201031751 六、發明說明： 【發明所屬之技術領域】 本發明係有關於製造新洋菜寡醣，簡稱 NAOS)的方法，尤指一種自龍鬚菜Sp)中分離出主要聚合度 (Degree of P〇lymerization ’簡稱Dp)為2_6的新洋菜寡醣的水解混合物 (lysate mixture) ’以大幅提高自龍鬚菜中分解出寡醣成份的生產率的製 造方法。 【先前技術】 按养酷類物貝一般被疋義為「益生物質」（prebi〇tics)，「益生 物質」是一種能夠選擇性促進「益生菌」（probiotics)生長的物質， 屬於-種食物原料’由於結構特殊，不能為人體的消化酵素分解利用， 故能通過上消化道，找地進人场，被腸⑽益生g獅性地發酵 利用。募醣係由2-10個單糖分子脫水縮合而成的聚合物，在自然界 中’寡醣、蔗糖、麥芽糖和乳糖等’係以游離狀態或糖苷（glyc〇side)狀 ❿態，存在於植物體和果實中，而具生理活性的寡酶，如：果寡酶 (toto-oligosacchrides)、異麥芽寡醣（is〇malt〇_〇lig〇saccharides)、半乳 寡糖（galacto-oligosacchrides)等’皆利用p形式的糖苷鍵來鍵結不同 的單醣分子’故難以被人體的消化道中用以分解α形式為主的酶解酵 素所分解，因驢_地通過人槪道，完整地軟大腸，提 供腸道内的纽菌所需’發酵生成有機酸，以獲得能量，且使腸道内 的pH值降低，以抑制腸道内腐敗菌生成對人體有害的物質，並防止 腸道微生物對膽汁酸裂解生成致癌物質。由此可知，寡釀類物質在人 201031751 體健康的轉及赫的漸上扮演著極為重要的肖色，然而，由新洋 菜寡酿的相關研究報告可知，既有技術都是以價格昂貴和高純度的洋 菜醣（agarose)，做為酵素(agarase)的分解基質，根據sigmaC〇報價， 其市售價格為美金1，330/公斤，導致其用以萃取寡酷的原料及生產成本 均居尚不T，以市f的畴(啲啊齡㈣)及六师eGagarGhexa〇se) 的募藤為例’每公克竟高達新台幣1〇〜32萬元，致寡聽類物質至今仍被 視為一種昂貴的食物原料。 • 纟灣四面環海，海洋資源豐富，是全世界首先且唯-以人工養殖 栽培龍鬚菜賴家，麵菜的產量在民國年即已達16,775嘴， 且加工外銷後，曾為台灣水產界增加不少受益。近年來，由於九孔養 瘦業興起’草食性的九孔需要產量充足而供應敎的_，以為成長 所需’由於’龍鬚菜的供應穩定’價格合理，故龍鬚菜已漸取代其他 蕩類’而成為養航孔的主要_，此即為現今龍鮮的主要用途， 藝至今只有少部分被當做觀洋菜補或供人仙，賴鮮的市場價 格始終很低’因此’如何善用龍鬚菜價廉物美且貨源充裕的特性，以 發揮其價值，即成鱗多研究單位及水產#者亟欲突破的—重要課題。 近年來，雖有研究單位已開始使用龍鬚菜製作寡酶水解產物，其 製作方法是分別以熱水與酵素（P_agarase)處理龍鬚菜的乾燥粉末，萃 取出龍鬚菜的㈣成份，並以傳統的複雜分離製程，自龍鬚菜的多醋 成份中分離出寡酶類物質，惟，其製作方法僅能獲得485%與4885% 的多糖萃取率，且傳統分離製程所分離出的寡醣水解產物，經測定其 還原醣（以galactose為代表)含量僅為U7至2 36毫克/毫升，故使 201031751 用前述傳統方法自賴菜中分解岭醣成份的生產率仍然偏低，而未 月I發揮其應有的效能。 有鑑於此，發明人乃針對龍鬚菜的生化特性，不斷進行研究實 驗’以期能透過簡單且低成本的製作過程，萃取出龍鬚菜的多聽成 份’並自龍鬚菜的多醣成份中分離出寡醋類物質，如此，搭配上龍鬚 菜本身價廉物美且貨祕裕的健，勢錢大幅降低募_物質的生 產成本’進而使募聽類物質成為一種便宜的食物原料，而能被添加在 鮝1多市售食品中，以維護人體健康，且達成有效預防疾病的目的同 知’為水產製造及食品加工業者帶來新的商機。 【發明内容】 本發明的主要目的’係在提供—種自龍鬚菜巾分離出主要聚合度 為2-6的新洋菜券聽水解混合物的製造方法，期能以簡單且有效率的製 程有效知:尚龍鬚菜乾燥粉末中多醣成份的萃取率，進而使得業者能 ❹在後續的製程中’自鮮糖成份中分離出更㈣寡麵物f，以大幅 提高自龍鬚菜中分解出募醣成份的生產率。 本發明的另一目的，係先將龍鬚菜的乾燥粉末與纖維素酶 (Cellulase) R-10及果膠酶（Macerozyme) r_i〇混合，進行酵素水解處 理後，再依序進行離心收集處理及凍結乾燥處理，以得到凍乾的龍鬚 菜的多醋萃取粉末，其萃取率高達57·8%，如此，即能以較為簡單且 有效率的製造方法，有效提高龍鬚菜乾燥粉末中多醣成份的萃取率。 本發明的又一目的，係針對該多醣萃取粉末，先後添加海洋細菌 MAiOS及MAEF108所生產之分解酵素(agarases)的粗酵素液，經水解 201031751 處理後’制S財驗’嗣’縣醣水解錢行濃縮處理，得到寡 醣濃縮液後，再對寡酶濃縮液進行膠濾層析處理，即可層析分離出全 醣量（A480nm)的分子質量為⑹.！至ul39Da·的單黯(半乳酶， galactose)、雙糖(新洋菜二醣，ne〇agar〇bi〇se)、四酶(新洋菜四酷， neoagarotetraose)及六醣(新洋菜六醣，ne〇agar〇hexa〇se)等新洋菜寡醣 (neoagarooligosaccharides ’ 簡稱 NA0S)的產物’產率分別為 〇 47%、 3.35%、2.64%、及4.18¼，如此，即能以較為簡單且有效率的製造方 ❿法，有效提高自該多酶成份中分離出更多的募_物質，以大幅提高 寡醣類物質的生產效率。 為便貴審查委員能對本發明的目的、技術特徵及其功效，做更 進一步的認識與瞭解，茲舉實施例配合圖式，詳細說明如下： 【實施方式】 本發明係一種自龍鬚菜中分離出主要聚合度（Dp)為2_6的新洋 e菜募醣水解混合物的製造方法，該方法係先將龍鬚菜的乾燥粉末與纖 維素it(Cellulase)R-l〇及果膠酶(Macerozyme)R-l〇混合，進行酵素水 解處理後’再依序進行離心收集處理及凍結乾燥處理，以得到束乾的 龍鬚菜的多醣萃取粉末，其萃取率高達57.8%，顯然，較前述傳統製 程中分别以熱水與酵素（agarase)處理龍鬚菜的乾燥粉末所得的龍鬚 菜的48.5%與48.85%多醣萃取率，提高了 8.5-9.0%。 另’由於海洋細菌MAEF108 (Jmwno肌及^ ve<s/cw/flr的的二菌株已完成基因體定序，而目前確气▲匕 201031751 進行多醣類分解反應的相關酵素，如：Agarase -- 16個基因、Alginate lyase — 5 個基因、Amylase — 20 個基因、Cellulase - 2 個基因、Xylanase -7個基因等，均可能存在於該二菌株的粗酵素液中。因此，本發明 乃採用海洋細菌MAEF108及MA103所生產之兩組分解酵素(agarases) 的粗酵素液，對龍鬚菜的多醣萃取物進行分解處理，茲詳述本發明備 製該二特有分解酵素的過程如下： (1) 海洋細菌MAEF108所生產之分解酵素的備製過程：係將海洋細 菌 MAEF108 菌株置入含 1〇〇 毫升 MMB-MAEF108 (modified marine broth for the MAEF108 strain ’ pH 6.0)培養基的 250 毫升 二角瓶中，接種1.0%已活化菌株的海洋細菌的菌 液’接者，在溫度20°C下’以轉速240 rpm的條件振盪培養48 小時後，收集發酵菌液，並在溫度4°c下，以3〇,7〇〇 X G (Gravity) 條件離心30分鐘後，收集上層液，即得到海洋細菌 所生產之分解酵素的粗酵素液，其中具有250單位/毫升的分解 酵素活性。 (2) 海洋細菌MA103所生產之分解酵素的備製過程：係將海洋細菌 MA103 菌株置入含 3.0 公升 MMB-MA103 Onodified marine broth for the strain MA103，pH 6·2)培養基的 5 公升發酵槽中， 接種1·0 %已活化菌株的海洋細菌以八103的菌液，接著在溫 度26°C下’以3·〇鹽酸(HCI)與3.〇氫氧化鈉將ρΗ 值控制在6.2’空氣流量維持在4.0公升/分鐘下，並以〇 孔徑大小膜過濾器過濾空氣，然後，以轉速15〇的條件振盪 201031751 培養48小時後’收集發酵菌液，在溫度代下，使用l4,3〇〇 XC7條件離心30分鐘後’收集上層液，即得到海洋細菌皿八1〇3 所生產之分解酵素的粗酵素液，其中具有5〇〇單位/毫升的分解 酵素活性。 ㈣’本發明針對龍鬚菜的多黯萃取物，先後添加海洋細菌退⑽ 及MAEF108所生產之分解酵素的粗酵素液，經水解處理後，即能得 到募醣水驗，嗣’轉时舰進行濃祕理，制寡料縮液後， 參再對寡醣漠縮液進行雜層析處理，即可檢測出其析出液的全糖量 (“ο副)的分子質量約為！阳至•，且能分離出單糖(半乳 醣galactose)、雙醣(新洋菜二酶，ne〇agaf〇bi〇se)、四_(新洋菜四畴， neoagarotetraose)及六膽(新洋菜六醣，ne〇agar〇hexa〇se)等新洋菜募酶 (neoagarooligosaccharides，簡稱 NA0S)的產物，產率分別為 〇47、 3.35、2.64 及 4.18%。 在本發明的實際製作過程中，自龍鬚菜中萃取出多酶成份的程 序’參閱第1圖所示’主要包括下列步驟，能有效提高多醣成份的萃取 率’以便業者能在後續的製程中，自多聽萃取物中分離出更多的寡醣 類物質： (101)將5〇〇公克的龍鬚隸燥粉末轉加人_水，其中龍鬚菜乾燥 粉末原料與蒸館水的重量比為i: 50，使二者均句混合，且加熱201031751 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a method for producing a New Zealand oligosaccharide, referred to as NAOS, and in particular to a main degree of polymerization (Degree of P) from Asparagus Sp. 〇lymerization 'abbreviated as Dp) is a lysate mixture of 2-6, which is a manufacturing method for greatly increasing the productivity of decomposing oligosaccharide components from Asparagus officinalis. [Prior Art] According to the conservation of the shellfish, it is generally referred to as "prebi〇tics", which is a substance that selectively promotes the growth of "probiotics". Due to the special structure, the raw material can not be decomposed and utilized by the digestive enzymes of the human body. Therefore, it can be used to enter the human body through the upper digestive tract, and is fertilized by the intestine (10). The sugar-collecting system is a polymer obtained by dehydration condensation of 2-10 monosaccharide molecules. In nature, 'oligosaccharides, sucrose, maltose, and lactose' are present in a free state or a glycoside-like state. Physiologically active oligozymes in plants and fruits, such as: too-oligosacchrides, is malt oligosaccharides, galacto-oligosacchrides ), etc., all use p-type glycosidic bonds to bind different monosaccharide molecules', so it is difficult to be decomposed by the enzymatic enzymes used in the digestive tract of the human body to decompose α-forms. The soft large intestine provides the fermentation of organic bacteria in the intestines to produce organic acids to obtain energy and lower the pH in the intestines to inhibit the production of harmful substances in the intestines and prevent intestinal microbes. Bile acid cleavage produces carcinogens. It can be seen that the illuminating substances play an extremely important role in the transformation of human health in 201031751. However, according to the research report of the new eucalyptus, the existing technology is expensive. And high-purity agarose, as a decomposition matrix of agarase, according to sigmaC〇, its commercial price is 1,330/kg, which leads to its extraction of raw materials and production costs. The average residence is not T, taking the domain of the city f (啲啊龄(四)) and the vine of the six divisions eGagarGhexa〇se) as an example. 'Each per gram is as high as NT$1 to $320,000. It is considered an expensive food ingredient. • Tsuen Wan is surrounded by the sea and is rich in marine resources. It is the first and only cultivated in the world. It is cultivated in the country. The output of noodles has reached 16,775 in the Republic of China. After processing and exporting, it was once the Taiwanese aquatic industry. Increased a lot of benefits. In recent years, due to the rise of the nine-hole thinning industry, the 'grass-like nine holes need sufficient supply and the supply of _ _, thinking that the growth required 'because of the stable supply of the sauerkraut' is reasonable, so the dragon mustard has gradually replaced other The main category of cultivating the hole is the main use of today's dragon oysters. This is only a small part of the art has been used as a garnish or a scent. The market price of Lai Xian is always low. Make good use of the characteristics of the long-term food and the abundant supply of the asparagus, in order to exert its value, that is, the research institutes and the aquatic products of the scales are the important subjects. In recent years, although the research unit has begun to use the asparagus to produce oligo-enzymatic hydrolysate, it is prepared by treating the dried powder of Asparagus with hot water and enzyme (P_agarase), and extracting the (4) component of Asparagus. The oligo-enzymes were isolated from the multi-vine components of Asparagus officinalis by traditional complex separation process. However, the preparation method can only obtain the extraction rate of polysaccharides of 485% and 4885%, and the isolation of the traditional separation process. The sugar hydrolysate, which has been determined to have a reducing sugar (represented by galactose), is only U7 to 2 36 mg/ml, so that the productivity of 201031751 using the aforementioned conventional method for decomposing the saccharide component from the lyophile is still low, but not I play its due role. In view of this, the inventors have been conducting research experiments on the biochemical characteristics of Asparagus, in order to extract the multi-sounding ingredients of Asparagus from a simple and low-cost production process and from the polysaccharides of Asparagus. Separating the vinegar-like substances, so that, with the good quality of the asparagus itself and the health of the goods, the money is greatly reduced, and the production cost of the substance is further reduced, thereby making the listening substance a cheap food raw material. It can be added to more than one commercially available food to maintain human health and achieve the goal of effective disease prevention. It brings new business opportunities for aquatic products manufacturing and food processing industry. SUMMARY OF THE INVENTION The main object of the present invention is to provide a method for producing a new amaranth scented hydrolyzed mixture having a main polymerization degree of 2-6, which is a simple and efficient process. Effectively know: the extraction rate of polysaccharides in the dried powder of Shanglong mustard, which in turn enables the industry to separate the more (4) oligosaccharides f from the fresh sugar ingredients in the subsequent process, in order to greatly improve the decomposition from the asparagus The productivity of the sugar component is raised. Another object of the present invention is to first mix the dried powder of Asparagus officinalis with Cellulase R-10 and Pectinase (Macerozyme) r_i〇, carry out enzymatic hydrolysis treatment, and then perform centrifugal collection and processing in sequence. And freeze-drying treatment to obtain the vinegar extract powder of lyophilized asparagus, the extraction rate is as high as 57.8%, so that the drying method can effectively improve the dry powder in the asparagus flower in a relatively simple and efficient manufacturing method. The extraction rate of the polysaccharide component. Another object of the present invention is to extract the crude enzyme liquid of the degrading enzyme (agarases) produced by marine bacteria MAiOS and MAEF108 for the polysaccharide extracting powder, and to treat the sugar hydrolysis of the county by hydrolysis after 201031751 treatment. After the concentrated treatment of the money to obtain the oligosaccharide concentrate, the oligo-enzyme concentrate is subjected to gel filtration chromatography, and the molecular mass of the whole sugar amount (A480nm) can be separated by chromatography (6). To ul39Da·single (galactose), disaccharide (new amaranth disaccharide, ne〇agar〇bi〇se), four enzymes (new aquarium, neoagarotetraose) and hexose (new amaranth) The yields of hexasaccharide, ne〇agar〇hexa〇se) and other products of neoanarooligosaccharides 'NA0S' are 47%, 3.35%, 2.64%, and 4.181⁄4, respectively. The simple and efficient manufacturing method effectively increases the separation of more substances from the multi-enzyme component to greatly increase the production efficiency of oligosaccharides. For the purpose of the present invention, the present invention can be further understood and understood. The embodiments are described in detail as follows: [Embodiment] The present invention is a kind from the asparagus. A method for producing a Xinyang e vegetable glycolysis mixture having a main polymerization degree (Dp) of 2_6 is isolated, which is a method in which a dry powder of Asparagus officinalis and a cellulase (Cellulase) R1 and a pectinase (Macerozyme) are firstly prepared. Rl〇 is mixed and subjected to enzyme hydrolysis treatment, and then subjected to centrifugal collection and freeze-drying treatment to obtain a dried polysaccharide extract of Asparagus officinalis, and the extraction rate is as high as 57.8%, which is obviously different from the above-mentioned conventional processes. The extraction rate of 48.5% and 48.85% polysaccharides of Asparagus officinalis obtained by treating the dried powder of Asparagus with hot water and agarase increased by 8.5-9.0%. In addition, due to the marine bacteria MAEF108 (Jmwno muscle and ^ ve < s / cw / flr of the two strains have completed the genetic sequencing, and now the gas ▲ 匕 201031751 for the decomposition of polysaccharides related enzymes, such as: Agarase - - 16 genes, Alginate lyase - 5 genes, Amylase - 20 genes, Cellulase - 2 genes, Xylanase - 7 genes, etc., may all be present in the crude enzyme solution of the two strains. Therefore, the present invention is The two groups of crude enzymes produced by marine bacteria MAEF108 and MA103 are used to decompose the polysaccharide extract of Asparagus. The process of preparing the two specific decomposing enzymes of the present invention is as follows: (1) The preparation process of the degrading enzyme produced by the marine bacteria MAEF108: the marine bacteria MAEF108 strain was placed in a 250 ml two-necked flask containing 1 ml of MMB-MAEF108 (modified marine broth for the MAEF108 strain 'pH 6.0) medium. The bacterial liquid of the marine bacteria inoculated with 1.0% of the activated strain was incubated at a temperature of 20 ° C for 48 hours at a rotational speed of 240 rpm, and then the fermentation broth was collected and After centrifugation at 3 ° C, 3 〇, 7 〇〇 XG (Gravity) conditions for 30 minutes, the supernatant liquid was collected to obtain a crude enzyme solution of a decomposition enzyme produced by marine bacteria, which had a decomposition enzyme of 250 units/ml. (2) Preparation process of decomposing enzymes produced by marine bacteria MA103: The marine bacteria MA103 strain was placed in a 5 liter fermentation medium containing 3.0 liters of MMB-MA103 Onodified marine broth for the strain MA103, pH 6·2) In the trough, the marine bacteria inoculated with 1.0% of the activated strain were treated with 8 103 bacteria, followed by a temperature of 26 ° C. The pH value of Η was controlled by 3% hydrazine hydrochloride (HCI) and 3. cesium hydroxide. 'The air flow rate is maintained at 4.0 liters/min, and the air is filtered with a membrane filter of 〇 pore size. Then, it is oscillated at a speed of 15 2010. 201031751 After culturing for 48 hours, 'the fermentation broth is collected, and under temperature, l4 is used. After centrifugation for 3 minutes under XC7 conditions, 'the supernatant liquid was collected, and the crude enzyme solution of the decomposing enzyme produced by the marine bacteria dish 8 1 , 3 was obtained, which had a decomposition enzyme activity of 5 〇〇 unit/ml. (4) 'The present invention is directed to the polysaccharide extract of Asparagus officinalis, and the crude enzyme solution of the decomposing enzyme produced by marine bacteria (10) and MAEF108 is added successively. After hydrolysis treatment, the sugar-receiving water can be obtained. After carrying out the thickening principle, after the sizing of the oligosaccharide, the oligosaccharide molybdenum solution can be detected by heterochromatography, and the total sugar content of the precipitate can be detected (the molecular weight of the "o pair" is about! •, and can separate monosaccharide (galactose galactose), disaccharide (new amaranth enzyme, ne〇agaf〇bi〇se), four _ (new cabbage four domains, neoagarotetraose) and six gallbladder (new ocean In the actual production process of the present invention, the yields of the products of neoanarooligosaccharides (NA0S), such as hexasaccharide, ne〇agar〇hexa〇se), are 〇47, 3.35, 2.64 and 4.18%, respectively. The procedure for extracting multi-enzyme components from Asparagus is shown in Fig. 1 'mainly including the following steps, which can effectively improve the extraction rate of polysaccharides' so that the manufacturer can separate from the extracts in the subsequent process. More oligosaccharides: (101) Transfer 5 gram grams of Longxu Li dry powder Add _ water, in which the asparagus is dried. The weight ratio of the powder raw material to the steaming water is i: 50, so that the two are mixed and heated.
至溫度100〇C，約4㈣後，將其冷卻至室溫(25。〇，以得到其萃 取溶液； A _在没度3rc下，將纖維素梅（Cellul㈣㈣及果_ 9 201031751 (MaCerozyme) R_10分別以6%的濃度[enzyme (酵素批ate (基材）’ E/S = 6%，w/w]，加入萃取溶液，進行水解處理； (103) 俟進行3小時水解處理後，以溫度85〇c對水解液加熱3〇分 鐘，使水解液失去活性後，將水解液冷卻至室溫(25。匸）； (104) 以3,500 X G離心力，對水解液進行3〇分鐘的離心處理後，收集 上層液’即能得到龍鬚菜的多醣萃取液； (105) 使用;東結乾燥機，在溫度_180C τ，對上層液進行束乾處理即 馨 能製成289公克的龍鬚菜的多醣萃取粉末(Polysaccharides extract in lyophilized powder) ’且保存於乾燥箱中供後續使用。 另在本發明的實際製作過程中，自前述多聽萃取粉末中分離出 寡醣水解物的程序，復參閱第i圖所示，主要包括下列步驟，能有效提 高寡醣成份的生產率： (106) 將289公克的龍鬚菜的多聽萃取物的乾燥粉末，置入5公升的發 酵槽中，以緩衝溶液將其回復成龍鬚菜的多醣溶液後，在每1〇〇 ® €升的龍鬚菜的多糖溶液中’分別添加5⑻酵素活性單位的海洋 細菌MA103及MAEF108所生產之分解酵素(agarases)的粗酵素 'm度為26°C (mcubationtemp.) ’龍鬚菜的反應基質濃度為 0.5%，緩衝溶液（Tris_HClbuffer，pH6 2)、氯化納及氯化 挪叫以重量比仏⑶：…混合咖值固定為阳⑽發酵 槽轉速(_哪(|)為25〇11)111’通氣量(挪沾〇11)為 4公升/分鐘， 及誘導分解酵素生成之添加物為〇3%重量比的洋菜(琴)等培養 條件下’對龍鬚菜的乡醣萃取物進行則、時的水解處理 201031751 (rehydration)’其水解效率高達到ι〇%以上，嗣，以溫度1⑻。c加 熱5分鐘，使酵素失活終止反應後，收集龍鬚菜的寡醣水解產 物’得到5.78公升的龍鬚菜的寡醣水解液’其測定還原醣（以 galactose為代表）含量為[π至2 49毫克/毫升； (107) 利用超過濾系統（disp〇sabie m〇dular tangential fl〇w __ 及 1’OOOdaltonsMWCO (molecular weight cut-off)膜，對5.78 公升 的龍鬚菜的养醣水解液進行漢縮處理，並經減 壓’濃縮至1，200毫升的龍鬚菜的募醣濃縮液； (108) 採用 BioGelP4 的膠濾層析(geipermeati〇nchr〇mat〇graphy，簡 稱GPC)法，分析^oo毫升的龍鬚菜的寡醣濃縮液的分子量分 佈情形，在本發明的實際製作過程中’係採用1〇〇公分高、2 6 公分直徑的管柱，内裝Sephadex G-10 (品牌名為Pharmacia^ USA) ’以無菌水當析出液（eluent)，在室溫下，進行寡醣的析出 及分離，再以分液收集器收集析出液。如此，龍鬚菜的多醣萃取 物經先後添加海洋細菌MA103及MAEF108所生產之分解酵素 的粗酵素液，在溫度26°C下’水解48小時，且經濃縮處理後， 所得到的寡醣濃縮液，經Sephadex G-10膠濾層析，檢測出其 析出液的全醣量（A480nm)的分子質量約為163.1至1，213.9 Da. ’且分離出不同分子質量的單醣(半乳酷，碑⑽⑽）、雙醣(新 洋菜二醣，neoagarobiose)、四醣(新洋菜四醣，ne〇agar〇tetTa〇se)及 /、聽（新洋菜六糖，neoagarohexaose)等新洋菜募酶 (neoagarooligosaccharide ’簡稱NAOS)產物，經收集凍乾處理， 11 201031751 即能得到各別純化的單一新洋菜寡醣製成品，其產量分別為 2.36、16.76、13.18 及 20.88 公克，其產率分別為 〇 47、3.35、 2.64及4.18%。發明人使用Sephadex G-10膠濾層析法，對單 醣、雙醣、四醣及六醣等醣類標準品混合液，進行全醣量 V 480^ 測定，得到第2圖所示的全醣量（八48^)的SephadexG_1〇膠濾 層析圖，另’發明人使用SephadexG-ΙΟ膠濾層析法，對本發明 的龍鬚菜多醣萃取物’經添加海洋細菌MAW3及所 生產之分解酵素，水解48小時後，所得到的寡酿濃縮液，進行 全醣量（A^Onm)測定，則得到第3圖所示的全醣量（A48〇 )的 ΦAfter the temperature is 100 ° C, about 4 (four), it is cooled to room temperature (25 ° 〇 to obtain its extraction solution; A _ in the absence of 3rc, the cellulose plum (Cellul (four) (four) and fruit _ 9 201031751 (MaCerozyme) R_10 The hydrolysis solution was added to the extraction solution at a concentration of 6% [enzyme (enzyme batch ate (substrate) E/S = 6%, w/w]; (103) 俟 was subjected to hydrolysis treatment for 3 hours, at a temperature 85〇c heat the hydrolyzate for 3 minutes, after the hydrolysate is deactivated, the hydrolyzate is cooled to room temperature (25. 匸); (104) After centrifugation at 3,500 XG, the hydrolyzate is centrifuged for 3 minutes. , collecting the supernatant liquid can get the polysaccharide extract of Asparagus officinalis; (105) use; East knot dryer, at the temperature _180C τ, the upper layer liquid is dried, that is, Xin can make 289 grams of Asparagus Polysaccharides extract in lyophilized powder' and stored in a dry box for subsequent use. Also in the actual production process of the present invention, the procedure for separating the oligosaccharide hydrolyzate from the above-mentioned multi-audio extraction powder is referred to As shown in Figure i, it mainly includes the following steps. To improve the productivity of the oligosaccharide component: (106) Put a dry powder of 289 g of the succulent extract of Asparagus officinalis into a 5 liter fermenter and return it to the polysaccharide solution of Asparagus officinalis with a buffer solution. Adding 5 (8) enzyme activity units of marine bacteria MA103 and MAEF108 to the polysaccharide solution of each of the 1 〇〇® € liter of Asparagus, the crude enzyme 'm degree is 26 ° C (mcubationtemp. ) 'The response substrate concentration of Asparagus is 0.5%, buffer solution (Tris_HClbuffer, pH6 2), sodium chloride and chloride mitigation to weight ratio 仏 (3): ... mixed coffee value is fixed to yang (10) fermentation tank speed (_ (|) is 25〇11) 111' ventilation (Nuozhao 11) is 4 liters/min, and the additive that induces the formation of decomposing enzymes is 〇3% by weight of agar (qin) and other culture conditions. The sugar extract of Asparagus is treated with the hydrolysis treatment of 201031751 (rehydration), which has a high hydrolysis efficiency of ι〇% or more, and is heated at a temperature of 1 (8) c for 5 minutes to deactivate the enzyme and terminate the reaction. The oligosaccharide hydrolysate of Asparagus 'a 5.78 liter of dragon's whisker The oligosaccharide hydrolysate's determination of reducing sugars (represented by galactose) is [π to 2 49 mg/ml; (107) using an ultrafiltration system (disp〇sabie m〇dular tangential fl〇w __ and 1'OOOdaltons MWCO) (molecular weight cut-off) membrane, Hantreat treatment of 5.78 liters of Asparagus nutrient hydrolysate, and concentrated to 1,200 ml of Asparagus's sugar concentrate after decompression; (108) The molecular weight distribution of the oligosaccharide concentrate of Asparagus edulis was analyzed by the gel filtration chromatography (Gipermeati〇nchr〇mat〇graphy, GPC for short) method of BioGelP4, which was used in the actual production process of the present invention. 1 〇〇 cm high, 2 6 cm diameter column with Sephadex G-10 (brand name Pharmacia^ USA) 'As a pure water eluent, oligosaccharide precipitation at room temperature Separate and collect the precipitate with a liquid separator. Thus, the polysaccharide extract of Asparagus is added to the crude enzyme solution of the decomposing enzyme produced by marine bacteria MA103 and MAEF108, and hydrolyzed at a temperature of 26 ° C for 48 hours, and after concentration treatment, the obtained oligosaccharide is concentrated. The liquid was detected by Sephadex G-10 gel filtration chromatography, and the molecular weight of the total sugar (A480nm) of the precipitate was about 163.1 to 1,213.9 Da. ' and the monosaccharides of different molecular masses were isolated (half milk) , monument (10) (10)), double sugar (new cabbage disaccharide, neoagarobiose), tetrasaccharide (new Chinese cabbage tetrasaccharide, ne〇agar〇tetTa〇se) and /, listening (new cabbage, six sugar, neoagarohexaose) and other new ocean The product of neoagrooligosaccharide (NAOS) was collected and lyophilized. 11 201031751 was able to obtain a single purified new oligosaccharide product with a yield of 2.36, 16.76, 13.18 and 20.88 g, respectively. The rates are 〇47, 3.35, 2.64 and 4.18%. The inventors used Sephadex G-10 gel filtration chromatography to measure the total sugar amount V 480^ for a mixture of sugar standards such as monosaccharide, disaccharide, tetrasaccharide and hexasaccharide, and obtained the full picture shown in Fig. 2 The amount of sugar (eight 48^) SephadexG_1 gelatin filter chromatogram, the other 'inventors use SephadexG-gelatin filter chromatography, the polysaccharide extract of Asparagus extract of the present invention' is added to the marine bacteria MAW3 and the decomposition of the production After the enzyme was hydrolyzed for 48 hours, the obtained oligosaccharide concentrate was measured for the total sugar amount (A^Onm), and the total sugar amount (A48〇) shown in Fig. 3 was obtained.
SephadexG_1G縣騎®。帛4圖麻战鬚菜乡料取物分 別經（A)海洋細菌MAi〇3所生產之分解酵素或⑻海洋細菌 MAEF108所生產之分解酵素進行分解後，與（c)單醣、雙醣、 四聽及六糖等酶類標準品混合液經薄層層析獅 chromatogr*，簡稱几〇分析後’所得到的分析結果。由前述 麟層析圖及分析結果，能清楚顯示，细本發_方法確實能 將聚合度（degree 〇f p〇lymerization，簡稱Dp)至少為2 6的新 洋菜募糖層析分離出，而得到各別純化的單一新洋菜寡醋製成 品。 由於，在本發明的前述實際製作過財，所_的公克的 龍鬚菜乾燥粉末，市售價格僅需美金3〇元/公斤，即可製得約憾公 克的’的新洋_，生產_1()%以上，其價值_喊 Co.⑽啊的報價，約為美金3,3〇〇元/公克，因此，本發明的價值 12 201031751 、 · 在於可充分利用最便宜與基礎的原材料，藉由組合海洋細菌隐⑽ 與MAEF 1〇8二菌株特有的粗酵素液，先後對龍鬚菜的多黯萃取物的 乾燥粉末進行轉域，再触分雜倾術，即可得到古 價值已純㈣料絲料品。料，市㈣分鱗素，依&卿C: 的報價，若其酵素活性為5，_單位/瓶，其售價約為美金伽元/瓶， 惟，在溫度40〇C下’以1〇彻(酵素活性單位)市售單一洋菜的分解 酵素水解洋菜醣（agar〇se，依沿脚心報價美金⑽元/公斤） =4 ^後，將得到第5圖所示的寡醣生產率示意圖，且能清楚看 =的分解酵素水解洋菜㈣*時後，僅能制約娜的水 ^新洋米寡_產品的生產率。而本發明於侧反應條件下，所得到的 7解新洋喊品即可_祕以上的生產效率(請參騎表^所 ^且透顺魏朗_及分赌果，顯林㈣確實能以更有效 -更低生產成本的方式，生產出高量且高單價的新洋菜募醋成品。 附表1、龍鬚菜寡醣的產率SephadexG_1G County Ride®.帛4 图麻战须菜乡物物物is decomposed by (A) decomposing enzyme produced by marine bacteria MAi〇3 or (8) decomposing enzyme produced by marine bacteria MAEF108, and (c) monosaccharide, disaccharide, The mixture of four enzymes and six hexasaccharides and other standard enzymes was analyzed by thin-layer chromatography of lion chromatogr*, abbreviated after several analyses. From the above-mentioned lining chromatogram and analysis results, it can be clearly shown that the fine _ method can indeed separate the degree of polymerization (degree 〇fp〇lymerization, Dp) at least 26, and extract the sugar by chromatography. A separately purified single new cabbage vinegar product was obtained. Because, in the above-mentioned actual production of the present invention, the gram of the dried sauerkraut dry powder, the commercial price of only 3 US dollars / kg, can be produced to the credit of the 'new ocean _, production _1 ()% or more, its value _ shout Co. (10) ah quote, about US$ 3,3 / / gram, therefore, the value of the invention 12 201031751, · in the use of the cheapest and basic raw materials By combining the marine bacteria hidden (10) with the crude enzyme solution unique to the MAEF 1〇8 strain, the dry powder of the polysaccharide extract of Asparagus officinalis was transferred to the field, and then the complex value was obtained, and the ancient value was obtained. Has been pure (four) wire products. Material, city (four) squama, according to & Qing C: quote, if its enzyme activity is 5, _ unit / bottle, its price is about US$ gamma / bottle, but at a temperature of 40 〇 C 1 〇 (Enzyme Active Unit) The commercially available single amaranth decomposition enzyme hydrolyzes the sugar (agar〇se, according to the foot of the heart quoted US dollars (10) yuan / kg) = 4 ^, will get the oligosaccharide shown in Figure 5 The productivity is schematic, and it can be clearly seen that the decomposition of the enzyme hydrolyzed agar (4)* can only limit the productivity of Na's water, new rice, and rice. However, under the side reaction conditions of the present invention, the obtained 7-solution Xinyang shouting product can be used for the production efficiency of the above-mentioned secrets (please refer to the riding table ^ and the smooth Weilang _ and the gambling fruit, the display forest (four) can indeed be more Efficient-lower production cost method to produce high-quality and high-priced new yam vinegar products. Schedule 1, Asparagus oligosaccharide yield
膠濾層析處理 (半乳醣等，Galactose) 二醣（新洋菜二醣 Neoagarobiose) 2.36公克（0.47^ 參 16.76公克（3.35%) 四醣（新洋菜四醣 Neoagarotetraose) 13.18公克（2.64%) 20.88公克（4·18%) 六醣（新洋菜六醣 Neoagarohexaose) 13 201031751 總計 53.18公克 00.64%) 據上所述，本發明顯然能以較為簡單且有效率的製造方法，有效 提高龍鬚菜乾燥粉末中多聽成份的萃取率，進而自多酵萃取物中分離 出更多的寡醣類物質，以大幅提高寡醣類物質的生產效率。 按，以上所述，僅為本發明最佳的一具體實施例，惟本發明的構 造特徵並不侷限於此，任何熟悉該項技藝者在本發明領域内，可輕易 思及的變化或修飾，皆可涵蓋在以下本案的專利範圍。 ❹ 【圖式簡單說明】 第1圖係本發明的製作方法的流程示意圖； 第2圖係使用SephadexG-ΙΟ膠濾層析法，對單醣、雙醣、四醣 及六醣等醣類標準品混合液，進行全醣量測定，所得到的 SephadexG-ΙΟ膠濾層析圖； 第3圖係使用SephadexG-ΙΟ膠濾層析法，對先後添加海洋細菌 參ΜΑ1〇3及MAEFl〇8所生產之分解酵素的龍鬚菜多酷萃取物，經水解 48小時所彳于到的养醣濃縮液，進行全醣量（a#^)測定，所得到的 SephadexG-ΙΟ膠濾層析圖； 第4圖係龍鬚菜多糖萃取物分別經⑷料細菌祖1〇3所生產 之分解酵素或（B)料細g ^^壓所生產之分解酵素進行分解 後’與（C)單聽、雙_、四糖及六酷等醣娜準品混合液經薄層層析 分析後，所得到的分析結果；及 第5圖係洋菜酶經1〇_6 〇酵素活性單位市售單一洋菜分解酵素 201031751 (agarase; Sigma Co.)降解12-24小時後所得之寡醣生產率。 【主要元件符號說明】Gel filtration chromatography (galactose, Galactose) Disaccharide (Newagorobiose) 2.36 g (0.47^ gins 16.76 g (3.35%) Tetrasaccharide (Neoagarotetraose) 13.18 g (2.64%) 20.88 g (4·18%) Hexaose (Neoagarohexaose) 13 201031751 Total 53.18 g 00.64%) According to the above, the present invention can effectively improve the dragon's whisker by a relatively simple and efficient manufacturing method. In the dry powder of the dish, the extraction rate of the components is multi-audited, and more oligosaccharides are separated from the poly-fermented extract to greatly increase the production efficiency of the oligosaccharide. The above description is only a preferred embodiment of the present invention, but the structural features of the present invention are not limited thereto, and any changes or modifications that can be easily conceived in the field of the present invention are familiar to those skilled in the art. , can be covered in the scope of the patent in this case below. ❹ [Simplified description of the drawings] Fig. 1 is a schematic flow chart of the production method of the present invention; Fig. 2 is a standard of sugars such as monosaccharide, disaccharide, tetrasaccharide and hexasaccharide using Sephadex G-gelatin filtration chromatography The mixed liquid is subjected to the determination of the total sugar amount, and the obtained Sephadex G-gelatin filter chromatogram; the third figure is the Sephadex G-gelatin filter chromatography, and the marine bacteria ginseng 1〇3 and MAEFl〇8 are successively added. The dried extract of Asparagus extract, which is produced by decomposing the enzyme, is subjected to hydrolysis of the sugar concentrate concentrated for 48 hours, and the whole sugar amount (a#^) is measured, and the obtained Sephadex G-gelatin filter chromatogram is obtained; 4 The extract of the polysaccharide of Asparagus officinalis is decomposed by the decomposition enzyme produced by (4) the bacterial progenitor 1〇3 or the decomposition enzyme produced by the (B) material fine g ^^ pressure, respectively, and (C) single listening, double The analysis results obtained after thin layer chromatography analysis of _, tetrasaccharide and Liukuo et al., and the fifth figure of acacia enzymes sold by a single 〇6 〇 enzyme active unit Degrading enzyme 201031751 (agarase; Sigma Co.) degraded the oligosaccharide productivity after 12-24 hours. [Main component symbol description]
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