1236501 玖、發明說明： 【明戶斤屬$員】 發明領域 本發明係有關於-種用以從動物組織萃取出可溶性膠 原蛋白的方法以及包含有由該方法所製得的可溶性膠原蛋 白的產物。 發明背景 10 15 20 膠原蛋白為動物體内最常見之—主要結構蛋白質，而 且它是硬骨（W)、„(e福age)、皮膚、肌腱⑽_ 與結缔組織中的主要蛋白質組份。呈天然形式的膠原蛋白 典型地是-堅硬的短桿狀分子。所有的膠原蛋白分子之決 定性性質是為三股螺旋(tdple helix),—種由三個多狀次單 位（或稱α鏈）所構成的盤曲螺旋物（c〇iled c〇ii)。有關第工 型、第Π型舆第㈣膠原蛋白的續之通式是為重複的 (Gly-X-Y)(其中X與γ通常為脯胺酸或經基脯㈣”在各個 端部處被連接以“端肽(telGptptide)”區域。該等鏈之端肤區 域是分子㈣主要交輕址，且料地與該蛋白質之免疫 原性有關聯。端肽領域可藉由酵素的處理方式來予以移除 而生成“無端肽（atel〇peptide)，，可溶性膠原蛋白。 在膠原蛋白生物化學上的快速進展促成了在許多疾病 [包括骨關節炎（osteoarthritis)、類風濕性關節炎（―㈣ arthnUs)、老化與癌症]之膠原蛋白病理學上之一增高的興 趣以及新的瞭解。此外’動物膠原蛋白具有一優良的生物 6 1236501 親和性、組織相容性與低抗原性，具有促進細胞分化與增 生之作用，具有一止血作用，而且在體内可被完全地分解 與吸收。膠原蛋白已被廣泛地使用在生物學的研究以及食 品、化妝品與藥學應用上，也被廣泛地用作為用在假體植 5 入物（prosthetic implant)、持續藥物釋放基質（sustained drug release matrix)、人工皮膚、傷 口敷料（wound dressing)、傷 口癒合基質（would healing matrix)、骨絡修補（bone repair) 與組織工程（tissue engineering)上之生物可相容的醫用材料 之一組份。 10 在本技藝中，膠原蛋白已從天然來源（例如牛的皮膚、 軟骨或骨骼）被分離出。骨骼通常被乾燥、脫脂、壓碎以及 去礦物化（demineralized)以供萃取膠原蛋白，而軟骨與皮膚 典型地被磨碎並以蛋白水解酵素（諸如胃蛋白酶與木瓜蛋 白酶）來消化。由於膠原蛋白對大多數的蛋白酶具有抗性惟 15 獨膠原酶（collagenase)例外，使用蛋白水解酵素之消化處理 來移除大多數的端肽以及污染性蛋白質的操作程序是極為 想要的，俾以純化膠原蛋白並使由膠原蛋白物質所致的免 疫反應之發生可能性減至最低。 目前已知有多種方法可被應用以從動物皮膚組織中萃 20 取出不溶性膠原蛋白。例如，不溶性膠原蛋白可藉由下列 方式而被分離出：以一機械力來破壞要被處理的動物皮 膚，而後將之置於一具適當離子強度的合適緩衝液内，俾 以溶出大部分的皮膚蛋白質，再以離心或過遽來回收蛋白 質產物。藉由此類方式所得到的產物大體上為不溶性蛋白 12365011236501 (1) Description of the invention: [Minghu genus $ member] Field of the invention The present invention relates to a method for extracting soluble collagen from animal tissue and a product containing the soluble collagen produced by the method . BACKGROUND OF THE INVENTION 10 15 20 Collagen is the most common structural protein in animals, and it is the main protein component of hard bone (W), (e), skin, tendon, and connective tissue. The natural form of collagen is typically a hard, short rod-shaped molecule. The decisive property of all collagen molecules is the triple helix (tdple helix), which is composed of three polymorphic subunits (also known as alpha chains) Coiled coils. The continuation formulas for type I and type II collagen are repeated (Gly-XY) (where X and γ are usually proline "Acids or glycoproteins" are linked at each end to "telGptptide" regions. The telomeres of these chains are the major sites of molecular ㈣, and are expected to have immunogenicity with the protein. Correlation. The telomere field can be removed by enzyme treatment to generate "atelopeptide, soluble collagen. Rapid advances in collagen biochemistry have contributed to many diseases [including bone and joints Osteoarthritis, rheumatoid Arthritis, aging and cancer] has increased interest and new understanding of collagen pathology. In addition, 'animal collagen has an excellent biological 6 1236501 affinity, histocompatibility and low antigen It has the function of promoting cell differentiation and proliferation, has a hemostatic effect, and can be completely broken down and absorbed in the body. Collagen has been widely used in biological research and food, cosmetics and pharmaceutical applications. It is widely used as a prosthetic implant, sustained drug release matrix, artificial skin, wound dressing, wound healing matrix, and bones Bone repair A component of biocompatible medical materials used in tissue engineering. 10 In this technique, collagen has been removed from natural sources (such as the skin, cartilage, or bones of cattle). Isolated. Bone is usually dried, defatted, crushed, and demineralized for collagen extraction, Cartilage and skin are typically ground and digested with proteolytic enzymes, such as pepsin and papain. Since collagen is resistant to most proteases, except collagenase, the only exception is the use of proteolytic enzymes. A digestion process to remove most telomeres and contaminating proteins is highly desirable in order to purify collagen and minimize the possibility of an immune response caused by collagen substances. Various methods are currently known to be applied to extract insoluble collagen from animal skin tissue. For example, insoluble collagen can be isolated by destroying the skin of the animal to be treated with a mechanical force, and then placing it in a suitable buffer with an appropriate ionic strength to dissolve most of the Skin protein is recovered by centrifugation or centrifugation. The product obtained by this method is generally insoluble protein 1236501
居似於上返的技術可參見，例如，私㈣·〇 n μ. ::山’ c〇 一“如“心其中揭示藉由將動物皮 胃句貝化（h〇m〇gemZatl〇n)繼而進行離心與深層過濾、（depth 5編1Qn胸化不溶性蛋白f。在這篇文獻中有探討使用 胃蛋白酶來進行萃取，但並沒有提到其他蛋白酶可被有效 =地應用於端肽”膠原蛋白的萃取以及移除。再者，此篇 疋亦未提到，在使用一蛋白水解酵素來切割“端肽，，膠原 史白蚪會有“無端肽”或可溶性膠原蛋白的生成。因此，要 的方法成為商業上有料，基本上需純到能容許 々所奴產物中移除掉“端肽”膠原蛋白與其他污染物的蛋白 水解酵素與方法。 4’140,537描述一種用以從一動物皮膚來收取膠原 史白的典型方法，其中動物皮膚被散浮於一適當的緩衝液 15内’该緩衝液含有一適於將邱值調整在2〜6 (較佳為〕）之間 的緩衝制’而動物皮膚在被散浮於該緩衝液内之同時亦藉 由-般常用的技術（例如機械性方法，譬如使用_均質機） 而被打碎。這樣的處理方法被認為可適用在從動物皮膚或 其他組織來萃取不溶性膠原蛋白，而且如該美國專利二其 i路内谷所提到的’可能只適用於收取具有微小改質的膠 原蛋白。 > 雖然US 4，140,537有提到使用一非為膠原酶之酵素（特 別是胃蛋白酶）來對動物皮膚或組織進行酵素水解處理以 私除具免疫原性的端肽（tel〇peptides)，此件美國專利前案沒 1236501 有明！或gizi — 口各 曰7F曰尤、囷水解S每（thermolysin)對於膠原蛋白會 產生何種作用。 另外在1^ 5,814,328所揭示的膠原蛋白製備方法中使 用到兩階段的酵素水解處理，而適用的酵素較佳地是木瓜 5 蛋白酶（papain)。 縱7現今業界已有多種方法可用於膠原蛋白的生產， 本技藝仍存在有一需要來發展一適合於產業上大規模製造 勺新方法以有效率地從動物皮膚萃取出有生物活性的可溶 性膠原蛋白，而非所欲之無生物活性的中間物之含量被減 10 少或消除。A technique that resembles the return can be found in, for example, private ㈣ · 〇n μ. :: 山 'c〇 一 "such as" Heart Revealed by the Stomach Stomach of Animals (h〇m〇gemZatl0n) Then proceeded to centrifugation and deep filtration, (depth 5 ed 1Qn thoracic insoluble protein f. In this literature, the use of pepsin for extraction is discussed, but it is not mentioned that other proteases can be effectively applied to telomere collagen Extraction and removal of proteins. Furthermore, it is not mentioned in this article that when a proteolytic enzyme is used to cut "telopeptides," collagen peptides will produce "endless peptides" or soluble collagen. Therefore, The desired method is commercially available, and it is essentially pure enough to allow proteolytic enzymes and methods to remove "telopeptide" collagen and other contaminants from the product. 4'140,537 describes a method used to remove an animal from an animal. A typical method of collecting collagen from the skin, in which the animal's skin is suspended in an appropriate buffer 15 'the buffer contains a buffer suitable for adjusting the Qiu value between 2 and 6 (preferably)) Control 'while animal skin is being It floats in the buffer and is also broken by common techniques (such as mechanical methods, such as using a homogenizer). Such a treatment method is considered to be suitable for extraction from animal skin or other tissues. Insoluble collagen, and as mentioned in the U.S. Patent No. 2 Wayne Valley, 'may only be used to collect collagen with minor modifications. ≫ Although US 4,140,537 mentions the use of a non-collagenase Enzymes (especially pepsin) are used to hydrolyze animal skin or tissues to remove the immunogenic telopeptides. This pre-US patent case is not clear in 1236551! Or gizi — 7F What kind of effect does hydrolysin have on collagen? In addition, the collagen preparation method disclosed in 1 ^ 5,814,328 uses two stages of enzyme hydrolysis treatment, and the applicable enzyme is preferably papaya. 5 Protease (papain). Even though there are many methods available in the industry today for the production of collagen, there is still a need in the art to develop a suitable for industrial production. A new method for large-scale manufacturing is to efficiently extract bioactive soluble collagen from animal skin, while reducing the content of non-bioactive intermediates as desired.
C 明内容I 發明概要 因此，在第一個方面，本發明提供一種用以從一富含 膠原蛋白的動物組織中萃取出可溶性膠原蛋白的方法，其 15 包含下列步驟： (a) 將一萄含膠原蛋白的動物組織浸泡於一 pH值為2〜 5的酸性水溶液内歷時一段充分的時間以使動物組 織膨脹； (b) 將步驟U)所得到之經膨脹的動物組織予以磨碎並散 ο π 浮於一緩衝液内； (c) 將步驟（b)所形成的混合物之pH值調整至一適合於 嗜熱菌水解酶（thermolysin)作用的pH值； (d) 將一適量的嗜熱菌水解酶加入至步驟（c)所形成的混 合物内並混合歷時一段充分時間，藉此，存在於該 1236501 此口物内之被料軸物組織被唁㈣水解酶水解 而釋出可溶性膠原蛋白； ()將々^⑷所形成的混合物之ρΗ值調整至低於5〇以 將嗜熱菌水解酶去活化；以及 ⑴將步‘（e)所形成的混合物離^以得到—含有可溶性 膠原蛋白的上澄液。 依據本發明的方法而得到的可溶性膠原蛋白是安定的 而且能提供經交聯的膠原蛋白原纖維。因此，在第二個方 面，本發明提供_種能供應用於生物相容性材料的製造之 丄〇膠原蛋白原纖維㈣agen fibril)，其係藉由依據本發明所揭 示的方法而製得的可溶性膠原蛋白經過膠原纖維形成 (fibrillogenesis)而被生成。 本發明之上述以及其他目的、特徵與優點在參照以下 之詳細說明與較佳實施例和隨文檢附之圖式後會變為明顯 15 可知，其中： 圖式簡單說明 第1圖是一凝膠電泳圖片，其中第丄行是_商業來源 之膠原蛋白標準品，而第2行與第3行分別為後面的實施 例4中藉由胃蛋白酶與嗜熱菌水解酶之萃取處理所得到的 20膠原蛋白，各以10吨的樣品來作SDS-PAGE分析。C. Description I. Summary of the Invention Accordingly, in a first aspect, the present invention provides a method for extracting soluble collagen from a collagen-rich animal tissue, which comprises the following steps: (a) a grape Collagen-containing animal tissues are immersed in an acidic aqueous solution with a pH of 2 to 5 for a sufficient period of time to swell the animal tissues; (b) the swollen animal tissues obtained in step U) are ground and dispersed ο π floats in a buffer solution; (c) adjusts the pH value of the mixture formed in step (b) to a pH value suitable for the action of thermolysin; (d) adjusts an appropriate amount of Thermobacteria hydrolase is added to the mixture formed in step (c) and mixed for a sufficient period of time, whereby the material shaft tissue present in the 1236501 mouthpiece is hydrolyzed by the hydrolytic enzyme to release soluble collagen () Adjust the ρΗ value of the mixture formed by 々 ^ ⑷ to less than 50 to deactivate the thermophilic hydrolase; and 离 separate the mixture formed in step '(e) to obtain-containing soluble collagen Protein on Liquid. The soluble collagen obtained by the method of the present invention is stable and can provide cross-linked collagen fibrils. Therefore, in a second aspect, the present invention provides a collagen fibril (agen fibril) that can be used for the manufacture of biocompatible materials, which is produced by the method disclosed in the present invention Soluble collagen is produced through fibrillogenesis. The above and other objects, features, and advantages of the present invention will become apparent after referring to the following detailed description and preferred embodiments and drawings attached to the text. Among them, the drawings are briefly explained. Gel electrophoresis picture, where the first line is a collagen standard of commercial origin, and the second and third lines are obtained in the following Example 4 by the extraction treatment of pepsin and thermolysin hydrolase 20 collagen, 10 tons of each sample for SDS-PAGE analysis.
I：實施方式]I 較佳實施例之詳細說明（發明的詳細說明） 用於食品、化 且'具有生物活 為了能夠在產業上大規模地製造適合應 妝品、藥品、醫用物品等方面之高度純化的 10 1236501 性之實質上無免疫原性的可溶性膠原蛋白，申請人經多方 研究而發現唁熱菌水解酶（thermolysin)非常適合供廡用在 膠原蛋白的萃取上。因此，本發明提供一種用以從一富含 膠原蛋白的動物組織中萃取出可溶性膠原蛋白的方法，其 5 包含下列步驟： (a) 將一富含膠原蛋白的動物組織浸泡於一 pH值為2〜 5的酸性水溶液内歷時一段充分的時間以使動物組 織膨脹； (b) 將步驟（a)所得到之經膨脹的動物組織予以磨碎並散 0 浮於一緩衝液内； (C)將步驟（b)所形成的混合物之pH值調整至一適合於 嗜熱菌水解酶（thermolySin)作用的pH值； (d) 將一適量的嗜熱菌水解酶加入至步驟（c)所形成的混 合物内並混合歷時一段充分時間，藉此，存在於該 〉 混合物内之被磨碎的動物組織被嗜熱菌水解酶水解 而釋出可溶性膠原蛋白； (e) 將步驟（d)所形成的混合物之pH值調整至低於5 〇以 將嗜熱菌水解酶去活化；以及 (f) 將步(e)所开》成的混合物離心以得到一含有可溶性 膠原蛋白的上澄液。 車乂佺地，在步驟（a)中所用的動物組織是從一選自下列 羊、且的動物身上所取得：牛、豬、羊，以及此等之一組合。 車乂侄地，在步驟（a)中，該動物組織是從一動物身上所 取传之選自下列群組的組織：軟骨、肌鍵、皮膚，以及此 1236501 等之一組合。更佳地，該動物組織是選自於下列群組：牛 皮豬皮、牛尾以及豬尾。在本發明的一個較佳具體例中， 被應用於步驟（a)中的動物組織是豬皮。 較佳地，被使用於步驟（a)中的動物組織在使用之前有 5 進行過脫脂處理（defatting treatment)。 較佳地，被使用於步驟（a)中的該酸性水溶液包含一選 自於下列所構成之群組中的酸··檸檬酸、乙酸、甲酸、鹽 酸。在本發明的一個較佳具體例中，該動物組織被浸泡於 一 pH值為3.0的酸性水溶液中歷時一段充分時間。在本發 月的個更佳具體例中，該動物組織被浸泡於一 值為 3.0的乙酸水溶液中。 15 20 乂佺地，被使用於步驟（b)中的缓衝液係選自於下列： 碌酸鹽緩衝的生理鹽水(PBS)缓衝液、Tds缓錢、魏鹽 緩衝液（b〇rate buffer)以及HEPES緩衝液。在本發明的一個 較佳具體例中，被使用於步驟⑻中的緩衝液是咖緩衝液。 較佳地，在步驟⑷中，步驟（b)所形成的混合物被調整 至具有- PH值落在5:0〜9.5之範圍内，更佳為pH 8〇。 較佳地’在步驟⑷中，步驟（b)所形成的混合物之pH值係 使用一含有-選自於由驗金相氫氧化物以及氫氧化錢所 構成之群組中的化合物之驗性水溶液來予以調整。在本發 、们m例中氫氧化納水溶液被用來將步驟 (b)所形成的混合物調整至pH值為8。 被使用於本發明方法的步驟⑷中的嗜熱菌水解酶是一 種具溫度安定性的金屬蛋白酶，於其每個分子上含有*個 12 1236501 詞離子。，水解峰會從蛋白質的n端疏水性胺基酸[例 胺酉义（leucine)、苯丙胺酸（phenylaianine)]來 切斷蛋白 貝的胜肽鍵。關於嗜熱g水料的使用量，熟習此項技術 σ、見所而來作選擇，例如，每公克的動物組織可以大約 〇训2卜㈣2 g的酵素來作處理。在本發明的-個較佳具 " 所使用的嗜熱菌水解峰是源自於桿菌屬（5⑽7/似) 的嗜熱菌水解酶（Sigma Chemieal Cg.，&· L—則， USA) 〇 10 15 20 位於緩衝液内之被弄碎的動物組織在經過嗜熱菌水解 每作用後，會貫質地溶出不溶性膠原蛋白並且嗜熱菌水解 S每冒切#彳端肽膠原蛋白而成為可溶性無端肽膠原蛋白。 l浮液内的可洛性膠原蛋白含量之量測可藉由本技藝 所詳知:常用方法，例如HPAGE、蛋白質分析法。 、車乂佺地，在本案方法的步驟（e)中是使用一含有一選自 々由才丁才豕酉夂、乙酸、甲酸、鹽酸所構成之群組中的酸之酸 夜來將步知（d)所形成的混合物之pH值調整至低於 在本I明的一個較佳具體例中，步驟〇)所形成的混合 物被β酸性水溶液調整至具有一 值為（5。在本發明的 更‘具體例中，一乙酸水溶液被用來將步驟（❼所形成 的此口物之pH值調整至45以將嗜熱菌水解酶去活化。 身又而。，在本發明方法的步驟（〇所形成的混合物可 “準的離心機在大約1?_至15，_ χ g (較佳為 使用 g)的離心力下被離心歷時一充分的時間，俾以移除 勺不/合性物質。離心時間可視要被離心的容積以及 13 1236501 選自於下列群組··氯化鈉（NaCl)、硫酸鈉以及硫酸銨。有 關使用NaCl作為一沉殿劑，可參見//〇,价仏_(9 α/. “外7人 J，Controlled Release 44 ° 任擇地，若藉由pH值的調整來引起蛋白質沉澱，可使 5 用一含有一選自於由鹼金屬的氫氧化物以及氫氧化銨所構 成之群組中的化合物之鹼性水溶液來將步驟⑴所得到的上 澄液之pH值調整為8並容許靜置歷時4〜24小時，即會發 生可溶性膠原蛋白沉澱。 較佳地，在本發明方法的步驟⑴中，步驟（…所得到的 沉澱物被重建於一含有一選自於由檸檬酸、乙酸、甲酸、 鹽酸所構成之群組中的酸之酸性水溶液内。在本發明之一 較佳具體例中，步驟（h)所得到的沉澱物被重建於一含有鹽 酸之酸性水溶液内。 車乂 t地在本叙明方法的步驟⑴所得到之不含不溶性 15 物質的溶液被進一 步進行無菌處理（sterilization)並冷束乾I: Embodiment] I Detailed description of the preferred embodiment (Detailed description of the invention) It is used in food, chemical and 'has biological activity, in order to be able to manufacture industrially suitable cosmetics, medicines, medical articles, etc. on a large scale. The highly purified 10 1236501 is a substantially non-immunogenic soluble collagen. The applicant has found through various studies that the thermolysin is very suitable for use in the extraction of collagen. Therefore, the present invention provides a method for extracting soluble collagen from a collagen-rich animal tissue, which comprises the following steps: (a) immersing a collagen-rich animal tissue in a pH value 2 ~ 5 acidic aqueous solution for a sufficient period of time to swell the animal tissue; (b) the swollen animal tissue obtained in step (a) is ground and dispersed in a buffer solution; (C) Adjusting the pH value of the mixture formed in step (b) to a pH value suitable for the action of thermolySin; (d) adding an appropriate amount of thermolysin hydrolyzing enzyme to the step (c) And the mixture is mixed for a sufficient period of time, whereby the ground animal tissue present in the mixture is hydrolyzed by a thermolysin to release soluble collagen; (e) formed in step (d) The pH of the mixture was adjusted to less than 50 to deactivate the thermolysing enzyme; and (f) the mixture prepared in step (e) was centrifuged to obtain a supernatant containing soluble collagen. The carcass, the animal tissue used in step (a) is obtained from an animal selected from the group consisting of sheep, cows, pigs, sheep, and combinations thereof. Che Yan, in step (a), the animal tissue is a tissue selected from an animal and selected from the group consisting of cartilage, muscle keys, skin, and one of these 1236501 combinations. More preferably, the animal tissue is selected from the group consisting of cowskin, pigskin, oxtail, and pigtail. In a preferred embodiment of the present invention, the animal tissue used in step (a) is pig skin. Preferably, the animal tissue used in step (a) has been subjected to a defatting treatment before use. Preferably, the acidic aqueous solution used in step (a) contains an acid selected from the group consisting of citric acid, acetic acid, formic acid, and hydrochloric acid. In a preferred embodiment of the present invention, the animal tissue is immersed in an acidic aqueous solution having a pH of 3.0 for a sufficient period of time. In a more specific example of this month, the animal tissue is immersed in an aqueous acetic acid solution of 3.0. 15 20 The buffer used in step (b) is selected from the following: buffered saline buffered saline (PBS) buffer, Tds buffer, Wei salt buffer (borate buffer) And HEPES buffer. In a preferred embodiment of the present invention, the buffer used in step ⑻ is a coffee buffer. Preferably, in step (i), the mixture formed in step (b) is adjusted to have a pH value in the range of 5: 0 to 9.5, more preferably pH 80. Preferably, in step (i), the pH value of the mixture formed in step (b) is a test aqueous solution containing a compound selected from the group consisting of metallurgical hydroxide and gold hydroxide. Be adjusted. In this example, the aqueous sodium hydroxide solution was used to adjust the mixture formed in step (b) to pH 8. The thermolysin hydrolase used in step ⑷ of the method of the present invention is a temperature-stable metalloproteinase, which contains * 12 1236501 word ions per molecule. The hydrolysis peak will cleave the peptide bond of the protein shell from the hydrophobic amino acid at the n-terminus of the protein [eg leucine, phenylaianine]. Regarding the amount of thermophilic g water, familiarize yourself with this technique σ, and choose from what you see. For example, about 2 g of enzyme per gram of animal tissue can be used for treatment. In a preferred embodiment of the present invention, the thermolysate hydrolysis peak used is a Thermolysin hydrolase (Sigma Chemieal Cg., L-then, USA) derived from Bacillus (5⑽7 / like). ) 〇10 15 20 The crushed animal tissues in the buffer solution will dissolve insoluble collagen consistently after each action of the thermophilic bacteria and the thermophilic bacteria will hydrolyze every time they cut # 彳 terminal peptide collagen to become Soluble acapeptide collagen. l The measurement of the content of coulosic collagen in the floating liquid can be known in detail by this technology: commonly used methods, such as HPAGE and protein analysis. In the step (e) of the method of the present case, the use of an acid containing an acid selected from the group consisting of Cai Dingcai, acetic acid, formic acid, and hydrochloric acid is used to learn the steps. (D) The pH value of the formed mixture is adjusted to be lower than that in a preferred embodiment of the present invention. The mixture formed in step 0) is adjusted to have a value of (5. In a more specific example, an aqueous solution of acetic acid is used to adjust the pH of the substance formed in the step (❼) to 45 to deactivate the thermolysin hydrolase. In turn, in the step of the method of the present invention ( 〇The resulting mixture can be centrifuged under a centrifugal force of about 1 ° to 15 °, preferably χ g (preferably using g) for a sufficient time to remove the incompatible material The centrifugation time depends on the volume to be centrifuged and 13 1236501 is selected from the following groups: · Sodium chloride (NaCl), sodium sulfate and ammonium sulfate. For the use of NaCl as an immersion agent, see // 〇, price 仏_ (9 α /. "Foreigner J, Controlled Release 44 ° Optionally, if the pH In order to cause protein precipitation, a basic aqueous solution containing a compound selected from the group consisting of alkali metal hydroxides and ammonium hydroxide can be used to convert the supernatant obtained in step ⑴ to 5 When the pH value is adjusted to 8 and allowed to stand for 4 to 24 hours, soluble collagen precipitation will occur. Preferably, in step ⑴ of the method of the present invention, the precipitate obtained in step (... is reconstituted in a An acidic aqueous solution of an acid selected from the group consisting of citric acid, acetic acid, formic acid, and hydrochloric acid. In a preferred embodiment of the present invention, the precipitate obtained in step (h) is reconstituted in a solution containing In an acidic aqueous solution of hydrochloric acid. The solution obtained in the step of the described method without insoluble 15 substance is further sterilized and cold-dried.
案方法所得到的產物包含有低免疫原性或無免 性或無免The product obtained by this method contains low immunogenicity or non-immunity or non-immunity.
genesis)而生成膠原 15 1236501 蛋白原纖維（collagen fibril)，而該膠原蛋白原纖維可被進一 步製作成為一選自於下列之形式：可注射凝膠（injectable gel)、膠原蛋白海綿（collagen sponge)、人工皮膚敷料 (artificial skin dressing)、燒燙傷敷料（burn dressings)、血 5 管假體（blood vessel prostheses)、皮内植入物（intradermal augmentation)、心瓣假體（heart value prostheses)、肌腱與 韋刃帶假體（tendon and ligament prostheses)、硬骨與軟骨假體 (bone and cartilage prostheses)、止血作用劑（hemostatic agents)以及藥物投遞系統（drug delivery system)。 10 此外，依據本發明法所生成的膠原蛋白原纖維是低免 疫原性或無免疫原性，因此可將之應用於與膠原蛋白原纖 維有關的疾病之治療上，這包括，例如，膠原蛋白血管疾 病（collagen vascular diseases)、骨關節炎（osteoarthritis)、 骨形成不全（osteogenesis imperfect)、牙周疾病（periodontal 15 diseases)、硬皮症（Scleroderma)以及尿失禁（urinary incontinence) 〇 本發明將參照下面的實施例來作更詳細的說明，該等 實施例被提供是為達例示說明之目的，而不意欲用來限制 本發明之範圍。 20 實施例1·從豬皮來萃取可溶性膠原蛋白 從豬隻身上取得新鮮豬皮，於刮除掉脂肪部分後秤取 100 g之經脫脂的豬皮（defatted porcine skin)並予以浸泡於 一 pH值為3.0的乙酸或檸檬酸酸性水溶液内歷時6〜12天。 在這個處理過程中，該浸泡用酸性水溶液被換過一次。於 16 1236501 室溫下，將經浸潰處理後被膨脹之豬皮取出磨碎並散浮於 10 L的50 mM Tris或PBS緩衝液（pH7.2〜7.4)中，並使用5N NaOH將所形成的懸浮液之pH值調整至8.0。之後，在室溫 下予以加入〇·3 g的嗜熱菌水解酶（thermolysin)(Sigma 5 Chemical Catalog No. P15 12)來消化該散浮液内之經磨碎 的膨脹豬皮歷時4〜6小時。接著，使用IN HC1來調整懸浮 液之pH值成為4.5，俾以將嗜熱菌水解酶去活化 (inactivate)。繼而將懸浮液離心（10,000 rpm，30分鐘），丟 棄不溶的沉澱塊，而得到一含有可溶性膠原蛋白之水性上 10 澄液。 如此所得到之水性上澄液可藉由下述純化操作而被進 一步純化。將NaCl加入至上述所得到之含有可溶性膠原蛋 白的水性上澄液内直至一最終濃度為0.5 Μ。所形成的混合 物被攪:拌以使可溶性膠原蛋白被鹽析沉殿出（salt out)，接 15 著進行離心（10,000卬111，30分鐘），俾以收集所形成的可溶 性膠原蛋白沉澱物，同時將存在於上澄液内之雜質移除。 之後，將該沉澱物重建於一 pH值為2〜3之乙酸或鹽酸水溶 液内，而所形成的溶液被過濾以移除不溶性物質，繼而藉 由通經一個0_22 //過濾、器（Whatman Poly cap)來進行無菌處 20 理（sterilization) 〇 如此所得到之經重建的含有可溶性膠原蛋白之溶液可 被進一步冷凍乾燥以供長期保存或留待後續製造上之應 用。將上述所得到之溶液無菌地分裝至小管或小瓶内，繼 而於液態氮内將該等小管或小瓶快速冷束，而後在高真空 17 1236501 下=~ /東乾燥室内將被冷滚的小管或小瓶進行;東乾處理 歷日守96小時。進行;東乾時的起始溫度為_4Gt：，而終止溫度 為28±2C。該等小管或小瓶於真空下被㈣，而後可在正 常條件下被保存於代下直到需要使用之時。 5 j述所得到之含有可溶性膠原蛋白之;東乾產物可供應 用於市釗、化妝品或保健食品等之製造。 在本實施例中，豬皮約可得到16g的可溶性膠 原蛋白。 實施例2·不同的蛋白水解酶在製備可溶性膠原蛋白上的比 10 較 在膠原蛋白的純化方法中，蛋白水解酶（pr〇te〇iytic enzyme)通g被用在“端肽”膠原蛋白的切割以供回收“無端 肽”可溶性膠原蛋白。該等酵素移除“端肽，，並提供可溶性膠 原蛋白的回收。本實施例進一步比對，在使用依據本發明 15的嗜熱菌水解酶與先前技術所慣用的胃蛋白酶這兩種酵素 下，對於可溶性膠原蛋白的回收會有何影響。 在本貫施例中，豬皮的熱菌水解酶處理係參照實施例工 中所述之步驟來進行，並使用〇 〇3 g嗜熱菌水解酶來消化 5 g的豬皮。而豬皮的胃蛋白酶處理係如下所述。 2〇 將去脂的豬皮（5§)浸泡於一 pH值為3.0的1 L醋酸鈉 溶液内歷時6〜12天。於室溫下，將經浸潰處理後被膨脹 之豬皮磨碎並加入0· 1 Μ醋酸溶液以使溶液總體積達5 L。 對所形成的懸浮液予以加入〇·〇3 g的胃蛋白酶（Sigma Chemical Catalog P-6887)來消化該懸浮液内之經磨碎的膨 18 1236501 脹豬皮，並予以攪拌歷時4〜6小時。 接著，使用5 N NaOH來調整懸浮液之pH值成為8 3 旅對懸浮液予以攪拌，俾以將胃蛋白酶去活化。之後，將 懸汙液之pH值調整至PH 4.3〜4.5，繼而將懸浮液離心 5 (10,000 rpm，3〇分鐘）’丟棄不溶的沉澱塊，而得到一含有 <溶性膠原蛋白之水性上澄液。 如此所得到之水性上澄液可藉由下述純化操作而被進 -步純化。將NaCl加人至上述所得到之含有可溶性膠原蛋 白的水性上澄液内直至一最終濃度為〇5M。所形成的混合 10物被攪拌以使可溶性膠原蛋白被鹽析沉澱出（salt out),接 著進行離心⑽00 rpm，3G分鐘），俾以收集所形成的可溶 性膠原蛋白沉殿物，同時將存在於上澄液内之雜質移除。 之後，將該沉殿物重建於500 ml的乙酸或鹽酸水溶液内。 在本貫施射，各f驗組係以各為g g …與胃蛋白酶來處理5g的豬皮，並經由bca(二= 酸’曰 blcinchoninic acid)蛋白質分析（Piecre Chemical)來測定 所得到的蛋白質數量與產率，所得結果被顯示於下面表1 中。 目％囷：^解酉毎與 白上的效用比較 π π衣W 1溶性膠原蛋genesis) to produce collagen 15 1236501 collagen fibril, and the collagen fibril can be further made into a form selected from the group consisting of: injectable gel, collagen sponge , Artificial skin dressing, burn dressings, blood vessel prostheses, intradermal implantation, heart value prostheses, tendons And tendon and ligament prostheses, bone and cartilage prostheses, hemostatic agents, and drug delivery systems. 10 In addition, the collagen fibrils generated according to the method of the present invention are low immunogenic or non-immunogenic, so they can be applied to the treatment of collagen fibril-related diseases, including, for example, collagen Vascular diseases, osteoarthritis, osteogenesis imperfect, periodontal 15 diseases, scleroderma, and incontinence. The present invention will refer to The following examples are provided for more detailed description. These examples are provided for the purpose of illustration and are not intended to limit the scope of the invention. 20 Example 1. Extracting soluble collagen from pig skin. Fresh pig skin was obtained from pigs. After scraping off the fat part, 100 g of defatted porcine skin was weighed and soaked in a pH. The acidic aqueous solution of acetic acid or citric acid with a value of 3.0 lasted 6 to 12 days. During this treatment, the acidic aqueous solution was changed once. At 16 1236501, at room temperature, take out the pig skin that has been swollen after being impregnated, grind and float it in 10 L of 50 mM Tris or PBS buffer (pH 7.2 ~ 7.4), and use 5N NaOH to The pH of the formed suspension was adjusted to 8.0. Thereafter, 0.3 g of thermolysin (Sigma 5 Chemical Catalog No. P15 12) was added at room temperature to digest the ground swollen pig skin in the suspension for 4 to 6 hours. hour. Next, the pH value of the suspension was adjusted to 4.5 using IN HC1 to inactivate the thermolysin hydrolase. The suspension was then centrifuged (10,000 rpm, 30 minutes), and the insoluble pellet was discarded to obtain an aqueous supernatant solution containing soluble collagen. The aqueous supernatant obtained in this manner can be further purified by the following purification operation. NaCl was added to the above-mentioned aqueous suspension containing the soluble collagen protein until a final concentration of 0.5 M was obtained. The resulting mixture is stirred: stir to dissolve the soluble collagen by salting out, then centrifuge out (10,000 离心 111, 30 minutes), and then collect the formed soluble collagen precipitate, At the same time, impurities existing in the upper liquid are removed. After that, the precipitate was reconstituted in an aqueous solution of acetic acid or hydrochloric acid having a pH of 2 to 3, and the formed solution was filtered to remove insoluble matters, and then passed through a 0_22 // filter, Whatman Poly Cap) for sterilization. The reconstituted solubilized collagen-containing solution thus obtained can be further freeze-dried for long-term storage or for subsequent manufacturing applications. Aseptically dispense the solution obtained above into a small tube or vial, and then quickly cool the small tube or vial in liquid nitrogen, and then under high vacuum 17 1236501 = ~ Or vial; Donggan treatment lasts 96 hours. Carry on; Donggan's starting temperature is _4Gt :, and the ending temperature is 28 ± 2C. These vials or vials are emptied under vacuum and can then be stored under normal conditions until replacement when needed. 5. The obtained collagen containing soluble collagen described in 5 j; Donggan products can be supplied for the production of Shizhao, cosmetics or health food. In this example, about 16 g of soluble collagen protein can be obtained from pig skin. Example 2: The ratio of different proteolytic enzymes to the preparation of soluble collagen. In the collagen purification method, proteolytic enzymes are used in the "telopeptide" collagen. Cleavage for recovery of "endcap" soluble collagen. These enzymes remove "telomeres" and provide recovery of soluble collagen. This example further compares the use of two enzymes, the thermolysin hydrolyzing enzyme according to the invention 15 and the pepsin enzyme conventionally used in the prior art. What effect will it have on the recovery of soluble collagen. In the present embodiment, the pyrolytic enzyme treatment of pig skin is performed according to the steps described in the example process, and it is hydrolyzed using 〇03g thermophilic bacteria. Enzyme to digest 5 g of pig skin. The pepsin treatment of pig skin is as follows. 20 Soak the degreased pig skin (5§) in a 1 L sodium acetate solution with a pH value of 3.0 for 6 ~ 12 days. At room temperature, the swollen pig skin that had been subjected to the immersion treatment was ground and added with a 0.1 M acetic acid solution to make the total solution volume 5 L. The resulting suspension was added with 0.03 g of pepsin (Sigma Chemical Catalog P-6887) to digest the ground swelled 18 1236501 swelled pig skin in this suspension and stir it for 4 to 6 hours. Next, use 5 N NaOH to adjust the suspension The pH becomes 8 3 Brigade agitate the suspension to stir the stomach The protease is deactivated. After that, the pH value of the suspension is adjusted to pH 4.3 ~ 4.5, and the suspension is centrifuged 5 (10,000 rpm, 30 minutes) to discard the insoluble pellet, and a soluble collagen is obtained. The aqueous supernatant solution obtained in this way can be further purified by the following purification operation. NaCl is added to the aqueous collagen solution containing soluble collagen obtained above until a final solution. The concentration is 0 5 M. The formed mixture 10 is stirred to dissolve the soluble collagen by salting out and then centrifuged (00 rpm, 3G minutes) to collect the formed soluble collagen sink. At the same time, the impurities existing in the upper liquid are removed. After that, the sunken object is rebuilt in 500 ml of acetic acid or hydrochloric acid aqueous solution. In the usual injection, each f test group is gg… and the stomach Protease was used to treat 5 g of pigskin, and the amount and yield of the obtained protein were determined by bca (b = inch 'blcinchoninic acid) protein analysis (Piecre Chemical), and the results are shown in Table 1 below. : ^ Solution and utility on every unitary white clothing W 1 Comparative π π insoluble collagen
相較於習知方法所用的胃蛋白酶，依據本發明所揭示 20 19 1236501 的方法使用嗜熱菌水解酶來製備可溶性膠原蛋白，不但效 率佳且收穫量亦高。 此外’為確認各實驗組所得到的蛋白質是膠原蛋白， 從各實驗組取樣品（10 來作SDS_PAGE分析[7%分離凝 5膠（separating gel)，25 mM甘胺酸緩衝液]，所得結果被顯示 於第1圖中。 ‘ 麥見第1圖，第1行係表示一商業來源之膠原蛋白標 準品[Cohesion Vitrogen，C-100828]，而第 2 與第 3 行係分 別為使用胃蛋白酶與嗜熱菌水解酶處理後所得到的膠原蛋 10白產物，在第1圖左侧所標示的，，α，，、 ，，/3，，與τ，，係表示 膠原蛋白的，，α，，、，，y?，，與τ，，次單位（subunits)之所在位置。 從第1圖所示結果可以看出，依據本案方法所製得的產物 確實含有可溶性膠原蛋白。 實施例3·嗜熱菌水解酶的使用量對於可溶性膠原蛋白的萃 15 取或回收之影響 本實施例進一步評估嗜熱菌水解酶的使用量對於可溶 性膠原蛋白的萃取會有何種影響。 在母個貫驗組中，以實施例丨中所述之相同操作步驟來 處理5 g的豬皮，但以不同數量（〇〇1 g、〇〇3 g以及〇〇6幻 2〇之嗜熱菌水解酶來作酵素水解處理。各實驗組所得到的可 >谷性膠原蛋白之數量係藉由BCA蛋白質分析來作測定，所 得結果被顯示於下面表2中。 白的回收之影響 豬皮（g) 20 1236501 水解酶（g) 質（g) 回收率（〇/0) 0.01 _0J_ΎΓ 0.03 0.75 ~V5 0.825 16.5 從表2所示結果可以看出，使用低量的嗜熱菌水解酶 (0.002 g酵素/g豬皮）即已能從豬皮有效地萃取出可溶性膠 原蛋白。 實施例4.使用嗜熱菌水解晦來大規模製備可溶性膠原蛋白 5 纟實施例進—步試驗，是否能以嗜熱菌水解崎來大規 模地製備可溶性膠原蛋白，俾達產業上的生產應用。 以實施例1中所述之相同操作步驟來進行本實施例之 貫驗，但各實驗組分別使用5 g、25 g與1〇〇 §的豬皮，並 配合使用0.03 g、〇·15 g以及〇·6 g的嗜熱菌水解酶。各實 10驗組所得到的可溶性膠原蛋白之數量係藉由BCA蛋白質分 析來作測定，所得結果被顯示於下面表3中。 表3 · 不同數量之豬皮以嗜熱菌水解酶作 到的膠原蛋白回收率 用後所得 豬皮膚組織（g) 5 ——一 __ 25 100 嗜熱菌水解酶（g) 0.03 0.15 0.6 總蛋白質（g) 0.75 3.75 16 回收率（％) 15 15 16 15 從表3所示結果可以看出，欲從動物組織來製備可溶 性膠原蛋白，在使用本發明所揭示的方法下是可以達到產 業上大規模生產的程度。 實施例5·可溶性膠原蛋白的膠原纖維形成（fibriU〇genesis) 由於只有具天然三股螺旋結構的膠原蛋白才能形成膠 原蛋白原纖維（collagen fibril)(_Sarkra价0心免少五 20 Eikenberry，“Characterization 〇f Fibrous forms of Collagen,” 21 1236501Compared with the pepsin used in the conventional method, the method disclosed in the present invention 20 19 1236501 uses a thermolysin hydrolyzing enzyme to prepare soluble collagen, which has high efficiency and high yield. In addition, in order to confirm that the protein obtained in each experimental group is collagen, a sample was taken from each experimental group (10 for SDS_PAGE analysis [7% separation gel, 25 mM glycine buffer], and the results were obtained. It is shown in Figure 1. '' See Figure 1, line 1 shows a commercial-derived collagen standard [Cohesion Vitrogen, C-100828], and lines 2 and 3 are using pepsin, respectively. The collagen protein 10 white product obtained after treatment with a thermolysin hydrolase is indicated on the left side of FIG. 1, α ,,, ,, / 3 ,, and τ, which represent collagen, α ,,,,, y? ,, and τ ,, subunits (subunits). From the results shown in Figure 1, it can be seen that the product prepared according to the method of this case does contain soluble collagen. Example 3 · Impact of the use of thermolysable hydrolase on the extraction or recovery of soluble collagen 15 This example further evaluates how the use of thermolysable hydrolase will affect the extraction of soluble collagen. In the test group, the example The same operation steps are described to treat 5 g of pig skin, but the enzymes were hydrolyzed with different amounts of thermolysin hydrolyzing enzymes (0.01 g, 0.003 g, and 0.05 g). Each experimental group The amount of gluten collagen obtained was determined by BCA protein analysis, and the results are shown in Table 2. The effect of white recovery on pig skin (g) 20 1236501 hydrolase (g) quality ( g) Recovery rate (〇 / 0) 0.01 _0J_ΎΓ 0.03 0.75 ~ V5 0.825 16.5 As can be seen from the results shown in Table 2, the use of a low amount of thermolysin hydrolase (0.002 g enzyme / g pig skin) can Soluble collagen is effectively extracted from the skin. Example 4. Large-scale preparation of soluble collagen using thermophilic bacteria hydrolyzed 5 纟 The example further tests whether it is possible to prepare soluble collagen on a large scale with thermophilic bacteria hydrolyzed. Protein, production and application in the field of industry. The same operation steps described in Example 1 were used to carry out the inspection of this example, but each experimental group used 5 g, 25 g, and 100 § pig skin. Use 0.03 g, 0.15 g, and 0.6 g of thermophile Bacteriolytic enzyme. The amount of soluble collagen obtained in each test group was determined by BCA protein analysis, and the results are shown in Table 3 below. Table 3 · Different numbers of pig skins were hydrolyzed by thermophilic bacteria The collagen recovery rate obtained by the enzyme is obtained from pig skin tissue (g) 5 ——__ 25 100 Thermolysin hydrolase (g) 0.03 0.15 0.6 Total protein (g) 0.75 3.75 16 Recovery rate (%) 15 15 16 15 From the results shown in Table 3, it can be seen that the use of the method disclosed in the present invention to prepare soluble collagen from animal tissues can achieve a large-scale industrial production. Example 5. Collagen fiber formation of soluble collagen (fibriUgenesis) Since only collagen with a natural three-helix structure can form collagen fibril (_Sarkra valence 0 heart free 5 20 Eikenberry, "Characterization 〇 f Fibrous forms of Collagen, ”21 1236501
Methods in Enzymology，Vol. 82, ρρ· 127-173, 1982、，本衰氣 例進一步測試’依據本發明所揭示的方法而得到的可溶性 膠原蛋白能否形成膠原蛋白原纖維，藉此來確認該可溶性 膠原蛋白是否具有三股螺旋結構。 5 將依據實施例1所製得的可溶性膠原蛋白以丨〜3Methods in Enzymology, Vol. 82, ρρ · 127-173, 1982, and this example of qi further tests whether the soluble collagen obtained according to the method disclosed in the present invention can form collagen fibrils, thereby confirming the Whether soluble collagen has a triple helix structure. 5 Soluble collagen prepared according to Example 1
mg/ml的〉辰度溶於1〇 mM HC1内。另外，以下列方式來製 備中和緩衝液·以氫氧化納來滴定填酸氫二納（dis〇dium phosphate)而形成一具有〇·2 M磷酸氫二鈉與J mM Na〇H 的/谷液（pH 11 _2)。在藉由中和反應來達成原纖維組合之 1〇前，將上述所準備的含有可溶性膠原蛋白之溶液以及中和 緩衝液存放在要用來進行操作的培育溫度於15它下歷時3〇 分鐘至數小時甚而到過夜亦可。在攪拌下將1/9容積的中 和緩衝液快速地加入至該含有可溶性膠原蛋白之溶液内並 快速地混合歷時約5分鐘，這會對該含有可溶性膠原蛋白 b之溶液提供-為7·2土〇·2的最終pH值以及一為2〇福的填 酸鹽濃度。所形成的混合物被快速地混合歷時約5分鐘， 接者予以緩緩地加入剩餘的中和緩衝液，並將所形成的混 合物培育在下歷時歷時3〇分鐘至數小時甚而到過夜 亦可。藉由在 l〇〜15t：下以 17，⑽0rpm(s〇vallRC5c GSA) 2〇來離心歷時15分鐘而收獲膠原蛋白原纖維。沉殺丸㈣㈣ 的蛋白質濃度係藉由BCA蛋白質分析來測定。 …另外，將依據實施例2中所述之使用胃蛋白酶處理而 …i々可岭性膠原蛋白來與依據本發明的可溶性膠原蛋白 進翁原纖維形成能力上比較，所得實驗結果被歸納於表4 22 1236501 中。 表= = 上胃蛋白酶的處理而得到的可 嗜熱菌水魅_酿 可溶性膠原蛋白（111§) 50 Too~~ ~~ — 150 原纖維（mg) 37.2 iTTTj ΓΊΪ275~ 形成百分比（°/〇) 74.4 73.1~ 75 100 74.8 從表4所示結果可以看出，相較於先前技術所用的胃 蛋白酶，依據本發明所揭示的方法使用嗜熱菌水解酶而襲 得的可溶性膠原蛋白確實具有膠原蛋白之天然三股螺旋結 構’故而能順利形成膠原蛋白原纖維。 本實施例所形成的膠原蛋白原纖維可依照本技藝已知 的技術被進-步配方而形成可注射凝膠（injectaMe㈣、膠 10 原蛋白海綿（C〇llagen sponge)、人工皮膚敷料㈣出㈣仙 dressing)等’或是被應用於形成各式各樣的經交聯的產品 上。 於本說明書中被引述之所有專利和文獻以其整體被併 15 入本案作為參考資料。若有所衝突時，本案詳細說明（包含 界定在内）將佔上風。 雖然本發明已參考上述特定的具體例被描述，明顯地 在不背離本發明之範圍和精神之下可作出报多的修改和變 化。因此意欲的是，本發明僅受如隨文檢附之申請專利範 圍所示者之限制。 20 【圖式簡單說明】 第1圖是-凝膠電泳圖片，其中第i行是一商業來源 之膠原蛋白標準品，而第2行與第3行分別為後面的實施 23 1236501 例4中藉由胃蛋白酶與嗜熱菌水解酶之萃取處理所得到的 膠原蛋白，各以10 gg的樣品來作SDS-PAGE分析。 【圖式之主要元件代表符號表】The concentration of mg / ml was dissolved in 10 mM HC1. In addition, a neutralization buffer was prepared in the following manner. Titration of disodium phosphate with sodium hydroxide was performed to form a / valley with 0.2 M disodium hydrogen phosphate and J mM NaOH. Liquid (pH 11 _2). Before the fibril combination is reached 10 by the neutralization reaction, the solution containing the soluble collagen and the neutralization buffer prepared above are stored at an incubation temperature of 15 for 30 minutes at a temperature for operation. It can be up to several hours or even overnight. Add 1/9 volume of neutralization buffer to the solution containing soluble collagen under stirring and quickly mix for about 5 minutes. This will provide the solution containing soluble collagen b-for 7 · 2 The final pH of soil 0.2 and the filling salt concentration of 20 fold. The resulting mixture is quickly mixed for about 5 minutes, and the remaining neutralization buffer is slowly added, and the resulting mixture is allowed to incubate for the next 30 minutes to several hours or even overnight. Collagen fibrils were harvested by centrifugation at 10 ~ 15t: 17, rpm (sovallRC5c GSA) 2 for 15 minutes. The protein concentration of Shensha Pill was determined by BCA protein analysis. … In addition, the use of pepsin treatment according to the description in Example 2 ... i々 ridge collagen is compared with the ability of the soluble collagen into fibril formation according to the present invention, and the experimental results obtained are summarized in the table 4 22 1236501. Table = = Thermophilic water charm obtained by treatment with upper pepsin _ brewed soluble collagen (111§) 50 Too ~~ ~~ — 150 fibril (mg) 37.2 iTTTj ΓΊΪ275 ~ formation percentage (° / 〇) 74.4 73.1 ~ 75 100 74.8 As can be seen from the results shown in Table 4, compared to the pepsin used in the prior art, the soluble collagen derived from the use of thermolysin according to the method disclosed in the present invention does have collagen The natural three-stranded spiral structure 'can smoothly form collagen fibrils. The collagen fibrils formed in this example can be further formulated in accordance with techniques known in the art to form injectable gels (injectaMe㈣, gel 10 sponges, artificial skin dressings)仙 dressing), etc. 'or is used to form a variety of cross-linked products. All patents and documents cited in this specification are incorporated by reference in their entirety. In case of conflict, the detailed explanation (including the definition) in this case will prevail. Although the invention has been described with reference to the specific examples described above, it will be apparent that many modifications and changes can be made without departing from the scope and spirit of the invention. Therefore, it is intended that the present invention is limited only by those indicated in the scope of patent applications attached to the document. 20 [Schematic description] Figure 1 is a gel electrophoresis picture, in which the i-th row is a collagen standard of commercial origin, and the second and third rows are respectively implemented later. 10 gg samples of collagen obtained from the extraction treatment of pepsin and thermolysin hydrolase were analyzed by SDS-PAGE. [Representation of the main components of the diagram]
24twenty four