TW202103583A - 源自飲食之高多醣利用性之新穎雙叉桿菌屬細菌 - Google Patents
源自飲食之高多醣利用性之新穎雙叉桿菌屬細菌 Download PDFInfo
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Abstract
本發明提供一種能夠利用木聚糖類之新穎雙叉乳酸桿菌菌株。
本發明之雙叉桿菌屬細菌於基因組上具有木聚糖酶基因。
Description
本發明係關於一種被分類為假鏈狀雙叉桿菌之雙叉桿菌屬細菌。
推測益生菌會對宿主之生理功能造成影響之原因之一在於益生菌代謝殘存於消化道內之糖源而產生乳酸、乙酸等短鏈脂肪酸。根據目前之研究可知，成人腸道中佔優勢之雙叉乳酸桿菌種之基因組中，相較於源自嬰兒之雙叉乳酸桿菌種，與源自植物之難消化性多醣、尤其是成人飲食中較多地含有之木聚糖類之代謝相關之基因較多。
因此，對於難消化性多醣利用性較高之雙叉乳酸桿菌菌株，期待於成人之腸內之增殖與較高之有機酸產生能力，因而認為其成為面向成人之益生菌之有力候補。
一直以來，作為能夠利用難消化性多醣類之雙叉乳酸桿菌，例如已知有可合成代謝澱粉之短型雙叉桿菌菌株(非專利文獻1)或雙岐雙叉桿菌菌株(非專利文獻2)、可合成代謝阿拉伯半乳糖假鏈狀雙叉桿菌菌株(非專利文獻3)等之存在。
然而，迄今為止，尚未知能夠分解作為飲食中所含有之主要難消化性多醣之木聚糖類之木糖骨架的雙叉乳酸桿菌。
[先前技術文獻]
[非專利文獻]
[非專利文獻1]BMC Genomics(2014)15:170
[非專利文獻2]Environmental Microbiology (2015) 17(7), 2515-2531
[非專利文獻3]日本食品微生物學會雜誌，Vol.24 No.4 Page.163-170
[發明所欲解決之問題]
本發明係關於提供一種能夠利用木聚糖類之新穎雙叉乳酸桿菌菌株。
[解決問題之技術手段]
本發明人等經過潛心研究，結果成功自人類之腸內單離、鑑定出可高效率地合成代謝木聚糖類之新穎微生物株。
即，本發明係以下1)～9)者。
1)一種雙叉桿菌屬細菌，其於基因組上具有木聚糖酶基因。
2)如1)之雙叉桿菌屬細菌，其中木聚糖酶基因係含有序列編號1所表示之鹼基序列之聚核苷酸或與該鹼基序列具有70%以上之同一性且編碼具有木聚糖酶活性之蛋白質之聚核苷酸。
3)如1)或2)之雙叉桿菌屬細菌，其具有合成代謝木聚糖類之能力。
4)如3)之雙叉桿菌屬細菌，其中木聚糖類係木聚糖及阿拉伯木聚糖。
5)如1)至4)中任一項之雙叉桿菌屬細菌，其具有合成代謝澱粉之能力。
6)如1)至5)中任一項之雙叉桿菌屬細菌，其中雙叉桿菌屬細菌被分類為假鏈狀雙叉桿菌。
7)一種假鏈狀雙叉桿菌YIT11027(NITE BP-02928)、假鏈狀雙叉桿菌YIT11055(NITE BP-02929)、假鏈狀雙叉桿菌YIT11057(NITE BP-02930)、假鏈狀雙叉桿菌YIT11952(NITE BP-02931)、假鏈狀雙叉桿菌YIT11954(NITE BP-02932)、假鏈狀雙叉桿菌YIT12989(NITE BP-02933)、或與該等近緣之菌株。
8)一種飲食品，其含有如1)至7)中任一項之雙叉桿菌屬細菌。
9)如8)之飲食品，其係醱酵乳飲食品。
[發明之效果]
本發明之新穎雙叉桿菌屬具有分解合成代謝成人飲食中較多地含有之木聚糖類、尤其是阿拉伯木聚糖之能力。因此，可期待本發明之假鏈狀雙叉桿菌於成人之腸內之增殖與較高之有機酸產生能力，可作為面向成人之益生菌而利用於醫藥品、食品等。
本發明之雙叉桿菌屬細菌只要為於基因組上具有木聚糖酶基因者，則其種類無特別限定。例如可為假鏈狀雙叉桿菌(Bifidobacterium. pseudocatenulatum)、短型雙叉桿菌(Bifidobacterium breve)、龍根雙叉桿菌(B.longum)、雙岐雙叉桿菌(B.bifidum)、動物雙叉桿菌(B.animalis)、豬雙叉桿菌(B.suis)、嬰兒雙叉桿菌(B.infantis)、青春雙叉桿菌(B.adolescentis)、鏈狀雙叉桿菌(B.catenulatum)、雷特氏雙叉桿菌(B.lactis)、球雙叉桿菌(B.globosum)、角雙叉桿菌(B.angulatum)、齒雙叉桿菌(B.dentium)等任一種，較佳可列舉假鏈狀雙叉桿菌。
一直以來，尚未知於基因組上具有木聚糖酶基因之雙叉乳酸桿菌，可認為本發明之雙叉桿菌屬細菌係新穎細菌。
所謂「木聚糖酶基因」，係指編碼被分類為GH10之木聚糖酶之基因(內切-1,4-β-木聚糖酶A，xynA)，作為本發明之雙叉桿菌屬細菌於其基因組上所保有之「木聚糖酶基因」，具體而言包含：含有序列編號1所表示之鹼基序列之聚核苷酸或與該鹼基序列具有70%以上、較佳為90%以上、更佳為95%以上、更佳為98%以上、更佳為99%以上之同一性且編碼具有木聚糖酶活性之蛋白質之聚核苷酸。
此處，含有序列編號1所表示之鹼基序列之聚核苷酸係指後述之YIT11057株所保有之木聚糖酶基因。
又，鹼基序列之同一性係將以應進行比較之2個核酸序列之鹼基儘可能多地一致之方式排列兩鹼基序列併用一致之鹼基數除以總鹼基數而得者以百分率表示者。若為從業者，則可適當地設定BLAST(Basic Local Alignment Search Tool，基礎區域排列搜尋工具)、ClustalX或Genetyx等軟體之參數，確定鹼基序列之同一性。
又，所謂木聚糖酶活性，意指以木聚糖類作為基質，將木聚糖類之木糖骨架水解而生成木寡糖及木糖之活性(木聚糖水解活性)。
此處，作為木聚糖類，可列舉：木聚糖、阿拉伯木聚糖、葡糖醛酸木聚糖、葡糖醛酸阿拉伯木聚糖、乙醯木聚糖等，較佳為木聚糖及阿拉伯木聚糖。
因此，換言之，本發明之於基因組上具有木聚糖酶基因之雙叉桿菌屬細菌係具有分解木聚糖類之木糖骨架之能力之雙叉桿菌屬細菌。
於本發明中，所謂「具有合成代謝木聚糖類之能力」，係指具有可以木聚糖類作為碳源進行增殖之能力。具體而言，例如係指可於以木聚糖類作為唯一碳源之培養基中進行增殖。例如指於僅以木聚糖類作為糖源之培養基中培養72小時後之濁度、例如OD600
增加量為0.1以上。濁度較佳為OD600
增加量為0.3以上，更佳為0.4以上。又，於不溶性組分較多之添加有木聚糖類之培養基等無法測定濁度之變化之情形時，係指培養72小時後之培養上清液中之短鏈脂肪酸之產生，例如乳酸、乙酸及甲酸之合計產生量為10 mM以上。短鏈脂肪酸產生量之合計較佳為20 mM以上，更佳為40 mM以上。
作為木聚糖類合成代謝能力之試驗中所使用之潛伏期培養基，使用於去除糖源之培養基中添加有木聚糖類者即可，去除糖源之培養基之組成可設為M-ILS培養基、蛋白腖-酵母(PY)培養基等組成。
添加於培養基中之木聚糖類之添加量於培養基中較佳為0.01～10品質%，更佳為0.05～5品質%，進而較佳為0.1～1品質%。再者，較理想為作為對照而於去除糖源之培養基中培養作為對象之雙叉桿菌屬細菌，並事先確認OD。
如此，由於本發明之於基因組上具有木聚糖酶基因之雙叉桿菌屬細菌具有分解合成代謝木聚糖類之能力，故可有助於因木聚糖類之分解而產生之乙酸、乳酸、甲酸等短鏈脂肪酸之供給。
本發明之雙叉桿菌屬細菌可藉由以木聚糖酶基因之存在、或上述合成代謝木聚糖類之能力作為指標，例如自存在於人類(例如，成人、嬰幼兒)之腸內之雙叉桿菌屬細菌、較佳為假鏈狀雙叉桿菌篩選而獲得。例如，可自假鏈狀雙叉桿菌挑選出後述實施例中所示之16株、進而YIT11027、YIT11055、YIT11057、YIT11952、YIT11954、YIT12989之6株。作為本發明之雙叉桿菌屬細菌，只要於基因組上具有木聚糖酶基因，則不限定於該等，包含於生物學上、遺傳學上與該等近緣之菌株。又，作為本發明之雙叉桿菌屬細菌，只要於基因組上具有木聚糖酶基因，則可為自然界中存在之天然株，亦可為天然株之變異種、或轉基因種。
YIT11027株、YIT11055株、YIT11057株、YIT11952株、YIT11954株、YIT12989株如下所示，於2019年3月25日寄存於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(千葉縣木更津市上總鐮足2-5-8 122號室)。
假鏈狀雙叉桿菌YIT11027(NITE BP-02928)、
假鏈狀雙叉桿菌YIT11055(NITE BP-02929)、
假鏈狀雙叉桿菌YIT11057(NITE BP-02930)、
假鏈狀雙叉桿菌YIT11952(NITE BP-02931)、
假鏈狀雙叉桿菌YIT11954(NITE BP-02932)、
假鏈狀雙叉桿菌YIT12989(NITE BP-02933)。
其中，就合成代謝木聚糖類之能力之方面、進而兼具澱粉合成代謝能力之方面而言，較佳為YIT11027株、YIT11057株、YIT11954株或其等之近緣之菌株。進而，就兼具具有酸、膽汁酸耐受性，且於基因組中未檢測出溶原噬菌體之特性之方面而言，更佳為YIT11057株或其近緣之菌株。
此處，所謂澱粉合成代謝能力，係指具有可以澱粉作為碳源進行增殖之能力。所謂酸、膽汁酸耐受性，係指人工胃酸及人工膽汁酸之連續暴露處理後之存活性。所謂不存在溶原噬菌體，係指使用作為噬菌體搜尋編程伺服器之PHASTER(http://phaster.ca/)，於基因組序列中未檢測到與既有噬菌體具有較高同源性之區域。
又，所謂近緣之菌株，係指根據對複數個管家基因序列進行評價之多位點序列分析(MLSA，Multilocus sequence analysis)法而序列一致之菌株。
具體而言，獲得作為適合於雙叉乳酸桿菌之MLSA解析之基因而報告(International Journal of Systematic and Evolutionary Microbiology(2006), 56, 2783-2792)之DNA解旋酶次單元B(DNA gyrase subunit B)(gyrB)、50S核糖體蛋白質L2(50S ribosomal protein L2)(rplB)、磷酸核糖醯胺轉移酶(Amidophosphoribosyltransferase)(purF)、DNA指導之RNA聚合酶次單元β基因(DNA-directed RNA polymerase subunit beta)(rpoB)、依賴ATP之Clp蛋白酶ATP結合次單元ClpC1(ATP-dependent Clp protease ATP-binding subunit ClpC1)(clpC)、延長因數G(Elongation factor G)(fusA)、異白胺酸-tRNA連接酶(Isoleucine-tRNA ligase)(ileS)之全長序列，對各菌株連結全部基因後，使用VSEARCH或BLAST等程式驗證比較物件株間之序列之一致性，於序列同一性為90%以上、較佳為95%以上、更佳為98%以上、更佳為99%以上之情形時，可判斷為係近緣種。
將YIT11057株中之gyrB基因、rplB基因、purF基因、rpoB基因、clpC基因、fusA基因及ileS基因之鹼基序列示於序列表中(gyrB：序列編號2、rplB：序列編號3、purF：序列編號4、rpoB：序列編號5、clpC：序列編號6、fusA：序列編號7、ileS：序列編號8)。
本發明之雙叉桿菌屬細菌之利用形態無特別限制，可為經冷凍乾燥者，或者亦能夠以含有該等細菌之培養物之形式利用，但於任何形態中，細菌均較佳為生菌之狀態。
本發明之雙叉桿菌屬細菌亦可與固體或液體之醫藥用無毒性載體混合，以慣用之醫藥品製劑之形態利用。作為此種製劑，例如可列舉：錠劑、顆粒劑、散劑、膠囊劑等固形劑；溶液劑、懸浮劑、乳劑等液劑；冷凍乾燥製劑等。該等製劑可利用製劑上之常規方法製備。作為上述醫藥用無毒性載體，例如可列舉：葡萄糖、乳糖、蔗糖、澱粉、甘露醇、糊精、脂肪酸甘油酯、聚乙二醇、羥乙基澱粉、乙二醇、聚氧乙烯山梨醇酐脂肪酸酯、胺基酸、明膠、白蛋白、水、生理鹽水等。又，亦可視需要適當添加穩定劑、濕潤劑、乳化劑、結合劑、等張劑、賦形劑等慣用之添加劑。
本發明之雙叉桿菌屬細菌不僅可製成如上所述之製劑，亦可於飲食品中調配使用。於調配於飲食品中之情形時，可直接含有或與各種營養成分一併含有。具體而言，於將本發明之雙叉桿菌屬細菌調配於飲食品中之情形時，可適當使用可用作飲食品之添加劑，使用慣用之方法，成形為適合於食用之形態，即顆粒狀、粒狀、錠劑、膠囊、糊等，又，亦可添加於各種各樣之食品，例如火腿、香腸等肉食加工食品；日式魚糕、竹輪等水產加工食品；麵包、甜食、黃油、奶粉中使用，或添加於水、果汁、牛奶、清涼飲料、茶飲料等飲料中使用。再者，飲食品中亦包括動物之飼料。
進而，作為飲食品，可適宜地使用以生菌之狀態含有本發明之雙叉桿菌屬細菌之醱酵乳飲食品、醱酵豆乳、醱酵果汁、醱酵蔬菜汁等醱酵飲食品，特佳為利用醱酵乳飲食品。醱酵乳飲食品之製造依照慣例即可，例如於製造醱酵乳之情形時，於經殺菌之乳培養基中單獨或與其他微生物同時地接種培養本發明之雙叉桿菌屬細菌，並對其進行均質化處理，獲得醱酵乳基底。其次，添加混合另行製備之糖漿溶液，利用均質機等將其均質化，進而添加香料，最後加工為最終製品即可。如此所獲得之醱酵乳飲食品亦可製成不含糖漿(甜味料)之原味型、鬆軟型、果味型、固形狀、液狀等任一形態之製品。
可於該醱酵乳飲食品中調配糖漿等甜味料、乳化劑、增黏(穩定)劑、各種維生素等任意成分。作為糖漿，可調配：葡萄糖、蔗糖、果糖、果糖葡萄糖液糖、葡萄糖果糖液糖、巴拉金糖、海藻糖、乳糖、木糖、半乳寡糖(GOS)、木寡糖(XOS)、阿拉伯木寡糖(AXOS)、木聚糖、阿拉伯木聚糖、阿拉伯寡糖(AOS)、聚阿拉伯糖、麥芽糖、蜂蜜、糖蜜等糖類；山梨糖醇、木糖醇、赤藻糖醇、乳糖醇、異麥芽糖醇(Palatinit)、還原飴糖、還原麥芽糖飴糖等糖醇；阿斯巴甜、索馬甜、蔗糖素、乙醯磺胺酸K、甜菊等高甜度甜味料；甘油脂肪酸酯、聚甘油脂肪酸酯、山梨醇酐脂肪酸酯、卵磷脂等乳化劑；瓊脂、明膠、鹿角菜膠、瓜爾膠、三仙膠、果膠、刺槐豆膠、結冷膠、羧甲基纖維素、大豆多糖類、褐藻酸丙二醇酯等增黏(穩定)劑。除此以外，亦可調配：維生素A、維生素B類、維生素C、維生素E類等維生素類；鈣、鎂、鋅、鐵、錳等礦物成分；檸檬酸、乳酸、乙酸、蘋果酸、酒石酸、葡萄糖酸等酸味劑；奶油、黃油、優酪乳油等乳脂肪；酸乳酪系、莓果系、柳橙系、花梨系、紫蘇系、柑橘系、蘋果系、薄荷系、葡萄系、杏系、梨、乳蛋糕乳脂、桃、甜瓜、香蕉、熱帶水果、草藥系、紅茶、咖啡系等香料類；草藥萃取物、紅糖萃取物等。
醱酵乳飲食品之製造中亦可併用本發明之雙叉桿菌屬細菌以外之微生物。作為此種微生物，例如可列舉：乾酪乳酸桿菌(Lactobacillus casei)、嗜酸乳酸桿菌(L.acidophilus)、植物乳酸桿菌(L.plantarum)、布氏乳酸桿菌(L.buchneri)、雞乳酸桿菌(L.gallinarum)、噬澱粉乳酸桿菌(L.amylovorus)、短乳酸桿菌(L.brevis)、鼠李糖乳酸桿菌(L.rhamnosus)、克菲爾乳酸桿菌(L.kefir)、副乾酪乳酸桿菌(L.paracasei)、捲曲乳酸桿菌(L.crispatus)、玉米乳酸桿菌(L.zeae)、瑞士乳酸桿菌(L.helveticus)、唾液乳酸桿菌(L.salivalius)、加氏乳酸桿菌(L.gasseri)、醱酵乳酸桿菌(L.fermentum)、洛德乳酸桿菌(L.reuteri)、德氏乳酸桿菌保加利亞亞種(L.delbrueckii subsp.bulgaricus)、德氏乳酸桿菌德氏亞種(L.delbrueckii subsp.delbrueckii)、約氏乳酸桿菌(L.johnsonii)等乳酸桿菌屬細菌；嗜熱鏈球菌(Streptococcus thermophilus)等鏈球菌屬細菌；雷特氏乳酸球菌雷特氏亞種(Lactococcus lactis subsp.lactis)、雷特氏乳酸球菌乳脂亞種(Lactococcus lactis subsp.cremoris)等乳酸球菌屬細菌；糞腸球菌(Enterococcus faecalis)、屎腸球菌(E.faecium)等腸球菌屬細菌；枯草桿菌(Bacillus subtilis)等桿菌屬細菌；釀酒酵母(Saccharomyses cerevisiae)、德氏有孢圓酵母(Torulaspora delbrueckii)、乳酒假絲酵母(Candida kefyr)等屬於酵母菌屬、有孢圓酵母菌屬、假絲酵母菌屬等之酵母。若與本發明之雙叉桿菌屬細菌一併併用選自乳酸桿菌屬細菌、鏈球菌屬細菌、乳酸球菌屬細菌之1種以上而製造醱酵乳飲食品，則可獲得較高之可口性，且飲食變得容易，故較佳。
使用本發明之雙叉桿菌屬細菌之情形時之投予量無嚴格之限制，其較佳之投予量以生菌數計，為每天105
cfu～1013
cfu，特佳為108
cfu～1012
cfu。
[實施例]
實施例1 具有合成代謝木聚糖類之能力之雙叉桿菌屬細菌之挑選
i)使用菌株
將自人類糞便單離並鑑定為假鏈狀雙叉桿菌之53株作為篩選對象。
ii)基質
將藉由木聚糖(源自燕麥)、阿拉伯木聚糖(源自小麥)、木聚糖之酵素處理而以工業方式製造之木寡糖(xylooligo-saccharide，XOS)使用於解析。關於木聚糖及阿拉伯木聚糖，利用HPLC(High Performance Liquid Chromatography，高效液相層析法)進行確認，結果未觀察到單糖之混入(未刊載資料)。又，將各構成單糖即木糖及阿拉伯糖、作為增殖確認用對照組之代表性之難消化性多醣即澱粉及其構成單糖之葡萄糖、以及乳糖亦供於解析。製備各基質之1%懸浮液後，使用高壓釜對木聚糖、阿拉伯木聚糖、澱粉進行滅菌，使用0.22 μm篩檢程式對其他基質進行過濾滅菌。
iii)合成代謝性試驗
為了防止因伴隨增殖之pH值降低導致之菌之增殖抑制，使用添加有100 mM PIPES(piperazine-N,N'-bis(2-ethanesulfonic acid)，哌𠯤-N,N'-雙(2-乙磺酸))之m-PY培養基。於使用之前於氮氣填充下冷藏保存之2倍濃縮m-PY培養基中等量混合1%基質溶液，用於培養試驗(最終濃度0.5%)。
將冷凍菌液植菌於改良GAM(Gifu Anaerobic Medium，岐阜厭氧培養基)瓊脂培養基中後，培養2～3天，將所獲得之菌落繼代培養於1%葡萄糖-乳糖添加改良GAM液體培養基中。於繼代培養時，注意刮取多數之菌落。於37℃下將其靜置培養一晚，翌日早晨將5%量之菌液再次繼代培養於新鮮之相同培養基中。培養6～9小時左右直至達到對數增殖期後，測定濁度。將培養液100 μl離心並去除上清液後，以成為OD600
＝0.2之方式添加未添加糖源之m-PY培養基，使顆粒物懸浮。於96孔盤中分注各培養基198 μl，添加菌液2 μl後，將50 μl之礦物油分層，於微盤讀取器PowerWave 340(Biotech公司)內，於37℃下進行培養。
每隔30分鐘測定一次濁度，監測自培養開始至60小時為止之濁度。以上培養操作全部於厭氧手套箱內進行。60小時後分取一部分培養液，進行去除蛋白質之處理後，供於HPLC測定短鏈脂肪酸產生量。再者，於在添加木聚糖之m-PY培養基中觀察到大量不溶性粒子之情形時，僅憑濁度之測定無法確認菌體之增殖，因此根據短鏈脂肪酸產生之有無判斷合成代謝性。使用HPLC，測定培養60小時後之培養上清液中之短鏈脂肪酸，確認到乳酸、乙酸及甲酸之合計產生量為10 mM以上。將關於各種基質可合成代謝多少株之結果示於表1。
[表1]
| 木聚糖 | 阿拉伯木聚糖 | XOS | 木糖 | 阿拉伯糖 | 澱粉 | 葡萄糖 | 乳糖 | |
| 合成代謝株數 | 16 | 16 | 53 | 39 | 36 | 28 | 51 | 53 |
| 非合成代謝株數 | 37 | 37 | 0 | 14 | 17 | 25 | 2 | 0 |
假鏈狀雙叉桿菌之全部菌株中確認到木寡糖之利用性，但鏈更長之木聚糖類之利用性於菌株間有所不同，挑選出合成代謝木聚糖及阿拉伯木聚糖之16株作為具有合成代謝木聚糖類之能力之雙叉桿菌屬細菌。
於利用木聚糖作為糖源之液體培養基中培養具有合成代謝木聚糖類之能力之雙叉桿菌屬細菌株，利用HPLC解析對數增殖期前期之培養上清液中之寡糖。其結果為，確認到生成有木糖及木寡糖。據此確認本細菌具有將木糖骨架切斷為木糖及木寡糖之活性。以使用上述16株之一之YIT11057株的解析結果為代表例，示於圖1。圖1中之X1～X6分別表示木糖及相應數量之具有木糖骨架之木寡糖，可知於培養基中未確認到之木糖及木寡糖藉由YIT11057株之培養而產生。
實施例2 YIT11027、YIT11055、YIT11057、YIT11952、YIT11954、YIT12989之6株之各性質
將自上述具有合成代謝木聚糖類之能力之16株中選擇之以下6株YIT11027、YIT11055、YIT11057、YIT11952、YIT11954、YIT12989之合成代謝性示於以下。YIT11027、YIT11057、YIT11954之3菌株中確認到木聚糖類之合成代謝性，亦確認到澱粉之合成代謝性(表2)。
[表2]
| 木聚糖 | 阿拉伯木聚糖 | XOS | 木糖 | 阿拉伯糖 | 澱粉 | 葡萄糖 | 乳糖 | |
| YIT 11027 | + | + | + | + | + | + | + | + |
| YIT 11055 | + | + | + | + | + | - | + | + |
| YIT 11057 | + | + | + | - | - | + | + | + |
| YIT 11952 | + | + | + | + | + | - | + | + |
| YIT 11954 | + | + | + | + | + | + | + | + |
| YIT 12989 | + | + | + | + | + | - | + | + |
| YIT 4037T (基準株) | - | - | + | + | + | + | + | + |
| +：有合成代謝性 -：無合成代謝性 |
實施例3 基因組解析
自菌體提取基因組DNA，使用下一代定序儀Miseq(Illumina公司)，確定基因組草圖。預測基因區域後，參照糖代謝基因資料庫dbCAN，自全部胺基酸序列提取糖代謝基因。搜尋分佈與木聚糖類之利用性一致之基因，結果具有屬於GH10之內切-1,4-β-木聚糖酶A基因(xynA)之株全部可利用木聚糖類。又，不具有相同基因之株均無法利用木聚糖類。
實施例4 YIT11057之各性質
關於YIT11057，對酸、膽汁酸耐受性、致病因數之綜合性搜尋、基因組中之溶原噬菌體之檢測進行評價。
1)酸、膽汁酸耐受性
i)人工液組成
＜人工胃液組成(pH值3.6)＞
胃蛋白酶 40 mg/L
朊間質蛋白腖 5 g/L
胃黏蛋白 1.5 g/L
NaCl 5 g/L
NaHCO3
3 g/L
KH2
PO4
1 g/L
3.6 N HCl pH值調整為3.6
＜人工腸液＞
NaCl 5 g/L
KCl 1 g/L
NaHCO3
3 g/L
3 M Na2
CO3
pH值調整為8.0
ii)將於1%葡萄糖-乳糖添加mGAM液體培養基中培養了一晚之菌液0.1 ml添加至人工胃液2 ml中，於37℃下保溫1小時。其後，加入人工腸液5.5 ml及16%膽汁酸0.5 ml，於37℃下靜置1小時。於酸處理前後、膽汁酸處理後採集菌液，利用生理鹽水將其稀釋後，將50 μl稀釋後之菌液塗佈於mGAM瓊脂培養基，測定CFU(Colony-forming unit，菌落形成單位)。其結果為，即便於膽汁酸處理後亦表現出0.1%程度之存活率(圖2)。於不具有耐受性之情形時，由於各處理後之CFU成為檢測下限值以下，故可知YIT11057具有相對較高之酸、膽汁酸耐受性。
2)致病因數之綜合性搜尋
向PATRIC伺服器(https://www.patricbrc.org/)(Wattam et al., 2017)輸入基因組草圖資訊，確認與致病因數資料庫之序列具有較高之同源性之基因之有無。其結果為，未檢測到致病因數基因。
3)基因組中之溶原噬菌體之檢測
關於菌株之基因組資訊，使用PHASTER伺服器(http://phaster.ca/)(Arndt et al., 2016)，嘗試檢測登錄於噬菌體資料庫之溶原噬菌體。其結果為，未檢測到溶原噬菌體。
圖1係YIT11057株之木糖骨架切割活性。
圖2係YIT11057株之酸、膽汁酸耐受性。
[主張利用生物材料1]
[寄存國家]JP日本
[寄存機構]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]2019/03/25
[寄存號碼]NITE BP-02928
[主張利用生物材料2]
[寄存國家]JP日本
[寄存機構]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]2019/03/25
[寄存號碼]NITE BP-02929
[主張利用生物材料3]
[寄存國家]JP日本
[寄存機構]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]2019/03/25
[寄存號碼]NITE BP-02930
[主張利用生物材料4]
[寄存國家]JP日本
[寄存機構]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]2019/03/25
[寄存號碼]NITE BP-02931
[主張利用生物材料5]
[寄存國家]JP日本
[寄存機構]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]2019/03/25
[寄存號碼]NITE BP-02932
[主張利用生物材料6]
[寄存國家]JP日本
[寄存機構]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]2019/03/25
[寄存號碼]NITE BP-02933
Claims (9)
- 一種雙叉桿菌屬細菌，其於基因組上具有木聚糖酶基因。
- 如請求項1之雙叉桿菌屬細菌，其中木聚糖酶基因係含有序列編號1所表示之鹼基序列之聚核苷酸或與該鹼基序列具有70%以上之同一性且編碼具有木聚糖酶活性之蛋白質之聚核苷酸。
- 如請求項1或2之雙叉桿菌屬細菌，其具有合成代謝木聚糖類之能力。
- 如請求項3之雙叉桿菌屬細菌，其中木聚糖類係木聚糖及阿拉伯木聚糖。
- 如請求項1至4中任一項之雙叉桿菌屬細菌，其具有合成代謝澱粉之能力。
- 如請求項1至5中任一項之雙叉桿菌屬細菌，其中雙叉桿菌屬細菌被分類為假鏈狀雙叉桿菌。
- 一種假鏈狀雙叉桿菌YIT11027(NITE BP-02928)、假鏈狀雙叉桿菌YIT11055(NITE BP-02929)、假鏈狀雙叉桿菌YIT11057(NITE BP-02930)、假鏈狀雙叉桿菌YIT11952(NITE BP-02931)、假鏈狀雙叉桿菌YIT11954(NITE BP-02932)、假鏈狀雙叉桿菌YIT12989(NITE BP-02933)、或與該等近緣之菌株。
- 一種飲食品，其含有如請求項1至7中任一項之雙叉桿菌屬細菌。
- 如請求項8之飲食品，其係醱酵乳飲食品。
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| US5405769A (en) * | 1993-04-08 | 1995-04-11 | National Research Council Of Canada | Construction of thermostable mutants of a low molecular mass xylanase |
| JP3364491B2 (ja) * | 2002-07-25 | 2003-01-08 | 株式会社アトリエ・ド・フロマージュ | ヨーグルトおよびその製造方法 |
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| EP2138186A1 (en) * | 2008-06-24 | 2009-12-30 | Nestec S.A. | Probiotics, secretory IgA and inflammation |
| CN101649303B (zh) * | 2009-05-08 | 2011-09-28 | 内蒙古蒙牛乳业（集团）股份有限公司 | 一种双歧杆菌及其应用和含有双歧杆菌的食品组合物 |
| CA2783418C (en) * | 2009-12-09 | 2019-08-13 | Novozymes A/S | Methods of producing gh8 xylanase variants |
| CN102703369B (zh) * | 2012-05-30 | 2013-11-06 | 邓启文 | 一种制备ev71疫苗的重组双歧杆菌及其制备方法和应用 |
| JP6470967B2 (ja) * | 2014-12-25 | 2019-02-13 | 花王株式会社 | 変異キシラナーゼ |
| CN104651337B (zh) * | 2015-01-28 | 2017-11-03 | 浙江大学 | 一种木聚糖酶、其编码基因xyn‑lxy及其应用 |
| EP3722413A4 (en) | 2017-12-08 | 2021-11-17 | Morinaga Milk Industry Co., Ltd. | NOVEL BIFIDOBACTERIA AND COMPOSITION WITH NOVEL BIFIDOBACTERIA |
| WO2019112054A1 (ja) | 2017-12-08 | 2019-06-13 | 森永乳業株式会社 | 新規ビフィドバクテリウム属細菌及び当該細菌を含む組成物 |
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| CA3133958A1 (en) | 2020-10-08 |
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| AU2020251432A1 (en) | 2021-11-11 |
| KR20210146295A (ko) | 2021-12-03 |
| BR112021019330A2 (pt) | 2021-12-07 |
| EP3949760A1 (en) | 2022-02-09 |
| JP7528057B2 (ja) | 2024-08-05 |
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