敏感肌改善方法组成的组中的至少一者，所述方法包括将利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到的发酵物经口给予需要所述发酵物的对象的步骤、或将所述发酵物应用于该对象的皮肤的步骤。
本发明的一个实施方式为利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到的发酵物在下述制剂的制造中的用途，所述制剂为选自由皮肤屏障功能改善剂、皮肤保湿剂、皮肤的弹力改善剂、皮肤的暗沉改善剂、皮肤的皱纹改善剂、肌肤粗糙改善剂、皮肤的抗老化剂、肌肤的肌理改善剂及敏感肌改善剂组成的组中的至少一者。
本实施方式涉及的发酵物可直接用作化妆品、医药品或准医药品，也可用作化妆品、医药品或准医药品的成分。化妆品、医药品或准医药品例如可为溶液、乳液、软膏、乳霜、膏(paste)、胶状物(jelly)、气雾剂、粉体、油、固态、泡沫等形态。上述发酵物可直接或通过滴落、散布、涂布等应用于皮肤，也可经口施予。本实施方式涉及的发酵物可为皮肤外用剂。作为上述化妆品或准医药品，可举出例如化妆水、洁面用品、乳液、乳霜、啫喱、精华液、美容液、涂抹式面膜、片状面膜、化妆油、软膏等基础化妆品；粉底、口红、腮红、眼影、眼线液(笔)、睫毛膏、散粉等彩妆化妆品、洁面用品、按摩剂、清洁剂、须后水、须前水、剃须膏、沐浴剂、身体乳、肥皂、洗发水、护发素、焗油膏、美黑乳液、防晒乳液、美黑霜、防晒霜、美黑啫喱、防晒啫喱、整发用品、毛发生长剂、生发剂、止汗剂、沐浴盐、含漱液、漱口水等。
将本实施方式涉及的发酵物用作化妆品、医药品或准医药品的成分的情况下，就发酵物的含量而言，例如在化妆品、医药品或准医药品中可为0.01～100％，优选为0.1～50％，更优选为0.3～3.0％、进一步优选为0.4～1.5％。
将本实施方式涉及的发酵物用作医药品或准医药品成分的情况下，就该医药品或准医药品而言，作为其他成分，还可包含例如药学上允许的成分(例如，赋形剂、粘结材料、润滑剂、崩解剂、乳化剂、表面活性剂、基剂、助溶剂、悬浮剂)。
医药品或准医药品可为固体、液体、膏等任意的形状，也可为片剂(包括未包衣片、糖衣片、泡腾片、薄膜包衣片、咀嚼片、含片等)、胶囊剂、丸剂、粉末剂(散剂)、细粒剂、颗粒剂、液剂、悬浮液、乳浊液、糖浆、糊剂、注射剂(包括使用时配合于蒸馏水或者氨基酸输注液或电解质输注液等输注液中而制备成液剂的情况)等剂型。上述各种制剂例如可通过将作为有效成分的上述发酵物和根据需要的其他成分混合并成型为上述剂型来制备。
本实施方式涉及的发酵物可直接作为食品使用，也可添加到食品中使用。通过摄取上述发酵物，从而能够期待上述的皮肤改善。作为食品，优选为突出了食品的第三功能(身体状况调节功能)的食品。作为突出了食品的第三功能的食品，可举出例如健康食品、功能性食品、营养组合物(nutritional composition)、营养辅助食品、补充剂、保健用食品、特定保健用食品、营养功能食品、或功能性标示食品等。
将上述发酵物添加于食品而使用的情况下，食品还可包含允许用作食品的成分。作为允许用作食品的成分，可举出例如矿物质类、维生素类、黄酮类、醌类、多酚类、氨基酸、核酸、必需脂肪酸、清凉剂、粘合剂、甜味剂、崩解剂、润滑剂、着色剂、香料、稳定剂、防腐剂、缓释调节剂、表面活性剂、溶解剂、润湿剂等。
作为食品，可举出以下食品，在上述制造工序中的中间制品、或最终制品中混合上述发酵物，从而可得到用于上述目的的食品：咖啡、果汁及茶饮料等清凉饮料、乳饮料、乳酸菌饮料、酸奶饮料、碳酸饮料、以及日本酒、洋酒、果酒及蜂蜜酒等酒等饮料；卡士达酱等涂抹酱；果泥等糊；巧克力、面包圈、馅饼(pie)、泡芙、口香糖、果冻(jelly)、糖果、曲奇、蛋糕及布丁等西点；糯米团、年糕、有馅面点、长崎蛋糕、豆沙水果凉粉及羊羹等日式点心；冰淇淋、冰棒及冰霜等冷点；咖哩、牛肉饭、粥、味噌汤、汤、肉酱、意大利面、腌渍品、果酱等预加工食品；调味汁、拌饭调味料、鲜味调味剂及汤料等调味料。
由本实施方式涉及的发酵物形成的食品、化妆品、医药品或准医药品、或者包含该发酵物的食品、化妆品、医药品或准医药品可用于选自由皮肤屏障功能改善用途、皮肤保湿用途、皮肤的弹力改善用途、皮肤的暗沉改善用途、皮肤的皱纹改善用途、肌肤粗糙改善用途、皮肤的抗老化用途、肌肤的肌理改善用途及敏感肌改善用途组成的组中的至少一者。另外，该食品、化妆品、医药品或准医药品可用于闭合蛋白生成促进用途、密封蛋白-1生成促进用途、密封蛋白-4生成促进用途、紧密连接蛋白-1生成促进用途、紧密连接蛋白-2生成促进用途、紧密连接蛋白-3生成促进用途、III型胶原蛋白α1链生成促进用途、IV型胶原蛋白生成促进用途、水孔蛋白3生成促进用途、丝氨酸棕榈酰转移酶2生成促进用途及/或TIMP2生成促进用途，也可用于编码上述蛋白质的基因的表达促进用途。另外，上述食品、化妆品、医药品或准医药品可用于白细胞介素-1α生成抑制用途、白细胞介素-1β生成抑制用途、环氧合酶2生成抑制用途及/或基质金属蛋白酶-9生成抑制用途，还可用于编码上述蛋白质的基因的表达抑制用途。
由本实施方式涉及的发酵物形成的、或包含该发酵物的食品、化妆品、医药品或准医药品还可标示下述内容：例如，紧密连接的改善；细胞旁途径的透过性改善；美肌；皮肤的保湿；防止皮肤弹力或张力的降低、皮肤干燥、肌肤粗糙、皱纹、老化；肌肤紧致；肌肤的肌理、暗沉的改善；敏感肌肤的改善；抗光老化。需要说明的是，抗光老化具体是指预防或改善由紫外线等光的影响而引起的色斑、皱纹等症状。
[实施例]
以下，基于实施例更具体地说明本发明。但是，本发明不限定于以下的实施例。
[蜂蜜及德国洋甘菊花粉末的发酵液的制备]
制备包含10％(w/w)的罗马尼亚产洋槐蜜(山田养蜂场株式会社)、1％(w/w)的德国洋甘菊花粉末的水溶液。将该水溶液于110℃处理5分钟进行杀菌，从而得到发酵前液。
将昆氏乳杆菌BPS402用MRS琼脂培养基于30℃静置培养48小时。从形成的菌落取一白金环，接种至发酵前液50mL，于30℃振荡培养24小时，从而得到预培养液。
在上述发酵基质含有液1000mL中接种1mL上述预培养液，于最适温度振荡培养24小时。将得到的发酵液于110℃处理5分钟进行杀菌后，过滤，添加苯氧乙醇使其成为0.5％的浓度，从而得到试验用的蜂蜜及德国洋甘菊花粉末的发酵液。
[DNA微阵列试验]
使用人皮肤三维模型试剂盒(EFT-412，仓敷纺绩株式会社制)进行DNA微阵列试验。依照实验方案，将皮肤模型移入6孔板，使用分析培养基，于37℃、5％CO2的条件下预培养一夜。预培养后，向角质层侧添加200μL上述发酵液，在真皮侧添加2.5ml分析培养基，持续培养3天。培养结束后，用PBS(-)洗涤皮肤组织，使用Derma punch(直径8mm)回收组织后，实施DNA微阵列分析。作为对照，使用添加了苯氧乙醇0.5％的纯化水，同样地进行分析。另外，利用MTT法评价由被检物质引起的对于皮肤三维模型的细胞毒性。
对42450个基因进行了全面分析，结果，与对照相比，经发酵液处理而表达上升至1.5倍以上的基因为3012个基因，表达减少至2分之1以下的基因为2826个基因。作为涉及皮肤功能的基因，如从其中提取了编码表1所示的15个蛋白质的基因。未确认到由上述发酵液引起的细胞毒性。
[表1]
如表1所示，就蜂蜜及德国洋甘菊花粉末发酵液而言，在编码紧密连接构成蛋白、胶原蛋白、水孔蛋白3、神经酰胺合成限速酶等与肌肤的水分保持或屏障功能的提高有关的蛋白质的基因中确认到较之纯化水而言为1.5倍以上的显著的表达上升。另一方面，对于编码参与炎症反应的白细胞介素1、报道了因紫外线而上升的环氧合酶2、及分解皮肤基底膜构成蛋白的MMP-9的各基因，可确认到蜂蜜及德国洋甘菊花粉末发酵液显著地抑制其表达。
[蛋白质生成促进试验]
作为被检物质，使用上述蜂蜜及德国洋甘菊花粉末发酵液、及10％蜂蜜水(罗马尼亚产洋槐蜜10％，纯化水90％)。进一步地，在以下的试验中使用将上述被检物质的浓度调节为1.0％或0.5％的水溶液。
＜闭合蛋白生成促进试验＞
将正常人新生儿表皮角质形成细胞(NHEK)使用正常人表皮角质形成细胞增殖培养基(KGM)进行培养后，通过胰蛋白酶处理来回收细胞。利用KGM将回收的细胞稀释为2×105个细胞/ml的浓度，然后接种于96孔板，每1个孔各接种100μL，培养一夜。培养结束后，在各孔中添加100μL用KGM溶解的被检试样，培养24小时。培养结束后，除去培养基，使细胞固定于板，通过使用了多克隆抗人闭合蛋白抗体的ELISA法测定在细胞表面表达的闭合蛋白的量。闭合蛋白生成促进率的计算方法如下所示。结果如表2所示。
闭合蛋白生成促进率(％)＝A/B×100
A:添加被检试样时的波长405nm处的吸光度
B:未添加被检试样时(对照)的波长405nm处的吸光度
[表2]
平均值±S.E.，n＝6，*：p＜0.05，***：p＜0.001
对于蜂蜜及德国洋甘菊花粉末发酵液而言，在0.5％及1.0％的浓度下确认到高的闭合蛋白生成促进作用。
＜密封蛋白-4生成促进试验＞
将正常人新生儿表皮角质形成细胞(NHEK)使用正常人表皮角质形成细胞增殖培养基(KGM)进行培养后，通过胰蛋白酶处理来回收细胞。利用KGM将回收的细胞稀释为2×105个细胞/ml的浓度，然后接种于96孔板，每1个孔各接种100μL，培养一夜。培养结束后，在各孔中添加100μL用KGM溶解的被检试样，培养24小时。培养结束后，除去培养基，使细胞固定于板，通过使用了单克隆抗人密封蛋白-4抗体的ELISA法测定在细胞表面表达的密封蛋白-4的量。密封蛋白-4生成促进率的计算方法如下所示。结果如表3所示。
密封蛋白-4生成促进率(％)＝A/B×100
A:添加被检试样时的波长405nm处的吸光度
B:未添加被检试样时(对照)的波长405nm处的吸光度
[表3]
平均值±S.E.，n＝6，*：p＜0.05，**：p＜0.01
对于蜂蜜及德国洋甘菊花粉末发酵液而言，在0.5％及1.0％的浓度下确认到密封蛋白-4生成促进作用。
＜紧密连接蛋白-2生成促进试验＞
将正常人新生儿表皮角质形成细胞(NHEK)使用正常人表皮角质形成细胞增殖培养基(KGM)进行培养后，通过胰蛋白酶处理来回收细胞。利用KGM将回收的细胞稀释为2×105个细胞/ml的浓度，然后接种于96孔板，每1个孔各接种100μL，培养一夜。培养结束后，在各孔中添加100μL用KGM溶解的被检试样，培养24小时。培养结束后，除去培养基，使细胞固定于板，通过使用了多克隆抗人ZO-2抗体的ELISA法测定在细胞表面表达的ZO-2的量。需要说明的是，紧密连接蛋白-2也被称作ZO-2。ZO-2生成促进率的计算方法如下所示。结果如表4所示。
ZO-2生成促进率(％)＝A/B×100
A：添加被检试样时的波长405nm处的吸光度
B：未添加被检试样时(对照)的波长405nm处的吸光度
[表4]
平均值±S.E.，n＝6，*：p＜0.05
对于蜂蜜及德国洋甘菊花粉末发酵液而言，在0.5％的浓度下确认到ZO-2生成促进作用。
如上文所述，对于蜂蜜及德国洋甘菊花粉末发酵液而言，在0.5％及1.0％的浓度下确认到闭合蛋白及密封蛋白-4的生成促进作用，在0.5％浓度下确认到ZO-2生成促进作用。另一方面，对于10％蜂蜜水而言，虽然在1.0％的浓度下使闭合蛋白生成增加，但对于密封蛋白-4及ZO-2的生成量未造成影响。
Claims (10)
1.发酵物，其是利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到的。
2.如权利要求1所述的发酵物，其中，所述德国洋甘菊或其处理物为德国洋甘菊花的干燥粉末。
3.如权利要求1或2所述的发酵物，其中，所述蜂蜜与所述德国洋甘菊或其处理物的质量比为26:1～4:1。
4.如权利要求1～3中任一项所述的发酵物，其用于选自由皮肤屏障功能改善用途、皮肤保湿用途、皮肤的弹力改善用途、皮肤的暗沉改善用途、皮肤的皱纹改善用途、肌肤粗糙改善用途、皮肤的抗老化用途、肌肤的肌理改善用途及敏感肌改善用途组成的组中的至少一者。
5.食品、化妆品、医药品或准医药品，其包含权利要求1～4中任一项所述的发酵物。
6.发酵物的制造方法，其包括利用昆氏乳杆菌将蜂蜜和德国洋甘菊或其处理物发酵而得到发酵物的工序。
7.如权利要求6所述的制造方法，其中，所述德国洋甘菊或其处理物为德国洋甘菊花的干燥粉末。
8.如权利要求6或7所述的制造方法，其中，所述蜂蜜与所述德国洋甘菊或其处理物的质量比为26:1～4:1。
9.如权利要求6～8中任一项所述的制造方法，其中，所述发酵物用于选自由皮肤屏障功能改善用途、皮肤保湿用途、皮肤的弹力改善用途、皮肤的暗沉改善用途、皮肤的皱纹改善用途、肌肤粗糙改善用途、皮肤的抗老化用途、肌肤的肌理改善用途及敏感肌改善用途组成的组中的至少一者。
10.食品、化妆品、医药品或准医药品的制造方法，其包括添加利用权利要求6～9中任一项所述的制造方法而得到的发酵物的工序。
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- 2017-12-21 CN CN201780079994.2A patent/CN110249055B/zh active Active
- 2017-12-21 WO PCT/JP2017/046026 patent/WO2018123828A1/ja unknown
- 2017-12-21 US US16/474,050 patent/US10933101B2/en active Active
- 2017-12-21 ES ES17886586T patent/ES2923931T3/es active Active
- 2017-12-21 JP JP2018559132A patent/JP6888835B2/ja active Active
- 2017-12-21 EP EP17886586.1A patent/EP3564382B1/en active Active
- 2017-12-28 TW TW106146186A patent/TW201828919A/zh unknown
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Also Published As
| Publication number | Publication date |
|---|---|
| ES2923931T3 (es) | 2022-10-03 |
| WO2018123828A1 (ja) | 2018-07-05 |
| EP3564382A4 (en) | 2020-09-02 |
| CN110249055B (zh) | 2023-01-03 |
| EP3564382B1 (en) | 2022-06-29 |
| US20190365828A1 (en) | 2019-12-05 |
| TW201828919A (zh) | 2018-08-16 |
| EP3564382A1 (en) | 2019-11-06 |
| JP6888835B2 (ja) | 2021-06-16 |
| JPWO2018123828A1 (ja) | 2019-10-31 |
| US10933101B2 (en) | 2021-03-02 |
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