Name: preparation of keratin hydrolysate from chicken feathers and its application in cosmetics
Uploaded: 2017-11-27T14:00:00
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这篇文章是在 Zlin 的托马斯巴塔大学 IGA/FT/2017/007 项目的支持下编写的。

我们大学的员工和学生中都招募了志愿者。该方法的选择是根据 "国际伦理准则的生物医学研究涉及人类的主题。日内瓦国际医学组织理事会 (2002)。是一种常见的化妆品成分, 用于护发产品 (洗发水、调理剂、等), 因此不需要机构审查委员会的批准。
1. 加工鸡毛
<ol><li>从家禽饲养场收集鸡毛。</li>
	<li>用足够多的新鲜跑步 (冷水) 冲洗鸡毛中的不溶性杂质和血液残渣。?...

<ol>
	<li>United States Department of Agriculture - National Agricultural Statistics Services. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Poultry Slaughter, 2016 Summary. , (2016).</li><li>McGovern, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+poultry+feathers:+more+bang+for+the+cluck.">Recycling poultry feathers: more bang for the cluck.</a> Environ.Health Perspect. 108 (8), A336-A339 (2000).</li><li>Gousterova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+keratin+and+collagen+containing+wastes+by+newly+isolated+thermoactinomycetes+or+by+alkaline+hydrolysis.">Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis.</a> Lett. 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Technol. 21 (4), 403-412 (2015).</li><li>Verdier-S&#233;vrain, S., Bont&#233;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+hydration:+a+review+on+its+molecular+mechanisms.">Skin hydration: a review on its molecular mechanisms.</a> J. Cosmet. Dermatol. 6 (2), 75-82 (2007).</li><li>Darlenski, R., Sassning, S., Tsankov, N., Fluhr, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+in+vivo+methods+for+investigation+of+the+skin+barrier+physical+properties.">Non-invasive in vivo methods for investigation of the skin barrier physical properties.</a> Eur. J. Pharm. Biopharm. 72 (2), 295-303 (2009).</li><li>Berardesca, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EEMCO+guidance+for+assessment+of+stratum+corneum+hydration:+electrical+methods.">EEMCO guidance for assessment of stratum corneum hydration: electrical methods.</a> Skin Res. Technol. 3 (2), 126-132 (1997).</li><li>Rogiers, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EEMCO+guidance+for+the+assessment+of+transepidermal+water+loss+in+cosmetic+sciences.">EEMCO guidance for the assessment of transepidermal water loss in cosmetic sciences.</a> Skin Pharmacol. Appl. Skin Physiol. 14 (2), 117-128 (2001).</li><li>Ali, S. 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碱性酶水解的优点是可以根据今后的应用进行改性。例如, 在头发护理化妆品应用中, 一个产品的轻微棕褐色不是一个障碍, 更高的温度在水解可以被应用导致更高的收益率。此外, 工艺过程中两个阶段的处理时间越长, 对整个工艺效率的影响越大--其收率上升到85%。
水合测量的结果表明, 在被监测的测量间隔 (1-48 h), 增加2% 的 OB 是最佳的, 因为它导致了 11-19% 增加的 SC 水合为男?...

屠宰场、食品工业和制革业每年生产大量的固体角蛋白 by-products--羊毛、羽毛、刚毛、蹄、爪、角等。根据最新的统计数据, 美国的鸡、火鸡、鸭和其他屠宰家禽的活体重为每年625亿磅1;在欧盟, 每年大约有287亿英镑。考虑到羽毛占家禽总重量的 8.5%, 美国每年单独生产大约53亿磅的废弃羽毛2。
角蛋白是一种表现出高耐化学性的蛋白质, 因为它与二硫化桥紧密交联, 使其加工变得困难。获得可溶性产品需要裂解交和可能进行水解的肽键3。二硫化物桥梁的劈裂可以通过硫醇阴离子的反应进行, 根据下面的模式4,5:
sa - + sbsc–↔sb+ s sc-
与一个非常高 pH 水平, 也出现的二硫化物桥梁的水解, 与样式符合6
–SS- +- → s- + –SOH
在温和的条件下 (pH 值约 8), 甚至 sulfitolysis 发生根据以下模式:
–SS– + HSO3- →–SH + –SSO3-
降解角蛋白最经济的方法是微生物分解, 其特点是加工过程中反应条件温和, 击穿效率高 (约 90%)7,8。Keratinases 是由一些从土壤和角蛋白废料中分离出来的细菌产生的9。微生物 keratinases 水解刚性和强交联的角蛋白结构10和所得到的所制备的含有丰富的可溶性蛋白质, 在它的基本氨基酸没有损失, 在它的检测11。
为了在化妆品制剂中加入一种蛋白质 (例如, 如、乳液、乳液和凝胶), 这些要求确保了这种蛋白质在水中是可溶的, 给定的系统是透明的, 并且避免了 re-aggregation 的多肽, 因为疏水相互作用。因此, 一个常见的做法是应用水解蛋白, 如水解胶原蛋白, 弹性蛋白, 和角蛋白。在化妆品乳液中加入水解物时, 采取步骤确保水解液首先溶解于水中。在某些情况下, 它是可取的蛋白质 (或水解物) 是可溶性的酒精或其他有机溶剂12。
通常在洗发水, 调理剂, 乳液, 和营养血清的头发, 以及睫毛, 指甲油, 和眼部化妆品的特点。所宣布的影响通常包括形成保护膜, 平滑头发或指甲结构, 提高可塑性和外观的处理形成, 调节产品的一致性, 并鼓励泡沫的形成13,14. 它也表明, 降低表面张力, 因此补充化妆品可以促进减少乳化剂添加到稳定霜。限制清洁剂 (表面活性剂) 对皮肤、眼睛和毛发的刺激作用, 从而减少清洁剂对组织的潜在副作用 (例如、皮肤脱水、硬度和降低屏障功能皮肤)。同时利用水解物的高缓冲能力来稳定化妆品的 pH 值;较短长度的多肽具有更大的缓冲效果15,16。虽然 KHs 已成为在头发和指甲化妆品的标准成分, 以及在产品中使用的护肤品, 研究的保湿作用, 不出现在当代文学。
碱性酶技术已经发展为加工角蛋白 by-products 成的, 并且活性测试是在过程中的一些化妆品添加剂的效果17,18,19,20,21,22. 二级碱性酶水解利用微生物蛋白酶对鸡羽毛的优越性在温和的反应条件下达到高效, 而在强酸中使用的水解液的质量很高。或碱。在第一阶段, 在碱性环境中, 羽毛会在较高的温度下孵化, 这会部分地扰乱角蛋白结构并使羽毛膨胀;在调整 pH 值后, 在第二阶段, 在温和的条件下, 用水解酶水解羽毛。透析具有高含量的蛋白质。
本文所描述的方法的目的是通过碱性酶水解处理家禽羽毛, 并检测其在化妆品乳液中的保湿性能。用非侵入性方法对体内的保湿性能进行了研究。最常用的测量皮肤水化和屏障功能的方法包括测量皮肤的电学特性 (电导或电容)。研究 SC 水合的不同方法包括近红外多光谱想象方法 (尼姆)、核磁共振光谱学、光学相干断层扫描或瞬态热传导23。sc 的阻隔作用与 sc 的 TEWL 性有关, 采用通风室法、通风室法和开放式室法测定24。
模型配方的性质是使用多探头适配器 MPA 5 和三种探针确定的。第一个, corneometer CM 825, 通过评估皮肤表面的电容量变化来测量皮肤水化;测量电容器显示 corneometric 单位的皮肤表面电容的变化。corneometer 只给出皮肤水化的相对评估25。对于 TEWL, 第二探针, tewameter TM 300, 用于测量水蒸发密度梯度 (在一个开放的房间仪器基于菲克的扩散定律) 从皮肤间接由两对传感器 (温度和相对湿度)表示每一个定义的区域和时间段内运输的水量 (克/米2/小时)。这种方法甚至可以检测到皮肤屏障功能的轻微破坏26。皮肤 pH 值是 SC27的屏障和抗菌功能的一个指标。皮肤地幔的酸度是由 (第三) 皮肤 PH 值905探头连接到 MPA 5 站测量的。这个专门设计的探头由一个平顶的玻璃电极组成, 用于全皮肤接触, 连接到电压表。该系统测量的潜在变化, 由于氢阳离子周围的非常薄层的半固态形式测试顶部的探针。电压的变化显示为 pH 值28。
本实验分为三部分: (1) 用两段碱性酶解法制备鸡羽毛中的水, 并通过透析 (除去盐和低分子分数) 纯化, (2) 化妆品的制备含有2、4和6% 的配方, 以及 (3) 通过测量皮肤水化、TEWL 和皮肤 pH 值来测试其性能。对10名平均年龄为27.2 岁的妇女和平均年龄为26.2 岁的10男子进行了试验。选择志愿者和测试本身的方法是按照国际生物医学研究的伦理原则进行的, 利用人体主体29;在纳入研究之前, 所有的人都给予了他们知情的同意。在测试开始之前, 志愿者们被要求填写一份关于他们健康状况的问卷。志愿者承诺在试验期间和测试期间, 避免在24小时内将任何化妆品应用于试验场和周边地区;此外, 他们只允许短暂的傍晚冲洗自来水。

<table>
	<tbody>
		<tr>
			<td>Material or chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIPEX 100T</td>
			<td>Novozymes</td>
			<td>LJP30020</td>
			<td>Lipex - enzyme produced by submerged fermentation of a genetically-modified microorganism, activity 100 KLU/g</td>
		</tr>
		<tr>
			<td>Savinase Ultra 16L</td>
			<td>Novozymes</td>
			<td>PXN40001</td>
			<td>Savinase - enzyme produced by submerged fermentation of a genetically-modified microorganism, activity 16 KNPU-S/g</td>
		</tr>
		<tr>
			<td>Potassium hydroxide, KOH</td>
			<td>Sigma-Aldrich</td>
			<td>302510289</td>
			<td>Potassium hydroxide, KOH, 97,0 %, Mr 56,11</td>
		</tr>
		<tr>
			<td>Phosphoric acid solution, H3PO4</td>
			<td>Sigma-Aldrich</td>
			<td>W290017</td>
			<td>Phosphoric acid solution, H3PO4, 85 wt. % concentration in water, Mr 98,00</td>
		</tr>
		<tr>
			<td>Sodium chloride physiological solution</td>
			<td>Sigma-Aldrich</td>
			<td>52455</td>
			<td>Tablets of BioUltra NaCl physiological solution; 1 tablet in 1000 mL of water yields 0.9 % NaCl</td>
		</tr>
		<tr>
			<td>Sodium hydroxide, NaOH</td>
			<td>Penta s.r.o.</td>
			<td>40216</td>
			<td>Sodium hydroxide, NaOH, 97,0 %, Mr 40,00</td>
		</tr>
		<tr>
			<td>AmiFarm (Cremor base-A)</td>
			<td>Fagron</td>
			<td>608425</td>
			<td>Hydrophilic oil in water (O/W) cream base; the composition: aqua, paraffin, paraffin liquid, cetearyl alkohol, Laureth 4, sodium hydroxide, carbomer, methylparaben, propylparaben.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IKA EUROSTAR POWER control-visc stirrers</td>
			<td>IKA-labortechnik</td>
			<td>Z404020</td>
			<td>Digital laboratory stirrer, for tasks up to the high viscosity range, 230V, 1/cs</td>
		</tr>
		<tr>
			<td>IKA Propeller stirrer, 3-bladed</td>
			<td>IKA-labortechnik</td>
			<td>R 1381</td>
			<td>Propeller stirrer, 3-bladed, stirrer &#216;: 45 mm, shaft &#216;: 8 mm, shaft length: 350 mm</td>
		</tr>
		<tr>
			<td>Dialysis tubing closures</td>
			<td>Sigma-Aldrich</td>
			<td>Z371017-10EA</td>
			<td>Dialysis tubing closures, red, size 110 mm</td>
		</tr>
		<tr>
			<td>Dialysis tubing cellulose membrane</td>
			<td>Sigma-Aldrich</td>
			<td>D9402-100FT</td>
			<td>Dialysis tubing cellulose membrane, average flat width 76 mm (3.0 in.)</td>
		</tr>
		<tr>
			<td>DOMO Pot with stailess, LCD</td>
			<td>DOMO Elektronic</td>
			<td>DO42325PC</td>
			<td>Preserving boiler stainless steel, 2000 W, 27-L container (diameter 37 cm, height 30 cm), temperature control 30-100 &#176; C, operation LCD display</td>
		</tr>
		<tr>
			<td>Hettich zentrifugen Universal 32</td>
			<td>Gemini bv</td>
			<td>2770 GS1R</td>
			<td>Mid bench centrifuge, speed 18000 rpm</td>
		</tr>
		<tr>
			<td>LT 3 shaking device</td>
			<td>Fischer Scientific</td>
			<td>6470.0002</td>
			<td>Orbital shaking device</td>
		</tr>
		<tr>
			<td>KERN 440-47N</td>
			<td>Kern</td>
			<td>440-47N</td>
			<td>Laboratory balance</td>
		</tr>
		<tr>
			<td>KERN 770</td>
			<td>Kern</td>
			<td>770 -N</td>
			<td>Laboratory analytical balance</td>
		</tr>
		<tr>
			<td>VENTICELL 222 - Komfort</td>
			<td>BMT, MMM Group</td>
			<td>C 131749</td>
			<td>Drying oven, temperature control 30-100 &#176; C, air circulation control</td>
		</tr>
		<tr>
			<td>Vacucell 55 - EVO</td>
			<td>BMT, MMM Group</td>
			<td>B 050328</td>
			<td>Vacuum drying oven, temperature control 30-100 &#176; C</td>
		</tr>
		<tr>
			<td>PULVERISETTE 19</td>
			<td>Fritsch</td>
			<td>19.1030.00</td>
			<td>Universal cutting mill, rotor with V-cutting edges and fixed knives</td>
		</tr>
		<tr>
			<td>Multi Probe Adapter System MPA 5</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td>10225237</td>
			<td>MPA 5 Station - equipment for measurement hydratation, TEWL and pH</td>
		</tr>
		<tr>
			<td>Skin pH-meter PH 905 probe</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td></td>
			<td>Probe to specifically measure the pH on the skin surface or the scalp</td>
		</tr>
		<tr>
			<td>Corneometer CM 825 probe</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td></td>
			<td>Probe to determine the hydration level of the skin surface (Stratum corneum).</td>
		</tr>
		<tr>
			<td>Tewameter TM 300</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td></td>
			<td>Probe for the assessment of the transepidermal water loss (TEWL)</td>
		</tr>
		<tr>
			<td>Heidolph RZR 2020</td>
			<td>Heidolph</td>
			<td>13-225-007-03-1</td>
			<td>Overhead stirrer, mechanical speed setting and stepless transmission; speed range 40-2000 rpm</td>
		</tr>
		<tr>
			<td>Heidolph mechanical stirrer BR 10</td>
			<td>Heidolph</td>
			<td>Z336688-1EA</td>
			<td>Blade impeller crossed stirrer</td>
		</tr>
		<tr>
			<td>Fagor FS 12</td>
			<td>Fagor</td>
			<td>BTT-138</td>
			<td>Laboratory refrigerator with freezer space</td>
		</tr>
		<tr>
			<td>WTW bench pH/mV meter</td>
			<td>WTW</td>
			<td>Z313165</td>
			<td>High-performance bench pH and pH/conductivity meters for routine and high precision laboratory measurements in research or quality control laboratories</td>
		</tr>
		<tr>
			<td>Container</td>
			<td>RPC Superfos</td>
			<td></td>
			<td>13-L plastic bucket, diameter 26 cm, height 26 cm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2010</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C+K software</td>
			<td colspan="2">Courage and Khazaka Electronic GmbH</td>
			<td>MPA 5 station operating software</td>
		</tr>
	</tbody>
</table>

preparation of keratin hydrolysate from chicken feathers and its application in cosmetics

角蛋白水解物 (KHs) 是建立在头发化妆品的标准成分。了解其保湿效果有利于护肤化妆品。该协议的目标是: (1) 用碱性酶水解法将鸡羽毛加工成一层, 用透析法纯化, (2) 如果将其加入软膏基 (OB), 可增加皮肤的水化, 改善皮肤屏障功能, 减少皮失水 (TEWL)。在碱性酶水解过程中, 在碱性环境下, 先在较高的温度下孵育, 然后在温和的条件下, 用水解酶水解。溶液的透析, 真空干燥, 研磨成细粉。在水乳剂中含有2、4和 6 weight% (基于 OB 重量) 的含油的化妆品配方。在10男性和10妇女的时间间隔内对其保湿性能进行测试, 1、2、3、4、24和 48 h. 试验配方在脱脂掌侧前臂部位分布。通过测量皮肤的电容来评估角质层 (SC) 的皮肤水化, 这是世界上使用最广泛和简单的方法之一。TEWL 的基础是测量每一个定义的区域和从皮肤的时间段的水运输量。这两种方法都是完全无创的。使一个优秀的闭塞;根据添加到 OB, 它带来约30% 减少 TEWL 后, 应用。它也起到了剂的作用, 因为它将水从表皮的下层层束缚到 SC;在 OB 的最佳的增加, 19% 上升在男性和22% 上升妇女发生。

根据这里所介绍的程序 (参见图 2) 是黄色的, 容易溶于高蛋白质含量的水中 (无机固体代表 < 2.0%); 1.0% 溶液的 pH 值为 5.3, 满足了化妆品级水解物。50克原料的产量约为30%。通过 SDS 页确定了其分子量分布, 如图 3所示。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56254/56254fig2.jpg"> <str...

Watch this Scientific Journal Video about Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics at JoVE.com

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

该协议的目的是用碱性酶水解法制备鸡羽毛中的角蛋白水解物, 并测试在化妆品软膏基中添加角蛋白水解液是否改善了皮肤屏障功能 (提高水化和减少皮水损失)。对男性和女性志愿者进行测试。

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

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