1331018 A7 B7 五、發明説明（· 1 .) 發明領域 本發明係有關一種含有益生菌之可食用產品且係有關一 種獲得該可食用產品之方法。 發明背景 益生性微生物係藉由改善寄主腸道微生物之平衡而有益 地影響寄主之微生物。一般咸信這些細菌抑制或影響腸道 中病原細菌之生長及/或代謝。吾人亦假定寄主免疫功能經 由益生性微生物活化。因此，已有許多不同的門徑將益生性 微生物包括在食品之中。 WO 98/10666 (SOCIETE DES PRODUITS NESTLE S.A.)揭示一種 製造含有活的益生性酸性細.菌之脫水食物組合物之方法， 其中將一食物組合物與一對氧敏感之益生性乳酸細菌培養 物共同地在熱空氣流下噴霧。 EP 0862863 (SOCIETE DES PRODUITS NESTLE S.A.)揭示一種 包含膠化之澱粉基質之乾燥即食穀物產品，其包括含有益 生性微生物之塗覆物或内銘。 US 4943437 (AB MEDIPHARM)揭示一種供給生物學活性物質 至基磔食物材料之方法，其中該生物學活性物質係經於惰 性載體中混成漿體，其中其係非水溶性的，以形成均質之懸 浮液，然後將該懸浮液施加至該基礎材料。 GB 2205476 (UNILEVER)揭示一種包含經細分之惰性麵粉載 體支持之細菌组合物，及一種具活力之微生物群之水性懸 浮液。然後此混合物經乾燥且合適於作為製備酸麵糰麵包 之乳酸細菌接種體。 -6 - 本纸張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 13310181331018 A7 B7 V. INSTRUCTIONS (1) Field of the Invention The present invention relates to an edible product containing probiotics and to a method of obtaining the edible product. BACKGROUND OF THE INVENTION Probiotic microorganisms beneficially affect host microorganisms by improving the balance of host gut microbes. It is generally believed that these bacteria inhibit or affect the growth and/or metabolism of pathogenic bacteria in the gut. We also assume that host immune function is activated by probiotic microorganisms. Therefore, there are many different pathways that include probiotic microorganisms in food. WO 98/10666 (SOCIETE DES PRODUITS NESTLE SA) discloses a method for producing a dehydrated food composition containing live probiotic acid bacteria, wherein a food composition is co-cultured with a pair of oxygen sensitive probiotic lactic acid bacteria cultures The ground is sprayed under a stream of hot air. EP 0862863 (SOCIETE DES PRODUITS NESTLE S.A.) discloses a dry ready-to-eat cereal product comprising a gelatinized starch matrix comprising a coating or a hint containing a probiotic microorganism. US 4,943,437 (AB MEDIPHARM) discloses a method of supplying a biologically active substance to a base food material, wherein the biologically active substance is mixed into a slurry in an inert carrier, wherein it is water-insoluble to form a homogeneous suspension The liquid is then applied to the base material. GB 2205476 (UNILEVER) discloses a bacterial composition comprising a finely divided inert flour carrier and an aqueous suspension of a viable microbial population. This mixture is then dried and suitably used as a lactic acid bacterial inoculum for preparing sourdough bread. -6 - This paper size applies to China National Standard (CNS) A4 specification (210X 297 mm) 1331018
發明説明（· 然而，將盈生性微生物（以下以“益生菌，，稱之）併入食品中 一伴隨淬多困難。首先要達成的一個目標是每天有適當之cfU (菌落形成單位）數目。若在該產品中之益生菌濃度不超過一 特定之閾值，則無法提供有益的作用。因此，以觀察到之有 政劑量之範圍是每人每天1〇9 為開端，且，假定消費者必 須在他/她每g攝取量之内攝取這些益生菌，則目標是在一 至二份内遞送此cfu量給消費者。 迄今，既有之門徑係利用經乾燥之益生菌，不論是其本身 或是與支持之载體物質一起乾燥。因此，在合適之培養基中 發酵後，益生菌通常經濃縮，例如藉由離心或過滤，且然後 經以噴霧乾燥法，流動床乾燥法或冷凍乾燥法乾燥。自乾燥 製私產生另一嚴重問題。即，益生菌以範圍在6〇，更常為9〇 至99%之Cfu蒙受實質之流失，其取決於所使用之技術，除非 採取特殊方法。不待說，這些乾燥步騾非常耗能。但高溫乾 燥法有其他缺點。其可破壞或傷害存在益生菌本身或存在 培植該等益生菌之發酵培養基中之代謝中間物。這類代謝 中間物會因此失去其有益之作用。同樣地，濃縮步驟之缺點 係流失存在發酵培養基中之代謝中間物。 然後藉由乾燥所獲得之粉末可施加至所需之食物產品。 根據以上引用之EP 0862863,例如，該經乾燥之益生菌經與液 態載體物質混合，其係油、水或蛋白質水解物。然後將此物 質喷灑至該食物產品上。 由於在單一餐食之内需要相對高數目之cfo而乾燥期間有 高流失量，故要有具有有效數Scfll之食物產品是一個難題。 本纸浪尺度適用中國國家標準(CNS) A4規格(210X297公釐) 1331018 A7 B7 五、發明説明（· 以上所引用之參考文獻中所提出之進一步問題係食物產品 上益生菌之長期安定性，即，該含有益生菌之食物產品必須 在週遭溫度下有貯存安定性。另一考量係該益生菌在胃及 腸道中之生存性。該益生菌必須足以抵抗胃中酸性環境及 抵抗膽酸以成功地移生於腸道中。此外，該包含益生菌之食 物產品對消費者而言必須是可口的。有需要於施加益生菌 至食物產品時不顯著影響其感官特性。 要獲得在口味，外觀及質地上與不含益生菌之相同產品 相較僅有很少或甚至沒有改變之含有益生菌之成品是個難 題。 本發明提出以上所述之問題。 發明概诚 因此，本發明提供一種包含益生菌之可食用產品，其中該 益生菌係新鮮地施加至該可食用產品。 在另一方面，本發明提供一種包含由益生菌產生之代謝 中間物之可食用產品，其中該代謝中間物係包含於自培植 於其中之益生菌分離出之發酵培養基。 相似地，根據本發明獲得包含益生菌之可食用產品之方 法包含藉由在液態培養基中發酵產生新鮮益生菌生質，並 直接地施加該新鮮生質至該可食用產品。 此外’在第四個方面，根據本發明獲得包含由益生菌產生 之代謝中間物之可食用產品之方法包含在液態培養基中培 植妓生菌，將該液態培養基與益生菌分離並直接施加該液 態培養基至可食用產品。 -8- 本纸張尺度適财@目家標準(CNS) Α4規格(21QX297公爱) 1331018 A7 B7 五、發明説明（..4· ·) 與合理預期相反的，頃發現的確可將衍生自發酵製程之 生質直接且新鮮地施加至可食用產品而不經高溫乾燥。以 此方式，獲得一含有益生菌之可食用產品，其具有優越之貯 存安定性且具有與不含益生菌之相似可食用產品之外觀及 感官特性相似之外觀及感官特性相似於。 此外，該可食用產品，若以預期或合理量食用，含有 量係足供有益的作用》 有利地’代謝中間物及微生物不再由於乾燥法及濃縮而 流失® 發明之詳細說明 在整份本申請書之說明中所表達之“可食用產品，，意指— 種例如由人類及/或由寵物如狗或描食用之產品。 關於本發明，“新鮮的益生菌’，或“新鮮施加之生質，，意指 在發酵製程後益生菌係未經乾燥的，例如藉由噴霧，流體化 床或冷凍乾燥。然而，不希望將“新鮮的益生菌，，瞭解為在特 定的時間限制範圍内施加該生質至可食用產品。無疑地可 能貯存該“新鮮的生質’，特定時間而無流失。若該生質亦可 經冷來特定時間並解;東而無實質之流失，則仍視之為新鮮 的《也有可能添加已知在應用之製程期間，例如在噴霧至可 食用產品上的期間，在貯存該產品期間亦且在該可食用產 品通過消化道期間增進，例如乳酸細菌，存活之保護劑至該 “新鮮的生質，、W0 98/1〇666提及某些具有這類作用之物質且 讀供先前技藝關於如何改善益生性微生物存活之大範圍 明細。儘管有這類添加物，仍可視該生質為“新鮮的生質”， -9- 本纸張尺度適用” S家標ijl(CNs)鐵格(_ 297公爱)------- I_ 1331018 A7 B7 五、發明説明（5 ) 因為無高溫乾燥製程。 為了本發明之目的，應瞭解詞彙“益生菌”，“益生性微生 物”或益生性生質係包括對於其寄主具有有益作用之任何微 生物，來自微生物之細胞内容物或代謝中間物。因此，可包 括酵母菌，黴菌及細菌βΕΡ 0862863列出某些目前已知之益 生菌實例。例如，可利用菌株詹森氏乳酸桿菌（CNCM I- 1225) 、乳酸雙歧桿菌（DSM20215)、嗜熱鏈球菌（ΤΗ4，克里斯金韓森 (Chr. Hansen)，丹麥）、或副酪蛋白乳酸桿菌（CNCM I- 2116)。克 里斯金韓森（Christian Hansen)生物系統 A/S (CHL)，10- Π Boge Α11έ，郵政信箱407，DK- 2970荷森（Horsholm)，丹麥，提供了對 不同益生性菌株之選擇。 為了本發明之目的，若無另行指示，則詞彙“益生菌’，更進 一步希望包括由該微生物在發酵製程期間產生之代謝中間 物。這些代謝中間物可經釋放至發酵培養基中或他們可貯 存於微生物内。大概這類代謝中間物是造成一特殊之益生 性微生物部分或所有有益作用之原因。 經意外地發現益生菌並非一定要經濃縮且無須在高溫下 乾燥而能直接且新鮮地施加至食物產品。因此本發明亦有 此大優點’其無傷害或甚至破壞由益生菌產生之代謝中間 物之效力之高溫處理^可略去濃縮步驟之事實之優點是不 會流失存在發酵培養基中之有效代謝中間物，例如由過濾 流失。 因此’經意外發現的確有可能提供一種含有益生菌之可 食用產品，其具有優越之貯存安定性且其具有外觀及感官 •10· 本紙蒗尺度適用中國囷家標準(CNS) A4規格(210X 297公爱） 1331018 A7 _ __B7_ 五、發明説明（~ *" —— 特性相似於一與其相似但不含益生菌之可食用產品之外觀 及感官特性。與所有的預期矛盾的，頃發現新鮮且直接應用 生f生生貧至可食用產品不引起或僅引起該含有益生菌之 成品極小之口味，外觀及質地改變。 與目前在益生菌食品技術中所認為相反的，也可嘴霧新 鮮生質至一乾燥之食物產品上，例如早餐穀物，而無須在應 用該生質前，期間或其後高溫乾燥之。在本發明目的範園内 ，僅須噴霧相對小量衍生自發酵製程之液體或漿體至乾燥 食物產品上。較佳地，持續發酵直到獲得相對高濃度之。 該食物產品會吸收大部分的水但該個別之食物產品並無實 質上增加之水活性。因為此理由，故亦不必使該包含益生菌 之可食用產品接受如文獻所建議之進一步乾燥製程或其他 處理。有趣地，迄今要添加大量益生菌且然後乾燥終產物仍 是難題。僅少數CfU在乾燥製程後仍正常存活。為了補償此流 失，必須施加高量益生菌，例如在水載體中。此接著使得必 須進行乾燥製程，特別是對希望最終是乾燥的產品。與此相 反地，本發明避免破壞性乾燥製程且因此不再需要施加高 量益生菌至可食用產品《其結果是，只需施加相對小量來自 發酵或包含益生菌之漿體或液體至可食用產品。當然，亦根 據本發明，仍可施加比較上些微大量之益生菌至可食用產 品以補償該產品貯存期間及通過消化道期間無可避免之流 失。 意外地，保存期限之研究顯示藉由直接應用生質所獲得 之食物產品上益生菌生存性極高。取決於所利用之益生性 -11 - 本紙張尺度適用中國國家標竿(CNS) A4規格(210X 297公釐) ------ 1331018 A7 B7 五、發明説明（7 ) 生物體’該益生菌保留其活性長達365天而無實質之流失。 此外，頃經意外發現施加至之食物產品益生菌顯示足以 抵抗胃及胃酸與膽酸之環境（活體外試驗），其取決於該益生 性生物體之菌種及菌株β 根據本發明所提供之可食用產品，可例如在應用該等益 生菌至可食用產品前添加至少一種保護劑至其中。 根據本發明之益生菌可藉由發酵獲得且可在發酵之後及 應用於可食用產品之前將其貯存防止益生菌c也實質流失之 時間及溫度，例如。顯然該生質在終止發酵或培植後，可經 貯存特定時間《在實驗中，不同益生菌之生質經於5t>c下貯 存4天而無可偵測到的流失。此外，對胃或膽酸（活體外試驗） 之抵抗亦不受貯存時間之影響。 為了進行本發明，可發酵益生菌直到終濃度達到每毫升 發酵，養基1〇6至5 x 1〇1〇，較佳地1〇7至3 χ 1〇1G，更佳地丨5 X⑼ 至心，甚至更佳地他至9.5 x 1〇9，特定言之2至9 χΐ〇9咖例 有可能，要施加至食物產品之益生菌係經濃縮至終濃产 為每毫升發酵培養基較佳地⑻至5χκ)1 二 1_5 X 108至1〇"，甚至更佳地1〇9至5 χ 1〇1Q咖例如。 可利用任何益生性微生物發酵。 m佩令贫 岡外J进目包含酵母菌，泰 =酵母菌屬，黴菌，較佳地_屬，細菌，較佳地乳_ ，雙歧桿菌屬’鍵球菌屬’腸球菌屬，及其混合物之^ 。'】如’可利用來自詹森氏乳酸桿菌、乳酸雙歧桿菌、嗜韵 1331018 A7 B7 五、發明説明（ 鏈球菌、或，副酪蛋白乳酸桿菌菌種之菌株。例如，若要生 產細菌性益生菌，菌株可選自乳酸桿菌屬、鏈球菌屬、雙歧 桿菌屬、類桿菌屬、梭絶桿菌屬、梭桿菌屬、蜂球菌屬、丙 酸桿菌屬、腸球制、乳球菌屬、„球菌屬、消化鍵球菌 屬、芽孢桿菌屬、片球菌屬、微球菌屬、明串球菌屬、 Weissella、氣球菌屬、歐諾球菌屬（〇en〇c〇ccus)。 因此，在本發明之-具體實施例中，一或多株益生性菌株 可選自包含乳酸雙歧桿菌（DSM2〇215)、詹森氏乳酸桿菌（ι· 1225 CNCM)、副酷蛋白乳酸桿菌（I_2116 CNCM)、嗜熱鍵球菌 (TH4，Chr· Hansen，DK)、其混合物、及亦包含其他益生性微 生物之混合物之群。 根據本發明，新鮮的益生菌生質添加至該可食用產品之 百分比可為以該可食用產品重量計之〇〇5至4%，較佳地〇1至 1.5%，最好 〇.2至 ι〇/。。 據此，施加至該可食用產品之益生菌終濃度可為丨〇6至ι 〇9 ’更佳地，1〇7至1〇8,最好2至8xl07 cfu/g該可食用產品總重。 根據該包含由益生菌產生之代謝中間物之可食用產品， 可直接施加該發酵培養基至該可食用產品。 根據本發明之方法，可保持持續之發酵直到達到終濃度 為每毫升發酵培養基106至5χ 1〇ω，較佳地1〇7至3?{ 1〇1G，更佳 地1_5 X 1〇7至1〇ι〇 ’甚至更佳地1〇8至9 5 X 1〇9，特定言之2至$ X 109個益生菌efu。 根據最終可食用產品所需之濃度及水活性(Aw)，本發明之 方法可包含，在施加該新鮮生質至可食用產品前，濃縮該生 -13· 本紙張尺_中_標兩s) A(摩297公登） 1331018 A7 B7 五、發明説明（ 11 質至終濃度為每毫升發酵培養基1〇7至1〇12，較佳地1〇8至5 χ 1〇 ，更佳地1.5 X 108至1011，甚至更佳地1〇9至5 χ 1〇1〇個cfil。 例如，該可食用產品在開始時及/或在保存期限期間之 係低於0.5。較佳地，其係低於0.4且更佳地其係小於〇 3。最好 ，該可食用產品之Aw係低於〇.2，例如，該可食用產品在保存 期限期間之Aw範圍係為〇_〇〇5至〇·3，或0.01至0.15。 該產品所具有之Aw取決於菌株在該特異條件下之存活能 力’其會隨不同之菌株而異。 較佳地，該可食用產品具有實質上限制自環境中攝取水 份之包裝《因此，該可食用產品包裝之〇2滲透率較佳地低於 4.2 ml/m2d’更佳地低於3.8 mi/m2d。同樣地，該可食用產品包 裝之水蒸氣傳送率（WVTR)較佳地低於3.5 g/m2d，更佳地低於3 g/m2d。凡熟習此項技藝者都能選擇具有這類特性之材料。例 如，該包裝可包含經共擠製之交聯定向延展之低密度聚乙 締（LDPE)。這些袋子可經密封，例如經熱封。 .如上特徵之包裝之目的係為在該可食用產品保存期限期 間维持較佳之八…值》該產品之保存期限可長達6個月，較佳 地長達12個月，更佳地長達18個月且最好長達兩年。 在另一具體實施例中，該方法可進一步包含，在發酵後， 將該新鮮生質貯存於防止益生菌efu實質流失之時間及溫度 在本發明之另一具體實施例中，該方法可進_步包含，在 產生新鮮益生菌生質之前，期間或之後，添加至少一^保護 劑至發酵培養基中或至該新鮮的益生性生質，例如。 -14 1331018 A7 _______B7_ 五、發明説明（10 ) 根據本發明之發酵製程可保持持續進行6小時至3天，較佳 地6至20小時’更佳地7至π小時，取決於所利用之益生性微 生物菌株。 根據本發明之方法’個別之包含益生菌可食用產品可利 用同一株或多株上述菌株。 有可能添加至該可食用產品之新鮮的益生菌生質百分比 為以該可食用產品重量計之〇 〇5至4〇/〇，較佳地〇 ^至i 5〇/〇，最 好0.2至1%。 因此，根據本發明之一具體實施例，施加至該可食用產品 之益生菌終濃度可為1〇6至109，更佳地，1〇7至1〇8，最好2至8 χ 1〇7，特定言之5 X 1〇7 Cfii/g可食用產品。 有可能，雖然非必要，濃縮該新鮮地衍生自發酵製程之生 質。例如，這類濃縮之達成可藉由離心或過濾。濃縮之程度 能使正確地於每克可食用產品投下cfU量。該濃縮亦可考慮 後續食物產品保存期限期間或通過消化道期間之流失。 可藉由喷霧或另外施加位經濃縮或經相對小量濃縮之生質 至可食用產品避免向溫乾燥製程，以使整體產品之水活性 不斷然增加。由於“吸收性乾燥，，故不須高溫乾燥製程；該 已乾燥（食物產品伴隨益生性生質迅速吸收水份且該水份 包含於該盈生性生質中。在應用之製程期間暴露於室溫係 足以防止終產品水活性之決定性増加。 右该生質經濃縮，不必丟棄由其獲得之上清液。在發酵益 生菌後培養基通常含有與益生菌本身相似有益作用之代謝 中間物。因此，在濃縮生質後該培養基之上清液亦可施加至 • 15- 本紙浪尺度it用中S目轉竿(CNS) A4規格(21G X 297公爱) ----- 1331018 五 、發明說明（ 11 可食用產品。 。為進行本發明方法，可利用所有種類之起始可食用產品。 I以益生菌富化人類食物及飲料及寵物之食&。當然，每個 i養配万之所有目的也都可提供益生菌。存在有極多種營 方例如用於運動者或運動員，用於有特殊營養需求的 亡如斜特定的天然食物組份過敏的人或有胃腸病症的人等 寺例如，亦可施加益生菌於巧克力或其他甜食◊事實上， =有種類之經擠製或經烹煮的或經其他製備之食物產品均 =Z、：<益生菌。例如，可利用乾燥產品，如乾燥之寵物食品 他乾燥之食物產品，像例如粉末，麵粉，牛奶或穀物粉 :又物薄片°可利用益生菌施加於所有種類之早餐穀物。亦 :以益生菌嘴霧可食用產品之組份，成分或原料。例如，一 或多主要包含澱粉材料之經烹煮穀物基質顆粒係合適的。 =烹煮的穀物基質之顆粒可為任何凡熟習此項技藝者所知 例如薄片穀物、經撕碎的全穀類、經㈣及其他經撕碎 的穀物:捲穀物，經膨化槍膨化之穀類，經烤箱膨化之穀物 榀擠氣之:k路化槍膨化穀物，薄片及/或經烹煮的擠製穀 ’經擠製〈發腺縠物，經烘培之早餐穀物，經壓縮之薄片 發脹穀物’經烘焙之早餐穀物，經壓縮之薄片比司吉(biscuits) 物料之製備可藉由以汁液烹煮穀物粗粒或毅粒，由所 又V U煮图塊形成小丸n培烤並可以糖塗覆他們 其他生產益生性生質之方法係在此項技藝中為人所熟知 。通常’利用有特別裝備之發酵單元或槽。雖然，理論上即 -16- 本-錄尺纽財關家料 1331018Disclosure of the Invention (· However, the incorporation of surplus microorganisms (hereinafter referred to as "probiotics,") into foods is accompanied by difficulty in quenching. One of the first goals to be achieved is to have an appropriate number of cfU (colony forming units) per day. If the concentration of probiotics in the product does not exceed a certain threshold, it will not provide a beneficial effect. Therefore, the range of observed political doses is 1〇9 per person per day, and it is assumed that the consumer must Ingesting these probiotics within his/her intake per g, the goal is to deliver this amount of cfu to the consumer in one to two servings. To date, the existing doorway utilizes dried probiotics, either by themselves or It is dried with the supporting carrier material. Therefore, after fermentation in a suitable medium, the probiotics are usually concentrated, for example by centrifugation or filtration, and then dried by spray drying, fluid bed drying or freeze drying. Another serious problem arises from drying and drying. That is, probiotics suffer a substantial loss of Cfu ranging from 6〇 to more than 9〇 to 99%, depending on the technology used. No special methods are used. Needless to say, these drying steps are very energy intensive. However, the high temperature drying method has other disadvantages, which can destroy or damage the presence of probiotic bacteria themselves or the presence of metabolic intermediates in the fermentation medium in which the probiotics are cultivated. Metabolic intermediates thus lose their beneficial effects. Similarly, the disadvantage of the concentration step is the loss of the metabolic intermediate present in the fermentation medium. The powder obtained by drying can then be applied to the desired food product. EP 0 862 863, for example, the dried probiotic is mixed with a liquid carrier material which is an oil, water or protein hydrolysate. This material is then sprayed onto the food product. Since a relative need is required within a single meal A high number of cfo and high loss during drying, so it is a problem to have a food product with a valid number of Scfll. This paper wave scale is applicable to China National Standard (CNS) A4 specification (210X297 mm) 1331018 A7 B7 V. Invention Note (· The further questions raised in the references cited above are the long-term stability of probiotics on food products, The food product containing the probiotic must have storage stability at ambient temperature. Another consideration is the viability of the probiotic in the stomach and intestine. The probiotic must be sufficient to resist the acidic environment in the stomach and resist bile acid for success. The food is transferred to the intestines. In addition, the food product containing the probiotic must be palatable to the consumer. It is necessary to apply the probiotic to the food product without significantly affecting its sensory properties. To obtain the taste, appearance and It is a problem to have a finished product containing probiotics with little or no change in texture compared to the same product without probiotics. The present invention addresses the above-mentioned problems. SUMMARY OF THE INVENTION Accordingly, the present invention provides a probiotic containing An edible product, wherein the probiotic strain is freshly applied to the edible product. In another aspect, the present invention provides an edible product comprising a metabolic intermediate produced by a probiotic, wherein the metabolic intermediate is included in A fermentation medium isolated from probiotics cultured therein. Similarly, a method of obtaining an edible product comprising a probiotic according to the present invention comprises producing fresh probiotic biomass by fermentation in a liquid medium and directly applying the fresh biomass to the edible product. Further, in a fourth aspect, a method for obtaining an edible product comprising a metabolic intermediate produced by a probiotic according to the present invention comprises cultivating a bacterium in a liquid medium, separating the liquid medium from the probiotic and directly applying the liquid medium To edible products. -8- This paper scale is suitable for the price @目家标准(CNS) Α4 specification (21QX297 public) 1331018 A7 B7 V. Invention description (..4· ·) Contrary to reasonable expectations, it is indeed derived from The fermentation process is applied directly and freshly to the edible product without drying at high temperatures. In this manner, an edible product containing probiotics having superior storage stability and having similar appearance and organoleptic properties similar to those of a similar edible product without probiotics is obtained. In addition, the edible product, if consumed in an expected or reasonable amount, is sufficient for beneficial effects. Advantageously, 'metabolic intermediates and microorganisms are no longer lost due to drying and concentration.>> Detailed description of the invention The term "edible product" as used in the description of the application means, for example, a product which is consumed by humans and/or by a pet such as a dog or a product. Regarding the present invention, "fresh probiotics", or "freshly applied" Biomass, meaning that the probiotic strain is not dried after the fermentation process, such as by spraying, fluidized bed or freeze-drying. However, it is not desirable to have "fresh probiotics," as defined in a specific time limit The biomass is applied to the edible product. Undoubtedly it is possible to store the "fresh biomass" for a specific period of time without loss. If the biomass can be cooled down for a specific period of time, and if there is no substantial loss in the east, it is still considered fresh. It is also possible to add It is known that during the application process, for example during spraying onto an edible product, during storage of the product and during the passage of the edible product through the digestive tract, for example lactic acid bacteria, a protective agent for survival to the "fresh" Biomass, W0 98/1〇666 refers to certain substances having such effects and is read in the prior art for a wide range of details on how to improve the survival of probiotic microorganisms. Despite the presence of such additives, the raw material can be regarded as "fresh biomass", -9- This paper scale applies to "S standard ijl (CNs) iron grid (_ 297 public) ------ - I_ 1331018 A7 B7 V. INSTRUCTIONS (5) Because there is no high temperature drying process. For the purposes of the present invention, it should be understood that the term "probiotics", "probiotic microorganisms" or probiotic germplasm systems have beneficial effects on their host. Any microorganism, derived from the cellular contents or metabolic intermediates of microorganisms. Therefore, yeast, mold and bacteria βΕΡ 0862863 may be listed as examples of some currently known probiotics. For example, the strain Z. johnsonii (CNCM) may be utilized. I-1225), Bifidobacterium lactis (DSM20215), Streptococcus thermophilus (ΤΗ4, Chr. Hansen, Denmark), or Lactobacillus paracasei (CNCM I-2116). Kriskin Christian Hansen Biosystems A/S (CHL), 10- Π Boge Α 11έ, PO Box 407, DK-2970 Horsholm, Denmark, offers a choice of different probiotic strains. Purpose, if not otherwise indicated, The term "probiotics" is further intended to include metabolic intermediates produced by the microorganism during the fermentation process. These metabolic intermediates can be released into the fermentation medium or they can be stored in the microorganism. Probably such metabolic intermediates are responsible for some or all of the beneficial effects of a particular probiotic microorganism. Surprisingly, probiotics have not been found to be concentrated and can be applied directly and freshly to food products without drying at elevated temperatures. Therefore, the present invention also has the great advantage that it has no harm or even destroys the efficacy of the metabolic intermediate produced by the probiotics. The advantage of the fact that the concentration step can be omitted is that it does not lose the effective metabolism in the fermentation medium. Things, such as being lost by filtration. Therefore, it has been found that it is possible to provide an edible product containing probiotics with superior storage stability and its appearance and sensory. 10· This paper size applies to China National Standard (CNS) A4 specification (210X 297) Public Love) 1331018 A7 _ __B7_ V. INSTRUCTIONS (~ *" —— The appearance and sensory characteristics of an edible product similar to a similar but not probiotic. All of the expected contradictions are found to be fresh and Direct application of raw foods to edible products does not cause or cause only the taste of the finished probiotics, the appearance and texture changes. Contrary to what is currently believed in probiotic food technology, it can also be used to produce fresh raw materials. To a dry food product, such as breakfast cereal, without the need to dry at a high temperature before, during or after the application of the biomass. In the scope of the present invention, it is only necessary to spray a relatively small amount of liquid or pulp derived from the fermentation process. Body to dry food product. Preferably, the fermentation is continued until a relatively high concentration is obtained. The food product absorbs most of the water but the individual The food product does not have substantially increased water activity. For this reason, it is not necessary to subject the edible product containing the probiotic to a further drying process or other treatment as suggested by the literature. Interestingly, a large number of probiotics have been added to date and The drying of the final product is then still a problem. Only a few CfUs survive normally after the drying process. To compensate for this loss, high amounts of probiotics must be applied, for example in water carriers. This then makes it necessary to carry out the drying process, especially for the hope In contrast, the present invention avoids a destructive drying process and therefore eliminates the need to apply high amounts of probiotics to the edible product. As a result, it is only necessary to apply a relatively small amount of pulp from the fermentation or containing the probiotic bacteria. Body or liquid to an edible product. Of course, according to the present invention, a relatively small amount of probiotics can still be applied to the edible product to compensate for the inevitable loss of the product during storage and through the digestive tract. Study of the term shows probiotic survival on food products obtained by direct application of raw materials Depends on the probiotics used -11 - This paper scale applies to China National Standard (CNS) A4 specification (210X 297 mm) ------ 1331018 A7 B7 V. Description of invention (7) Organisms' Probiotics retain their activity for up to 365 days without substantial loss. In addition, the probiotics applied to the food product by accident have been shown to be resistant to the stomach and stomach acid and bile acid environment (in vitro test), depending on the probiotic Strain and Strain of Sexual Organisms According to the edible product provided by the present invention, at least one protective agent can be added thereto, for example, before applying the probiotics to the edible product. The probiotic according to the present invention can be fermented by the present invention. It is obtained and can be stored after fermentation and before being applied to the edible product to prevent the time and temperature at which the probiotic c is also substantially lost, for example. Obviously, after the termination of fermentation or cultivation, the biomass can be stored for a specific time. In the experiment, the biomass of different probiotics was stored under 5t>c for 4 days without detectable loss. In addition, resistance to gastric or bile acids (in vitro tests) is also unaffected by storage time. For carrying out the invention, the probiotics can be fermented until the final concentration reaches a fermentation rate of from 1 to 6 x 5 x 1 〇 1 〇, preferably from 1 〇 7 to 3 χ 1 〇 1 G, more preferably X 5 X (9) to the final concentration. Heart, even better, he is 9.5 x 1〇9, specifically 2 to 9 χΐ〇9 coffee case is possible, the probiotics to be applied to the food product are concentrated to the final concentration for each milliliter of fermentation medium. Ground (8) to 5χκ)1 2 1_5 X 108 to 1〇", even better 1〇9 to 5 χ 1〇1Q coffee, for example. Any probiotic microbial fermentation can be utilized. m 佩 贫 贫 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 包含 , 包含 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , The mixture ^. '] such as 'can be used from the strain of Lactobacillus, Bifidobacterium lactis, Rhythm 1331018 A7 B7 V, invention description (Streptococcus, or, casein Lactobacillus strains. For example, to produce bacterial Probiotics, the strain may be selected from the group consisting of Lactobacillus, Streptococcus, Bifidobacterium, Bacteroides, Fusarium, Fusobacterium, Neisseria, Propionibacterium, Enterococcus, Lactococcus, „Citrus, Digestive genus, Bacillus, Pediococcus, Micrococcus, Streptococcus, Weissella, Balloon genus, Eucalyptus (〇en〇c〇ccus). Therefore, in the present invention In a specific embodiment, the one or more probiotic strains may be selected from the group consisting of Bifidobacterium lactis (DSM2〇215), Lactobacillus johnsonii (M. 1225 CNCM), Lactobacillus paracasei (I_2116 CNCM), A population of Streptococcus thermophilus (TH4, Chr. Hansen, DK), a mixture thereof, and also a mixture of other probiotic microorganisms. According to the present invention, the percentage of fresh probiotic biomass added to the edible product may be The edible product is heavy 〇〇 5 to 4%, preferably 〇1 to 1.5%, preferably 〇.2 to ι〇/. Accordingly, the final concentration of probiotics applied to the edible product may be 丨〇6 to ι 〇9 'better, 1〇7 to 1〇8, preferably 2 to 8xl07 cfu/g of total weight of the edible product. According to the edible product containing the metabolic intermediate produced by the probiotic, it can be directly applied The fermentation medium is passed to the edible product. According to the method of the present invention, continuous fermentation can be maintained until a final concentration of 106 to 5 χ 1 〇 ω, preferably 1 〇 7 to 3 { 1 〇 1 G, per minute of the fermentation medium is reached. More preferably 1_5 X 1〇7 to 1〇ι〇' or even better 1〇8 to 9 5 X 1〇9, specifically 2 to $ X 109 probiotics efu. According to the final edible product Concentration and water activity (Aw), the method of the present invention may comprise, prior to applying the fresh biomass to the edible product, concentrating the raw-13. The paper ruler _ medium _ standard two s) A (Motor 297) 1331018 A7 B7 V. INSTRUCTIONS (11 The final concentration is 11〇1 to 1〇12 per ml of fermentation medium, preferably 1〇8 to 5χ1〇, more preferably 1.5×108至1011, or even more Preferably, the edible product has a cfil of less than 0.5 at the beginning and/or during the shelf life. Preferably, it is less than 0.4 and more preferably It is less than 〇3. Preferably, the edible product has an Aw of less than 〇.2, for example, the Aw range of the edible product during the shelf life is 〇_〇〇5 to 〇·3, or 0.01 to 0.15. The Aw of this product depends on the viability of the strain under this specific condition', which will vary with different strains. Preferably, the comestible product has a package that substantially limits the intake of moisture from the environment. Thus, the 〇2 permeability of the edible product package is preferably less than 4.2 ml/m2d', preferably less than 3.8 mi. /m2d. Similarly, the water vapor transmission rate (WVTR) of the edible product package is preferably less than 3.5 g/m2d, more preferably less than 3 g/m2d. Anyone who is familiar with this skill can choose materials with such characteristics. For example, the package may comprise a cross-linked, directionally stretched low density polyethylene (LDPE) that is coextruded. These bags can be sealed, for example by heat sealing. The purpose of the above-described packaging is to maintain a better value during the shelf life of the edible product. The shelf life of the product can be as long as 6 months, preferably as long as 12 months, preferably as long as 18 months and preferably up to two years. In another embodiment, the method may further comprise, after the fermentation, storing the fresh biomass in a time and temperature for preventing substantial loss of the probiotic efu. In another embodiment of the invention, the method is The step comprises, prior to, during or after the production of the fresh probiotic biomass, adding at least one protectant to the fermentation medium or to the fresh probiotic biomass, for example. -14 1331018 A7 _______B7_ V. INSTRUCTION DESCRIPTION (10) The fermentation process according to the present invention can be maintained for 6 hours to 3 days, preferably 6 to 20 hours, more preferably 7 to π hours, depending on the probiotics utilized. Sexual microbial strain. The method according to the present invention 'individually containing the probiotic edible product can use the same strain or a plurality of the above strains. The percentage of fresh probiotics that may be added to the edible product is 〇〇5 to 4〇/〇, preferably 〇^ to i5〇/〇, preferably 0.2 to the weight of the edible product. 1%. Thus, in accordance with an embodiment of the present invention, the final concentration of probiotics applied to the edible product may range from 1 to 6 to 109, more preferably from 1 to 7 to 1, and preferably from 2 to 8 to 1 . 7, specific words 5 X 1〇7 Cfii / g edible products. It is possible, if not necessary, to concentrate the freshly derived biomass from the fermentation process. For example, such concentration can be achieved by centrifugation or filtration. The degree of concentration enables the correct amount of cfU to be administered per gram of edible product. This concentration can also be considered during the shelf life of the subsequent food product or during the digestive tract. The water-drying process can be avoided by spraying or otherwise applying a concentrated or relatively small amount of concentrated biomass to the edible product to continuously increase the water activity of the overall product. Due to "absorbent drying, there is no need for a high temperature drying process; this is already dry (the food product is accompanied by probiotic biomass to rapidly absorb moisture and the moisture is contained in the surplus biomass. Exposure to the chamber during the application process) The warming system is sufficient to prevent the decisive addition of the water activity of the final product. The right biomass is concentrated and the supernatant is not discarded. The medium after fermentation of the probiotic usually contains a metabolic intermediate similar to the beneficial effect of the probiotic itself. After concentrating the biomass, the supernatant above the medium can also be applied to the - 15 paper wave scale. It is used in the S-eye transfer (CNS) A4 specification (21G X 297 public) ----- 1331018 V. Invention Description (11 edible products. For the method of the invention, all kinds of starting edible products can be utilized. I probiotics enrich human food and beverages and pet food & of course, each i raises 10,000 Probiotics are also available for all purposes. There are many types of camps, such as athletes or athletes, for people with special nutritional needs who are allergic to specific natural food components or have gastrointestinal conditions. For example, people can also apply probiotics to chocolate or other sweets. In fact, there are types of extruded or cooked or otherwise prepared food products = Z,: < Probiotics. For example, Dry products such as dry pet foods such as powders, flours, milk or cereal flours can be used, such as powders, flakes, and probiotics can be applied to all kinds of breakfast cereals. Also: probiotic fungus A component, ingredient or ingredient of an edible product. For example, one or more cooked cereal base granules comprising primarily a starch material are suitable. = The granules of the cooked cereal substrate can be known to those skilled in the art. For example, flakes of cereals, shredded whole grains, (4) and other shredded cereals: rolled cereals, cereals puffed with puffing guns, cereals smashed by ovens, kneaded, puffed cereals, flakes and / or cooked extruded valley 'squeezed <fat glands, baked breakfast cereals, compressed flakes swelled cereals' baked breakfast cereals, compressed flakes biscuits materials It can be prepared by cooking the grain coarse grains or the granules with juice, forming the pellets by the VU boiled tablets, and baking the sugar and coating them with other methods for producing probiotic raw materials. Well-known. Usually 'utilize the fermentation unit or tank with special equipment. Although, in theory, the -16- Ben-recorded Newton Guanjia material 1331018
使是包含培養基之無菌槽都適於培植微生物。培養基組合 物义選擇係根據特定的益生性菌株之特殊偏好 。對於一特 殊盈生性菌株之最適培養基組合物—般而言係與購自供應 商之益生性菌（朊)生物體一起供應。在完成發酵後，可直接 將該生質施加至可食用產品。也有可能將其貯存一特定時 間而不改變其應用於可食用產品之適合性。特別是若運輸 該益生性生質至該可食用產品之生產處是強制性的，則該 益生性生質亦可經慣用之冷凍，以預防益生性〇加之流失。 在施加該生質至可食用產品之前，可濃縮該生質。該濃縮 步驟，雖非強制性的，但例如若即使些微增加之水含量在終 產物都必須避免，則是適當的。例如，若該產品上益生菌之 終濃度必須要特別高，即使因為僅在小量之單一份該可食 用產品中就必須包含足夠數目之cfu，即使為了其他理由都 可亦可進行濃縮。該濃縮方法亦在此項技藝中為人所熟知 的般而言，可選擇之方法係過濾或離心。 -最後，將該益生性生質，不論是否經濃縮，施加至該可食 用產品。此應用之進行可根據一般塗覆食物產品之規則。例 如，該生質之施加可在當在輸送帶上運輸該產品時，或者在 塗覆鼓輪中。在噴霧系統之設計上可有數種選擇，由摺疊管 到旋轉盤。某些產品適於在塗覆鼓輪中處理，例如在旋轉二 豉輪中。該塗覆鼓輪可同時供作混合機及將穀物暴露於噴 霧下之機構。可利用商業之兩相（空氣/液體）噴嘴將生質噴灑 於旋轉中之穀物上般而言，可利用與塗覆維生素溶液相 同之噴霧系統。這些技術係在此項技藝中為人所熟知。 17-The sterile tank containing the medium is suitable for cultivating microorganisms. Media Combinations The choice of sense is based on the specific preferences of a particular probiotic strain. The optimum medium composition for a particular probiotic strain is generally supplied with a probiotic bacteria (朊) organism purchased from a supplier. After the fermentation is completed, the biomass can be directly applied to the edible product. It is also possible to store it for a specific period of time without changing its suitability for use in edible products. In particular, if it is compulsory to transport the probiotic raw material to the production site of the edible product, the probiotic raw material may also be frozen by conventional use to prevent loss of probiotics. The biomass can be concentrated prior to application of the biomass to the edible product. This concentration step, although non-mandatory, is suitable, for example, if even slightly increased water content must be avoided in the final product. For example, if the final concentration of probiotics on the product must be particularly high, even if only a small amount of the edible product must contain a sufficient amount of cfu, it can be concentrated for other reasons. The concentration method is also well known in the art, and the alternative method is filtration or centrifugation. - Finally, the probiotic biomass, whether concentrated or not, is applied to the edible product. This application can be carried out according to the general rules for coating food products. For example, the application of the biomass can be when transporting the product on a conveyor belt, or in a coating drum. There are several options in the design of the spray system, from folding the tube to rotating the disc. Some products are suitable for handling in a coating drum, such as in a rotating two-wheel. The coating drum can be used as both a mixer and a mechanism for exposing the grain to spray. A commercial two-phase (air/liquid) nozzle can be used to spray the biomass onto the rotating grain. As in the case of a vitamin solution, the same spray system can be utilized. These techniques are well known in the art. 17-
本纸浪又度適用中國國家標準(CNS) Α4規格(21〇χ 297公茇） 1331018 A7 B7 五、發明説明（13 ) 可以考慮無實質cfU流失之方式將已包含益生菌之食物產 品暴露於周遭溫度或上升之溫度下，其取決於特質及偏好。 亦有可能冷凍該食物產品，取決於其特性及最終食物產品 之目的。當然，可對該可食用產品進行其他進一步之處理或 加工，取決於該終產物或該可食用產品之目的。實例之一是 以惰性氣體或氣體混合物如&或N2/C02對終產品通氣》 根據本發明之方法及產品係藉由例證經極詳細說明於以 下實例中。 實例 實例中所利用之菌株如下： -乳酸雙歧桿菌* : DSM20215 (德國培養物收藏品） -嗜熱鏈球菌（TH4)* -詹森氏乳酸桿菌：1- 1225 (CNCM) -副酪蛋白乳酸桿菌：I- 2116 (CNCM) *獲自克里斯金韓森（Christian Hansen)生物系統A/S (CHL)， 10-12 Boge Α11έ，郵件信箱 407，DK- 2970荷森(Horsholm)，丹麥。 利用幼兒穀物產品，早餐穀物薄片，穀物/乳點心及嬰兒 穀物粉末於實驗。下表1顯示這些產品組合物及其製備方法。 表1 :在實例中所指之組合物及可食用產品之生產 產品 產品類型 组合物 密度g/1 幼兒穀物產品 含有糖/蜜糖塗 覆物之擦製穀物 圈 穀物（小麥、燕麥及 大麥）、.糖、蜜糖、 麥芽糊精、維生素 及礦物質 115 早餐穀物薄片 含有薄的糖塗覆 物之經傳統烹煮 全麥、糖、精製糖 蒙、麥芽、鹽、蜜糖 135 -18- 本纸張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1331018 A7 _ B7 五、發明説明（14 ) 之小麥薄片 、葡萄糖、維生素 及礦物質 蠢物/乳點心 含高量乳類之擠 製水果形狀之穀 物 小麥麵粉、奶粉、 糖、香蕉濃縮物、 麥芽糖、澱粉、鹽、 維生素及礦物質、 香料 130 嬰兒穀物粉末 以小麥為基礎之 嬰兒穀物配方 小麥麵粉、糖、卵 轉脂、香草越、維 生素及礦物質 315 复到1 :施加乳酸雙歧择菌生皙至不同產品： 發酵乳酸雙歧桿菌然後以離心濃縮之，該發酵之詳細說 明係示於下表2及3中。添加標準保護劑至該濃縮物。以實驗 室-規模添加此生質至不同之商購穀物產品（參看上表〇。 於實驗室-規模之應用，將1.5- 2 kg之穀物產品置入旋轉批 式塗覆鼓輪中且利用具有兩相（空氣/液體）喷嘴之商業噴霧槍 將該生質喷霧至旋轉中之穀物上。在喷霧前後小心地稱重 該含有生質之噴霧槍以估計施加至榖物上之生質實際量。 所有的事例中均添加總穀物量之〇 5〇/〇。 盘2 :乳酸雙歧桿菌之培養基組合物（實例1)a 培養基组合物 成分 份量（S/D 乳清渗透物(permeate) — ^ 14 葡萄糖 25 消泡劑 1 乳清蛋白質水解物 5 酵母菌萃戒物 28 肉（腺） 4 果糖 14 緩衝鹽 10 -19- 本纸張尺度適用中國國家標準(CNS) A4規格(210X 297公袭) 1331018 A7 B7 五、發明説明（15 )This paper wave is again applicable to China National Standard (CNS) Α4 specification (21〇χ 297 茇) 1331018 A7 B7 V. Invention Description (13) Exposure of food products containing probiotics can be considered in the form of no substantial cfU loss. At ambient temperatures or rising temperatures, it depends on traits and preferences. It is also possible to freeze the food product, depending on its characteristics and the purpose of the final food product. Of course, the edible product may be subjected to further processing or processing depending on the end product or the purpose of the edible product. One example is the venting of the final product with an inert gas or gas mixture such as & or N2/C02. The methods and products according to the present invention are illustrated in greater detail by way of illustration in the following examples. The strains used in the examples are as follows: - Bifidobacterium lactis*: DSM20215 (German culture collection) - Streptococcus thermophilus (TH4)* - Lactobacillus johnsonii: 1- 1225 (CNCM) - Paracasein Lactobacillus: I-2116 (CNCM) * Obtained from Christian Hansen Biosystems A/S (CHL), 10-12 Boge Α11έ, mail box 407, DK-2970 Horsholm, Denmark . Use early childhood cereal products, breakfast cereal flakes, cereal/milk snacks and baby cereal powders for experimentation. Table 1 below shows these product compositions and methods for their preparation. Table 1: Compositions of the compositions and edible products referred to in the examples Product Type Composition Density g/1 Infant cereal products Mopped cereal ring cereals (wheat, oats and barley) containing sugar/honey coatings , sugar, honey, maltodextrin, vitamins and minerals 115 Breakfast cereal flakes containing a thin sugar coating of traditionally cooked whole wheat, sugar, refined sugar, malt, salt, honey 135 - 18- This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 mm) 1331018 A7 _ B7 V. Invention Description (14) Wheat flakes, glucose, vitamins and minerals Milk-extruded fruit-shaped cereals Wheat flour, milk powder, sugar, banana concentrate, maltose, starch, salt, vitamins and minerals, spices 130 Baby cereal powder Wheat-based infant cereal formula Wheat flour, sugar, eggs Trans fat, vanilla, vitamins and minerals 315 Recombined 1: Apply lactic acid bifidobacteria to different products: Fermentation of Bifidobacterium lactis and concentration by centrifugation, details of the fermentation The details are shown in Tables 2 and 3 below. A standard protective agent is added to the concentrate. Add this biomass to a different commercial grain product on a laboratory-scale basis (see table above. For laboratory-scale applications, 1.5-2 kg of cereal product is placed in a rotary batch coating drum with A two-phase (air/liquid) nozzle commercial spray gun sprays the biomass onto the rotating grain. Carefully weigh the germ-containing spray gun before and after spraying to estimate the biomass applied to the waste. Actual amount. The total grain amount was added to 〇5〇/〇 in all cases. Disk 2: Medium composition of Bifidobacterium lactis (Example 1) a Medium composition component amount (S/D whey permeate (permeate) ) — ^ 14 Glucose 25 Defoamer 1 Whey Protein Hydrolysate 5 Yeast Extracts 28 Meat (Gland) 4 Fructose 14 Buffer Salt 10 -19- This paper scale applies to China National Standard (CNS) A4 specification (210X 297 public attacks) 1331018 A7 B7 V. Description of invention (15)
奶粉 _ 0.8 表3 :乳酸雙歧桿菌之發酵參數（實例】 )。 發酵規模 2001蜂養基 溫度 37〇C 培養時間 14小時 發酵結束時之具活力菌數 1 X 10JU cfli/ml 離心及添加保護劑後之具活力菌數 9 x 1〇^ cfu/ml 表4:應用試驗之結果 產品 產品上具活力之菌數（cfo/g) 成品上之 幼兒穀物產品 1.5χ 10δ 0.15 早餐穀物薄片 8.8 χ ΙΟ' 0.3 穀物/乳點心 Ϊ.5χ10ύ 0.1 嬰兒穀物粉末 1.1 χ 10δ Γ 0.3 如表4明確顯示，每公克可食用產品獲得高量具活力之菌 數。就貯存之目的而言’水活性保持在可接受之範圍内。 實例2 :施加乳酸雙歧桿菌’詹森氏轧酸棵茴，副酪杏白轧 酸桿菌，嗜熱鏈球菌生質至幼兒縠物產品 發酵不同菌株（發酵之詳細說明係經示於表5至12)並以離 心濃縮之。添加標準保護劑至該濃縮物。以產物總童量計之 0.5%並以實驗室-規模添加不同生質至一可商購之幼兒毅物 產品。（如實例1相同之方法） 表5 :乳酸雙歧桿菌之培養基組合物（實例2)。 培養基组合物 成分 份量（g/1) 乳清滲透物 14 葡萄糖 25 消泡劑 1 乳清蛋白質水解物 ' 5 _酵母菌萃取物 28 肉（腺） 4 -20- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐） 1331018 A7 B7 五、發明説明（16 ) 果糖 14 緩衝鹽 10 奶粉 0.8 表6 :乳酸雙歧桿菌之發酵參數（實例2). 發酵規模 200 1培養基 溫度 37〇C 培養時間 14小時 發酵結束時之具活力菌數 1 X 1010 cfu/ml 離心及添加保護劑後之具活力菌數 9 X ΙΟ10 cfu/ml 表7 :詹森氏乳酸桿菌之培養基组合物（實例2)。 成分 份量（g/1) 乳清滲透物 15 葡萄糖 15 消泡劑 1 乳清蛋白質水解物 5 酵母菌萃取物 30 肉（腺） 5 果糖 15 緩衝鹽 10 奶粉 10 表8:詹森氏乳酸桿菌之發酵參數(實例2)。 發酵規模 2000 1培養基 溫度 40°C 培養時間 14小時 發酵結束時之具活力菌數 7 X 109 cfu/ml 離心及添加保護劑後之具活力菌數 5 X 101ϋ cfu/ml 表9:嗜熱鏈球菌之培養基組合物（實例2)。 成分 份量（g/1) 乳清渗透物 50 消泡劑 1 乳清蛋白質水解物 5 酵母菌萃取物 20 肉（腺） 5 -21 - 本纸張尺度適用中國國家標準(CNS) A4規格(210X 297公釐) 1331018Milk powder _ 0.8 Table 3: Fermentation parameters of Bifidobacterium lactis (example). Fermentation scale 2001 bee nutrient temperature 37 〇 C culture time 14 hours at the end of fermentation with number of viable bacteria 1 X 10JU cfli / ml Centrifugation and addition of protective agent with viable bacteria number 9 x 1 〇 ^ cfu / ml Table 4: Results of the application test The number of viable bacteria on the product product (cfo/g) The infant cereal product on the finished product 1.5χ 10δ 0.15 Breakfast cereal flakes 8.8 χ ΙΟ' 0.3 Cereals/dairy snacks. 5χ10ύ 0.1 Baby cereal powder 1.1 χ 10δ Γ 0.3 As shown in Table 4, the number of viable bacteria per gram of edible product is obtained. The water activity is kept within an acceptable range for storage purposes. Example 2: Application of Bifidobacterium lactis 'Jensen's rolling acid fennel, A. bacillus, A. faecalis, different strains of fermentation of S. thermophilus to children's products (the detailed description of fermentation is shown in Table 5). To 12) and concentrate by centrifugation. A standard protective agent is added to the concentrate. 0.5% of the total product was added and different raw materials were added on a laboratory-scale basis to a commercially available child's product. (The same method as in Example 1) Table 5: Medium composition of Bifidobacterium lactis (Example 2). Medium composition component (g/1) whey permeate 14 glucose 25 defoamer 1 whey protein hydrolysate ' 5 _ yeast extract 28 meat (gland) 4 -20- This paper scale applies to Chinese national standards ( CNS) A4 size (210X 297 mm) 1331018 A7 B7 V. Description of invention (16) Fructose 14 Buffer salt 10 Milk powder 0.8 Table 6: Fermentation parameters of Bifidobacterium lactis (Example 2). Fermentation scale 200 1 Medium temperature 37〇 C Culture time 14 hours The number of viable bacteria at the end of fermentation 1 X 1010 cfu/ml Number of viable bacteria after centrifugation and addition of protective agent 9 X ΙΟ10 cfu/ml Table 7: Medium composition of Lactobacillus johnsonii (example) 2). Ingredients (g/1) Whey Permeate 15 Glucose 15 Antifoaming Agent 1 Whey Protein Hydrolysate 5 Yeast Extract 30 Meat (Gland) 5 Fructose 15 Buffer Salt 10 Milk Powder 10 Table 8: Lactobacillus Fermentation parameters (Example 2). Fermentation scale 2000 1 medium temperature 40 ° C culture time 14 hours number of viable bacteria at the end of fermentation 7 X 109 cfu / ml number of viable bacteria after centrifugation and addition of protective agent 5 X 101 ϋ cfu / ml Table 9: thermophilic chain Culture medium composition of cocci (Example 2). Ingredients (g/1) Whey Permeate 50 Defoamer 1 Whey Protein Hydrolysate 5 Yeast Extract 20 Meat (Gland) 5 -21 - This paper scale applies to China National Standard (CNS) A4 specification (210X 297 mm) 1331018
-- 5 緩衝鹽 5 开L囷体亦顯不足夠之具活^ 菌數及低的終水活性。 、'’ im:含直直森氏乳皇限結果 -22- 本紙張尺度適財關家料(CNS) A4規格(21GX 297公爱) ----------- I酵規模 -^ΚΆ_ζί£1__ pa 度 —-—i 200 1培養基 3養時間 ~----- 40°C 6小時 一贫醉結束時之具活力菌數 2 X 109 cfu/ml 離心及添加保護劑後之具活力茴势 4 X ΙΟ10 cfii/ml 合物（實例 2) 份量_ 一成分 黃豆（腺） i泡齊丨j~ .酵母菌萃取物 10 15-- 5 Buffer salt 5 Open L 囷 body is also not enough to have a live number of bacteria and low final water activity. , '' im: with straight Sens milky limited results - 22 - This paper scale is suitable for wealth (CNS) A4 specifications (21GX 297 public) ----------- I fermentation scale -^ΚΆ_ζί£1__ pa degree---i 200 1 medium 3 raising time ~----- 40 °C 6 hours at the end of the intoxication with the number of viable bacteria 2 X 109 cfu / ml after centrifugation and adding a protective agent Vigorous anger potential 4 X ΙΟ10 cfii/ml compound (Example 2) Serving amount _ One ingredient soy bean (gland) i bubble 丨 j~ . Yeast extract 10 15
裝 果糖 緩衝鹽 30 -------—1__ /·) 溫度 ---— 200 1培卷基 37〇C 食时丨bj -發酵結束時之具活力菌數“ - ___17小時 9 X 109 cfU/ml 離心方添Λσ你雄添,1多名士 a :丄丄斗丄—*-- 4夂/ί]、、"ϊμ木设失Π傻 < 异活力茼數 9 X 1〇ιυ cfu/ml 成品上之Aw ±f ---- /14 ^ 瓶 產品上之具活力 菌數 icfU/g") _乳酸雙歧桿菌 1.5 X 1〇8 詹森氏乳酸桿菌 2·5 X 108 — 嗜熱鏈球菌 2.8 X 1〇8 副酪蛋白乳酸桿菌 2χ 1〇8 ^ 施加至該幼兒穀物產品之其他菌抶十 0.15 <0.1 <0.1 訂Fructose-loaded buffering salt 30 --------1__ /·) Temperature---- 200 1 卷 基 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - cfU/ml Centrifugal Fang Λ 你 雄 雄 雄 ,, more than 1 celebrity a: 丄丄 丄 丄 * * * * * * * * * * * * * * * * * * ί ί ί ί ί ί ί 9 9 9 9 9 9 9 9 9 9 9 9 9 9 Cfu/ml Aw ±f on the finished product ---- /14 ^ The number of viable bacteria on the bottle product is icfU/g") _ Bifidobacterium lactis 1.5 X 1〇8 Lactobacillus jensei 2·5 X 108 — Streptococcus thermophilus 2.8 X 1〇8 Lactobacillus casein 2χ 1〇8 ^ Other fungi applied to the cereal product of the child Ten 0.15 <0.1 <0.1
k. 1331018 A7 __ B7 五、發明説明（18 ) 發酵詹森氏乳酸桿菌且然後以離心濃縮之（發酵之詳細說 明參看表14及15)。添加標準保護劑至該濃縮物。以試驗生產 規模添加此生質至一幼兒穀物產品。 於試驗生產規模之應用，將100 kg/h幼兒穀物產品導入一 連續式糖衣鼓輪中。以一連_兩相（空氣/液體）噴嘴將0.5 kg/h 之詹森氏乳酸桿菌生質噴霧至該穀物上。 將成品包裝於鋁襯裡並使其在2(TC下進行保存期限研究 (結果參看表16)。 表Η :詹森氏乳酸桿菌培養基組合物（實例3) 成分 份量ig/1) 乳清滲透物 15 葡萄糖 15 消泡劑 1 乳清蛋白質水解物 5 酵母菌萃取物 30 肉（腺） 5 果糖 15 緩衝鹽 10 奶粉 10 表15 :詹森氏乳酸桿菌之發酵參數（實例3) 發酵規模 2000 1培卷某 溫度 4(TC 培養時間 14小時 發酵結束時之具活力菌數 3 X 10? cfu/ml 離心及添加保護劑後之具活力菌數 1 X 10ivl cfu/ml 表16 :在幼兒縠，產品上之應用及保存期限結果 在20°C下之天數 產品上之具活力菌數 成品上之 開始 1.3 X 108 <0.1 90 1.6 X 108 <0.1 180 1.1 X 108 <0.1 -23- 本纸張尺度適用中國國家標準(CNS) A4規格(210X 297公爱) 1331018 A7 B7 五、發明説明（19 270 1.3 X 108 365 9.5 X 107 <0.1 <0.1 保存期限研究顯示貯存期長達一年不實質上降低該產品 上之cfii數目。 實例4 :直接及在4天貯存期後添加經濃縮及辛經澧缩夕y _ 雙歧桿菌生質至幼兒縠物產品 發酵乳酸雙歧桿菌（詳細說明係示於表17及18)，直接利用 一部分生質且以離心濃縮另一部分並添加標準保護劑。以 實驗室規模添加兩種生質至幼兒穀物產品。另一系列試驗 之進行係以相同之生質但在應用前經於5。(：貯存4天。 於實驗室規模之應用，將2 kg穀物產品置入一旋轉批式塗 覆鼓輪中並利用具有兩相（空氣/液體）噴嘴之商業噴霧槍將該 生質噴霧至該旋轉中之穀物上。在所有的事例中均添加該 穀物總重之0.5%。在一狀況下，該生質係於發酵後直接地而 在其餘狀況下其係經濃縮的且然後重複相同步驟且在生質 在應用前經貯存4天（5°C )。 亦在活體外分析成品對胃道抵抗力。 表17 :乳酸雙歧桿菌之培養基组合物（實例4)。 成分 份量（g/1) 乳清滲透物 14 葡萄糖 25 消泡劑 1 乳清蛋白質水解物 5 酵母菌萃取物 28 肉（腺） 4 果糖 14 緩衝鹽 10 -24- 本紙張尺度適用中國國家標準(CMS) A4規格(210X297公爱) 1331018 A7 B7 五、 發明説明（20 ) 0.8 奶粉 表18 :乳酸雙歧桿菌之發酵參數(實例4) 發酵規模 200 1培養基 溫度 37〇C 培養時間 14小時 發酵結束時之具活力菌數 9 X 109 cfu/ml 取自發酵及在5°C下貯存4天之樣品之具活 力菌數 5 X 109 cfb/ml 離心及添加保護劑後之具活力菌數 8 X 101ϋ cfu/ml 離心及添加保護劑及在5°C下貯存4天後之 具活力菌數 6 X 101ϋ cfu/ml 表19 :在幼兒穀物產品上應用試驗之結果 乳酸雙歧桿菌生質 產品上之具活力 菌數(cfii/g) 在胃腸道之總 log流失（在活體外） 未經濃縮 3x 107 0.4 未經濃縮但經貯存 2 xlO7 0.4 經濃縮 4χ 108 0.2 經濃縮且經貯存 7χ 108 0.3 如表19所顯示，在模擬腸道環境中之流失係在可接受之範 圍内。 -25- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐)k. 1331018 A7 __ B7 V. INSTRUCTION DESCRIPTION (18) Fermentation of Lactobacillus johnsonii and subsequent concentration by centrifugation (see Tables 14 and 15 for details of fermentation). A standard protective agent is added to the concentrate. This raw material is added to a baby cereal product on a trial production scale. For the trial production scale application, a 100 kg/h infant cereal product was introduced into a continuous sugar coating drum. 0.5 kg/h of Lactobacillus johnsii biomass was sprayed onto the grain with a two-phase (air/liquid) nozzle. The finished product was packaged in an aluminum lining and subjected to a shelf life study at 2 (TC (see Table 16 for results). Table Η: Lactobacillus juniper medium composition (Example 3) Ingredient ig/1) Whey permeate 15 Glucose 15 Defoamer 1 Whey Protein Hydrolysate 5 Yeast Extract 30 Meat (Gland) 5 Fructose 15 Buffer Salt 10 Milk Powder 10 Table 15: Fermentation parameters of Lactobacillus johnsonii (Example 3) Fermentation scale 2000 1 Petri Volume 4 (TC culture time 14 hours at the end of fermentation, the number of viable bacteria 3 X 10? cfu / ml Centrifugation and the addition of protective agent after the number of viable bacteria 1 X 10ivl cfu / ml Table 16: In children, products Application and shelf life results The number of days on the product at 20 °C The number of viable bacteria on the product begins 1.3 X 108 <0.1 90 1.6 X 108 <0.1 180 1.1 X 108 <0.1 -23- The paper Zhang scale applies Chinese National Standard (CNS) A4 specification (210X 297 public) 1331018 A7 B7 V. Invention description (19 270 1.3 X 108 365 9.5 X 107 < 0.1 < 0.1 shelf life study shows storage period up to one year Does not substantially reduce the number of cfii on the product. Example 4: Directly and after the 4-day storage period, the concentrated and symbiotic y _ _ Bifidobacterium biomass was added to the baby sputum product fermentation Bifidobacterium lactis (details are shown in Tables 17 and 18), and the direct utilization part Prepare and concentrate another part by centrifugation and add standard protectant. Add two kinds of raw materials to the infant cereal product on a laboratory scale. The other series of experiments are carried out with the same biomass but before application 5 (: Storage for 4 days. For laboratory scale applications, 2 kg of cereal product was placed in a rotary batch coating drum and sprayed to the rotation using a commercial spray gun with a two-phase (air/liquid) nozzle In the case of cereals, 0.5% of the total weight of the grain is added in all cases. In one case, the biomass is concentrated directly after fermentation and in the remaining conditions and then the same steps are repeated and The biomass was stored for 4 days (5 ° C) before application. The product was also analyzed for resistance to the stomach in vitro. Table 17: Medium composition of Bifidobacterium lactis (Example 4). ) whey permeate 14 glucose 25 Defoamer 1 Whey Protein Hydrolysate 5 Yeast Extract 28 Meat (Gland) 4 Fructose 14 Buffer Salt 10 -24- This paper scale applies to Chinese National Standard (CMS) A4 Specification (210X297 public) 1331018 A7 B7 Five , invention description (20) 0.8 milk powder table 18: fermentation parameters of Bifidobacterium lactis (Example 4) fermentation scale 200 1 medium temperature 37 〇 C culture time 14 hours at the end of fermentation, the number of viable bacteria 9 X 109 cfu / ml Number of viable bacteria from fermentation and storage at 5 °C for 4 days 5 X 109 cfb/ml Number of viable bacteria after centrifugation and addition of protective agent 8 X 101ϋ cfu/ml Centrifugation and addition of protective agent at 5° Number of viable bacteria after storage for 4 days at C 6 X 101ϋ cfu/ml Table 19: Results of application test on infant cereal products Vibrant bacteria on bifidobacterium lactate product (cfii/g) in the gastrointestinal tract Total log loss (in vitro) Not concentrated 3x 107 0.4 Not concentrated but stored 2 xlO7 0.4 Concentrated 4χ 108 0.2 Concentrated and stored 7χ 108 0.3 As shown in Table 19, in a simulated intestinal environment Loss is within acceptable limits. -25- This paper size is applicable to China National Standard (CNS) A4 specification (210X 297 mm)