TWI777107B - Composition with anti-oxidation strains or postbiotics of lactic acid bacterium and uses thereof - Google Patents
Composition with anti-oxidation strains or postbiotics of lactic acid bacterium and uses thereof Download PDFInfo
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- TWI777107B TWI777107B TW108142643A TW108142643A TWI777107B TW I777107 B TWI777107 B TW I777107B TW 108142643 A TW108142643 A TW 108142643A TW 108142643 A TW108142643 A TW 108142643A TW I777107 B TWI777107 B TW I777107B
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- acid bacteria
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Abstract
Description
本發明是有關一種組合物及其用途，特別是一種用以抗氧化之含乳酸菌菌株或其發酵物之組合物及其用途。 The present invention relates to a composition and its use, in particular to a composition containing a lactic acid bacteria strain or its fermented product for anti-oxidation and its use.
老化是人體器官最大的殺手，器官老化會導致慢性腎病、失智症、心血管疾病、糖尿病、癌症或其他慢性疾病，嚴重時可導致個體死亡。而身體所產生的自由基(free radicals)則是造成器官老化的重要元凶，身體所產生的自由基主要分為活性氧類(reactive oxygen species)以及活性氮類(reactive nitrogen species)。活性氧類是生物正常代謝中會產生的副產品，包含一些氧離子、過氧化氫等等，這些活性氧物質在細胞信號傳導、對抗微生物感染和保持機體恆常性中扮演著很重要的角色。而活性氮類則是在病原菌入侵時，大量被免疫細胞產生，主要用於殺死入侵的病原菌。血管內皮細胞也會分泌少量的活性氮物質，來促進血管擴張及訊息傳導。因此，調節並維持良好的自由基平衡對人體非常重要。 Aging is the biggest killer of human organs. Organ aging can lead to chronic kidney disease, dementia, cardiovascular disease, diabetes, cancer or other chronic diseases. In severe cases, it can lead to individual death. The free radicals produced by the body are an important culprit in the aging of organs. The free radicals produced by the body are mainly divided into reactive oxygen species and reactive nitrogen species. Reactive oxygen species are by-products produced in the normal metabolism of organisms, including some oxygen ions, hydrogen peroxide, etc. These reactive oxygen species play an important role in cell signaling, fighting microbial infection and maintaining body homeostasis. Reactive nitrogen is produced by immune cells in large quantities when pathogens invade, and is mainly used to kill the invading pathogens. Vascular endothelial cells also secrete a small amount of reactive nitrogen species to promote vascular dilation and signal transmission. Therefore, it is very important for the human body to regulate and maintain a good free radical balance.
當身體調節自由基的機轉喪失或失衡，過量的自由基產生則會破壞細胞DNA，改變細胞內蛋白質結構，攻擊細胞膜，最後導致體細胞的死亡。 慢性腎病(chronic kidney disease)的其中一個原因則是因為慢性發炎而腎臟累積了過多的活性氧物質，造成腎細胞的死亡，使得腎器官逐漸喪失其生理功能，到了末期患者只能洗腎度日。阿茲海默症則是腦內類澱粉的堆積造成慢性發炎，而發炎所產生的自由基誘發神經元細胞死亡，最後造成失智症。皮膚老化也是因為長時間暴露於紫外線(UV exposure)或熱源(heat exposure)下，誘發活性氧的量急劇增多，導致皮膚暗沉、皮膚癌等疾病。 When the body's mechanism of regulating free radicals is lost or unbalanced, excessive free radical production will damage cellular DNA, change the structure of intracellular proteins, attack cell membranes, and eventually lead to the death of somatic cells. One of the causes of chronic kidney disease is that due to chronic inflammation, the kidneys accumulate too much reactive oxygen species, causing the death of kidney cells, causing the kidneys to gradually lose their physiological functions. . Alzheimer's disease is a chronic inflammation caused by the accumulation of amyloid in the brain, and the free radicals produced by the inflammation induce neuronal cell death, and finally cause dementia. Skin aging is also caused by prolonged exposure to ultraviolet (UV exposure) or heat (heat exposure), which induces a sharp increase in the amount of reactive oxygen species, leading to diseases such as dull skin and skin cancer.
綜上所述，發展出一種安全且可長期使用，以抗氧化之營養補充品有其迫切性。而乳酸菌一般來說是安全的，因此，找出具有抗氧化功能的乳酸菌菌株或其發酵物便是目前極需努力的目標。 To sum up, it is urgent to develop a nutritional supplement that is safe and can be used for a long time with antioxidants. Lactic acid bacteria are generally safe, therefore, finding out lactic acid bacteria strains with antioxidant function or their fermented products is the goal that needs to be worked hard at present.
本發明提供一種含乳酸菌菌株或其發酵物之組合物，其具有抗氧化效果，因而能夠減少自由基濃度，以抑制器官老化。 The present invention provides a composition containing a lactic acid bacteria strain or a fermented product thereof, which has an anti-oxidative effect, thereby reducing the concentration of free radicals and inhibiting organ aging.
本發明一實施例之用以抗氧化之含乳酸菌菌株或其發酵物之組合物包含具有抗氧化活性效果之乳酸菌菌株或其發酵物以及賦形劑、稀釋劑或載體，其中經分離之乳酸菌菌株包含羅伊氏乳酸桿菌(Lactobacillus reuteri)GL-104菌株(寄存編號為BCRC 910404)、唾液乳酸桿菌(Lactobacillus salivarius subsp.salicinius)AP-32菌株(寄存編號為BCRC 910437)以及鼠李糖乳酸桿菌(Lactobacillus rhamnosus)bv-77菌株(寄存編號為BCRC 910533)，上述乳酸菌菌株寄存於財團法人食品工業發展研究所。 The composition containing lactic acid bacteria strains or their fermented products for anti-oxidation according to an embodiment of the present invention comprises lactic acid bacteria strains with antioxidant activity or their fermented products and excipients, diluents or carriers, wherein the isolated lactic acid bacteria strains Including Lactobacillus reuteri GL-104 strain (accession number BCRC 910404), Lactobacillus salivarius subsp. salicinius AP-32 strain (accession number BCRC 910437) and Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) bv-77 strain (the deposit number is BCRC 910533), the above-mentioned lactic acid bacteria strain is deposited in the Food Industry Development Research Institute.
本發明另一實施例之乳酸菌菌株或其發酵物在製備抗氧化功效之組合物之用途，其中組合物包含具有抗氧化活性效果之乳酸菌菌株或其發酵物以及賦形劑、稀釋劑或載體，且經分離之乳酸菌菌株包含羅伊氏乳酸桿菌 (Lactobacillus reuteri)GL-104菌株(寄存編號為BCRC 910404)、唾液乳酸桿菌(Lactobacillus salivarius subsp.salicinius)AP-32菌株(寄存編號為BCRC 910437)以及鼠李糖乳酸桿菌(Lactobacillus rhamnosus)bv-77菌株(寄存編號為BCRC 910533)，上述乳酸菌菌株寄存於財團法人食品工業發展研究所。 Another embodiment of the present invention is the use of a lactic acid bacteria strain or a fermented product thereof in preparing a composition with antioxidant activity, wherein the composition comprises a lactic acid bacteria strain with antioxidant activity or a fermented product thereof and an excipient, diluent or carrier, And the isolated lactic acid bacteria strains include Lactobacillus reuteri ( Lactobacillus reuteri ) GL-104 strain (accession number BCRC 910404), Lactobacillus salivarius subsp. salicinius AP-32 strain (accession number BCRC 910437) and Lactobacillus rhamnosus bv-77 strain (the deposit number is BCRC 910533), the above-mentioned lactic acid bacteria strain is deposited in the Food Industry Development Research Institute.
本發明又一實施例之乳酸菌菌株或其發酵物在製備抗氧化功效之食品組合物之用途，其中食品組合物包含具有抗氧化活性效果之乳酸菌菌株或其發酵物以及賦形劑、稀釋劑或載體，且經分離之乳酸菌菌株包含羅伊氏乳酸桿菌(Lactobacillus reuteri)GL-104菌株(寄存編號為BCRC 910404)、唾液乳酸桿菌(Lactobacillus salivarius subsp.salicinius)AP-32菌株(寄存編號為BCRC 910437)以及鼠李糖乳酸桿菌(Lactobacillus rhamnosus)bv-77菌株(寄存編號為BCRC 910533)，上述乳酸菌菌株寄存於財團法人食品工業發展研究所。 Another embodiment of the present invention is the use of a lactic acid bacteria strain or a fermented product thereof in preparing a food composition with antioxidant activity, wherein the food composition comprises a lactic acid bacteria strain with antioxidant activity or a fermented product thereof and an excipient, diluent or vector, and the isolated lactic acid bacteria strains include Lactobacillus reuteri GL-104 strain (accession number BCRC 910404), Lactobacillus salivarius subsp. salicinius AP-32 strain (accession number BCRC 910437) ) and Lactobacillus rhamnosus bv-77 strain (the deposit number is BCRC 910533), and the above-mentioned lactic acid bacteria strains are deposited in the Food Industry Development Research Institute.
以下藉由具體實施例配合所附的圖式詳加說明，當更容易瞭解本發明之目的、技術內容、特點及其所達成之功效。 The following detailed description will be given in conjunction with the accompanying drawings through specific embodiments, so as to make it easier to understand the purpose, technical content, characteristics and effects of the present invention.
圖1為本發明之乳酸菌活性菌株自由基清除能力分析的試驗結果。 Fig. 1 is the test result of the free radical scavenging ability analysis of the active strain of lactic acid bacteria of the present invention.
圖2為本發明之乳酸菌菌株之發酵物自由基清除能力分析的試驗結果。 FIG. 2 is the test result of the analysis of the free radical scavenging ability of the fermentation product of the lactic acid bacteria strain of the present invention.
圖3為本發明之乳酸菌活性菌株還原能力分析的試驗結果。 Fig. 3 is the test result of the reduction ability analysis of the active strain of lactic acid bacteria of the present invention.
圖4為本發明之乳酸菌之發酵物還原能力分析的試驗結果。 FIG. 4 is the test result of the analysis of the reducing ability of the fermentation product of the lactic acid bacteria of the present invention.
圖5為本發明之乳酸菌菌株誘發人類腸上皮細胞產生超氧化物歧化酶之活性分析結果。 Figure 5 is the result of the activity analysis of the lactic acid bacteria strain of the present invention inducing the production of superoxide dismutase in human intestinal epithelial cells.
圖6為本發明之乳酸菌菌株誘發人類腸上皮細胞產生過氧化氫酶之活性分析結果。 FIG. 6 shows the results of the activity analysis of the lactic acid bacteria strains of the present invention inducing the production of catalase in human intestinal epithelial cells.
圖7為本發明之10%乳酸菌發酵物所含的超氧化物歧化酶之活性分析結果。 Fig. 7 is the result of the activity analysis of superoxide dismutase contained in the 10% lactic acid bacteria fermentation product of the present invention.
以下將詳述本發明之各實施例，並配合圖式作為例示。除了這些詳細說明之外，本發明亦可廣泛地施行於其它的實施例中，任何所述實施例的輕易替代、修改、等效變化都包含在本發明之範圍內，並以申請專利範圍為準。在說明書的描述中，為了使讀者對本發明有較完整的瞭解，提供了許多特定細節；然而，本發明可能在省略部分或全部特定細節的前提下，仍可實施。此外，眾所周知的步驟或元件並未描述於細節中，以避免對本發明形成不必要之限制。圖式中相同或類似之元件將以相同或類似符號來表示。特別注意的是，圖式僅為示意之用，並非代表元件實際之尺寸或數量，有些細節可能未完全繪出，以求圖式之簡潔。 The various embodiments of the present invention will be described in detail below, with the accompanying drawings as examples. In addition to these detailed descriptions, the present invention can also be widely implemented in other embodiments, and any easy substitutions, modifications, and equivalent changes of any of the embodiments are included within the scope of the present invention, and the scope of the patent application is allow. In the description of the specification, numerous specific details are provided in order to provide the reader with a more complete understanding of the present invention; however, the present invention may be practiced without some or all of the specific details. Also, well-known steps or elements have not been described in detail to avoid unnecessarily limiting the invention. The same or similar elements in the drawings will be represented by the same or similar symbols. It should be noted that the drawings are for illustrative purposes only, and do not represent the actual size or number of components, and some details may not be fully drawn for the sake of simplicity in the drawings.
本發明所述之乳酸菌菌株之冷凍乾燥培養物已寄存於財團法人食品工業發展研究所，地址30062新竹市食品路331號。寄存之詳細資料如表1所示：
如表1所列已寄存的乳酸菌菌株中，長雙歧桿菌長雙歧亞種OLP-01菌株及其發酵物、短雙歧桿菌Bv-889菌株及其發酵物、長雙歧桿菌嬰兒亞種BLI-02菌株及其發酵物、動物雙歧桿菌乳亞種CP-9菌株及其發酵物、兩歧雙歧桿菌Bf-688菌株及其發酵物、羅伊氏乳酸桿菌GL-104菌株及其發酵物、唾液乳酸桿菌AP-32菌株及其發酵物、鼠李糖乳酸桿菌bv-77菌株及其發酵物被發現具有抗氧化以及清除自由基的活性效果。因此，表1所列之乳酸菌菌株或其發酵物可作為抗氧化以及清除自由基之用途。 Among the registered lactic acid bacteria strains listed in Table 1, Bifidobacterium longum subsp. longum OLP-01 strain and its fermented product, Bifidobacterium breve Bv-889 strain and its fermented product, Bifidobacterium longum subsp. longum subsp. BLI-02 strain and its fermented product, Bifidobacterium animalis subsp. lactis strain CP-9 and its fermented product, Bifidobacterium bifidum strain Bf-688 and its fermented product, Lactobacillus reuteri GL-104 strain and its fermented product The fermented product, Lactobacillus salivarius AP-32 strain and its fermented product, and Lactobacillus rhamnosus bv-77 strain and its fermented product were found to have antioxidant and free radical scavenging activities. Therefore, the lactic acid bacteria strains listed in Table 1 or their fermentation products can be used for antioxidant and free radical scavenging purposes.
本發明一實施例之用於抗氧化及清除自由基之含乳酸菌菌株或發酵物之組合物包含乳酸菌菌株或其所發酵產生之發酵物以及賦形劑、稀釋劑或載體。該乳酸菌菌株選自以下至少其中之一經分離的乳酸菌菌株：長雙歧桿菌長雙歧亞種OLP-01菌株(寄存編號為BCRC 910875)、短雙歧桿菌Bv-889菌株(寄存編號為BCRC 910844)、長雙歧桿菌嬰兒亞種BLI-02菌株(寄存編號為 BCRC 910812)、動物雙歧桿菌乳亞種CP-9菌株(寄存編號為BCRC 910645)、兩歧雙歧桿菌Bf-688菌株(寄存編號為BCRC 910902)、羅伊氏乳酸桿菌GL-104菌株(寄存編號為BCRC 910404)、唾液乳酸桿菌AP-32菌株(寄存編號為BCRC 910437)以及鼠李糖乳酸桿菌bv-77菌株(寄存編號為BCRC 910533)。上述乳酸菌菌株寄存於財團法人食品工業發展研究所。於一實施例中，賦形劑、稀釋劑或載體可為生理上可接受之賦形劑、稀釋劑或載體，使本發明之含乳酸菌菌株或發酵物之組合物作為一食品組合物使用。或者，賦形劑、稀釋劑或載體可為醫藥上可接受之賦形劑、稀釋劑或載體，使本發明之含乳酸菌菌株或發酵物之組合物作為一醫藥組合物使用。或者，賦形劑、稀釋劑或載體可為藥妝上可接受之賦形劑、稀釋劑或載體，使本發明之含乳酸菌菌株或發酵物之組合物作為一藥妝品組合物使用。 The composition containing lactic acid bacteria strains or fermented products for antioxidation and free radical scavenging according to an embodiment of the present invention comprises lactic acid bacteria strains or fermented products produced by fermentation thereof, and excipients, diluents or carriers. The lactic acid bacteria strain is selected from at least one of the following isolated lactic acid bacteria strains: Bifidobacterium longum subsp. longum OLP-01 strain (accession number BCRC 910875), Bifidobacterium breve Bv-889 strain (accession number BCRC 910844 ), Bifidobacterium longum infantum subsp. BLI-02 strain (the deposit number is BCRC 910812), Bifidobacterium animalis subsp. lactis strain CP-9 (accession number BCRC 910645), Bifidobacterium bifidum Bf-688 strain (accession number BCRC 910902), Lactobacillus reuteri GL-104 strain ( accession number BCRC 910404), Lactobacillus salivarius strain AP-32 (accession number BCRC 910437) and Lactobacillus rhamnosus strain bv-77 (accession number BCRC 910533). The above-mentioned lactic acid bacteria strains are deposited in the Food Industry Development Research Institute. In one embodiment, the excipient, diluent or carrier can be a physiologically acceptable excipient, diluent or carrier, so that the composition containing the lactic acid bacteria strain or the fermented product of the present invention can be used as a food composition. Alternatively, the excipient, diluent or carrier can be a pharmaceutically acceptable excipient, diluent or carrier, so that the lactic acid bacteria strain-containing or fermented composition of the present invention can be used as a pharmaceutical composition. Alternatively, the excipient, diluent or carrier may be a cosmeceutically acceptable excipient, diluent or carrier, so that the composition containing the lactic acid bacteria strain or fermented product of the present invention can be used as a cosmeceutical composition.
於食品組合物之實施例中，生理上可接受之賦形劑、稀釋劑或載體可為一食品。舉例而言，食品可包含但不限於乳製飲品、茶、咖啡、口香糖、潔牙糖(例如***片、咀嚼錠、軟糖等)或以上之組合，其中乳製飲品可包含發酵乳、優格、乳酪或乳製飲品乳粉等。醫藥組合物可為口服劑型或皮膚外用劑型。舉例而言，口服劑型可為錠劑、膠囊、溶液劑及粉劑等。 In embodiments of food compositions, the physiologically acceptable excipient, diluent or carrier may be a food product. For example, the food can include, but is not limited to, dairy drinks, tea, coffee, chewing gum, toothpaste (such as lozenges, chewable lozenges, gummies, etc.) or a combination of the above, wherein the dairy drinks can include fermented milk, Yogurt, cheese or dairy drink milk powder, etc. The pharmaceutical composition may be an oral dosage form or a topical dosage form for skin application. For example, oral dosage forms can be lozenges, capsules, solutions, powders, and the like.
於藥妝品組合物之實施例中，藥妝上可接受之賦形劑、稀釋劑或載體可為：1)液狀化妝品，例如沐浴液、洗髮液、化妝水、香水等；2)乳液狀化妝品；3)膏霜狀化妝品，例如潤面霜、粉底霜、洗髮膏；4)粉狀化妝品，例如香粉、爽身粉；5)塊狀化妝品，例如粉餅、化妝盒；6)棒狀化妝品，例如口紅、髮蠟。 In the embodiment of the cosmeceutical composition, the cosmeceutically acceptable excipient, diluent or carrier can be: 1) liquid cosmetics, such as body wash, shampoo, lotion, perfume, etc.; 2) Emulsion-like cosmetics; 3) Cream-like cosmetics, such as moisturizing cream, foundation cream, shampoo; 4) Powdered cosmetics, such as fragrance powder, talcum powder; cosmetic products such as lipstick and hair wax.
於含乳酸菌菌株之組合物之實施例中，乳酸菌菌株為具有活性的菌株。舉例而言，乳酸菌菌株之數量為106CFU以上；較佳者，乳酸菌菌株之數量為1010CFU以上。於含乳酸菌發酵物之組合物之實施例中，發酵物可包含去活 性菌株或去除菌體的發酵液或其乾燥粉末。舉例而言，發酵液可為發酵上清液或乳清發酵液等。於一實施例中，乳酸菌發酵物之粉劑含量為0.5%以上；或者，乳酸菌發酵物之溶液含量為2.5%以上。 In the embodiment of the composition containing lactic acid bacteria strains, the lactic acid bacteria strains are active strains. For example, the number of lactic acid bacteria strains is more than 10 6 CFU; preferably, the number of lactic acid bacteria strains is more than 10 10 CFU. In the embodiment of the composition containing the lactic acid bacteria fermentation product, the fermentation product may comprise deactivated bacterial strains or bacterial cell-removed fermentation broth or dry powder thereof. For example, the fermentation broth can be a fermentation supernatant or a whey fermentation broth or the like. In one embodiment, the powder content of the lactic acid bacteria fermentation product is more than 0.5%; or the solution content of the lactic acid bacteria fermentation product is more than 2.5%.
實施例1：本發明之乳酸菌菌株之形態學以及一般性質 Example 1: Morphology and general properties of the lactic acid bacteria strains of the present invention
根據16S rDNA序列分析以及API細菌鑑定系統分析結果來確認菌株在分類學上的特徵。上述菌株在形態學及一般性質上的特徵詳細列於表2：
實施例2：乳酸菌菌株之收集、培養與保存 Example 2 : Collection, cultivation and preservation of lactic acid bacteria strains
申請人所收集之乳酸菌菌株是以20%甘油保存於-80℃。使用前，以含有0.05% cysteine之MRS broth(DIFCO)，37℃下活化(24小時)二次後使用。使用於研究之本發明乳酸菌菌株中，長雙歧桿菌長雙歧亞種OLP-01菌株以及羅伊氏乳酸桿菌GL-104菌株分離自人類腸道；短雙歧桿菌Bv-889菌株、長雙歧桿菌嬰兒亞種BLI-02菌株、動物雙歧桿菌乳亞種CP-9菌株、兩歧雙歧桿菌Bf-688菌株以及鼠李糖乳酸桿菌bv-77菌株分離自人類母乳；唾液乳酸桿菌AP-32菌株分離自人類糞便。本發明之乳酸菌菌株所發酵產生之發酵物是以上述乳酸菌菌株至少其中之一進行發酵以產生其發酵物，再經由離心、過濾、加熱殺菌等步驟，最後純化出發酵液。依據需求，發酵液可進一步乾燥成乳酸菌發酵物粉末。發酵液之粉劑或水溶液可保存於常溫。 The Lactobacillus strains collected by the applicant were stored at -80°C with 20% glycerol. Before use, use MRS broth (DIFCO) containing 0.05% cysteine at 37° C. (24 hours) for two times before use. Among the lactic acid bacteria strains of the present invention used for research, Bifidobacterium longum subsp. longum OLP-01 strain and Lactobacillus reuteri GL-104 strain were isolated from human intestinal tract; Bifidobacterium breve Bv-889 strain, Bifidobacterium longum strain Bifidobacterium infantis strain BLI-02, Bifidobacterium animalis subsp. lactis strain CP-9, Bifidobacterium bifidum strain Bf-688, and Lactobacillus rhamnosus strain bv-77 were isolated from human breast milk; Lactobacillus salivarius AP The -32 strain was isolated from human feces. The fermented product produced by the lactic acid bacteria strain of the present invention is fermented by at least one of the above-mentioned lactic acid bacteria strains to produce its fermented product, and then through the steps of centrifugation, filtration, heat sterilization, etc., the fermentation liquid is finally purified. According to demand, the fermentation broth can be further dried into lactic acid bacteria fermented powder. The powder or aqueous solution of the fermentation broth can be stored at room temperature.
實施例3：乳酸菌菌株之自由基清除能力分析 Example 3 : Analysis of free radical scavenging ability of lactic acid bacteria strains
DPPH(di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium)是穩定自由基分子，DDPH自由基在甲醇溶液中在波長517nm下有最高的吸收值。當DPPH自由基與抗氧化物質作用後，抗氧化物質提供氫質子而清除自由基，而DPPH自由基就會失去本身藍紫色的特性而造成吸光值的下降。利用測定OD517值的下降來測定受測乳酸菌菌株之自由基清除能力。 DPPH (di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium) is a stable free radical molecule, and DDPH free radical has the highest absorption value at wavelength 517nm in methanol solution. When the DPPH free radical interacts with the antioxidant substance, the antioxidant substance provides hydrogen protons to scavenge the free radical, and the DPPH free radical will lose its own blue-violet characteristics, resulting in a decrease in the absorbance value. The free radical scavenging ability of the tested lactic acid bacteria strains was determined by measuring the decrease in OD517 value.
乳酸菌菌株之自由基清除能力的檢測方法如下。將本發明之乳酸菌菌株之菌株懸浮液(約為2×109CFU)、參考乳酸菌菌株：副乾酪乳酸桿菌(Lactobacillus paracasei)GL-156菌株、嗜酸乳酸桿菌(Lactobacillus acidophilus)TYCA06菌株、約氏乳酸桿菌(Lactobacillus johnsonii)MH-68菌株以及鼠李糖乳酸桿菌(Lactobacillus rhamnosus)F-1菌株之菌株懸浮液(約為2×109CFU)、2.5μg/ml維生素C(陽性控制組，positive control)、不具抗氧化活性效果之嗜熱鏈 球菌(Streptococcus thermophiles)SY-66菌株(約為2×109CFU，陰性控制組，negative control)及二次水(空白組，blank)分別與0.2mM DPPH在甲醇溶液中1：1混合。混和均勻後，在黑暗室溫下反應30分鐘。接著於4℃離心(12000rpm，2min)後，取200μl到96孔盤中並測定OD517值。自由基清除能力的計算公式如下：自由基清除能力=(ODblank-ODsample)/ODblank * 100% The detection method of the free radical scavenging ability of lactic acid bacteria strains is as follows. The strain suspension of the lactic acid bacteria strain of the present invention (about 2×10 9 CFU), the reference lactic acid bacteria strains: Lactobacillus paracasei GL-156 strain, Lactobacillus acidophilus TYCA06 strain, Yokohama Lactobacillus johnsonii MH-68 strain and strain suspension of Lactobacillus rhamnosus F-1 strain (about 2×10 9 CFU), 2.5 μg/ml vitamin C (positive control group, positive control), Streptococcus thermophiles SY-66 strain without antioxidant activity (about 2×10 9 CFU, negative control group, negative control) and secondary water (blank group, blank) were compared with 0.2 mM DPPH was mixed 1:1 in methanol solution. After mixing uniformly, the reaction was carried out at room temperature in the dark for 30 minutes. Then, after centrifugation (12000rpm, 2min) at 4°C, 200μl was taken into a 96-well plate and the OD517 value was determined. The calculation formula of free radical scavenging ability is as follows: free radical scavenging ability=(OD blank -OD sample )/OD blank * 100%
其中ODsample為受測樣品的吸光值，ODBlank為空白組的吸光值。 OD sample is the absorbance value of the tested sample, and OD Blank is the absorbance value of the blank group.
請參照圖1，以說明本發明之乳酸菌菌株清除自由基能力分析(DPPH assay)之試驗結果，其中符號***表示p值<0.005；符號**表示p值<0.01，亦即統計學上具有非常顯著的差異；符號NS表示無明顯差異。由圖1之試驗結果可知，相較於不具抗氧化能力之嗜熱鏈球菌SY-66菌株，本發明之長雙歧桿菌長雙歧亞種OLP-01菌株、短雙歧桿菌Bv-889菌株、長雙歧桿菌嬰兒亞種BLI-02菌株、動物雙歧桿菌乳亞種CP-9菌株、兩歧雙歧桿菌Bf-688菌株、羅伊氏乳酸桿菌GL-104菌株、唾液乳酸桿菌AP-32菌株及鼠李糖乳酸桿菌bv-77菌株皆具有明顯較高的清除自由基的能力。 Please refer to FIG. 1 to illustrate the test results of the free radical scavenging ability analysis (DPPH assay) of the lactic acid bacteria strains of the present invention, wherein the symbol *** indicates p value <0.005; the symbol ** indicates p value < 0.01, that is, statistically There is a very significant difference; the symbol NS indicates no significant difference. It can be seen from the test results in FIG. 1 that, compared with the Streptococcus thermophilus SY-66 strain without antioxidant capacity, the Bifidobacterium longum subsp. longum OLP-01 strain and Bifidobacterium breve Bv-889 strain of the present invention , Bifidobacterium longum infantum subsp. BLI-02 strain, Bifidobacterium animalis subsp. lactis CP-9 strain, Bifidobacterium bifidum Bf-688 strain, Lactobacillus reuteri GL-104 strain, Lactobacillus salivarius AP- Both the 32 strains and the Lactobacillus rhamnosus bv-77 strains had significantly higher ability to scavenge free radicals.
實施例4：乳酸菌發酵物之自由基清除能力分析 Example 4 : Analysis of free radical scavenging ability of lactic acid bacteria fermentation product
乳酸菌發酵物之自由基清除能力的檢測方法如下。分別將本發明之乳酸菌發酵物粉劑1%水溶液、參考乳酸菌菌株：副乾酪乳酸桿菌GL-156菌株、嗜酸乳酸桿菌TYCA06菌株、約氏乳酸桿菌MH-68菌株以及鼠李糖乳酸桿菌F-1菌株之發酵物粉劑1%水溶液、8.5μg/ml維生素C(陽性控制組，positive control)、不具抗氧化活性效果之嗜熱鏈球菌SY-66菌株之發酵物粉劑1%水溶液(陰性控制組，negative control)及二次水(空白組，blank)分別與0.2mM DPPH在甲醇溶液中1：1混合。混和均勻後，在黑暗室溫下反應30分鐘。接著於4℃離心 (12000rpm，2min)後，取200μl到96孔盤中並測定OD517值。自由基清除能力的計算公式如前所述。 The detection method of the free radical scavenging ability of lactic acid bacteria fermentation product is as follows. The 1% aqueous solution of the lactic acid bacteria fermented product powder of the present invention, the reference lactic acid bacteria strains: Lactobacillus paracasei GL-156 strain, Lactobacillus acidophilus TYCA06 strain, Lactobacillus johnsonii MH-68 strain and Lactobacillus rhamnosus F-1 1% aqueous solution of fermented powder of strains, 8.5 μg/ml vitamin C (positive control group, positive control), 1% aqueous solution of fermented powder of Streptococcus thermophilus SY-66 strain without antioxidant activity (negative control group, negative control) and secondary water (blank, blank) were mixed 1:1 with 0.2 mM DPPH in methanol solution, respectively. After mixing uniformly, the reaction was carried out at room temperature in the dark for 30 minutes. Then, after centrifugation (12000rpm, 2min) at 4°C, 200μl was taken into a 96-well plate and the OD517 value was determined. The formula for calculating the free radical scavenging capacity is as described above.
請參照圖2，以說明本發明之乳酸菌發酵物(0.5%)清除自由基能力分析(DPPH assay)之試驗結果，其中符號***表示p值<0.005；符號**表示p值<0.01，亦即統計學上具有非常顯著的差異。由圖2之試驗結果可知，相較於嗜熱鏈球菌SY-66菌株之發酵物，本發明之長雙歧桿菌長雙歧亞種OLP-01菌株、短雙歧桿菌Bv-889菌株、長雙歧桿菌嬰兒亞種BLI-02菌株、動物雙歧桿菌乳亞種CP-9菌株、兩歧雙歧桿菌Bf-688菌株、羅伊氏乳酸桿菌GL-104菌株、唾液乳酸桿菌AP-32菌株及鼠李糖乳酸桿菌bv-77菌株之發酵物皆具有明顯較高的清除自由基的能力。 Please refer to FIG. 2 to illustrate the test results of the free radical scavenging ability analysis (DPPH assay) of the lactic acid bacteria fermented product (0.5%) of the present invention, wherein the symbol *** indicates p value <0.005; the symbol ** indicates p value < 0.01, That is, there is a statistically significant difference. It can be seen from the test results in FIG. 2 that, compared with the fermentation product of Streptococcus thermophilus SY-66 strain, the Bifidobacterium longum subsp. longum OLP-01 strain, Bifidobacterium breve Bv-889 strain, Bifidobacterium infantis subsp. BLI-02 strain, Bifidobacterium animalis subsp. lactis strain CP-9, Bifidobacterium bifidum strain Bf-688 strain, Lactobacillus reuteri GL-104 strain, Lactobacillus salivarius AP-32 strain The fermented product of Lactobacillus rhamnosus bv-77 strain has significantly higher ability to scavenge free radicals.
實施例5：乳酸菌菌株之還原力分析。 Example 5 : Analysis of reducing power of lactic acid bacteria strains.
抗氧化還原力活性分析(Ferric-reducing ability of power，FRAP assay)是一種普遍用來測試抗氧化劑還原力活性的一種方法，以樣品整體的還原能力作為抗氧化力。於酸性環境(pH 3.6以下)下，FRAP試劑中的三價鐵(Fe3+)會被抗氧化物如維生素C還原成二價鐵(Fe2+)，而造成顏色的改變。利用TPTZ(2,4,6-Tri-(2-pyridyl)-5-triazine)的呈色特性可測得樣品的還原能力。當Fe3+-TPTZ複合物被還原成Fe2+-TPTZ時，會由黃色轉為藍色，藍色越深表示抗氧化力越強。因此，檢測OD593值即可計算出抗氧化劑的還原能力，也就是抗氧化能力。 Ferric-reducing ability of power (FRAP assay) is a method commonly used to test the reducing power of antioxidants, and the overall reducing ability of the sample is used as the antioxidant power. In an acidic environment (below pH 3.6), the ferric iron (Fe 3+ ) in the FRAP reagent will be reduced to ferrous iron (Fe 2+ ) by antioxidants such as vitamin C, resulting in a color change. The reducing ability of the sample can be measured by the color development properties of TPTZ (2,4,6-Tri-(2-pyridyl)-5-triazine). When the Fe 3+ -TPTZ complex is reduced to Fe 2+ -TPTZ, it will turn from yellow to blue, and the darker the blue, the stronger the antioxidant capacity. Therefore, the reducing capacity of antioxidants, that is, antioxidant capacity, can be calculated by detecting the OD 593 value.
本次實驗是將本發明乳酸菌之菌株懸浮液(約為2×109CFU)、參考乳酸菌菌株：副乾酪乳酸桿菌GL-156菌株、嗜酸乳酸桿菌TYCA06菌株、約氏乳酸桿菌MH-68菌株以及鼠李糖乳酸桿菌F-1菌株之菌株懸浮液(約為2×109CFU)、1.25μg/ml維生素C(陽性控制組，positive control)、不具抗氧化活性效果之嗜熱鏈球菌SY-66菌株之菌株懸浮液(約為2×109CFU，陰性控制組，negative control)和Fe3+-TPTZ做反應，並檢測OD593值。所測的值和所製備的已知濃度的FeSO4標準液混合FRAP試劑所得到的標準檢量線做比對。利用檢量線公式計算出含乳酸菌菌株懸浮液之還原力(μg/ml,Fe2+)。 In this experiment, the strain suspension of lactic acid bacteria of the present invention (about 2×10 9 CFU), reference lactic acid bacteria strains: Lactobacillus paracasei GL-156 strain, Lactobacillus acidophilus TYCA06 strain, Lactobacillus johnsonii strain MH-68 And the strain suspension of Lactobacillus rhamnosus F-1 strain (about 2×10 9 CFU), 1.25 μg/ml vitamin C (positive control group, positive control), Streptococcus thermophilus SY without antioxidant activity The strain suspension of -66 strain (about 2×10 9 CFU, negative control group, negative control) was reacted with Fe 3+ -TPTZ, and the OD 593 value was detected. The measured value is compared with the standard calibration line obtained by mixing FRAP reagent with FeSO 4 standard solution of known concentration. The reducing power (μg/ml, Fe 2+ ) of the suspension containing lactic acid bacteria was calculated using the calibration curve formula.
請參照圖3，其為本發明之乳酸菌菌株還原力分析(FRAP assay)之試驗結果，其中符號***表示p值<0.005，符號**表示p值<0.01，亦即統計學上具有非常顯著的差異；符號*表示p值<0.05，亦即統計學上具有顯著的差異；符號NS表示無明顯差異。由圖3之試驗結果可知，相較於嗜熱鏈球菌SY-66菌株，本發明之長雙歧桿菌長雙歧亞種OLP-01菌株、短雙歧桿菌Bv-889菌株、長雙歧桿菌嬰兒亞種BLI-02菌株、動物雙歧桿菌乳亞種CP-9菌株、兩歧雙歧桿菌Bf-688菌株、羅伊氏乳酸桿菌GL-104菌株、唾液乳酸桿菌AP-32菌株及鼠李糖乳酸桿菌bv-77菌株皆具有明顯較高的還原的能力。 Please refer to FIG. 3 , which is the test result of the lactic acid bacteria strain reducing power analysis (FRAP assay) of the present invention, wherein the symbol *** represents p value<0.005, and the symbol ** represents p value<0.01, that is, statistically significant Significant difference; symbol * indicates p -value < 0.05, ie statistically significant difference; symbol NS indicates no significant difference. It can be seen from the test results in FIG. 3 that, compared with the Streptococcus thermophilus SY-66 strain, the Bifidobacterium longum subsp. longum OLP-01 strain, Bifidobacterium breve Bv-889 strain, Bifidobacterium longum strain of the present invention Bifidobacterium infantis BLI-02 strain, Bifidobacterium animalis subsp. lactis strain CP-9, Bifidobacterium bifidum Bf-688 strain, Lactobacillus reuteri GL-104 strain, Lactobacillus salivarius AP-32 strain and rhamnosus Lactobacillus saccharobacterium bv-77 strains all have significantly higher reducing ability.
實施例6：乳酸菌發酵物之還原力分析 Example 6 : Analysis of reducing power of lactic acid bacteria fermentation product
本次實驗是將本發明之乳酸菌菌株之發酵物粉劑0.5%水溶液、參考乳酸菌菌株：副乾酪乳酸桿菌GL-156菌株、嗜酸乳酸桿菌TYCA06菌株、約氏乳酸桿菌MH-68菌株以及鼠李糖乳酸桿菌F-1菌株之發酵物粉劑0.5%水溶液、5μg/ml維生素C(陽性控制組，positive control)、不具抗氧化活性效果之嗜熱鏈球菌SY-66菌株之發酵物粉劑0.5%水溶液(陰性控制組，negative control)和Fe3+-TPTZ做反應，並檢測OD593值。所測的值和所製備的已知濃度的FeSO4標準液混合FRAP試劑所得到的標準檢量線做比對。利用檢量線公式計算出乳酸菌發酵物的還原力(μg/ml,Fe2+)。 In this experiment, 0.5% aqueous solution of fermented product powder of lactic acid bacteria strains of the present invention, reference lactic acid bacteria strains: Lactobacillus paracasei GL-156 strain, Lactobacillus acidophilus TYCA06 strain, Lactobacillus johnsonii MH-68 strain and rhamnose 0.5% aqueous solution of fermented material powder of Lactobacillus F-1 strain, 5 μg/ml vitamin C (positive control group, positive control), 0.5% aqueous solution of fermented material powder of Streptococcus thermophilus SY-66 strain without antioxidant activity ( Negative control group, negative control) and Fe 3+ -TPTZ were reacted, and the OD 593 value was detected. The measured value is compared with the standard calibration line obtained by mixing FRAP reagent with FeSO 4 standard solution of known concentration. Using the calibration curve formula, the reducing power (μg/ml, Fe 2+ ) of the lactic acid bacteria fermentation product was calculated.
請參照圖4，以說明本發明之乳酸菌發酵物還原力分析試驗(FRAP assay)的試驗結果，其中符號***表示p值<0.005；符號**表示p值<0.01，亦即統 計學上具有非常顯著的差異。由圖4之試驗結果可知，相較於嗜熱鏈球菌SY-66菌株，本發明之長雙歧桿菌長雙歧亞種OLP-01菌株、短雙歧桿菌Bv-889菌株、長雙歧桿菌嬰兒亞種BLI-02菌株、動物雙歧桿菌乳亞種CP-9菌株、兩歧雙歧桿菌Bf-688菌株、羅伊氏乳酸桿菌GL-104菌株、唾液乳酸桿菌AP-32菌株及鼠李糖乳酸桿菌bv-77菌株之發酵物皆具有明顯較高的還原能力。 Please refer to FIG. 4 to illustrate the test results of the lactic acid bacteria fermentation product reducing power analysis test (FRAP assay) of the present invention, wherein the symbol *** represents p value<0.005; the symbol ** represents p value<0.01, that is, statistically have very significant differences. It can be seen from the test results in FIG. 4 that, compared with the Streptococcus thermophilus SY-66 strain, the Bifidobacterium longum subsp. longum OLP-01 strain, Bifidobacterium breve Bv-889 strain, Bifidobacterium longum strain of the present invention Bifidobacterium infantis BLI-02 strain, Bifidobacterium animalis subsp. lactis strain CP-9, Bifidobacterium bifidum Bf-688 strain, Lactobacillus reuteri GL-104 strain, Lactobacillus salivarius AP-32 strain and rhamnosus The fermented products of Lactobacillus saccharobacterium bv-77 strains have obviously higher reducing ability.
實施例7：乳酸菌菌株誘發腸上皮細胞表現抗氧化酵素活性分析 Example 7 : Analysis of Antioxidant Enzyme Activity in Intestinal Epithelial Cells Induced by Lactobacillus Strain
身體內對於太多的自由基(oxidative stress)會有所謂的調控機制，體細胞會產生抗氧化劑(antioxidant)來因應，如合成穀胱甘肽(glutathione)、泛醇(ubiquinol)和尿酸(uric acid)等物質來吸收游離電子，另一方面從食物中攝取抗氧化劑如維生素C、維生素E等，也能抑制自由基的產生。身體細胞的另一個抗氧化系統為抗氧化酵素(antioxidant enzymes)網絡。超氧化物歧化酶(superoxide dismutase，SOD)是一種重要的抗氧化酵素，可以將超氧化物通過歧化反應轉化為氧氣和過氧化氫，人體內有三種超氧化物歧化酶，分別存在於胞外、細胞質(cytoplasm)及粒線體(mitochondria)中。另一種酵素為過氧化氫酶(catalase)是可以將超氧化物歧化酶所產生的過氧化氫轉化為氧氣和水。在飽和的狀態下，一個過氧化氫酶分子每秒能將四千萬個過氧化氫分子轉化為水和氧氣。 There is a so-called regulation mechanism in the body for too many free radicals (oxidative stress). Somatic cells will produce antioxidants (antioxidants) in response, such as the synthesis of glutathione (glutathione), panthenol (ubiquinol) and uric acid (uric acid). Acid) and other substances to absorb free electrons, on the other hand, intake of antioxidants such as vitamin C, vitamin E, etc. from food can also inhibit the production of free radicals. Another antioxidant system of body cells is the network of antioxidant enzymes. Superoxide dismutase (superoxide dismutase, SOD) is an important antioxidant enzyme, which can convert superoxide into oxygen and hydrogen peroxide through dismutation reaction. There are three kinds of superoxide dismutase in the human body, which exist in extracellular , cytoplasm (cytoplasm) and mitochondria (mitochondria). Another enzyme is catalase, which converts hydrogen peroxide produced by superoxide dismutase into oxygen and water. At saturation, one catalase molecule can convert 40 million hydrogen peroxide molecules per second into water and oxygen.
Caco-2細胞為人類結腸腺癌細胞上皮細胞，其細胞結構和功能相似於分化的小腸上皮細胞。Caco-2細胞具有微絨毛等結構並含有與小腸刷狀緣上皮相關的酶系，因而被廣泛應用在模擬體內腸細胞生理活動模式。在細胞培養系統中，Caco-2細胞可以長成單一層細胞，細胞彼此排列緊密，不僅在形態學上和小腸上皮細胞相似，且具有同樣的胞飲作用、Tight junction等結構。 Caco-2 cells are human colon adenocarcinoma epithelial cells, and their cellular structure and function are similar to differentiated small intestinal epithelial cells . Caco-2 cells have structures such as microvilli and contain enzymes related to the brush border epithelium of the small intestine, so they are widely used to simulate the physiological activity pattern of intestinal cells in vivo. In the cell culture system, Caco-2 cells can grow into a single layer of cells, and the cells are closely arranged with each other, which is not only morphologically similar to small intestinal epithelial cells, but also has the same structures such as pinocytosis and Tight junction.
本實驗在Caco-2細胞的培養系統中加入本發明之乳酸菌之活性菌株(實驗組)、嗜熱鏈球菌SY-66菌株(對照組)及不加入任何活性菌株只有培養液 作為控制組(Control)，以比例1：100(cells：probiotics)共同培養16小時後，洗掉乳酸菌菌株，再將Caco-2細胞打破萃取蛋白質，以檢測細胞內的超氧化物歧化酶(superoxide dismutase，SOD)活性(其試驗結果如圖5所示)及過氧化氫酶(catalase)活性(其試驗結果如圖6所示)。SOD活性分析是使用SOD Assay Kit(Cayman Cat.706002)，而catalase活性分析是使用Catalase Assay Kit(Cayman Cat.707002)。所有實驗流程皆依照kit的說明書建議進行分析。 In this experiment, the active strain of the lactic acid bacteria of the present invention (experimental group), the Streptococcus thermophilus SY-66 strain (control group) were added to the culture system of Caco-2 cells, and no active strain was added, only the culture medium As a control group (Control), after co-cultivation at a ratio of 1:100 (cells: probiotics) for 16 hours, the lactic acid bacteria strains were washed away, and then Caco-2 cells were broken down to extract proteins to detect intracellular superoxide dismutase (superoxide dismutase). dismutase, SOD) activity (the test results are shown in Figure 5) and catalase (catalase) activity (the test results are shown in Figure 6). The SOD activity assay was performed using the SOD Assay Kit (Cayman Cat. 706002), and the catalase activity assay was performed using the Catalase Assay Kit (Cayman Cat. 707002). All experimental procedures were analyzed in accordance with the kit's instructions.
以下請參照圖5以及圖6，以說明本發明之乳酸菌菌株誘發腸上皮細胞表現抗氧化酵素之效果，其中符號***表示p值<0.005；符號**表示p值<0.01，亦即統計學上具有非常顯著的差異；符號*表示p值<0.05，亦即統計學上具有顯著差異；符號NS表示無明顯差異。由圖5以及圖6之試驗結果可知，相較於嗜熱鏈球菌SY-66菌株，本發明之長雙歧桿菌長雙歧亞種OLP-01菌株、短雙歧桿菌Bv-889菌株、長雙歧桿菌嬰兒亞種BLI-02菌株、動物雙歧桿菌乳亞種CP-9菌株、兩歧雙歧桿菌Bf-688菌株、羅伊氏乳酸桿菌GL-104菌株、唾液乳酸桿菌AP-32菌株及鼠李糖乳酸桿菌bv-77菌株可以誘發人類腸上皮細胞株Caco-2細胞增加抗氧化酵素(SOD及catalase)的表現，以分解過多的體內自由基，並達到抗氧化的效果。 5 and 6, to illustrate the effect of the lactic acid bacteria strain of the present invention inducing the expression of antioxidant enzymes in intestinal epithelial cells, wherein the symbol *** indicates p value <0.005; the symbol ** indicates p value < 0.01, that is, statistics There is a very significant difference in science; the symbol * means p value < 0.05, that is, there is a statistically significant difference; the symbol NS means no significant difference. It can be seen from the test results in FIG. 5 and FIG. 6 that compared with the Streptococcus thermophilus SY-66 strain, the Bifidobacterium longum subsp. longum OLP-01 strain, the Bifidobacterium breve Bv-889 strain, the long Bifidobacterium infantis subsp. BLI-02 strain, Bifidobacterium animalis subsp. lactis strain CP-9, Bifidobacterium bifidum strain Bf-688 strain, Lactobacillus reuteri GL-104 strain, Lactobacillus salivarius AP-32 strain And Lactobacillus rhamnosus bv-77 strain can induce human intestinal epithelial cell line Caco-2 cells to increase the expression of antioxidant enzymes (SOD and catalase) to decompose excessive free radicals in the body and achieve antioxidant effects.
實施例8：乳酸菌發酵物之抗氧化酵素活性分析 Example 8 : Antioxidant enzyme activity analysis of lactic acid bacteria fermentation product
本實驗是將本發明之乳酸菌菌株以及嗜熱鏈球菌SY-66菌株之發酵物乾燥粉末配置成10%水溶液進行超氧化物歧化酶(SOD)活性分析。另以未經乳酸菌菌株發酵之培養基作為控制組。SOD活性分析是使用SOD Assay Kit(Cayman Cat.706002)。所有實驗流程皆依照kit的說明書建議進行分析。 In this experiment, the dry powder of the lactic acid bacteria strain of the present invention and the fermentation product of the Streptococcus thermophilus SY-66 strain was prepared into a 10% aqueous solution for superoxide dismutase (SOD) activity analysis. In addition, the medium without fermentation of lactic acid bacteria was used as the control group. The SOD activity assay was performed using the SOD Assay Kit (Cayman Cat. 706002). All experimental procedures were analyzed in accordance with the kit's instructions.
請參照圖7，以說明本發明之乳酸菌發酵物之抗氧化酵素之試驗結果，其中符號***表示p值<0.005，即統計學上具有非常顯著的差異；符號*表 示p值<0.05，即統計學上具有顯著的差異。由圖7之試驗結果可知，相較於嗜熱鏈球菌SY-66菌株，本發明之長雙歧桿菌長雙歧亞種OLP-01菌株、短雙歧桿菌Bv-889菌株、長雙歧桿菌嬰兒亞種BLI-02菌株、動物雙歧桿菌乳亞種CP-9菌株、兩歧雙歧桿菌Bf-688菌株、羅伊氏乳酸桿菌GL-104菌株、唾液乳酸桿菌AP-32菌株及鼠李糖乳酸桿菌bv-77菌株之發酵物具有明顯較強的SOD抗氧化酵素活性，其可分解過多的體內自由基，以達到抗氧化的效果。 Please refer to Fig. 7, to illustrate the test results of the antioxidant enzymes of the lactic acid bacteria fermented product of the present invention, wherein the symbol *** represents p value<0.005, that is, there is a very significant difference in statistics; symbol * represents p value<0.05, That is, there is a statistically significant difference. It can be seen from the test results in FIG. 7 that, compared with the Streptococcus thermophilus SY-66 strain, the Bifidobacterium longum subsp. longum OLP-01 strain, Bifidobacterium breve Bv-889 strain, Bifidobacterium longum strain of the present invention Bifidobacterium infantis BLI-02 strain, Bifidobacterium animalis subsp. lactis strain CP-9, Bifidobacterium bifidum Bf-688 strain, Lactobacillus reuteri GL-104 strain, Lactobacillus salivarius AP-32 strain and rhamnosus The fermentation product of Lactobacillus saccharobacterium bv-77 strain has significantly stronger SOD antioxidant enzyme activity, which can decompose excessive free radicals in the body to achieve antioxidant effect.
需注意的是，乳酸菌對身體健康的功能在於菌株(strain)的特異性，而非菌種(species)，此種對於人之身體健康有特殊功效之菌株稱為功能性益生菌(Guidelines for the evaluation of probiotics in food；Report of joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food；London Ontario,Canada April 30 and May 1,2002：1-7)。舉例而言，根據2018年發表於Scientific Reports的一篇論文(PMID：30013208)提到，野生種Bifidobacterium longum subsp.infantis在有氧的環境下，由於菸鹼醯胺腺嘌呤二核甘酸磷酯(nicotinamide adenine dinucleotide phosphate，NADPH)氧化酶的作用，會在菌體內產生過多的活性氧自由基(H2O2)。因此，野生種Bifidobacterium longum subsp.infantis似乎無法有效清除自由基，並造成菌體的死亡或抑制其生長。而本發明之長雙歧桿菌嬰兒亞種(Bifidobacterium longum subsp.infantis)BLI-02菌株具有清除自由基的功效，相較於野生種Bifidobacterium longum subsp.infantis具有其獨特性。此外，在超氧化物歧化酶(SOD)的表現上，中興大學的一篇研究中(http：//hdl.handle.net/11455/50980)發現Bifidobacterium longum B6菌株以及15708菌株，不具SOD活性。由上述2份研究可知，本發明之長雙歧桿菌嬰兒亞種BLI-02菌株及長雙歧桿菌長雙歧亞種OLP-01菌株為具有抗氧化活性效果的獨特性菌株。 It should be noted that the function of lactic acid bacteria on physical health lies in the specificity of the strain, not the species. Such strains with special effects on human health are called functional probiotics (Guidelines for the evaluation of probiotics in food; Report of joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food; London Ontario, Canada April 30 and May 1, 2002: 1-7). For example, according to a paper published in Scientific Reports in 2018 (PMID: 30013208), the wild species Bifidobacterium longum subsp. infantis in an aerobic environment, due to nicotinamide adenine dinucleotide phosphate ( The action of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase will produce excessive reactive oxygen radicals (H 2 O 2 ) in the bacteria. Therefore, the wild species Bifidobacterium longum subsp. infantis does not seem to be able to effectively scavenge free radicals and cause bacterial cell death or inhibit its growth. The Bifidobacterium longum subsp. infantis BLI-02 strain of the present invention has the effect of scavenging free radicals, which is unique compared to the wild species Bifidobacterium longum subsp. infantis . In addition, in the performance of superoxide dismutase (SOD), a study by Zhongxing University ( http://hdl.handle.net/11455/50980 ) found that Bifidobacterium longum B6 strain and 15708 strain did not have SOD activity. It can be seen from the above two studies that the Bifidobacterium longum subsp. infantis BLI-02 strain and the Bifidobacterium longum subsp. longum OLP-01 strain of the present invention are unique strains with antioxidant activity.
根據Jayamanne V.S.及Adams M.R.的研究(PMID：16478503)，其實驗中使用Bifidobacterium longum NCTC11818、Bifidobacterium breve NCIMB702258、Bifidobacterium longum biotype infantis NCIMB702205、Bifidobacterium adolescentis NCIMB702204、Bifidobacterium bifidum NCIMB702203與H2O2氧化自由基相互作用。發現以上所列之乳酸菌菌株並沒有抗氧化的特性。因此，相較於Jayamanne V.S.及Adams M.R.的研究，本發明之兩歧雙歧桿菌Bf-688菌株以及短雙歧桿菌Bv-889菌株為具有抗氧化活性效果的獨特性菌株。此外，於Oberg T.S.et al研究中(PMID：23772066)，發現同為Bifidobacterium animalis subsp.lactis BL-04菌株以及DSM 10140菌株表現了不同的抗氧化能力，故以此推知並非所有的Bifidobacterium animalis subsp.lactis都具有抗氧化特性。因此，本發明之動物雙歧桿菌乳亞種CP-9菌株為具有抗氧化活性效果的獨特性菌株。 According to the research of Jayamanne VS and Adams MR (PMID: 16478503), Bifidobacterium longum NCTC11818, Bifidobacterium breve NCIMB702258, Bifidobacterium longum biotype infantis NCIMB702205, Bifidobacterium adolescentis NCIMB702204, Bifidobacterium bifidum NCIMB702203 interact with H 2 O 2 free radicals in their experiments. The lactic acid bacteria strains listed above were found to have no antioxidant properties. Therefore, compared with the research of Jayamanne VS and Adams MR, the Bifidobacterium bifidum Bf-688 strain and the Bifidobacterium breve Bv-889 strain of the present invention are unique strains with antioxidant activity. In addition, in Oberg TSet al research (PMID: 23772066), it was found that Bifidobacterium animalis subsp. lactis BL-04 strain and DSM 10140 strain showed different antioxidant capacity, so it is inferred that not all Bifidobacterium animalis subsp. lactis strains All have antioxidant properties. Therefore, the CP-9 strain of Bifidobacterium animalis subsp. lactis of the present invention is a unique strain with antioxidant activity.
根據Chooruk A等人的研究(PMID：28474851)，SOD酵素的活性及生產，在直接從口腔內所分離出來的野生型L.salivarius及L.rhamnosus並不高(約0.1-0.2U之間)。而本發明之唾液乳酸桿菌AP-32菌株以及鼠李糖乳酸桿菌bv-77菌株的發酵物中就有約1.5-1.75U左右的SOD活性，顯示本發明之唾液乳酸桿菌AP-32菌株以及鼠李糖乳酸桿菌bv-77菌株為具有抗氧化活性效果的獨特性菌株。又根據Narciza O等人的研究(PMID：30807829)發現，L.reuteri需要白藜蘆醇(resveratrol)來誘發表現dhaT基因才能得到抗氧化的特性，而本發明之羅伊氏乳酸桿菌GL-104菌株直接就具有抗氧化活性效果，顯示本發明之羅伊氏乳酸桿菌GL-104菌株為具有抗氧化活性效果的獨特性菌株。 According to the study of Chooruk A et al. (PMID: 28474851), the activity and production of SOD enzymes are not high in wild-type L. salivarius and L. rhamnosus directly isolated from the oral cavity (between about 0.1-0.2U) . The fermented products of Lactobacillus salivarius AP-32 strain and Lactobacillus rhamnosus bv-77 strain of the present invention have SOD activity of about 1.5-1.75U, indicating that the Lactobacillus salivarius AP-32 strain of the present invention and the mouse The Lactobacillus Lactobacillus bv-77 strain is a unique strain with antioxidant activity. According to the research of Narciza O et al. (PMID: 30807829), it was found that L. reuteri needs resveratrol (resveratrol) to induce the expression of the dhaT gene to obtain antioxidant properties, and the L. reuteri GL-104 of the present invention The strain directly has antioxidant activity, which shows that the Lactobacillus reuteri GL-104 strain of the present invention is a unique strain with antioxidant activity.
綜合上述，相較於其它受測的乳酸菌菌株，本發明之長雙歧桿菌長雙歧亞種OLP-01菌株、短雙歧桿菌Bv-889菌株、長雙歧桿菌嬰兒亞種BLI-02菌株、動物雙歧桿菌乳亞種CP-9菌株、兩歧雙歧桿菌Bf-688菌株、羅伊氏乳酸 桿菌GL-104菌株、唾液乳酸桿菌AP-32菌株、鼠李糖乳酸桿菌bv-77菌株以及上述乳酸菌菌株之發酵物具有較佳的自由基清除能力以及還原能力，且能誘發Caco-2細胞增加抗氧化酵素的表現。因此，本發明之乳酸菌菌株以及其發酵物具有抗氧化之活性效果，且能夠減少自由基濃度，以抑制或延緩器官老化。 To sum up the above, compared with other tested lactic acid bacteria strains, the Bifidobacterium longum subsp. longum OLP-01 strain, Bifidobacterium breve Bv-889 strain, and Bifidobacterium longum infantum subsp. subsp. BLI-02 strain of the present invention , Bifidobacterium animalis subsp. lactis CP-9 strain, Bifidobacterium bifidum Bf-688 strain, Roy's lactic acid Bacillus GL-104 strain, Lactobacillus salivarius AP-32 strain, Lactobacillus rhamnosus bv-77 strain and the fermentation products of the above lactic acid bacteria strains have better free radical scavenging ability and reducing ability, and can induce the increase of Caco-2 cells The performance of antioxidant enzymes. Therefore, the lactic acid bacteria strain of the present invention and its fermented product have the active effect of anti-oxidation, and can reduce the concentration of free radicals to inhibit or delay the aging of organs.
以上所述之實施例僅是為說明本發明之技術思想及特點，其目的在使熟習此項技藝之人士能夠瞭解本發明之內容並據以實施，當不能以之限定本發明之專利範圍，即大凡依本發明所揭示之精神所作之均等變化或修飾，仍應涵蓋在本發明之專利範圍內。 The above-mentioned embodiments are only to illustrate the technical idea and characteristics of the present invention, and its purpose is to enable those who are familiar with the art to understand the content of the present invention and implement it accordingly. It should not be used to limit the patent scope of the present invention. That is, all equivalent changes or modifications made according to the spirit disclosed in the present invention should still be covered within the patent scope of the present invention.
1.財團法人食品工業發展研究所、2019年02月22日、BCRC 910875 1. Food Industry Development Research Institute, February 22, 2019, BCRC 910875
2.財團法人食品工業發展研究所、2018年07月20日、BCRC 910844 2. Food Industry Development Research Institute, July 20, 2018, BCRC 910844
3.財團法人食品工業發展研究所、2018年01月18日、BCRC 910812 3. Food Industry Development Research Institute, January 18, 2018, BCRC 910812
4.財團法人食品工業發展研究所、2014年08月21日、BCRC 910645 4. Food Industry Development Research Institute, August 21, 2014, BCRC 910645
5.財團法人食品工業發展研究所、2019年06月12日、BCRC 910902 5. Food Industry Development Research Institute, June 12, 2019, BCRC 910902
6.財團法人食品工業發展研究所、2008年10月29日、BCRC 910404 6. Food Industry Development Research Institute, October 29, 2008, BCRC 910404
7.財團法人食品工業發展研究所、2009年07月30日、BCRC 910437 7. Food Industry Development Research Institute, July 30, 2009, BCRC 910437
8.財團法人食品工業發展研究所、2011年12月27日、BCRC 910533 8. Food Industry Development Research Institute, December 27, 2011, BCRC 910533
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