CN109415422B - 超低分子角蛋白肽的制备方法及其的利用 - Google Patents
超低分子角蛋白肽的制备方法及其的利用 Download PDFInfo
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- CN109415422B CN109415422B CN201680062991.3A CN201680062991A CN109415422B CN 109415422 B CN109415422 B CN 109415422B CN 201680062991 A CN201680062991 A CN 201680062991A CN 109415422 B CN109415422 B CN 109415422B
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Abstract
本发明涉及超低分子角蛋白肽的制备方法及其的利用。上述超低分子角蛋白肽的制备方法利用包含角蛋白的培养基中的具有角蛋白分解活性的微生物的培养、超滤法、离子交换色谱法及凝胶过滤色谱法。根据本发明的角蛋白肽制备方法,可实现废弃资源的环保生物学处理及抗老化功能性超低分子角蛋白肽的有效的纯化及回收。并且,本发明的角蛋白肽通过分解胶原蛋白,来具有作为引起皮肤老化的酶的基质金属蛋白酶‑1的表达抑制能力和活性抑制能力,防止皮肤老化及改善皮肤皱纹的效果优秀,对皮肤细胞无毒性,从而适合用作皮肤皱纹的预防、改善或治疗用化妆品、药学、食品组合物,从而可有用地适用于高附加值功能性化妆品材料的有效且快速的制备及开发。
Description
技术领域
本发明涉及超低分子角蛋白肽的制备方法及其的利用。具体地,本发明涉及利用包含角蛋白的培养基中的具有角蛋白分解活性的微生物的培养、超滤、离子交换色谱及凝胶过滤色谱的超低分子角蛋白肽制备方法,以及通过上述方法制备而成的肽及包含其的皮肤老化或皮肤皱纹预防或改善用化妆品、食品组合物。
背景技术
随着男女老少均对具有弹力且美丽的皮肤的关注度增加,在化妆品市场中,从现有的以健康为中心的化妆品越来越趋向可满足对美丽的渴望且有助于皱纹改善的功能性材料的发掘。尤其,角蛋白肽作为形成毛发、指甲、皮肤等形成上皮结构的基本的天然的氨基酸聚合物(50个以下氨基酸),由于与生物体内蛋白质的相互作用非常优秀,因此具有多种生理活性。
最近,因化妆品制备技术的均衡化,含有差别化的天然成分的新材料开发在化妆品产业中成为核心的话题,从而对作为天然材料的角蛋白肽的功能性高度关注。但是,在韩国国内,因对全量进口的功能性肽的原料的价格负担(每1kg为2亿韩元~30亿韩元)功能性肽化妆品开发非常慢,大部分情况下即使含有肽,也只不过含有极少量。
在以人类或家畜的疾病治疗为目的所使用的医疗用肽(包括疫苗)的情况下,尽管是极微量,但大部分都是高价产品。即使是功效非常优秀的肽,也大部分为大小由15个以上的氨基酸组成的聚合物(分子量为3kDa以上),从而皮肤角质渗透性向细胞内部的吸收不容易。因此,在肽新材料开发中最核心的要素为1)肽的功效、2)肽的大小(分子量)、3)确定生产成本的选择性分离及高纯度纯化技术。
当前,为了产业上利用的低分子肽的分离及制备,依赖于超滤膜,在高价的医药用肽的情况下,通过复杂的色谱法进行纯粹分离而使用。通常,为了对通过合成方法或重组方法生产的高分子进行纯化,如肽及蛋白质等,典型地,为利用疏水性相互作用的反相液相色谱法(“RP-LC”:reverse phase liquid chromatography)及反相高效液相色谱法(“RP-HPLC”:reverse phase high-performance liquid chromatography)。
反相液相色谱法及反相高效液相色谱法可有效地分离密切相关的杂质,并且可对多个多种分子进行纯化,尤其,利用为用于制备产业规模的蛋白质的大量纯化工序。尤其,为了纯化肽蛋白纯化,在反相色谱法中,作为媒质最普遍使用附着于二氧化硅表面的C-4、C-8及C-18烷基链。在这种反相液相色谱法中纯化的核心为固定相的树脂粒子的形状、大小、缓冲液类、流量、pH等。在蛋白质纯化中,虽然进行这种技术改善,但是在一部分经纯化的蛋白质中仍然存在相当量的杂质(比如,具有相反离子)。尤其,为了开发不是医药用的功能性化妆品材料,利用如上所述的高价的设备的分离方法在经济性及生产性方面非常低,从而在实际产业的适用中存在其局限。因此,为了开发来源于天然的功能性化妆品肽新材料,非常需要可有效且迅速地取得具有功效的功能性超低分子肽,同时需要经济、简单的色谱纯化工序的开发。
发明内容
对此,本发明人锐意努力开发从难分解性角蛋白分解产物可回收、分离具有皮肤抗老化功效的功能性角蛋白肽的工序,并开发纯化工序,选择性地对无需传递***可有效且自由地渗透皮肤角质层和细胞膜的水平的超低分子肽进行分离。
因此,本发明的一实施方式提供用于制备皮肤老化或皮肤皱纹预防用角蛋白肽的方法,在包含角蛋白的培养基中,培养具有角蛋白分解活性的微生物,并利用超滤法、离子交换色谱法及凝胶过滤色谱法。
并且,本发明的一实施方式提供由上述方法制备而成的角蛋白肽及包含其的皮肤老化或皮肤皱纹预防或改善用化妆品组合物、食品组合物。
并且,本发明的一实施方式提供由选自由序列1至序列9组成的组中的一种以上的序列表示的肽及包含上述肽的皮肤老化或皮肤皱纹预防或改善用化妆品组合物、食品组合物。
本发明的一实施方式提供皮肤老化或皮肤皱纹预防用角蛋白肽混合物的制备方法,上述皮肤老化或皮肤皱纹预防用角蛋白肽混合物的制备方法包括:步骤(1),在包含角蛋白的培养基中,通过培养具有角蛋白分解活性的微生物,来取得角蛋白水解产物;步骤(2),在从上述步骤(1)取得的角蛋白水解产物中分馏分子量为100Da至1000Da的蛋白质;步骤(3),通过对从上述步骤(2)取得的分馏产物进行离子交换色谱、凝胶过滤色谱或其的组合来纯化蛋白质;以及步骤(4),通过对从步骤(3)取得的经纯化的蛋白质进行凝胶过滤色谱,来对蛋白质进行纯化及脱盐。
以下详细说明本发明。
本发明的发明人在作为营养源来添加难分解性家禽类羽毛的最小限度培养基中,通过超高温厌氧发酵有效取得角蛋白分解产物,从而大量获得具有皮肤抗老化功效的角蛋白肽。并且,通过使用超滤(ultrafiltration),来对水解产物来分子量进行分馏,从而大量回收超低分子(1kDa以下)角蛋白肽。并且,利用离子交换色谱及凝胶过滤色谱对这种超低分子角蛋白肽进行分离及纯化,进而,通过简化纯化步骤,来仅利用凝胶过滤色谱法分离及纯化超低分子角蛋白肽。并且,确认这种肽不呈现细胞毒性,并具有基质金属蛋白酶-1(MMP-1)的活性抑制能力和表达抑制能力,这些肽具有抗老化功能性,皮肤角质渗透性优秀,因而确认可利用于皮肤皱纹或皮肤老化的预防或改善。
以下,对本发明的步骤进行详细说明。
本发明的步骤(1)为如下步骤:在包含角蛋白的培养基中,通过培养具有角蛋白分解活性的微生物,来取得角蛋白水解产物。
在本发明中,“具有角蛋白分解活性的微生物”无限制地包含具有可分解角蛋白的活性的微生物,优选地,可以为超高温微生物。
在本发明中使用的术语“超高温微生物”是指作为极端生物(extremophile)的一种,在相对高的温度,即,在约60℃以上中具有最优生育温度的微生物,并不限制其种类。优选地,可以为闪烁杆菌属(Fervidobacterium)。作为上述闪烁杆菌属可以为岛青霉素(islandicum)、青霉普兰(pennivorans)、长白昆(changbaicum)、坤德瓦斯(gondwanense)种。最优选地,可利用海岛闪烁杆菌(Fervidobacterium islandicum)AW-1(KCTC4680)。
在本发明中使用的术语“角蛋白(keratin)”作为结构蛋白的一种,是形成细胞骨架的主要组成成分。角蛋白根据半胱氨酸含量区分为软角蛋白(soft keratin)和硬角蛋白(hard keratin)。软角蛋白的半胱氨酸为约10~14%,是二硫化(disulfide)键多的头发、指甲、羊毛、鸡毛等。角蛋白的α-螺旋(α-角蛋白)或β-折叠(β-角蛋白)的结构相互缠绞,从而蛋白链非常坚固地堆积,在聚肽物之间存在很多二硫键、氢键及疏水性相互作用,从而非常稳定,并对蛋白分解酶的抵抗性大。
本发明的角蛋白可来源于鸟类或哺乳类的羽毛、毛发、皮革、指甲、爪、角或蹄。优选地,可在培养基中包含鸟类的羽毛来利用。
优选地,在培养基中包含5~15g/L的上述鸟类的羽毛,更优选地,包含8g/L。在以上述的范围包含其含量的情况下,可在1~2日内实现通过厌氧培养的角蛋白肽的生产,并更有利于取得适当的大小的功能性角蛋白肽。
上述培养可以为60℃以上的高温培养。上述培养温度无其限制,优选地,可以为60℃至90℃。
上述步骤(1)的培养可以为厌氧培养。并不特别限制上述厌氧培养的方式,可以为厌氧静置培养。
在本发明的具体实施例中,在具有表1的组成的生育培养基中,作为氮源添加8g/L的鸡毛,在70℃的温度下,对通过海岛闪烁杆菌AW-1进行厌氧培养取得的角蛋白肽水解产物进行过滤。接着,对经过滤的上述角蛋白肽水解产物以10000×g进行离心分离,来沉淀回收菌体,并进行过滤后回收上清液。
本发明的步骤(2)为如下步骤:在从上述步骤(1)取得的角蛋白水解产物中分馏分子量为100Da至1000Da的蛋白质。
上述步骤(2)可通过超滤进行,在本发明中,用于超滤的超滤膜的细孔(pore)大小可以为10kDa至1kDa,优选地,最终可利用1kDa的过滤膜。通过使用如上所述的过滤膜,可取得残留有1kDa以下的肽的角蛋白肽分馏产物。
本发明的步骤(3)为如下步骤:通过对从上述步骤(2)取得的分馏产物进行离子交换色谱、凝胶过滤色谱或其的组合来纯化蛋白质。
通过对在上述步骤(2)中进行过滤并经洗脱的1kDa以下的大小的蛋白质产物进行离子交换色谱或凝胶过滤色谱或其的组合来纯化蛋白质。
在上述离子交换色谱(Ion Exchange Chromatography)中,作为树脂可使用交联的聚苯乙烯(cross-linked polystrene)或葡聚糖(dextran)。并不限制上述树脂的微珠大小,但是可以为20μm至100μm,优选地,可以为34μm至50μm。
在本发明的具体实施例中,离子交换色谱包括:步骤a),向离子交换色谱柱填充由缓冲液中的50mM浓度的三(羟甲基)氨基甲烷(Tris-HCl)(pH 7.5)得到平衡化的琼脂糖或基于葡聚糖的聚合物树脂;步骤b),使角蛋白肽试样以每分钟(minute)约1ml以下的流量进入上述柱相;步骤c),利用与在上述a)步骤中相同的缓冲液对上述柱进行清洗;以及步骤d),通过实施 0 M至1 M 浓度的食盐(Sodium chloride)的线性梯度,洗脱从上述柱得到纯化的产物。
并不限制其缓冲液的种类,优选地,可利用三羟甲基氨基甲烷(Tris)溶剂。并且,缓冲液可具有20mM至50mM的浓度。
在本发明的具体实施例中,凝胶过滤色谱包括:步骤a),向离子交换色谱柱填充由缓冲液中的0.1摩尔浓度的食盐(Sodium chloride)得到平衡化的琼脂糖或基于葡聚糖的聚合物树脂;步骤b),使角蛋白肽试样以每分钟(minute)约0.7ml以下的流量进入上述柱相;步骤c),利用与在上述a)步骤中相同的缓冲液对上述柱进行清洗;以及步骤d),利用与上述步骤a)相同的缓冲液,洗脱从上述柱得到纯化的产物。
并不限制其缓冲液的种类,优选地,可利用三羟甲基氨基甲烷(Tris)溶剂。并且,缓冲液可具有20mM至50mM的摩尔浓度。
本发明的步骤(4)为如下步骤:对从步骤(3)取得的经纯化的蛋白质进行凝胶过滤色谱,来对蛋白质进行纯化及脱盐。
在本发明的具体实施例中,实施如下步骤:步骤a),向离子交换色谱柱填充由缓冲液中的水(water)得到平衡化的琼脂糖或基于葡聚糖的聚合物树脂;步骤b),使角蛋白肽试样以每分钟(minute)约0.5ml以下的流量进入上述柱相;步骤c),利用与上述步骤a)相同的缓冲液对上述柱进行清洗;以及步骤d),利用与上述步骤a)相同的缓冲液,对从上述柱得到纯化的产物进行洗脱。
在本发明中“脱盐”为去除包含于试样中的盐类,并不限制其方式,可使用离子交换法、排阻色谱、透析或凝胶过滤液体色谱等。在本发明中,为了完全去除有可能包含于试样的盐类,最终使用利用水的凝胶过滤液体色谱法。
并且,本发明的另一实施方式提供通过上述方法生产的角蛋白肽混合物。
在本发明中,角蛋白肽混合物以均包含通过上述方法取得的角蛋白肽的集合或由此可鉴定的一个以上的角蛋白肽的意义来使用。
在本发明中生产的角蛋白肽通过分解胶原蛋白,来具有作为引起皮肤老化的酶的基质金属蛋白酶-1的表达抑制能力,并不具有细胞毒性,从而适合于用作皮肤老化、皮肤皱纹的预防、改善或治疗用化妆品、药学、食品组合物。
尤其,本发明的角蛋白肽作为分子量为100Da至1000Da的角蛋白肽,是超低分子肽,因而具有皮肤角质渗透性优秀的特征。
并且,本发明的另一实施方式提供包含上述角蛋白肽混合物的皮肤老化或皮肤皱纹预防或改善用化妆品组合物。
本发明的角蛋白肽具有基质金属蛋白酶-1的表达抑制能力,因而呈现皮肤老化或皮肤皱纹预防效果。
本发明的角蛋白肽具有基质金属蛋白酶-1的活性抑制能力,因而呈现皮肤老化或皮肤皱纹预防效果。
本发明的角蛋白肽具有预防由紫外线诱发的皮肤老化或皮肤皱纹的效果。
优选地,相对于化妆品组合物的总重量,包含0.001~50重量百分比的本发明的角蛋白肽,但并不限定于此。
除了上述有效成分之外,本发明的化妆品组合物还可包含通常的辅助剂及载体,如通常使用的抗氧化剂,稳定剂,增溶剂,维生素,颜料,香料等。例如,上述化妆品组合物还可包含辅助成分,如甘油,丁二醇,聚氧乙烯氢化蓖麻油,醋酸生育酚,柠檬酸,泛醇,角鲨烷,柠檬酸钠、尿囊素等。
本发明的化妆品组合物基本涂敷于皮肤,因而可参照本发明所属领域中的化妆品组合物,来制备成通常制备的任何剂型。例如,可剂型化为溶液、悬浮液、乳浊液、糊剂、凝胶、乳霜、乳液、粉、肥皂、含有表面活性剂的洁面剂、油、粉状粉底、乳浊液粉底、蜡粉底及喷雾剂等,但不局限于此。更详细地,可制备成柔肤化妆水、营养化妆水、营养霜、按摩霜、精华素、眼霜、洁面霜、洗面奶、卸妆水、面膜、喷雾剂或粉的剂型。
在本发明的剂型为糊剂、乳霜或凝胶的情况下,可利用动物性油、植物性油、蜡、石蜡、淀粉、胺黄树胶、纤维素衍生物、聚乙二醇、硅、膨润土、二氧化硅、滑石或氧化锌等作为载体成分。
在本发明的剂型为粉或喷雾剂的情况下,可包含乳糖、滑石、二氧化硅、氢氧化铝、硅酸钙或聚酰胺粉作为载体成分,尤其,在喷雾剂的情况下,还可包含氯氟烃、丙烷/丁烷或二甲醚等的推进剂。
在本发明的剂型为溶液或乳浊液的情况下,可包含溶剂、增溶剂或乳化剂作为载体成分,具体地,可包含水、乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、丙二醇、1,3-丁基乙二醇油、甘油脂肪酸酯、聚乙二醇、山梨糖醇的脂肪酸酯。
在本发明的剂型为悬浮液的情况下,作为载体成分可包含液体稀释剂,如水、乙醇、丙二醇等;悬浮剂,如乙氧基化异硬脂醇,聚氧乙烯山梨醇酯、聚氧乙烯山梨糖醇酐酯等;微晶纤维素、偏氢氧化铝、膨润土、琼脂、黄蓍胶等。
在本发明的剂型为含有表面活性剂的洁面剂的情况下,可包含脂肪醇硫酸盐、脂肪醇醚硫酸盐、磺基琥珀酸单酯、羟乙基磺酸盐、咪唑啉衍生物、甲基牛磺酸盐、肌氨酸盐、脂肪酸酰胺醚硫酸盐、烷基酰胺甜菜碱、脂肪醇、脂肪酸甘油酯、脂肪酸二乙醇酰胺、植物性油、羊毛脂衍生物或乙氧基化甘油脂肪酸酯等作为载体成分。
并且,本发明的另一实施方式提供包含上述角蛋白肽化合物的皮肤老化或皮肤皱纹预防或改善用食品组合物。
在本发明的角蛋白肽用作食品添加物的情况下,可按照通常的方法适当地使用,如可直接添加上述角蛋白肽,或与其他食品或食品成分混合而使用等。
并且,作为有效成分的上述角蛋白肽的混合量可根据使用目的(预防、保健或治疗处置)适当地进行变更,相对于食品组合物重量可包含0.001~50重量百分比的上述角蛋白肽,但并不限定于此。
作为具体例,当制备食品或饮料的情况下,相对于原料添加15重量百分比以下的量,优选地,添加10重量百分比以下的量。但是将保健及卫生为目的或保健调节为目的长时间摄取的情况下,上述量可以为上述范围以下,在安全性方面不存在任何问题,因而有效成分还可使用上述范围以上的量。
上述食品的种类并不特别限制。作为可添加本发明的角蛋白肽的食品的例有包含肉类、香肠、面包、巧克力、糖类、快餐类、饼干类、比萨、方便面、其他面类、口香糖类、冰淇淋类的乳制品、各种汤、饮料、茶、清凉剂、酒精饮料及维生素复合剂等,并且均包含通常的意义上的健康食品。
在本发明的食品组合物制备成饮料的情况下,如通常的饮料可包含各种香味剂或天然碳水化物等追加成分。作为上述天然碳水化合物可使用单糖,如葡萄糖、果糖等;二糖,如麦芽糖、蔗糖等;天然甜味剂,如糊精、环糊精等;或合成甜味剂,如糖精,阿斯巴甜等。相对于本发明的食品组合物总重量,包含0.01~10重量百分比的上述天然碳水化物,优选地,包含0.01~0.1重量百分比。
除上述之外,本发明的食品组合物可包含各种营养剂、维生素、电解质、风味剂、着色剂、果胶酸及其盐、藻酸及其盐、有机酸、保护胶体增稠剂、pH调节剂、稳定剂、防腐剂、甘油、乙醇、适用于碳酸饮料的碳酸化剂等。另外,本发明的组合物可以包含用于制备天然果汁,果汁饮料及蔬菜饮料的果肉。这些成分可独立使用或组合使用。上述添加剂的比例没有特别限制,但是优选地,相对于本发明的食品组合物的总重量,包含在0.01〜0.1重量百分比范围内。
并且,本发明可提供包含上述角蛋白肽混合物作为有效成分的皮肤老化或皮肤皱纹预防或治疗用药学组合物。
包含于本发明的皮肤老化或皮肤皱纹预防或治疗用药学组合物的角蛋白肽的含量可根据治疗剂的使用方法、服用人的状态、疾病的种类及重症程度适当地调节。优选地在本发明的药学组合物中,包含0.001~50重量百分比的角蛋白肽,但并不必须限定于此。
本发明的组合物还可包含通常在制备药学组合物中所使用的适当的载体、赋形剂及稀释剂。并且,根据通常的方法可剂型化为口服型剂型,如散剂、颗粒剂、片剂、胶囊剂、悬浮液、乳剂、糖浆、气雾剂等,外用剂、坐剂、及灭菌注射溶液的形态来使用。优选地,在本发明所属技术领域中众所周知的适当的制剂使用在文献(Remington's PharmaceuticalScience,最近,Mack Publishing Company, Easton PA)中公开的制剂。作为可包含的载体、赋形剂及稀释剂有乳糖、右旋糖、蔗糖、山梨糖醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、***胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、羟苯甲酯、羟苯丙酯、滑石、硬脂酸镁、矿物油等。在对上述组合物制剂化的情况下,通过使用的稀释剂或赋形剂来配制,如填充剂、增量剂、粘合剂、润湿剂、崩解剂、表面活性剂等。作为用于口服给药的固体制剂包含片剂、丸剂、散剂、颗粒剂、胶囊剂等。这种固体制剂通过在上述混合物中混合至少一种以上的赋形剂来配制,如淀粉、碳酸钙(calciumcarbonate)、蔗糖、乳糖、明胶等。并且,除了简单的赋形剂之外,还使用硬脂酸镁、滑石等润滑剂。作为用于口服的液体制剂相应的有悬浮剂,内用液剂,乳剂、糖浆等,除了作为通常所使用的稀释剂的水、液体石蜡之外,可包含各种赋形剂如润湿剂,甜味剂,芳香剂和保鲜剂。作为用于非口服给药的制剂包括灭菌的水溶液,非水溶性溶剂,悬浮剂,乳剂,冷冻干燥制剂,坐剂等。作为非水性溶剂、悬浮剂可使用植物油,如丙二醇(propylene glycol),聚乙二醇、橄榄油等;酯,如可进行注射的油酸乙酯等。作为坐剂的基质可使用合成脂肪酸酯(witepsol)、聚乙二醇、吐温61、可可脂、月桂酸甘油酯、甘油明胶等。
在本发明中使用的术语“给药”是指以任意适当的方法向个体提供规定的本发明的组合物。
本发明的药学组合物能够以由研究人员、兽医、医师或其他通过临床想到的、在组织类、动物或人中诱导生物学或医学反应的有效成分或药学组合物的量、即,作为诱导所治疗的疾病或障碍的缓解的量的治疗上的有效量给药。对于本发明的药学组合物的治疗上的有效给药量及给药次数根据所需的效果变化,这是理所当然的。因而,可根据本发明所属技术领域的普通技术人员容易确定所给药的最优给药量,可根据疾病的种类、疾病的重症度、包含于组合物中的有效成分及其他成分的含量、剂型的种类及患者的年龄、体重、一般健康状态,性别、饮食、给药时间、给药途径、组合物的分泌率、治疗期间,包括同时使用的药物的多种因子进行调节。为了优选的效果,本发明的药学组合物能够以1~10000mg/kg/天(day)的量进行给药,优选地,能够以1mg/kg/天至200mg/kg/天的量给药,可以一天给药一次,还可分数次进行给药。
本发明的药学组合物可通过多种途径给药至个体。可预计给药的全部方式,例如可通过口服、直肠或静脉、肌肉,皮下、子宫内硬膜或脑血管内注射给药。
并且,本发明的另一实施方式提供由选自由序列1至序列9组成的组中的一种以上的序列表示的肽。
由上述序列1至序列9表示的肽为对通过基于本发明的角蛋白肽的制备方法制备的、具有抗老化功能的角蛋白肽,通过液相色谱串联质谱法(LC-MS/MS)进行鉴定而合成的氨基酸序列。更具体地,上述肽可利用作为靶物质的角蛋白的序列和可分解上述角蛋白的菌株,例如,利用基于AW-1菌株的角蛋白分解酶及肽的切割部位构建的数据库取得,在上述数据库中,通过进行液相色谱串联质谱法,来鉴定肽,从而以所取得的序列为基础,可利用本领域的普通技术合成。上述肽具有1kDa以下的超低分子量,并呈现抑制由紫外线B诱导的基质金属蛋白酶-1的表达的效果及抑制基质金属蛋白酶-1的活性的效果,因而可有效地使用于皮肤皱纹的预防或改善。
并且,本发明的另一实施方式提供皮肤老化或皮肤皱纹预防或改善用化妆品组合物,上述皮肤老化或皮肤皱纹预防或改善用化妆品组合物包含由选自由序列1至序列9组成的组中的一种以上的序列表示的肽。
并且,本发明的另一实施方式提供皮肤老化或皮肤皱纹预防或改善用食品组合物,上述皮肤老化或皮肤皱纹预防或改善用食品组合物包含由选自由序列1至序列9组成的组中的一种以上的序列表示的肽。
根据本发明的角蛋白肽制备方法,可实现废弃资源的环保生物学处理及抗老化功能性超低分子角蛋白肽的有效的纯化及回收。并且,本发明的角蛋白肽通过分解胶原蛋白,来具有作为引起皮肤老化的酶的基质金属蛋白酶-1的表达抑制能力和活性抑制能力,皮肤老化防止及皮肤皱纹改善效果优秀,对皮肤细胞无毒性,从而适合用作皮肤皱纹的预防、改善或治疗用化妆品、药学食品组合物,从而可有用地适用于高附加值功能性化妆品材料的有效且迅速的制备及开发。
附图说明
图1为角蛋白肽制备方法的简图。
图2为表示1kDa以下的角蛋白肽的离子交换色谱及凝胶过滤色谱的分馏结果的图。
图3为表示利用1kDa以下的角蛋白肽的凝胶过滤色谱法的分馏结果的图。
图4表示本发明的在超角蛋白肽的人类皮肤成纤维细胞中的毒性试验(A)及在人类皮肤成纤维细胞中,利用紫外线B(UVB)诱导的基质金属蛋白酶-1的表达抑制效果的结果(B)的图。泳道1为无处理对照组、泳道2为20mJ/cm2的紫外线B(UVB)的独立处理组、泳道3、泳道4、泳道5、泳道6、泳道7、泳道8为与20mJ/cm2的紫外线B(UVB)一同以5μg/mL、10μg/mL、20μg/mL处理本发明的角蛋白肽混合物(No.2、KP7)的实验组。
图5表示表3的合成角蛋白肽的基质金属蛋白酶-1活性抑制试验(A)、在人类皮肤成纤维细胞中的毒性试验(B)及对在人类皮肤成纤维细胞中利用紫外线B(UVB)诱导的基质金属蛋白酶-1的表达抑制效果进行测定的结果(C)的图。泳道1为无处理对照组、泳道2为20mJ/cm2的紫外线(UVB)的独立处理组、泳道3、泳道4为与20mJ/cm2的紫外线B(UVB)一同以10μM处理合成角蛋白肽的实验组。
具体实施方式
以下,通过实施例来进一步详细地说明本发明。然而,这些实施例是用于示例性地说明本发明,本发明的范围不限定于这些实施例。
实施例1.制备角蛋白肽
1.1培养及角蛋白水解产物的制备
为了制备角蛋白肽混合物,如下表1所示,利用氮源,在70℃温度条件下,在添加有鸡毛的生长培养基内对作为鸡毛分解菌株的海岛闪烁杆菌(Fervidobacteriumislandicum)AW-1(KCTC4680)进行厌氧培养,得到了角蛋白水解产物。
表1
之后,对上述角蛋白水解产物进行如下操作:利用过滤纸(5μm、No.20、HyundaiMicro、Korea)将经分解的鸡毛残渣进行初次过滤之后,在4℃温度条件下,以10000×g离心分离20分钟,并回收了上清液。经回收的上清液作为试样利用于功能性超低分子角蛋白肽分离及纯化过程中。
1.2角蛋白水解产物的纯化
为了简便地从上述试样分馏出1kDa以下的超低分子角蛋白肽,利用了超滤法(Ultrafiltration)。在超滤中,利用了具有10kDa及1kDa的细孔(pore)大小的膜来作为过滤膜。具体地,使试样向设置有过滤膜的过滤模块流入,并施加约10~30psi的压力,使得试样一边通过过滤膜一边进行过滤。首先,利用10kDa的超滤膜,分离了10kDa以上的蛋白质,对于从过滤模块流出的10kDa以下的蛋白质试样,利用1kDa的超滤膜分馏了1kDa以上的试样与1kDa以下的试样。
对经分馏的1kDa以下的角蛋白肽试样,利用两种方法来将角蛋白肽进行了纯化:i)通过离子交换色谱的纯化。通过利用生物双流快速蛋白质液相层析***(Biologic duo-flow FPLC system)(美国伯乐公司(Bio-rad)出品)、离子交换色谱(Macro-prep DEAEsupport,美国伯乐公司(Bio-rad)出品)的离子交换色谱,根据离子性分离了上述经分馏的试样。进行了纯化(50mM的三(羟甲基)氨基甲烷缓冲液(Tris-HCl buffer)、pH7.5、0M-1M的NaCl)并将其结果在图2中示出。如图2所示,在测定210nm时,确认除了两个峰(peak),对阳离子性相对强的Fr.4-47进行合并(pooling)之后,进行了冷冻干燥。为了对经冷冻干燥的上述试样进行根据脱盐及分子量的分离,利用美国伯乐公司(Bio-rad)出品的生物双流快速蛋白质液相层析***(biologic duo-flow FPLC system)、复合凝胶(superdex 30pg)(美国通用公司(GE)出品),进行了凝胶过滤色谱(20mM的三(羟甲基)氨基甲烷缓冲液(Tris-HCl buffer)、pH7.0、100mM的NaCl),在测定210nm时,确认了4个峰,将与标准物质相比预测包含角蛋白肽的Fr.16-29合并之后,进行了冷冻干燥。以下,将由上述方法取得的角蛋白肽称为“No.2”。
ii)利用凝胶过滤色谱的纯化。利用与上述i)中所描述的方法相同的方法进行了凝胶过滤色谱。将进行的结果在图3中示出,如图3所示,合并Fr.17-26之后,进行了冷冻干燥。以下,将由上述方法取得的角蛋白肽称为“KP7”。
进行色谱时,为了测定蛋白质及肽的量,以280nm及210nm测定了试样的吸光度。
像这样,对了最终确认经分离及纯化的No.2、KP7试样的脱盐及分子量,利用凝胶过滤预装柱(Superdex peptide 10/300 GL)(美国通用公司(GE)出品),进行了凝胶过滤色谱,在确认了确实为具有1kDa以下的分子量的超低分子角蛋白肽(图2及图3)之后,利用了以下实施例。
实施例2.角蛋白肽的人皮肤成纤维细胞中的细胞毒性试验及由紫外线B诱导的基 质金属蛋白酶-1的表达抑制效果
为了检验通过上述实施例1的方法制备的角蛋白肽的细胞毒性,利用人皮肤成纤维细胞(human dermal fibroblast),来进行了MTT分析法。
首先,将人成纤维细胞分注于96-孔板中,使得成为5×103cell/well,利用混合有分别含有10%的胎牛血清(fetal bovine serum;FBS)与青霉素-链霉素(GIBCOInvitrogen、Auckland、NZ)的达尔伯克改良伊格尔培养基(Dulbecco's Modified Eagle'sMedium,DMEM),在37℃温度、5%的CO2的条件下,培养了6小时。将200mg的3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)粉末溶于40ml的磷酸缓冲盐溶液(PBS)中,然后过滤,由此准备了5mg/mL的MTT溶液。分注6小时之后,添加含有制备的角蛋白肽的培养基,72小时之后,添加了5mg/mL的MTT溶液各20μl。培养3小时之后,去除培养基与MTT溶液,加入二甲亚砜(DMSO,dimethyl sulfoxide)各200μL,在室温条件下混合了30分钟。利用酶标仪(microplatereader;Sunrise-Basic Tecan,Austria),对反应混合物在570nm测定了吸光度,并且根据下列数学式1计算出了细胞生存率。细胞生存率的计算结果在图4A中示出。
数学式1
细胞生存率(%)=100-((无处理对照组的吸光度-试样添加组的吸光度)/无处理对照组的吸光度×100)
如图4A所示,确认到了本发明的角蛋白肽直到100μg/mL的浓度未呈现细胞毒性。
并且,为了测定细胞中基质金属蛋白酶-1的表达水平,向经培养的上述源自人类的人皮肤成纤维细胞(human dermal fibroblast)中,将本发明的角蛋白肽溶于无菌水中,并处理成5μg/mL、10μg/mL、20μg/mL浓度。接着,通过利用凝胶成像***(Vilber Lourmat,BioLink Crosslinker,France),以20mJ/cm2照射紫外线B,来培养24小时之后,回收了细胞蛋白质。此时,在照射紫外线之前去除培养基,然后用磷酸缓冲盐溶液(PBS,phosphatebuffered saline)溶液清洗,使得去除培养基内血清成分,然后照射了紫外线B。为了回收蛋白质,用磷酸缓冲盐溶液溶液清洗2次之后,利用细胞溶解液(lysis buffer)来回收附着于底部的细胞,并在14000rpm条件下离心分离10分钟。回收各自的上清液之后,以牛血清白蛋白(BSA,bovine serum albumin)为标准物质,利用蛋白质定量检测试剂盒(proteinassay kit、Bio-Rad、USA),根据其的使用方法对各个细胞上清液的蛋白质浓度进行了定量。从各个蛋白质提取物将相当于约10mg的蛋白质变性分离为8%的凝胶十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE,gel sodium dodecyl sulphate-polyacrylamide gelelectrophoresis)凝胶,以120mA向聚偏氟乙烯膜(PVDF膜,Polyvinylidene fluoridemembrane)转移2小时。然后,浸渍于含有5%的脱脂牛奶(skim milk)的三乙醇胺缓冲盐水溶液吐温(TBS-T)中反应6小时,使得阻断(blocking)非特异性蛋白质,分别用蒸馏水 清洗1次,用三乙醇胺缓冲盐水溶液吐温清洗两次。此时,将作为抗体的基质金属蛋白酶-1(MMP-1,Matrix Metalloprotease-1)单克隆抗体(NeomArkers公司出品,美国加利福尼亚州弗里蒙特(Fremont,CA))以1:1000的比率稀释于三乙醇胺缓冲盐水溶液吐温溶液中来使用,在4℃温度条件下反应过夜。一次抗体反应结束后,用三乙醇胺缓冲盐水溶液吐温清洗15分钟各2次,将结合有辣根过氧化物酶(HRP,horseradishperoxidase)的抗兔IgG(anti-rabbitIgG,美国圣克鲁斯生物技术(Santacruz,USA)出品)以1:5000的比率稀释并用作2次抗体,并且在常温下反应了1小时30分钟。反应结束后,利用三乙醇胺缓冲盐水溶液吐温清洗5分钟4次,并与电致化学发光(ECL)基质(英国安马西亚公司(Amersham TM,UK)出品)反应5分钟之后,使曝光于X射线(X-ray)胶片,来对各个细胞的有关细胞老化的蛋白质的表达变化进行了分析。将上述试验结果在图4B中示出。
如图4B所示,当将No.2处理为10μg/mL浓度以上,将KP7处理为5μg/mL浓度以上时,确认到了显著地抑制了相对于UVB独立处理组的基质金属蛋白酶-1的表达的情况。
因此,由于本发明的角蛋白肽具有对皮肤细胞无毒性,并且在紫外线照射条件下也具有可抑制基质金属蛋白酶-1的表达的优秀的效果,因而确认了可用于预防、改善及治疗皮肤老化及皮肤皱纹的化妆品或食品组合物。尤其,确认到了可用于因UVB的皮肤老化及皮肤皱纹的改善。
并且,在现有专利(申请号:10-2014-0092000)中,在分别以200μg/mL、400μg/mL的浓度处理1kDa以下的角蛋白肽组中,呈现出了相对于UVB独立处理组的约12%、41%的基质金属蛋白酶-1的表达抑制功效,但可确认到的是,与以往相比,No.2、KP7可在更低的浓度下抑制基质金属蛋白酶-1的表达。这表现出利用本发明的实施例1的超低分子角蛋白肽的纯化方法制备而成的角蛋白肽对皮肤老化及皮肤皱纹的预防、改善及治疗效果更为有效。
实施例3.角蛋白肽的氨基酸序列鉴定及合成
如上所述,为了弄清抗老化能及皱纹改善能得以确认的No. 2、KP7试样中呈现出这种活性的角蛋白肽的氨基酸序列,进行了利用LC-MS/MS方法的肽鉴定。
具体地,从基因库(Genbank)中获得了鸡(Gallus gallus ver. 5.0)的羽毛角蛋白序列(feather keratin sequence),以便用于角蛋白肽作图(mapping)的数据库构建,并且通过AW-1菌株的转录物分析,确认了涉及鸡毛分解的蛋白酶的选定及肽切割位点,在此基础上构建了用于LC-MS/MS结果分析的数据库。利用电喷雾四级杆飞行时间(ESI-Q-TOF,美国赛默飞(戴安)公司(Thermo(Dionex))出品,型号:UHPLC Ultimate 3000,操作***:ABsciex Triple TOF 5600+)来作为质谱仪,通过上述方法进行了分离、纯化及评估有抗老化能力的角蛋白肽的鉴定,并弄清了源自No.2的16种、源自KP7的8种的作为具有1kDa以下(最小445.3Da至最大710.3Da)的分子量的超低分子肽,并在下列表2中示出。
表2
并且,如下列表3所示,为了弄清具有抗老化能的肽序列,基于肽鉴定结果,合成了9种肽。
表3
实施例4.作为合成肽的体外分析法的基质金属蛋白酶-1抑制酶活性的评价
为了在上述实施例3中合成的肽中评价基质金属蛋白酶-1(Matrixmetalloproteinase-1)的酶活性抑制能力,使用了Enzo公司的基质金属蛋白酶-1荧光法药物筛选试剂盒(MMP-1 Fluorometric Drug Discovery Kit)。作为阳性对照组,使用了已知的用于抑制基质金属蛋白酶-1活性的N-异丁基-N-(4-甲氧基苯磺酰基)甘氨酰异羟肟酸(NNGH,N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid),N-异丁基-N-(4-甲氧基苯磺酰基)甘氨酰异羟肟酸以0.65μM的浓度进行了处理。作为试样添加组,将基质金属蛋白酶-1与角蛋白肽混合成1μg/mL、5μg/mL、10μg/mL的混合液在37°C温度条件下反应了5分钟。向上述反应混合物加入基质,在Ex/Em=540/590nm中测定了荧光,并且根据下列数学式2来评价了基质金属蛋白酶-1活性抑制能力。在图5A中示出了基质金属蛋白酶-1活性抑制能力的评价结果。
数学式2
MMP-1活性抑制能力(%)=试样添加组的荧光强度/无处理对照组的荧光强度×100
如5A所示,确认了如下的情况:合成肽在10μM的浓度下,相对于基质金属蛋白酶-1酶独立处理组,在2n7-2、2n7-4、2n7-5、KP7-1、KP7-2、2-1及2-2中抑制了基质金属蛋白酶-1的酶活性,尤其,肽2n7-4、KP7-1、KP7-2、2-1及2-2具有显著的基质金属蛋白酶-1活性抑制效果。
实施例5.由人成纤维细胞中的细胞毒性试验及紫外线B诱导的基质金属蛋白酶-1
的表达抑制效果
为了检验在上述实施例3中合成的肽的细胞毒性,利用人皮肤成纤维细胞(humandermal fibroblast),与上述实施例2的方法相同地,进行了MTT分析法。在图5B中示出了试验结果。
如图5B所示,确认了本发明的合成肽直到100μg/mL的浓度未呈现出细胞毒性。
并且,为了在细胞测定基质金属蛋白酶-1的表达,在经培养的上述源自人类的人皮肤成纤维细胞(human dermal fibroblast)中,将合成肽2-1及2-2溶于DMSO,处理为10μM的浓度,并以与上述实施例2的方法相同地进行。在图5C中示出了测定基质金属蛋白酶-1表达的结果。
如图5C所示,确认了当将合成肽2-1、2-2处理为10μM的浓度时,相对于UVB独立处理组,抑制了基质金属蛋白酶-1的表达约24%及35%。
序列表
<110> 庆北大学校产学协力团(Kyungpook National University Industry-Academic Cooperation Foundation)
<120> 超低分子角蛋白肽的制备方法及其的利用
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Claims (10)
1.一种皮肤老化或皮肤皱纹预防或改善用角蛋白肽混合物的制备方法,其特征在于,包括:
步骤(1),在包含角蛋白的培养基中,通过培养具有角蛋白分解活性的保藏编号为KCTC4680的海岛闪烁杆菌(Fervidobacterium islandicum)AW-1,来取得角蛋白水解产物;步骤(2),通过超滤在从上述步骤(1)取得的角蛋白水解产物中分馏分子量为100Da至1000Da的蛋白质;
步骤(3),通过对从上述步骤(2)取得的分馏产物进行离子交换色谱、凝胶过滤色谱或其的组合来纯化蛋白质;以及
步骤(4),通过对从步骤(3)取得的经纯化的蛋白质进行凝胶过滤色谱,来对蛋白质进行纯化及脱盐,
其中角蛋白肽混合物包含由选自由序列4、序列6至序列9组成的组中的至少一种序列表示的肽。
2.根据权利要求1所述的皮肤老化或皮肤皱纹预防或改善用角蛋白肽混合物的制备方法,其特征在于,上述步骤(1)的角蛋白来源于鸟类或哺乳类的羽毛、毛发、皮革、指甲、爪、角或蹄。
3.根据权利要求1所述的皮肤老化或皮肤皱纹预防或改善用角蛋白肽混合物的制备方法,其特征在于,上述步骤(1)的培养为在60℃至90℃的温度条件下进行的厌氧培养。
4.一种角蛋白肽混合物,其特征在于,通过权利要求1至3中任一项所述的皮肤老化或皮肤皱纹预防或改善用角蛋白肽混合物的制备方法生产。
5.一种皮肤老化或皮肤皱纹预防或改善用化妆品组合物,其特征在于,包含权利要求4所述的角蛋白肽混合物。
6.根据权利要求5所述的皮肤老化或皮肤皱纹预防或改善用化妆品组合物,其特征在于,上述皮肤老化或皮肤皱纹是由紫外线诱发的。
7.根据权利要求5所述的皮肤老化或皮肤皱纹预防或改善用化妆品组合物,其特征在于,上述角蛋白肽具有基质金属蛋白酶-1表达抑制能力或活性抑制能力。
8.一种皮肤老化或皮肤皱纹预防或改善用食品组合物,其特征在于,包含权利要求4所述的角蛋白肽混合物。
9.由选自由序列2、序列4、序列6至序列9组成的组中的至少一种序列表示的肽在制备预防或改善皮肤老化或皮肤皱纹的化妆品组合物的用途。
10.由选自由序列2、序列4、序列6至序列9组成的组中的至少一种序列表示的肽在制备预防或改善皮肤老化或皮肤皱纹的食品组合物的用途。
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