1274757 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（1 ) 產業上的應用領域 本發明提出一種生產高純度異麥芽寡糖的方法，產品爲 機能性保健食品，用途相當廣泛，可應用於保健食品、飮 料、製酒等產業。 發明背景 異麥芽寡糖（isomalto-oligosaccharides，簡稱 IMO)與果 寡糖、半乳糖寡糖、大豆寡糖等同屬於機能性寡糖，具有 多種機能特性，能滋化腸內比福多菌（Bifidus)的增殖、不 易引起蛀牙等。寡糖的生理機能特性主要有（1)改善腸內 微生物族群；（2)降低腸內腐敗物質的產生；（3)改善 血內脂質過多，減少膽固醇；（4 )減少便秘現象·，（ 5 ) 防止蛀牙；（6 )免疫賦活；以及（7 )促進鈣質吸收等等。 1997年在日本，各類寡糖的需求量約30000公噸，其中異 麥芽寡糖佔1 1000公噸[譚靜芬，食品工業月刊，（1999)， 1-8]，各類寡糖主要用途是添加在淸涼飮料.、糖果之中。 而異麥芽寡糖由於具良好的加工特性，使其占有寡糖銷售 量達30%以上。由於機能性寡糖在日本已核准在商品中標 示爲特定保健用食品，因此預期需求量將持續地增加， 葡萄糖分子間至少含有一個a -D-(l，6)鏈結的雙糖和寡 糖統稱爲異麥芽寡糖，其主要成分是異麥芽糖 (isomaltose)、盼樂糖（panose, 62_0-a-glucosyl-maltose)、 異麥芽三糖（isomaltotriose)等，異麥芽寡糖存在於傳統的 發酵食品，如淸酒、味噌、醬油中，大量生產可以利用酵 -4- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐) (請先閱讀背面之注意事項再填寫本頁) - j --------訂·！------· 經濟部智慧財產局員工消費合作社印製 1274757 A7 ___ B7_ 五、發明說明（2 ) 素製造出純度爲50% — 60%乾重的異麥芽寡糖。傳統從澱 粉生產異麥芽寡糖糖漿的方法是先以酵素α-澱粉酶（α-amylase) (EC 3·2·1·1)、支鏈澱粉酶（pUilulanase) (EC 3 ·2 · 1·41)、β-澱粉酶（β-amylase) (EC 3.2.1.2)將澱粉水解得 到α-(1^4)鍵結的葡萄糖寡聚合物（α-D-gluco-oligosaccharides)，再利用 a-D 苷酶（a-D-glucosidase) (EC 3.2·1 ·20)的葡萄苷轉移功能獲得α-( 1—6)-鏈結的寡糖 [Takaku，H·，Handbook of amylases and related enzymes, Ed· The Amylase Research Society of Japan, Pergamon Press, Oxford，（1988)，215-217]。具有葡萄苷轉移功能的 α-D-脊酶也叫做葡萄糖基轉移酵素（transglucosidase)(簡 稱 TG)或 D-葡萄糖基轉移酵素（D-glucosyltransferase) (EC 2.4.1.24)。如日本專利61 _2 12296所述爲例，30°/。的玉米、 馬鈴薯、或樹薯澱粉以熱安定性之α-澱粉酶液化爲聚合度 (DP)在6到10之間的液化糖漿（lidnefact)，此液化糖漿在 pH 5與60 °C之下與β-澱粉酶及TG浸泡進行糖化48-72 小時，即得異麥芽寡糖糖漿。酵素TG會催化α-D-葡萄糖 寡聚合物（a-D-gluco-oligosaccharides)的水解及葡萄糖苔 的轉移反應，通常是轉移至葡萄糖的6-OH上，例如將葡 萄糖苷轉移至D-葡萄糖可得到異麥芽糖，轉移至麥芽糖上 可以得到盼樂糖（panose)。經由TG酵素作用之後，產物即 含有一大類具有α-( 1 —6)鍵結的葡萄糖寡聚合物稱爲異麥 芽寡糖，此外產物中亦含有大量的葡萄糖及α-(1^4)的寡 糖（主要是麥芽糖）。TG主要發現於黴菌，但Aspergillus 本紙張尺度適用中國國家標準（CNS)A4規格（21〇 x 297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂· 線· 1274757 A7 B7 五、發明說明（3) (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 niger的TG只作用在低DP的葡萄糖寡聚合物，其它黴菌 如 Aureobasidium pullulans [Yun et al·，Biotechnol. Lett. 16 (1994) 1 145-1 150]、Aspergillus carbonarious [Duan et al.? Biotechnol. Lett. 1 6 (1 994) 1 1 5 1 -1 1 56]、Aspergillus nidulans [Kato et al·，Appl. Environ. Microbiol· 68 (2002) 1250-1256]等也發現有這個酵素功能存在。Kuriki等人 [Kuriki et al·，Appl. Environ. Microbiol. 59 (1993) 953-959]發現來自Bacillus subitlis的酵素新支鏈殿粉酶 (neopullulanase)也具有oc-(l->6)-葡萄昔轉移功能 (1^118£1)^035^^011)的功能，因此以0^澱粉酶搭配新支鏈激 粉酶（neopullulanase)也可從澱粉生成異麥芽寡糖。以上從 澱粉生產異麥芽寡糖的各種方法，得到的異麥芽糖佔產物 總糖的量最高在60%左右，其餘是葡萄糖及麥芽糖等α-(1^4)鍵結的寡糖。除澱粉之外，直接以高濃度的麥芽糖 爲原料，也可以經由TG的酵素作用生成異麥芽寡糖[Yun et al·，Biotechnol· Lett· 16 (1994) 1145-1150]，不管是用酵 素或菌體細胞爲催化劑最終的異麥芽寡糖比率最高也是 60%左右。國內學者也有硏究使用固定或未固定的α-D-苷 酶（glucosidase)(葡萄糖基轉移酵素）來生產異麥芽寡糖， 所得產物純度也是如此[許垤綦等人，中國農業化學會誌， 3 1 (1993) 740-75 1 ;黃金義，1995，大同工學院碩士論文； 林建宏，2000，大同大學碩士論文]。利用固定的新支鏈澱 粉酶（neopullulanase)(來自 Bacillus stearothermophilus， 具有TG功能）可以轉化支鏈澱粉（pullulan)爲盼樂糖 -6 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 __ B7 _ 五、發明說明（4) (請先閱讀背面之注意事項再填寫本頁) (panose)，但純度最高也只有達到84%，其它成份仍爲葡 萄糖與麥芽糖[Kuriki et al·, J. Ferment· Bioeng. 73 (1992) 198-202] 〇 美國專利6025168與日本專利1 1-004700爲同一發明， 係使用固定的TG將澱粉水解液轉化爲異麥芽寡糖糖漿， 澱粉水解液的DP介於4到70之間，最好是20到60之間， 可來自酵素或酸水解，爲使DP降低，有時ex-澱粉酶與支 鏈澱粉酶也搭配使用。這各方法連續生產所得異麥芽寡糖 糖漿，其異麥芽寡糖含量是40-45%。 日本專利01-098601提出一個提高異麥芽寡糖純度的 方法，即將酵素法所得異麥芽寡糖糖漿經過鹼性金屬型強 酸性之陽離子交換樹酯，可將90%以上的葡萄糖分離，得 到純度80%以上的異麥芽寡糖，此產品之固體成分仍有麥 芽糖（DP爲2)。 經濟部智慧財產局員工消費合作社印製 美國專利5612203提到將糖類分子還原端的碳修飾如 氧化成羧酸或胺化，修飾過的糖即可吸附在離子交換樹酯 上，再用適當的酵素如β-澱粉酶將糖分子切下來，這個方 法雖可提高產物純度，但若原料爲異麥芽寡糖時有些產物 就不再是異麥芽寡糖，如原料中有異麥芽糖分子其產物是 葡萄糖。 國內尙無有關生產異麥芽寡糖的專利，含有α-D-寡葡 萄糖基的寡糖則有兩項專利，分別是中華民國專利編號 083 105867與專利086102774,皆與異麥芽寡糖大不相同。 在生產異麥芽寡糖方面，國內的廠商有環泰企業桃園廠和 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 B7 五、發明說明（5) 碩泰實業高雄廠，所用之原料均採用進口之高純度澱粉， 引進日本技術，使用自由酵素法，即使用未固定之TG，生 產之異麥芽寡糖含量達55-60%，其餘成份爲葡萄糖與麥芽 糖。 綜合以上所述，不管原料來源是澱粉或麥芽糖利用TG 酵素轉移所得到的異麥芽寡糖純度一般約在60%左右，其 餘成份主要是葡萄糖與麥芽糖，限於技術已無法再提高異 麥芽寡糖純度。爲突破此一極限，本發明乃利用異麥芽寡 糖之不被發酵性，以釀酒所用之酵母菌，將異麥芽寡糖漿 中之葡萄糖與麥芽糖等發酵成酒精，可提高異麥芽寡糖純 度接近100%，酒精可蒸餾去除或回收。 發明要點 本發明係一種生產高純度異麥芽寡糖的方法，以釀酒所 用之酵母菌，將澱粉或麥芽糖經葡萄糖基轉移酵素 (transglucosidase)轉化所得之異麥芽寡糖漿中之可發酵糖 (或稱可消化糖，digestible sugar，簡稱DS)，即葡萄糖與 麥芽糖等發酵成酒精，可得到純度98%以上的異麥芽寡 糖，其中的酒精可蒸餾去除或回收。 實例的硏究結果顯示：酵母菌除糖時，吟釀淸酒酵母 (Saccharomyces cervisiae)或底層啤酒酵母（Saccharomyces carlsbergensis)以3 0°C震盪（150 rpm)培養24小時之對數期 酵母最具除可消化糖效率，培養40小時之穩定期酵母菌數 濃度較高，雖然除（可消化）糖效果稍差，但一樣可以完全 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閱讀背面之注意事項再填寫本頁) . --線· 經濟部智慧財產局員工消費合作社印製 1274757 A7 __B7 五、發明說明（8 ) 或稱transglucosidase簡稱TG)作用過的潑粉液或麥芽糖所 (請先閱讀背面之注意事項再填寫本頁) 得到之異麥芽寡糖漿，其中異麥芽寡糖純度60%左右或以 下，以酵母菌發酵將其中之可發酵糖消化去除，得到高純 度之異麥芽寡糖。 發酵除糖步驟主要將異麥芽寡糖漿中的可發酵（可消 化）糖，即葡萄糖與麥芽糖等發酵成酒精，異麥芽寡糖的各 個成份因不會被發酵利用，所以產物中各寡糖的相對比例 維持不變。不同的澱粉原料來源，在適當的液化、糖化及 TG的作用之下，皆可得到濃度高到60%的異麥芽寡糖糖 漿，並利用本發明的方法得到高純度之異麥芽寡糖。 例如利用澱粉價爲68.45%之米屑，調成35%乾重懸濁 液，經液化、糖化及添加0.1%葡萄糖基轉移酵素（TG，日 本Amano產品）進行葡萄糖基之轉移作用之後，所得之米 屑總糖重30.21%，內含異麥芽寡糖（IMO)的總重爲 53.19%。 經濟部智慧財產局員工消費合作社印製 利用各種澱粉原料製備異麥芽寡糖，受液化程度、酵 素加入方式、反應時間與pH之控制，改變不同條件就有 不同之異麥芽寡糖組成。例如依實例一所述以米屑所產生 之異麥芽寡糖，其組成份異麥芽糖（IG2)含量較多，其次爲 異麥芽三糖（IG3)與盼樂糖（panose)。改變不同澱粉原料而 以糯米、玉米及米麴等爲原料，所得之異麥芽寡糖由HPLC 圖譜比對，差異性不大，與米屑異麥芽寡糖組成相當接近。 異麥芽寡糖製造，控制高濃度的麥芽糖（G2)爲反應基 質可產生高異麥芽糖含量之異麥芽寡糖；生產高盼樂糖 -11 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 _B7_ 五、發明說明（9 ) (panose)之異麥芽寡糖則需要高濃度直鏈寡糖Gn (n=3〜 4)當反應基質。 (請先閱讀背面之注意事項再填寫本頁) 利用食用澱粉（樹薯粉）爲原料，液化溫度90°C，液化 0.5小時，糖化酵素（Fungamyl)與異構化酵素（TG)同時間加 入，在pH 5.0，55 °C下轉化13小時，可得到盼樂糖含量 最高之異麥芽糖。 食用澱粉（樹薯粉）爲原料所得異麥芽寡糖（環泰公司 的產品）純度57.18%，與米屑來源之異麥芽寡糖純度 53.19%比較，相當接近，惟二者異麥芽寡糖組成份不同， 前者異麥芽寡糖則以盼樂糖（panose)爲主，後者以IG2爲 主。二者分別利用酵母菌在30°C、振盪80 rpm之下發酵 72小時，可完全除去可消化糖，最終產物爲接近100%的 高異麥芽寡糖糖漿。 各種釀酒用酵母皆可用來發酵除糖，但效果及控制條 件不同，其中吟釀淸酒酵母及底層啤酒酵母去除可消化糖 (DS)的效果佳，這二株酵母菌以30°C震盪（150 rpm)培養24 小時之對數期酵母最具除糖效率，培養40小時之穩定期酵 母雖然菌數濃度較高，除糖效果反而差一些。 經濟部智慧財產局員工消費合作社印製 在探討反應基質濃度對除糖之影響時發現，高濃度 時，即總糖重量濃度35% (w/v)的樹薯粉來源之異麥芽寡 糖與30% (w/v)的米屑來源之異麥芽寡糖前者採用吟釀淸 酒酵母及底層啤酒酵母分段添加效果最佳，後者則直接加 入底層啤酒酵母菌體爲優；在低濃度時，如重量濃度17.5% (w/v)的樹薯粉來源之異麥芽寡糖及1 5% (w/v)的米屑來源 -12- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 ___B7___ 五、發明說明（10 ) 之異麥芽寡糖，均可採用底層啤酒酵母倂同麥汁進行發 酵，操作上較爲簡便有效。 (請先閲讀背面之注意事項再填寫本頁) 各種不同澱粉原料如糯米、玉米、米麴及食用澱粉等， 經轉化後之異麥芽寡糖及去除DS之高純度異麥芽寡糖， 在pH 3、1 2 1°C下加熱1小時仍不會變質或分解，用於保 健食品、酒類、淸涼飮料、高溫處理或低pH之食品，相 信皆可保持其特性及機能。 酵母發酵除可發酵糖，生產高純度異麥芽寡糖之步驟 如下： 1 ·調配適當濃度之低純度異麥芽寡糖水溶液； 2·加入已培養之酵母菌進行發酵；及選擇性的 3·中和、脫色等後處理，將酒精蒸餾去除或回收，除 水濃縮。 酵母培養方法 1·麴汁培養法：由斜面培養之吟釀淸酒酵母接種於試 管培養24小時（麹汁5 mL，3 0°C，靜置培養），之後於三 角瓶擴大培養24小時或40小時（麹汁1 00 mL，30°C，1 50 rpm振擾培養）。 經濟部智慧財產局員工消費合作社印製 2·麥汁培養法：由斜面培養之底層啤酒酵母接種於試 管培養24小時（麥汁5 mL，30°C，靜置培養），之後於三 角瓶擴大培養24小時或40小時（麥汁100 mL，30°C，150 rpm振盪培養）。 酵母發酵除糖（去除異麥芽寡糖漿中之可消化糖）方法： 於低純度的異麥芽寡糖溶液中，添加酵母可以發酵除 -13 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 ^__B7 五、發明說明（11 ) (請先閱讀背面之注意事項再填寫本頁) 去可發酵之葡萄糖、麥芽糖、以及麥芽三糖等，適當控制 酵母的添加方式與發酵條件，最終可以得到純度達98%以 上的異麥芽寡糖，以下舉例數種方法於實例中使用： 第1法（酵母菌體法）：取酵母培養液100 mL，以5000 rpm離心20分鐘，去上層液，再以蒸餾水淸洗、離心，取 其菌體加入所配製之100 mL糖液中，在30°C，80 rpm下， 振盪發酵72小時。 第2法（酵母倂麥汁法）：取酵母培養液50 mL，直接 加入50 mL加倍濃度之糖液中，同上述第1法發酵去除 DS 〇 第3法（分段添加相同酵母法）：第一天同酵母菌體法， 24小時後追加同等量且相同菌體。 第4法（分段添加不同酵母法）：第一天先加入吟釀淸 酒酵母菌體，24小時後追加底層啤酒酵母同等量菌體，其 餘操作與第3法相同。 實際應用上，本發明所提酵母發酵方法不受限於這幾 個特別的酵母添加方法。 寡糖分析 經濟部智慧財產局員工消費合作社印製 基質及產物的分析與定量以高效液相層析爲之，所用 管柱爲 Supelcosil LC-NH2，移動相爲 80%丙烯 (acetonitrile)與 20%純水，30°C、流速 1·2 mL/min 之下操 作，RI偵測器。從層析圖可分別定量聚合度1至4的寡糖 以及聚合度大於4的寡糖（Gn)，葡糖糖爲聚合度爲1之單 糖，聚合度爲2的糖類（DP2)可分別分析定量麥芽糖與異麥 -14- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 B7 五、發明說明（12) 芽糖，聚合度爲3之寡糖（DP3)可分別分析麥芽三糖、盼樂 糖（panose)、以及異麥芽三糖。 (請先閲讀背面之注意事項再填寫本頁) 實例一 經濟部智慧財產局員工消費合作社印製 澱粉源爲米屑，係蓬萊米以連續精米機精白後產生的 下腳品，澱粉含量約68%，其它成份爲粗蛋白8%，粗脂肪 3%以及灰份1 %。首先米屑經以下懸濁液調配、液化、糖 化與TG異構化等步驟之後得到純度53.19%的異麥芽寡糖 糖漿··（1).懸濁液調配：取1996克米屑（含水份12.35%)， 加水至5L，總重5379.5克，即爲35g/100 mL (乾重）懸濁 液，以均質機攪拌均勻，供液化、糖化及異構化用。（2)· 液化：於35g/100 mL米屑懸濁液，添加〇·5 g/(kg-米屑） 之液化酵素（Novo公司產品，品名Termamyl 120 L)與30 ppm Ca + + (CaCl2，H20)，以電磁爐加熱至95°C，澱粉糊化 後再追加1.0 g/(kg-米屑）之液化酵素，於93-95°C維持1-2 小時，以碘液檢測澱粉液化程度，在碘反應藍紫色消失呈 紅色時停止液化，降溫至55°C，加酸調整pH爲5.0-5.5， 加水補足加熱蒸發損失之重量。（3).糖化與異構化：米屑 液化液移入55°C水浴，分別添加0.05% (w/w)的去支鏈酵 素（Novo 公司產品，品名 Promozyme 400 L)與 0.1% (w/w) 的冷-澱粉酶或〇」％ (w/w)的糖化酵素（Novo公司產品，品 名Fungamyl 8 00 L)，於55°C糖化24小時後，再加0.1% TG 異構化24小時，取出加熱至85°C，5分鐘破壞酵素後冷卻， 再依原重補足水分，即得異麥芽寡糖漿，總糖含量30% -15- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 B7 五、發明說明（13) (W/V)，其中異麥芽寡糖濃度佔總糖重量的53.19%。 取上述30% (w/v)之異麥芽寡糖水溶液100 mL，以第4 法加入酵母發酵除去可消化糖，即加入1 〇 〇 mL去麥汁之 吟釀淸酒酵母菌體（麥汁培養40小時之菌體，細胞數目濃 度爲2·7χ108菌株/mL)，在3(TC，80 rpm下發酵，24小時 後追加底層啤酒酵母同等量菌體（麴汁培養40小時之菌 體，細胞數目濃度爲1·8 χΙΟ8菌株/mL)。異麥芽寡糖水溶 液中葡萄糖、麥芽糖、以及麥芽三糖被發酵去除比例隨時 間的變化如圖一，每日的變化如表一。 表一顯示，第一天加入吟釀淸酒酵母，發酵二十四小時 內可完全消耗掉葡萄糖，第二天加入底層啤酒酵母，在缺 乏葡萄糖碳源之下，可在第二天之內消化95.5%的麥芽糖 與90%的麥芽三糖。圖一顯示酵母可分別於第60與第72 小時將麥芽糖與麥芽三糖完全消化完，因此根據HPLC分 析的結果，所得到的異麥芽糖爲佔總糖1 00%的高純度異麥 芽寡糖，成份包括異麥芽糖、盼樂糖、異麥芽三糖、以及 聚合度爲四之異麥芽寡糖。 (請先閱讀背面之注意事項再填寫本頁) II---- I 訂· -----I--· 經濟部智慧財產局員工消費合作社印製 -16- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 B7 五、發明說明（14) 表一 時間 麥芽糖消耗 率(％) 麥芽三糖消 耗率(％) 發酵去除之總 糖量(g/100 mL) 異麥芽寡糖 純度(％) 葡萄糖消耗 率(％) 24 h 100 0 0 48 h 100 95.5 90 72 h 100 100 100 14.14 100 (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 實例二 以米屑爲澱粉源，液化、糖化及TG異構化後得純度 53.19%的異麥牙寡糖’總糖含量30% (w/v)，其製作方法 如實例一。取50 mL此異麥芽寡糖水溶液，以第2法加入 酵母發酵除去可消化糖，即加入50 mL含麥汁之底層啤酒 酵母菌體（培養40小時之菌體，細胞數目濃度爲1.8xl08 菌株/mL)，在30°C，80 rpm下發酵72小時。因兩者以1 ·· 1混合，因此發酵開始時總糖含量爲15% (w/v)之水溶液。 發酵前後異麥芽寡糖水溶液中各糖類的濃度以HPLC分析 定量，層析圖如圖二與圖三。異麥芽寡糖水溶液中葡萄糖、 麥芽糖、以及麥芽三糖被發酵去除比例隨時間的變化如圖 四，每日的變化如表二。 圖二與圖三顯示發酵前後異麥芽寡糖溶液中葡萄糖 (G1)、麥芽糖（G2)、麥芽三糖（G3)、異麥芽糖（IG2)、盼樂 糖（P)、異麥芽三糖（IG3)、以及聚合度爲四之寡糖（G4)等 -17- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 _____B7_五、發明說明（) HPLC分析圖譜。比較發酵前後結果發現可消化糖（含葡萄 糖、麥芽糖、以及麥芽三糖）已完全被酵母利用去除，而異 麥芽寡糖部份則沒有改變，顯示此酵母發酵法可提高異麥 芽寡糖的純度但不影響異麥芽寡糖中異麥芽糖、盼樂糖、 異麥芽三糖等之分佈比例。 表二與圖四顯示，在米屑來源之粗異麥芽寡糖液中，因 營養充足，底層啤酒酵母可輕易地在48小時內即將所有可 消化糖去除。雖然添加酵母時同時也將培養酵母的麥汁也 一起加入，麥汁中尙殘留有麥芽糖與麥芽三糖等，圖四顯 示原來存在於粗異麥芽寡糖液中及麥汁夾帶進來的麥芽糖 與麥芽三糖等可消化糖很快就被酵母代謝利用，麥芽糖第 一天就幾乎被消化完，麥芽三糖也在第二天幾乎被利用 光。發酵三天之後，HPLC分析的結果顯示，所得到的異 麥芽糖爲佔總糖100%的高純度異麥芽寡糖。因米屑來源之 粗異麥芽寡糖液中的營養充足，無論採用何種方法，發酵 三天之後菌體染色率未見明顯上升，即死細胞無明顯增 加，除糖效果佳。 (請先閱讀背面之注意事項再填寫本頁) -------訂i 經濟部智慧財產局員工消費合作社印製 ___________________ 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐〉 A7 1274757 ______ Β7 五、發明說明（16 ) 表二 時間 葡萄糖消耗 率(％) 麥芽糖消耗 率(％) 麥芽三糖消 耗率(％) 發酵去除之總 糖量(g/100mL) 異麥芽寡糖 純度(％) 24 h 100 91.9 50.4 48 h 100 100 96 72 h 100 100 100 7.07 100 實例三 食用澱粉（樹薯粉）經液化、糖化及TG酵素異構化後可 得純度接近60%的異麥芽寡糖。購自環泰之異麥芽寡糖即 以食用澱粉製造而來，每35克乾重的異麥芽寡糖含3.35 g 異麥芽糖、11.7 lg盼樂糖、2.66 g異麥芽三糖、以及2.3 g DP4之寡糖，可消化糖含量則分別爲8.3 g葡萄糖、5.44 g 麥芽糖、以及1.25g麥芽三糖。取環泰異麥芽寡糖，與蒸 餾水調配，得總糖重量濃度爲35 % (w/v)之異麥芽寡糖水 溶液，異麥芽寡糖之純度爲57.18%。取100 mL此異麥芽 寡糖水溶液，以第4法加入酵母發酵除去可消化糖，即加 入100 mL去麥汁之吟釀淸酒酵母菌體（培養24小時之菌 體，細胞數目濃度爲2·〇χ108菌株/mL)，在30°C，80 rpm 下發酵，24小時後追加底層啤酒酵母同等量菌體（培養24 小時之菌體，細胞數目濃度爲1.5 XI 08菌株/mL)。異麥芽 寡糖水溶液中葡萄糖、麥芽糖、以及麥芽三糖被發酵去除 -19- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公€ (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 ϋ n 1 I ϋ ·1 n^OJI ϋ I ·1 ϋ ·ϋ I I I n 1 ϋ n 1 n -ϋ ϋ n -ϋ n -^1 -ϋ n , 1274757 A7 B7 五、發明說明（17) 比例隨時間的變化如表三。 樹薯粉爲來源之粗異麥芽寡糖水溶液，第一天以吟釀淸 酒酵母發酵即可將葡萄糖完全消化去除，但麥芽糖與麥芽 三糖完全未被利用。第二天加入底層啤酒酵母之後麥芽糖 與麥芽三糖開始被消化，至第二天終了，麥芽糖完全被消 化，麥芽三糖也去除了八成。因缺乏氮源，第二天之後的 底層啤酒酵母已無活力，第三天呈停滯狀態。雖然麥芽三 糖沒有繼續被消化，發酵三天終了收成時，所得到的異麥 芽糖仍是佔總糖98.77%的高純度異麥芽寡糖。 與實例一以米屑爲來源之粗異麥芽寡糖水溶液的發酵 結果比較，發酵策略（方法）雖然相同，酵母在以樹薯粉爲 來源之粗異麥芽寡糖水溶液的存活度顯然偏差’代表死細 胞比率的染色率第三天終了高達80%。其原因就是以米屑 爲來源之粗異麥芽寡糖水溶液中存有少許的氮源及其它營 養品，酵母細胞得以繼續繁殖與存活，這個優點在以樹薯 粉爲來源之粗異麥芽寡糖水溶液則沒有，但這可以靠添加 少量的氮源等來補足。 (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 -20- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 ____B7 五、發明說明（18 ) 表三 時間 葡萄糖消耗 率(％) 麥芽糖消耗 率(〇/〇) 麥芽三糖消 耗率(％) 發酵去除之總 糖量(g/100 mL) 異麥芽寡糖 純度(％) 24 h 100 0 0 48 h 100 100 79.37 72 h 100 100 80 14.74 98.77 (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 實例四 食用澱粉（樹薯粉）來源的低純度異麥芽寡糖，如實例三 所示。取環泰異麥芽寡糖，與蒸餾水調配，得總糖重量濃 度爲35% (w/v)之異麥芽寡糖水溶液。取5〇 mL此異麥芽 寡糖水溶液，以第2法加入酵母發酵除去可消化糖，即加 入50 mL含麥汁之底層啤酒酵母菌體（培養40小時之菌 體，細胞數目濃度爲1.8χ108菌株/mL)，在30°C，80 rpm 下發酵72小時。因兩者以1 : 1混合，因此發酵開始時總 糖含量爲17.5% (w/v)之水溶液。異麥芽寡糖水溶液中葡萄 糖、麥芽糖、以及麥芽三糖被發酵去除比例隨時間的變化 如圖五，每日的變化如表四。 調配總糖濃度較低的粗異麥芽寡糖水溶液，酵母消化去 除可消化糖的速度會快一些。因此使用單一菌種底層啤酒 酵母，連同培養時的麥汁第一天一起加入後即不再追加酵 母，發現酵母死亡率明顯降低，第一天除了將葡萄糖完全 -21 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 B7_ 五、發明說明（19 ) (請先閱讀背面之注意事項再填寫本頁) 消化之外，也利用了大部份的麥芽糖，並開始消化麥芽三 糖。第二天可以將麥芽糖全部消化，第三天終了麥芽三糖 也消化了 80%以上，發酵三天終了收成時，所得到的異麥 芽糖是佔總糖98.96%的高純度異麥芽寡糖。 與實例二比較，一樣可以發現酵母在以樹薯粉爲來源之 粗異麥芽寡糖水溶液的存活度較差，因此後期代謝消化麥 芽三糖的速度即較慢，但最終還是可以得到純度98%以上 的異麥芽寡糖。 表四 時間 葡萄糖消耗 麥芽糖消耗 麥芽三糖消 發酵去除之總 異麥芽寡糖 率(％) 率(％) 耗率(％) 糖量(g/100mL) 純度(％) 24 h 100 81.6 24.4 48 h 100 100 67.5 72 h 100 100 83.5 7.39 98.96 實例五 經濟部智慧財產局員工消費合作社印製 食用澱粉（樹薯粉）來源的低純度異麥芽寡糖，如實例三 所示。取環泰異麥芽寡糖，與蒸餾水調配，得總糖重量濃 度爲17.5 % (w/v)之異麥芽寡糖水溶液。取100 mL此異麥 芽寡糖水溶液，以第3法加入酵母發酵除去可消化糖，即 加入100 mL去麥汁之底層啤酒酵母菌體（培養24小時之菌 -22- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐〉 7 75 74 12 A7B7 i、發明說明（20) (請先閱讀背面之注意事項再填寫本頁) 體，細胞數目濃度爲1.5xl〇8菌株/mL)，在30°C，80 rpm 下發酵，24小時後追加底層啤酒酵母同等量菌體（培養24 小時之菌體，細胞數目濃度爲1.5 xlO8菌株/mL)。異麥芽 寡糖水溶液中葡萄糖、麥芽糖、以及麥芽三糖被發酵去除 比例隨時間的變化如表五。 與實例四比較，加入無麥汁之底層啤酒酵母於樹薯粉來 源的低純度異麥芽寡糖水溶液，因無氮源及其它營養品補 充，細胞活力不足代謝消化能力較差’第一天只能消化75% 的葡萄糖，第二天追加等量的底層啤酒酵母之後才消化其 餘的葡萄糖及全部的麥芽糖，雖然發酵三天之後仍有16% 的麥芽三糖，但所得到的異麥芽糖則是佔總糖99.06%的高 純度異麥芽寡糖。 表五 時間 麥芽糖消耗 率(％) 麥芽三糖消 耗率(％) 發酵去除之總 糖量(g/100mL) 異麥芽寡糖 純度(％) 葡萄糖消耗 率(％) 24 h 75 0 0 48 h 100 100 59.03 72 h 100 100 84 7.40 99.06 經濟部智慧財產局員工消費合作社印製 實例六 食用澱粉（樹薯粉）來源的低純度異麥芽寡糖’如實例三 -23- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明（21) 所示。取環泰異麥芽寡糖，與蒸餾水調配，得總糖重量濃 度爲17.5 % (w/v)之異麥芽寡糖水溶液。取1〇〇 mL此異麥 芽寡糖水溶液，以第4法加入酵母發酵除去可消化糖，即 加入100 mL去麥汁之吟釀淸酒酵母菌體（培養24小時之菌 體，細胞數目濃度爲2.0χ108菌株/mL)，在30°C，80 rpm 下發酵，24小時後追加底層啤酒酵母同等量菌體（培養24 小時之菌體，細胞數目濃度爲1.5 χΙΟ8菌株/mL)。異麥芽 寡糖水溶液中葡萄糖、麥芽糖、以及麥芽三糖被發酵去除 比例隨時間的變化如表六。 與實例五的差別是第一天發酵的是用吟釀淸酒酵母，其 葡萄糖的代謝較底層啤酒酵母爲佳，因此第一天即可將葡 萄糖全部消化，第二天加入的底層啤酒酵母則消化麥芽糖 與麥芽三糖，發酵三天終了收成時，所得到的異麥芽糖爲 佔總糖99.35%的高純度異麥芽寡糖。 表六 時間 葡萄糖消耗 率(％) 麥芽糖消耗 率(％) 麥芽三糖消 耗率(％) 發酵去除之總 糖量(g/100mL) 異麥芽寡糖 純度(％) 24 h 100 0 0 48 h 100 100 82.61 72 h 100 100 89.30 7.43 99.35 -24- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ----- - ---—--I 丨皿訂— !丨！ 線-^11- (請先閱讀背面之注意事項再填寫本頁) 1274757 A7 _____ B7_五、發明說明（22 ) 實例七 以米屑爲澱粉源，液化、糖化及TG異構化後得純度 53.19%的異麥芽寡糖，總糖含量30% (w/v)，其製作方法 如實例一。取100 mL此異麥芽寡糖水溶液，以第1法加 入酵母發酵除去可消化糖，即加入100 mL已去除麥汁之 底層啤酒酵母菌體（培養24小時之菌體，細胞數目濃度爲 1·6χ108菌株/mL)，在30°C，80 rpm下發酵72小時。異麥 芽寡糖水溶液中葡萄糖、麥芽糖、以及麥芽三糖被發酵去 除比例隨時間的變化如圖六，每日的變化如表七。 由於米屑來源之異麥芽寡糖含有少量氮源及其它營養 品，因此加入底層啤酒酵母發酵，不管有無倂入麥汁，並 不影響酵母菌的存活及代謝活性，因此葡萄糖、麥芽糖、 以及麥芽三糖等皆可快速消化。圖六顯示葡萄糖、麥芽糖、 以及麥芽三糖三者分別在第24、28、以及36即可全部消 化光，因此根據HPLC分析的結果，所得到的異麥芽糖爲 佔總糖100%的高純度異麥芽寡糖。 (請先閱讀背面之注意事項再填寫本頁) -丨丨！ ---訂--II--I--- 經濟部智慧財產局員工消費合作社印製 -25 - 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） 1274757 A7 B7 五、發明說明（23) 表七 時間葡萄糖消耗麥芽糖消耗麥芽三糖消發酵去除之總糖異麥芽寡糖 率(％) 率(％) 耗率(％) 量(g/100mL) 純度(％) 24 h 100 97.1 78.5 48 h 100 100 96 72 h 100 100 100 14.14 100 (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 實例八 以米屑爲澱粉源，液化、糖化及TG異構化後得純度 53.19%的異麥芽寡糖，總糖含量30% (w/v)，其製作方法 如實例一。以蒸餾水1 : 1稀釋，即總糖重量濃度爲15% (w/v)之異麥芽寡糖水溶液。取1〇〇 mL此異麥芽寡糖水溶 液’以第1法加入酵母發酵除去可消化糖，即加入100 mL 已去除麥汁之底層啤酒酵母菌體（培養24小時之菌體，細 胞數目濃度爲1·6χ108菌株/mL)，在3(TC， 80 rpm下發酵 72小時。異麥芽寡糖水溶液中葡萄糖、麥芽糖、以及麥芽 三糖被發酵去除比例隨時間的變化如圖七，每日的變化如 表八。 本實例與實例七完全相同，只是米屑爲澱粉源之低純度 異麥芽寡糖水溶液的總糖重量濃度減半，結果顯示在較稀 的糖液中，酵母發酵速率較快。如圖七所示，葡萄糖、麥 芽糖、以及麥芽三糖三者分別在第16、20、以及24即可 -26- 本紙張尺¥適用中國國家標準（CNS)A4規格⑽x 297公釐) 一-- «III — 線 1274757 A7 B7 五、發明說明（24 ) 全部消化光，因此根據HPLC分析的結果，所得到的異麥 芽糖爲佔總糖1 〇〇%的高純度異麥芽寡糖。本實例與實例七 發酵速率較快的另外一個原因是所添加的底層啤酒酵母是 在活性較高時的對數期，培養24小時之菌體是在對數期， 而培養40小時之後酵母應該是在繁殖過程的靜止期。 表八 時間 葡萄糖消耗 率(％) 麥芽糖消耗 率(％) 麥芽三糖消 耗率(％) 發酵去除之總 糖量(g/100mL) 異麥芽寡糖 純度(％) 24 h 100 100 100 48 h 100 100 100 72 h 100 100 100 7.07 100 (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 -27- 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）1274757 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperatives Printing V. Invention Description (1) Industrial Applicability Field The present invention provides a method for producing high-purity isomalto-oligosaccharide, which is a functional health food and has a wide range of uses. It can be applied to health food, beverage, wine and other industries. BACKGROUND OF THE INVENTION Isomalto-oligosaccharides (IMO) are equivalent to fructooligosaccharides, galactose oligosaccharides, and soybean oligosaccharides, and are functional oligosaccharides, which have various functional properties and can nourish intestinal bacteria. The proliferation of Bifidus) is not easy to cause tooth decay. The physiological functions of oligosaccharides mainly include (1) improving the intestinal microflora; (2) reducing the production of spoilage substances in the intestine; (3) improving excessive lipids in the blood and reducing cholesterol; (4) reducing constipation phenomenon, (5) Preventing tooth decay; (6) immunization; and (7) promoting calcium absorption and the like. In 1997, the demand for various oligosaccharides in Japan was about 30,000 metric tons, of which isomalto-oligosaccharides accounted for 1 1,000 metric tons [Tan Jingfen, Food Industry Monthly, (1999), 1-8]. The main uses of various oligosaccharides are Add in the 淸 飮 .. Among the sweets. The isomalto-oligosaccharide has a good processing property, which makes it account for more than 30% of the sales of oligosaccharides. Since functional oligosaccharides have been approved in Japan as specific health food products in the commodity, it is expected that the demand will continue to increase, and the glucose molecules contain at least one a-D-(l,6) chain of disaccharides and oligosaccharides. Glycosides are called isomalto-oligosaccharides, and their main components are isomaltose, panose (62_0-a-glucosyl-maltose), isomaltotriose, etc. In traditional fermented foods, such as alcohol, miso, and soy sauce, mass production can be carried out using the yeast -4- paper scale applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back first) Fill in this page again) - j -------- order! ------· Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 1274757 A7 ___ B7_ V. Description of invention (2) Produce isomalt oligosaccharides with a purity of 50% - 60% dry weight. The traditional method for producing isomalto-oligosaccharide syrup from starch is to first use enzyme α-amylase (EC 3·2·1·1) and pullulanase (pUilulanase) (EC 3 ·2 · 1). · 41), β-amylase (β-amylase) (EC 3. 2. 1. 2) The starch is hydrolyzed to obtain α-(1^4)-bonded glucose oligosaccharide (α-D-gluco-oligosaccharides), and then a-D-glucosidase (EC 3. 2·1 · 20) glucoside transfer function to obtain α-(1-6)-linked oligosaccharides [Takaku, H., Handbook of amylases and related enzymes, Ed· The Amylase Research Society of Japan, Pergamon Press, Oxford, (1988), 215-217]. The α-D-pinease with aglycone transfer function is also called transglucosidase (abbreviated as TG) or D-glucosyltransferase (EC 2. 4. 1. twenty four). For example, as described in Japanese Patent No. 61 _2 12296, 30°/. Corn, potato, or tapioca starch is liquefied by heat-stable alpha-amylase into a liquefied syrup having a degree of polymerization (DP) between 6 and 10, which is at pH 5 and 60 °C. Saccharification with β-amylase and TG for 48-72 hours gives isomalto-oligosaccharide syrup. Enzyme TG catalyzes the hydrolysis of α-D-gluco-oligosaccharides and the transfer of glucose moss, usually to 6-OH of glucose, for example, the transfer of glucoside to D-glucose Isomaltose, transferred to maltose to obtain panose. After the action of TG enzyme, the product contains a large class of glucose oligosaccharides with α-(1 - 6) linkages called isomalt oligosaccharides, and the product also contains a large amount of glucose and α-(1^4). Oligosaccharides (mainly maltose). TG is mainly found in mold, but Aspergillus paper size is applicable to China National Standard (CNS) A4 specification (21〇x 297 mm) (please read the note on the back and fill out this page). Order · Line · 1274757 A7 B7 V. Description of the invention (3) (Please read the note on the back and then fill out this page.) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed niger TG only acts on low DP glucose oligopolymer, other molds such as Aureobasidium pullulans [Yun et Al·, Biotechnol. Lett. 16 (1994) 1 145-1 150], Aspergillus carbonarious [Duan et al. ? Biotechnol. Lett. 1 6 (1 994) 1 1 5 1 -1 1 56], Aspergillus nidulans [Kato et al., Appl. Environ. Microbiol· 68 (2002) 1250-1256] also found that this enzyme function exists. Kuriki et al [Kuriki et al., Appl. Environ. Microbiol. 59 (1993) 953-959] found that the new branch of the enzyme from the Bacillus subitlis, neopullulanase, also has oc-(l->6)- grapevine transfer function (1^118£1)^035^^ The function of 011), therefore, isoamyl oligosaccharides can also be produced from starch by the combination of 0^amylase and neopullulanase. The above various methods for producing isomalt oligosaccharides from starch have the highest total sugar content of about 60%, and the rest are α-(1^4)-bonded oligosaccharides such as glucose and maltose. In addition to starch, direct use of high concentrations of maltose as raw material can also produce isomalto-oligosaccharides via the action of TG enzymes [Yun et al., Biotechnol· Lett. 16 (1994) 1145-1150], regardless of the enzyme used. Or the bacterial cell as the catalyst, the final ratio of isomalto-oligosaccharide is also about 60%. Domestic scholars have also studied the use of fixed or unfixed α-D-glucosidase (glucosyltransferase) to produce isomalt oligosaccharides, and the purity of the obtained products is also the same [Xu Wei et al., Chinese Agricultural Chemistry Association Zhi, 3 1 (1993) 740-75 1 ; Huang Yiyi, 1995, Master thesis of Datong Institute of Technology; Lin Jianhong, 2000, Datong University Master thesis]. Using a fixed new pullulanase (from Bacillus stearothermophilus, with TG function) can transform amylopectin (pullulan) into Pancred-6 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1274757 A7 __ B7 _ V. Invention description (4) (Please read the note on the back and fill out this page) (panose), but the purity is only up to 84%, the other ingredients are still glucose and maltose [ Kuriki et al·, J. Ferment· Bioeng. 73 (1992) 198-202] 〇 US Patent No. 6025168 and Japanese Patent No. 1-004700 are the same invention, using a fixed TG to convert a starch hydrolyzate into an isomalto-oligosaccharide syrup, the DP of the starch hydrolysate is between 4 and Between 70, preferably between 20 and 60, may be derived from enzymes or acid hydrolysis, and in order to lower DP, sometimes ex-amylase is used in combination with pullulanase. Each of these methods continuously produces the resulting isomalto-oligosaccharide syrup having an isomalt oligosaccharide content of 40 to 45%. Japanese Patent No. 01-098601 proposes a method for improving the purity of isomalto-oligosaccharide, which is obtained by separating the isomalt oligosaccharide syrup obtained by the enzyme method through a basic metal type strongly acidic cation exchange resin, and separating more than 90% of glucose. Isomalt oligosaccharide with a purity of more than 80%, and the solid component of this product still has maltose (DP is 2). The Ministry of Economic Affairs, Intellectual Property Office, Staff and Consumers Cooperatives, US Patent 5,612,203, mentions the carbon modification of the reducing end of sugar molecules, such as oxidation to carboxylic acid or amination, the modified sugar can be adsorbed on the ion exchange resin, and the appropriate enzyme is used. If the β-amylase cuts the sugar molecule, this method can improve the purity of the product. However, if the raw material is isomalto-oligosaccharide, some products are no longer isomalto-oligosaccharides, such as the product of isomaltose in the raw material. It is glucose. There are no patents related to the production of isomalto-oligosaccharides in China, and there are two patents for oligosaccharides containing α-D-oligoglucose. They are the Republic of China Patent No. 083 105867 and the patent 086102774, both of which are related to isomalto-oligosaccharides. Not the same. In the production of isomaltoligosaccharides, the domestic manufacturers have the Huantai Enterprise Taoyuan Plant and the paper scale applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1274757 A7 B7 V. Invention Description (5) Shuotai Industrial Kaohsiung Plant, the raw materials used are imported high-purity starch, imported Japanese technology, using free enzyme method, that is, using unfixed TG, the production of isomalto-oligosaccharide content is 55-60%, the remaining ingredients are glucose and maltose. In summary, the purity of the isomalto-oligosaccharide obtained by the transfer of TG enzymes from starch or maltose is generally about 60%, and the remaining components are mainly glucose and maltose, which is limited by technology and can no longer improve isomalto-oligosaccharides. Sugar purity. In order to break through this limit, the present invention utilizes the non-fermentability of isomalto-oligosaccharide to ferment glucose and maltose in isomalto-oligosaccharide syrup into alcohol for use in brewing, thereby improving isomalto-oligosaccharide The sugar purity is close to 100% and the alcohol can be distilled off or recycled. SUMMARY OF THE INVENTION The present invention is a method for producing high-purity isomalto-oligosaccharide, which is a fermentable sugar in an isomalt-oligosaccharide syrup obtained by transforming starch or maltose with a transglucosidase by using a yeast for brewing ( Or digestible sugar (referred to as DS), that is, glucose and maltose are fermented into alcohol, and isomalto-oligosaccharides with a purity of more than 98% can be obtained, and the alcohol can be distilled or recovered. The results of the study showed that the yeast was degreased, and the Saccharomyces cervisiae or Saccharomyces carlsbergensis was shaken at 30 °C (150 rpm) for 24 hours. Digestible sugar efficiency, the concentration of yeast in the stable period of 40 hours of cultivation is higher, although the effect of digestible sugar is slightly worse, the same can be applied to the Chinese National Standard (CNS) A4 specification (210 X 297 public). PCT) (Please read the notes on the back and fill out this page). --Line · Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1274757 A7 __B7 V. Invention description (8) or transglucosidase referred to as TG) powder or maltose (please read the back of the note before you fill in this Page) The obtained malt oligosaccharide syrup, wherein the isomalt oligosaccharide has a purity of about 60% or less, and the fermentable sugar therein is digested and removed by yeast fermentation to obtain a high-purity isomalto-oligosaccharide. The fermentation and sugar removal step mainly ferments the fermentable (digestible) sugar in the isomalt oligosaccharide syrup, that is, glucose and maltose into alcohol, and the components of the isomalto-oligosaccharide are not fermented, so the products are The relative proportion of sugar remains unchanged. Different sources of starch raw materials, under the action of appropriate liquefaction, saccharification and TG, can obtain isomalto-oligosaccharide syrup with a concentration of up to 60%, and obtain high-purity isomalto-oligosaccharide by the method of the invention. . For example, the price of starch is 68. 45% rice crumb, adjusted to 35% dry weight suspension, liquefied, saccharified and added 0. After 1% glucosyltransferase (TG, Japanese Amano product) was subjected to glucose-based transfer, the total sugar weight of the obtained rice was 30. 21%, the total weight of isomalto-oligosaccharide (IMO) is 53. 19%. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives. The use of various starch raw materials to prepare isomalto-oligosaccharides is controlled by the degree of liquefaction, the way the enzyme is added, the reaction time and pH, and the different conditions are different. For example, isomalto-oligosaccharide produced by rice flour according to Example 1 has a higher content of isomaltose (IG2), followed by isomaltose (IG3) and panose. Different starch raw materials were used to make glutinous rice, corn and rice bran as raw materials. The obtained isomalt oligosaccharides were compared by HPLC, and the difference was not significant, which was quite close to that of rice stalk malt oligosaccharide. Isomalt oligosaccharide production, control of high concentration of maltose (G2) as a reaction substrate can produce high isomaltose content of isomalto-oligosaccharide; production of high tropose -11 - this paper scale applies to China National Standard (CNS) A4 Specifications (210 X 297 mm) 1274757 A7 _B7_ V. Description of the invention (9) (panose) isomalto-oligosaccharides require a high concentration of linear oligosaccharides Gn (n = 3 to 4) as the reaction matrix. (Please read the notes on the back and fill out this page.) Use edible starch (shushu powder) as raw material, liquefaction temperature 90 ° C, liquefaction 0. 5 hours, the saccharification enzyme (Fungamyl) and isomerase (TG) are added at the same time, at pH 5. Conversion at 0,55 °C for 13 hours gave the highest maltose content. The purity of isomalto-oligosaccharide (product of Huantai Company) obtained from edible starch (shushu powder) was 57. 18%, the purity of the malt oligosaccharide with the source of rice shavings 53. 19% comparison is quite close, but the composition of the two different malt oligosaccharides is different. The former isomalt oligosaccharide is mainly panose, and the latter is mainly IG2. The two were separately fermented by yeast at 30 ° C for 80 hours under shaking at 80 rpm to completely remove the digestible sugar, and the final product was nearly 100% high isomalt oligosaccharide syrup. Various brewing yeasts can be used for fermentation and sugar removal, but the effects and control conditions are different. Among them, the brewing yeast and the bottom beer yeast remove the digestible sugar (DS), and the two strains are shaken at 30 ° C ( 150 rpm) The most glucose-removing efficiency of the log phase yeast cultured for 24 hours, and the stable phase of the yeast cultured for 40 hours, although the bacterial concentration is higher, the sugar removal effect is worse. Printed by the Intellectual Property Office of the Intellectual Property Office of the Ministry of Economic Affairs, when discussing the effect of the concentration of the reaction substrate on the sugar removal, it was found that the high concentration, ie, the total sugar weight concentration of 35% (w/v) of the yam powder derived from the malt oligosaccharide It is best to use the 30% (w/v) rice-wheat-derived malto-oligosaccharide in the former, and the bottom-adding brewer's yeast is the best. The latter is directly added to the bottom beer yeast. At a concentration, such as a weight concentration of 17. 5% (w/v) of potato ash source of isomalto-oligosaccharide and 1 5% (w/v) of rice swarf source-12- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 1274757 A7 ___B7___ V. Inventive Description (10) The isomalt oligosaccharides can be fermented with the bottom beer yeast and the wort, which is simple and effective. (Please read the notes on the back and fill out this page.) Different starch raw materials such as glutinous rice, corn, rice bran and edible starch, transformed isomalt oligosaccharides and high purity isomalto-oligosaccharides from DS. It will not deteriorate or decompose after heating at pH 3, 1 2 1 °C for 1 hour. It is believed to maintain its characteristics and functions for foods such as health food, alcohol, desiccant, high temperature treatment or low pH. Yeast fermentation In addition to fermentable sugars, the steps for producing high-purity isomalto-oligosaccharides are as follows: 1) formulating an appropriate concentration of low-purity isomalt oligosaccharide aqueous solution; 2. adding cultured yeast for fermentation; and selective 3 · Post-treatment such as neutralization, decolorization, etc., alcohol is distilled off or recovered, and concentrated in water. Yeast culture method 1. Juice culture method: Inoculate the brewing yeast from the slant culture in a test tube for 24 hours (5 mL of mash juice, 30 ° C, static culture), and then expand the culture in a flask for 24 hours or 40 hours (1 00 mL of mash, 30 ° C, 1 50 rpm shaking culture). Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumers Co., Ltd. 2. Wort culture method: The bottom layer of brewer's yeast cultured in slant culture is inoculated in a test tube for 24 hours (wort juice 5 mL, 30 ° C, static culture), and then expanded in a triangular flask. Incubate for 24 hours or 40 hours (100 mL of wort, 30 ° C, shaking culture at 150 rpm). Yeast fermentation and sugar removal (removal of digestible sugar in isomalto-oligosaccharide syrup) Method: In low-purity isomalt oligosaccharide solution, yeast can be fermented to remove -13 - This paper scale applies to China National Standard (CNS) A4 Specifications (210 X 297 mm) 1274757 A7 ^__B7 V. Description of invention (11) (Please read the notes on the back and fill out this page). Fermentable glucose, maltose, maltotriose, etc. The addition method and fermentation conditions can finally obtain isomalto-oligosaccharides with a purity of more than 98%. The following examples are used in the examples: Method 1 (yeast method): Take 100 mL of yeast culture solution to After centrifugation at 5000 rpm for 20 minutes, the supernatant was removed, washed with distilled water, centrifuged, and the cells were added to the prepared 100 mL of sugar solution, and shaken at 30 ° C, 80 rpm for 72 hours. The second method (yeast buckwheat juice method): take 50 mL of the yeast culture solution, directly add 50 mL of the double-concentration sugar solution, and the same method as the above-mentioned first method to remove the DS 〇 method 3 (adding the same yeast method in stages): On the first day, the yeast cell method was added, and the same amount and the same cells were added after 24 hours. The fourth method (additional yeast method in stages): The yeast yeast was added to the yeast yeast on the first day, and the same amount of the bottom beer yeast was added after 24 hours, and the remaining operation was the same as in the third method. In practical applications, the yeast fermentation method of the present invention is not limited to these particular yeast addition methods. Analysis and quantification of printed substrates and products by the Intellectual Property Office of the Intellectual Property Office of the Ministry of Economics, oligosaccharide analysis, high performance liquid chromatography, the column used was Supelcosil LC-NH2, and the mobile phase was 80% acetonitrile and 20%. Pure water, operating at 30 ° C, flow rate of 1 · 2 mL / min, RI detector. From the chromatogram, the oligosaccharide with a degree of polymerization of 1 to 4 and the oligosaccharide (Gn) having a degree of polymerization greater than 4, the monosaccharide having a degree of polymerization of 1 and the saccharide having a degree of polymerization of 2 (DP2) can be respectively determined. Analytical quantification of maltose and iso-mai-14- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1274757 A7 B7 V. Description of invention (12) Bud sugar, oligosaccharide with a degree of polymerization of 3 (DP3) ) Maltotriose, panose, and isomaltose can be analyzed separately. (Please read the precautions on the back and then fill out this page.) Example 1 Ministry of Economic Affairs Intellectual Property Bureau employees consumption cooperatives printed starch source for rice shavings, which is the lower foot product produced by Penglai rice with continuous fine rice machine, the starch content is about 68%. The other ingredients are 8% crude protein, 3% crude fat and 1% ash. First, the rice chips are purified by the following suspension preparation, liquefaction, saccharification and TG isomerization. 19% isomalto-oligosaccharide syrup·(1). Suspension preparation: Take 1996 grams of rice shavings (water content 12. 35%), add water to 5L, total weight 5379. 5 g, which is a suspension of 35 g/100 mL (dry weight), is evenly stirred by a homogenizer for liquefaction, saccharification and isomerization. (2)· Liquefaction: Add 〇·5 g/(kg-米屑) liquefied enzyme (Novo product, product name Termamyl 120 L) and 30 ppm Ca + + (CaCl2) in 35g/100 mL rice crumb suspension. , H20), heated to 95 ° C in an induction cooker, and then added to the starch after gelatinization. 0 g / (kg - rice) liquefied enzyme, maintained at 93-95 ° C for 1-2 hours, the degree of starch liquefaction detected by iodine solution, stop liquefaction when the iodine reaction blue purple disappeared red, cooling to 55 ° C , add acid to adjust the pH to 5. 0-5. 5, add water to make up the weight of the heating evaporation loss. (3). Saccharification and isomerization: rice liquefaction liquid is transferred to a 55 ° C water bath, respectively added 0. 05% (w/w) of de-branched enzyme (Novo product, product name Promozyme 400 L) and 0. 1% (w/w) of cold-amylase or 〇"% (w/w) saccharification enzyme (Novo product, name Fungamyl 8 00 L), saccharified at 55 ° C for 24 hours, plus 0. 1% TG isomerized for 24 hours, taken out and heated to 85 ° C, 5 minutes to destroy the enzyme and then cooled, and then make up the water according to the original weight, that is, the isomalt oligosaccharide syrup, the total sugar content is 30% -15- This paper scale applies China National Standard (CNS) A4 Specification (210 X 297 mm) 1274757 A7 B7 V. Description of Invention (13) (W/V), in which the concentration of isomalto-oligosaccharide is 53. 19%. Take 100 mL of the above 30% (w/v) isomalt oligosaccharide aqueous solution, and add the yeast fermentation to remove the digestible sugar by the fourth method, that is, add 1 〇〇mL to the wort and brew the yeast yeast (wheat The cells cultured in the juice for 40 hours, the cell number concentration was 2·7χ108 strain/mL), and the fermentation was carried out at 3 (TC, 80 rpm, and the same amount of the bottom beer yeast was added after 24 hours (the bacteria cultured in the juice for 40 hours) The cell number concentration was 1·8 χΙΟ8 strain/mL. The proportion of glucose, maltose, and maltotriose in the aqueous solution of isomalt oligosaccharide was changed as shown in Figure 1. The daily change is shown in Table 1. Table 1 shows that the first day of adding brewed yeast, the fermentation can completely consume glucose within 24 hours, the next day to add the bottom beer yeast, in the absence of glucose carbon source, can be digested in the next day 95. 5% maltose with 90% maltotriose. Figure 1 shows that the yeast can completely digest maltose and maltotriose at 60 and 72 hours, respectively. Therefore, according to the results of HPLC analysis, the obtained isomaltose is a high-purity isomalto-oligosaccharide which accounts for 100% of the total sugar. Sugar, the ingredients include isomaltose, pancreatic sugar, isomaltotriose, and malto-oligosaccharide having a degree of polymerization of four. (Please read the notes on the back and fill out this page) II---- I Book · -----I--· Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed-16- This paper scale applies to Chinese national standards (CNS) A4 size (210 X 297 mm) 1274757 A7 B7 V. Description of invention (14) Table 1 time maltose consumption rate (%) Maltose trisaccharide consumption rate (%) Total amount of sugar removed by fermentation (g/100 mL) Isomalt oligosaccharide purity (%) Glucose consumption rate (%) 24 h 100 0 0 48 h 100 95. 5 90 72 h 100 100 100 14. 14 100 (Please read the notes on the back and fill out this page.) Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives. Example 2 Using rice chips as the starch source, the purity is obtained after liquefaction, saccharification and TG isomerization. 19% of the heterodental oligosaccharide' total sugar content was 30% (w/v), and its preparation method was as in Example 1. Take 50 mL of this aqueous solution of isomalto-oligosaccharide, and add the yeast fermentation to remove the digestible sugar by the second method, that is, add 50 mL of the bottom beer yeast containing the wort (the cells cultured for 40 hours, the cell number concentration is 1. 8xl08 strain/mL), fermentation at 30 ° C, 80 rpm for 72 hours. Since the two are mixed with 1··1, the total sugar content at the start of fermentation is 15% (w/v). The concentration of each saccharide in the aqueous solution of isomalto-oligosaccharide before and after fermentation was quantified by HPLC analysis, and the chromatograms are shown in Fig. 2 and Fig. 3. The changes in the proportion of fermentation of glucose, maltose, and maltotriose in the aqueous solution of isomalto-oligosaccharide over time are shown in Figure 4. The daily changes are shown in Table 2. Figure 2 and Figure 3 show glucose (G1), maltose (G2), maltotriose (G3), isomaltose (IG2), pantole (P), and isomalt in the isomalt oligosaccharide solution before and after fermentation. Sugar (IG3), and oligosaccharide (G4) with a degree of polymerization -17- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1274757 A7 _____B7_5, invention description () HPLC Analyze the map. Comparing the results of the pre- and post-fermentation results, it was found that the digestible sugars (including glucose, maltose, and maltotriose) were completely removed by yeast, while the isomalto-oligosaccharide fraction was not changed, indicating that the yeast fermentation method can improve the isomalto-oligosaccharides. The purity of the sugar does not affect the distribution ratio of isomaltose, pancreatic sugar, isomaltose, and the like in the isomalto-oligosaccharide. Table 2 and Figure 4 show that in the crude malt oligosaccharide solution of rice shavings, the bottom beer yeast can easily remove all digestible sugars within 48 hours due to sufficient nutrition. Although the yeast wort is also added together with the addition of yeast, maltose and maltotriose remain in the wort, and Figure 4 shows that it is present in the crude malt oligosaccharide solution and the wort is entrained. The digestible sugars such as maltose and maltotriose are quickly metabolized by yeast. Maltose is almost completely digested on the first day, and maltotriose is almost used up the next day. After three days of fermentation, the results of HPLC analysis showed that the obtained isomaltose was a high-purity isomalto-oligosaccharide which was 100% of total sugar. Due to the sufficient nutrients in the crude malt oligosaccharide solution from rice sources, no matter which method was adopted, the staining rate of the cells did not increase significantly after three days of fermentation, that is, the dead cells did not increase significantly, and the sugar removal effect was good. (Please read the note on the back and fill out this page) ------- Book i Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Print ___________________ This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 A7 1274757 ______ Β7 V. Description of invention (16) Table 2 Time glucose consumption rate (%) Maltose consumption rate (%) Maltotriose consumption rate (%) Total amount of sugar removed by fermentation (g/100mL) Malt oligosaccharide purity (%) 24 h 100 91. 9 50. 4 48 h 100 100 96 72 h 100 100 100 7. 07 100 Example 3 Edible starch (tree cassava) can be obtained by liquefaction, saccharification and isomerization of TG enzyme to obtain isomalt oligosaccharides with a purity close to 60%. The isomalto-oligosaccharide purchased from Huantai is made from edible starch, and each 35 grams of dry weight of isomalto-oligosaccharide contains 3. 35 g of isomaltose, 11. 7 lg Pan Le Sugar, 2. 66 g of isomaltose, and 2. 3 g of DP4 oligosaccharides, the digestible sugar content was 8. 3 g glucose, 5. 44 g maltose, and 1. 25g maltotriose. The mixture was mixed with distilled water to obtain a solution of isomalto-oligosaccharide in a total sugar concentration of 35 % (w/v). The purity of isomalto-oligosaccharide was 57. 18%. Take 100 mL of this isomalt oligosaccharide aqueous solution, and add the yeast fermentation to remove the digestible sugar by the fourth method, that is, add 100 mL of the wort to brew the yeast cell (the cells are cultured for 24 hours, and the cell number concentration is 2·〇χ108 strain/mL), fermented at 30 ° C, 80 rpm, and after 24 hours, the same amount of bacteria in the bottom of the brewer's yeast was added (the cells were cultured for 24 hours, and the cell number concentration was 1. 5 XI 08 strain / mL). Glucose, maltose, and maltotriose in fermented isomalt oligosaccharide solution are removed by fermentation-19- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 km € (please read the notes on the back and fill in the form) On this page) Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed ϋ n 1 I ϋ · 1 n^OJI ϋ I ·1 ϋ ·ϋ III n 1 ϋ n 1 n -ϋ ϋ n -ϋ n -^1 -ϋ n 1274757 A7 B7 V. INSTRUCTIONS (17) The ratio of time to time is shown in Table 3. The potato powder is the source of the crude malt oligosaccharide solution. The first day, the brewing of the yeast can be used to completely digest the glucose. Removed, but maltose and maltotriose were completely unused. After the addition of the bottom beer yeast, maltose and maltotriose began to be digested. By the end of the next day, maltose was completely digested and maltotriose was removed. Eighty percent. Due to the lack of nitrogen source, the bottom beer yeast has no vitality after the next day, and it is stagnant on the third day. Although the maltotriose does not continue to be digested, the isomaltose obtained is still harvested after three days of fermentation. Accounting for total sugar 98. 77% high purity isomalt oligosaccharides. Compared with the fermentation results of the crude malt oligosaccharide solution with the rice crumbs as the source, the fermentation strategy (method) is the same, and the survival of the yeast aqueous solution of the crude malt oligosaccharide from the potato powder is obviously biased. The staining rate representing the ratio of dead cells was as high as 80% at the end of the third day. The reason is that there is a little nitrogen source and other nutrients in the crude malt oligosaccharide aqueous solution from rice chips, and the yeast cells can continue to multiply and survive. This advantage is based on the crude malt from the tapioca powder. The oligosaccharide aqueous solution is not, but it can be supplemented by adding a small amount of nitrogen source or the like. (Please read the notes on the back and then fill out this page.) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed -20- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1274757 A7 ____B7 V. DESCRIPTION OF THE INVENTION (18) Table 3 Time glucose consumption rate (%) Maltose consumption rate (〇/〇) Maltotriose consumption rate (%) Total amount of sugar removed by fermentation (g/100 mL) Isomalt oligosaccharide purity ( %) 24 h 100 0 0 48 h 100 100 79. 37 72 h 100 100 80 14. 74 98. 77 (Please read the note on the back and then fill out this page.) Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives Example 4 Low-purity isomalto-oligosaccharides from edible starch (tree flour), as shown in Example 3. The mixture was mixed with distilled water to obtain an aqueous solution of isomalto-oligosaccharide having a total sugar weight concentration of 35% (w/v). Take 5 mL of this isomalt oligosaccharide aqueous solution, and add the yeast fermentation to remove the digestible sugar by the second method, that is, add 50 mL of the bottom layer of the brewer's yeast containing the wort (the cells cultured for 40 hours, the cell number concentration is 1 . 8χ108 strain/mL), fermentation at 30 ° C, 80 rpm for 72 hours. Since the two are mixed at 1:1, the total sugar content at the start of fermentation is 17. 5% (w/v) aqueous solution. The proportion of glucose, maltose, and maltotriose in the aqueous solution of isomalto-oligosaccharide was measured by time as shown in Figure 5. The daily changes are shown in Table 4. The aqueous solution of crude malt oligosaccharide with a lower total sugar concentration is prepared, and the yeast digestion can remove the digestible sugar faster. Therefore, the use of a single strain of bottom beer yeast, together with the first day of the wort culture, no longer added yeast, found that the yeast mortality was significantly reduced, the first day in addition to the glucose completely - the paper scale applies to China Standard (CNS) A4 specification (210 X 297 mm) 1274757 A7 B7_ V. Invention description (19) (Please read the note on the back and fill out this page) In addition to digestion, most of the maltose is also used, and Start digesting maltotriose. The maltose can be completely digested the next day. At the end of the third day, the maltotriose is also digested by more than 80%. When the fermentation is finished for three days, the obtained isomaltose is 98% of the total sugar. 96% high purity isomalto-oligosaccharide. Compared with the second example, it can be found that the yeast has a poor viability in the aqueous solution of the crude malt oligosaccharide from the tapioca powder, so the rate of metabolic digestion of maltotriose is slower, but the purity can be finally obtained. More than % of malto-oligosaccharides. Table 4 Time Glucose consumption Maltose consumption Maltotriose elimination Fermentation removal Total isomalt oligosaccharide rate (%) Rate (%) Consumption rate (%) Sugar amount (g/100mL) Purity (%) 24 h 100 81. 6 24. 4 48 h 100 100 67. 5 72 h 100 100 83. 5 7. 39 98. 96 Example 5 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed Low-purity isomalto-oligosaccharides from edible starch (saspberry flour), as shown in Example 3. Take the singular malto-oligosaccharide and mix it with distilled water to obtain a total sugar weight concentration of 17. 5 % (w/v) aqueous solution of isomalto-oligosaccharide. Take 100 mL of this isomalt oligosaccharide aqueous solution, add the yeast fermentation to remove the digestible sugar by the third method, that is, add 100 mL of the bottom layer of the brewer's yeast cell (the cultured bacteria for 24 hours - this paper scale applies to China) National Standard (CNS) A4 Specification (210 X 297 mm) 7 75 74 12 A7B7 i, Invention Description (20) (Please read the note on the back and fill out this page) Body, the cell number concentration is 1. 5xl〇8 strain/mL), fermented at 30 ° C, 80 rpm, and after 24 hours, the same amount of bacteria in the bottom of the brewer's yeast was added (the cells were cultured for 24 hours, and the cell number concentration was 1. 5 xlO8 strain/mL). The proportions of glucose, maltose, and maltotriose in the aqueous solution of isomalto-oligosaccharides were removed by fermentation as shown in Table 5. Compared with the fourth example, the non-wort bottom beer yeast is added to the low-purity isomalt oligosaccharide aqueous solution derived from the tree potato powder. Due to the lack of nitrogen source and other nutrient supplements, the cell viability is insufficient and the metabolic digestion ability is poor. It can digest 75% of glucose, and the same amount of bottom beer yeast is added the next day before digesting the remaining glucose and all maltose. Although there are still 16% maltotriose after three days of fermentation, the obtained isomaltose is Is the total sugar 99. 06% high purity isomalt oligosaccharides. Table 5 Time maltose consumption rate (%) Maltotriose consumption rate (%) Total sugar content by fermentation (g/100mL) Isomalt oligosaccharide purity (%) Glucose consumption rate (%) 24 h 75 0 0 48 h 100 100 59. 03 72 h 100 100 84 7. 40 99. 06 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing example Six low-purity isomalto-oligosaccharides derived from edible starch (tree powder) 'Example 3-23- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1274757 A7 B7 Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printing 5, Invention Description (21). Take the singular malto-oligosaccharide and mix it with distilled water to obtain a total sugar weight concentration of 17. 5 % (w/v) aqueous solution of isomalto-oligosaccharide. Take 1 〇〇mL of this isomalt oligosaccharide aqueous solution, and add the yeast fermentation to remove the digestible sugar by the fourth method, that is, add 100 mL of the wort to brew the yeast cell (the cells cultured for 24 hours, the number of cells The concentration is 2. 0χ108 strain/mL), fermented at 30 ° C, 80 rpm, and after 24 hours, the same amount of bacteria in the bottom of the brewer's yeast was added (the cells were cultured for 24 hours, and the cell number concentration was 1. 5 χΙΟ8 strain/mL). The proportions of glucose, maltose, and maltotriose in the aqueous solution of isomalto-oligosaccharides were removed by fermentation as shown in Table 6. The difference from the fifth case is that the first day of fermentation is brewing yeast, the metabolism of glucose is better than that of the bottom beer yeast, so the whole day can digest the glucose, and the bottom beer yeast added the next day. Digestion of maltose and maltotriose, the fermentation of three days after the harvest, the resulting isomaltose is 99. 35% high purity isomalto-oligosaccharide. Table 6 Time glucose consumption rate (%) Maltose consumption rate (%) Maltotriose consumption rate (%) Total amount of sugar removed by fermentation (g/100mL) Isomalt oligosaccharide purity (%) 24 h 100 0 0 48 h 100 100 82. 61 72 h 100 100 89. 30 7. 43 99. 35 -24- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) ----- - ------I 丨 订 — 丨 丨! Line-^11- (Please read the note on the back and then fill out this page) 1274757 A7 _____ B7_5, invention description (22) Example 7 uses rice chips as the starch source, purity after liquefaction, saccharification and TG isomerization 53. 19% isomalto-oligosaccharide with a total sugar content of 30% (w/v), as described in Example 1. Take 100 mL of this isomalt oligosaccharide aqueous solution, and add the yeast fermentation to remove the digestible sugar by the first method, that is, add 100 mL of the bottom layer of the brewer's yeast cells (the cells cultured for 24 hours, the cell number concentration is 1 • 6χ108 strain/mL), fermentation at 30 ° C, 80 rpm for 72 hours. The proportion of glucose, maltose, and maltotriose in the aqueous solution of isomalto-oligosaccharide was changed by time as shown in Fig. 6. The daily changes are shown in Table 7. Since the maltose-derived malt oligosaccharide contains a small amount of nitrogen source and other nutrients, the fermentation of the bottom beer yeast, whether or not it is in the wort, does not affect the survival and metabolic activity of the yeast, so glucose, maltose, and Maltose trisaccharides can be quickly digested. Figure 6 shows that glucose, maltose, and maltotriose can completely digest light at 24, 28, and 36, respectively. Therefore, according to the results of HPLC analysis, the obtained isomaltose is 100% high in total sugar. Isomalt oligosaccharides. (Please read the notes on the back and fill out this page) - Hey! ---Order--II--I--- Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing-25 - This paper scale applies China National Standard (CNS) A4 specification (210 X 297 mm) 1274757 A7 B7 V. DESCRIPTION OF THE INVENTION (23) Table 7 Time Glucose Consumption Maltose Consumption Maltose Trisaccharide Elimination of Total Sugar Immersed Maltose Oligosaccharide Rate (%) Rate (%) Amount (%) Amount (g/100mL) Purity (%) 24 h 100 97. 1 78. 5 48 h 100 100 96 72 h 100 100 100 14. 14 100 (Please read the notes on the back and fill out this page) #印部Intelligent Property Bureau employee consumption cooperative printing Example 8 Using rice chips as starch source, purity after liquefaction, saccharification and TG isomerization 53. 19% isomalto-oligosaccharide with a total sugar content of 30% (w/v), as described in Example 1. Diluted with distilled water 1:1, that is, an aqueous solution of isomalto-oligosaccharide having a total sugar weight concentration of 15% (w/v). 1 mL of this aqueous solution of isomalto-oligosaccharide was added to the yeast to remove the digestible sugar by the first method, that is, 100 mL of the bottom layer of the brewer's yeast (the cells cultured for 24 hours, cell number concentration) was added. For 1·6χ108 strain/mL), fermentation was carried out at 3 (TC, 80 rpm for 72 hours). The proportion of glucose, maltose, and maltotriose in the isomalt oligosaccharide aqueous solution was changed by time as shown in Fig. 7. The change of the day is shown in Table 8. This example is identical to the example 7 except that the total sugar weight concentration of the low-purity isomaltoligosaccharide aqueous solution containing rice starch is halved, and the result shows that in the thinner sugar liquid, yeast fermentation The rate is faster. As shown in Figure 7, glucose, maltose, and maltotriose can be used at the 16th, 20th, and 24th respectively. -26- This paper size is applicable to China National Standard (CNS) A4 specification (10) x 297 1) «III — Line 1274757 A7 B7 V. Inventive Note (24) All digested light, so according to the results of HPLC analysis, the obtained isomaltose is a high-purity isomal malt of 1% by weight of total sugar. Oligosaccharides. Another reason for the faster fermentation rate of this example and the seventh example is that the added bottom beer yeast is in the logarithmic phase when the activity is high, the bacteria cultured for 24 hours is in the log phase, and the yeast should be in the culture after 40 hours of cultivation. The stationary phase of the reproductive process. Table 8 Time glucose consumption rate (%) Maltose consumption rate (%) Maltotriose consumption rate (%) Total sugar amount by fermentation (g/100mL) Isomalt oligosaccharide purity (%) 24 h 100 100 100 48 h 100 100 100 72 h 100 100 100 7. 07 100 (Please read the note on the back and fill out this page.) Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative -27- This paper scale applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm)