TW201801615A - 特殊膳食用乾穩定益生菌組合物 - Google Patents
特殊膳食用乾穩定益生菌組合物 Download PDFInfo
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Abstract
本發明提供乾穩定益生菌組合物。該組合物包含一或多種活益生菌微生物、一或多種水解蛋白質、一或多種二醣、一或多種寡醣及一或多種多醣,但不包含海藻糖。該組合物在40℃之溫度及33%之相對濕度下3個月後具有至少1×1010CFU/g之活力及小於1 log單位/g之活力損失。亦提供製備該乾穩定益生菌組合物之方法。
Description
本發明係關於特殊膳食(例如,嬰兒配方)用穩定益生菌組合物。
目前存在各種用於補充動物(包括人類)胃腸道之益生菌微生物(亦稱為益生菌)。該等微生物可調節動物腸內之天然微生物群落用於期望生物效應。 將有效量之益生菌(probiotic bacteria)提供至宿主之挑戰之一係在典型工業製造製程之嚴苛條件下及高溫度及濕度下之長期儲存下保存其活力。儘管業內在關於用於將生物材料遞送至動物之消化系統中之囊封及調配技術方面已有發展,但在製造製程、分配及儲存期間保護益生菌之活力之囊封或穩定化技術方面之發展極少。業內需要使得益生菌在暴露於各種嚴苛環境、尤其與高溫度及濕度相關聯之彼等下時能夠存活之穩定技術。 另外,益生菌產品本身之固有水分形成另一挑戰,此乃因益生菌通常對水活性敏感,尤其在與高溫結合時。迄今為止,尚未鑑別出當併入至諸如營養產品之產品中時在分配及儲存期間之中間水分條件(即,約0.2及更高、或高達約0.4或更高之水活性)及高溫(例如,至少約30℃、或高達約40℃或更高之溫度)下提供對益生菌之顯著保護之技藝或技術。因此,業內需要適於在益生菌之活力可受損之各種地理位置(包括熱帶氣候中之彼等)中分配之穩定益生菌組合物。 其他挑戰包括對於在適於由諸如嬰兒、幼兒及老人等人消耗之特殊膳食調配物中使用習用食品成份之法規限制。不推薦將習用合成囊封及穩定化化合物及甚至一些天然化合物(例如阿拉伯樹膠(gum acacia)、海藻酸鹽、乳蛋白質及某些糖(例如海藻糖))用於該等特殊膳食調配物中。容許用於特殊膳食用之營養化合物之推薦清單係由聯合FAO/WHO國際食品法典委員會(FAO/WHO Codex Alimentarius Commission)監管。 因此,期望適於特殊膳食用之穩定益生菌組合物,其包含益生菌微生物(例如益生菌)及其他成份;及用於製造此等組合物之穩定化技術。
本發明提供特殊膳食用之乾穩定益生菌組合物及其製備方法。 根據本發明之一個態樣,提供乾組合物。組合物包含一或多種活益生菌微生物、一或多種水解蛋白質、一或多種二醣、一或多種寡醣及一或多種多醣。組合物不包含海藻糖。組合物在40℃之溫度及33%之相對濕度下3個月後具有至少1×1010
CFU/g之活力、小於1 log單位/g之活力損失。 組合物可於特殊膳食產品中對宿主提供益生菌益處。特殊膳食產品可選自由以下組成之群:嬰兒配方、較大嬰兒配方、基於經加工穀物之食品、罐裝嬰幼兒食品、動物補充劑或治療劑及/或用於醫療目的之特殊食品。 在特定實施例中,特殊膳食產品係嬰兒配方。 活益生菌微生物可選自由以下組成之群:活的益生菌、真菌及酵母。 組合物可包含基於組合物之總乾重量至少50%之一或多種水解蛋白質。該一或多種水解蛋白質可選自由以下組成之群:乳蛋白質、植物蛋白質及其組合。該一或多種水解蛋白質可選自由以下組成之群:水解酪蛋白、水解乳清蛋白質、水解豌豆蛋白質、水解大豆蛋白質及其組合。 組合物可包含基於組合物之總乾重量少於20%之一或多種二醣。該一或多種二醣可選自由以下組成之群:蔗糖、乳糖及其組合。 組合物可包含基於組合物之總乾重量5-30%之一或多種寡醣。該一或多種寡醣可選自由以下組成之群:菊糖、麥芽糖糊精、右旋糖酐、果寡醣(FOS)、半乳寡醣(GOS)、甘露寡醣(MOS)及其組合。 組合物可包含基於組合物之總乾重量1-10%之一或多種多醣。該一或多種多醣可選自由以下組成之群:角叉菜膠、瓜爾膠(guar gum)、阿拉伯樹膠、刺槐豆膠、澱粉、改質澱粉及其組合。 組合物可進一步包含一或多種額外試劑。組合物可包含基於組合物之總重量0.5-10%之一或多種額外試劑。該一或多種額外試劑可選自由以下組成之群:羧酸鹽、生育酚及其組合。羧酸鹽可選自由抗壞血酸鹽及檸檬酸鹽組成之群。該一或多種額外試劑可包含重量比為1:4至4:1之一或多種生育酚及一或多種羧酸鹽。較佳地,一或多種額外試劑包含重量比為4:1之維生素E及抗壞血酸鈉。 根據本發明之另一態樣,提供用於製備本發明之組合物之方法。方法包含一或多種選自由以下組成之群之乾燥方法:空氣乾燥、真空乾燥、流化床乾燥及噴霧乾燥。 根據本發明之另一態樣,提供用於製備本發明之組合物之方法。該方法包含:(a) 將一或多種活益生菌微生物、一或多種水解蛋白質、一或多種二醣、一或多種寡醣及一或多種多醣合併於鹼性水溶劑中以形成漿液;(b) 將該漿液在液氮中快速冷凍以形成呈珠粒、微滴或串形式之固體冷凍顆粒;(c) 固體冷凍顆粒藉由在真空下蒸發之一次乾燥步驟,同時維持顆粒之溫度高於其冷凍溫度之,藉此形成一次乾燥調配物;及(d) 在全強度真空(full strength vacuum)及20℃或更高之熱源溫度下將一次乾燥調配物二次乾燥達足以使一次乾燥調配物之水活性降低至0.3 Aw或更低之時間。由此,製得本發明之組合物。方法可進一步包含在步驟(a)之前將一或多種水解蛋白質、一或多種二醣、一或多種寡醣及一或多種多醣進行滅菌。方法可進一步包含將組合物切割、壓碎、碾磨或粉碎成自由流動之粉末。粉末之粒度可小於約1000 μm。 在本發明之方法中,組合物可包含有效量之一或多種活益生菌微生物用於在特殊膳食產品中向宿主提供益生菌益處。方法可進一步包含利用組合物製作特殊膳食產品。特殊膳食產品可選自由以下組成之群:嬰兒配方、較大嬰兒配方、基於經加工穀物之食品、罐裝嬰幼兒食品、動物補充劑或治療劑及/或用於醫療目的之特殊食品。在特定實施例中,特殊膳食產品較佳係嬰兒配方。
本申請案涉及並主張於2015年7月29日提出申請之標題為STABLE DRY PROBIOTIC COMPOSITIONS FOR SPECIAL DIETARY USES
之美國臨時申請案第62/198,220號之益處,該臨時申請案之內容以引用的方式併入本文中。 本發明提供較佳用於特殊膳食用之新穎乾穩定益生菌組合物及製作此等組合物之方法。該等組合物為益生菌微生物提供期望穩定性及保護。益生菌微生物可在用於製造可消耗產品之製造製程期間、整個分配通路及在極端儲存條件下受到保護。大多數益生菌調配者在其產品中利用極高細菌細胞計數,其有時可較有效劑量高10倍且甚至100倍,此係基於以下理解:顯著數量之細胞在製造製程、運輸及儲存期間最終喪失活力並死亡。 如本文中所使用之術語「特殊膳食用」係指向為宿主製作或向宿主施加特殊膳食產品。較佳地,特殊膳食產品由聯合FAO/WHO國際食品法典委員會在標題為「Standard For Infant Formula and Formulas For Special Medical Purposes Intended for Infants, CODEX STAN 72-1981」 (「美國標準法典72」)之文件中推薦。特殊膳食產品之實例包括嬰兒配方、較大嬰兒配方、基於經加工穀物之食品、罐裝嬰幼兒食品及用於醫療目的之特殊食品。較佳地,特殊膳食產品係嬰兒配方。 宿主可係任何動物,包括魚、禽類(例如,雞)或哺乳動物(例如反芻動物、豬或伴侶動物(例如馬、犬或貓))。在特定實施例中,哺乳動物係人類。人類宿主可係嬰兒、兒童或老人。較佳地,人類宿主係嬰兒。 如本文中所使用之術語「嬰兒」係指自出生至約12個月大的人。 如本文中所使用之術語「兒童」係指自約12個月大至約12歲的人。 如本文中所使用之術語「老人」係指至少約55歲、60歲、65歲或70歲、較佳地至少約65歲的人。 術語「益生菌微生物」及「益生菌」在本文中可互換使用,且係指當以有效量投與宿主時,對宿主提供或賦予益生菌益處之活微生物。如本文中所使用之術語「有效量」係指當經由(例如)膳食產品(例如特殊膳食產品)投與宿主時,足以在宿主中達成期望益生菌益處之益生菌微生物之量。益生菌微生物可選自由以下組成之群:活的益生菌、真菌及酵母。期望益生菌益處可係任何有益之健康或營養效應,例如,維持健康之胃腸菌群、增強免疫力、預防過敏及感冒並保護免於腹瀉、特應性皮炎及尿路感染。 如本文中所使用之術語「活力」係指組合物中之益生菌微生物在適於益生菌微生物生長之營養介質上形成菌落之能力,且可表示為菌落形成單位(CFU)除以組合物之重量,例如,CFU/g。 如本文中所使用之術語「相對濕度(RH)」係指通常在既定溫度下空氣中之水蒸氣之量。相對濕度通常小於使空氣飽和所需之濕度,且通常以飽和濕度之百分比表示。 如本文中所使用之術語「乾」係指經脫水或無水(例如,實質上缺少液體)之物質(例如,本發明之組合物)之物理狀態。物質(例如,本發明之組合物)可藉由一或多種乾燥製程進行乾燥,例如,空氣乾燥、真空乾燥、流化床乾燥、噴霧乾燥及凍乾。 如本文中所使用之術語「水活性(Aw)」係指物質(例如,本發明之組合物)中之水之可用性,其表示物質中水之能量狀態。其可定義為物質上方水的蒸氣壓除以相同溫度下純水之蒸氣壓。純蒸餾水具有恰好為1之水活性,即,Aw=1.0。乾物質可具有約0.5或更低、較佳地約0.3或更低、更佳地約0.2或更低、最佳地約0.1或更低之Aw。 提供乾組合物。組合物包含一或多種活益生菌微生物、一或多種水解蛋白質、一或多種二醣、一或多種寡醣及一或多種多醣。組合物具有至少1×109
CFU/g、1×1010
CFU/g、1×1011
CFU/g或1×1012
CFU/g、較佳地1×1010
CFU/g之初始活力。組合物在預定條件下在預定時期之後具有小於1 log單位/g之活力損失。較佳地,組合物不包含海藻糖。 組合物可包含有效量之一或多種活益生菌微生物用於在特殊膳食產品中向宿主提供益生菌益處。特殊膳食產品可係嬰兒配方、較大嬰兒配方、基於經加工穀物之食品、罐裝嬰幼兒食品或用於醫療目的之特殊食品,較佳地嬰兒配方。 預定時期可係約1週、2週、3週或4週,或1個月、2個月、3個月、4個月、5個月、6個月、12個月、18個月、24個月或36個月,較佳為約1個月、2個月或3個月,更佳地1個月或3個月。指定時期可包括在指定時期10%內之更短或更長時期。如本文中所使用之術語「3個月」係指約84-90天之時期。如本文中所使用之術語「2個月」係指約56-60天之時期。如本文中所使用之術語「1個月」係指約28-30天之時期。 預定條件可包括預定溫度及預定相對濕度(RH)。預定溫度可係至少約25℃、37℃、40℃、45℃、50℃或55℃。預定相對濕度(RH)可係至少約10%、20%、30%、33%、35%、40%、50%、60%、70%或80%。 預定條件可係加速儲存條件。舉例而言,預定條件可包括至少約40℃且至少約33% RH或至少約45℃且至少約33% RH。 組合物在約40℃及33% RH下約3個月之後或在約45℃及33% RH下1個月之後可具有小於1 log單位/g之活力損失。 組合物可包含基於組合物之總乾重量約1-30%、10-25%、10-20%或15-20%之一或多種活益生菌微生物。適宜益生菌微生物包括(但不限於)酵母,例如酵母菌屬(Saccharomyces
)、德巴利酵母菌屬(Debaromyces
)、假絲酵母菌屬(Candida
)、畢赤酵母菌屬(Pichia
)及球擬酵母菌屬(Torulopsis
);黴菌,例如麯黴屬(Aspergillus
)、根黴屬(Rhizopus
)、毛黴屬(Mucor
)、青黴屬(Penicillium
)及球擬酵母菌屬;及細菌,例如以下屬:雙歧桿菌屬(Bifidobacterium
)、梭菌屬(Clostridium
)、梭形桿菌屬(Fusobacterium
)、蜜蜂球菌屬(Melissococcus
)、丙酸桿菌屬(Propionibacterium
)、鏈球菌屬(Streptococcus
)、腸球菌屬(Enterococcus
)、乳球菌屬(Lactococcus
)、葡萄球菌屬(Staphylococcus
)、消化鏈球菌屬(Peptostreptococcus
)、芽孢桿菌屬(Bacillus
)、片球菌屬(Pediococcus
)、微球菌屬(Micrococcus
)、白色念珠菌屬(Leuconostoc
)、魏斯氏菌屬(Weissella
)、氣球菌屬(Aerococcus
)、酒球菌屬(Oenococcus
)及乳桿菌屬(Lactobacillus
)。適宜益生菌微生物之特定實例可由以下物種表示,且包括彼等物種內之所有培養生物型:黑麴菌(Aspergillus niger
)、
米麴菌(A. oryzae
)、凝結芽孢桿菌(Bacillus coagulans
)、遲緩芽孢桿菌(B. lentus
)、地衣芽孢桿菌(B. licheniformis
)、腸膜芽孢桿菌(B. mesentericus
)、短小芽孢桿菌(B. pumilus
)、枯草桿菌(B. subtilis
)、納豆芽孢桿菌(B. natto
)、嗜澱粉擬桿菌(Bacteroides amylophilus
)、多毛擬桿菌(Bac. capillosus
)、棲瘤胃擬桿菌(Bac. ruminocola
)、豬擬桿菌(Bac. suis
)、青春雙歧桿菌(Bifidobacterium adolescentis
)、動物雙歧桿菌(B. animalis
)、短雙歧桿菌(B. breve
)、兩歧雙歧桿菌(B. bifidum
)、嬰兒雙歧桿菌(B. infantis
)、乳雙歧桿菌(B. lactis
)、長雙歧桿菌(B. longum
)、假長雙歧桿菌(B. pseudolongum
)、嗜熱雙歧桿菌(B. thermophilum
)、品托勒佩斯假絲酵母(Candida pintolepesii
)、丁酸梭菌(Clostridium butyricum
)、乳脂腸球菌(Enterococcus cremoris
)、二丁酮腸球菌(E. diacetylactis
)、屎腸球菌(E. faecium
)、中間腸桿菌(E. intermedius
)、乳酸腸球菌(E. lactis
)、蒙氏腸球菌(E. mundtii
)、嗜熱腸球菌(E. thermophilus
)、大腸埃希氏桿菌(Escherichia coli
)、脆壁克魯維氏酵母(Kluyveromyces fragilis
)、嗜酸乳桿菌(Lactobacillus acidophilus
)、消化乳桿菌(L. alimentarius
)、食澱粉乳桿菌(L. amylovorus
)、捲曲乳桿菌(L. crispatus
)、短小乳桿菌(L. brevis
)、乾酪乳桿菌(L. case
)、彎曲乳桿菌(L. curvatus
)、纖維二醣乳桿菌(L. cellobiosus
)、德氏乳桿菌保加利亞亞種(L. delbrueckii ss. bulgaricus
)、香腸乳桿菌(L farciminis
)、發酵乳桿菌(L. fermentum
)、加氏乳桿菌(L. gasseri
)、瑞士乳桿菌(L. helveticus
)、乳酸乳桿菌(L. lactis
)、植物乳桿菌(L. plantarum
)、約氏乳桿菌(L. johnsonii
)、羅伊氏乳桿菌(L. reuteri
)、鼠李糖乳桿菌(L. rhamnosus
)、清酒乳桿菌(L. sakei
)、唾液乳桿菌(L. salivarius
)、腸系膜狀白色念珠菌(Leuconostoc mesenteroides
)、啤酒(有害)片球菌(P. cereviseae (damnosus)
)、乳酸片球菌(Pediococcus acidilactici
)、戊醣片球菌(P. pentosaceus
)、費氏丙酸桿菌(Propionibacterium freudenreichii
)、薛曼納氏丙酸桿菌(Prop. shermanii
)、釀酒酵母(Saccharomyces cereviseae
)、肉葡萄球菌(Staphylococcus carnosus
)、木糖葡萄球菌(Staph. xylosus
)、嬰兒鏈球菌(Streptococcus infantarius
)、唾液鏈球菌嗜熱亞種(Strep. salivarius. thermophilus
)、嗜熱鏈球菌(Strep. Thermophilus
)及乳鏈球菌(Strep. Lactis
)。較佳地,益生菌係乳酸細菌及雙歧桿菌。 組合物可包含基於組合物之總乾重量至少約40%或50%、較佳地至少約50%之一或多種水解蛋白質。舉例而言,組合物可包含約40-80%、40-70%、50-70%或50-60%、較佳40-80%之水解蛋白質。 術語「水解蛋白質」及「蛋白質水解產物」在本文中可互換使用,且係指藉由水解或消化分解成較短肽片段及/或胺基酸之蛋白質。水解或消化可藉由強酸、強鹼、酶或其組合實施。水解蛋白質可來自動物或植物,較佳地來自哺乳動物,更佳地來自乳源。水解蛋白質可係乳蛋白質、植物蛋白質或其混合物。 水解蛋白質可係部分或廣泛水解,較佳地廣泛水解。水解蛋白質可係多肽與胺基酸之混合物。在一些實施例中,至少約60%、70%、80%、90%、95%或99%、較佳地至少約70%之水解蛋白質之分子量低於約100,000道耳頓(Dalton)、75,000道耳頓、50,000道耳頓、25,000道耳頓、10,000道耳頓、5,000道耳頓、1,000道耳頓或500道耳頓、較佳地約50,000道耳頓、更佳地約10,000道耳頓、更佳地約2,000道耳頓。例如,至少約50%、60%、70%、80%或90%、較佳地至少約70%之水解蛋白質之分子量低於約2,000道耳頓。 適於製作本發明之組合物之水解蛋白質之蛋白質包括卵蛋白質、明膠、乳蛋白質、酪蛋白、乳清蛋白質、白蛋白、大豆蛋白質、豌豆蛋白質、大米蛋白質、小麥蛋白質及其他植物蛋白質。較佳地,蛋白質係推薦用於特殊膳食用之彼等。 水解蛋白質之實例包括水解酪蛋白、水解乳清蛋白質、水解豌豆蛋白質、水解大豆蛋白質及其組合。在一個較佳實施例中,水解蛋白質包含水解酪蛋白或豌豆蛋白質,其至少約80%具有小於約2,000道耳頓之分子量。 組合物包含二醣、寡醣及多醣之碳水化合物混合物,其中益生菌微生物包埋於其中。不受理論約束,據信,藉由合併如本文中所述之碳水化合物混合物及廣泛水解之蛋白質所形成之基質容許更快之乾燥並有助於所得乾組合物之期望非晶形及剛性結構。 組合物可包含基於組合物之總乾重量少於約30%、20%或10%、較佳地少於20%之一或多種二醣。舉例而言,組合物可包含約1-30%、1-20%、1-10%、5-30%、5-20%、5-10%、10-20%、10-15%或10-20%、較佳地約5-30%之二醣。 二醣較佳係推薦用於特殊膳食用之彼等。二醣可係乳糖、蔗糖、麥芽糖、果糖或其組合,較佳乳糖或蔗糖、更佳乳糖。二醣較佳不係海藻糖。在一些較佳實施例中,本發明之組合物不包含海藻糖。 組合物可包含基於組合物之總乾重量約1-30%、1-20%、1-10%、5-30%、5-20%、5-10%、10-20%、10-15%或10-20%之一或多種寡醣。較佳地,組合物包含5-30%之寡醣。 寡醣係在營養應用中通常視為益菌生(prebiotics)之可溶性纖維。有利地,可溶性纖維經過胃而不消化並可藉由腸微生物群落消化而變得可用。可溶性纖維之併入亦可有助於保護益生菌免受消化酶及胃之高酸性。 寡醣較佳係推薦用於特殊膳食用之彼等。寡醣可係菊糖、麥芽糖糊精、右旋糖酐、果寡醣(FOS)、半乳寡醣(GOS)、甘露寡醣(MOS)或其組合,較佳係麥芽糖糊精或菊糖,更佳係菊糖。 組合物可包含基於組合物之總乾重量約0.1-40%、0.5-30%、1-30%、1-20%、1-10%、1-5%或5-10%之一或多種多醣。較佳地,組合物包含1-10%之多醣。多醣較佳係推薦用於特殊膳食用之彼等。多醣可係角叉菜膠、瓜爾膠、阿拉伯樹膠、刺槐豆膠、澱粉、改質澱粉或其組合,較佳係刺槐豆膠或瓜爾膠,更佳係刺槐豆膠。較佳地,多醣不係海藻酸鹽或幾丁聚醣。在一些較佳實施例中,組合物不包含海藻酸鹽或幾丁聚醣。在一些其他較佳實施例中,組合物不包含海藻糖或海藻酸鹽。 在一些實施例中,組合物包含基於組合物之總乾重量0.1-20%之多醣、5-30%之寡醣及1-20%之二醣。具體而言,組合物可包含基於組合物之總乾重量0.1-20%之刺槐豆膠、5-30%之菊糖及1-20%之乳糖。 本發明之組合物可進一步包含一或多種額外試劑。額外試劑可為益生菌微生物、宿主或該二者提供額外益處。舉例而言,額外試劑可為宿主提供治療性或免疫原性效應。額外試劑可選自由以下各項組成之群:維生素、抗氧化劑、微量元素、固醇、硬脂酸鎂、發煙二氧化矽、表面活性劑、肽及類固醇及其組合。 組合物可包含基於組合物之總重量之0.1-20%、0.5-20%、1-20%、0.1-10%、0.5-10%、1-10%或1-5%之額外試劑。額外試劑較佳係推薦用於特殊膳食用之試劑。 額外試劑可選自由以下組成之群:羧酸鹽、生育酚及其組合。羧酸鹽可選自由抗壞血酸鹽及檸檬酸鹽組成之群。在一些實施例中,額外試劑包含重量比為4:1至1:4之一或多種生育酚及一或多種羧酸鹽。舉例而言,額外試劑包含重量比為4:1之維生素E及抗壞血酸鈉。 在一些實施例中,組合物包含基於組合物之總重量40-80%之水解蛋白質、5-30%之二醣、5-30%之寡醣及1-10%之多醣。在較佳實施例中,組合物包含基於組合物之總重量54%之水解豌豆蛋白質、8%之乳糖、14%之菊糖及3%之刺槐豆膠。組合物可進一步包含基於組合物之總重量4%之額外試劑,該額外試劑包含重量比為4:1之維生素E及抗壞血酸鈉。 本發明之組合物可藉由此項技術中已知之技術製備。製備方法可包括諸如以下之方法:混合、冷凍、冷凍乾燥、環境空氣乾燥、真空乾燥、噴霧乾燥或其組合。所得益生菌組合物無論單獨還是整合於特殊膳食產品中,當暴露於寬範圍之溫度及濕度條件下時具有增強之活力。 用於製備組合物之益生菌微生物較佳係濃縮成固體含量為約5-30% w/v之濕糊狀稠度之發酵收穫物。益生菌濃縮物在與其他成份合併之前可呈濕的、冷凍或解凍糊狀物之形式。以呈乾燥形式之益生菌微生物開始係替代方案。 穩定益生菌組合物之製備可包括濃縮所選擇之益生菌,將成份與所濃縮之益生菌混合以形成漿液,在液氮中快速冷凍漿液以形成呈微滴、串或珠粒形式之顆粒,藉由在減壓方案下蒸發顆粒中之水分同時將熱供應至顆粒來乾燥顆粒,並然後將所得之穩定益生菌組合物包裝或組合成特殊膳食產品,其可係營養產品,例如嬰兒配方。 一種適宜混合方法可係將組合物中除益生菌微生物外之所有成份之乾混合物直接添加至包含益生菌微生物之濃縮培養物或介質溶液中以形成漿液。可將乾混合物在20-80℃下預先溶解於以濃鹼性溶液(例如,1M或5M氫氧化鈉(NaOH)溶液)調整至pH為8-9之水溶液中。在漿液中,益生菌微生物之乾重質量可佔約5-30% w/v,而乾混合物可佔約70-95%或80-90% w/v。漿液中之總固體含量可係約20-60%或30-50%。可調整乾混合物中多醣之量以達成漿液之期望黏度,以容許有效乾燥,同時避免乾燥期間可發生之橡膠樣形成或過度發泡。漿液之期望密度可藉由此項技術中已知之任何方式達成,例如,藉由在真空下脫氣或注射氣體(例如空氣、氮、二氧化碳或氬)。 可將漿液快速冷凍至約-30℃至約-180℃,或藉由霧化、滴落或注射至液氮浴中在液氮中快速冷凍。可收集呈珠粒、串或微滴形式之所得顆粒並在冷凍乾燥器或真空乾燥器中進行乾燥,或另一選擇為儲存於深冷凍器(例如,介於-30℃與-80℃之間)中以冷凍形式用於隨後使用或例如藉由冷凍乾燥或真空乾燥用於隨後乾燥。 一般而言,可用之乾燥方法技術包括在真空烘箱或離心蒸發器中冷凍乾燥或蒸發乾燥解凍之漿液,同時維持漿液或乾燥產品之溫度高於其冷凍溫度(例如,-20℃至-5℃),隨後碾磨至期望粒度。較佳地,益生菌微生物由顆粒中未結晶之非晶形材料包覆。以非晶形狀態之材料包覆益生菌微生物之優點在於增加顆粒之物理穩定性並降低顆粒內有害結晶形成。應注意,達成未結晶之非晶形結構並非長期穩定性之先決條件,此乃因一些微生物在更結晶之狀態下可能更佳。在適宜例示性實施例中,可將快速冷凍漿液以約0.1 kg/sqft至約1.5 kg/sqft之負載容量加載至托盤上,並然後立即轉移至真空乾燥室,在其中乾燥製程可以包括以下之三個主要步驟進行:(a)在小於<1000毫托(mTORR)之真空壓力下冷凍顆粒之可選短的溫度適應及結構穩定步驟,(b) 在真空及在高於顆粒冷凍點之顆粒溫度下一次乾燥或一次蒸發乾燥,及(c) 在全強度真空壓力及升高之熱源溫度下二次乾燥足以使所得乾組合物之水活性降至(例如) 0.3 Aw或更低之時間。所得乾組合物可係玻璃狀非晶形的。 術語「凍乾(lyophilization)」及「冷凍乾燥(freeze drying)」在本文中可互換使用,且係指藉由快速冷凍並在冷凍狀態下脫水(有時稱為昇華)製備呈乾燥形式之組合物。凍乾係在使得組合物中之成份結晶之溫度下進行。 如本文中所使用之術語「一次乾燥」係指在實質上低於熱源之溫度(即,熱源溫度或擱板溫度)之產物溫度下乾燥產物,以製得經一次乾燥之產物。通常,大多數一次乾燥可藉由廣泛蒸發來實施,同時產物溫度保持顯著低於熱源之溫度。 如本文中所使用之術語「二次乾燥」係指在接近熱源之溫度(即,熱源溫度或擱板溫度)之產物溫度下乾燥經一次乾燥之產物,以製得乾燥產物。此製程可在足以降低所得乾燥產物之水活性之真空下進行。在典型乾燥製程中,二次乾燥步驟將調配物之水活性降至(例如) 0.3或更低之Aw。 在一個實施例中,本發明之組合物係藉由包含以下各項之方法製備:(a) 將一或多種活益生菌微生物、一或多種水解蛋白質、一或多種二醣、一或多種寡醣及一或多種多醣合併於鹼性水溶劑中以形成漿液;(b) 將漿液在液氮中快速冷凍以形成呈珠粒、微滴或串形式之固體冷凍顆粒;(c) 固體冷凍顆粒藉由在真空下蒸發之一次乾燥步驟,同時維持顆粒之溫度高於其冷凍溫度,藉此形成一次乾燥調配物;及(d) 在全強度真空及20℃或更高之熱源溫度下將一次乾燥調配物二次乾燥達足以使一次乾燥調配物之水活性降至0.3 Aw或更低之時間,藉此製得組合物。 方法可進一步包含在步驟(a)之前使一或多種水解蛋白質、一或多種二醣、一或多種寡醣及一或多種多醣滅菌。滅菌可藉由此項技術中已知之方法達成。例如,在步驟(a)之前將水解蛋白質、二醣、寡醣及多醣之混合物在壓力下加熱,並隨後冷卻。 方法可進一步包含在步驟(a)之前使一或多種水解蛋白質、一或多種二醣、一或多種寡醣及一或多種多醣溶解。 方法可進一步包含將組合物切割、壓碎、碾磨或粉碎成自由流動之粉末。粉末之粒度可小於約10,000 μm、1,000 μm、500 μm、250 μm或100 μm,較佳小於約1,000 μm,更佳小於約250 μm。 本發明之乾組合物可直接作為薄片使用,或研磨成粉末並篩分至約1-10,000 μm、較佳地10-1,000 μm之平均粒度。 本發明之組合物可作為濃縮粉末或重構液體(例如,飲料)投與。其亦可以薄片或粉末形式併入至現有食物產品中。 方法可進一步包含用本發明之組合物製作特殊膳食產品,本發明之組合物包含在特殊膳食產品中用於向宿主提供益生菌益處之有效量之一或多種活益生菌微生物。特殊膳食產品之實例可包括嬰兒配方、較大嬰兒配方、基於經加工穀物之食品、罐裝嬰幼兒食品及用於醫療目的之特殊食品。較佳地,特殊膳食產品係嬰兒配方。 包含益生菌之所得乾穩定粉末可與熔融脂肪團聚。可將乾粉末置於40℃下之行星式混合器中並可將熔融脂肪(例如可可油、天然蠟或棕櫚油、硬脂酸、硬脂或其混合物)緩慢添加至溫熱粉末。可將混合物冷卻至低於脂肪之熔融溫度,同時繼續混合直至達成視覺上均勻大小之團聚粉末為止。所得組合物中熔融脂肪之重量質量可係約20-70%、較佳地30-50%。實例 1. 乾燥及穩定組合物之製備
將水解豌豆蛋白質(65 g, Marcor, Carlstadt, NJ)溶解於100 ml溫熱蒸餾水(75℃)中。使用20%濃NaOH溶液將豌豆溶液之pH調整至8.5。將刺槐豆膠(3 g, Tic gum, Belcamp, MD)、乳糖(10 g, Foremost Farms, Rothschild, WI)、菊糖(17 g, Cargill Minneapolis, MN)、4:1 w/w (5 g)維生素E與抗壞血酸鈉之混合物進行乾摻和並利用葉輪混合器在500 rpm連續混合下添加至豌豆溶液。在連續混合下將溶液冷卻並維持溫度介於35℃與40℃之間。 此所得穩定組合物係半透明的,具有糖漿之稠度及琥珀色。將糖漿樣溶液轉移至裝配有控制溫度夾套之雙行星式混合器(DPM, lqt, Ross Engineering, Inc. Savannah, GA)。混合器夾套溫度為37℃。在45 rpm混合下添加冷凍細菌(100g,雙歧桿菌屬)並持續2-3分鐘,或直至所有細菌充分解凍並均勻分佈。將益生菌混合物冷卻至4℃並在此溫度下保持30-60分鐘。將混合物滴入液氮浴中並於其中快速冷凍以形成冷凍珠粒,將其自液氮收穫並儲存在-80℃下用於隨後乾燥。 為進行乾燥,將冷凍珠粒以800 g/sqft之負載容量散佈於預先冷卻之托盤(-20℃)上並然後立即置於冷凍乾燥器(Model 25 SRC, Virtis, Gardiner, NY)中之擱板上。然後將真空調整至介於1800-2200毫托之間並將擱板溫度升至+20℃。將該等溫度及真空壓力設定維持12小時。在一次乾燥之前,視情況藉由施加約1000毫托之真空壓力以使冷凍珠粒之溫度適應至約-20℃,以使冷凍珠粒之溫度適應約10分鐘。然後,在一次乾燥步驟之後,將真空壓力調整至2000-2700毫托並將擱板溫度調整至+20℃。將該等溫度及真空壓力設定維持12小時。然後在全強度真空(150-200毫托)下進行二次乾燥步驟,並將擱板溫度在40℃下再維持12小時。調配物經完全乾燥,且如藉由Hygropalm Awl儀器(Rotonic Instrument Corp., Huntington, NY)所量測,其水活性為Aw 0.23。然後將乾燥材料碾磨且篩分至粒度≦ 250 μm並儲存於4℃下。實例 2. 乾益生菌組合物之儲存穩定性
將包含來自實例1之益生菌之乾穩定組合物或於10%海藻糖中之通常冷凍乾燥之細菌懸浮液之樣品置於40℃及33% RH之加速儲存條件下之乾燥器中。使用標準微生物稀釋及LMRS瓊脂平板程序,定期取樣品進行微生物CFU評價。圖1顯示在40℃及33% RH之加速儲存條件下之儲存穩定性。未保護之益生菌在加速儲存條件下之前幾週內完全喪失其活力,而本發明之益生菌之乾組合物在40℃及33% RH下84天後僅失去0.70 log單位/g。實例 3. 碳水化合物及蛋白質對儲存穩定性之效應
本發明之組合物不需要根據聯合FAO/WHO國際食品法典委員會容許用於特殊膳食用之營養化合物之推薦清單中不包括之彼等碳水化合物穩定劑。a. 包含麥芽糖糊精之組合物 :
製備含有以下之組合物:50 g豌豆蛋白質水解產物(Marcor, Carlstadt, NJ)、33 g麥芽糖糊精(Tate & Lyle, Decatur, Il)、l0 g菊糖(Cargill, Minneapolis, MN)、2 g刺槐豆膠(Tic gum, Belcamp, MD)及5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)。將豌豆蛋白質水解產物溶解於100克蒸餾水中並將pH調整至7.5。乾摻和碳水化合物並添加至豌豆蛋白質溶液。將混合物加熱至70℃以溶解所有化合物並將溶液冷卻至37℃。將鼠李糖乳桿菌之冷凍珠粒(100 g)添加至溶液並如實例1中所述乾燥漿液。乾組合物中活細菌之初始計數係9.92 log CFU/g。將此產物之樣品置於如實例1中所述之加速穩定性挑戰下。樣品在1個月後具有1.19 log單位/g損失,因此未能通過挑戰。b. 包含小麥蛋白質分離物之組合物 :
製備包含以下之另一組合物:25 g豌豆蛋白質水解產物
(Marcor, Carlstadt, NJ)、33 gProlite
200 (小麥蛋白質分離物,Archer Daniels Midland Company Decatur, IL)、25 g麥芽糖糊精(Tate & Lyle, Decatur, IL)、l0 g菊糖(Cargill, Minneapolis, MN)、2 g刺槐豆膠(Tic gum, Belcamp, MD)及5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)之。如上文實例1及2中所述製備含有鼠李糖乳桿菌屬之組合物並進行乾燥。乾組合物中活細菌之初始計數係10.17 log CFU/g。將此產物之樣品置於如實例1中所述之加速穩定性挑戰下。樣品在2週後產生1.71 log單位/g之損失,因此未能通過挑戰測試。c. 包含 乳清蛋白質分離物之組合物:
製備含有以下之另一組合物:25 g豌豆蛋白質水解產物(Marcor, Carlstadt, NJ)、25 g乳清蛋白質分離物(Davisco, Eden Prairie, MN)、33 g麥芽糖糊精(Tate & Lyle, Decatur, IL)、10 g菊糖(Cargill, Minneapolis, MN)、2 g刺槐豆膠(Tic gum, Belcamp, MD)及5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)。如上文實例1及2中所述製備含有鼠李糖乳桿菌屬之組合物並進行乾燥。乾組合物中活細菌之初始計數係10.40 log CFU/g。將此產物之樣品置於如實例1中所述之加速穩定性挑戰下。樣品在2週後產生1.30 log單位/g之損失,因此未能通過挑戰測試。d. 包含乳糖之組合物 :
製備含有以下之另一組合物:50 g豌豆蛋白質水解產物(Marcor, Carlstadt, NJ)、33 g乳糖(Foremost Farms, Rothschild, WI)、10 g菊糖(Cargill, Minneapolis, MN)、2 g刺槐豆膠(Tic gum, Belcamp, MD)及5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)。如上文實例1及2中所述製備含有鼠李糖乳桿菌屬之組合物並進行乾燥。乾組合物中活細菌之初始計數係9.77 log CFU/g。將此產物之樣品置於如實例1中所述之加速穩定性挑戰下。樣品通過第1個月之穩定性但在2個月後具有1.89 log單位/g之損失,因此未能通過挑戰測試。e. 本發明之組合物:
製備含有以下之組合物:65 g豌豆蛋白質水解產物
(Marcor, Carlstadt, NJ)、l0 g乳糖(Foremost Farms, Rothschild, WI)、17 g菊糖(Cargill, Minneapolis, MN)、3 g刺槐豆膠(Tic gum, Belcamp, MD)及5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)。如上文實例1及2中所述製備含有雙歧桿菌屬之組合物並進行乾燥,唯將豌豆蛋白質水解產物溶液之pH調整至pH 8.5。乾組合物中活細菌之初始計數係10.87 log CFU/g。將此產物之樣品置於加速穩定性挑戰下。樣品在3個月後僅損失0.70 log單位/g,因此,通過挑戰測試。實例 4. 菊糖對於儲存穩定性之效應
美國標準法典72容許在嬰兒配方中使用寡醣(例如菊糖及麥芽糖糊精)。評估組合物中0%至30%菊糖含量之效應。製備含有以下之組合物:50 g豌豆蛋白質水解產物(Marcor, Carlstadt, NJ)、43 g乳糖(Foremost Farms, Rothschild, WI)、2 g刺槐豆膠(Tic gum, Belcamp, MD)、5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)及0 g、10 g、15 g或30 g之菊糖(菊糖之添加量係自組合物中乳糖之量減去)。如上文實例1及2中所述製備含有嗜酸乳桿菌屬之組合物並進行乾燥。包含0 g、10 g、15 g及30 g菊糖之乾組合物中活細菌之初始計數分別係9.42、9.57、9.59及9.80 log CFU/g。將每一組合物之樣品置於加速穩定性挑戰下。包含0 g菊糖(無寡醣)之樣品在2個月後損失1.14 log單位/g且在3個月後損失2.25 log單位/g,而包含15 g及更高之菊糖之樣品在3個月後損失小於2 log單位/g,因此,確定本發明之組合物中之菊糖之最小量必須高於10%。實例 5. 二醣對於儲存穩定性之效應
美國標準法典72在嬰兒配方中限制海藻糖之使用但允許使用蔗糖及乳糖。以下實例展示,根據本發明之組合物成功地以增加量之水解豌豆蛋白質及僅少量二醣乳糖替代海藻糖。a. 包含海藻糖之組合物:
製備含有以下之組合物:25 g豌豆蛋白質水解產物(Marcor, Carlstadt, NJ)、62 g 海藻糖
(Cargill, Minneapolis, MN)、5 g菊糖(Cargill, Minneapolis, MN)、3 g刺槐豆膠(Tic gum, Belcamp, MD)及5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)。如上文實例1及2中所述製備含有嗜酸乳桿菌屬之組合物並進行乾燥。乾組合物中活細菌之初始計數係9.49 log CFU/g。將組合物之樣品置於加速穩定性挑戰下。樣品在3個月後損失0.83 log單位/g,因此,展示相對良好之穩定性。然而,在嬰兒配方中不允許使用海藻糖。b. 以乳糖替代海藻糖之效應:
製備含有50 g豌豆蛋白質水解產物(Marcor, Carlstadt, NJ)、2 g刺槐豆膠(Tic gum, Belcamp, MD)、5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)及33 g
乳糖(Foremost Farms, Rothschild, WI)、10 g菊糖(Cargill, Minneapolis, MN)或13 g及30 g菊糖之組合物。如上文實例1及2中所述製備含有嗜酸乳桿菌屬之組合物並進行乾燥。包含33 g或13 g乳糖之乾組合物中活細菌之初始計數分別係9.57 log CFU/g及9.80 log CFU/g。將每一組合物之樣品置於加速穩定性挑戰下。包含33 g乳糖之樣品在1個月之後損失1.25 log單位/g,而包含13 g乳糖之樣品持續2個月,因此,確定穩定化組合物中13%之乳糖且伴有50%之水解豌豆蛋白質可有效替代海藻糖。c. 不含海藻糖及海藻酸鹽之組合物之穩定性:
為評估在具有超過50%二醣之組合物中替代不期望用於嬰兒配方之成份(例如,海藻糖及海藻酸鹽)之效應,製備含有以下之三(3)種組合物:25 g豌豆蛋白質水解產物(Marcor, Carlstadt, NJ)、5 g菊糖(Cargill, Minneapolis, MN)、5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)、3 g海藻酸鹽(ISP Corp., Wayne, NJ)或3 g刺槐豆膠(Tic gum, Belcamp, MD)及62 g海藻糖(Cargill, Minneapolis, MN)或62 g乳糖
(Foremost Farms, Rothschild, WI) (表1)。如上文實例1及2中所述製備含有鼠李糖乳桿菌屬之組合物並進行乾燥。三種乾組合物中活細菌之初始計數係約10 log CFU/g (表1)。使每一組合物之樣品經受加速穩定性挑戰。僅包含海藻酸鹽及海藻糖二者之組合物在84天之後展示小於1 log單位/g之損失,而另兩種其中海藻糖及海藻酸鹽分別經乳糖及刺槐豆膠替代之組合物僅28天之後即損失大於1 log單位/g。因此,為達成在40℃及33%相對濕度下3個月之後小於1 log單位/g之損失之穩定性要求,海藻糖及海藻酸鹽在通常具有超過50%二醣之組合物中係必要成份。 表1. 海藻糖及海藻酸鹽替代對於益生菌穩定性之效應 實例 6. 多醣對於儲存穩定性之效應
多醣在益生菌組合物中提供必要之結構支撐。美國標準法典72在嬰兒配方中限制若干種多醣(例如海藻酸鹽)之使用,但允許使用瓜爾膠、刺槐豆膠及澱粉。海藻酸鈉、瓜爾膠、刺槐豆膠及澱粉係商業購得之多醣,且對其在穩定化本發明之組合物中之效應進行評估。a. 包含瓜爾膠之組合物:
製備含有36 g酪蛋白水解產物(DMV International Nutritionals, Delhi, NY)、25 g菊糖(Cargill, Minneapolis, MN)、 36 g 蔗糖
(Sigma)及3 g瓜爾膠(Tic gum, Belcamp, MD)之組合物。如上文實例1及2中所述製備含有鼠李糖乳桿菌屬之組合物並進行乾燥。乾組合物中活細菌之初始計數係9.90 log CFU/g。將此產物之樣品置於如實例1中所述之加速穩定性挑戰下。樣品通過第1個月之穩定性,但在2個月之後具有1.10 log單位/g之損失,因此未能通過挑戰測試。b. 包含海藻酸鈉之組合物:
製備含有17 g酪蛋白水解產物(DMV International Nutritionals, Delhi, NY)、5 g菊糖(Cargill, Minneapolis, MN)、75 g蔗糖(Sigma)及3 g海藻酸鈉(ISP Corp., Wayne, NJ)之組合物。如上文實例1及2中所述製備含有鼠李糖乳桿菌屬之組合物並進行乾燥。乾組合物中活細菌之初始計數係9.90 log CFU/g。將此產物之樣品置於如實例1中所述之加速穩定性挑戰下。樣品通過第1個月之穩定性,但在2個月之後具有1.25 log單位/g之損失,因此未能通過挑戰測試。c. 包含刺槐豆膠之組合物:
製備含有以下之組合物:25 g豌豆蛋白質水解產物(Marcor, Carlstadt, NJ)、5 g菊糖(Cargill, Minneapolis, MN)、62 g海藻糖(Cargill, Minneapolis, MN)、3 g刺槐豆膠(Tic Gums, Belcamp, MD)及5 g維生素E與抗壞血酸鈉之混合物(4:1 w/w)。如上文實例1及2中所述製備含有鼠李糖乳桿菌屬之組合物並進行乾燥。乾組合物中活細菌之初始計數係9.93 log CFU/g。將此產物之樣品置於如實例1中所述之加速穩定性挑戰下。樣品通過2個月之穩定性,其中具有0.81 log單位/g損失。此確定刺槐豆膠可在穩定化之益生菌組合物中替代海藻酸鹽功能。實例 7. 嬰兒配方
根據實例1製備包含雙歧桿菌屬之乾穩定組合物,之後篩分為兩個粒度群組(高於50 μm及低於250 μm)。藉由將99.9 g嘉寶(Gerber Good Start) (Nestle Infant Nutrition, Florham Park, NJ.)與0.1 g大小範圍介於50 μm與250 μm之間之乾組合物顆粒混合來製備包含益生菌之嬰兒配方。最終產物含有約108
CFU之鼠李糖乳桿菌(Lactobacillus
GG) / 100 g嬰兒配方。將益生菌嬰兒配方裝入180 cc HDPE瓶中,並暴露於40℃/33% RH之控制溫度/濕度。產物每個月進行微生物學穩定性測試經12個月之時期,或直至觀察到分析計數減少低於5 × 107
/單位劑量為止。實例 8. 益生菌補充劑
根據實例1製備包含嗜酸乳桿菌之乾穩定組合物並調配為口服劑型(例如錠劑、膜衣錠或膠囊)。藉由在旋轉機器上使用1/2"圓形標準凹形工具直接壓縮來製備含有99.9 g壓縮劑(右旋糖)及0.1 g大小範圍介於50 μm與250 μm之間之乾調配物顆粒之橙味錠劑。最終產物含有約108
CFU/單位劑量。錠劑之硬度在8-10 kp範圍內且崩解時間係大約20秒。將壓縮錠劑以每瓶100片錠劑裝入180 cc HDPE瓶中並暴露於40℃/33% RH之控制溫度/濕度。產物每個月進行微生物學穩定性測試經12個月之時期,或直至觀察到分析計數減少低於1 × 106
/單位劑量為止。實例 9. 功能性飲料
根據實例1製備包含嗜酸乳桿菌之乾穩定組合物並調配為含有以下各項(重量%)之乾混合物:71%蔗糖、14%麥芽糖糊精、10%菊糖、2%右旋糖、1%無水檸檬酸、0.3%阿拉伯樹膠、0.3%矯味劑、0.3%磷酸三鈣及0.1%大小範圍介於50 μm與250μm之間之乾益生菌組合物顆粒(嗜酸乳桿菌)。最終產物含有約109
cfu/單位劑量(30 g乾混合物)。將產物裝入小鋁箔袋(30 g單位劑量/袋)中,藉由攪拌於340毫升水中飲用。飲料乾混合物中之益生菌之穩定性每個月進行微生物學穩定性測試經12個月之時期,或直至觀察到分析計數減少低於1 × 107
/單位劑量為止。實例 10. 多種維生素 / 益生菌錠劑
如實例1中所述製造十(10) g乾粉末組合物。為製錠,將乾燥及穩定之益生菌組合物(100 mg)與400 mg含有2% w/w硬脂酸鎂及2% w/w親水性發煙二氧化矽(AEROSIL® 200, Evonik Industries)之商業購得之綜合維生素粉末(Centrum®, Pfizer)混合並在手持式壓丸設備(使用½"錠劑直徑外殼)中壓縮。每一錠劑含有約107
cfu/錠劑。將錠劑以每瓶100片錠劑裝入180 cc HDPE瓶中並暴露於40℃/33% RH之控制溫度/濕度。瓶每個月進行微生物學穩定性測試經12個月之時期或直至觀察到分析計數減少低於1 × 106
/錠劑為止。 當提及可量測值(例如量、百分比及諸如此類)時,如本文中所使用之術語「約」意欲涵蓋自指定值±20%或±10%、更佳地±5%、甚至更佳地±1%且再更佳地±0.1%之變化,此乃因此等變化係適當的。 本文中所引用之所有文件、書籍、手冊、論文、專利、公開之專利申請案、指南、摘要及其他參考文獻均以整體引用的方式併入。熟習此項技術者將在考量本說明書及實踐本文中所揭示之本發明後明瞭本發明之其他實施例。意欲將本說明書及實例僅視為例示性的,其中本發明之真正範圍及精神係由以下申請專利範圍指示。
圖1顯示實例2之樣品在40℃及33% RH之加速儲存條件下之儲存穩定性。
Claims (29)
- 一種乾組合物,其包含一或多種活益生菌微生物、一或多種水解蛋白質、一或多種二醣、一或多種寡醣及一或多種多醣,其中該組合物不包含海藻糖,其中該組合物具有至少1×1010 CFU/g之活力,且其中該組合物在40℃之溫度及33%之相對濕度下3個月後具有小於1 log單位/g之活力損失。
- 如請求項1之組合物,其中該組合物在特殊膳食產品中向宿主提供益生菌益處。
- 如請求項2之組合物,其中該特殊膳食產品係選自由以下組成之群:嬰兒配方、較大嬰兒配方、基於經加工穀物之食品、罐裝嬰幼兒食品及用於醫療目的之特殊食品。
- 如請求項2之組合物,其中該特殊膳食產品係嬰兒配方。
- 如請求項1之組合物,其中該活益生菌微生物係選自由以下組成之群:活的益生菌、真菌及酵母。
- 如請求項1之組合物,其中該組合物包含基於該組合物之總乾重量至少50%之該一或多種水解蛋白質。
- 如請求項1之組合物,其中該一或多種水解蛋白質係選自由以下組成之群:乳蛋白質、植物蛋白質及其組合。
- 如請求項1之組合物,其中該一或多種水解蛋白質係選自由以下組成之群:水解酪蛋白、水解乳清蛋白質、水解豌豆蛋白質、水解大豆蛋白質及其組合。
- 如請求項1之組合物,其中該組合物包含基於該組合物之總乾重量少於20%之該一或多種二醣。
- 如請求項1之組合物,其中該一或多種二醣係選自由以下組成之群:蔗糖、乳糖及其組合。
- 如請求項1之組合物,其中該組合物包含基於該組合物之總乾重量5%至30%之該一或多種寡醣。
- 如請求項1之組合物,其中該一或多種寡醣係選自由以下組成之群:菊糖、麥芽糖糊精、右旋糖酐、果寡醣(FOS)、半乳寡醣(GOS)、甘露寡醣(MOS)及其組合。
- 如請求項1之組合物,其中該組合物包含基於該組合物之總乾重量1%至10%之該一或多種多醣。
- 如請求項1之組合物,其中該一或多種多醣係選自由以下組成之群:角叉菜膠、瓜爾膠、阿拉伯樹膠、刺槐豆膠、澱粉、改質澱粉及其組合。
- 如請求項1之組合物,其進一步包含一或多種額外試劑。
- 如請求項15之組合物,其中該組合物包含基於該組合物之總重量0.5%至10%之該一或多種額外試劑。
- 如請求項15之組合物,其中該一或多種額外試劑係選自由以下組成之群:羧酸鹽、生育酚及其組合。
- 如請求項17之組合物,其中該羧酸鹽係選自由抗壞血酸鹽及檸檬酸鹽組成之群。
- 如請求項15之組合物,其中該一或多種額外試劑包含重量比為1:4至4:1之一或多種生育酚及一或多種羧酸鹽。
- 如請求項15之組合物,其中該一或多種額外試劑包含重量比為4:1之維生素E及抗壞血酸鈉。
- 一種製備如請求項1之組合物之方法,其包含一或多種選自由以下各項組成之群之乾燥方法:空氣乾燥、真空乾燥、流化床乾燥及噴霧乾燥。
- 一種製備如請求項1之組合物之方法,其包含: (a) 將該一或多種活益生菌微生物、該一或多種水解蛋白質、該一或多種二醣、該一或多種寡醣及該一或多種多醣合併於鹼性水溶劑中以形成漿液; (b) 將該漿液在液氮中快速冷凍以形成呈珠粒、微滴或串形式之固體冷凍顆粒; (c) 該等固體冷凍顆粒藉由在真空下蒸發同時維持該等顆粒之溫度高於其冷凍溫度之一次乾燥步驟,藉此形成一次乾燥調配物;及 (d) 在全強度真空及20℃或更高之熱源溫度下將該一次乾燥調配物二次乾燥足以使該一次乾燥調配物之水活性降低至0.3 Aw或更低之時間,藉此製備如請求項1之組合物。
- 如請求項22之方法,其進一步包含在步驟(a)之前將該一或多種水解蛋白質、該一或多種二醣、該一或多種寡醣及該一或多種多醣滅菌。
- 如請求項22之方法,其進一步包含將該組合物切割、壓碎、碾磨或粉碎成自由流動之粉末。
- 如請求項24之方法,其中該粉末之粒度係小於約1000 μm。
- 如請求項22之方法,其中該組合物有效量之該一或多種活益生菌微生物用於在特殊膳食產品中向宿主提供益生菌益處。
- 如請求項26之方法,其進一步包含利用該組合物製作該特殊膳食產品。
- 如請求項26之方法,其中該特殊膳食產品係選自由以下各項組成之群:嬰兒配方、較大嬰兒配方、基於經加工穀物之食品、罐裝嬰幼兒食品及用於醫療目的之特殊食品。
- 如請求項26之方法,其中該特殊膳食產品係嬰兒配方。
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EP3328215A4 (en) | 2019-03-06 |
BR112018001784A2 (pt) | 2018-09-11 |
TWI705766B (zh) | 2020-10-01 |
CN108347982A (zh) | 2018-07-31 |
EP3328215B1 (en) | 2021-07-07 |
MY194231A (en) | 2022-11-23 |
AU2016297986B8 (en) | 2020-06-11 |
US20180221422A1 (en) | 2018-08-09 |
ES2881809T3 (es) | 2021-11-30 |
WO2017019273A1 (en) | 2017-02-02 |
JP2018521672A (ja) | 2018-08-09 |
PT3328215T (pt) | 2021-08-03 |
KR20180080177A (ko) | 2018-07-11 |
AU2016297986A1 (en) | 2018-02-22 |
BR112018001784B1 (pt) | 2022-05-10 |
AR105464A1 (es) | 2017-10-04 |
EP3328215A1 (en) | 2018-06-06 |
CA2994112C (en) | 2023-08-08 |
US10953050B2 (en) | 2021-03-23 |
CA2994112A1 (en) | 2017-02-02 |
JP6889699B2 (ja) | 2021-06-18 |
AU2016297986B2 (en) | 2020-05-28 |
DK3328215T3 (da) | 2021-09-13 |
PL3328215T3 (pl) | 2021-12-13 |
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