TWI805932B - 抗氧化多層包埋益生菌顆粒及其製造方法 - Google Patents
抗氧化多層包埋益生菌顆粒及其製造方法 Download PDFInfo
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Abstract
本發明有關於一種抗氧化多層包埋益生菌顆粒及其製造方法,其係利用短鏈極性分子層之複數個分子嵌設於菌體核之益生菌的細胞壁表面,再依序利用醣類分子層及雙極性抗氧化乳化蛋白層包覆二者。所得的抗氧化多層包埋益生菌顆粒可有效提升益生菌的抗吸濕潮解性、儲存安定性、耐氧化性、胃酸耐受性及酵素分解耐受性。
Description
本發明是有關於一種包埋益生菌顆粒及其製造方法,特別是有關於一種提升益生菌抗吸濕潮解性、儲存安定性、耐氧化性、胃酸耐受性及酵素分解耐受性之抗氧化多層包埋益生菌顆粒及其製造方法。
益生菌(Probiotics)為對宿主要益處之菌株,通常指活性乳酸菌株、比菲德氏菌或酵母。在生理上,可藉由體內攝取之碳水化合物轉換為短鏈脂肪酸,如醋酸、丙酸、丁酸及乳酸。在全球市場上,益生菌每年以10~25%的速度快速成長,因此帶動了一連串相關產業與技術的精進,常見的產品有粉劑、膠囊錠劑及液態發酵奶或固態發酵乳,為現今食品產業中,十分重要的一個區塊,更是代表了潔淨、健康與希望的產品。
以乳酸菌為例,例如中華民國發明專利公告第587098號,揭示一種增進免疫能力之乳酸菌,包含刺激免疫性濃度之下列任一或多種生物性純培養物:鼠李糖乳桿菌HN001 (CCRC 910107);鼠李糖乳桿菌HN067 (CCRC 910108);乳酸雙歧桿菌(Bifidobacterium lactis
)HN019 (CCRC 910110)或嗜酸乳桿菌(Lactobacillus acidophilus
)HN017 (CCRC 910109),及生理上可接受的賦形劑或稀釋劑,其中該賦形劑或該稀釋劑為食品,而該食品為醱酵乳、優格、乳酪、乳製飲品或乳粉。另外,如中華民國發明專利公告第I451871號,揭示一種食品組合物,用以抑制發炎反應,包含:多株乳酸菌菌株,其包含唾液乳酸桿菌(Lactobacillus salivarius
)AP-32菌株,寄存編號BCRC 910437、路乳酸桿菌(Lactobacillus reuteri
)TE-33菌株,寄存編號BCRC 910441、嗜酸乳酸桿菌(Lactobacillus acidophilus
)F-1菌株,寄存編號BCRC 910469及鼠李糖乳酸桿菌(Lactobacillus rhamnosus
)CT-53菌株,寄存編號BCRC 910468,其中該些乳酸菌菌株具有胃酸、膽鹽及抑霉劑克黴唑(Clotrimazole)耐受性;以及生理上可接受的一賦形劑或一稀釋劑,其中該賦形劑或該稀釋劑為醱酵乳、優格、乳酪、乳製飲品乳粉、茶或咖啡。
在目前的技術中,益生菌可概分為未經包埋處理的液劑或粉劑,一般常見需要維持低溫保存或短效期之產品,以降低環境中氧氣或溫度的傷害,提升保存期限。然而,儲藏環境一旦出現差異或失溫,容易導致益生菌大量死亡,而失去原有的機能特性。
因此,在市場需求下,研發人員開發出各種提升乳酸菌存活率之技術。以改善益生菌酸鹼耐受性不佳為例,如中華民國發明專利公告第M384660號,其揭示一種可抑制體內多餘脂肪生長之LS66膠囊組合結構,包含一本體、一個以上之LS66孢子乳酸菌以及一塗佈層,該本體包含一上膠囊與下膠囊,而上膠囊與下膠囊結合後內部形成一容置空間,該LS66孢子乳酸菌係填設於上述容置空間內,該塗佈層塗佈於本體內部表面,其中該塗佈層係為一活化劑。乳酸菌利用硬膠囊包埋後,雖可保護菌體通過酸性的胃部環境,但硬膠囊於腸道不易溶解,使得釋出菌體的時機不穩定,且同樣有不耐久存的問題。
除了以硬膠囊包埋改善益生菌酸鹼耐受性不佳其次,以上,科學化開發益生菌機能性特點之應用專利及改善益生菌在液劑或粉劑中儲藏或加工安定性特點之外,更有大顆粒晶球包覆性益生菌材料之案例。
益生菌可利用單一極性層(如脫脂奶粉、糖質類之親水極性材料,或如油脂類之疏水非極性材料) 進行固定化或包埋。其次,益生菌亦可利用多層式醣質壁材(或稱包覆材)包覆,可提供多層次緩釋劑型,例如由外至內依序利用胃酸中酸分解壁材、膽鹽鹼性解離壁材及腸道中分解壁材進行包覆。由此所得的多層次緩釋劑型雖可有效提升益生菌之酸鹼耐受性,但常因酸鹼解離不完全而進入下一階段時,無法持續解離,導致在體內無法完全解離釋放包覆在其中益生菌之困擾且包埋價格昂貴與包埋後菌體存活率低之缺點,而需大量攝取,容易造成糖分或脂質攝取過量的嚴重缺點。
因此,本發明之一態樣是在提供一種抗氧化多層包埋益生菌顆粒(multilayer antioxidant coating lactic acid bacteria microencapsule,MAOC-LAB),其利用短鏈極性分子層之複數個分子嵌設於菌體核之益生菌的細胞壁表面,再依序利用醣類分子層及雙極性抗氧化乳化蛋白層包覆二者,以形成複數層同心立體結構。
本發明之另一態樣係在提供一種抗氧化多層包埋益生菌顆粒之製造方法,由此製得的抗氧化多層包埋益生菌顆粒可有效提升益生菌的抗吸濕潮解性、儲存安定性、耐氧化性、胃酸耐受性及酵素分解耐受性。
根據本發明之上述態樣,提出一種抗氧化多層包埋益生菌顆粒。在一實施例中,上述抗氧化多層包埋益生菌顆粒可包含菌體核,嵌設於菌體核細胞壁表面的短鏈極性分子層,設於菌體核及短鏈極性分子層上的醣類分子層,以及設於醣類分子層上的雙極性抗氧化乳化蛋白層。在上述實施例中,菌體核係由至少一益生菌所組成,而短鏈極性分子層之複數個分子可嵌設於至少一益生菌之細胞壁表面。在上述實施例中,雙極性抗氧化乳化蛋白層可由蛋白質與油溶性抗氧化劑所組成。
依據本發明一實施例,上述至少一益生菌可包括但不限於乳桿菌、乳球菌、比菲德氏菌及酵母菌及上述之任意組合。在此實施例中,上述至少一益生菌可包括但不限於乾燥菌體、含菌醱酵液或含菌濃縮醱酵液及上述之任意組合。在其他例子中,上述至少一益生菌之菌體密度可例如為每毫升1×1010
菌落形成單位(colony forming unit,cfu)至1×1011
cfu/mL。
依據本發明一實施例,上述短鏈極性分子層可包括但不限於胺基酸鹽類、磷酸鹽類、檸檬酸酯類、醇類化合物、胡蘿蔔素或茄紅素及上述之任意組合。
依據本發明一實施例,上述短鏈極性分子層可包括但不限於胺基酸鹽類、磷酸鹽類、檸檬酸酯類、醇類化合物、胡蘿蔔素或茄紅素及上述之任意組合。
依據本發明一實施例,上述醣類分子層可包括但不限於單醣、雙醣、寡醣及多醣及上述任意組合,且醣類分子層可例如為結晶態。
依據本發明一實施例,上述蛋白質可包括但不限於脫脂奶粉、全脂奶粉、大豆蛋白粉、乳清蛋白粉、酪蛋白粉、豌豆蛋白粉、酵母蛋白粉及上述之任意組合。
依據本發明一實施例,上述油溶性抗氧化劑可包括但不限於維生素E、蝦紅素、葉黃素、芝麻油、米糠油、脂肪酸類及上述之任意組合。在一些例子中,前述脂肪酸類可包括但不限於脂肪酸甘油酯、檸檬酸甘油酯、酒石酸甘油酯、乳酸甘油酯、乙氧基甘油酯、磷酸甘油酯、琥珀酸甘油酯、脂肪酸聚合甘油酯、交酯化蓖麻酸聚合甘油酯、脂肪酸蔗糖酯、脂肪酸山梨醇酐酯、脂肪酸丙二醇酯、單及雙脂肪酸甘油二乙醯酒石酸酯、脂肪酸鹽類及上述之任意組合。
依據本發明一實施例,上述菌體核、短鏈極性分子層、醣類分子層及雙極性抗氧化乳化蛋白層可構成複數層同心立體結構。
根據本發明之另一態樣,提出一種抗氧化多層包埋益生菌顆粒之製造方法,包含提供菌體核,將短鏈極性分子層之複數個分子嵌設於菌體核細胞壁表面,將醣類分子層包覆菌體核及短鏈極性分子層,將雙極性抗氧化乳化蛋白層包覆醣類分子層,以及進行乾燥步驟,以形成抗氧化多層包埋益生菌顆粒。在一實施例中,上述菌體核可由至少一益生菌所組成,且至少一益生菌可包括但不限於乳桿菌、乳球菌、比菲德氏菌及酵母菌及上述之任意組合,而短鏈極性分子層之複數個分子則嵌設於至少一益生菌之細胞壁表面。在此實施例中,上述雙極性抗氧化乳化蛋白層可由蛋白質與油溶性抗氧化劑進行均質乳化步驟所形成。在此實施例中,菌體核、短鏈極性分子層、醣類分子層及雙極性抗氧化乳化蛋白層可構成複數層同心立體結構。
依據本發明一實施例,上述乾燥步驟包括冷凍乾燥處理、真空乾燥處理或噴霧乾燥處理。
應用本發明之抗氧化多層包埋益生菌顆粒及其製造方法,所製得的益生菌顆粒具有複數層同心立體結構,可有效提升益生菌的抗吸濕潮解性、儲存安定性、耐氧化性、胃酸耐受性及酵素分解耐受性。
承前所述,本發明提供一種抗氧化多層包埋益生菌顆粒(multilayer antioxidant coating lactic acid bacteria microencapsule,MAOC-LAB),其利用短鏈極性分子層之複數個分子嵌設於菌體核之益生菌的細胞壁表面,再依序利用醣類分子層及雙極性抗氧化乳化蛋白層包覆二者,以形成複數層同心立體結構。
請參照圖1,其係繪示根據本發明一實施例之抗氧化多層包埋益生菌顆粒之剖面示意圖。在一實施例中,上述抗氧化多層包埋益生菌顆粒100可包含菌體核110、嵌設於菌體核110細胞壁115表面的短鏈極性分子層121、設於菌體核110及短鏈極性分子層121上的醣類分子層123、設於醣類分子層123上的雙極性抗氧化乳化蛋白層125。在上述實施例中,菌體核110係由至少一益生菌所組成,而短鏈極性分子層之複數個分子可嵌設於至少一益生菌之細胞壁115表面。在上述實施例中,雙極性抗氧化乳化蛋白層可由蛋白質與油溶性抗氧化劑所組成。
在上述實施例中,至少一益生菌的種類並無特別限制,可例如乳桿菌、乳球菌、比菲德氏菌及酵母菌及上述之任意組合。在此實施例中,上述至少一益生菌的狀態亦無特別限制,可例如乾燥菌體、含菌醱酵液或含菌濃縮醱酵液及上述之任意組合。在其他例子中,上述至少一益生菌在使用時以濃縮為宜,其光學密度可例如為至少OD600
100,而其菌體密度相當於每毫升1×1010
菌落形成單位(colony forming unit,cfu)至1×1011
cfu/mL。
上述益生菌包含乳酸菌,隸屬於革蘭氏陽性菌,其細胞壁115由肽聚糖組成,而細胞膜111則兼具親水性及親油性之磷脂雙層(phospholipid bilayer)薄膜結構,細胞壁115與細胞膜111之間則為壁膜間隙113。革蘭氏陽性菌缺乏革蘭氏陰性菌的外膜跟脂多醣層,較易受外界環境影響。習知技術在針對上述益生菌進行固定化包埋處理時,大多利用單層的極性親水性壁材包埋益生菌。惟此等單層包埋結構經乾燥後,非極性脂肪酸容易產生大範圍的裂解或破損,進而傷害菌體甚至降低乾燥後的存活率。
因此,在上述實施例中,抗氧化多層包埋益生菌顆粒可包含短鏈極性分子層,其複數個分子可嵌設於益生菌之細胞壁表面,藉此改善習知單層包埋技術的缺點。申言之,在上述實施例中,短鏈極性分子層可包括但不限於胺基酸鹽類、磷酸鹽類、檸檬酸酯類、醇類化合物、胡蘿蔔素或茄紅素及上述之任意組合。由於上述短鏈極性分子具可穿透性,可嵌設於益生菌之細胞壁表面,在益生菌後續的乾燥過程中,有助於穩定包埋結構,避免益生菌之脂雙層結構裂解、崩壞而死亡。倘若抗氧化多層包埋益生菌顆粒未使用上述短鏈極性分子層嵌設於益生菌之細胞壁表面,則由此包埋的益生菌經乾燥後,所得的結構較不穩定,容易導致脂雙層結構裂解、崩壞甚至提高乾燥後的死亡率。
在上述實施例中,包覆短鏈極性分子層之醣類分子層可包括但不限於單醣、雙醣、寡醣及多醣及上述任意組合,且醣類分子層以結晶態為較佳。
在一些具體例中,上述單醣包含葡萄糖、甘露糖及山梨糖。在一些具體例中,上述雙醣包含蔗糖、乳糖及海藻糖。在一些具體例中,上述寡醣包含異麥芽寡糖。在一些具體例中,上述多醣包含澱粉及糊精。由於短鏈極性分子層可嵌設於益生菌之細胞壁表面,有助於醣類分子層包覆於細胞壁上,改變細胞壁之親水性。
在一實施例中,抗氧化多層包埋益生菌顆粒包含雙極性抗氧化乳化蛋白層,可設於醣類分子層上。在此實施例中,雙極性抗氧化乳化蛋白層可由蛋白質與油溶性抗氧化劑所組成。
在上述實施例中,適合的蛋白質可包括但不限於脫脂奶粉、全脂奶粉、大豆蛋白粉、乳清蛋白粉、酪蛋白粉、豌豆蛋白粉、酵母蛋白粉及上述之任意組合。由於蛋白質可與親水性醣類分子層具良好親和性並結合,在胃酸環境中又具凝膠性,可降低胃酸中溶解度,提高乳酸菌在胃酸中存活率。
在上述實施例中,適合的油溶性抗氧化劑可包括但不限於維生素E、蝦紅素、葉黃素、芝麻油、米糠油、脂肪酸類及上述之任意組合。在一些例子中,前述脂肪酸類可包括但不限於脂肪酸甘油酯、檸檬酸甘油酯、酒石酸甘油酯、乳酸甘油酯、乙氧基甘油酯、磷酸甘油酯、琥珀酸甘油酯、脂肪酸聚合甘油酯、交酯化蓖麻酸聚合甘油酯、脂肪酸蔗糖酯、脂肪酸山梨醇酐酯、脂肪酸丙二醇酯、單及雙脂肪酸甘油二乙醯酒石酸酯、脂肪酸鹽類及上述之任意組合。由於油溶性抗氧化劑能阻絕環境中水氣與氧氣,可延緩吸濕性。
依據本發明一實施例,上述菌體核、短鏈極性分子層、醣類分子層及雙極性抗氧化乳化蛋白層可構成複數層同心立體結構,有效提升益生菌的抗吸濕潮解性、儲存安定性、耐氧化性、胃酸耐受性及酵素分解耐受性。
本發明另提出一種抗氧化多層包埋益生菌顆粒之製造方法。在一實施例中,首先,提供菌體核。在此實施例中,菌體核可由至少一益生菌所組成,關於益生菌的種類及菌體密度已如前所述,此處不另贅言。
接著,在一些實施例中,短鏈極性分子層之複數個分子可先嵌設於菌體核之細胞壁表面後,再將醣類分子層包覆菌體核及短鏈極性分子層。關於短鏈極性分子層及醣類分子層的種類如前悉述,不再贅言。在其他實施例中,短鏈極性分子層亦可與醣類分子層混合後,再與菌體核混合,以包覆菌體核。舉例而言,0.5 w/v%~30 w/v%之短鏈極性分子可先與濃度30 w/v%~60 w/v%醣類分子溶於水中混合均勻,將酸鹼值調整至pH3~pH8後,將混合溶液加熱至60°C~120°C,持溫15分鐘至120分鐘,再與益生菌之菌體核在0 kg/cm2
至0.25kg/cm2
的壓力下混合15分鐘至120分鐘。在這些實施例中,不論短鏈極性分子層及醣類分子層是先後包覆抑或同時包覆,短鏈極性分子層之複數個分子皆可先嵌設於益生菌細胞壁表面,而醣類分子層則包覆於益生菌細胞壁及短鏈極性分子層上。
之後,雙極性抗氧化乳化蛋白層可包覆醣類分子層。在一些實施例中,雙極性抗氧化乳化蛋白層可由蛋白質與油溶性抗氧化劑進行均質乳化步驟所形成。關於蛋白質及油溶性抗氧化劑的種類已如前所述,此處不贅。在一些實施例中,前述之均質乳化步驟可使用2 w/v%~20 w/v%之蛋白粉、0.05 v/v %~8 v/v %油脂及油溶性抗氧化劑混合後進行。在這些例子中,均質乳化步驟可使用習知製程條件,或使用例如0 巴(Bar)至12 Bar之乳化壓力、例如4°C至120°C之溫度、進行5分鐘至90分鐘。
而後,將包埋短鏈極性分子層、醣類分子層及雙極性抗氧化乳化蛋白層之菌體核進行乾燥步驟,以形成抗氧化多層包埋益生菌顆粒。上述乾燥步驟可使用習知方式進行,例如冷凍乾燥處理、真空乾燥處理或噴霧乾燥處理等,惟本發明不限於上述所舉。在一些實施例中,上述冷凍乾燥處理可使用習知製程條件,或將上述包埋多層之菌體核置於-35°C或以下的溫度進行冷凍乾燥處理,以獲得抗氧化多層包埋益生菌顆粒。抗氧化多層包埋益生菌顆粒可於40°C以下的溫度儲放,以避免多層包埋結構之層次分離。
由此所得的抗氧化多層包埋益生菌顆粒,菌體核、短鏈極性分子層、醣類分子層及雙極性抗氧化乳化蛋白層可構成複數層同心立體結構,經實驗證實,可有效提升益生菌的抗吸濕潮解性、儲存安定性、耐氧化性、胃酸耐受性及酵素分解耐受性。
補充說明的是,相較於習知包覆材料及製程,本發明之抗氧化多層包埋益生菌顆粒及其製造方法藉由特定組成及特定製程,有效降低壁材的體積及含量。如此一來,不僅提高抗氧化多層包埋益生菌顆粒經乾燥後之活菌數,製程中又無需使用流動床乾燥機,成本較習知包埋方式(例如晶球)更加容易、降低成本且容易量產。
以下利用數個實施例以說明本發明之應用,然其並非用以限定本發明,本發明技術領域中具有通常知識者,在不脫離本發明之精神和範圍內,當可作各種之更動與潤飾。
實施例一:製備抗氧化多層包埋益生菌顆粒
製備例1
首先,將嗜酸乳桿菌(Lactobacillus acidophilus
)之菌體核(至少OD600
100)依序利用6 w/v%之短鏈極性分子層與35 w/v%之醣類分子層的混合溶液進行包埋,再利用20 w/v%之雙極性抗氧化乳化蛋白層進行包埋。前述短鏈極性分子層與醣類分子層混合溶液的酸鹼值為pH 4.0±0.5,包覆時的加熱溫度為90°C±5、持溫時間30分鐘、壓力為0.15kg/cm2
。雙極性抗氧化乳化蛋白層則是混合30 w/v%的脫脂奶粉(skim milk)以及0.2 w/v%的維生素E(VIT. E)後,在2 bar的壓力及90°C的溫度下進行均質乳化30分鐘而形成。之後,上述多層包埋顆粒於-40°C冷凍24小時,再於-30°C進行冷凍乾燥處理達72小時後,經20目篩網磨粉過篩,以製得抗氧化多層包埋益生菌顆粒。
製備例2
製備例2的製程條件同製備例1,不同之處在於製備例2的菌體核為雷特氏B菌(Bifidobacterium lactis
)。
比較例1
首先,將含有6 w/v%之短鏈極性分子、35 w/v%之醣類分子及20 w/v%之雙極性抗氧化乳化蛋白之混合溶液,對嗜酸乳桿菌(Lactobacillus acidophilus
)之菌體核(至少OD600
100)進行單層包埋,其中單層包覆的酸鹼值、加熱溫度為90°C±5、持溫時間、壓力等製程條件與製備例1相同,此處不另贅述。之後,上述單層包埋顆粒於-40°C冷凍24小時,再於-30°C進行冷凍乾燥處理達72小時後,經20目篩網磨粉過篩而製得單層包埋益生菌顆粒。
比較例2
比較例2的製程條件同比較例1,不同之處在於比較例2的菌體核為雷特氏B菌(Bifidobacterium lactis
)。
實施例二:評估實施例一的抗氧化多層包埋益生菌顆粒的保護效果
1. 評估抗吸濕潮解性
首先,將實施例一之製備例1與製備例2的抗氧化多層包埋益生菌顆粒,以及比較例1與比較例2的單層包埋益生菌顆粒,分別置於培養皿(直徑9 cm)中,精秤其初始重量。接著,將製備例1、製備例2、比較例1與比較例2的包埋益生菌顆粒暴露於濕度75%±5%之環境中,每隔5分鐘,精秤其各時間點測得之重量,共連續測量20分鐘。然後,根據式(I)評估各時間點的吸水率,其結果如表1所示。×100% (I)
表1
組別 | 初始重量(g) | 5分鐘 吸水率 | 10分鐘 吸水率 | 15分鐘 吸水率 | 20分鐘 吸水率 |
製備例1 | 9.4238 | 1.46% | 2.89% | 4.11% | 5.71% |
製備例2 | 9.4210 | 1.32% | 2.91% | 5.16% | 6.62% |
比較例1 | 9.3231 | 1.78% | 4.10% | 5.83% | 8.62% |
比較例2 | 9.5107 | 2.11% | 5.31% | 8.14% | 10.23% |
由表1結果可知,以20分鐘吸水率為例,相較於比較例1,製備例1的抗氧化多層包埋益生菌顆粒可降低33.7%的相對吸水量 [相對吸水量=(製備例1-比較例1)/比較例1×100%]。同樣以20分鐘吸水率為例,相較於比較例2,製備例2的抗氧化多層包埋益生菌顆粒可降低36%的相對吸水量。由此結果證實,不論以嗜酸乳桿菌或雷特氏B菌為菌體核,實施例一的抗氧化多層包埋益生菌顆粒確實可有效降低吸水率及相對吸水量,具有抗吸濕潮解的效果,進而提高產品加工的穩定性。
2.評估儲存安定性
實施例一的抗氧化多層包埋益生菌顆粒及單層包埋益生菌顆粒在不同溫度下儲存一段時間後,分別測量其活菌數,以評估不同包埋技術對益生菌安定性的差異。
首先,將實施例一之製備例1與製備例2的抗氧化多層包埋益生菌顆粒,以及比較例1與比較例2的單層包埋益生菌顆粒,分別包裝於鋁箔夾鏈袋中,然後儲存於不同的溫度區間,例如低於-10°C、低於7°C、25°C及40°C,或合併高濕環境(65%±5%RH,75%±5%RH)中。經儲存三個月後,統計各組活菌數,其結果如表2所示。
表2
組別 | 初始菌數(cfu/g) | <-10°C 三個月 (cfu/g) | <7°C 三個月 (cfu/g) | 25°C ±2°C 65%±5%RH 三個月 (cfu/g) | 40°C ±2°C 75%±5%RH 三個月 (cfu/g) |
製備例1 | 1.2 ×1011 | 1.3 ×1011 | 1.2 ×1011 | 1.1 ×1011 | 6.0 ×109 |
製備例2 | 3.5 ×1011 | 3.3 ×1011 | 3.4 ×1011 | 2.5 ×1011 | 4.3 ×109 |
比較例1 | 1.4 ×1011 | 1.3 ×1011 | 1.1 ×1011 | 7.0 ×1010 | 3.0 ×108 |
比較例2 | 3.6 ×1011 | 3.9 ×1011 | 3.7 ×1011 | 1.4 ×1011 | 8.0 ×107 |
由表2結果可知,不論是製備例1與製備例2之抗氧化多層包埋益生菌顆粒,或比較例1與比較例2的單層包埋益生菌顆粒,在低於7°C儲存三個月後,二種包埋技術皆可提供益生菌良好的保護。然而,在25°C及40°C儲存三個月後,可發現製備例1與製備例2之抗氧化多層包埋益生菌顆粒的活菌數,顯著高於比較例1與比較例2的單層包埋益生菌顆粒。相較於比較例1,製備例1的抗氧化多層包埋益生菌顆粒儲放在40°C中,可提高嗜酸乳桿菌20倍的活菌數。同樣地,相較於比較例2,製備例2的抗氧化多層包埋益生菌顆粒可提高雷特氏B菌超過50倍之活菌數。上述結果證明,實施例一之抗氧化多層包埋益生菌顆粒確實有助於提高益生菌在不同溫度下之儲存安定性。
3.評估耐氧化性
此實施例是在流動床機乾燥機中通入高量空氣,以提供強烈氧化的環境,藉此評估不同包埋技術對益生菌耐氧化性的差異。
首先,在流動床乾燥機的艙體內,分別置入50公斤的製備例1與製備例2的抗氧化多層包埋益生菌顆粒,以及比較例1與比較例2的單層包埋益生菌顆粒。接著,啟動流動床造粒模式,其中通氣量為800m3
/hr,入口風相對溼度為小於7%,入口風溫度為50°C,全程共1小時。在過程中,每隔15分鐘取樣檢測活菌數,觀察在高溫、低濕、高量空氣的強氧化環境下,不同包埋技術對益生菌活菌數的變化,其結果如表3所示。
表3
組別 | 初始菌數(cfu/g) | 噴霧時間: 15分鐘 | 噴霧時間: 30分鐘 | 噴霧時間: 45分鐘 | 噴霧時間: 60分鐘 |
製備例1 | 1.2 ×1011 | 1.2 ×1011 | 8.9 ×1010 | 7.2 ×1010 | 5.2 ×1010 |
製備例2 | 3.5 ×1011 | 2.4 ×1011 | 1.1 ×1011 | 6.5 ×1010 | 2.5 ×1010 |
比較例1 | 1.4 ×1011 | 1.1 ×1011 | 6.3 ×1010 | 2.1 ×1010 | 7.0 ×109 |
比較例2 | 3.6 ×1011 | 1.9 ×1011 | 7.7 ×1010 | 3.1 ×1010 | 8.1 ×109 |
由表3結果可知,在高溫、低濕、高量空氣的強氧化環境下,的確會降低益生菌的存活率。不過,以噴霧時間60分鐘的活菌數為例,製備例1的抗氧化多層包埋益生菌顆粒與比較例1之單層包埋益生菌顆粒,二者嗜酸乳桿菌之活菌數差異可達7.4倍。同樣地,製備例2的抗氧化多層包埋益生菌顆粒與比較例2之單層包埋益生菌顆粒,二者雷特氏B菌之活菌數差異可達3倍。由此結果證實,在高氧化傷害的環境下,實施例一的抗氧化多層包埋益生菌顆粒確實可有效提高益生菌的耐氧化性。
4. 評估胃酸耐受性
前述實施例一的抗氧化多層包埋益生菌顆粒及單層包埋益生菌顆粒分別浸泡於十倍重量之酸化磷酸緩衝鹽溶液中,經3個小時反應後,以模擬胃酸處理的環境。分別測量不同包埋技術之益生菌在不同pH值之環境下的活菌數,藉此評估不同包埋技術對益生菌之胃酸耐受性,結果如下表4所示。
表4
組別 | 初始菌數(cfu/g) | pH 7.2 | pH 3.2 | pH 2.4 | pH 2.0 |
製備例1 | 1.2 ×1011 | 1.3 ×1011 | 1.2 ×1011 | 8.2 ×1010 | 2.7 ×109 |
製備例2 | 3.5 ×1011 | 3.4 ×1011 | 3.2 ×1011 | 2.3 ×1011 | 5.5 ×109 |
比較例1 | 1.4 ×1011 | 1.3 ×1011 | 1.1 ×1011 | 6.1 ×1010 | 3.0 ×108 |
比較例2 | 3.6 ×1011 | 3.3 ×1011 | 3.3 ×1011 | 8.1 ×1010 | 4.9 ×107 |
由表4結果可知,不論是製備例1與製備例2之抗氧化多層包埋益生菌顆粒,或比較例1與比較例2的單層包埋益生菌顆粒,在中性pH 7.2及pH 3.2的環境下,各組間的活菌數無顯著差異。然而,當環境酸鹼值降低至pH 2.4或pH 2.0時,製備例1與製備例2之抗氧化多層包埋益生菌顆粒的活菌數,顯著高於比較例1與比較例2的單層包埋益生菌顆粒,其差異最多可達117倍。上述結果證實,抗氧化多層包埋益生菌顆粒確實有助於提高益生菌(不論嗜酸乳桿菌或雷特氏B菌)的胃酸耐受性。
5. 評估酵素分解耐受性
此實施例是將製備例1與製備例2的抗氧化多層包埋益生菌顆粒,以及比較例1與比較例2的單層包埋益生菌顆粒,分別添加至0.1%之不同酵素水溶液中,藉此評估不同包埋技術對益生菌之酵素分解耐受性的差異。
首先,將100克之製備例1與製備例2的抗氧化多層包埋益生菌顆粒,以及比較例1與比較例2的單層包埋益生菌顆粒,分別加入含有乳糖分解酶、胰蛋白酶、葡萄糖氧化酶、木瓜酵素等水溶液中(酵素濃度為50,000U/mL),在40°C之溫度下反應10分鐘後,計算各組活菌數,其結果如表5所示。
表5
組別 | PBS | 乳糖酶 | 胰蛋白酶 | 葡萄糖氧化酶 | 木瓜酵素 |
製備例1 | 1.2 ×1011 | 9.2 ×1010 | 9.1 ×1010 | 1.1 ×1011 | 7.7 ×1010 |
製備例2 | 3.1 ×1011 | 3.4 ×1011 | 3.2 ×1011 | 2.4 ×1011 | 1.1 ×1011 |
比較例1 | 1.1 ×1011 | 8.4 ×1010 | 7.1 ×1010 | 1.2 ×1011 | 4.0 ×1010 |
比較例2 | 2.8 ×1011 | 2.2 ×1011 | 7.6 ×1010 | 2.1 ×1011 | 7.9 ×1010 |
由表5結果可知,不同的酵素處理對益生菌的活菌數產生程度不等之影響。比較二種糖類分解酵素(即乳糖分解酶、葡萄糖氧化酶)及二種蛋白酶(即胰蛋白酶、木瓜酵素)之結果可知,不論是製備例1與製備例2之抗氧化多層包埋益生菌顆粒,或比較例1與比較例2的單層包埋益生菌顆粒,經醣類分解酵素處理後,對於嗜酸乳桿菌或雷特氏B菌的活菌數並無顯著差異。然而,經蛋白酶處理後,製備例1與製備例2之抗氧化多層包埋益生菌顆粒的活菌數,明顯高於比較例1與比較例2的單層包埋益生菌顆粒。上述結果證明,實施例一之抗氧化多層包埋益生菌顆粒確實有助於提高益生菌的對蛋白酵素分解耐受性,尤其是富含蛋白消化酵素之胃液。
綜言之,由上述數個實施例證實,本發明之抗氧化多層包埋益生菌顆粒及其製造方法,所製得的益生菌顆粒具有複數層同心立體結構,確實可有效提升益生菌的抗吸濕潮解性、儲存安定性、耐氧化性、胃酸耐受性及酵素分解耐受性。
需補充的是,本發明雖以特定的製程或特定的分析方法作為例示,說明本發明之抗氧化多層包埋益生菌顆粒及其製造方法,惟本發明所屬技術領域中任何具有通常知識者可知,本發明並不限於此,在不脫離本發明之精神和範圍內,本發明之抗氧化多層包埋益生菌顆粒及其製造方法亦可使用其他製程或其他的分析方法進行。
由上述實施例可知,本發明的抗氧化多層包埋益生菌顆粒及其製造方法,其優點在於所製得的益生菌顆粒具有複數層同心立體結構,可有效提升益生菌的抗吸濕潮解性、儲存安定性、耐氧化性、胃酸耐受性及酵素分解耐受性。
雖然本發明已以數個實施例揭露如上,然其並非用以限定本發明,在本發明所屬技術領域中任何具有通常知識者,在不脫離本發明之精神和範圍內,當可作各種之更動與潤飾,因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。
100:抗氧化多層包埋益生菌顆粒
110:菌體核
111:細胞膜
113:壁膜間隙
115:細胞壁
121:短鏈極性分子層
123:醣類分子層
125:雙極性抗氧化乳化蛋白層
為讓本發明之上述和其他目的、特徵、優點與實施例能更明顯易懂,所附圖式之詳細說明如下:
[圖1]係繪示根據本發明一實施例之抗氧化多層包埋益生菌顆粒之剖面示意圖。
無
100:抗氧化多層包埋益生菌顆粒
110:菌體核
111:細胞膜
113:壁膜間隙
115:細胞壁
121:短鏈極性分子層
123:醣類分子層
125:雙極性抗氧化乳化蛋白層
Claims (15)
- 一種抗氧化多層包埋益生菌顆粒,包含:一菌體核,其中該菌體核係由至少一益生菌所組成;一短鏈極性分子層,其中該短鏈極性分子層之複數個分子係嵌設於該至少一益生菌之一細胞壁表面,且該短鏈極性分子層係選自於由胺基酸鹽類、磷酸鹽類、檸檬酸酯類、醇類化合物、胡蘿蔔素或茄紅素及上述之任意組合所組成之一族群,該胺基酸鹽類係選自於由L-組胺酸鹽酸鹽、DL-蛋胺酸、L-離胺酸鹽酸鹽、L-麩酸鈉及上述之任意組合所組成之一族群,該磷酸鹽類係選自於由磷酸鹽、偏磷酸鹽、焦磷酸鹽、磷酸鈣、磷酸一鉀、磷酸二鈉、磷酸三鈉、磷酸三鈣、三聚磷酸鈉、四聚磷酸鈉及上述之任意組合所組成之一族群,該檸檬酸酯類係選自於由檸檬酸、檸檬酸鈉、檸檬酸鈣、檸檬酸亞鐵鈉、檸檬酸鉀、檸檬酸鐵及上述之任意組合所組成之一族群,且該醇類化合物係選自於由D-甘露醇、生育醇、麥芽糖醇丙二醇、D-山梨醇、乳糖醇、肌醇、D-木糖醇及上述之任意組合所組成之一族群;一醣類分子層,設於該菌體核及該短鏈極性分子層上,其中該醣類分子層係選自於由單醣、雙醣、寡醣及多醣及上述任意組合所組成之一族群,且該醣類分子層為一結晶態;以及一雙極性抗氧化乳化蛋白層,設於該醣類分子層上,其中該雙極性抗氧化乳化蛋白層係由一蛋白質與一油溶性抗 氧化劑所組成,該油溶性抗氧化劑係選自於由維生素E、蝦紅素、葉黃素、芝麻油、米糠油、脂肪酸類及上述之任意組合所組成之一族群,且其中該菌體核、該短鏈極性分子層、該醣類分子層及該雙極性抗氧化乳化蛋白層係組成複數層同心立體結構。
- 如請求項1所述之抗氧化多層包埋益生菌顆粒,其中該至少一益生菌係選自於由乳桿菌、乳球菌、比菲德氏菌及酵母菌及上述之任意組合所組成之一族群。
- 如請求項1所述之抗氧化多層包埋益生菌顆粒,其中該至少一益生菌係選自於由一乾燥菌體、一含菌醱酵液、一含菌濃縮醱酵液及上述之任意組合所組成之一族群。
- 如請求項1所述之抗氧化多層包埋益生菌顆粒,其中該至少一益生菌之一菌體密度為每毫升1×1010菌落形成單位(colony forming unit,cfu)至1×1011cfu/mL。
- 如請求項1所述之抗氧化多層包埋益生菌顆粒,其中該單醣包含葡萄糖、甘露糖及山梨糖,該雙醣包含蔗糖、乳糖及海藻糖,該寡醣包含異麥芽寡糖,且該多醣包含澱粉及糊精。
- 如請求項1所述之抗氧化多層包埋益生菌顆粒,其中該蛋白質係選自於由脫脂奶粉、全脂奶粉、大豆蛋白粉、乳清蛋白粉、酪蛋白粉、豌豆蛋白粉、酵母蛋白粉及上述之任意組合所組成之一族群。
- 如請求項1所述之抗氧化多層包埋益生菌顆粒,其中該脂肪酸類係選自於由脂肪酸甘油酯、檸檬酸甘油酯、酒石酸甘油酯、乳酸甘油酯、乙氧基甘油酯、磷酸甘油酯、琥珀酸甘油酯、脂肪酸聚合甘油酯、交酯化蓖麻酸聚合甘油酯、脂肪酸蔗糖酯、脂肪酸山梨醇酐酯、脂肪酸丙二醇酯、單及雙脂肪酸甘油二乙醯酒石酸酯、脂肪酸鹽類及上述之任意組合所組成之一族群。
- 一種抗氧化多層包埋益生菌顆粒之製造方法,包括:提供一菌體核,其中該菌體核係由至少一益生菌所組成,且該至少一益生菌係選自於由乳桿菌、乳球菌、比菲德氏菌及酵母菌及上述之任意組合所組成之一族群;將一短鏈極性分子層之複數個分子嵌設於該至少一益生菌之一細胞壁表面,其中該短鏈極性分子層係選自於由胺基酸鹽類、磷酸鹽類、檸檬酸酯類、醇類化合物、胡蘿蔔素或茄紅素及上述之任意組合所組成之一族群,該胺基酸鹽類係選自於由L-組胺酸鹽酸鹽、DL-蛋胺酸、L-離胺酸 鹽酸鹽、磷酸一鉀、L-麩酸鈉及上述之任意組合所組成之一族群,該磷酸鹽類係選自於由磷酸鹽、偏磷酸鹽、焦磷酸鹽、磷酸鈣、磷酸二鈉、磷酸三鈉、磷酸三鈣、三聚磷酸鈉、四聚磷酸鈉及上述之任意組合所組成之一族群,該檸檬酸酯類係選自於由檸檬酸、檸檬酸鈉、檸檬酸鈣、檸檬酸亞鐵鈉、檸檬酸鉀、檸檬酸鐵及上述之任意組合所組成之一族群,且該醇類化合物係選自於由D-甘露醇、生育醇、麥芽糖醇丙二醇、D-山梨醇、乳糖醇、肌醇、D-木糖醇及上述之任意組合所組成之一族群;將一醣類分子層包覆該菌體核及該短鏈極性分子層,其中該醣類分子層係選自於由單醣、雙醣、寡醣及多醣及上述任意組合所組成之一族群,且該醣類分子層為一結晶態;將一雙極性抗氧化乳化蛋白層包覆該醣類分子層,其中該雙極性抗氧化乳化蛋白層是由一蛋白質與一油溶性抗氧化劑進行一均質乳化步驟所形成,且該油溶性抗氧化劑係選自於由維生素E、蝦紅素、葉黃素、芝麻油、米糠油、脂肪酸類及上述之任意組合所組成之一族群;以及進行一乾燥步驟,以形成該抗氧化多層包埋益生菌顆粒,其中該菌體核、該短鏈極性分子層、該醣類分子層及該雙極性抗氧化乳化蛋白層係構成複數層同心立體結構。
- 如請求項8所述之抗氧化多層包埋益生菌顆粒之製造方法,其中該至少一益生菌係選自於由乳桿菌、 乳球菌、比菲德氏菌及酵母菌及上述之任意組合所組成之一族群。
- 如請求項8所述之抗氧化多層包埋益生菌顆粒之製造方法,其中該至少一益生菌為一乾燥菌體、一含菌醱酵液或一含菌濃縮醱酵液。
- 如請求項8所述之抗氧化多層包埋益生菌顆粒之製造方法,其中該至少一益生菌之一菌體密度為1×1010cfu/mL至1×1011cfu/mL。
- 如請求項8所述之抗氧化多層包埋益生菌顆粒之製造方法,其中該單醣包含葡萄糖、甘露糖及山梨糖,該雙醣包含蔗糖、乳糖及海藻糖,該寡醣包含異麥芽寡糖,且該多醣包含澱粉及糊精。
- 如請求項8所述之抗氧化多層包埋益生菌顆粒之製造方法,其中該蛋白質係選自於由脫脂奶粉、全脂奶粉、大豆蛋白粉、乳清蛋白粉、酪蛋白粉、豌豆蛋白粉、酵母蛋白粉及上述之任意組合所組成之一族群。
- 如請求項8所述之抗氧化多層包埋益生菌顆粒之製造方法,其中該脂肪酸類係選自於由脂肪酸甘油酯、檸檬酸甘油酯、酒石酸甘油酯、乳酸甘油酯、乙氧基甘油 酯、磷酸甘油酯、琥珀酸甘油酯、脂肪酸聚合甘油酯、交酯化蓖麻酸聚合甘油酯、脂肪酸蔗糖酯、脂肪酸山梨醇酐酯、脂肪酸丙二醇酯、單及雙脂肪酸甘油二乙醯酒石酸酯、脂肪酸鹽類及上述之任意組合所組成之一族群。
- 如請求項8所述之抗氧化多層包埋益生菌顆粒之製造方法,其中該乾燥步驟包括冷凍乾燥處理、真空乾燥處理或噴霧乾燥處理。
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