TW574035B - Immunostimulating composition - Google Patents
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- TW574035B TW574035B TW89117246A TW89117246A TW574035B TW 574035 B TW574035 B TW 574035B TW 89117246 A TW89117246 A TW 89117246A TW 89117246 A TW89117246 A TW 89117246A TW 574035 B TW574035 B TW 574035B
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Landscapes
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- Chemical & Material Sciences (AREA)
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- Communicable Diseases (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Description
574035 五、發明説明(1 ) 本發明係有關於-種免疫刺激组成物及干擾素1生成 促進組成物者。 按,皮膚與黏膜係生命體與外界接觸之境界φ,特別是 腸道黏膜等消化道黏膜,更將接觸到許多騎、細菌、寄生蟲、 病原性抗原、食物抗原等異物。因此,此等皮膚及黏膜乃具有 防止該等異物侵入體内以保護生命體之免疫系統。免疫球蛋白 A(IgA)則為人所周知之免疫物質,具有防止前述異物進入之作 用。該J g A在細菌、病毒等的中和、抑制細菌附著於組織、抑 制食物抗原所引起的過敏反應等之上,扮演著極重要的角色。 而,存在於腸道的淋巴組織,即,集合淋巴結淋巴細胞則與該 IgA之產生有關。 消化道黏膜中之生命體防禦體系,即免疫機能,若降低 的話,則微生物將於消化道中固定下來並繁殖,致引起消化道 感染症、細菌性腹簿等,並發生發燒、唱吐、腹厲、腹痛等症 狀而’過度勞累、緊張等引起之食您不振症也會造成前述免 疫機能降低。 特別是幼兒、老人、體力衰弱者,因免疫力變弱,故對 ;兩原菌等的外部入钕’抵抗力低。所以,人們乃期望能特別對 這一人及刖述4化道感染症患者等,投與具有可促進之產 生(IgA產生亢進作用),並可將之維持於高含量值之作用的 物貝,尤其是能以飲料等食品之形態來投與之物質。 然而’具有該IgA產生充進作用,並可有效預防乃至治療 :肖化道免疫機能降低、細菌性消化道感染症等之藥物,目前則 尚未為人所知。而對細菌性腹瀉症,迄今便只有對脫水症部份 本紙張尺度適用巾國國家標準(CNS) A4規格(210X297公釐) (請先閲讀背面之注意事項再填寫本頁) 訂丨 -4- J/4U35 A7 ^----------- B7 五、發明説明(2 ) --— 進行輸液注A,以及投與抗生素而已。 因此本發明之目的係在提供一種新穎的消化道免疫刺 成物忒消化暹免疫刺激組成物不但可有效預防及治療前 迷消化道感染症等,並可特別以飲料等食品形態來提供。 本案t月人為達成上述目的,經過專心的研究,結果發 現-新事實,即:乳果募糖可對人體發揮預定之免疫賦活效 果而,藉投與該物,可治療及預防消化道感染症等;依此, 終完成本發明。 即,本發明提供了一種同時含有乳果寡糖及載體之免疫 刺激組成物者。 此外,本案發明人另外並發現一項新事實,即:藉由投 與前述組成物,可促進干擾素—7(IFN— 產生能。 所以,本發明亦提供了一種同時含有乳果募糖及載體之 IFN— γ生成促進組成物。 本發明組成物必須含有有效成分之乳果募糖(以下「LS」 稱之)之有效成分。在此,乳果寡糖係可以下述構造式表示之 A丟之物質,其化合物名為Q 一冷一 D — σ辰喃半乳糠基—(1 — 4)一0 — a —D—哌喃葡萄·糖基—(1 —2)一万—D一果糖呋喃 糖苷。 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) (請先閲讀背面之注意事項再填寫本頁) •、可丨
574035 ----- 五、發明説明(3
LS可依各種方法製造。該方法則可例舉出以下之例:如 曰本專利公報特公昭第57-58905號公報所揭示者,其係將產氣 氣#菌屬菌起源之果聚糖生成酶放至含有蔗糖及乳糖之溶液 内使之起作用之方法;及,如日本專利公開公報特開昭第 64 85090號A報所揭示者,則係將諸如獨特擲孢酵母等屬於擲 孢酵母屬之菌體及其處理液放至含有蔬糖及乳糖之溶液内使 之起作用之方法;以及,如日本專利公開公報特開平第2_35〇95 號公報所揭不者,係將Rahnella aquatilis等屬於屬之 菌體及其處理液放至含有蔗糖及乳糖之溶液内使之起作用之 方法。 本發明組成物可利用依照前述各方法所得之以Ls為主之 反應混合物及以其精製之LS精製品。 另外,LS為雙叉乳桿菌之腸内繁殖所必須 里要營養素 (糖源)。 本發明組成物中之LS的調和量,通常都為〇 5〜7〇^丨⑼ 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) (請先閲讀背面之注意事項再填寫本頁) 、^11 -6- 574035 A7 B7 五、發明説明(4 ) 之程度,但宜從5〜30g/l00g之程度範圍來選擇。 本發明之免疫刺激組成物及IFN— 了生成促進組成物只 要是可服用乃至可攝取,其形態並無特別之限制,可作成各種 形態,譬如塊狀、液狀、糖衆狀、粉末狀等。更具體之形態則 可例舉如下,# :液狀或粉狀之甜味料形態;清涼飲料、牛乳 飲料、碳酸飲料等飲料形態(保健飲料);麵包類、餅乾、糖果 等點心類形態;或其他的健康食品形態等之各種食品形態;以 及粕劑、散劑、懸濁劑、錠劑、起泡製劑等醫藥品形態。其中 尤以飲料形態、發泡劑形態等為理想之形態。 前述各種型態之本發明組成物,除有效成分之“外,可 配合其形態,添加適當之食品載體及製藥學上所容許之醫藥品 載脰而a周製之。食品載體係各種可食性添加劑,譬如增量劑、 甜味劑、其他的糖質、維他命類、香料、著色劑等。 更洋細的說,例如本發明組成物係食品形態時,考慮到 平均一天的LS攝取量(有效量)為之程度,且為使該組成 物可以該有效量而輕易的攝取,故將LS與食品載體共同配和 调製。食品形態之代表例,如飲料,一般來說,可調製成含有 有效成分之乳果寡糖〇·5〜70g/l〇〇ml,尤其是調製成含有 5〜30g/l〇〇ml乳果寡糖之水溶液形態。另外,該飲料宜使用適 當之pH調節劑乃至緩衝劑等,將之調整成pH4〇〜6.5之程度, 尤其是調至4.5〜6.0之程度。 在此,具代表性的pH調整劑乃至緩衝劑,可舉例如下, 即:檸檬酸、酒石酸、蘋果酸、乳酸、碳酸等之弱酸,及其鹽 類’譬如檸檬酸鈉、擰檬酸銨、酒石酸鈉、蘋果酸鈉、乳酸鈉、 本紙張尺度適用中國國家標準(CNS) A4规格(210X297公釐) (請先閲讀背面之注意事項再填寫本頁) -訂— 乳酸鈣、碳酸鈉、碳酸氮納等 ^ ^ 蝌酼風鈉也可作為前述pH調 “至緩衝劑來制。該㈣及其鹽類可單獨使用,亦可2 1 川幵用。其調和之比例則可依所得飲料能保持於前述適當 PH範圍内之範圍來作適宜之決 士曰 通$ ’約為組成物重量之2 重1%以下,但宜為〇 〇5〜〇 3重量%。 又’和-般的飲料等相同,前述飲料等食品形態之本發 明組成物中可添加調和各種糖質乃至甜味料。糖質可例舉如 下,即:葡萄糖、果糖等單糖類;麥芽糖、薦糖等二糖類;糊 精、壤糊精等多糖類(但LS除外);木糖醇、赤蘚醇、山梨糖醇 等糖醇類;及,糖醋等。甜味料則可舉例如下,即·天然甜味 料(果糖二苔A等斯替維亞屬(stevia)萃取物、了甜蛋白、甘草甜 素等)’以及合成甜咮料(糖精、阿斯巴甜等)。此等糖質乃至 甜味料之調和比例,通常約為所得飲料之15重量%以下,但宜 為13重量%以下。 進而’食品形態之本發明組成物依照需要,可添加調和 如以下1種或2種以上之添加劑。該添加劑包含有:譬如葡萄 袖、蘋果、橘子、檸檬、鳳梨、香蕉、梨子等各種果汁(濃縮 果汁、粉末果汁等亦可);維他命類及維生素原(棕櫚酸維生素 Α、雙長效維生素Bi、維生素I、鹽酸吡哆醇、維生素812、抗 環血酸鈉、菸驗酸醯胺、泛酸鈣、葉酸、維生素H、維生素D、 重酒石酸膽驗、維生素E、卜胡、蘿蔔素等水溶性及脂溶性維 他命類);香味料(檸檬香味、橘子香味、葡萄柚香味、香草精 等);氨基酸、核酸及其鹽類(谷氨酸、谷氨酸鈉、甘氨酸、丙 氨酸、天冬醯胺酸、天冬醯胺酸鈉、次黃苷酸等);食物纖維(聚 574035 A7 _______B7__ 五、發明説明(6 ) 右旋糖、果膠、蒼耳膠、阿拉伯膠、藻酸等);礦物質乃至微 量元素(氣化鈉、醋酸鈉、硫酸鎂、氯化鉀、氣化鎂、碳酸鎂、 氣化鈣、磷酸二鉀、磷酸一鈉、磷酸甘油鈣、檸檬酸第一鐵鈉、 檸檬酸鐵胺、檸檬酸鐵、硫酸錳、硫酸銅、碘化鈉、山梨酸鉀、 鋅、錳、銅、碘、鈷等)。 本發明組成物係混合前述各成分調製而成者。該調製方 法並無特別之限定,可同時混合所有的必要成分,又,油性成 分與水性成分兩者皆使用時,預先於適當之油劑内溶解油性成 分’就可用乳化劑將之與水溶性成分之水溶液乳化。該乳化操 作係依一般之方法,通常都使用如均勻混合器、高壓均勻混合 器等乳化分散機,可按照完全通過方式或循環方式實施。進 而’前述各成分之混合乃至乳化通常都在常溫下實施,但亦可 採用加溫操作。將如此調製而成之本發明組成物填充至適當容 器後’依照常用之方法,即藉由加熱滅菌、無菌過濾等消菌, 做成製品。 另外’本發明組成物也可調製成在使用前能先溶解分散 於水中之適當形態的發泡製劑,如錠劑、顆粒劑、散劑、膠囊 等。該發泡製劑之發泡成分係使用碳酸氫鈉及(或)碳酸鈉,以 及中和劑共同調製而成。此處之中和劑,可舉例如下,即:棒 棣酸、酒石酸、延明索酸、抗環血酸、乳酸、蘋果酸等。前述 發泡成分及中和劑之調和比例,通常宜從全組成物中之發泡成 分8〜60重量%及中和劑1〇〜70重量%之範圍來選擇。如此,發 泡製劑可按照需要進而使用預定量之碳酸鉀,並可依照一般的 方法來調製,譬如直接粉末壓縮法或乾式、或者濕式顆粒壓縮 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐)' -- -9- (請先閲讀背面之注意事項再填寫本頁)
574035 五、發明説明(7 法 本發明組成物之攝取乃至服用量係按照使用目的,或是 適用,物之適用者之年齡、性別、體重、疾病程度等來做適宜 之決定’並無特別之限定。本發明組成物係固劑時,—般來說, 平均一次服用之固劑大約含有1〜3〇g之有效成分,更佳者則是 將該固劑溶解於3〇〜2〇〇1水中 1之jc中再服用。另外,本發明組成 物係飲料等液劑形能0车 屏^ d心&、日才,取好平均一次服用大約3〇〜2〇〇ml。 本發明組成物之平均—天的攝取乃至服用量,其合適之攝取、 服用量約為其中含有此有效成分G.5〜心者,尤幻,為佳, 3〜20g則更佳。按照此攝取、服用量,前述關、飲料等可於 天内分為數次服用。 本發明組成物藉由其服用乃至攝取,可發揮免疫賦活及 圓-r產生能充進作用,且提高消化道中之生命體防紫體系 (免疫機能)’對消化道感染症等有顯著之預防及治療效果。 第1圖之圖表係表示實驗例2中對攝取本發明組成物之實 驗對象的顆粒性白血球部份所求測得之嗜食率。 第2圖之圖表係表示實驗例3中對被餵食本發明組成物之 實驗白老鼠的集合淋巴結.淋巴細胞之IFN — r產生量(pg/m" 所測得之結果。 接下來,為更詳細說明本發明,先舉出本發明組成物之 所進行之實驗例。又,各例中之份及%都是重量基準。 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐)
、tr— (請先閲讀背面之注意事項再填寫本頁) 10- 574035 A7 B7 五、發明説明(8 ) 實施例1〜6 將表1所載組成之各原料化合物投入水中、、曰人 τ此合、溶解,調 製成本發明組成物。並可於各實施例之組成物中淮一本μ ^ τ延一步调和適 當之香料及/或維他命類。又’各實施例係以水調和至全部重 量為 1 000ml。 表1 實施例No. 1 2 3 4 5 6 陽離子 (mEq/1) Na K+ Ca2+ Mg2 + 計 21 5 1 0.5 27.5 15 5 1 0.5 21.5 21 5 1 0.5 27.5 15 5 2 0.5 22.5 8 4 1 0.5 13.5 27 5 1 0.5 33.5 陰離子 (mEq/1) cr 檸檬酸離子(3-) 乳酸離子(-) 酒石酸離子(2_) 蘋果酸離子(2-) 計 16.5 10 1 0 0 27.5 10.5 10 1 0 0 21.5 16.5 8 1 1 1 27.5 10.5 10 2 0 0 22.5 6.5 4 1 1 1 13.5 17.5 11 1 2 2 33.5 果糖二苷A (mg/1) 80 75 83 73 70 85 糖分 果糖 (g/1) 葡萄糖 (g/1) 20 2 18 1 17 2 16 3 15 2 22 1 乳果募糖(g/1) 100 50 10 60 30 200 實施例7 混合下述成分(全部重量5g),並直接打錠進行調製(旋 劑),或秤量混合各成分予以分包(散劑),或是秤量混合各成 分後予以造粒並乾燥後,予以分包(散劑),而調製成各種製劑 形態。 LS55P 34% (採用LS時 18.7%) 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) (請先閲讀背面之注意事項再填寫本頁) .、可| -11 - 574035 A7 B7 五、發明説明(9 ) L —抗環血酸 21% L 一酒石酸 20% 甜味料 適量 碳酸氫納 21% 氣化納 適量 碳酸鉀 0.5% 香料·著色料 微量 合計 100% (請先閲讀背面之注意事項再填窝本頁) 又,該「LS5 5P」係含有LS5 5%之粉末。 實施例8〜10 和實施例7相同,調製含有下述成分之錠劑、散劑、及顆 粒劑形態之本發明組成物。 <實施例8處方> LS55P 40% (採用LS時 22%) L —抗環血酸 10% L —酒石酸 23% 甜味料 適量 碳酸氫鈉 22% 檸檬酸鈉 適量 碳酸鉀 0.4% 香料·著色料 微量 合計 100%(全部重量5g) <實施例9處方> 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) -12- 574035 A7 B7 發明説明(10 ) LS55P 40% (採用LS時 22%) L —抗環血酸 11% L —酒石酸 23% 甜味料 適量 碳酸氫鈉 22% 擰檬酸銨 0.8% 維生素b12 微量 檸檬酸鈉 微量 碳酸鉀 0.4% 香料·著色料 微量 合計 100%(全部重量4.6g) <實施例10處方〉 LS55P 40% (採用LS時 22%) L —酒石酸 29% 甜味料 適量 碳酸氫鈉 24% 檸檬酸鐵銨 3.6% 維生素b12 微量 碳酸钟 0,5% 香料·著色料 微量 合計 100%(全部重量4g) 實施例11〜1 8 (請先閲讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) -13- 574035
、發明説明(11 ) 與實施例7〜1〇相同,調製如下述表2所示之組成的發泡錠 劑形態之本發明組成物。 2 表 構成成分 _實施> ί列 No. 11 12 13 14 「15 16 17 18 LS55P (%) (採用LS時) 40 (22) 30 (16.5) 40 (22) 60 (33) 50 (27.5) 35 (19.3) 45 (24.8) 35 (19.3) L —抗環血酸 (〇/0) 11 16 10 8 10 10 10 13 _!^二酒石酸 (%) 23 23 23 13 19 20 20 25 味料 (%) 適量 適量 適量 適量 適量 適量 適量 適量 碳酸氫鈉 (%) 22 22 23 15 1 5 20 20 23 ϋ"酸鐵錄 (%) 0.8 0.8 1.0 0.8 0.8 0.7 檸檬酸第1鐵鈉(%) — — — — 一 1.2 __ 檸檬酸鐵 (%) - — — — 一 0.8 _ 酸鈉 (%) 適量 適量 適量 適量 適量 適量 適量 適量 全部重量 (g) 4.6 4.6 4.7 4.6 4.6 4.7 4.7 5.4 (請先閲讀背面之注意事項再填寫本頁) 實施例19〜25 混合下述表3所示之各成分(mg),並藉由直接壓縮法,調 製成可嚼食之錠劑(發泡錠)形態之本發明組成物。 表3 構成成分 實施例N 〇. 19 20 21 22 23 24 25 LS75P (採用LS時 (mg) mg) - — -' 700 (525) 3330 (2498) 600 (450) 350 (263) LS55P (採用LS時 (mg) mg) 1800 (990) 360Ό (1980) 4620 (2541) - — - — 糖酯 (mg) 40 80 841 20 80 15 7 聚右旋糖 (mg) 150 400 300 100 300 60 60 蔗糖 (mg) 100 200 200 20 200 80 20 維他命C (mg) 150 320 200 20 200 — — 橘子果汁粉末(mg) 80 150 — — 100 20 5 檸檬果汁粉末(mg) — — 84 40 — 10 5 酒石酸 (mg) — — — — 460 95 50 NaHC03 (mg) — — — — 500 100 50 K2CO, (mg) 30 40 40 40 30 8 8 香料·甜味料(mg) 適量 適量 適量 適量 適量 適量 適量 全部重量 (mg) 2400 4950 5580 1000 5200 1000 5000 又,上述「LS75P」係含有75%LS之粉末。 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) -14- 574035 A7 B7 五、發明説明(12 ) 實施例26〜34 混合下述表4所示之各成分,加水使全部重量為1〇〇〇nU, 調製成健康飲料形態之本發明成物。 表4 構成成分 (1000ml 中) /3 -胡蘿蔔素(mg) _ 實施例Ν ο . 26 — 27 28 29 30 31 32 33 34 3 5 10 15 1 2 30 5 3 聚右旋糖 (mg) 5 3 5 7 4 2 10 20 20 乳化劑 (mg) 油劑 (m g) 果糖 (mg) 一 6 10 20 30 5 6 20 15 8 100 90 80 120 50 50 200 70 "60~ — 15 10 — 15 15 15 5 10 檸檬酸 (mg) 200 400 100 300 50 20 一 — 酒石酸 (mg) - - 50 - 50 10 100 200 一 乳酸 (mg) ~ 一 — - 50 10 100 — 200 抗環血酸 (mg) 300 200 100 50 30 150 200 1000 50 維生素E (mg) 10 5 10 20 20 0.5 20 10 5 LS55P (g) (採用LS mg) 2 (1.1) 5 (2.75) 10 (5.5) 3 (1.65) 8 (4.4) 7 (3-85) 5 (2.75) 12 (6.6) 10 (5.5) 香料·調味料(mg) 適量 適量 適量 適量 適量 適量 適量 適量 適ί 並且,使用丙二醇脂肪酸酯作為乳化劑,使用紅花油作 為油劑。 實施例35〜45 混合下述表5所示之各成分,加水使全部重量為1 〇〇〇mh 調製成飲料形態之本發明組成物。 並且’表中之氣體谷積值係表不以溶解與溶液同體積之 二氧化碳氣體為1時所算出之二氧化碳含有量之指標,該數值 越大就表示含有的二氧化碳量越多。 本紙張尺度適用中國國家標準(CNS) A4规格(210X297公釐) (請先閲讀背面之注意事項再填寫本頁)
-15- 574035 五、發明説明(13
(請先閲讀背面之注意事项再填寫本頁} 訂— 實驗例1 此實驗係利用與安慰劑飲料作對照的雙盲試驗群間比較 法,來檢討飲料形態之本發明組成物之攝取對腸内細菌叢與免 疫忐所帶來之影響,如下述進行之。 1.實驗對象:成年男性28名 2·實驗設計··與安慰劑飲料.作對照之雙盲試驗群間比較法。 3 ·試驗群之構成: 本發明群:本發明飲料攝取群(n=14) 對照群:安慰劑飲料攝取群(n=14) 4.實驗物: 本發明飲料: 將乳果募糖(LS)5g溶解於50ml之水中,調製而成之溶液 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) 16- 574035 A7 ___ B7 五、發明説明(14 ) 瓶)。 安慰劑飲料: 將本發明飲料中之LS以蔗糖取代,依照同樣方法調製而 成者。 5. 用法·用量:i天】瓶(總共5〇ml) ’就寢前飲用。飲用期 間為6週。 6. 測定項目:腸内細菌叢及糞便中IgA濃度 7·糞便採集方法: 採集實驗對象於飲用前與飲用6週後之新鮮糞便。將採集曰 起床後之排泄物採集於可密閉之聚乙烯製袋中,密閉袋子時, 需注意不使聚乙烯製袋内有空氣殘留,其後,再將之放入聚丙 烯製之糞便採集容器内並冷藏。測量採集到之糞便的重量,均 勻混合後,進行腸内細菌叢及IgA量之測定。且,一部分糞便 以-80°C保存之,供測定IgA量之用。 8.腸内細菌叢測定法: 糞便細菌叢之檢索係以光岡等人之方法(光岡知足、腸内常 在菌叢、臨床檢查、23(4广320-334(1979))為依據,使用3種 非選擇培養基(BL洋菜、EG洋菜、胰蛋白酶解酪蛋白 「Trypticase」大丑血洋菜)作為分離用培養基,以及丨丨種選擇 培養基(BS洋菜、NBGT洋菜、ES洋菜、變法VS洋菜、新黴素 (Neomycin) Nagler 洋菜、變法 LBS 洋菜、DHL洋菜、TATAC 洋菜、PEES洋菜、馬鈴薯葡萄糖洋菜、GE洋菜)來實施。此等 各培養基組成和腸内常在細菌叢【光岡知足、臨床檢查、 23(4) : 320-334(1979)】所載者相同。 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公楚) (請先閲讀背面之注意事項再填寫本頁)
-17- 574035 A7 ---------B7 五、發明説明(15 ) ' "~' — 、里出大、力1,〇g之新鮮糞便,在厭氧性稀釋液(依照前述 文獻所記載之方法調製)9ml中懸濁後,在二氧化碳環境下以該 種稀釋液依次稀釋10倍,調製1〇1〜1〇8倍稀釋液。在bl洋菜及 EG洋菜培養基内塗抹〇 5ml之以前述方法所調製之、Μ?及 1〇8倍稀釋液’在胰蛋白酶解路蛋白大豆金洋菜培養基内塗抹 〇.5ml之以前述方法所調製之1〇5、1〇6及1〇7倍稀釋液,在其他 的培養基内塗抹G.5ml之以前述方法所調製之1〇1、ι〇3、ι〇5及 1〇7倍稀釋液。 -後BL洋菜、EG洋菜、BS洋菜、NBGT洋菜、ES洋菜、 變法VS洋菜、新黴素Nagler洋菜及變法lbs洋菜係放入厭氧熱 欠瓶中和一氧化碳置換後,利用鋼絲棉法以37°c進行48小時 之厭氧培養。胰蛋白酶料蛋白大豆血洋菜及瓶洋菜係们7 ^養24小時,❿,TATAC洋菜、卿S洋菜、馬鈴薯葡萄糖 洋菜及GE洋菜則同樣都培養48小時。 。養後測里各培養基上之菌落數,藉由顯微鏡進行形態學 上之觀察,對菌落性狀、革蘭氏染色性、有無芽胞、需氧發育 十生進行菌群層次之鑑定’並以平均lg糞便之菌數的對數值 (l〇glOCFU/g乾便)來表示鑑定之結果。 9.糞便中IgA濃度測定法 (1) 調整糞便中IgA測定用之樣本 凍結乾燥以-8(TC保存之糞便樣本,於〇 2g乾燥糞便加入 0.1M碳酸緩衝液(pH9.6)2.0ml且充分攪拌後,以15〇〇迴轉/分離 心10分鐘,將上層澄清液調整至ρΗ7·5,作為IgA測定用樣本。 (2) 測定糞便中IgA測定用樣本中的igA濃度 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公爱)
、可I (請先閲讀背面之注意事項再填寫本頁) -18- 574035 A7 "" -----— _ 幻 五、發明説明(16 ) 依路木等人(露木重雄、山崎省二、上村裕、木村昌伸、 川島拓司、上田雄幹等之利用酵素抗體法(elisa)測定老鼠膽 十及!細内谷物中之IgA抗體及總IgA ;雙又乳桿菌;2 ·· 9,13(1988))之方法,以EUSA測定IgA濃度。
IgAk體之測定係以如下方式實施。即使用源於非標示老 乳之k人體IgA(2 5" g/ml,200 // 1)作為抗原,將該抗原放入組 織培養盤Θ ’在4°C下靜置一夜。將前述抗原改為放入同量之 緩衝液’作為對照組織培養盤。 隔天,在吸收了溶液的各個組織培養盤内加入1%bsa溶液 (0.1M碳酸緩衝液,2〇〇/zl)以作為間隔質,並於37它下反應6〇 分鐘。接下來將糞便中IgA測定用樣本(4〇/zl)放入抗原處理及 對照組織培養盤内,並於37它下反應45分鐘。反應後,加入以 過氧化酶標不之源於老鼠之抗人體1§入(1〇〇〇倍稀釋,# 1), 並於37°C反應45分鐘。接著,加入SIGMA FAST 〇Dp (sigma 公司’ 200 //1)發色n分鐘後,以3M HCI(5〇ml)停止反應,測 定吸光度(490nm)。 樣本中之IgA濃度係以sIgA標準品(經純化之人類分泌性 5mg; Capple公司)做成檢量線,將由該檢量線算出之樣本中的 igA濃度換算成平均1§之乾燥糞便的濃度且表示之。 1〇·統計解析: 針對實驗物飲用前與飲用6週後之值,算出各群的平均值與 標準偏差。群間之比較係以反覆加味進行偏差分析,以危險率 5%以下為有效者。下述表6係顯示前述實驗之結果。 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) " '~一— -19- 574035 五、發明説明(17 飲料攝 η 平均土SD 飲用前 飲用6週後 安3劑飲料 η 平均士SD 飲用前 飲用6週後 糞便中IgA濃度 (mg/g乾重量) 腸内細菌數 0〇glOCFU/g 乾便) 腸内細菌 佔有率 (%) 14 3.9 士 3.2 6.9±5.4 14 5.6士7.0 4.4±3.1 13 9.5±〇.7 1〇.1士0.7 12 9.8±〇. 6 9.8±0.6 13 19.6 士 15.2 32.1 土 15.2 12 23.5士12.6 18.8 土 9.2 (請先閲讀背面之注意事項再填寫本頁) 由表6所示之結果可判斷出以下之事項。 1.本發明飲料攝取群藉由攝取本發明飲料而使雙叉桿菌屬 (Bifidobacterium)之菌數增加(9 5土〇 7—1〇1 土〇 7 1〇gl〇 CFU/g 乾便、成對t試驗;p=0.013)。相對於此,安慰性飲料攝取群則 看不出有變化(9.8^0.6-^9.8:1:0.6 log 10 CFU/g乾便)。因此, 可看出群間之雙又桿菌屬菌數之變化的有效差。 •本發明飲料攝取群藉由攝取本發明飲料而使雙叉桿菌屬之 佔有率亦增加(19.6±·15.2—32.1土15.2%、成對t試驗;Ρ = 〇·〇〇1)。 相對於此,安慰劑飲料攝取群則有降低之傾向(23 5土126〜 1 8·8±_9·2 /〇、成對t試驗;ρ = 〇丨1〇),亦可看出群間之雙叉桿菌 屬佔有率之變化的有效差。 3.本發明攝取群之糞便中IgA濃度有意義的上昇(3 9土3 2〜 69±5·4 m§/§乾重量、成對t試驗;P = 〇.〇14)。相對於此, 安慰劑飲料攝取群則無變化(5·6±7·〇— 4.4土3.1 mg/g乾重 量)’可看出群間之糞便中IgA濃度變化的有效差。 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) 訂· -20- 574035 A7 " --------B7____ 五、發明説明^—P ~ 由以上所述可知,實驗之本發明飲料係藉由攝取使糞便中 g A /辰度有思義的上昇。因此,可知本發明飲料能促進消化 道中之IgA產生能。 又’本案發明人並證實,藉由攝取和前述相同之LS,天 竺鼠的盲腸内之IgA濃度會顯著上昇。 實驗例2 此實驗係檢討健康者在攝取本發明組成物時,本發明組成 物(有效成分LS)對其免疫指標之影響,如下述實施之。 1·實驗對象: 以過去1個月内未服用抗生物質之健康成年人20人為對 象。 2 ·實驗物及攝取條件: 實驗物係本發明飲料,攝取條件係1天攝取本發明飲料(將 LS5g溶解於50ml水中之溶液)2次(上午1〇點及下午3點),連續 攝取4週。 3. 實驗方法: 讓貫驗對象攝取實驗物,在攝取開始日之當日(〇週)及攝 取4週後(4週後)各進行抽血。又,抽血及採便的前一日從晚間 9點後就不進食。實驗期間之飲食,避免暴飲暴食及攝取發酵 食品。並且,實驗期間也控制藥物的服用。 4. 評價項目及評價法: (1) 血清轉鐵球蛋白:利用濁度測定法測定。 (2) 顆粒性白血球部份之嗜食率: 在20 // 1之螢光珠粒(以2从m螢光標示之珠粒、型號 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) (請先閲讀背面之注意事項再填寫本頁) 、可| -21- 574035 A7 __ _B7_ 五、發明説明(19 )
Polyscience # 18604,Lot No. 425844)上添加丄 m j 之葡 萄糖媒體(將下述溶液A、溶液B及消毒水以1 ··丨:8之容量比 混合之物),以1 l〇〇〇rpm離心洗淨30分鐘。 葡萄糖媒體組成 A溶液:1.01M NaCl 0.31M CH3COONa 0.04M KC1 0.04M CaCl2 0.02M MgCl2 B溶液:0.07M 葡萄糖 將所得沉澱放入相同之葡萄糖媒體lml内懸濁,藉由超音波 處理得到一均勻的珠粒液。將上述螢光珠粒1〇〇 # 1添加至由實 驗對象肝素抽血所得之末梢血100//:[,於”艽下一邊分別振盪 〇、5、1〇及30分鐘,一邊培養之。反應結束後,加入將溶血劑 溶解於等量緩衝液(Facs溶解緩衝液,Bect0I1 Dickinson公 司Cat. No. 349202)所形成之溶液2m卜待紅血球完全溶解 後’並於冷藏後再測定。樣本以流動細胞計數器(C〇uLTER EPICS XL-MCL System II)·檢測。其時係設定柵門碰觸顆粒性 白血球部份時發出螢光之嗜食細胞可以波峰擷取者,而解析其 嗜食率。 (3)嗜酸性白血球:利用顯微鏡檢查法測定。 5 ·統計處理: 以平均值士S.D·來表示所有的資料,利用隨機區劃實驗法分 割後’再藉由Dunnett之多重比較進行檢定,判斷有效差。以 本紙張尺度適用中國國家標準(CNjS) A4規格(21〇χ297公釐) (請先閲讀背面之注意事項再填寫本頁) .訂· .f, -22- 574035 A7 --—-------------B7 五、發明説明(20 ) 危險率5%以下為有效水準。 6.結果 (1)血清轉鐵球蛋白: 以表7顯示所得之結果。 表7 ----- 攝取開始日(0週) 攝取4週後(4週後) 血中轉鐵球蛋白 (mg/dl) 285士30 295 士 34·》 Mean士S.D. ’ n=20(0週),17(4週) (請先閲讀背面之注意事項再填寫本頁) :P<0.05 vs Oif 由表7所示之結果可判斷以下之事項,即:攝取4週後之血清 轉球蛋白〉辰度與〇週相比,顯示出危險率5 %以下達顯著水準之 高含量。 (2) 顆粒性白血球部份之嗜食率: 結果如第1圖(縱軸:顆粒性白血球部份之嗜食率(%)、橫軸: 實驗期間(週))所示,顆粒性白血球部份之嗜食率的平均值 士S.D.(30分鐘反應值)在攝取〇週及攝取4週後,各為5〇8土1〇8% 及63.6 士 14.1%,攝取4週後與〇週相比,顯示出危險率以下 具顯示水準之高含量。 (3) 血中嗜酸性白血球: 結果如第8圖所示。 又’表8中連同時進行之血液一般檢查(白血球細胞數、嗜中 性白血球細胞數、嗜鹼性白血球細胞數、淋巴細胞數及單核白 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) -23- 574035 A7 ___B7 五、發明説明(21 ) 血球數)之結果也一併紀錄。 表8 攝取開始日(0週) 攝取4週後(4週後) 白血球細胞(計數/" 1) 5443±1604 5536±2370 嗜中性白血球 (%) 59.9±1 1.0 59.9±6.7 嗜鹼性白血球 (%) 1.0±0.7 1.0±0.5 嗜酸性白血球 (%) 4.7±3.9 3.2±2.0& 淋巴細胞 (%) 28.6±9.5 29.2 土 5.8 早核白血球 (% ) — 5.9 士 2.1 6.7 士 1.2 Mean士S.D·,n=2〇(〇週),17(4週) x· : P<0.05vs0週 (請先閲讀背面之注意事項再填寫本頁) 由該表所示之結果,顯示攝取4週後之血中嗜酸性白血球數 與0週相比為危險率5%以下達顯著水準之低含量。 7,考察 •、可| .籲- 由以上之結果可判斷,藉攝取本發明組成物,可增加構成感 染防禦關鍵所在之顆粒性白血球的嗜食能及血清轉鐵球蛋 白’因此’本發明組成物有可提高宿主之感染防禦能力的作 用。又’藉攝取本發明組成物,血液中之嗜酸性白血球數會降 低’由此,表示本發明組成物有抑制過敏反應之可能性。 實驗例3 此例係檢討以遺傳性糖尿病發病之動物模型作為本發明組 成物之餵食對象時對腸道免疫性之影響,如下述實施之。 1.實驗動物 使用4週齡之LETO家鼠及OLETF家鼠(各10隻)。又,OLETF 家鼠授精卵於1 994年8月24曰委託美國血型培養收集中心
(American Type Culture Collection)代為保管,編號 ATCC 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) -24- 574035 A7 B7 五、發明説明(22 )
No.7201 6。LETO家鼠則於大塚製藥工廠德島實驗動物管理室 以可分讓之狀態保存。 (請先閲讀背面之注意事項再填寫本頁) 2.飼養條件 在搬進動物後,進行為期1週之檢疫。檢疫期間係處於自由 攝取mf固形飼料【才y工y夕少酵母社(直譯:東洋酵母公司) 製】之條件下,且,飲用水係自由攝取自來水。係使用不銹鋼 製鐵絲網籠子飼養,以平均1個籠子1隻之飼養條件進行。明暗 周期則以7 : 00 — 19 : 00為明期。 實驗期間以 AIN76 組成(American Institue of Nutrition(1977) Report of the American Institute Nutrition ad hoc committee on standards for nutrition studies.,J. Nutr.,107 : 1340 — 1348)為依據,調製低纖 維(3%)、高脂肪(5%玉米油+10%牛油)之飼料為對照飼料,將 該對照飼料中之蔗糖/玉米澱粉混合物的一部份與LS置換,調 製成使飼料平均含有5%LS之本發明飼料(含有LS飼料)。各飼 料之組成如下述表9所示。 表9 成分(g/100g飼料) 對照飼料 本發明飼料 酪蛋白 20.00 20.00 玉米澱粉/蔗糖(3 : 10)- 57.20 52.20 玉米油 5.00 5.00 牛油 10.00 10.00 纖維素 3.00 3.00 AIN76礦物混合物 3.50 3.50 AIN76維他命混合物 1.00 1.00 DL—蛋氨酸 0.30 0.30 乳果寡糖(LS) — 5.00 kcal 444 424 3.實驗群 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) -25 - 574035
、發明説明(23 貝驗群(供予實驗之動物、飼料、動物數目)係如以下所示 君羊 動物 第1群LETO家鼠 第2群LETO家鼠 第3群 OLETF家鼠 第4群OLETF家鼠 4. 貫驗時間表 檢疫結束後開始以對照飼料飼養2週。將飼養後之各群平均 體重相同者的LETO家鼠及〇LETF家鼠各自分為如上表的群 組。分群後交換實驗飼料,開始飼養(7週大),以實驗飼料飼 養22週。 5. 解剖 為期22週之飼養結束後(29週大時),讓家鼠在乙二醚麻醉下 將之剖腹,由腹部下腔靜脈抽血後,將之放血宰殺。下腔靜脈 血以3000迴轉離心分離15分鐘後,將血清均分以—8〇t:保存。 宰殺後’小腸(各群3隻)係用於製造淋巴細胞。 6. 製造淋巴細胞 小腸集合淋巴結淋巴細胞之製造,係使用源於桿菌之蛋白 (使用商品名Dispase、Xs — 'J V力' —7^/、/么社(音譯· 林哥曼哈以姆公司)製造之「Dispase Π」),並以酵素法進行 之。集合淋巴細胞之分離係依照福瑞賈斯奇等人之方 (Fragaski,et al.’Jouraal of Immunological Methods,48,33-44(1989))。宰 後,在無菌狀態下摘取十二指腸上部至迴腸尾部。以消毒生理 食鹽水徹底洗淨後,用注射器將消毒生理食鹽水送至腸道内部 飼料 對照飼料 本發明飼料 對照飼料 本發明飼料 動物數目(隻) 10 10 10 10 酶 貝 法 木又 C請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) -26 - 574035 A7 -----— B7 _____ 五、發明説明(24 ) ^ — 清除内容物。於鋪在冰上之消毒紗布上切割集合淋巴結。將切 割後之集合淋巴結置於不完全RPMI164〇培養基(含MW/瓜丨健 大黴素【gentamycin】之RPMI164〇)中。集合淋巴結全部取出 後,在含有 DisPaSel.5mg/ml之培養基(joklik-m〇dified Mem) 液(酵素液)中,一邊以攪拌器攪拌,一邊於37t下進行酵素處 理40分鐘。回收開始分離之細胞,加入新的同樣的酵素液。此 刼作反覆3〜4次。將完全分離之細胞於4它下離心分離35〇g\ 分鐘,以PBS洗淨後,在預定的培養基内懸濁,以血球計算板 測定細胞數。 集中各群3隻的集合淋巴結,進行淋巴細胞之製造。 7 ·培養淋巴細胞 淋巴細胞係以RPMI1 640(含有2mM L —楚醯胺酸、5〇μΜΜ基 乙醇、100U/ml青黴素、i〇(^g/mi鏈黴素及1〇%FCS)將細胞數 調整至lx 106細胞/nU。將lml/井之淋巴細胞懸濁液置於細胞培 養用之24組織培養盤感光板(FalCON 3047)中,以ConA(伴 刀豆球蛋白A ;源於Canavalia ensiformis【(夕于于夕7>)音 譯:塔奇那塔豆】' シク、、フ社(音譯:西古碼公司)製)5〇pg/ml 作為促細胞分裂劑,在ConA之刺激下進行培養。 抗體產生能實驗係於37°C、5%C02下實施培養7天,細胞激 素產生能實驗亦於同樣條件下實施培養3天。培養脾臟的淋巴 細胞培養時,1樣本培養1井,而培養集合淋巴結淋巴細胞時, 1樣本培養3井。 培養結束後將感光板以16 0 0迴轉/分離心1 〇分鐘,回收上層 澄清液。回收之上層澄清液以—20°C凍結保存直到做各項目之 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) (請先閲讀背面之注意事項再填窝本頁) 訂— _纛, -27- 574035 A7 -------B7 _ 五、發明説明(25 ) " ' ' :~1 測定。 8 ·測定細胞激素 (請先閱讀背面之注意事項再填寫本頁} 淋巴細胞組織培養上層澄清液中之圓^係使用市售之 試劑套組,並以ELISA法測定。 9 ·統計處理 結果以平均值±偏差標準來表示。細胞激素濃度在進行各群 間之一元配置分散分析後,進行費歇爾防護咖㈣心
Protected LSD)之多重比較。而集合淋巴結細胞因為使用第中 各群之樣本而未進行統計處理。並以危險率不滿5%為顯著值。 10.結果 第2圖係表不所求得之集合淋巴結淋巴細胞培養中的IFN — T產生能之結果。 由此結果可知,本發明組成物飼料攝取群(2群及4群)與對照 飼料攝取群(1群集3群)相比,不管是LET〇家鼠及〇letf家 鼠,都顯示出其IFN — ^濃度具高含量值。 本紙張尺度適用中國國家標準(CNS) Α4規格(21〇χ297公釐) >28-
Claims (1)
- 574035申請專利範圍 •修正 { 第一號專利申請案情簡.„.2〇 卜種飲料型態之干擾素·,生成促進組成物,其中在每 i〇〇g組成物中,含有G.5_7Gg乳果寡糖並且含有載體。 2·如申請專利範圍第i項之飲料型態之干擾素1生成促進 組成物’其中乳果寡糖係供每天攝取量為Ug。 3 ·種I人料型態之消化道感染症之預防及治療劑之醫藥組 成物’其中在每啊組成物中,含有〇.5-7〇g乳果募糖並 且含有載體。 4.如申請專利範圍第3項之飲料型態之消化道感染症之預 防及/π療劑之醫藥組成物,其中乳果募糖係供每天攝取 量為l-3〇g。 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) 29
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US7087564B2 (en) * | 2004-03-05 | 2006-08-08 | Air Liquide America, L.P. | Acidic chemistry for post-CMP cleaning |
US9101160B2 (en) | 2005-11-23 | 2015-08-11 | The Coca-Cola Company | Condiments with high-potency sweetener |
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US8017168B2 (en) | 2006-11-02 | 2011-09-13 | The Coca-Cola Company | High-potency sweetener composition with rubisco protein, rubiscolin, rubiscolin derivatives, ace inhibitory peptides, and combinations thereof, and compositions sweetened therewith |
KR101615173B1 (ko) | 2008-05-02 | 2016-04-25 | 가부시기가이샤하야시바라 | β-프락토푸라노시드 결합을 갖는 비환원성 올리고당과 함께 환원성 당을 함유하는 시럽 상태 감미료의 착색 억제 방법과 그 이용 |
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