TWI755617B - 益生菌組合物之製造方法及其用途 - Google Patents
益生菌組合物之製造方法及其用途 Download PDFInfo
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- TWI755617B TWI755617B TW108125679A TW108125679A TWI755617B TW I755617 B TWI755617 B TW I755617B TW 108125679 A TW108125679 A TW 108125679A TW 108125679 A TW108125679 A TW 108125679A TW I755617 B TWI755617 B TW I755617B
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- Prior art keywords
- solution
- probiotic composition
- strains
- calcium
- feed
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Abstract
本發明公開了一種益生菌組合物之製造方法,包括:一菌液,係希瓦氏菌之菌液,該菌液與該海藻酸鈉溶液或海藻酸溶液混合;及加入一鈣離子溶液中,使其固定成形。本發明功效主要顯現在於:本技術具有在室溫下長期保存和耐高溫的功效,可適用於一般菌株,而不只侷限於可形成內孢子的少數菌種或是必須將菌株冷凍保存再活化。本發明之方法可應用於製造水產飼料、動物飼料或補充人類所需之益生菌。
Description
本發明涉及一種繁殖,維持或保存微生物或其組合物之方法,尤其是一種保存或維持活的微生物;另涉及一種動物飼料的成型或建立,尤其是透過聚結成型;透過製粒成型。
一種乳酸菌及其用途,可以於養殖環境中維持生長、有助於魚類之生長及免疫力提升及提供一種乳酸益生菌於水產養殖之用途,係將乳酸益生菌用於提升養殖生物之生長及免疫力,以提升養殖魚類之收成,唯仍有實際使用上的限制,由於益生菌無法耐高溫,無法將益生菌菌株直接放入飼料的製程,而須待飼料製造後再進行後噴,將益生菌噴灑於飼料的表面上,但這是費時又費工,且會增加飼料的成本,且也需要考量當飼料一但碰到水時,其噴灑在表面的益生菌會有多少保留在飼料上面,及室外酷熱環境等因素,正是目前需要解決的重要問題。
於養殖環境中,抗生素濫用會使致病菌的耐藥性增加,嚴重破壞了養殖水體中正常細菌相的平衡,無論是在水產養殖生產和水產品質安全上皆帶來極大的憂慮。若為了減少因氨氮及亞硝氮等危害,在養殖過程中而採用的方式有換水、曝氣等來處理,不過由於這些方法的作用效果持續時間短且成本高,具有很大的侷限性。
若要長期室溫保存益生菌,目前有2種方法,一是可形成內孢子的細菌,只有少數菌種可以,二是冷凍乾燥,此種方法需要保護劑,但不是所有的細菌都有適合的冷凍保護劑可以使用。
一般飼料在製作過程,至少要加熱至80℃以上,甚至到120℃以上,益生菌無法耐高溫,可將益生菌誘導形成內孢子,再進入製程,但只有少數菌種可以達到。
透過益生菌與藻酸鹽基質,再利用植物油和聚山梨醇酯80(Tween-80)將此混合液微膠囊化,此微膠囊僅可用於增強在酸和膽汁中以及短暫加熱的存活。
本發明所欲解決的問題:
1.益生菌長期保存的問題:若要室溫長期保存益生菌,目前有2種方法,一是可形成內孢子的細菌,只有少數菌種可以,二是冷凍乾燥,此種方法需要保護劑,但不是所有的細菌,都有適合的保護劑可以使用。
2.益生菌耐高溫的問題:飼料在製作過程,至少要加熱至80℃以上,甚至到120℃以上,因此若要在飼料中加入益生菌,目前有2種方法,一是將益生菌誘導形成內孢子,再進入製程,但只有少數菌種可以達到,二是飼料製作完成後,才再將益生菌噴在飼料上面,但這是費時又費工,並且會增加飼料的成本。
3.在水產養殖方面的問題,影響水產養殖產量的重要因素為疾病,此原因使得造成大量損失,因此當養殖業面臨疾病問題時,養殖業常使用抗生素
加以治療,但可能會導致具有抗藥性的菌株出現,來自養殖生物的特定抗藥性的基因能夠以特殊方式進入與人體健康有關的細菌中,後續並透過食物鏈的方式,具有抗藥性的細菌也會危害到食物鏈後端的人類們,因此進而造成人類的健康問題,故必須尋求其他方式以維持養殖生物之健康,而益生菌是一項值得關注的方向。
為達成上述的目的,本發明公開了一種益生菌組合物之製造方法,包括:一菌液,係細菌或布拉酵母菌一種或一種以上菌液,該菌液與該海藻酸鈉溶液或海藻酸溶液係以5:1至1:10之間的重量百分比混合;及加入一鈣離子溶液中,直至使其固定成形。
如上所述,其中該海藻酸鈉或海藻酸佔該溶液比例為1%~10%重量百分比。
如上所述,其中鈣離子固形物佔該鈣離子溶液比例為0.5%~5%重量百分比。
如上所述,其中該細菌係希瓦氏菌(Shewanella sp.)、潘朵拉菌(Pantoea sp.)、假單胞菌(Pseudomonas sp.)、光合菌、硝化菌、乳酸菌(Lactic acid bacteria)、雙歧桿菌(Bifidobacterium sp.)或芽孢桿菌(Bacillus sp.)。
如上所述,其中該鈣離子溶液係氯化鈣溶液、乳酸鈣溶液、碳酸鈣溶液、醋酸鈣溶液、檸檬酸鈣溶液或草酸鈣溶液其中一種以上之任意組合。
為達成上述的目的,本發明另公開了一種益生菌組合物,係
由上述益生菌組合物之製造方法所製得之物。
如上所述,該益生菌組合物一種用於飼料的用途,經製造為粒狀物後加入一般飼料製程中製作成為飼料。
如上所述,該益生菌組合物一種用於口服益生菌的用途,經製造成形為粒狀物供人類食用。
如上所述,該益生菌組合物於常溫下保存菌株的用途,其中益生菌組合物菌株於常溫15~35℃下保存36個月,仍有菌株存活。
如上所述,該益生菌組合物於高溫處理下保存菌株的用途,經121℃處理6分鐘、95℃處理10分鐘或65℃處理24小時,其中益生菌組合物菌株經高溫處理後仍有菌株存活。
本發明的功效主要顯現在於:在先前技術中,僅能讓可形成內孢子的少數菌種之益生菌具有在室溫下長期保存和耐高溫的特性,其他一般菌株皆須以冷凍並輔以抗凍劑做保存其菌株存活。而本發明克服先前技術中存在的問題點或困難性,可以讓所有細菌菌株及布拉酵母菌具有可在室溫下長期保存和耐高溫的特性。
水產養殖環境大多高溫,夏季溫度有可能高到40度,許多菌株放在室溫後,菌種就會死亡,透過本發明之益生菌組合物除了應用於可形成內孢子的細菌外,亦可使用於不具內孢子的細菌,並且經由本發明的益生菌組合物之製造方法使得益生菌不僅可在常溫保存36個月,亦可克服在飼料製作需80℃以上,更甚至到120℃以上的加熱程序,又或者是酷熱的室外環境,亦不需使用冷凍保存。因此,應用本發明將益生菌組合物混入
飼料作為活微生物飼料的添加物,或經由讓水產動物透過食入帶有益生菌之活微生物飼料的添加物,比現有方式可更有效用來增強動物或人們的免疫力、調整腸道機能、減少抗生素及藥物使用。
S100~S400:步驟
圖1、本發明益生菌組合物之製造方法流程圖
表1、益生菌在水產養殖中的應用
表2、液態希瓦氏菌菌液及希瓦氏菌乾燥菌球釋放存活菌數之測試
飼料營養是增進水中生物成長及維持其健康的重要因素,必須營養素如產能營養素(如蛋白質與胺基酸、碳水化合物、脂質與必需脂肪酸)、維生素(維生素A、維生素C、維生素E)、礦物質(磷、鐵、硒),另有其他非營養物質如免疫賦活劑(疫苗、佐劑、葡萄聚醣、核苷酸、動物性萃取物、植物性萃取物、左美素)及益生菌。
微生物製劑由單一或複合式的益生菌組成,益生菌(probiotics),是可以促進腸道菌種的平衡,增加宿主健康的活的微生物,目前廣泛的定義為應用到人類或其他動物,藉由改善腸內微生物相平衡而有益於宿主的活菌,不論是單一或混合菌株,都可視為益生菌。
益生菌可用做為一種生物製劑,藉由投餵方式來改善腸胃道的菌相、酵素消化作用、病源微生物的抑制、抗突變和抗癌的活化、增加免疫反應等。
表1中列出水中的益生菌(Martinez Cruz et al.,2012),定義為能分泌有利於宿主的抗菌物質、與病原菌互相競爭排斥、並且能改善飼料利用率及提供營養價值,也運用在改善水質及增加抗病能力等方面。目前可用在水產養殖上的益生菌種類有許多,並有研究表示特定的益生菌株甚至可穩定水質,讓養殖生物可以增加其換肉率,或提高抗病能力等效果。
在大面積養殖環境中,飼料中添加抗生素是一種控制疾病最有效的策略,施以藥物可用於診斷、預防或治療疾病。
本發明提供一種益生菌組合物之製造方法,將細菌或布拉酵母菌一種或一種以上菌液與一海藻酸鈉或海藻酸溶液,菌液與海藻酸鈉溶液或海藻酸溶液係以5:1至1:10之間的重量百分比混合(S100),及加入
一鈣離子溶液(S200),持續滴加至鈣離子溶液中直至使其固定成形(S300),亦可進一步進行乾燥程序(S400)。
實施例1
本發明提供一種益生菌組合物之製造方法,將布拉酵母菌菌液與一海藻酸鈉溶液,其中海藻酸鈉佔該海藻酸鈉溶液比例為1%~10%重量百分比,鈣離子固形物佔該鈣離子溶液比例為0.5%~5%重量百分比,該菌液與該海藻酸鈉溶液係以2:1的重量百分比混合,持續滴加至鈣離子溶液中直至使其固定成形,並進行乾燥程序。
實施例2
本發明提供一種益生菌組合物之製造方法,將潘朵拉菌、乳酸菌及酵母菌布拉酵母菌菌液與一海藻酸鈉溶液,該三種菌液混合後與該海藻酸溶液係以1:3的重量百分比混合,另外添加養殖所需的產能營養素(礦物質磷)及其他非營養物質(動物性萃取物,如魚粉、肉骨粉、扇貝萃取物),持續滴加至鈣離子溶液中直至使其固定成形,亦可進一步進行乾燥程序。
本發明的益生菌組合物之製造方法,其中海藻酸鈉或海藻酸佔該溶液比例為1%~10%重量百分比,較佳為1%~6%重量百分比,最佳為1%~5%。
本發明的益生菌組合物之製造方法,其中鈣離子固形物佔該鈣離子溶液比例為0.5%~5%重量百分比,較佳為1~3%重量百分比,最佳為1.5~2.5%重量百分比。
目前商業用途較常使用的益生菌,包含細菌類有二種,即嗜酸性乳酸菌(Lactobacillus acidophilus)及比菲德氏菌(Bifidobacterium,即Bifidus,又稱雙叉桿菌或是雙歧桿菌);另外,酵母菌(yeast)則有一種有益的酵母菌屬的布拉酵母菌(學名:Saccharomyces boulardii)可以用來治療一些由細菌所導致的腹瀉和感染性腸炎。
本發明所述的菌液係包含適用於所有的細菌,另適用於布拉酵母菌,因此可將任一種細菌與布拉酵母菌菌液混合使用,或任兩種細菌混合使用,又或者是將任兩種以上細菌與布拉酵母菌菌液混合使用。
如上所述,較佳為使用希瓦氏菌、潘朵拉菌、假單胞菌、光合菌、硝化菌、乳酸菌、雙歧桿菌、芽孢桿菌及布拉酵母菌。
希瓦氏菌(Shewanella sp.),為革蘭氏陰性桿菌,屬兼性厭氧菌,無孢子產生,周身纖毛且具單鞭毛,長度2~3μm,直徑0.4~0.7μm。
本發明的益生菌組合物之製造方法,其中該鈣離子溶液係氯化鈣溶液、乳酸鈣溶液、碳酸鈣溶液、醋酸鈣溶液、檸檬酸鈣溶液或草酸鈣溶液其中一種以上之任意組合,因此益生菌組合物之製造方法的過程中可同時混合使用不止一種鈣溶液,使得該菌液與該海藻酸鈉溶液或海藻酸溶液經混合之海藻酸鈉菌液或海藻酸菌液後,滴入混合之鈣溶液時可固定成形,亦可進一步進行乾燥程序。
人體的健康與腸道內的益生菌群結構息息相關,菌群中不同種類之間,菌群與宿主之間,菌群、宿主與環境之間,某一種或複數種微生物當餵食予人類或動物時可增進其腸內菌叢之品質,若菌群結構相對穩
定,本發明益生菌組合物中為優勢菌株,對在宿主中的表現有提高作用。
該鈣離子溶液較佳實施方式可另經過殺菌程序以提高本發明益生菌組合物在宿主中為優勢菌株。
如上所述,該鈣離子溶液較佳可選用氯化鈣溶液。
LB培養基(Luria-Bertani broth,LB),是微生物學實驗中最常使用之營養性培養基,用於培養大腸桿菌等細菌,分為液態培養基和加入瓊脂而製成的固態培養基。
磷酸鹽緩衝生理鹽水(Phosphate buffered saline,簡稱為PBS),具有維持滲透壓的能力,且對細胞無毒性,是生物學研究中常用的緩衝液,10倍(10X)PBS為常見的磷酸鹽緩衝生理鹽水濃縮配製方法。
菌液包埋方式為透過將菌液及海藻酸鈉溶液或海藻酸溶液之混合溶液,利用重力、噴霧或其他一般方式向下滴入鈣離子溶液後可固定成形為顆粒狀,或也可利用滴入鈣離子溶液中之特殊模具而固定成形為不特定形狀,另也可先將菌液及海藻酸鈉溶液或海藻酸溶液之混合溶液先裝入模具後再置入鈣離子溶液固定成形。
如上所述,菌液及海藻酸鈉溶液或海藻酸溶液之混合溶液較佳可選用調控液滴大小滴下至鈣離子溶液固定成形。
如上所述,菌液及海藻酸鈉溶液或海藻酸溶液之混合溶液較佳可選用等速滴下至鈣離子溶液固定成形。
實施例3
本發明將3%海藻酸鈉溶液和水產用益生菌液以重量百分
比1:2的比例混合均勻,益生菌液如:希瓦氏菌、潘朵拉菌、假單胞菌、乳酸菌、雙歧桿菌、酵母菌或芽孢桿菌等,再滴入2.5%氯化鈣溶液中,使其固定成形後進行乾燥,加入一般飼料製程中製作成為水產養殖飼料。
實驗方法
希瓦氏菌菌液培養
(1)藥品配製:
實驗藥品:LB培養基、瓊脂(Agar)、海藻酸鈉、氯化鈣、PBS(10X)pH 7.4
LB培養基:用一次水配製含有10g/L胰蛋白腖(Tryptone)和5g/L酵母粉(Yeast extract)及5g/L氯化鈉(NaCl)。
(2)實驗步驟:
挑希瓦氏菌單一菌落置入5ml LB培養基,以37℃震盪培養16小時,將5ml倒入1L LB培養基,37℃震盪培養16小時,以分光光度計於波長600nm,利用LB培養基將菌液稀釋至吸光值為1.8,即得菌液。
益生菌菌液包埋
(1)藥品配製:
(a)海藻酸鈉溶液:取30g海藻酸鈉溶於1000ml一次水中。
(b)氯化鈣溶液:取25g氯化鈣溶於1000ml一次水中。
(2)實驗步驟:
各取出菌液及海藻酸鈉溶液以1:2重量百分比混合均勻後,利用重力滴入下方氯化鈣溶液中,使其固定成形,以37℃烘乾,做進
行測試。
耐熱性測試
(1)藥品配置:
(a)LB培養基:用一次水配置含有10g/L胰蛋白腖和5g/L yeast酵母粉及5g/L氯化鈉的LB培養基。
(b)LB平盤培養基:8g LB培養基與6g瓊脂混合,以一次水定量至400ml,滅菌後倒入培養皿後備用。
(c)1×PBS:利用10X PBS(1.37M NaCl、0.1M Na2HPO4、27mM KCl、18mM KH2PO4 pH7.4)以一次水稀釋至1倍。
(2)實驗步驟:
將經過乾燥後的希瓦氏菌乾燥菌球在室溫存放一年後,將一顆透過益生菌菌液包埋的乾燥菌球及1mL的菌液以121℃處理6分鐘後,將乾燥菌球置入1mL 1×PBS,分別於0、0.5、1、1.5、2、2.5、3、3.5及4小時,分別取100μL塗LB平盤培養基,以37℃培養24小時後,計算菌落數。
希瓦氏菌(液):為液態希瓦氏菌菌液;希瓦氏菌(乾):為希瓦氏菌乾燥菌球。
實驗結果
實施例4
透過本發明上述益生菌組合物之製造方法,透過一菌液,係細菌或布拉酵母菌一種或一種以上菌液,該菌液與該海藻酸鈉溶液或海藻酸溶液係以5:1至1:10之間的重量百分比混合;及加入該鈣離子溶液中,直至使其固定成形可製造而成本發明之益生菌組合物。
實施例5
由本發明益生菌組合物之製造方法,經製造為益生菌組合物後可添加入水產飼料或動物飼料的一般常見製程中製作成為飼料。
實施例6
由本發明益生菌組合物之製造方法,經製造為益生菌組合物後可添加入人類食品或保健品中供人類食用。
經實施例3實驗結果顯示,該益生菌組合物於常溫下保存菌株,其中益生菌組合物菌株於常溫15~35℃下保存36個月,仍有菌株存活。
經實施例3實驗結果顯示,該益生菌組合物於高溫處理下保存菌株,經121℃處理6分鐘、95℃處理10分鐘或65℃處理24小時,其中益生菌組合物菌株經高溫處理後仍有菌株存活。
本發明為呈現解決問題所採用的技術手段較佳實施方式或實施例而已,並非用來限定本發明專利實施之範圍,即凡與本發明專利申請範圍文義相符,或依本發明專利範圍所作的均等變化與修飾,皆為本發明專利所涵蓋。
S100~S400:步驟
Claims (9)
- 一種益生菌組合物之製造方法,包括:一菌液,係希瓦氏菌之菌液;與一海藻酸鈉溶液或海藻酸溶液,該菌液與該海藻酸鈉溶液或海藻酸溶液係以5:1至1:10之間的重量百分比混合;及加入一鈣離子溶液中,直至使其固定成形經乾燥為一乾燥菌球。
- 如請求項1所述之益生菌組合物之製造方法,其中該海藻酸鈉或海藻酸佔該溶液比例為1%~10%重量百分比。
- 如請求項1所述之益生菌組合物之製造方法,其中鈣離子固形物佔該鈣離子溶液比例為0.5%~5%重量百分比。
- 如請求項1所述之益生菌組合物之製造方法,其中該鈣離子溶液係氯化鈣溶液、乳酸鈣溶液、碳酸鈣溶液、醋酸鈣溶液、檸檬酸鈣溶液或草酸鈣溶液其中一種以上之任意組合。
- 一種益生菌組合物,係由請求項1~4任一項所述之益生菌組合物之製造方法所製得之物。
- 一種如請求項5所述之益生菌組合物用於飼料的用途,其中該益生菌組合物經製造為粒狀物後加入一般飼料製程中製作成為飼料。
- 一種如請求項5所述之益生菌組合物用於口服益生菌的用途,其中該益生菌組合物經製造成形為粒狀物供人類食用。
- 一種如請求項5所述之益生菌組合物於常溫下保存菌株的用途,其中該益生菌組合物菌株於常溫15~35℃下保存36個月,仍有菌株存活。
- 一種如請求項5所述之益生菌組合物於高溫處理下保存菌株的用途,經121℃處理6分鐘、95℃處理10分鐘或65℃處理24小時,其中該益生菌組合物菌株經高溫處理後仍有菌株存活。
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