517088 經濟部智慧財產局員工消費合作社印製 A7 B7 i、發明説明（） 本發明係有關一種利用複合細胞系統生產高純度果 寡糖之方法，且更特定言之，有關一種利用有Θ -岐喃果糖 苷酷活性的真菌菌絲和有蔔萄糖去氫醏的活菌細胞同時與 蔗糖溶液反應以生產高純度，例如大於80%乾重的果寡糖 之方法。 ， 4 最近在食品加'工界開發一些寡糖的材料，具有多種 機能特性。如能滋化腸內5///心^菌增殖，不易引起蛀牙 等。适類的寡糖包括果寡糖（F r u c t ο ο 1 i g 〇 s a c c h a r i d e s)、半 乳糖寡糖（Galactooligosaccharides)、大豆寡糖（Soybean oligosaccharides)。以上除了大豆.寡糖是由大豆直接抽取 外，其它都是利用酵素法合成。果寡糖廣泛存在於天然的 植物中，如蘆筍、洋蔥、牛蒡等。果寡糖的工業化大量生 產乃是以蔗糖爲原料利用/3 -吱喃果糖苷S每（冷-fructofuriinosidase)進行果糖轉移反應產生含50〜60%果寡 糖成份，其它成份爲30〜40%的葡萄糖和10〜20%的蔗糖之 產物。 在過去的硏究中，發現果寡糖生成反應受到其反應. 生成物葡萄糖的抑制，而大大的降低反應速率（K. JL Duan et al·，“ Kinetics studies and mathematical model for enzymatic production of fructooligosaccharides from sucrose”，Enzyme Microb· Technol. 1994，16, p. 3 3 4 - 339.)，因此若能在反應進行中同時除去葡萄糖，則可大 大提高酵素反應效率，並且形成高純度果寡糖。高純度果 寡糖可作爲糖尿病患者的代用糖，添加在糖果中，可防止 小孩蛀牙，添加於嬰兒奶粉內，可滋化腸內⑴/Mw ，抑 制腐敗菌的增殖，降低斷奶或進用副食品時腹瀉的可能。 果寡糖爲蔗果三糖（Ι-kestose )，蔗果四糖 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297厶釐） (請先閱讀背面之注意事項再填寫本頁)517088 Printed by A7, B7, Consumer Cooperatives, Intellectual Property Bureau, Ministry of Economic Affairs i. Description of the Invention The present invention relates to a method for producing high-purity fructooligosaccharides using a composite cell system, and more specifically, to an A method in which fructoside is a highly active fungal hypha and viable dextrose-containing viable bacterial cells are simultaneously reacted with a sucrose solution to produce high-purity, such as greater than 80% dry weight fructooligosaccharides. 4 Recently, some oligosaccharide materials have been developed in the food processing industry, with various functional properties. If it can promote the proliferation of 5 /// heart bacteria in the intestine, it is not easy to cause tooth decay. Suitable oligosaccharides include fructo-oligosaccharides (F r u c t ο ο 1 i g 0 s a c c h a r d e s), galactooligosaccharides, and soybean oligosaccharides. Except for soy and oligosaccharides, which are directly extracted from soybeans, others are synthesized by the enzyme method. Fructo-oligosaccharides are widely found in natural plants, such as asparagus, onions, and burdock. The industrialized mass production of fructooligosaccharides is based on the use of sucrose as the raw material. The fructose transfer reaction is carried out with / 3-fructofraninoside S (cold-fructofuriinosidase) to produce 50 ~ 60% fructooligosaccharides, and the other ingredients are 30 ~ 40%. The product of glucose and 10 ~ 20% sucrose. In the past research, it was found that the fructo-oligosaccharide production reaction is affected by its reaction. The product glucose is inhibited, and the reaction rate is greatly reduced (K. JL Duan et al., "Kinetics studies and mathematical model for enzymatic production of fructooligosaccharides from "sucrose", Enzyme Microb · Technol. 1994, 16, p. 3 3 4-339.), so if the glucose can be removed at the same time as the reaction proceeds, the enzyme reaction efficiency can be greatly improved, and a high-purity fructooligosaccharide can be formed. High-purity fructooligosaccharide can be used as a substitute sugar for diabetic patients. It can be added to candy to prevent tooth decay in children. When added to infant milk powder, it can nourish intestinal crest / Mw, inhibit the proliferation of spoilage bacteria, and reduce weaning or consumption. Possible diarrhea when food. Fructo-oligosaccharides are cane-fructose (I-kestose) and cane-fructose. This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297%) (Please read the precautions on the back before filling this page)
517088 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明説明（） · (nystose)和蔗果五糖（fructosyl nystose)之統稱，其 化學構造如第一圖所示。 對於高純度果寡糖的生產一直是多方努力的目標。 歸納以前的方法如下： 1 ·利用葡萄糖氧化_ ( g 1 u c 〇 s e ο X i d a s e )氧化果寡糖水飴 中的葡萄糖成爲葡萄糖酸，再用離子交換樹脂，將葡萄 糖酸吸附分離下來，而得到高純度果寡糖。[Κ· H· Jung e t al., “ Production of high fructooligosaccharides syrup with two enzyme system of fructosyi-transferase and . glucose oxidase ’’ ， Biotech. Letters 1993，15(1)，p.65-70 ； J. W. Yun et a 1., “ Batch production of high-content fructo- oligosaccha rides from sucrose by the mixed-enzyme system of β-fructofuranosidase and glucose oxidase “， J· Ferment. Bioeng·，1 994，7 7 (2) , p · 1 5 9 -1 6 3 ·】 2·利用離子交換樹脂層析法，直接分離果寡糖和葡萄糖。 [楊博文、黃喜玲、王隆輝、劉曜東，19 94，“離子交 換樹脂分離管柱爾業-化-生-產90%果寡糖之硏究“，台灣 糖業硏究所硏究彙報，146， p.33-46 ； Meiji Seika , 1984 ,日本專利 59 - 95895 ·】 利用一特殊菌-皴摺青黴菌（rugulosum ) 的釀酵液，內含果糖轉移醅、菊糖醅（inulinase )、轉化 SI ( invertase )作用於蔗糖，而產生約84 %純度的果寡 糖。[ C· Barthomeuf and H. Pourrat , “Production of high-content fructooligosaccharides by an enzymatic system from Penicillium rugulosum.^ Biotech. Letters 1995，17(9) , p.911-916.】 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297^釐） (請先閱讀背面之注意事項再填寫本頁)517088 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. General description of inventions () and (fructosyl nystose), the chemical structure of which is shown in the first figure. The production of high-purity fructooligosaccharides has been the goal of many efforts. The previous methods are summarized as follows: 1. Oxidation of glucose in fructo-oligosaccharides to make gluconic acid using glucose oxidation (g 1 uc ο X idase), and then using ion exchange resin to adsorb and separate gluconic acid to obtain high Purity fructooligosaccharide. [KH Jung et al., "Production of high fructooligosaccharides syrup with two enzyme system of fructosyi-transferase and. Glucose oxidase", Biotech. Letters 1993, 15 (1), p.65-70; JW Yun et a 1., "Batch production of high-content fructo- oligosaccha rides from sucrose by the mixed-enzyme system of β-fructofuranosidase and glucose oxidase", J. Ferment. Bioeng ·, 1 994, 7 7 (2), p · 1 5 9 -1 6 3 ·] 2. Direct separation of fructooligosaccharides and glucose using ion exchange resin chromatography. [Yang Bowen, Huang Xiling, Wang Longhui, Liu Yidong, 19 94, "Ion exchange resin separation tube column industry -Chemical-bio-production of 90% fructooligosaccharides ", Research Report of Taiwan Sugar Research Institute, 146, p.33-46; Meiji Seika, 1984, Japanese Patent 59-95895 -Fructose fermentation solution of rugulosum, which contains fructose transfer 醅, inulinase, and SI (invertase) to act on sucrose, and produces about 84% pure fructooligosaccharide. [C · Barthomeuf and H. Pourrat, "Production of high-content fructooligosaccharides by an enzymatic system from Penicillium rugulosum. ^ Biotech. Letters 1995, 17 (9), p. 911-916.] This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 ^ cent) (please (Read the notes on the back before filling out this page)
、1T % 517088 經濟部智慧財產局員工消費合作社印製 A7 ， B7 _五、發明説明（） 質，例如 CARBOSEP ( Tech-Sep，France ) 。 3·中空纖 維，例如 Hollow Fiber Module (Minikros，USA) 。 4· 板框式濾膜，例如 Plate and Frame Design ( DDS， Denmark)。過濾孔徑最小 2000 Daltons (分子量）至 最大〇·45微米。微過濾系統可用苛性鈉、過氧化氫、次氯 酸鈉或70%酒精溶液循環滅菌。 於本發明方法中，係利用碳酸鈣或石灰來控制酸鹼 度到pH4.0-PH8.0。而於工廠規模生產時，是用酸鹼度自 動控制系統輸送碳酸鈣或石灰的粉末或乳液直接添加於生 物反應槽中，以中和酸鹼度至pH 5.5，同時生成溶解度很 低的葡萄糖酸鈣。從微過濾系統濾出的高純度果寡糖經用 高效液相層析儀（HPLC)分析，得到果寡糖濃度在80 % 以上，且無葡萄糖酸鈣被檢驗出來。 於本發明一實施例中係利用一複合細胞生物反應 器，外接一中空纖維膜微過濾系統，如第二圖所示設備， 來生產高純度果寡糖。其中，滅菌的蔗糖水溶液（2)經蠕動 幫浦（3)輸送到5公升醱酵槽（9)，內含2公升蔗糖-果寡糖反 應生成液和適量的曰本曲黴（ CCRC 93007 )菌絲以及氧化葡糖酸桿菌（G/MCMoAada CCRC 14104)之活菌細胞。壓縮空氣（6)經空氣過 濾器（7)和空氣流量計（8)，以10升/分的流速進入5公升釀酵 槽（9)，而由排氣口（4)排出。5公升醱酵槽（9)的操做條件控 制在攪拌速度300 rpm和溫度30°C。pH電極（5)經電線（11) 將訊號傳送到pH控制器（15)，pH控制在5.5以上，當pH低 於5.5時，則pH控制器（15)即將訊號經電線（10)傳送到蠕 動幫浦（14)，以啓動蠕動幫浦（14)而將碳酸鈣懸浮液輸送 到5公升釀酵槽（9)中，直到pH高於5.5時才停止。電磁攪拌 本紙張尺度適用中國國家標準(CNS ) A4規格（210X297名釐） (請先閲讀背面之注意事項再填寫本頁) # 訂 517088 A7 B7 五、發明説明（) 器（13)則不斷地轉動上方容器（12)中的攪拌子，以避免碳 酸鈣沉澱下來。在連續反應過程中，高壓送液幫浦（16)不 斷地將5公升釀酵槽內的蔗糖-果寡糖反應生成液輸送到微 過濾器（17) ’部份的反應生成液透過濾膜而流入容器 (19)，其它反應生成液則流回5公升醱酵槽（9)。控制蔗糖-果寡糖反應生成液流到容器（19)和蔗糖水溶液（2)經蠕動幫 浦（3)輸送到5公升釀酵槽（9)兩者的流速相同時，即構成連 續反應生成果寡糖的裝置。 圖式說明： 第一圖爲果寡糖的主要成分：蔗果三糖（1-kestose )，蔗果四糖（nystose )和蔗果五糖（fructosyl nystose)之化學結構式。 (請先閲讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 第二圖爲本發明利用複合細胞系統生產果寡糖的方 法一實施例流程圖， 其中： (1)空氣過濾器 - 防止吸入空氣中的微生物，避免蔗 糖溶液被雜菌污染。 (2)入料的容器 - 盛裝滅菌之蔗糖溶液。 (3)蠕動幫浦 一 輸送蔗糖溶液至生物反-應槽-。 (4)空氣出口 - 生物反應槽的排氣口。 (5) pH電極 - 測定生物反應槽中的反應液的pH 値。 (6)壓縮空氣 一 來自空氣壓縮機的壓縮空氣。 (7)空氣過濾器 - 過濾孔徑0.2微米以下，濾掉壓縮空 氣中的微生物 (8)空氣入口 - •生物反應槽的入氣口，附空氣流量 計，可調節空氣流量。 (9)生物反應器 - 5公升醱酵槽。可控制溫度、攪拌速 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297j釐） 517088 經濟部智慧財產局員工消費合作社印製 A7 f _B7____五、發明説明（） — 度、空氣流量。可裝上pH電極、溶 氧電極，以及若干個液體出口和入 口。裡面有2公升反應液，含有菌 體、菌絲、蔗糖和反應生成物。 (10) 訊號發出的電線-當生物反應器中的反應液低於設定 '之pH値時，由pH控制器發出電流， 啓動蠕動幫浦（14 )。 (11) 訊號接受的電線-pH控制器與pH電極的連線，讀出Ph 値。 -盛裝滅菌之碳酸鈣懸浮液，用來中 和反應中所產生的葡萄糖酸。 -驅動碳酸鈣容器中的攪拌子，以攪 動碳酸鈣懸浮液。 -使生物反應槽中的液體輸送至碳酸 鈣容器，造成反應液中的葡萄糖酸 和碳酸鈣中和。 - pH控制器接受pH電極的信號，記錄 pH値，並且根據設定的pH値，發出 電流以啓動蠕動幫浦（14 )。 -將生物反應槽中的液體輸送至限外 過濾器，由回流的流量控制閥調整 壓力，最高可達100 psi。 -微過濾器可採用各式裝置：1.陶瓷 材質，例如 KERASEP ( Tech-Sep, France) 。2.碳材質，例如 CARBOSEP ( Tech-Sep, France) 〇 3.中空纖維，例如 Hollow Fiber (12)碳酸鈣容器 (13)電磁攪拌器 (14)蠕動幫浦 (15) pH控制器 (16)高壓送液幫浦 .(17)微過濾器 (請先閲讀背面之注意事項再填寫本頁)1T% 517088 Printed by A7, B7 of the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 5. The description of the invention () quality, such as CARBOSEP (Tech-Sep, France). 3. Hollow fiber, such as Hollow Fiber Module (Minikros, USA). 4. Plate and frame filters, such as Plate and Frame Design (DDS, Denmark). Filtration pore sizes range from 2000 Daltons (molecular weight) to 0.45 microns. The microfiltration system can be sterilized by cycling with caustic soda, hydrogen peroxide, sodium hypochlorite, or a 70% alcohol solution. In the method of the present invention, calcium carbonate or lime is used to control the pH to pH 4.0 to pH 8.0. For factory-scale production, an automatic pH control system is used to deliver calcium carbonate or lime powder or emulsion directly to the bioreactor to neutralize the pH to pH 5.5, while producing calcium gluconate with very low solubility. High-purity fructooligosaccharides filtered from the microfiltration system were analyzed by high performance liquid chromatography (HPLC), and the fructooligosaccharide concentration was above 80%, and no calcium gluconate was detected. In one embodiment of the present invention, a composite cell bioreactor is used in conjunction with a hollow fiber membrane microfiltration system, as shown in the second figure, to produce high-purity fructooligosaccharides. Among them, the sterilized aqueous sucrose solution (2) is transported to the 5 liter fermentation tank (9) through the peristaltic pump (3), which contains 2 liters of sucrose-fructo-oligosaccharide reaction solution and an appropriate amount of Aspergillus japonicum (CCRC 93007) bacteria. Silk and viable cells of Gluconobacter oxydans (G / MCMoAada CCRC 14104). The compressed air (6) passes through the air filter (7) and the air flow meter (8), enters the 5 liter fermentation tank (9) at a flow rate of 10 liters per minute, and is discharged through the exhaust port (4). The operating conditions of the 5 liter fermentation tank (9) were controlled at a stirring speed of 300 rpm and a temperature of 30 ° C. The pH electrode (5) transmits the signal to the pH controller (15) through the wire (11), and the pH is controlled above 5.5. When the pH is lower than 5.5, the pH controller (15) transmits the signal to the pH controller (15). The peristaltic pump (14) is used to start the peristaltic pump (14) and transfer the calcium carbonate suspension to the 5 liter fermentation tank (9), and it will not stop until the pH is higher than 5.5. The size of this paper applies the Chinese National Standard (CNS) A4 specification (210X297 centimeters) (please read the precautions on the back before filling this page) # 517088 A7 B7 5. The description of the invention () device (13) is continuously Turn the stir bar in the upper container (12) to prevent the calcium carbonate from settling down. In the continuous reaction process, the high-pressure liquid feeding pump (16) continuously sends the sucrose-fructo-oligosaccharide reaction production liquid in the 5 liter fermentation tank to the microfilter (17). While flowing into the container (19), the other reaction products are returned to the 5 liter fermentation tank (9). Control the sucrose-fructo-oligosaccharide reaction to flow to the container (19) and the sucrose aqueous solution (2) to the 5 liter brewing tank (9) via the peristaltic pump (3). Device that produces oligosaccharides. Description of the drawings: The first diagram is the chemical structural formula of the main components of fructooligosaccharides: 1-kestose, nystose, and fructosyl nystose. (Please read the notes on the back before filling this page) The second picture printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs is a flowchart of an embodiment of a method for producing fructooligosaccharides using a composite cell system according to the present invention, where: (1) Air filter-Prevents inhalation of microorganisms in the air and prevents contamination of the sucrose solution with germs. (2) Feeding container-hold sterilized sucrose solution. (3) Peristaltic pumps-Transfer the sucrose solution to the biological reaction tank. (4) Air outlet-The exhaust port of the biological reaction tank. (5) pH electrode-measures the pH of the reaction solution in the biological reaction tank. (6) Compressed air-Compressed air from an air compressor. (7) Air filter-filter pore size below 0.2 micron, filter out microorganisms in compressed air. (8) Air inlet-• Air inlet of biological reaction tank, with air flow meter, can adjust air flow. (9) Bioreactor-5 liter fermentation tank. Temperature and stirring speed can be controlled. This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210X297j centigrade) 517088 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 f _B7____ 5. Description of the invention (degrees), air flow It can be equipped with a pH electrode, a dissolved oxygen electrode, and several liquid outlets and inlets. It contains 2 liters of reaction solution containing mycelium, mycelium, sucrose, and reaction products. (10) Signal wire-When the reaction solution in the bioreactor is lower than the set pH, a current is sent from the pH controller to start the peristaltic pump (14). (11) The wire that the signal accepts-the connection between the pH controller and the pH electrode, read Ph 値. -Holds a suspension of sterilized calcium carbonate to neutralize the gluconic acid produced in the reaction. -Drive the stir bar in the calcium carbonate container to stir the calcium carbonate suspension. -The liquid in the biological reaction tank is transferred to the calcium carbonate container, which causes the neutralization of gluconic acid and calcium carbonate in the reaction liquid. -The pH controller receives the signal from the pH electrode, records the pH 値, and sends a current based on the set pH 値 to activate the peristaltic pump (14). -Transfer the liquid in the bioreactor to the out-of-limits filter and adjust the pressure by the return flow control valve, up to 100 psi. -Micro-filter can use various devices: 1. Ceramic material, such as KERASEP (Tech-Sep, France). 2. Carbon material, such as CARBOSEP (Tech-Sep, France) 〇3. Hollow fiber, such as Hollow Fiber (12) calcium carbonate container (13) electromagnetic stirrer (14) peristaltic pump (15) pH controller (16) High pressure liquid pump. (17) Micro filter (Please read the precautions on the back before filling this page)
本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 517088 A7 B7 五、發明說明（.）This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 517088 A7 B7 V. Description of the invention (.)
Module ( Minikros，USA)。 4·板 (請先閱讀背面之注意事項再填寫本頁) 框式濃膜，例如 Plate and Frame D e s i g n ( D D S，D e n m a r k )。過濃孔 徑最小2000 Daltons (分子量）至 最大0.45微米。 (18) 產物出口 -反應生成液透過微過濾器而由此處 流出。 (19) 產物接收容器-收集反應生成濾液，內含高純度果 寡糖。 第三圖爲本發明實施例一所得果寡糖產物之高效液相層 析（HPLC)分析圖。 第四圖爲本發明實施例二所得果寡糖產物之高效液相層 析（HPLC )分析圖… 實施例： 本發明要用下面的實施例予以進一步闡述。不過要了解 者，這些實施例僅具闡釋目的而絕非用以限制本發明範圍。 氧化葡糖酸桿菌（)菌體的生產： 經濟部智慧財產局員工消費合作社印製 氧化葡糖酸桿菌CCRC 14104購自食品工業發展硏究所 .菌種中心（新竹）。取洋菜培養基（含2.5 %甘露醇、0.5 % 酵母抽出物、1.5 %洋菜）上的氧化葡糖酸桿菌，接種於500 毫升三角瓶中裝有200毫升培養基（含0.5 %、酵母抽出物、5 %葡萄糖），於30°C 振盪培養24小時，然後接種於5公升醱 酵槽，內有2公升培養基（含0.5 %酵母抽出物、10%葡萄 糖、0.2%磷酸鉀' 0.02%硫酸鎂、pH 6·0 )，於醱酵條件： 溫度，30°C ; 攪拌速度，300 rpin ; 通氣，5升/分；自 動控制PH5.5 ; 培養30小時。將培養液冷卻至4°C，離心， 大約可收取25 - 35公克濕菌體。 9 本紙張尺度適用中國國豕標準（CNS)A4規格（210 χ 297公釐） 517088 經濟部智慧財產局員工消費合作社印製 A7 — _ B7 _ 五、發明說明（） 日本曲黴（)菌絲的生產： 日本曲黴，寄存於食品工業發展硏究所菌種中心（新 竹），CCRC 93007，屬於專利菌（中華民國專利：發明第 076598號）。取洋菜培養基（含1 %酵母抽出物、1 %麥芽 抽出物 、1.5 %洋菜）上的孢子接種於500毫升三角瓶中 裝有200毫升培養基（含1 %酵母抽出物、1 %麥芽抽出 物、5 %蔗糖），於30°C，振盪培養24 30小時。然後接 種於5公升醱酵槽，內有3公升培養基（含1 %酵母抽出物、1 %麥芽抽出物 、20 %蔗糖 、pH 6.5 )，於醱酵條件：溫 度，30°C ; 攪拌速度，200 rpm ; 通氣，3升/分；培 養30小時。將培養液冷卻至，離心，大約可收取65 - 80 公克濕菌絲。 石-喃果糖洛"比的活性測定： 取適量的溼菌絲，加入100毫升的10% ( W/V )蔗糖溶 液中，蔗糖溶液是以0·1 Μ醋酸鈉緩衝液（pH 5.4)配製而 成。在37°C恆溫水槽中反應60分鐘，反應後立即置於沸水浴 中10分鐘，將沒-喃果糖咯北失活，以終止反應。每單位 活性定義爲在上述條件下，每分鐘生成1微莫耳的葡萄糖。 葡萄糖氧化活性的測定： 取適量的溼菌細胞，加入100毫升的1 % ( w/v)葡萄糖 溶液中，葡萄糖溶液是以〇·1 Μ醋酸鈉緩衝液（pH 5.5)配 製而成。在37°C下恆溫振盪反應60分鐘，反應結束後立即置 於沸水浴中10分鐘，以終止反應。細胞的葡萄糖氧化活性定 義爲在上述條件下，每分鐘氧化1微莫耳葡萄糖爲一單位活 性。 醣類分析的方法： 醣類的分析是以HPLC。分析管柱是SUPELCO LC- 10 本紙張尺度適用中國國家標準（CNS〉A4規格（210 X 297公釐） 1^1 —----— ΙΦΜ • »s n ft— n n 1 n 0 fm 1« ϋ I .1 I n I 争 (請先閱讀背面之注意事項再填寫本頁) 517088 A7B7 五、發明說明（） ΝΉ2 ，條件：偵測器是 Waters 410 Differential ilefraetoiiieter，移動相是乙 /水（75:25)，壓力是 1500 psi，移動相流速是 1毫升/分。 實施例一： 以複合細胞生物反應器連續生產高純度果寡糖 在5公升生物反應槽中，加入2公升30 %蔗糖水溶液進行 滅菌，加入日本曲黴CCRC 93007菌絲，每公克蔗糖反應物i 其肩-喃果糖4吨的活性用量爲1.5單位活性，加入氧化葡 糖酸桿菌CCRC 14104之活菌細胞，每公克蔗糖反應物之葡 萄糖氧化活性用量爲3單位活性。在30 °C 恆溫，通氣量10 升/分，攪拌速度300 rpm之下，酸鹼度用碳酸蔣乳液控制 在ρΗ5·5。整個反應系統裝置如第二圖所示。最初的2公 升的蔗糖反應液在反應 12小時後，開始用微過濾系統 (Minikros Hollow Fiber Module、濾膜孔徑 0.2 微米、過 濾面積 335平方公分、操作壓力15 pSi )，連續濾出產 物，每小時可濾出70毫升反應生成液，並以等流量同步加入 30 %之蔗糖反應液，如此進行4天，分析其濾出液之各種糖 份如表一所示，三種果寡糖之濃度總和爲80%以上。 (請先閱讀背面之注意事項再填寫本頁) -ilmu ^·!--Module (Minikros, USA). 4 · Plate (Please read the precautions on the back before filling this page) Frame type thick film, such as Plate and Frame D e s i g n (D D S, De n m a r k). Excessive pore diameters range from 2000 Daltons (molecular weight) to 0.45 microns. (18) Product outlet-The reaction product flows out through the microfilter. (19) Product receiving container-collect the reaction to produce a filtrate, which contains high-purity fructooligosaccharides. The third figure is a HPLC analysis chart of the fructooligosaccharide product obtained in Example 1 of the present invention. The fourth figure is a high-performance liquid phase analysis (HPLC) analysis chart of the fructooligosaccharide product obtained in Example 2 of the present invention ... Examples: The present invention will be further illustrated by the following examples. It should be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Production of Gluconobacter oxydans () bacteria: Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Gluconobacter oxidans CCRC 14104 was purchased from the Food Industry Development Research Institute. Strain Center (Hsinchu). Gluconobacter oxydans on agar culture medium (containing 2.5% mannitol, 0.5% yeast extract, 1.5% agar), inoculated into a 500ml triangle flask containing 200ml culture medium (containing 0.5%, yeast extract , 5% glucose), shaking culture at 30 ° C for 24 hours, and then inoculated in a 5 liter fermentation tank, which contains 2 liters of culture medium (containing 0.5% yeast extract, 10% glucose, 0.2% potassium phosphate '0.02% magnesium sulfate , PH 6.0), under fermentation conditions: temperature, 30 ° C; stirring speed, 300 rpin; aeration, 5 l / min; automatic control of pH 5.5; cultivation for 30 hours. Cool the culture to 4 ° C and centrifuge to collect about 25-35 grams of wet bacteria. 9 This paper size is in accordance with China National Standard (CNS) A4 (210 x 297 mm) 517088 Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 — _ B7 _ 5. Description of the invention () Aspergillus niger () Mycelium Production: Aspergillus japonicus, deposited in the Institute of Food Industry Development Research Center (Hsinchu), CCRC 93007, belongs to the patent bacteria (Republic of China Patent: Invention No. 076598). Take spores from agar culture medium (containing 1% yeast extract, 1% malt extract, 1.5% agar) and inoculate a 500ml triangle bottle with 200ml culture medium (containing 1% yeast extract, 1% wheat). Bud extract, 5% sucrose), cultured at 30 ° C with shaking for 24 to 30 hours. Then inoculated in a 5 liter fermentation tank containing 3 liters of culture medium (containing 1% yeast extract, 1% malt extract, 20% sucrose, pH 6.5), in fermentation conditions: temperature, 30 ° C; stirring speed , 200 rpm; aeration, 3 l / min; culture for 30 hours. The culture is cooled down and centrifuged to collect approximately 65-80 grams of wet mycelium. Determination of stone-fructose Luo's specific activity: Take an appropriate amount of wet hyphae and add it to 100 ml of 10% (W / V) sucrose solution. The sucrose solution is 0.1 M sodium acetate buffer (pH 5.4). Formulated. The reaction was carried out in a constant temperature water bath at 37 ° C for 60 minutes, and immediately after the reaction, it was placed in a boiling water bath for 10 minutes. Activity per unit is defined as the production of 1 micromole of glucose per minute under the conditions described above. Determination of glucose oxidation activity: Take an appropriate amount of wet bacteria cells and add 100 ml of 1% (w / v) glucose solution. The glucose solution is prepared with 0.1 M sodium acetate buffer (pH 5.5). The reaction was shaken at 37 ° C for 60 minutes. After the reaction was completed, it was placed in a boiling water bath for 10 minutes to stop the reaction. The glucose oxidizing activity of a cell is defined as oxidizing 1 micromole of glucose per minute as a unit of activity under the above conditions. Method for sugar analysis: Sugar analysis is by HPLC. The analysis column is SUpelco LC-10. The paper size is applicable to Chinese national standard (CNS> A4 specification (210 X 297 mm) 1 ^ 1 —----— ΙΦΜ • »sn ft— nn 1 n 0 fm 1« ϋ I.1 I n I (please read the notes on the back before filling this page) 517088 A7B7 V. Description of the invention () Ή2, Condition: The detector is Waters 410 Differential ilefraetoiiieter, and the mobile phase is B / water (75: 25), the pressure is 1500 psi, and the mobile phase flow rate is 1 ml / min. Example 1: Continuous production of high-purity fructooligosaccharides in a composite cell bioreactor In a 5 liter bioreactor, add 2 liters of a 30% sucrose aqueous solution. Sterilize, add Aspergillus japonicum CCRC 93007 mycelium, each gram of sucrose reactant i has an active dosage of 4 tons of fructosaccharose 1.5 units of activity, add viable bacterial cells of Gluconobacter oxydans CCRC 14104, per gram of sucrose The amount of glucose oxidation activity is 3 units of activity. At a constant temperature of 30 ° C, aeration volume of 10 liters per minute, and a stirring speed of 300 rpm, the pH is controlled at ρΗ5 · 5 with a carbonated carbonate emulsion. The whole reaction system device is shown in the second figure .most After reacting for the first 2 liters of sucrose reaction solution for 12 hours, a microfiltration system (Minikros Hollow Fiber Module, filter membrane pore size 0.2 micron, filter area 335 cm2, operating pressure 15 pSi) was used to continuously filter out the product every hour 70 ml of reaction solution can be filtered out, and 30% sucrose reaction solution is added simultaneously at the same flow rate. This is performed for 4 days. The sugar content of the filtered solution is shown in Table 1. The total concentration of the three fructooligosaccharides is 80% or more. (Please read the notes on the back before filling this page) -ilmu ^ ·!-
n Ifl ϋ n I 經濟部智慧財產局員工消費合作社印製 表一： 時間 葡萄糖 + 果糖 蔗糖 蔗果 三糖 蔗果 四糖 蔗果 五糖 總寡糖 (乾基） (天） (毫克/ 毫升） (毫克/ 毫升） (毫克/ 毫升） (毫克/ 毫升） (毫克/ 毫升） (%) 1 9.4 9.1 100 82.5 5.8 86.9 11 本紙張尺度適用中國國家標準（CNS)A4規袼（210 X 297公釐） ΚΙ Β7 2 9.4 9.5 96.8 83.6 5.9 86.6 9.4 9.6 94· 4 84.3 5.9 86:4 4 9.4 10.8 91.7 85.6 6.0 85.3 在第48小時之糖成份的HPLC分析如第三圖所示。 517088 五、發明說明() 實施例二： 以複合細胞生物反應器連續生產高純度果寡糖 在5公升生物反應槽中，加入2公升40 %蔗糖水溶液進行 滅菌，加入黑曲黴ATCC 20611菌絲，每公克蔗糖反應物其 点-呋喃果糖苷酶的活性用量爲1·5單位活性，加入氧化葡糖 酸桿菌CCRC 14104之活菌細胞，每公克蔗糖反應物之葡萄 糖氧化活性用量爲3單位活性。在301C 恆溫，通氣量10升/ 分，攪拌速度300 rpm之下，酸鹼度用碳酸鈣乳液控制在 pH5.5。整個反應系統裝置如第二圖所示。最初的 2公升 的蔗糖反應液在反應 12小時後，開始用微過濾系統 (Minikros Hollo w Fiber Mo dule、濾膜孔徑 0.2 微米、過 濾面積 335平方公分、操作壓力15 psi )，連續濾出產 物，每小時可濾出55毫升反應生成液，並以等流量同步加入 40 %之蔗糖反應液，如此進行3天，分析其濾出液之各種糖 份如表二所示，三種果寡糖之濃度總和爲80%以上。 ! ΊΙ^1Ι — — — — — ! - I I ! I I I I ^ *11111111 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 表二： 葡萄糖 蔗果 蔗果 蔗果 總寡糖 時間 + 果糖 蔗糖 三糖 四糖 五糖 (乾基） (天） (毫克/ 毫升） (毫克/ 毫升） (毫克/ 毫升） (毫克/ 毫升） (毫克/ 毫升） (%) 12 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） 517088 五2發明說明() A7 — _______B7 1 19.1 11.0 9 9 102 ,. 17 . 8 8,5 2 22.6 12.4 103 104 18 86·5 3 21.8 14.4 104 100 19 86.1 在第48小時之糖成份的HPLC分析如第四圖所示。 綜上所示，本發明利用複合細胞系統生產果寡糖的方法 具有下列特點和動機： 1. 利用有沒-呋喃果糖苷酶活性的日本曲黴（震i//«s CCRC 93007 )和有葡萄糖去氫吨活性的氧 化葡糖酸桿菌（6/|1€£?1106以/€/<^:>)；心115，€!〇1€；141〇4) 在懸浮的蔗糖反應液系統，不需要將細胞固定化，即可 連續式生產，可免除基質與溶氧的質傳阻力。 2. 使用的蔗糖溶液可達1〇〜60 %之間的濃度》可連續生 產80%純度以上的果寡糖，大大的降低生產成本，提 高產物的價値。 3. 副產物葡萄糖酸鈣很難溶於水，因此容易從果寡糖溶液 中分離出來。 4·由於反應中能去除葡萄糖的抑制，因此反應速率加快， 蔗糖轉換率提高。 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 13 本紙張又度適用_國國家標準（CNS)A4規格（210 X 297公釐）n Ifl ϋ n I Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Table 1: Time Glucose + Fructose Sucrose Cane Fruit Triose Cane Fruit Tetraose Cane Fruit Pentasaccharide Total Oligosaccharides (Dry) (days) (mg / ml) (Mg / ml) (mg / ml) (mg / ml) (mg / ml) (mg / ml) (%) 1 9.4 9.1 100 82.5 5.8 86.9 11 This paper size applies Chinese National Standard (CNS) A4 Regulations (210 X 297 mm) ) K1 B7 2 9.4 9.5 96.8 83.6 5.9 86.6 9.4 9.6 94 · 4 84.3 5.9 86: 4 4 9.4 10.8 91.7 85.6 6.0 85.3 The HPLC analysis of the sugar component at the 48th hour is shown in the third figure. 517088 5. Description of the invention () Example 2: Continuous production of high-purity fructo-oligosaccharides in a composite cell bioreactor In a 5 liter bioreactor, add 2 liters of a 40% sucrose aqueous solution for sterilization, add Aspergillus niger ATCC 20611 mycelium, The amount of dot-fructofuranosidase activity per gram of sucrose reactant is 1.5 units of activity, and the amount of glucose oxidizing activity per gram of sucrose reactant is 3 units of activity when viable cells of Gluconobacter oxydans CCRC 14104 are added. At a constant temperature of 301C, aeration volume of 10 liters per minute, and a stirring speed of 300 rpm, the pH was controlled at 5.5 with a calcium carbonate emulsion. The whole reaction system device is shown in the second figure. After reacting for the first 2 liters of sucrose reaction solution for 12 hours, a microfiltration system (Minikros Hollo w Fiber Mo dule, filter membrane pore size 0.2 micron, filtration area 335 cm 2, operating pressure 15 psi) was used to continuously filter out the product. 55 ml of reaction solution can be filtered out every hour, and 40% sucrose reaction solution is added simultaneously at the same flow rate. This is performed for 3 days. The sugar content of the filtered solution is shown in Table 2. The concentrations of three fructooligosaccharides The total is over 80%. ! ΊΙ ^ 1Ι — — — — —!-II! IIII ^ * 11111111 (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Table 2: Glucose cane fruit cane fruit cane fruit Total oligosaccharide time + fructose sucrose trisaccharide tetrasaccharide pentasaccharide (dry basis) (days) (mg / ml) (mg / ml) (mg / ml) (mg / ml) (mg / ml) (%) 12 books Paper size applies Chinese National Standard (CNS) A4 specification (210 x 297 mm) 517088 May 2 Invention Description () A7 — _______B7 1 19.1 11.0 9 9 102,. 17. 8 8, 5 2 22.6 12.4 103 104 18 86 · 5 3 21.8 14.4 104 100 19 86.1 The HPLC analysis of sugar components at 48 hours is shown in the fourth figure. In summary, the method for producing fructooligosaccharides using the composite cell system of the present invention has the following characteristics and motives: 1. Utilization of Aspergillus japonicum (Zhen i // «s CCRC 93007) with glucose-fructofuranosidase activity and glucose Dehydrotonne-active Gluconobacter oxidans (6 / | 1 € £ 1106 to / € / < ^: >); Heart 115, €! 〇1 €; 14104) in suspension of sucrose The system can be continuously produced without immobilizing cells, and the mass transfer resistance of matrix and dissolved oxygen can be eliminated. 2. The sucrose solution used can reach a concentration between 10% and 60%. It can continuously produce fructooligosaccharides with a purity of more than 80%, which greatly reduces the production cost and increases the price of the product. 3. The by-product calcium gluconate is difficult to dissolve in water and is therefore easily separated from the fructooligosaccharide solution. 4. As the inhibition of glucose can be removed during the reaction, the reaction rate is accelerated and the sucrose conversion rate is increased. (Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 13 This paper is again applicable _ National Standard (CNS) A4 (210 X 297 mm)