201231666 四、發明說明： 【發明所屬之技術領域】 本發明係運賴蕈8絲體之液體發酵培養，成功地將海藻糖及甘露糖聚合而成 絲甘m«衫雜，郷之於目_轉及活化細胞修復。 【先前技術】 海蕩糖⑽▲)是—種非還原性雙糖，由兩綱雜分子經由^ 鍵結所組成（脳_.，細）。正式化學名稱為⑽·喊隨綠α如比喃 ^^^(a-D-glucopyranosyla-D.gluc〇py„ 〇1^2〇11 〇 廣泛發現於細菌、酵母g、真菌、_及—些高物及無脊椎動物中。海藻糖能 直接舆生物細《接觸來維雜祕，社因乾_造成的廳合與通紐的改變 (Crowe & C卿e，2G00 ; RiehaKis ei β/_，雇）。海藻糖不易被酸及水解酵素 所水解，且溶於水令時性質穩定，溶解度及渗透壓與麥芽糖相似，同 時具低吸祕’在相舰度92%，其水分含量仍可轉在9 54%，由此可知海藻 糖是-個化性穩定的糖類（Birch“·，1%3 ; Richards_,，2〇〇2)。201231666 IV. Description of the invention: [Technical field to which the invention pertains] The present invention is a liquid fermentation culture of a scorpion scorpion, which successfully polymerizes trehalose and mannose into a succulent m-shirt. Transfer and activate cell repair. [Prior Art] The sea sugar (10) ▲) is a non-reducing disaccharide composed of two kinds of heteromolecules via a ^ bond (脳_., fine). The official chemical name is (10)·Call with green α such as 喃^^^(aD-glucopyranosyla-D.gluc〇py„ 〇1^2〇11 〇 widely found in bacteria, yeast g, fungi, _ and some high objects And invertebrates. Trehalose can directly smear the biological details of the contact, and the change of the hall and the connection between the two (Crowe & C, e, 2G00; RiehaKis ei β/_, hired Trehalose is not easily hydrolyzed by acid and hydrolyzed enzymes, and is stable when dissolved in water. The solubility and osmotic pressure are similar to those of maltose. At the same time, it has a low sorption threshold of 92% at the phase of the ship, and its moisture content can still be transferred. 9 54%, it can be seen that trehalose is a stable sugar (Birch "·, 1% 3 ; Richards_,, 2〇〇2).
在生物體中海藻糖具有能量健存之功能，如處於加熱、冷康、乾燥或高渗透壓 之情況下’海_糖能有保護蛋白質和細胞膜的功能（他職肪心，1993 );對許多 種類的細射’雜糖可穩定細胞壁組成的魏（Riehards^.，纖）；對動植物 體有儲存能量的魏及保存_魏，並且可提高在乾燥環境的生長、能增加 酵母菌對環境改變的適應性（Yoshida⑽” 1998 )，包括乾羊、渗透壓的變化、熱 休克以及對酒精的耐受性等。此外，海驗對於食品、藥物（Higashiy_，細）及 生物科技上皆有魏(Spriess&IvelU搬），添加適當量的海，可提升動 物細胞的柄率（&_❹/.，)。嫉賴狀雜賊其仙姐，通過 美國食品及騎檢驗局認證㈣應哚職办以牌dedasSafe)之安全健康食品， 所以目前已廣泛地應用於食品、化妝品與醫藥方面。以其安定性而言，已利用於酵 素、蛋白質、生物量（biomass)、藥物製備與器官移植方面做為安定劑；疫苗與哺 乳動物細胞巾做。以其保水性而言，與微脂粒結合做為化妝品保水劑 (Schiraldiera/.，2002)。此外，於生物體中利用基因重組技術生成海藻糖，亦能有效 發揮其作用。甘雜（Mannose)參與許乡基本細胞雜，缺乏會造成身體許多問 201231666 二。甘露糖激發了抗發炎作用及組織再生，在活化巨修胞扮演一個積極角色，巨 匕可X/月除造成發炎的碎物’巨嗤細胞至少有四個可結合甘露糖的不同接受器， 意味其在發炎碎物收集可發揮適當功能。甘露糖在傷口癒合時的具抗發炎力，討 刺激纖維母細胞製造更多的膠原和蛋白聚畴，讓癒合速度更快，減少疼痛。甘露糖 被視為自然的抗發炎物f，可以修正過度活躍財性—球以減少被誤導的發炎。 2細胞都含有甘露糖’故其可影響身體所有器官和喊。甘露齡視網膜的光接 又器上亦有4現，其在視力上扮演重要角色’甘露糖的補充可以用在連接其他療法 1糖尿病患者白内障的纽。甘露糖具有保存細胞膜新陳代謝物的能力，此代謝物 對水晶體有保護作用。 μ 述本么明培養複合海溪甘露糖多糖體的優點，如海藻糖可以讓多醣體 0 κ °物更穩定，目秘祕耐高溫’且可以降低梅納反應、防止蛋白質變性、讓聚 衫賴轉紋、保錄高；蝴㈣__最細_，可以產生純度 兩、品質佳之高分子多醣體成分；另甘露糖可激發抗發炎作用及組織再生，在活化 巨仙胞扮演-個積極角色，巨嗟細胞可以清除造成發炎的碎物，巨伽胞至少有 個可、.Da甘路糖的不同射||，意味其在發炎碎物收集可發揮適當魏。甘露糖 具有保存細_新陳代職的能力，此代謝物對水晶財保護侧。即甘鎌可輔 助及強化多·體在發炎碎物清除及水晶體保護之功能。 眼表層淚旗失衡、眼表細胞暴露於高渗或高張力環境、或眼驗異常等情況均會 引七淚液W減少’眼㈣上皮細胞乾燥，眼睛出現賴、搔癢、灼傷、異物感等 錄’即魏症之病症’是眼床社要疾狀-，目«界已提出許多組合物 絲應·治療及處理魏症候群，如人H添加賴糖成分之舰液、或特 疋雇1/月劑成刀之組合物等。其中「海藻糖」（trehal〇se)之高保水特性，已將立鹿用 至眼科臨床治療，如直接將海藻糖成分添加於眼藥水中，可治療乾眼症病人 (MatSU° <獅2 );2叫5月8日公告之日本特許獻專利公«祕麵8 旒中揭露多驗合物應用於眼科時，具有提高藥物的組織轉移性的效果，已知多酿 類組合物作為滴眼劑的基質是有用的，其中以環脂（黎）作為滴眼劑的基質，可 作為促進藥物向眼内轉移的滴眼劑；·年9月n曰公告之曰本特許廉之專利公 報第雇-腦6號以離子性隱形眼鏡，吸附聚乙㈣略細，中和料電荷，來 穩定淚液層的系統，該系統適用於隱形眠鏡用滴目艮劑或配戴液。即過去之滴眼液多 直接取用多醣類應用’雖然可保水扣不姓々„ lL4;K, v 什JC仁不持久’且此類小分子糖對眼部細胞並無免疫 201231666 活化作用。但本發明之複合多酿體是海藻糖與甘露糖在雜發酵培養天數下_輩 多醣體所聚合喊的安全、穩定、f優的複合_贿合物，具持久保水性及因富 含β-Gh^n及活性胜肽，其可用於活化眼部轉細胞；消除眼部細胞殘邊及因= 體免疫過高所產生的蛋白質沉_，促進細胞再生；也可_抗氡化，而減少眼部 細胞老化，這是一般小分子多醣類及其組合物所無之功效。 【發明内容】 鑑於上述習知相關技術之缺失，本發明之主要目的雜供—種絲甘露糖複合 多醣體的製财法，獅基本的培養基與分雜化步驟，赠得絲甘露糖複合多 釀體供本發明所用。本發明之另-目的係提供一種能有效與眼部水分結合之安全、 穩定之高分子多酿體聚合物，為達上述目的： 本發明物製備過程係將5%的甘露糖(mann〇se)加5%的海藻糖扣―)加入！ %的酵母萃取物(yeastextract)混合均勻後，經高溫、高壓滅菌3min分鐘(Aut〇dave 121 C ’ 1.51b) ’於至溫下冷卻後、再將靈芝(Gan〇derma 〗ucidum)、樟芝(a血〇dia comphorata)巴西磨知（Agaricus brazil)、雲芝（Coriolus versicolor)及裂稽菌 (Schizophyllum commune)等五種菇蕈的菌絲體(Mycelium)、分別加入培養，且不 同的菇蕈種類，其最佳的培養時間亦不同，各自最佳的培養時間如下所示：In the organism, trehalose has the function of energy storage, such as heating, cold, dry or high osmotic pressure. 'Sea sugar can protect protein and cell membrane function (He is fat, 1993); Many kinds of fine shots of 'heterosaccharides can stabilize the cell wall composed of Wei (Riehards ^., fiber); for the animal and plant body to store energy and preserve _ Wei, and can improve the growth in a dry environment, can increase the yeast to the environment Adaptability of change (Yoshida (10) 1998), including dry sheep, changes in osmotic pressure, heat shock and tolerance to alcohol, etc. In addition, Hai test has Wei for food, medicine (Higashiy_, fine) and biotechnology. (Spriess & IvelU moved), adding the appropriate amount of sea, can increase the animal cell handle rate (& _ ❹ /.,). 嫉 状 杂 其 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙 仙It is widely used in food, cosmetics and medicine for its safe and healthy foods. It has been used in enzymes, proteins, biomass, and pharmaceutical preparations for its stability. It is used as a stabilizer for organ transplantation; vaccines are used with mammalian cell towels. In terms of water retention, they are combined with microlipids as a cosmetic water-retaining agent (Schiraldiera/., 2002). In addition, genes are used in organisms. The recombinant technology produces trehalose, which can also effectively play its role. Mannose participates in the basic cell miscellaneous of Xuxiang, and the lack of it will cause many body problems 201231666. 2. Mannose stimulates anti-inflammatory and tissue regeneration, in the activation of giant cells Playing an active role, the giant scorpion can be X/month in addition to causing inflamed debris. The giant scorpion cells have at least four different receptors that can bind mannose, meaning that they can function properly in the collection of inflammatory debris. Mannose is in the wound. It has anti-inflammatory ability when healed, and stimulates fibroblasts to make more collagen and protein domains, which makes healing faster and reduces pain. Mannose is regarded as a natural anti-inflammatory substance, which can correct excessive active property. - The ball is used to reduce the misleading inflammation. 2 The cells all contain mannose, so it can affect all organs of the body and shout. The nectar of the retina has 4 now. It plays an important role in vision. The supplement of mannose can be used to link cataracts in other patients with diabetes. Mannose has the ability to preserve the metabolism of cell membranes. This metabolite has a protective effect on crystals. The advantages of compound Haixi mannose polysaccharide, such as trehalose, can make the polysaccharide 0 κ ° more stable, and the secret is resistant to high temperature' and can reduce the Mena reaction, prevent protein denaturation, let the poly shirt turn, and record High; butterfly (four) __ the finest _, can produce high purity two high quality polysaccharide components; another mannose can stimulate anti-inflammatory effects and tissue regeneration, play an active role in the activation of giant celestial cells, giant scorpion cells can be cleared Inflamed debris, at least one of the giant gamma cells, .Da Ganlu sugar different shots ||, meaning that it can play properly in the collection of inflammatory debris. Mannose has the ability to preserve fine _ new generations of substitutes, this metabolite on the side of crystal wealth protection. That is, Ganzi can assist and strengthen the function of multiple bodies in the removal of inflamed debris and crystal protection. Eye surface tear flag imbalance, ocular surface cells exposed to hypertonic or high tension environment, or abnormal eye examination will lead to seven tears to reduce 'eye (four) epithelial cells dry, eyes appear rash, itching 'The disease of Wei's disease is the condition of the eye-bed society.-------There are many compositions that have been proposed to treat and treat Wei syndrome, such as the addition of lysine to humans, or special employment 1/ The composition of the monthly agent into a knife and the like. Among them, the high water retention characteristics of trehal〇se have been used in the clinical treatment of ophthalmology. For example, the trehalose component can be directly added to the eye drops to treat dry eye patients (MatSU° <lion 2 2) The Japanese patent application entitled "Secret 8" announced on May 8th reveals that the multi-agent is used in ophthalmology and has the effect of improving the tissue transferability of the drug. The multi-flavored composition is known as the eye drop. The matrix of the agent is useful, wherein the cyclolipid (Li) is used as a matrix for eye drops, and can be used as an eye drop for promoting the transfer of the drug into the eye; Employ-Brain No. 6 with ionic contact lenses, adsorption polyethylene (four) slightly fine, neutralizing material charge, to stabilize the tear layer system, the system is suitable for invisible sleep mirrors with eye drops or wearing liquid. That is to say, the past eye drops are more directly used in the application of polysaccharides. Although the water retention is not the same as 々 „ lL4; K, v is not persistent J' and the small molecule sugar is not immune to the eye cells 201231666 Activation However, the composite multi-breast of the present invention is a safe, stable, and excellent composite compound of trehalose and mannose which is polymerized under the mixed fermentation days, has a long-lasting water retention and is rich in β-Gh^n and active peptide, which can be used to activate ocular transfected cells; eliminate ocular residual cells and protein precipitation caused by excessive immunity; promote cell regeneration; The invention reduces the aging of the eye cells, which is a function of the general small molecule polysaccharides and the compositions thereof. SUMMARY OF THE INVENTION In view of the above-mentioned related art, the main purpose of the present invention is to provide a compound-filament mannose complex. The method of making a polysaccharide, the basic medium of the lion and the step of multi-hybridization, and the mannose-composite multi-brew are provided for use in the present invention. Another object of the present invention is to provide a safe and effective combination with eye moisture. Stable macropolysaccharide polymer In order to achieve the above object: The preparation process of the present invention is to add 5% mannose (mann〇se) plus 5% trehalose buckle-)! After the yeast extract (yeastextract) is uniformly mixed, it is sterilized by high temperature and high pressure. 3min minutes (Aut〇dave 121 C '1.51b) 'After cooling to the temperature, Ganoderma lucidum (Gan〇derma ucidum), Astragalus lucidum (a blood dia comphorata), Brazil (Agaricus brazil), Yunzhi The mycelium (Mycelium) of five kinds of mushroom, such as (Coriolus versicolor) and Schizophyllum commune, are separately added to the culture, and the different culture time of the different mushroom species is different, and the best culture is different. The time is as follows:
靈芝(Ganoderma lueidum) 約 13-15 天 樟芝(Antrodia comphorata) 約 20-22 天 巴西磨兹(Agaricus brazil) 約 13-15 天 雲芝(Coriolus versicolor) 約 20-22 天 裂褶菌(Schizophy 11 um commune) 約 4-6天 上述五種菇蕈菌絲體是以振盪培養方法將菌絲分別培養，使各種菇簟菌絲能在培養 液中，合成大量的多醣體，再將五種菇蕈菌絲培養液一同混合均勻後，含菌絲的培 養液用Polytron PT6000(Switzerland)均質機，以3〇,〇〇〇rmP的轉速擊碎、均勻混合 3分鐘，將該混合液先經200網目/平方毫米的過濾網過濾後，再應用TAMI Industries(France)出品的50ΚΧ)(0·30μηι之孔隙)的橫掃式陶瓷過濾膜，充分的去除分 子量在5萬以下的小分子後’則為菇蕈多醣體液，此液體再以1 : 4的量倒入純酒 201231666 精中，於室溫下靜置2〜3科，待職後，取出結晶狀沉殿物，再以 C聰冷象乾燥航分乾燥，之後將乾燥粉末回溶於水中， 混合均勾後’則可製成滴眼液所應用之海藻糖複合轉多醣體原料。本海薄糖複人 轉多醣縣料可單_賴較，亦可添加於各觀有親液巾，達騎化㈣ _、促進細胞再生 '保濕、、抗氣化、減少乾眼症的不適感，對眼睛的保健上^ 【實施方式】 本發明之另一目的係提供一種能有效與眼部水分結合之安全、穩定之高分子多 醣體聚合物，為達上述目的：本發明物製備過程係將5%的甘露糖(義n〇=)刀加= 的海藻糖(trehalose)加入1%的酵母萃取物(yeastextract^Btj合均勻後，經高溫、 滅菌3πήη分鐘(Autoclave 121t，h5Ib)，於室溫下冷卻後、再將靈 lucidmn)、樟芝(Antrodia comphorata)、巴西磨菇(八牌丨咖^邱雲芝(c〇r⑹诹 versicolor)及裂褶菌(Schizophyi lum commune)等五種縣的菌絲體⑽制㈣ 別加入培養’且不同的!！蕈種類，其最佳的培養時間亦不同，各自最佳的培養時二 如下所示： 。、曰 靈芝(Ganoderma lucidum) 約 13-15 天 樟芝(Antrodia comphorata) 約 20-22 天 巴西磨兹(Agaricus brazil) 約 13-15 天 雲芝(Coriolus versicolor) 約 20-22 天 裂權菌(Schizophy 11 um commune) 約 4-6天 上述五種蒜蕈菌絲體是以振盈培養方法將菌絲分別培養，使各種蒜簟菌絲能在土立 養液中，合成大量的多醣體，再將五種菇蕈菌絲培養液以相同體積混合均勻後， 經萃取純化後’形成複合海藻甘露糖多醣體聚合物之粉末。取得具免疫促進功效 的海藻甘露糖複合多醣體，含β-Glucan及活性胜肽，其可用於活化眼部的吞噬細 胞；消除眼部細胞殘渣及因自體免疫過高所產生的蛋白質沉殿物，促進細胞再生； 也可經由抗氧化’而減少眼部細胞老化；且此高分子多醣體，可有效與眼部水分 結合，達到保濕效果，減少乾眼症的不適感，對眼睛的保健上具有更大效果。 本海藻糖複合菇蕈多醣體原料可單獨製成滴眼液，達到活化眼部細胞、促進細胞再 201231666 分析 生、保濕、抗氧化、減少乾眼症的不職，對眼睛祕健上具錢大效果。 實施例1:本海藻糖複合益蕈多酷體原料可單獨製成滴眼液，本滴眼液之hplc (一)檢測儀器·· RI2000 detector曲折率檢知器 曰期 2010年12月26曰 溫度 室溫23〜25°C 管柱 KS-G 移‘相 ddH2〇 KS-804 流速 0.5ml/min KS-805 注射體積 20μ1 總壓力值 450 psi 、樣品前處理：取111111^^1>叩樣品溶液以〇4化111濾膜過濾後等機 、HPLC檢定條件 (二）標準品 名稱 分子量(M.W.) 名稱 分子量(M."W.) STD-P800 800,000 STD-P50 50,000 STD-P400 400,000 STD-P20 20,000 STD-P100 100,000 Lactose 360 (三）檢量線 〇Ganoderma lueidum about 13-15 days Antrodia comphorata about 20-22 days Agaricus brazil about 13-15 days Coriolus versicolor about 20-22 days of Schizophy 11 Um commune) About 4-6 days, the above five kinds of mushroom mycelium are cultured by shaking culture method, so that various mushroom mycelium can synthesize a large amount of polysaccharide in the culture solution, and then five kinds of mushrooms After the mycelial culture solution was uniformly mixed, the mycelium-containing culture solution was crushed with a Polytron PT6000 (Switzerland) homogenizer at a speed of 3 Torr, 〇〇〇rmP, and uniformly mixed for 3 minutes, and the mixture was passed through 200. After filtering the mesh/mm2 filter, apply a 50 ΚΧ (0·30μηι pore) cross-sweeping ceramic filter from TAMI Industries (France) to fully remove small molecules with a molecular weight below 50,000. Mushroom sputum polysaccharide body fluid, this liquid is poured into the pure wine 201231666 in a quantity of 1: 4, and it is allowed to stand at room temperature for 2~3 subjects. After waiting for the job, take out the crystal-like sinking matter, and then take C Cong cold elephant Dry the airdrops and then dry the powder back into the water. After the mixture is hooked, the trehalose composite trans-polysaccharide raw material applied to the eye drops can be prepared. This sea thin sugar complex can be transferred to the polysaccharide county can be _ 赖, can also be added to each view has a lyophilic towel, Daqihua (four) _, promote cell regeneration 'moisturizing, anti-gasification, reduce dry eye discomfort Sense of health care for the eye ^ Embodiments Another object of the present invention is to provide a safe and stable polymer polysaccharide polymer which can effectively combine with eye moisture, in order to achieve the above object: preparation process of the present invention Add 5% mannose (=n〇=) knife plus trehalose to 1% yeast extract (yeastextract^Btj and homogenize, then sterilize for 3πήη minutes (Autoclave 121t, h5Ib). After cooling at room temperature, lucidmn), Antrodia comphorata, Brazilian mushroom (Eight 丨 丨 ^ 邱 Qiu Yunzhi (c〇r (6) 诹versicolor) and Schizophyi lum commune) The mycelium (10) (4) is not added to the culture and the different!! 蕈 type, the best culture time is also different, the best culture time is as follows: 、, Ganoderma lucidum about 13- 15 days Antrodia comphorata about 20-22 days Brazilian Moz (A Garicus brazil) About 13-15 days Coriolus versicolor About 20-22 days Schizophy 11 um commune About 4-6 days The above five kinds of garlic mycelium are hyphae by vibrating culture method Cultured separately, so that various garlic mycelium can synthesize a large number of polysaccharides in the soil culture liquid, and then mix the five kinds of mushroom mycelial culture liquid in the same volume, and then extract and purify to form a composite seaweed mannose. A powder of polysaccharide polymer. Acquires an immunization-promoting algae mannose complex polysaccharide containing β-Glucan and an active peptide, which can be used to activate phagocytic cells in the eye; eliminate ocular cell debris and autoimmune The high protein produced by the temple can promote cell regeneration; it can also reduce the aging of the eye cells through antioxidants; and the high molecular polysaccharide can effectively combine with the eye water to achieve a moisturizing effect and reduce dry eye syndrome. Discomfort, it has a greater effect on the health of the eyes. The trehalose compound mushroom 蕈 polysaccharide material can be separately made into eye drops, to achieve activation of eye cells, promote cells and then 201231666 analysis of raw, moisturizing Anti-oxidation, reduce the dryness of dry eye, and have a great effect on eye secrets. Example 1: This trehalose compound benefits more and more cool raw materials can be made into eye drops alone, the eye drops of hplc (1) Detection instrument·· RI2000 detector tortuosity detection device period December 2010 26曰 temperature room temperature 23~25°C column KS-G shift 'phase ddH2〇KS-804 flow rate 0.5ml/min KS- 805 injection volume 20μ1 total pressure value 450 psi, sample preparation: take 111111^^1> 叩 sample solution filtered with 〇4 111 filter, equivalence, HPLC assay conditions (2) standard name molecular weight (MW) (M."W.) STD-P800 800,000 STD-P50 50,000 STD-P400 400,000 STD-P20 20,000 STD-P100 100,000 Lactose 360 (3) Checking line 〇
,Μ·ΙΛΙ J-J^E PJepussJoanle/v^o-1,Μ·ΙΛΙ J-J^E PJepussJoanle/v^o-1
25 30 35 40 45 5025 30 35 40 45 50
Retention time(min) 201231666 (四）分析結果Retention time(min) 201231666 (IV) Analysis results
滯留時間 圖··多醣溶液HPLC層析圖 實施例2 : 〇 本海藻糖複合菇蕈多醣體原料亦可添加於各種現有滴眼液中 __ 組成物 -含量百分比(重量/體積） _ Cyanocobalamin - — · ————— 0.00005 Chlorobutmiol 1.5 Boric acid 10 _ Treha-Manno Glucan 15 ___ EDTA disodium 0.2 一 Sodium borate 2 Water 適量Retention time chart·········· — · ————— 0.00005 Chlorobutmiol 1.5 Boric acid 10 _ Treha-Manno Glucan 15 ___ EDTA disodium 0.2 A sodium borate 2 Water