200407193 玖、發明說明： 【發明所屬之技術領域】 本發明係關於磷酸鈣奈米顆粒和至少一種蛋白質之膠綠 分散液。本發明亦關於由前述分散液凍乾獲得的奈米顇粒 及由烺燒經凍乾奈米顆粒獲得的顆粒。 最後，本發明亦關於此等分散液和此等奈米顆粒之製備 方法及用途。 【先前技術】 在很多產物中用加入鈣化合物源以使該產物富含鈣，而 作為消費者需要之機能。在尋求用鈣補充食品、飲料或食 品製備物時，這特別為食品領域之情況。在以（例如）預防骨 質疏鬆症或治療骨症為目標將鈣化合物源加入錠劑或溶液 時，這亦為醫藥領域之情況。 用舞補充產物的已知技術在於加入可溶性鈣鹽，例如， 葡糖酸鈣。 用磷酸鈣作為鈣源亦為已知習慣做法，特別以分散液形 式。 然而’在使用磷酸鈣之例中，所得分散液不穩定。 此外，難以獲得為具有各向同性形態學之羥磷灰石之前 驅體之磷酸舞粉末。實際上，巍磷灰石習知顯示各向異性 形態學。 然而’目前技術不能夠獲得穩定且其形態學受控制之化 合物。 為滿足工業公司需要，已有必要發現提供可控制形態學 85420 -6- 200407193 且為小尺寸穩定化合物的其他方法。 【發明内容】 ，因此’本發明提出解決的問題為提供-種穩定、奈米, '、且具有經控制形態學之化合物，特別以分散液形式。 太伞匕為目的根據第—具體實施例，本發明提供鱗酸杂 :米顆粒和至少-種蛋白質之膠態分散液，其中該奈米菊 ^大小係介0和300奈米之間，且其中該奈米顆粒之用 怨為球形。 顆粒，該奈米 Ο ，顆粒特徵為 根據第二具體實施例，本發明亦提供奈米 顆粒特徵為它們由上述膠態分散液凍乾獲T'得 根據第三具體實施例，本發明係提供顆粒 它們由烺燒上述奈米顆粒獲得。 本發明一個主題為亦為製備磷酸鈣奈米顆粒和至少一種 蛋白質之膠態分散液之方法，其特徵為包括以下步驟： 形成一種包含鈣錯合劑和鈣源之混合物， (b) 將至少一種蛋白質加入自步驟⑷得到的媒介物， (c) 將磷源加入自步騾（b)得到的媒介物， (d) 加熱自步驟（c)得到的媒介物。 本發明一個主題亦為具有磷酸鈣奈米顆粒和至少一種蛋 白質之膠態分散液用於食品、化妝品及醫藥工業之用途。 本發明另一主題亦為奈米顆粒或經烺燒奈米顆粒用於食 品、化妝品及醫藥工業之用途。 本發明亦具有提供可為羥磷灰石[CaiG(p〇4M〇H)2]前驅 體之鈣源之優點，羥磷灰石為構造骨骼期間沈積於骨基質 85420 200407193 上的礦物質。 本發明亦具有提供磷酸鈣之膠態分散液及/或奈米顆粒 之優點，膠態分散液及/或奈米顆粒可用於牙科領域以避免 脫礦質，特別用於牙膏或漱口劑。 本發明亦具有不使用酪蛋白之優點。 本發明的另一優點為提供膠態分散液及/或奈米顆粒，二 者可提供以尚濃度約加入它們的產物。 本發明亦具有提供經烺燒顆粒之優點，例如，具有球形 各向同性形態之羥磷灰石，這可有（例如）更大色譜優點。y 在閱讀純以非限制方式給出的以下說明和實例時，本發 明的其他優點和特徵更變得清晰明顯。 x 【實施方式】 本發明首先關於具有磷酸鈣奈米顆粒和至少一種蛋白質 <膠態分散液，其中該奈米顆粒之大小係介於50和300奈米 之間且其中該奈米顆粒之形態為球形。 所根據本發明奈米顆粒之膠態分散液包含i少一種蛋白 貝最好為至少一種較佳可溶於水性相之蛋白質，特別可 溶於水。 根據本發明’”至少一種蛋白質，，亦指至少一種多肽。 根據本發明之蛋白質可為變性蛋白質形式。 ”較佳利用天然源蛋白質、經改質以改良其在水性相的溶 "之天”卷源蛋白質和天然及/或改質多肽，但酪蛋白排除 在：此等蛋白質可或可不含以磷為主的基團。 u作為蛋白質特別提到的有至少一種選自由溶菌酶、大 85420 200407193 豆蛋白、自可溶乳相衍生的蛋白 吐从疋丄 員以及自各種抗乳血清衍 生的蛋白質所組成之群組之蛋白質。 目仃 作為植物源蛋白質實例可提 從j的有源自观且、蠶豆、羽 m 蠶且、小扁豆、小麥、女夫m水 盎夫釦，丄 大麥、黑麥、玉米、稻米、 為H 大豆、落花生、蔡花、油菜籽、野芥子（特別 =質挪子(乾挪子肉)、㈣、葬麻、馬铃著和甜菜根 作為動物蛋白源以非限制方式提料有，有乳蛋白質（如 =白’例如’卵清蛋白）和自牛、豬或魚的骨及/或皮得到的 蛋白質（例如，明膠）。 作為改質天然源蛋白質提到的有大豆蛋白質，如由國際 蛋白質技術公司（Protein Technologies Internati〇nai)或杜邦 耐默斯公司（Dupont de Nemours)銷售者。 根據本發明之奈米顆粒之膠態分散液不含酪蛋白。這意 味在根據本發明製備此等奈米顆粒膠態分散液期間不加Z 2蛋白或酸蛋白鹽。另一方面，由於用至少一種衍生自可 乳相、各種類型抗乳血清或任何其他部分之蛋白質，可 有微量酪蛋白或其鹽存在於奈米顆粒膠態分散液中。 根據本發明之奈米顆粒膠態分散液亦可包含至少一種鈣 -錯合劑。 該鈣錯合劑可有利具有至少一個羧酸鹽官能，較佳至少 三個羧酸鹽官能，更佳至少四個羧酸鹽官能。 錯合劑最佳具有特徵為pKa值（酸度常數餘對數）在〗5和8 之間的各種酸官能及特徵為pKc值（錯合常數的餘對數）在2 85420 200407193 和8之間（較佳在2和6之間）的具有鈣離子錯合物之離解常 數。 根據本發明之較佳鈣錯合劑為式（NaCOO)CH2CH2_ CH(C00Na)N(CH2C00Na)2之N，N_二乙酸穀胺酸鈉鹽。 在某些例中，鈣錯合劑可在組成膠態分散液的奈米顆粒 之内及/或表面，但亦可在分散液的連續相或同時在二 内。 根據本發明之膠態分散液之奈米顆粒一般由3和20奈米 間直徑的基本微晶組成。 基本微晶’，指為奈米顆粒組分之微晶。此等基本微晶最 佳具有3和20奈米大小之間的各向同性形態，且可由透射電 子顯微法見到。 由X-射線衍射觀察，根據本發明膠態分散液奈米顆粒中 所含的磷酸鈣之結構一般為無定形。這一射線衍生特徵 對由根據本發明凍乾膠態分散液獲得的粉末進行。利用P3! 固體NMR··，此等磷酸妈特徵為，主線化學移動在2 5和4 ppm間，較佳在3.4 ppm左右，而附線化學移動在5和7 ppm 之間’較佳在6.4 ppm左右。3·4 ppm左右的線一般歸因於碳 酸化輕磷灰石。 根據一較佳具體實施例，根據本發明膠態分散液之奈米 顆粒主要具有球形形態。它們最佳具有奈米大小，直徑在 50和300奈米之間。 ’’球形形態’’指具有由R=大直徑/小直徑比界定的形狀指 數特徵之形態學。 85420 -10 - 200407193 在本發明之例中，這一比R最佳趨向！至15，較佳1至 1·25，更佳 1 至 1.1。 根據本發明膠體分散液的此等奈米顆粒以大於或等於6〇 %數f平均尤單一化奈米顆粒單一化，較佳大於或等於8〇 %，更佳大於或等於90%。 該大小分佈一般為單分散。 π單分散’’指奈米顆粒大於均勻，且其特徵為r=(d9G_di〇)/2 to比在〇·〇5和〇·5之間，較佳在〇 〇5和〇 25之間，最佳在〇 〇5 和0.125之間。 可用冷凍ΤΕΜ技術測定基本顆粒的大小、單分散性及聚 集態。這使由透射電子顯微法（ΤΕΜ)觀察在天然媒介物（為 水）中冷凍保持的樣品成為可能。冷凍在液態乙烷中對约％ 至100奈米薄膜進行。利用4;東ΤΕΜ，顆粒的分散態充分保 持，且為真實媒介物呈現之代表。 根據本發明膠態分散液之奈米顆粒展示0 5/1和6/1間之 磷酸鈣/蛋白質比，表示為磷酸鈣質量/蛋白質質量。 根據本發明膠態分散液之奈米顆粒有時具有均勻鱗酸躬 :蛋白質《組合物；即，例如，奈米顆粒由均勻產生的磷 鉍鈣基本微晶和蛋白質之組合組成，或在奈米顆粒周圍具 有較高磷酸鈣微晶濃度之不均勻核_殼類型組合組成。組合 句勻度可由透射冷凍顯微法顯示（杜伯查特法（D汕⑻het 麵d))。磷酸鈣的更大對比由與蛋白質組分比較觀察。 根據本發明之奈米顆粒之膠態分散液最佳具有4和8間之 PH ’較佳在5和7.5之間。 85420 200407 或ΓΓΓΓ奈米顆粒之表面電荷依蛋白質性質可為正性 同PHW Λ在6範圍之ΡΗ，膠態分散液之奈米顆粒在相 蛋白質_電荷相同的表面電荷。當蛋 = 容相蛋白或如大豆蛋白之蛋白質時，膠態分散液之 奈未顆粒顯現負電荷。者 從 時，它們顯現正電荷。Γ匕等表面=為溶菌酶類型蛋白質 數输製電泳電勢曲線決定 廷何可由（例如）作為阳函200407193 (1) Description of the invention: [Technical field to which the invention belongs] The present invention relates to a colloidal green dispersion of calcium phosphate nanoparticle and at least one protein. The present invention also relates to nano particles obtained by lyophilization of the aforementioned dispersion liquid and particles obtained by roasting and lyophilizing the nano particles. Finally, the present invention also relates to a method for preparing these dispersions and these nano particles and uses thereof. [Prior art] In many products, a calcium compound source is added to make the product rich in calcium, and as a function required by consumers. This is particularly the case in the food sector when seeking to supplement foods, beverages or food preparations with calcium. This is also the case in the medical field when calcium sources are added to lozenges or solutions with the goal of, for example, preventing osteoporosis or treating osteoporosis. A known technique for supplementing products with dance consists in adding a soluble calcium salt, for example, calcium gluconate. It is also known practice to use calcium phosphate as a calcium source, particularly in the form of a dispersion. However, in the case of using calcium phosphate, the obtained dispersion was unstable. In addition, it is difficult to obtain a phosphoric acid dance powder which is a precursor of hydroxyapatite having an isotropic morphology. In fact, the apatite habit shows anisotropic morphology. However, 'current technology is unable to obtain compounds which are stable and whose morphology is controlled. To meet the needs of industrial companies, it has been necessary to find other ways to provide controllable morphology 85420 -6- 200407193 and small size stable compounds. [Summary of the Invention] Therefore, the problem addressed by the present invention is to provide a stable, nanometer, and controlled morphology compound, especially in the form of a dispersion. For the purpose of the parasol, according to the first embodiment, the present invention provides a colloidal dispersion of linoleic acid: rice particles and at least one protein, wherein the size of the nanochrysanthemum is between 0 and 300 nanometers, and Among them, the nano particles are spherical. The granules, the nano-particles, are characterized according to the second embodiment, and the present invention also provides the nano-particles, characterized in that they are lyophilized from the above colloidal dispersion to obtain T ′. According to the third embodiment, the present invention provides Particles They are obtained by sintering the above-mentioned nano particles. A subject of the present invention is also a method for preparing a colloidal dispersion of calcium phosphate nanoparticle and at least one protein, characterized in that it comprises the steps of: forming a mixture comprising a calcium complexing agent and a calcium source, (b) adding at least one The protein is added to the vehicle obtained in step (i), (c) a phosphorus source is added to the vehicle obtained in step (b), and (d) the vehicle obtained in step (c) is heated. A subject of the present invention is also the use of a colloidal dispersion with calcium phosphate nano particles and at least one protein for the food, cosmetic and pharmaceutical industries. Another subject of the present invention is also the use of nano particles or braised nano particles for the food, cosmetics and pharmaceutical industries. The present invention also has the advantage of providing a calcium source that can be a precursor of hydroxyapatite [CaiG (PO4MOH) 2], which is a mineral deposited on bone matrix 85420 200407193 during bone construction. The present invention also has the advantage of providing a colloidal dispersion of calcium phosphate and / or nano particles. The colloidal dispersion and / or nano particles can be used in the dental field to avoid demineralization, especially for toothpaste or mouthwash. The invention also has the advantage of not using casein. Another advantage of the present invention is to provide a colloidal dispersion and / or nano particles, both of which can provide a product in which they are added at about a concentration. The invention also has the advantage of providing calcined particles, for example, hydroxyapatite with a spherical isotropic morphology, which may have, for example, greater chromatographic advantages. y Other advantages and features of the present invention will become more apparent when reading the following descriptions and examples given purely in a non-limiting manner. [Embodiment] The present invention firstly relates to a calcium phosphate nanoparticle and at least one protein < colloidal dispersion, wherein the size of the nanoparticle is between 50 and 300 nanometers and the nanoparticle is The shape is spherical. The colloidal dispersion of the nanoparticle according to the present invention contains at least one protein, preferably at least one protein which is preferably soluble in the aqueous phase, and particularly soluble in water. According to the present invention "" at least one protein, also refers to at least one polypeptide. The protein according to the present invention may be in the form of a denatured protein. "It is preferred to use naturally derived proteins, modified to improve their solubility in the aqueous phase." "Volume-derived proteins and natural and / or modified polypeptides, but casein is excluded: these proteins may or may not contain phosphorus-based groups. U As a protein, at least one selected from lysozyme, 85420 200407193 Soy protein, protein derived from soluble milk phase, vomiting proteins from groups and proteins derived from various anti-milk serums. As an example of plant-derived proteins, extracts derived from j Kanji, fava beans, feather m, faba beans, lentils, wheat, husband and wife, water snails, barley, rye, corn, rice, H soybean, groundnut, Caihua, rapeseed, wild mustard (Special = Chinois (dried chili meat), crickets, burlaps, horse bells, and beetroots are extracted in an unrestricted manner as animal protein sources, including milk proteins (such as = white ', such as' ovalbumin'), and from cattle Protein (eg, gelatin) derived from the bones and / or skin of pigs or fish. Soy protein is mentioned as a modified natural source protein, such as by Protein Technologies Internationai or DuPont Nemes (Dupont de Nemours) seller. The colloidal dispersion of nano particles according to the present invention does not contain casein. This means that no Z 2 protein or acid protein is added during the preparation of these nano particle colloidal dispersions according to the present invention. Salt. On the other hand, trace amounts of casein or a salt thereof may be present in the nanoparticle colloidal dispersion due to the use of at least one protein derived from a milkable phase, various types of anti-milk serum, or any other part. According to the present invention The nanoparticle colloidal dispersion may also contain at least one calcium-complexing agent. The calcium complexing agent may advantageously have at least one carboxylate function, preferably at least three carboxylate functions, more preferably at least four carboxylate functions Functionality. The complex has the best pKa value (residual logarithm of acidity constant) between 5 and 8, various acid functions and pKc value (remainder logarithm of complexation constant) of 2 85420 200407193 and 8 (preferably between 2 and 6) have dissociation constants with calcium ion complexes. A preferred calcium complexing agent according to the present invention is the formula (NaCOO) CH2CH2_CH (C00Na) N (CH2C00Na) N, N_diacetic acid glutamine sodium salt. In some cases, the calcium complexing agent may be within and / or on the surface of the nano particles that make up the colloidal dispersion, but may also be in the continuous phase of the dispersion Or at the same time. The nano particles of the colloidal dispersion according to the present invention are generally composed of basic crystallites with a diameter between 3 and 20 nanometers. "Basic crystallites" refer to the microcrystals of the nanoparticle components. The isobasic crystallites preferably have an isotropic morphology between 3 and 20 nanometers in size and can be seen by transmission electron microscopy. As observed by X-ray diffraction, the structure of calcium phosphate contained in the colloidal dispersion nano particles according to the present invention is generally amorphous. This ray-derived feature is performed on powders obtained from lyophilized colloidal dispersions according to the present invention. Using P3! Solid NMR ··, these phosphates are characterized by a mainline chemical shift between 25 and 4 ppm, preferably around 3.4 ppm, and a linear chemical shift between 5 and 7 ppm ', preferably at 6.4 About ppm. Lines around 3.4 ppm are generally attributed to carbonized light apatite. According to a preferred embodiment, the nano particles of the colloidal dispersion according to the present invention mainly have a spherical morphology. They are preferably nano-sized with diameters between 50 and 300 nanometers. "'Spherical morphology'" refers to a morphology having a shape index characteristic defined by R = large diameter / small diameter ratio. 85420 -10-200407193 In the example of the present invention, this ratio R is the best trend! To 15, preferably 1 to 1.25, and more preferably 1 to 1.1. These nano particles according to the colloidal dispersion of the present invention are singularized with an average number of 60% or more. The nano particles are singularized, preferably 80% or more, and more preferably 90% or more. The size distribution is generally monodisperse. `` π monodisperse '' means that the nano particles are larger than uniform, and is characterized by a ratio of r = (d9G_di〇) / 2 to between 0.05 and 0.5, preferably between 0.05 and 0.25, The best is between 0.05 and 0.125. Freeze TEM technology can be used to determine the basic particle size, monodispersity and aggregation state. This makes it possible to observe transmission frozen samples in natural media (as water) by transmission electron microscopy (TEM). Freezing is performed on liquid to about 100 to 100 nm films in liquid ethane. With 4; East TEM, the dispersed state of the particles is fully maintained, and it is representative of the representation of real media. Nanoparticles of a colloidal dispersion according to the present invention exhibit a calcium phosphate / protein ratio between 0 5/1 and 6/1, expressed as calcium phosphate mass / protein mass. Nanoparticles of colloidal dispersions according to the present invention sometimes have a uniform phosphonate: protein composition; that is, for example, the nanoparticle is composed of a uniformly produced combination of basic microcrystals of phosphorous bismuth calcium and protein, or The composition of heterogeneous core-shell type with high calcium phosphate crystallite concentration around rice grains. The uniformity of the combined sentence can be displayed by transmission freezing microscopy (Dubachart method (D Shan⑻het surface d)). A larger contrast of calcium phosphate is observed by comparison with the protein component. The colloidal dispersion of nano particles according to the present invention preferably has a pH 'between 4 and 8 and preferably between 5 and 7.5. 85420 200407 or ΓΓΓΓ The surface charge of the nanoparticle can be positive depending on the nature of the protein. PHW Λ is in the range of 6; the nanoparticle of the colloidal dispersion has the same surface charge as the protein_charge. When egg = phase-contained protein or protein such as soy protein, the nanoparticle of the colloidal dispersion appears negatively charged. They appear positive when charged. The surface of Γ dagger etc. is determined by the lysozyme type protein. The electrophoresis potential curve is determined by Ting He (for example) as a positive function.
旦發明奈米顆粒之膠態分散液最佳具有作為 里敬度表示的〇.2和2_間之畴酸舞濃度。 I 能膠態分散液可由後濃縮或超_縮。膠 …i夜可具有作為約表示的大於_弓之濃产。 發明之膠態分散液可包含自舞和韻 :含締合陰離子和陽離子生成的殘餘鹽，如_、 白1、而4而3、如或咖3。它們亦可包含殘餘 貝鈣離子/或游離態的磷酸根離子。 液:始=:蛋白質溶液中的化合物村存在於膠態分散‘ 散液界疋化合物的殘餘濃度可由超過滤純化膠態分 二二，主二本發明關於由上述膠態分散_獲得 奈米顆粒Γ又其餘邵分中’它們有時被稱為經准乾 較發明的此等奈米顆粒係根據習知隸技術製備， 在由超過濾欲經凍乾的膠態分散液清洗後。 85420 -12- 200407193 顆粒可再分散於水，以得到膠態 根據本發明的這些奈米 分散液。 Ώ此自根據本發明〈奈米顆粒，可製備最佳具有上述 分散特徵的膠態分散液。Once the colloidal dispersion of nano particles is invented, it is preferable to have a domain acid concentration between 0.2 and 2 ° as expressed by Rex. I can colloidal dispersions can be post-concentrated or super-shrink. Gum ... i may have a concentration greater than _ Gongzhi as expressed by approx. The colloidal dispersion of the invention may contain self-dancing and rhyme: containing residual salts generated by associating anions and cations, such as _, white 1, and 4 and 3, such as or cacao 3. They may also contain residual calcium ions and / or free phosphate ions. Liquid: Start =: Compounds in protein solution exist in colloidal dispersion 'Residual concentration of 疋 compounds in the liquid boundary can be purified by ultrafiltration. The colloidal fraction can be divided into two. The present invention relates to obtaining nano particles from the colloidal dispersion. Γ and the rest, they are sometimes called quasi-dried. These nano particles are invented according to conventional techniques and are washed by ultrafiltration colloidal dispersion to be lyophilized. 85420 -12- 200407193 The particles can be redispersed in water to obtain a colloidal nano dispersion according to the invention. In this way, according to the present invention, nanoparticle, a colloidal dispersion having the above-mentioned dispersion characteristics can be prepared optimally.
由簡單使此等隸奈米㈣再分散於水獲得的分散液之 膠體穩定性較佳大於1夭，& & L 〜 、天較佳大於1星期，較佳在無菌觀 祭分散液條件完全超時穩定。 在分散液之例中，此等奈米顆粒包括至少—種具有與以 上界定者相同特徵之蛋白質。 根據本發明第_王題之奈米顆·粒中所含磷酸_之結構與 對本發明第一主題所述相同。 凍乾顆粒亦可包含鹽，如Naa、NaN〇3、NH4N〇3、NH4C1、 KN〇3 或 KC1 〇 象乾顆粒亦可包含水。百分比以重量計，水之量在01和 10%之間，較佳1和8%之間。 根據-較佳具體實施例，根據本發明之奈米顆粒具有主 要球形形H，為奈米大小，具有單分散分佈，且有時具有 均勻磷酸#5和蛋白質之組合物，如以上關於分散液奈米顆 粒之描述。 磷酸鈣/蛋白質比與對分散液奈米顆粒所述者相同。 最後，本發明亦關於特徵為由再懸浮經凍乾奈米顆粒獲 得的膠態分散液。 根據第二王題，本發明關於由烺燒上述奈米顆粒獲得且 自身由凍乾上述膠態分散液獲得的顆粒。 85420 -13- 200407193 懷燒較佳在空氣或惰性氣氛域後在空氣下進行，較佳 以大於或等於4Gn:/分鐘之溫度梯度進行。 :佳適當進行閃馈燒’即，引入在娘燒溫度預熱的烘箱 在此懷燒溫度保持2分鐘至1小時時間。 800〇。〇車乂佳門在2〇〇C和1 〇〇(Γ(：間溫度進行懷燒，較佳在3〇0和 ”：=製造具有各向同性形態之馈燒粉末，較佳使用具高 -巧.蛋自質”質量比（即，2:1至6:1範圍質量 可再分散粉末。 較佳故燒具有大於或等於3之_酸蛋白質比之經康乾 奈米顆粒。 據本發明之經娘燒顆粒最佳為視需要凝 形態，且較佳為奈米大小。 ,此等_燒顆粒在㈣後具有鱗灰石類龍構，亦稱為 ㈣灰石’較佳為式cw(p〇4)6(〇h)2、Ca2P2〇7、CaHP〇4或 4)2之4型，或另外為單獨無定形續酸鈣或作為混合 根據本發明之經烺燒顆粒可包含Naa、NaN〇3、ΝΗ4Να 或NH4CI類型鹽。 根據本發明之經烺燒顆粒可且在很少情況下包含微量蛋 或夕肤或錯合劑或其源自蛋白質或多肽或錯合劑熱解 的殘餘物。 μ 根據本發明之經烺燒顆粒一般無水。The colloidal stability of the dispersion obtained by simply re-dispersing these nanometers in water is preferably greater than 1%, & & L ~, and the day is preferably greater than 1 week, preferably under the conditions of a sterile dispersion solution Full timeout stability. In the case of a dispersion, these nano particles include at least one protein having the same characteristics as those defined above. According to the present invention, the structure of the phosphoric acid contained in the nano particles and grains is the same as that described for the first subject of the present invention. The lyophilized particles may also contain salts, such as Naa, NaNO3, NH4NO3, NH4C1, KNO3, or KCI1. The dry particles may also include water. The percentages are by weight and the amount of water is between 01 and 10%, preferably between 1 and 8%. According to a preferred embodiment, the nanoparticle according to the present invention has a predominantly spherical shape H, is the size of a nanometer, has a monodisperse distribution, and sometimes has a composition of uniform phosphoric acid # 5 and protein, as described above for the dispersion Description of nano particles. The calcium phosphate / protein ratio is the same as described for the dispersion nanoparticle. Finally, the invention also relates to a colloidal dispersion characterized by resuspending lyophilized nanoparticle. According to the second title, the present invention relates to particles obtained by calcining the above-mentioned nanoparticle and itself obtained by lyophilizing the above-mentioned colloidal dispersion. 85420 -13- 200407193 It is preferred to carry out scorching in air or in an inert atmosphere, preferably under a temperature gradient of 4 Gn: / min or more. : It is better to perform flash-feeding, that is, to introduce an oven that is preheated at the simmering temperature, and keep the simmering temperature for 2 minutes to 1 hour. 800. 〇Che Jia Jiamen burns at 2000C and 100 (Γ (:), preferably at 3000 and ": = to produce feed powder with isotropic morphology, preferably with high -Qiao. Egg self-quality "mass ratio (ie, mass redispersible powder in the range of 2: 1 to 6: 1. It is better to burn Kanggan Nano particles with an acid-protein ratio of 3 or greater. According to the present The invented Jingniang granules are preferably in the form of coagulation as needed, and are preferably the size of nanometers. These sintered granules have a limestone-like dragon structure after sacrifice. cw (p〇4) 6 (〇h) 2, Ca2P207, CaHP〇4 or 4) 2 type 4 or additionally amorphous calcium continuous acid alone or as a mixture of calcined particles according to the present invention may comprise Naa, NaNO3, ΝΗ4Να or NH4CI type salts. The burned granules according to the present invention may and, in rare cases, contain traces of eggs or skin or complexing agents or residues derived from the pyrolysis of proteins or peptides or complexing agents The calcined particles according to the invention are generally anhydrous.
根據本發明之經烺燒顆粒最佳展現各向同性，且比值R 85420 -14- 200407193 (R=大直徑（L)/小直徑⑴）最佳趨向1至1.5，較佳丨至丨.3 β 根據第四主題，本發明關於—種製備根據本發明之膠態 分散液之方法，其包括以下步驟： U)形成一種包含鈣錯合劑和鈣源之混合物， (b)將至少一種蛋白質加入自步驟⑷得到的媒介物， (C)將磷源加入自步騾（b)得到的媒介物， (d)加熱自步騾（c)得到的媒介物。 步驟⑷中所用㈣最佳為_鹽或氫氧化避或碳酸药。可 特別提到的有_氯化_、硝酸約、熟石灰及碳酸約。 ㈣度在步驟⑷生成的混合物中有利最多1M，較佳最多 〇·5Μ 〇 根據本發明製備方法之步驟⑷特徵為，最佳調節自步驟 ⑷得到媒介物之ρΗ，以獲得5和7間之ρΗβρΗ習知由加入Ηα 或hno3調節。 万法《步驟（a)以最佳在01和3間之莫耳比Ra進行，Ra= 鈣錯合劑/鈣。 在步驟（b)中，所用蛋白質溶液一般由簡單在軟化水中分 散蛋白質獲得。 步騾0)中所用磷源最佳為磷酸鹼金屬或銨鹽。可特別提 到的有磷酸二氫鈉（NaHJO4)或磷酸單氫鈉（Na2Hp〇4)或磷 故一氫铵（NH4(H2P〇4))或磷酸單氫銨（(而4)2(Hp〇4))。 在根據本發明製備期間及加入根據步驟（c)之磷源前，最 佳將媒介物之pH調節到4.5和8間之值。 方法之步驟（c)以最佳在〇·3和6間之質量比灿進行，Rb= 85420 -15- 200407193 磷酸鈣/蛋白質。 方法之步驟（c)以最佳在1和4間之莫耳比Rc進行，Rc二鈣 莫耳數/磷莫耳數。 加入反應劑在控制速率或較佳暫態且在環境溫度進行。 在万法之步驟（d)過程將自步騾（C)得到的媒介物加熱到 取佳在20 c和90°C間之溫度。此步騾根據習知熱處理方法 進行。 幸乂佳將自步騾（c)得到的媒介物引入在熱處理溫度預熱的 恒溫箱。熱處理時間一般在2小時和24小時之間，較佳在6 小時和20小時之間。 然後可由超過濾用軟化水清洗使膠態分散液純化。由超 匕濾/目洗此夠除去殘餘游離鹽及錯合劑。膠態分散液亦可 由超過濾濃縮。 研可將抗菌劑加入所得膠態分散液。 根據本發明第五主題’本發明關於根據本發明之膠態分 散液或用根據本發明製備方法獲得的膠態分散液用於食 品、化妝品及醫藥工業之用途。 本發明亦關於根據本發日月之隸奈米顆粒和根據本發明 《經炮燒奈米顆粒用於食品、化妝品及醫藥工業之用途。 本發明π關於$乾奈米顆粒或經愤燒顆粒之膠態分散液 用於調節和/或貯存蛋白質、多肽或任何其他分子之用途。 本發明耗於經娘境奈米顆粒用作色譜所用载體之用 迷〇 以下實例說明本發明，但未限制其範圍。 85420 -16 - 200407193 實例 1/製備膠態分散液 1-1/實例1-1 :磷酸鈣、錯合劑及大豆蛋白質之膠態分散液 步騾（a)：將 2.75克 CaCl2· 2H20 (MW 二 147.02克，即，18.7 毫莫耳Ca)溶於軟化水，並加入1.4M濃度26.5釐米3結構式 (NaCOO)(CH2CH2)-CH-(COONa)N(CH2COONa)2 之錯合劑 GBS 5 (即，37.4毫莫耳GBS 5)，由此得到含Ca2+離子之溶液 A。該混合物具有13之pH。用1M HC1將其調節到pH 6。最 終體積為35釐米3。 步驟0>)：將1.87克蛋白質加到軟化水，由此得到含大豆 蛋白質[由杜邦蛋白質技術國際公司銷售（Protein Technologies International，Dupont)]品質FP 940之溶液B。用軟化水使體 積達到25釐米3。將溶液B即刻倒入新製備的溶液A。 步騾（c) ： ^0.88^Να2ΗΡΟ4 (MW = 141.96^ ^ 即，6·23 毫 莫耳Ρ)加到25釐米3，由此獲得含磷酸鈉離子之溶液C。用 1Μ HC1將該溶液調節到pH 6。將溶液C即刻加入前述混合 物。將反應混合物再調節到pH 6。將混合物攪拌5分鐘。 步騾（d)：將反應混合物放入一在50°C預熱的恒溫箱。分 散液在50°C 16小時後具有膠態外觀。 1-2/實例1-2 :磷酸鈣、錯合劑及溶菌酶蛋白質之膠態分散液 步騾（a) ··將 2.75克 CaCl2· 2H20 (MW 二 147.02克，即，18·7 毫莫耳Ca)溶於軟化水，並加入1.4Μ濃度26.5釐米3結構式 NaCOO(CH2CH2)-CH-(COONa)N(CH2COONa)2之錯合劑 GBS 5 (即，37.4毫莫耳GBS 5)，由此得到含Ca2+離子之溶液A。 85420 -17- 200407193 混合物具有13之pH。用1M HC1將其調節到PH 6 。最終體 積為35釐米3。 免驟⑻:將L87克菌酶加到軟化水，由此得到含溶菌酶 之溶液B。用軟化水使體積達到25釐米3。將溶液B即刻倒入 新製備的溶液A。 免驟⑷:將 0 88 克 Na2HP〇4 (MW = 141.96克，即，6 23 毫 莫耳P)加到25釐米3，由此獲得含磷酸鈉離子之溶液c。用 1M HC1將該溶液調節到pH 6。將溶液C即刻加入前述混合 物。將反應混合物再調節到pH 6。將混合物攪拌5分鐘。 童^⑷··將反應混合物放入一在50°C預熱的恒溫箱。分 散液在50°C 16小時後具有膠態外觀。 1-3/實例1-3 :磷酸鈣、錯合劑及乳蛋白質之膠態分散液 步驟(a) ·將 2.75克 CaCl2 · 2H2O (MW = 147.02克，即，18.7 毫莫耳Ca)溶於軟化水，並加入1 4M濃度26 5釐米3結構式The calcined particles according to the present invention exhibit the best isotropy, and the ratio R 85420 -14- 200407193 (R = large diameter (L) / small diameter ⑴) optimally tends to 1 to 1.5, preferably 丨 to 丨 .3 β According to a fourth subject, the present invention relates to a method for preparing a colloidal dispersion according to the present invention, comprising the steps of: U) forming a mixture comprising a calcium complexing agent and a calcium source, (b) adding at least one protein The vehicle obtained in step (i) is (C) a phosphorus source is added to the vehicle obtained in step (i) (b), and (d) the vehicle obtained in step (c) is heated. The step ㈣ used in step ㈣ is most preferably a salt or a hydroxide avoiding or a carbonate. Special mention may be made of chlorination, nitric acid, slaked lime and carbonic acid. The degree of advantage in the mixture produced in step 1 is preferably at most 1M, preferably at most 0.5M. The step according to the preparation method of the present invention is characterized by optimally adjusting the ρ of the medium obtained from step Η to obtain a value between 5 and 7. ρΗβρΗ is known to be adjusted by adding Ηα or hno3. The method "step (a) is performed with a molar ratio Ra between 01 and 3, Ra = calcium complexing agent / calcium. In step (b), the protein solution used is generally obtained by simply dispersing the protein in demineralized water. The phosphorus source used in step 骡 0) is preferably an alkali metal phosphate or ammonium salt. Special mention may be made of sodium dihydrogen phosphate (NaHJO4) or sodium monohydrogen phosphate (Na2Hp04) or ammonium phosphate (NH4 (H2P04)) or ammonium monohydrogen phosphate ((and 4) 2 (Hp 〇4)). The pH of the vehicle is preferably adjusted to a value between 4.5 and 8 during the preparation according to the invention and before adding the phosphorus source according to step (c). Step (c) of the method is performed at an optimal mass ratio between 0.3 and 6, Rb = 85420-15-200407193 calcium phosphate / protein. Step (c) of the method is performed with a molar ratio Rc optimal between 1 and 4, Rc dicalcium molar number / phosphor molar number. The addition of reactants occurs at a controlled rate or preferably transiently and at ambient temperature. In step (d) of the method, the medium obtained in step (C) is heated to a temperature preferably between 20 c and 90 ° C. This step is performed according to a conventional heat treatment method. Fortunately, the media obtained in step (c) was introduced into a thermostat preheated at the heat treatment temperature. The heat treatment time is generally between 2 hours and 24 hours, preferably between 6 hours and 20 hours. The colloidal dispersion can then be purified by ultrafiltration with washing with demineralized water. Ultrafiltration / eyewashing is sufficient to remove residual free salts and complexing agents. The colloidal dispersion can also be concentrated by ultrafiltration. An antibacterial agent may be added to the obtained colloidal dispersion. According to the fifth subject of the present invention, the present invention relates to the use of the colloidal dispersion liquid according to the present invention or the colloidal dispersion liquid obtained by the preparation method according to the present invention for the food, cosmetics and pharmaceutical industries. The present invention also relates to the use of nano particles according to the present sun and moon, and the use of the burned nano particles in the food, cosmetics, and pharmaceutical industries according to the present invention. The present invention relates to the use of colloidal dispersions of dried nano particles or scorched particles for the regulation and / or storage of proteins, polypeptides or any other molecule. The present invention is used for the use of cationic nano particles as a carrier for chromatography. The following examples illustrate the present invention without limiting its scope. 85420 -16-200407193 Example 1 / Preparation of colloidal dispersion 1-1 / Example 1-1: Colloidal dispersion of calcium phosphate, complexing agent and soybean protein Step (a): 2.75 g of CaCl 2 · 2H20 (MW 2 147.02 grams, that is, 18.7 millimolar Ca) dissolved in demineralized water, and added 1.4M concentration 26.5 cm 3 structural formula (NaCOO) (CH2CH2) -CH- (COONa) N (CH2COONa) 2 GBS 5 (ie , 37.4 millimoles GBS 5), thereby obtaining a solution A containing Ca2 + ions. The mixture has a pH of 13. It was adjusted to pH 6 with 1M HC1. The final volume is 35 cm3. Step 0 >): 1.87 g of protein was added to demineralized water, thereby obtaining a solution B of soybean protein-containing protein [sold by Protein Technologies International, Dupont] quality FP 940. Use demineralized water to make the volume 25 cm3. Solution B was immediately poured into freshly prepared solution A. Step (c): ^ 0.88 ^ Να2ΗΡΟ4 (MW = 141.96 ^^, that is, 6.23 millimolar P) was added to 25 cm3, thereby obtaining a solution C containing sodium phosphate ion. This solution was adjusted to pH 6 with 1M HC1. Solution C was added immediately to the aforementioned mixture. The reaction mixture was readjusted to pH 6. The mixture was stirred for 5 minutes. Step (d): Put the reaction mixture in a pre-heated incubator at 50 ° C. The dispersion had a colloidal appearance after 16 hours at 50 ° C. 1-2 / Example 1-2: Colloidal dispersion of calcium phosphate, complexing agent and lysozyme protein Step (a) · 2.75 grams of CaCl2 · 2H20 (MW 2147.02 grams, ie, 18.7 millimoles Ca) is dissolved in demineralized water, and 1.4M concentration 26.5 cm 3 of the structural formula NaCOO (CH2CH2) -CH- (COONa) N (CH2COONa) 2 is added as a compound GBS 5 (ie, 37.4 millimoles GBS 5), thereby A solution A containing Ca2 + ions was obtained. 85420 -17- 200407193 The mixture has a pH of 13. It was adjusted to pH 6 with 1M HC1. The final volume is 35 cm3. Elimination of leaching: Add 87 grams of lysozyme to demineralized water to obtain solution B containing lysozyme. Use demineralized water to make the volume 25 cm3. Solution B was immediately poured into freshly prepared solution A. Free from turbulence: 0 88 g of Na2HP04 (MW = 141.96 g, ie, 6 23 mmol) was added to 25 cm3, thereby obtaining a solution c containing sodium phosphate ions. This solution was adjusted to pH 6 with 1M HC1. Solution C was added immediately to the aforementioned mixture. The reaction mixture was readjusted to pH 6. The mixture was stirred for 5 minutes. Tong ^ 将 · Put the reaction mixture in a pre-heated incubator at 50 ° C. The dispersion had a colloidal appearance after 16 hours at 50 ° C. 1-3 / Example 1-3: Colloidal dispersion of calcium phosphate, complexing agent and milk protein Step (a) • 2.75 g of CaCl2 2H2O (MW = 147.02 g, ie, 18.7 millimolar Ca) are dissolved in softening Water and add 1 4M concentration 26 5 cm 3 structural formula
NaCOO(CH2CH2)_CH-(COONa)N(CH2COONa)2之錯合劑 GBS 5 (即，37.4毫莫耳GBS 5)，由此得到含Ca2+離子之溶液a。 混合物具有13之pH。用1MHC1將其調節到PH 6。最終體積 為35釐米3。 免驟(b) ••將1 ·87克乳蛋白質加到軟化水，由此得到含乳 蛋白質之溶液B。用軟化水使體積達到25釐米3。將溶液b即 刻倒入新製備的溶液A。 免驟⑷:將 0·88克 Na2HP04 (MW = 141.96克，即，6,23 毫 莫耳P)加到25楚:米3，由此獲得含磷酸鋼離子之溶液c。用 1M HC1將該溶液調節到pH 6。將溶液C即刻加入前述混合 85420 -18 - 物。將反應混合物再調節到pH 6。將混合物攪拌5分鐘。 堂遵丄虹：將反應混合物放入一在50°C預熱的恒溫箱。分 散液在50°C 16小時後具有膠態外觀。 2 ’ 2_1/實例2_1 : (HAP ··大豆蛋白質）=(1:1)重量比，pH 6 將實例1_1中獲得的膠態分散液凍乾。 由p固體NMR觀察對應3.4 ppm化學轉移之峰。 紅/東乾粉末可再分散於軟化水。利用透射電子顯微法， 由束乾顯微法[杜伯查特（Dub〇ehet)法]觀察完全單一化物 體’物體具有具約15〇奈米球形形態大小之單分散顆粒大小 分佈。 電泳電勢曲線顯示等電點在pH4，且負電荷在約_19毫伏 分散液之pH。 2_2/實例2_2: (HAP:溶菌酶蛋白質）=(1:1)重量比，pH6 將實例1-2中獲得的膠態分散液凍乾。 由P固體NMR觀察對應3.4 ppm化學轉移之峰。 經凍乾粉末可再分散於軟化水。利用透射電子顯微法， 由凍乾顯微法（杜伯查特）法觀察完全單一化物體，物體具有 具約150奈米球形形態大小之單分散顆粒大小分佈。 電泳電勢曲線顯示等電點在pH 9，且正電荷在約+5毫伏 分散液之pH。 2-3/實例2_3 : (HAP: WPC蛋白質）=(1:1)重量比，pH6 將實例1-3中獲得的膠態分散液凍乾。 由31P固體NMR觀察對應3.4ppm化學轉移之争。 85420 -19- 200407193 、、’二滚乾&末可再分散於軟化水。利用透射電子顯微法， 由凍乾顯微法（杜伯查特）法觀察完全單一化物體，物體具有 具約15G奈米球形形態大小之單分散顆粒大小分佈。 電冰電勢曲線顯示等電點在pH4，且負電荷在約_18毫伏 分散液之pH。 3/麗備經烺燒. 3-1/實例3_1 :用（HAP : wpc蛋白質）=(3⑴重量比試驗， 如實例1-3製備膠態分散液，但使用〇 62克乳蛋白質。將 該分散液凍乾。然後在900。〇進行閃烺燒。 所得經烺燒顆粒粉末由磷酸鈣組成，且由TEM展示各向 同性，其比R為1.4。 由X-射線觀察具有羥磷灰石Ca3(P〇4)2和Ca2P2〇7之混合 物之結構。 ° 85420 20-The complex agent GBS 5 of NaCOO (CH2CH2) _CH- (COONa) N (CH2COONa) 2 (ie, 37.4 millimoles GBS 5), thereby obtaining a solution a containing Ca2 + ions a. The mixture had a pH of 13. Adjust it to pH 6 with 1MHC1. The final volume is 35 cm3. Step (b) •• Add 1.87 g of milk protein to demineralized water to obtain milk protein-containing solution B. Use demineralized water to make the volume 25 cm3. Solution b was immediately poured into freshly prepared solution A. Free from sudden addition: Add 0. 88 grams of Na2HP04 (MW = 141.96 grams, that is, 6,23 millimolar P) to 25 Chu: m3, thereby obtaining a solution c containing phosphate steel ions. This solution was adjusted to pH 6 with 1M HC1. Solution C was added immediately to the aforementioned mixed 85420-18-. The reaction mixture was readjusted to pH 6. The mixture was stirred for 5 minutes. Don Zunhong: Put the reaction mixture in a pre-heated incubator at 50 ° C. The dispersion had a colloidal appearance after 16 hours at 50 ° C. 2 '2_1 / Example 2_1: (HAP ·· soy protein) = (1: 1) weight ratio, pH 6 The colloidal dispersion liquid obtained in Example 1_1 was lyophilized. A peak corresponding to 3.4 ppm chemical transfer was observed from p-solid NMR. Red / East dry powder can be redispersed in demineralized water. The transmission electron microscopy method was used to observe the completely monolithic body's object by the beam-drying microscopy method [DubOehet method]. The object had a monodisperse particle size distribution having a spherical shape size of about 150 nm. The electrophoretic potential curve shows that the isoelectric point is at pH 4 and the negative charge is at the pH of the dispersion of about -19 mV. 2_2 / Example 2_2: (HAP: lysozyme protein) = (1: 1) weight ratio, pH 6 The colloidal dispersion obtained in Example 1-2 was lyophilized. A peak corresponding to a 3.4 ppm chemical transfer was observed from P solid NMR. The lyophilized powder can be redispersed in demineralized water. Using transmission electron microscopy, the lyophilized microscopy (Dubachart) method was used to observe a completely singularized object. The object had a monodisperse particle size distribution with a spherical shape size of about 150 nanometers. The electrophoretic potential curve showed an isoelectric point at pH 9 and a positive charge at the pH of the dispersion of about +5 mV. 2-3 / Example 2_3: (HAP: WPC protein) = (1: 1) weight ratio, pH 6 The colloidal dispersion liquid obtained in Example 1-3 was lyophilized. Observation from 31P solid NMR corresponds to a dispute of 3.4 ppm chemical transfer. 85420 -19- 200407193, ′ two tumble dry & can be redispersed in demineralized water. The transmission electron microscopy method was used to observe a completely singularized object by freeze-drying microscopy (Dubochat). The object had a monodisperse particle size distribution with a spherical morphology of about 15G nanometers. The electric ice potential curve shows that the isoelectric point is at pH 4 and the negative charge is at the pH of the dispersion of about -18 millivolts. 3 / Lai Bei Jingyao. 3-1 / Example 3_1: (HAP: wpc protein) = (3⑴ weight ratio test, as in Example 1-3 to prepare a colloidal dispersion, but using 062 grams of milk protein. This The dispersion was lyophilized. Then the flash-firing was performed at 900 °. The resulting fire-firing particle powder was composed of calcium phosphate and exhibited isotropy by TEM with a ratio R of 1.4. It was observed by X-rays with hydroxyapatite Structure of a mixture of Ca3 (P〇4) 2 and Ca2P207. ° 85420 20-