TW202229542A - 抑制il-17產生之雙歧乳酸桿菌 - Google Patents
抑制il-17產生之雙歧乳酸桿菌 Download PDFInfo
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- TW202229542A TW202229542A TW110132581A TW110132581A TW202229542A TW 202229542 A TW202229542 A TW 202229542A TW 110132581 A TW110132581 A TW 110132581A TW 110132581 A TW110132581 A TW 110132581A TW 202229542 A TW202229542 A TW 202229542A
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- bifidobacterium
- production
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- lactobacillus
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Abstract
本發明之目的在於提供一種抑制IL-17產生且可適用於飲食品之源自嬰兒腸內之雙歧乳酸桿菌、及含有該雙歧乳酸桿菌之飲食品。
本發明提供一種抑制IL-17產生之雙歧桿菌屬長雙歧桿菌種之雙歧乳酸桿菌。又,該雙歧乳酸桿菌為長雙歧桿菌N714株(NITE BP-03004)。又,本發明提供一種含有該雙歧乳酸桿菌之飲食品。藉此,能夠緩解因IL-17增加所引起之慢性炎症性疾病。
Description
本發明係關於一種抑制IL-17產生之源自嬰兒腸內之雙歧乳酸桿菌之菌株、及含有該菌體之飲食品。尤其是關於一種長雙歧桿菌。
習知,作為參與免疫之細胞,已知有參與細胞性免疫之1型輔助T(Th1)細胞、及參與體液性免疫之Th2細胞。而且,於近年來之研究中,除了Th1細胞、Th2細胞以外,還發現了被稱為Th17細胞之新T細胞亞群。可知該Th17細胞於過敏反應或自體免疫、一部分感染防禦等中發揮中心作用。又,Th17細胞係因產生特徵性之介白素17(Interleukin-17:IL-17)而得以命名。
此處,介白素(interleukin:IL)係淋巴細胞或單核球、巨噬細胞等免疫活性細胞所產生之生理活性物質之統稱,且係介導與免疫反應相關之細胞間交互作用之蛋白性物質。已知其中之IL-17主要由Th17細胞產生,會對纖維母細胞或上皮細胞、血管內皮細胞、巨噬細胞等細胞發揮作用,誘導炎症性細胞激素或趨化因子、細胞黏著因子等各種因子而引起炎症(參照非專利文獻1、2)。
IL-17有數個亞群,由Th17細胞產生IL-17A及IL-17F。IL-17A及IL-17F具有以胺基酸水準計為50%之同源性,且對應之受體亦相同,因此認為許多功能重複。又,亦已知於人Th17細胞中,IL-17F之產生量多於IL-17A。
另一方面,亦可知IL-17之增加與包括類風濕性關節炎、牛皮癬及多發性硬化症之一些慢性炎症性疾病相關(參照非專利文獻3、4)。因此,作為慢性炎症性疾病之治療,正在摸索一種以IL-17為靶之治療方法。
[先前技術文獻]
[非專利文獻]
[非專利文獻1]Aggarwal S, Gurney AL. IL-17: prototype member of an emerging cytokine family. J Leukoc Biol 2002; 71: 1-8.
[非專利文獻2]Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity 2004; 21: 467-76.
[非專利文獻3]Nakae S, Nambu A, Sudo K, et al. Suppression of immune induction of collagen-induced arthritis in IL-17-deficient mice. J Immunol 2003; 171: 6173-7.
[非專利文獻4]Komiyama Y, Nakae S, Matsuki T, et al. IL-17 plays an important role in the development of experimental autoimmune encephalomyelitis. J Immunol 2006; 177: 566-73.
(發明所欲解決之問題)
此次,本發明人等對能否藉由飲食來抑制IL-17產生進行了研究。而且,新發現於以嬰兒糞便作為分離源之雙歧乳酸桿菌中存在抑制IL-17產生之雙歧乳酸桿菌,從而完成了本發明。
(解決問題之技術手段)
為了解決上述課題,本發明之特徵在於,其係抑制IL-17產生之屬於雙歧桿菌屬之雙歧乳酸桿菌。
又,為了解決上述課題,本發明係一種雙歧乳酸桿菌,其特徵在於,上述雙歧乳酸桿菌為長雙歧桿菌N714株(NITE BP-03004)。
進而,為了解決上述課題,本發明之特徵在於,其係含有上述雙歧乳酸桿菌之飲食品。
(對照先前技術之功效)
本發明之雙歧乳酸桿菌抑制IL-17產生。藉此,有能夠緩解因IL-17增加所引起之慢性炎症性疾病的可能性。
以下,對本發明詳細地進行說明。
1.長雙歧桿菌N714株(NITE BP-03004)
本發明之雙歧乳酸桿菌為長雙歧桿菌(
Bifidobacterium longum)。本發明中之記號N714係日清食品控股股份有限公司自行賦予菌株之編號。本發明之菌株係本發明人自嬰兒糞便中首次分離所得者。
本發明之長雙歧桿菌N714株係於下述條件下寄存。
(1)寄存機關名稱:獨立行政法人製品評價技術基盤機構 專利微生物寄存中心
(2)聯繫方式:〒292-0818 千葉縣木更津市上總鐮足2-5-8 122號室
(3)寄存編號:NITE BP-03004
(4)用於識別之顯示:N714
(5)寄存日:2019年7月10日
本發明之長雙歧桿菌N714株之菌學性質係如以下表1及表2所示。本菌學性質係基於伯傑氏系統細菌學手冊(Bergey's manual of systematic bacteriology)Vol. 2 (1986)中所記載之方法。表1表示與本菌株相關之形狀等,表2表示藉由API 50CH及API CHL(Biomerieux)所進行之生理-生物化學性狀試驗之結果。於表1及2中,「+」表示陽性,「-」表示陰性。
[表1]
培養溫度(℃) | 37℃ | |
細胞形態 | 桿菌 | |
0.5×2.0~4.0 μm | ||
革蘭氏染色性 | + | |
有無芽孢 | - | |
運動性 | - | |
菌落形態 | 培養基 | GAM瓊脂培養基 |
培養時間 | 24小時 | |
直徑 | 0.5~2.0 mm | |
色調 | 奶油色 | |
形狀 | 圓形 | |
隆起 | 透鏡狀 | |
周緣 | 全緣 | |
表面 | 平滑 | |
透明度 | 不透明 | |
黏稠度 | 黃油狀 | |
生長溫度試驗 | 30℃ | + |
45℃ | - | |
觸酶反應 | - | |
氧化酶反應 | - | |
自葡萄糖產生酸/氣體(酸之酸性/氣體之酸性) | +/- | |
O/F測試(氧化/醱酵) | +/+ | |
+:陽性,-:陰性 |
[表2]
*醱酵性試驗
+:陽性 -:陰性
0 | 對照 | - | 25 | 馬栗樹皮苷* | + | |
1 | 甘油* | - | 26 | 柳苷* | - | |
2 | 赤藻糖醇* | - | 27 | 纖維雙糖* | - | |
3 | D-阿拉伯糖* | - | 28 | 麥芽糖* | + | |
4 | L-阿拉伯糖* | + | 29 | 乳糖* | + | |
5 | 核糖* | + | 30 | 蜜二糖* | + | |
6 | D-木糖* | + | 31 | 蔗糖* | + | |
7 | L-木糖* | - | 32 | 海藻糖* | - | |
8 | 核糖醇* | - | 33 | 菊糖* | - | |
9 | β-甲基-D-木糖* | - | 34 | 蜜三糖* | - | |
10 | 半乳糖* | + | 35 | 棉子糖* | + | |
11 | 葡萄糖* | + | 36 | 澱粉* | - | |
12 | 果糖* | + | 37 | 肝糖* | - | |
13 | 甘露糖* | + | 38 | 木糖醇* | - | |
14 | 山梨糖* | - | 39 | 龍膽二糖* | - | |
15 | 鼠李糖* | - | 40 | D-松二糖* | + | |
16 | 半乳糖醇* | - | 41 | D-來蘇糖* | - | |
17 | 肌醇* | - | 42 | D-塔格糖* | - | |
18 | 甘露醇* | + | 43 | D-岩藻糖* | - | |
19 | 山梨糖醇* | + | 44 | L-岩藻糖* | - | |
20 | α-甲基-D-甘露糖苷* | - | 45 | D-阿拉伯糖醇* | - | |
21 | α-甲基-D-葡萄糖苷* | - | 46 | L-阿拉伯糖醇* | - | |
22 | N-乙醯葡糖胺* | - | 47 | 葡萄糖酸鹽* | - | |
23 | 苦杏仁苷* | - | 48 | 2-酮葡萄糖酸鹽* | - | |
24 | 熊果苷* | - | 49 | 5-酮葡萄糖酸鹽* | - |
2.IL-17產生抑制試驗
如下述實驗例所示,本發明之長雙歧桿菌N714株具有較高之IL-17產生抑制能力。藉由以下試驗方法進行IL-17產生抑制能力之確認。
<IL-17產生抑制試驗所使用之試樣之製備>
IL-17產生抑制試驗所使用之試樣係藉由將雙歧乳酸桿菌於表3所示之GAM培養基(日水)中於37℃下培養24小時而獲得。繼而,使用離心分離機收集經增殖之菌體。用殺菌水將收集之菌體洗淨,藉由離心分離機進行收集菌體。將洗淨及收集菌體反覆進行3次。其後,實施加熱殺菌,使用冷凍乾燥機將加熱殺菌體進行冷凍乾燥,獲得乾燥菌體粉末。
[表3]
蛋白腖 | 10.00 g |
大豆蛋白腖 | 3.00 g |
朊蛋白腖 | 10.00 g |
消化血清粉末 | 13.50 g |
酵母萃取物 | 5.00 g |
肉萃取物 | 2.20 g |
肝臟萃取物 | 1.20 g |
葡萄糖 | 3.00 g |
磷酸二氫鉀 | 2.50 g |
氯化鈉 | 3.05 g |
可溶性澱粉 | 5.00 g |
L-半胱胺酸鹽酸鹽 | 0.30 g |
硫代乙醇酸鈉 | 0.30 g |
蒸餾水 | 1000 mL |
<IL-17產生抑制試驗>
於IL-17產生抑制試驗中,使用人周邊血液單核細胞(hPBMC,CTL)作為細胞。使用添加有10%胎牛血清、4 mM之GlutaMAX(Gibco)之Iscove改良Dulbecco培養基(IMDM,Iscove's Modified Dulbecco's Medium)(Sigma),將細胞以1.0×10
6cells/well接種於96孔板,將以上所獲得之試樣以10 ng/well接種於96孔板。向其中添加作為Th17細胞分化刺激劑之25 ng/mL之rhIL-6(BioLegend)、12.5 ng/mL之rhIL-23(BioLegend)、25 ng/mL之rhIL-21(BioLegend)、2 ng/mL之rhTGF-β1(BioLegend)、25 ng/mL之rhIL-1β(BioLegend),於37℃、5%CO
2之條件下培養96小時。培養後,回收hPBMC之培養上清液,藉由市售之免疫測定套組(R&D)測定上清液中之IL-17F。
3.飲食品
本發明之雙歧乳酸桿菌可包含於飲食品中來使用。本發明之雙歧乳酸桿菌尤其適用於飲料,例如考慮醱酵乳及乳酸菌飲料。於現行之與乳及乳製品之成分標準等相關之政府機關指令中,作為成分標準,若為醱酵乳(無脂乳固形份為8.0%以上者)或乳製品乳酸菌飲料(無脂乳固形份為3.0%以上者),則必須為1.0×10
7cfu/mL以上,若為乳酸菌飲料(無脂乳固形份未滿3.0%者),則必須為1.0×10
6cfu/mL以上,藉由於乳等醱酵液中增殖,或以最終製品之形態增殖,可實現上述菌數。又,除了添加有雙歧乳酸桿菌之醱酵乳及乳酸菌飲料以外,亦可用於黃油等乳製品、蛋黃醬等蛋加工品、乳酪餅等小甜麵包類等。又,亦可適用於泡麵或餅乾等加工食品。除了上述以外,本發明之食品亦可為將上述雙歧乳酸桿菌與視需要之適當載體及添加劑一起添加並進行製劑化而成之形態(例如粉末、顆粒、膠囊、錠劑等)。
本發明之雙歧乳酸桿菌除了包含於一般之飲料或食品中有用以外,包含於特定保健用食品、營養輔助食品等中亦有用。又,本發明之雙歧乳酸桿菌除了應用於食品以外,亦可應用於化妝水等化妝品領域;整腸劑等醫藥品領域;潔牙粉等日用品領域;青貯料、動物用餌料、植物液體肥料等動物飼料•植物肥料領域。
(產業上之可利用性)
本發明之雙歧乳酸桿菌(長雙歧桿菌N714株)對IL-17產生抑制具有較高之活性。
[實施例]
以下,表示本發明之實施例,但本發明並不限定於以下實施例。
<試驗例1>IL-17產生抑制試驗
對本發明之長雙歧桿菌N714株及本公司持有之長雙歧桿菌比較株實施IL-17產生抑制試驗。
首先,對於本發明之菌株、下述標準株及比較菌株,分別於表3所示之GAM培養基(日水)中於37℃下培養24小時。培養時使用厭氧產氣袋(AnaeroPack)(三菱瓦斯化學),於厭氧條件下培養。其次,將經增殖之菌體進行離心分離並收集菌體。用殺菌水將所收集之菌體洗淨3次,加熱殺菌後進行冷凍。其後,使用冷凍乾燥機進行冷凍乾燥,獲得乾燥菌體粉末。使所獲得之乾燥菌體粉末懸浮於磷酸鹽緩衝液(PBS,Phosphate Buffered Saline)(-)中,將所得者作為雙歧乳酸桿菌懸浮液。
其次,將hPBMC以1.0×10
6cells/well接種於96孔板。其次,添加各試樣群,使最終濃度成為10 ng/mL。又,作為Th17細胞分化刺激劑,每孔添加25 ng/mL之rhIL-6(BioLegend)、12.5 ng/mL之rhIL-23(BioLegend)、25 ng/mL之rhIL-21(BioLegend)、2 ng/mL之rhTGF-β1(BioLegend)、25 ng/mL之rhIL-1β(BioLegend),於37℃、5%CO
2之條件下培養96小時。培養後,回收hPBMC之培養上清液,藉由市售之免疫測定套組(R&D)測定上清液中之IL-17。再者,將雙歧乳酸桿菌懸浮液及Th17細胞分化刺激劑均未添加者記為『無刺激』,將僅添加了Th17細胞分化刺激劑者記為『刺激』。
將添加了各試樣群之情形時培養上清液中所包含之IL-17F濃度(pg/mL)示於圖1。
如圖1所示可知,於雙歧乳酸桿菌中,若存在抑制IL-17F產生者,則亦存在未抑制IL-17F產生者。其中,本發明之N714株雖然IL-17F之產生高於無刺激,但與其他雙歧乳酸桿菌進行比較,對IL-17F產生抑制表現出最高之活性。再者,認為無刺激之IL-17F濃度較低之原因在於未分化成Th17細胞。此處,N714株中IL-17F產生受到抑制之原因雖不明,但推測如下。推測可能性之一在於,因添加了N714株而向Th17細胞之分化本身受到抑制,結果IL-17F產生受到抑制。推測另一可能性在於,雖然分化成Th17細胞,但因某種原因而IL-17產生受到抑制。推測因該等原因之任一者或兩者,進而因該等以外之原因,IL-17產生受到抑制。
另外,本試驗中使用IL-17F作為IL-17之評價指標之原因在於,IL-17亞群中IL-17F之產生量最多,容易檢測出。另一方面,於IL-17F以外之亞群中,產生量非常少,難以檢測出。因此,於本試驗中將IL-17F作為評價指標。然而,基於上述推測,推測IL-17F以外之亞群亦藉由本發明之長雙歧桿菌N714而產生受到抑制。
<試驗例2>長雙歧桿菌N714之優勢評價
其次,為了確認本發明之長雙歧桿菌N714株之優勢,藉由與作為標準株之長雙歧桿菌JCM1217之比較來進行評價。試驗係藉由與試驗例1相同之方法進行3次,使用其平均値與刺激進行比較。將結果示於圖2。
根據圖2亦可知,添加了長雙歧桿菌N714株之群與刺激群進行比較,IL-17產生受到顯著抑制。
如以上所說明,可知本發明之長雙歧桿菌N714株具有有效地抑制IL-17產生之功能。
圖1係將本發明之菌株與比較菌株之IL-17F產生抑制效果進行比較之圖。
圖2係將本發明之菌株與標準株之IL-17F產生抑制效果進行比較之圖。
寄存國家 JP日本
寄存機機 獨立行政法人製品評價技術基盤機構特許微生物寄託中心
寄存日期 2019/07/10
寄存號碼 BP-03004
Claims (3)
- 一種雙歧桿菌屬長雙歧桿菌種之雙歧乳酸桿菌,其抑制IL-17產生。
- 如請求項1之雙歧乳酸桿菌,其中,上述雙歧乳酸桿菌為長雙歧桿菌N714株(NITE BP-03004)。
- 一種飲食品,其含有請求項1或2之雙歧乳酸桿菌。
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