TWI824110B - 新穎乳酸菌及含有其之組成物 - Google Patents
新穎乳酸菌及含有其之組成物 Download PDFInfo
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- TWI824110B TWI824110B TW109104887A TW109104887A TWI824110B TW I824110 B TWI824110 B TW I824110B TW 109104887 A TW109104887 A TW 109104887A TW 109104887 A TW109104887 A TW 109104887A TW I824110 B TWI824110 B TW I824110B
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- Prior art keywords
- strain
- bacteria
- composition
- bacterial flora
- lactobacillus crispatus
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Abstract
本發明關於一種捲曲乳桿菌(Lactobacillus crispatus)菌株,該菌株之16s rRNA基因的核苷酸序列是具有與序列識別號1所示之核苷酸序列至少95%的相同性。
Description
發明領域
本發明關於一種新穎乳酸菌及含有其之組成物。
發明背景
習知,所謂腸內細菌叢一般是指存在於糞便或內容物的細菌。不過在近年,附著於宿主黏膜之細菌的生理上意義,及在黏膜內所形成之黏膜附著細菌叢的存在變得明朗。
例如有報告:炎症性腸道疾病(IBD)及克羅恩氏病(Crohn’s disease)的患者是有形成與身心健全者不同的黏膜附著細菌叢(非專利文獻1)。此外,已知悉:可包含於腸內細菌叢的細菌之中,毛螺旋菌科(Lachnospiraceae)細菌及不動桿菌屬(Acinetobacter)細菌、擬桿菌屬(Bacteroides)細菌、普雷沃氏菌屬(Prevotella)細菌,在腸管或全身發炎時在腸內會增加(非專利文獻2~8)。
在另一方面,已知於母乳是含有嬰兒發育所需之各種各樣的營養素。此外,近年,已有報告:於母乳亦含有乳酸菌,且有助於新生兒的腸內細菌叢形成(非專利文獻9)。進一步,亦有針對母乳的細菌叢與孩子發育之相關性進行報告(非專利文獻10)。
不過,依母體的體質及育兒環境,有難以實施母乳育兒的情況。此外,母乳的細菌叢有個體差異,因此當母親的細菌叢很差的情況,孩子的黏膜附著細菌叢及腸內細菌叢亦有蒙受其影響的情況。
[先前技術文獻]
[非專利文獻]
[非專利文獻1] The Japanese Society of Gastroenterology,Nishino et al., DOI 10.1007/s00535-017-1384-4, 2017
[非專利文獻2] The Journal of Nutrition, Genomics, Proteomics, and Metabolomics, Preidis et al., 940-948, First published online April 6, 2016
[非專利文獻3] The ISME Journal 6, Berry et al., 2091-2106, (2012)
[非專利文獻4] Neuroscience, Aldy et al., 1-12, 03 August 2017
[非專利文獻5] Gut Microbes, Volume 6, Issue 1, Tang et al., 48-56, 2015
[非專利文獻6] Setoyama et al. Microbes Infect. 2003 Feb;5(2):115-22
[非專利文獻7] Scher et al. eLife 2013;2:e01202.DOI:10.7554/eLife.01202
[非專利文獻8] Arai et al. Japanese Society of Allergology, 67(1), 24-27, 2018
[非專利文獻9] SCIENTIFIC Reports, 7, 9940, DOI:10.1038/s41598-017-09278 -y, Drell et al., 30 August 2017
[非專利文獻10] Frontiers in Microbiology, Volume 8, Article 2100, Toscano et al., October 2017。
發明概要
發明所欲解決之課題
本發明一實施態樣的一個目的是在於:提供一種新穎乳酸菌,該乳酸菌能夠有效地調節在常居細菌叢中的細菌構成均衡。
用以解決課題之手段
本發明人等,發現一種新穎乳酸菌,其能夠有效地調節以黏膜附著細菌叢或腸內細菌叢為首之常居細菌叢的細菌構成均衡。本發明是基於這樣的知識。
本發明的一實施態樣包含以下之態樣。
[1]一種捲曲乳桿菌(Lactobacillus crispatus)菌株,該菌株之16s rRNA基因的核苷酸序列具有與序列識別號1所示之核苷酸序列至少95%的相同性。
[2]如[1]記載之菌株,其中前述相同性為98%以上。
[3]如[1]或[2]記載之菌株,其係寄存在寄存編號NITE BP-02869下的捲曲乳桿菌(Lactobacillus crispatas) CFF No.2031。
[4]一種組成物,包含如[1]~[3]中任一項記載的菌株而成。
[5]如[4]記載之組成物,其係使用來作為醫藥、飲食品、化妝品或飼料。
[6]如[4]或[5]記載之組成物,其係嬰幼兒或未成年用。
[7]如[4]~[6]中任一項記載之組成物,其係用以調節常居細菌叢中所含細菌的構成均衡。
[8]如[7]記載之組成物,其中前述常居細菌叢中所含細菌是選自於毛螺旋菌科細菌、不動桿菌屬細菌、黏液螺旋菌(Mucispirillum)屬細菌、普雷沃氏菌屬細菌及擬桿菌屬細菌之至少1個。
[9]如[7]或[8]記載之組成物,其中前述常居細菌叢為黏膜附著細菌叢或腸內細菌叢。
[10]如[4]~[9]中任一項記載之組成物,其係用以抑制與炎症相關的疾病或症狀。
[11]如[4]~[10]中任一項記載之組成物,其係用以保護消化道。
[12]如[4]~[11]中任一項記載之組成物,其係用以抑制體重減少。
[13]一種捲曲乳桿菌菌株之用途,該用途係製造常居細菌叢中所含細菌之構成均衡的調節用組成物中,且該菌株之16s rRNA基因的核苷酸序列是具有與序列識別號1所示之核苷酸序列至少95%的相同性。
[14]一種調整常居細菌叢中所含細菌之構成均衡的方法,包含下述而成:將捲曲乳桿菌菌株的有效量投予至需要其之對象,且前述菌株的16s rRNA基因的核苷酸序列是具有與序列識別號1所示之核苷酸序列至少95%的相同性。
發明效果
依據本發明的一實施態樣的話,能夠有效地調節在以黏膜附著細菌叢或腸內細菌叢為首之常居細菌叢中的細菌構成均衡。依據本發明之一實施態樣的話,能夠有利地促進健全的常居細菌叢形成。本發明之乳酸菌株,能夠有利地利用於改善起因於炎症之疾病或狀態方面。此外,依據本發明之一實施態樣的話,乳酸菌株是能夠有利地利用在使得未成年或嬰幼兒等孩子攝取而預防成長後的疾病方面。
用以實施發明之形態
以下,詳細地說明本發明。
依據本發明之一實施態樣的話,提供一種捲曲乳桿菌CFF No.2031菌株,其係屬於捲曲乳桿菌的乳酸菌的新穎菌株。
捲曲乳桿菌 CFF No.2031菌株(以下亦簡稱為「CFF No.2031菌株」或「寄存菌株」。)是以人類母乳作為分離源所分離出。該菌株菌學的性質是顯示在後述的實施例中。該菌株是於2019年1月29日,寄存在獨立行政法人製品評估技術基盤機構(National Institute of Technology and Evaluation)專利微生物寄存中心(〒292-0818 千葉縣木更津市Kazusa鎌足2-5-8 122號室),寄存編號NITE BP-02869之下。
本發明的乳酸菌株,可為上述寄存菌株或實質上與其同等的菌株。上述寄存菌株或實質上與其同等的菌株是屬於捲曲乳桿菌的菌株,其16S rRNA基因的核苷酸序列是具有與序列識別號1所示之上述寄存菌株的16S rRNA基因的核苷酸序列至少95%的相同性。此外,上述相同性較佳為具有至少98%以上,更佳為具有至少99%以上,進一步較佳為具有至少99.9%以上,又更進一步較佳為100%的相同性。
在本發明中,所謂2個核苷酸序列間的「相同性」相同性,意指在被比較的2個序列間,在最優比對(best alignment)(最佳比對(optimal alignment))後所獲得之相同的核苷酸的百分比,該百分比僅為統計性的,且2個序列間的差異是跨度該等的全長無規地分布。2個核苷酸序列間的序列比較,通常是在利用最適合的方法進行比對後透過比較該等序列而進行,此情況時,前述比較是以每個片段、或每個「比較窗」進行即可。用於比較之序列的最佳比對是能夠透過電腦軟體(BLAST P比較軟體)進行。
此外,上述實質上同等的菌株,較佳為與上述寄存菌株在功能上同等。上述實質上同等的菌株是否與上述寄存菌株功能上同等,是能夠進行與後述之實施例的例1及例2同樣的試驗,透過統計的手法藉著是否是沒有顯著差異的範圍而判定。
上述實質上同等的菌株,較佳為與上述寄存菌株同等地具有在常居細菌叢中所含細菌之構成均衡的調節功能。此外,依據更佳的實施態樣的話,上述常居細菌叢中所含之細菌是毛螺旋菌科細菌(包含毛螺旋菌科(Lachnospiraceae),未分類的毛螺旋菌科(Unclassified Lachnospiraceae))、不動桿菌屬細菌(Acinetobacter)、黏液螺旋菌屬細菌(Mucispirillum)、普雷沃氏菌屬細菌(包含普雷沃氏菌屬(Prevotella),[普雷沃氏菌屬] ([Prevotella]))、或擬桿菌屬細菌(Bacteroides)。於此處,上述菌株的分類是視為按照軟體phyloseq(https://joey711.github.io/phyloseq)及該軟體參照的Greengenes 的分類。此外,依據進一步較佳的實施態樣的話,上述實質上同等的菌株是與上述寄存菌株同等地具有減少在上述常居細菌叢中之毛螺旋菌科細菌、不動桿菌屬細菌、普雷沃氏菌屬細菌或擬桿菌屬細菌的活性。此外,依據又另一個較佳的實施態樣的話,上述實質上同等的菌株是與上述寄存菌株同等地,具有增加在上述常居細菌叢中之黏液螺旋菌屬細菌的活性。此外,依據另一個較佳的實施態樣的話,上述實質上同等的菌株是與上述寄存菌株同等地,具有在投予過其之對象中抑制起因於腸管發炎時的腹瀉等所致的體重減少之活性。此外,依據另一更佳的實施態樣的話,上述實質上同等的菌株,是與上述寄存菌株同等地,具有在投予過其之對象中抑制腸炎發病時的DAI分數之活性。此外,上述常居細菌叢較佳為黏膜附著細菌叢或腸內細菌叢。
在本發明的乳酸菌株來說,只要不損及本發明效果,亦包含從寄存菌株或實質上與其同等的菌株,透過突變處理、基因重組、自然突變株的選擇等所育種出來的菌株。
本發明之乳酸菌株,例如,能夠透過培養該菌株而容易地使增殖。培養的方法只要是CFF No.2031菌株能夠增殖,未被特別限定,能夠依需要適宜修正通常可使用於乳酸菌的培養的方法而使用。例如,培養溫度為25~50℃即可,較佳為35~42℃。又,培養可在好氧條件下,及厭氧條件下之任一條件下進行,較佳為在厭氧條件下進行,例如,能夠一邊通氣碳酸氣等厭氧氣體一邊培養。此外,亦能夠在液體靜置培養等微好氧條件下進行培養。
就培養本發明之乳酸菌株的培養基而言,未被特別限定,能夠依需要適宜修正通常可使用於乳酸菌的培養的培養基而使用。即,作為碳源,能夠因應同化性使用例如:半乳糖、葡萄糖、果糖、甘露糖、纖維雙糖、麥芽糖、乳糖、蔗糖、海藻糖、澱粉、澱粉水解物、廢糖蜜等糖類。作為氮源,能夠使用例如:氨、硫酸銨、氯化銨、硝酸銨等銨鹽類或硝酸鹽類。此外,作為無機鹽類,能夠使用例如:氯化鈉、氯化鉀、磷酸鉀、硫酸鎂、氯化鈣、硝酸鈣、氯化錳、硫酸亞鐵等。此外,亦能夠使用蛋白腖、大豆粉、脫脂大豆粕、肉萃、酵母萃等有機成分。此外,就已調製完成之培養基而言,例如能夠合適地使用MRS培養基。
就本發明之乳酸菌株而言,培養後,能夠照原樣使用所獲得之培養物,亦能夠進行稀釋或濃縮而使用,亦能夠使用回收自培養物的菌體。此外,只要不損及本發明效果,在培養後能夠進行加熱、及冷凍乾燥等種種的追加操作。
本發明之乳酸菌株,較佳為生菌,但為死菌亦可。
本發明之乳酸菌株可照原樣使用,亦能夠依期望,與天然或其它的添加物一起以組成物的形態使用。
本發明之乳酸菌株,能夠利用來作為在對象中之常居細菌叢中所含細菌之構成均衡的調節機劑。因此,包含本發明之乳酸菌株而成之組成物,較佳為可使用來作為在對象中之在常居細菌叢中所含細菌之構成均衡的調節用組成物。此外,依據更佳的實施態樣的話,上述組成物可用以使在上述常居細菌叢中之毛螺旋菌科細菌、不動桿菌屬細菌、普雷沃氏菌屬細菌、或擬桿菌屬細菌減少。此外,依據另一更佳的實施態樣的話,上述組成物能夠用以增加在上述常居細菌叢中之黏液螺旋菌屬細菌。此外,依據又另一個較佳的施態樣的話,上述組成物是能夠用以抑制在投予過其之對象中起因於腸炎發病時的腹瀉所致的體重減少。此外,依據又另一個較佳的實施態樣的話,上述組成物能夠用以抑制活性在投予過其之對象中腸炎發病時之DAI分數。此外,依據又另一更佳的實施態樣的話,上述組成物可用以抑制在投予過其之對象中的黏膜的炎症,並抑制腹瀉或糞便出血。
此外,本發明之乳酸菌株,能夠利用來用以使得常居細菌叢中與炎症有關之細菌的量減少。因此,依據另一實施態樣的話,本發明之組成物能夠用以抑制炎症相關之疾病或症狀。
在本發明中之常居細菌叢較佳為黏膜附著細菌叢或腸內細菌叢。
本發明之組成物較佳為能夠作為醫藥、飲食品、化妝品或飼料而使用。
醫藥是只要含有本發明之乳酸菌株未特別受限制。就醫藥而言,亦可將本發明之乳酸菌株與藥學上可允許的載體一起進行製劑化而使用。
本發明之醫藥的劑型未受特別限制,具體地,可例示:錠劑、丸劑、粉劑、液劑、懸浮劑、乳劑(emulsion)、顆粒劑、膠囊劑、糖漿劑、栓劑、注射劑、軟膏劑、貼附劑、點眼劑及點鼻劑等。此外,在製劑化之際,能夠使用一般作為製劑載體所使用之賦形劑、結合劑、崩解劑、潤滑劑、穩定劑、矯味矯臭劑、稀釋劑、界面活性劑或注射劑用溶劑等添加劑。
本發明之醫藥中乳酸菌的含量是依劑型、用法、患者的年齡、性別、疾病的種類、疾病的程度、及其它的條件等而適宜設定,通常較佳為1×106
~1×1012
cfu/g或1×106
~1×1012
cfu/ml的範圍內,更佳為1×107
~1×1011
cfu/g或1×107
~1×1011
cfu/ml的範圍內。當CFF No.2031菌株為死菌的情況,cfu/g或cfu/ml可替換為個細胞/g或個細胞/ml。
飲食品是只要含有本發明之乳酸菌株,未受特別限制,就飲食品而言,可例示:清涼飲料、碳酸飲料、營養飲料、果汁飲料、及乳酸菌飲料等飲料(包含該等飲料的濃縮原液及調製用粉末);冰淇淋、雪果霜、刨冰等冰製點心;糖果、口香糖(chewing gum)、硬糖、樹膠(gum)、巧克力、糖果錠、零食、餅乾、果凍、果醬、鮮奶油、及烘焙點心等點心類;加工乳、乳飲料、發酵乳、優酪飲品、及奶油等乳製品;麵包;經腸營養食品、流質食品、育兒用乳、運動飲料;其它功能性食品等。此外,飲食品亦可為補充劑,例如可為錠狀的補充劑。當為補充劑的情況來說,關於每日用餐量及攝取卡路里能夠不受其它的食品影響地攝取CFF No.2031菌株。
飲食品能夠透過將本發明之乳酸菌株添加於飲食品的原料而製造,在添加本發明之乳酸菌株以外是能夠與一般的飲食品同樣地製造。本發明之乳酸菌株的添加可於飲食品製造步驟的任一階段進行。此外,飲食品亦可歷經已添加之本發明之乳酸菌株所致之發酵步驟而製造。就那樣的飲食品而言,可舉:乳酸菌飲料及發酵乳等。
就食品或飲料的原料而言,能夠使用可用於一般之飲料及食品的原料。
在本發明之飲食品來說,亦包含用以製造飲食品的原料、及食品添加物等,在飲食品的製造步驟或製造後被添加至飲食品者。例如,本發明之乳酸菌株能夠使用來作為發酵乳製造用菌元。此外,亦可於已被製造出之發酵乳中後續添加本發明之乳酸菌株。
在飲食品中本發明之乳酸菌株的含量,依飲食品的態樣而適宜設定,通常,在飲食品中較佳為1×106
~1×1012
cfu/g或1×106
~1×1012
cfu/ml的範圍內,更佳為1×107
~1×1011
cfu/g或1×107
~1×1011
cfu/ml的範圍內。
本發明之飲食品,能夠作為標示有下述用途之飲食品而販賣:用於調節在常居細菌叢中所含細菌之構成均衡,用於減少在常居細菌叢中之毛螺旋菌科細菌、不動桿菌屬細菌、擬桿菌屬細菌或普雷沃氏菌屬細菌,用於增加在黏膜附著細菌叢或腸內細菌叢中之黏液螺旋菌屬細菌,用於抑制體重減少,用於抑制DAI分數,用於抑制腹瀉或糞便出血,用於減少與炎症有關之菌株,用於抑制炎症相關之疾病或症狀等用途。
此外,在本發明的飲食品來說,能夠進行「用於調節在常居細菌叢中所含細菌的構成均衡」、「用於減少在常居細菌叢中之毛螺旋菌科細菌、不動桿菌屬細菌、擬桿菌屬細菌或普雷沃氏菌屬細菌」、「用於增加在常居細菌叢中之黏液螺旋菌屬細菌」、「用於抑制體重減少」、「用於抑制DAI分數」、「用於抑制腹瀉或糞便出血」、「用於減少與炎症有關之細菌」、「用於抑制炎症相關之疾病或症狀」等標示。又,除此之外,是表示透過組成物使用在上述用途而次要產生的效果之辭句的話,也可使用而毋須贅言。就這般之辭句而言,例如,可舉:「整頓腸胃之狀態」、「改善胃部或腸部不適感」等。
前述所謂「標示」是意指用以使需要者知悉上述用途之所有行為,是能夠使想到/類推上述用途般的標示的話,無論標示的目的、標示的內容、標示的對象物及媒體等,全部相當於本發明的「標示」。不過,較佳為透過需要者能夠直接地認知上述用途般的表現進行標示。
具體地,可例示:在本發明之飲食品涉及的商品或商品的包裝記載上述用途的行為,把在商品或商品的包裝記載有上述用途者進行轉讓、交付,為了轉讓或者交付而進行展示、輸入的行為,在關於商品的廣告、價格表或者交易文件上記載上述用途而展示、或者發布,或在以該等為內容的資訊記載上述用途而透過電磁性的(網際網路等)方法進行提供的行為等,特佳為對於在包裝、容器、型錄、小冊子、POP等販賣現場的宣傳材料、其它的文件等的標示。
此外,就標示而言,較佳為由行政機關等所許可的標示(例如,基於行政機關制定之各種制度而受到認可,並以基於那樣之認可的態樣進行的標示)。可例示例如:作為健康食品、功能性食品、經腸營養食品、特別用途食品、營養功能食品、準藥品等的標示,其它由厚生勞動省所認可的標示,可例示例如:特定保健用食品、以類似於其之制度所認可的標示。就後者之例而言,可例示:作為特定保健用食品的標示、作為附條件之特定保健用食品的標示、旨在影響身體的結構或功能的標示、減低疾病風險的標示等。進一步詳細地說,可例示:作為制定在健康促進法施行細則(平成15年4月30日日本國厚生勞動省令第86號)的特定保健用食品的標示(特別是保健用途的標示),及與此類似的標示等。
本發明之化妝品亦可準據上述醫藥或飲食品進行調製而作為內服用美容劑使用,但亦可作為局部用或全身用的皮膚外用劑。就皮膚外用劑的合適之例而言可舉:化妝水、乳液、乳霜 、軟膏、洗劑(lotion)、油劑、面膜等基礎化妝料、固體皂、液體皂、洗手液等洗面乳或潔膚乳、按摩用劑、卸妝用劑、除毛劑、脫毛劑、剃鬚處理物、鬍後乳液、鬍前乳液、刮鬍霜、粉底、口紅、腮紅、眼影、眼線、睫毛膏等妝飾化妝品、香水類、美甲劑、指甲油、指甲油去除劑、敷劑、硬膏劑、貼劑、片劑、貼附劑、氣溶膠劑等。
在本發明之化妝品中CFF NO.2031菌株的含量,是依化妝品的態樣及投予對象而適宜設定,較佳為1×106
~1×1012
cfu/g或1×106
~1×1012
cfu/ml的範圍內,更佳為1×107
~1×1011
cfu/g或1×107
~1×1011
cfu/ml的範圍內。
此外,在化妝品來說,能夠因應必要任意地選擇・併用例示於以下的添加成分而製造。就這般的添加成分而言,未被特別限定,可舉:油脂類(可可脂、洋甘菊油、胡蘿蔔油、黃瓜油、牛油脂肪酸、石栗油、紅花油、乳油木果油等)、蠟類(蜂蠟、巴西棕櫚蠟、鯨蠟、羊毛脂、液狀羊毛脂、還原羊毛脂、硬質羊毛脂、小燭樹蠟、褐煤蠟、蟲膠蠟、米糠蠟等)、礦物油(流動石蠟、凡士林、石蠟、地蠟(ozokerite)、純地蠟(ceresin)、微晶蠟等)、脂肪酸類(月桂酸、肉豆蔻酸、棕櫚酸、硬脂酸、山萮酸、油酸、亞麻油酸、次亞麻油酸、二十二碳六烯酸、二十碳五烯酸、12-羥基硬脂酸、十一碳烯酸、妥爾油、羊毛脂脂肪酸等天然脂肪酸、異壬酸、己酸、2-乙基丁酸、異戊酸、2-甲基戊酸、2-乙基己酸、異戊酸等合成脂肪酸)、醇類(乙醇、異丙醇、月桂醇、鯨蠟醇、硬脂醇、油醇、羊毛脂醇、膽固醇、植固醇、苯氧基乙醇等天然醇、2-己基癸醇、異硬脂醇、2-辛基十二烷醇等合成醇等)、各種維生素類(維生素A群:視網醇、視黃醛(維生素A1)、脫氫視黃醛(維生素A2)、胡蘿蔔素、茄紅素(原維生素A)、維生素B群:鹽酸硫胺素、硫酸硫胺素(維生素B1)、核黃素(維生素B2)、吡哆醇(維生素B6)、氰鈷胺素(維生素B12)、葉酸類、菸鹼酸類、泛酸類、生物素類、膽鹼、肌醇類、維生素C群:維生素C酸或其衍生物、維生素D群:麥角鈣化醇(維生素D2)等)、高分子化合物(阿拉伯膠、安息香膠、丹馬膠(dammar gum)、癒創木樹脂、殼聚醣(chitosan)、玻尿酸或其鹽、硫酸軟骨素或其鹽、乙基纖維素、甲基纖維素、羧甲基纖維素、羧乙基纖維素、羧乙基纖維素鈉、羥基乙基纖維素、羥基丙基纖維素、硝基纖維素、結晶纖維素、聚乙烯醇、聚乙烯基甲基醚、聚乙烯基吡咯啶酮(polyvinylpyrrolidone)、聚乙烯基甲基丙烯酸酯、聚丙烯酸鹽、聚環氧乙烷及聚環氧丙烷等聚環氧烷或其交聯聚合物、羧乙烯聚合物、聚乙亞胺等)等。
就本發明之飼料而言,可例示:寵物食物、家畜飼料、及飼育魚飼料等。本發明之飼料,能夠透過將CFF NO.2031菌株混合至一般的飼料,例如:穀類、粕類、糠類、魚粉、骨粉、油脂類、脫脂奶粉、乳清、礦物質飼料、或酵母類等而製造。此外,例如如青貯料般,歷經由已添加之CFF NO.2031菌株所致之發酵步驟而製造飼料亦可。所製造出之飼料是能夠經口投予至一般的哺乳動物、家畜類、飼育魚類、及玩賞動物等。
在本發明之飼料中CFF NO.2031菌株的含量,依飼料的態樣及投予對象而適宜設定,但較佳為1×106
~1×1012
cfu/g或1×106
~1×1012
cfu/ml的範圍內,更佳為1×107
~1×1011
cfu/g或1×107
~1×1011
cfu/ml的範圍內。
此外,依據另一態樣的話,提供一種方法,其係調整在常居細菌叢所含之細菌之構成均衡的方法,包含對需要其之對象投予本發明之乳酸菌株的有效量而成。依據本發明之另一個較佳的態樣的話,上述方法是用以使得與炎症有關的菌(例如,在常居細菌叢中之毛螺旋菌科細菌、不動桿菌屬細菌、擬桿菌屬細菌或普雷沃氏菌屬細菌等)減少的方法。此外,依據另一更佳的態樣的話,上述方法是用以增加在常居細菌叢中之黏液螺旋菌屬細菌的方法。此外,依據另一進一步較佳的態樣的話,上述方法是用以抑制選自於起因於腸炎發病之腹瀉所致的體重減少、DAI分數、腹瀉或糞便出血之至少一個症狀的方法。
此外,依據另一態樣的話,提供一種方法,其係炎症相關之疾病或症狀的抑制方法,包含對需要其之對象投予本發明之乳酸菌株的有效量而成之方法。於此處,在「抑制」來說,不僅是治療或改善已確立的病態,亦包含預防將來有確立之可能性的病態。
就上述炎症相關之疾病或症狀而言,可舉:炎症性腸道疾病(IBD),具體地可舉:潰瘍性結腸炎(UC)及克羅恩氏病(CD)等。
此外,依據一個態樣的話,本發明的方法是排除醫療行為。依據另一態樣的話,本發明之方法是非治療性方法。
本發明之乳酸菌株的有效量,只要不妨礙本發明效果,未被特別限定,例如,能夠設為1.0×104
cfu/次~1.0×1012
cfu/次。依據一個態樣的話,CFF NO.2031菌株的有效量是令為每一餐的攝取量。
本發明之乳酸菌株的有效量能夠1日1次或分為多次進行投予,此外,隔數天或數週進行1次投予亦可。此外,因應對象的狀態等,能夠適宜選擇投予時期。
本發明之對象較佳為人類,更佳為15歲以下的未成年,進一步較佳為嬰幼兒。對嬰幼兒應用本發明之乳酸菌株是在作為母乳的代替品而調節於黏膜附著細菌叢或腸內細菌叢所含之細菌的構成均衡方面特別有利。
此外,本發明之乳酸菌株,可不限於一個時期持續地投予,亦可在嬰幼兒期的一個時期投予。將本發明之乳酸菌株在嬰幼兒期投予一事,在預防成長後的疾病方面亦特別有利。
此外,對象可為身心健全者,亦可為具有與在胃腸管的炎症相關之疾病或症狀者,或者亦可為具有起因於前者之身體狀況不佳者。
此外,在本發明中之投予方法,較佳為經口。不過只要不妨礙本發明效果,未被特別限定,例如,亦可藉由經管、經腸、經皮(塗布等)等其它的方法對對象投予本發明之乳酸菌株。
此外,依據另一態樣的話,提供一種本發明之乳酸菌株的用途,其係在常居細菌叢所含之細菌構成均衡調整用組成物的製造中的用途。依據較佳的態樣的話,上述組成物,是用於減少與炎症有關之菌(例如,在常居細菌叢中之毛螺旋菌科細菌、不動桿菌屬細菌、普雷沃氏菌屬細菌或擬桿菌屬細菌等)的組成物。此外,依據另一個較佳的態樣的話,上述組成物是用於增加在常居細菌叢中之黏液螺旋菌屬細菌的組成物。此外,依據另一更佳的態樣的話,上述組成物是用以抑制選自於起因於腸炎發病之腹瀉所致的體重減少、DAI分數、腹瀉或糞便出血之至少一個症狀的組成物。
此外,依據另一個較佳的態樣的話,提供一種本發明之乳酸菌株的用途,其係在用於抑制炎症相關之疾病或症狀的組成物的製造中的用途。
[實施例]
以下說明本發明的實施例,但本發明並非被限定於該等實施例。
例1:對黏膜附著細菌叢及腸內細菌叢投予乳酸菌之影響的確認試驗
將購入自日本SLC之2週齡的C57BL/6N小鼠進行7天的設施馴化之後,在3週齡使斷奶。其次,以使得體重成為均等的方式,將小鼠分為對照組(C組)及捲曲乳桿菌 CFF No.2031株投予組(L組)之2組(n=6)進行了飼育管理。斷奶後的小鼠是使自由地攝取小鼠/大鼠/倉鼠用Labo MR Stock(日本農產工業)並且使自由地攝取未滅菌的水。飼育室以8時點燈,20時關燈的12小時明暗周期,並且室溫是設定於25±2℃。
自斷奶日的次日起起始各受試物質的經口投予,進行了14天經口投予。C組是經口投予PBS(-),L組是經口投予捲曲乳桿菌 CFF No.2031株。進行經口投予之際的液量視小鼠的體重調節,若體重低於12.5g的話,令為100μl,若為12.5以上低於17.5g的話,令為150μl。
就經口投予用的器具而言是使用了1ml注射器(TERUMO)。再者,在經口投予時,1ml注射器是切斷為適當的長度,並把前端烘烤加工成圓形的CHUKOH FLO PTFE管AWG-26(中興化生工業)進行安裝,並安裝了經高壓釜滅菌過的23G針(TERUMO)。
經口投予至小鼠的含有捲曲乳桿菌 CFF No.2031株之液是如以下般調製。將被保存在-80℃之捲曲乳桿菌(L.crispatas) CFF No.2031株植菌於10ml的MRS培養基,並在厭氧條件下進行37℃24小時培養,作成了前置培養液。冷藏保存該前置培養液,使用於調製毎日的含乳酸菌株之液。含菌株之液是在經口投予的前一日從前置培養液取出200μl在新的10ml的MRS培養基進行繼代培養,使用了以與前置培養相同條件下進行了培養者。培養結束後,以10,000×g在室溫下進行3分鐘離心分離,將菌體作成丸粒之後,以PBS(-)洗淨,並以濁度(OD595)為基準,以使得成為5×109
cfu/ml的方式懸浮於PBS(-),作成了含有乳酸菌株之液。
把經口投予結束後7天間令為受試物質的洗除期間(washout period)(停止期間),不進行經口投予地飼育。
洗除期間結束後(小鼠到達6週齡之後),解剖全部的小鼠,分別採取了盲腸黏膜與盲腸內容物、及結腸黏膜與結腸內容物。
使用QuickGene DNA tissue kit S(KURABO)從採取到之各組織的黏膜及內容物萃取出細菌DNA。把萃取出之細菌基因體DNA作為模板,透過使用了KAPA HiFi HS Ready Mix(NIPPON Genetics)之PCR反應將16S rRNA基因之V3-V4區域進行了擴增。在此反應來說,2ndPCR用的序列是使用附有尾部的前置引子(forward primer)341F(序列識別號2:5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’),及反置引子(reverse primer)805R(序列識別號3:5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’)。將12.5μl之KAPA HIFI HS Ready Mix、2.5μl之模板DNA、各0.05μl之引子、9.9μl之自由水(free water)之總量為25μl作為反應液來進行了PCR。95℃ 3分鐘的初期變性之後,進行25循環: 95℃ 30秒鐘、55℃ 30秒鐘、72℃ 30秒鐘的溫度循環,並在最後進行了72℃ 5分鐘的伸長反應。PCR反應結束後,使用1%瓊脂糖凝膠(tris acetate EDTA :TAE)進行電泳,確認了目標基因經擴增。其後,使用NucleoFast 96 PCR Clean up plate (TAKARA BIO)來純化了PCR產物。透過使用了負載有Index及Illumina sequencing adapter之引子的第2次的PCR反應,進行了純化產物的標籤附加。
使用SequalPrepTMNormalization Plate Kit(Invitrogen)進行了藉PCR所獲得之產物的純化及濃度調製。接著,藉由使用了KAPA SYBR Fast qPCR Kit(NIPPON Genetics)的real-time PCR來進行了純化產物之濃度的定量。把可充分地獲得濃度之樣本每1樣本各混合10μl,並使用Agencouet AMPure XP(BECKMAN COULTER)進行了純化/濃縮。
按照KAPA Library Quantification Kit(NIPPON Genetics)的標準程序算出了所獲得之文庫(library)的濃度。其後,根據求出之濃度,利用10 mM Tris Buffer將樣本稀釋為4nM。其後的操作是按照16S Metagenomic Sequencing Library Preparation (Illumina)進行了最終調製。把調製出之樣本上樣至Miseq Reagent Kit V3(600PE)的試劑筒,進行了由MiSeq(Illumina)進行的定序。所獲得之定序數據是在基於QIIME之一次分析之後,使用STAMP進行了二次分析。
結果是如圖1~4所示。
圖1A及圖1B是基於盲腸的黏膜附著細菌叢總體基因體分析的主座標分析之結果。在上述分析來說是使用了phyloseq(參照https://joey711.github.io/phyloseq/)。因此,於圖2A、圖3A、圖4A~圖4H所示之菌的分類是按照phyloseq及該軟體參照的Greengenes之分類。基於總體基因體分析的主座標分析之結果,與對照組(C組)相比較,在捲曲乳桿菌 CFF No.2031株投予組(L組)來說,是於盲腸的黏膜附著細菌叢的構成確認到變化(PERMANOVA p=0.002)。
基於盲腸之黏膜附著細菌叢總體基因體分析的菌叢結構分析結果是如圖2A所示。此外,關於盲腸的黏膜附著細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中毛螺旋菌科細菌(未分類的毛螺旋菌科(Unclassified Lachnospiraceae))的占有率,與在對照組中之該占有率(序列的比例(proportion of sequences)(%))相比較是顯著地低(圖2B,Wilcoxon/Kruskal-Wallis的檢定(等級和(rank sum)),p<0.05)。在另一方面,在捲曲乳桿菌 CFF No.2031株投予組(L組)中黏液螺旋菌屬細菌的占有率,與在對照組中之該占有率相比較是顯著地高(圖2C,Wilcoxon/Kruskal-Wallis的檢定(等級和),p<0.05)。
基於結腸之黏膜附著細菌叢總體基因體分析之菌叢結構分析結果是如圖3A所示。關於結腸的黏膜附著細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中不動桿菌屬細菌的占有率,與在對照組(C組)中之該占有率相比較是顯著地低(圖3B,Wilcoxon/Kruskal-Wallis的檢定(等級和),p<0.05)。
已知毛螺旋菌科細菌及不動桿菌屬細菌在腸管發炎時會增加(非專利文獻2~5)。因此,從透過投予捲曲乳桿菌 CFF No.2031株而該等細菌減少這樣的結果來看,認為捲曲乳桿菌 CFF No.2031株具有能夠抑制消化道發炎的效果。此外, 黏液螺旋菌(Mucispirillum)是會同化黏液的細菌。從該細菌是增加的這樣的結果來看,認為透過投予捲曲乳桿菌 CFF No.2031株,消化道黏膜會充分地被黏液充滿。
由以上來看,認為捲曲乳桿菌 CFF No.2031株是具有提高消化道黏膜的阻障功能的效果。
基於盲腸內容物之細菌叢總體基因體分析之菌叢結構分析結果,是如圖4A所示。此外,關於盲腸內容物的細菌叢,是如圖4B所示,在捲曲乳桿菌 CFF No.2031株投予組(L組)中擬桿菌屬(Bacteroides)的占有率,與對照組相比較是顯著地低(學生t檢定,p=0.0484)。此外,如圖4C所示,與對照組相比較,在L組中之[普雷沃氏菌屬]細菌([Prevotella])的占有率是顯著地低(學生t檢定,p=0.0430)。此外,如圖4D所示,與對照組相比較,在L組中之毛螺旋菌科細菌(未分類的毛螺旋菌科(Unclassified Lachnospiraceae))的占有率是顯著地低(學生t檢定,p<0.0001)。進一步,與對照組相比較,在L組中之乳桿菌屬(Lactobacillus)的占有率是顯著地高(學生t檢定,p=0.0409)。
基於結腸內容物之細菌叢總體基因體分析的菌叢結構分析結果是如圖4E所示。關於結腸內容物的細菌叢,如圖4F所示,與對照組相比較,在L組中之擬桿菌屬細菌(Bacteroides)的占有率是顯著地低(學生t檢定,p=0.0004)。此外,如圖4G所示,與對照組相比較,在L組中之普雷沃氏菌屬(Prevotella)的占有率是顯著地低(Wilcoxon/Kruskal-Wallis的檢定(等級和),p=0.0131)。此外,如圖4H所示,與對照組相比較,在L組中之[普雷沃氏菌屬]細菌([Prevotella])的占有率是顯著地低(學生t檢定,p=0.0072)。
已知在腸管及全身發炎時,普雷沃氏菌屬細菌及擬桿菌屬細菌會增加(非專利文獻6~8)。因此,從透過投予捲曲乳桿菌 CFF No.2031株而該等細菌會減少這樣的結果來看,認為捲曲乳桿菌 CFF No.2031株具有能夠抑制消化道發炎的效果。從以上來看,認為捲曲乳桿菌 CFF No.2031株具有抑制消化道黏膜發炎的效果。
例2:因乳酸菌投予所致之消化道阻障功能增強試驗
將自查理斯河實驗室日本公司(Charles River Laboratories Japan, Inc.)與母小鼠一併購入之2胎(各胎6隻)2週齡之C57BL/6N小鼠,導入本研究室的飼育室,進行了7天的設施適應。在3週齡使仔小鼠斷奶,並以使得體重成為均等的方式分為對照組(C組)、捲曲乳桿菌 CFF No.2031株投予組(L組)這2組(N=6)進行了飼育管理。與例1同樣地自斷奶日的次日起,對DC組是起始PBS(-)的經口投予,對DL組是起始捲曲乳桿菌 CFF No.2031株的經口投予,且進行了14天經口投予。把自經口投予結束後起的7天設為經投予之菌的洗除(wash out)期間,不進行經口投予地飼育。在洗除(wash out)期間結束而小鼠到達6週齡的時點,在例2來說是將1.5%DSS(以15 g/l將葡聚糖硫酸鈉鹽(Dextran Sulfate Sodium (DSS) salt)、結腸炎等級(Colitis grade) (MP Biomedicals M.W 36-50kDa)溶解於蒸餾水)換為自來水作為飲水,使自由攝取。在DSS投予第11日將所有小鼠供至解剖。
再者,斷奶後沒有特別記載的期間是使自由地攝取小鼠/大鼠/倉鼠用Labo MR Stock(日本農產工業),並使自由地攝取未滅菌的自來水。飼育室是以8時點燈,20時關燈的12小時明暗周期,室溫是設定在25±2℃,濕度是設定在40~70%。
自投予DSS起始日(d22)至最終日的前一日(d31)為止,毎日進行了小鼠的體重測定及糞便性狀(硬度及出血)的評分。惟,最終日(d32)是在即將解剖前進行了小鼠的體重測定及糞便性狀的評分。從體重減少率及糞便性狀算出是表示腸炎重症度的指數之DAI,進行了腸炎症狀的評價。再者,此評價方法是準據Mennigen et al.(2009)(表2-1)。
[表1]
此外,如圖5A所示,在投予DSS第11日(d11),與對照組(C組:86.87%)相比較,在捲曲乳桿菌 CFF No.2031株投予組(L組:92.26%)中觀察到體重減少抑制效果(t-test,p=0.05)。此外,如圖5B所示,在投予DSS第11日(d11),與對照組(C組:DAI分數9.07)相比較,在捲曲乳桿菌 CFF No.2031株投予組(L組::DAI分數7.00)中DAI分數的惡化亦受到抑制(t-test,p=0.07)。
當把在本試驗使用之小鼠的投予時期與週齡換算為人類的情況(是將小鼠的壽命設為2年,將人類壽命設為60~80歲進行換算的情況),在本試驗來說,將乳酸菌對1歲半~2歲半左右的兒童進行1年~1年半期間投予的結果,是變得會預防了可能在4~5歲左右發生的炎症性疾病。從此結果來看,認為將本發明之乳酸菌株在嬰幼兒期進行投予一事能夠利用於預防成長後的疾病。
[產業上之可利用性]
依據本發明的話,提供一種新穎乳酸菌,其能夠有效地調節在以黏膜附著細菌叢或腸內細菌叢為首之常居細菌叢中的細菌構成均衡。
[圖1A]是顯示進行了主座標分析之結果的圖表:在例1中,關於對照組(C組)及捲曲乳桿菌 CFF No.2031株投予組(L組)進行了基於盲腸的黏膜附著細菌叢之總體基因體(metagenome)分析的主座標分析(縱軸參數:Axis 2,橫軸Axis 1)。
[圖1B]是顯示進行了主座標分析之結果的圖表:在例1中,關於對照組(C組)及捲曲乳桿菌 CFF No.2031株投予組(L組)進行了基於盲腸的黏膜附著細菌叢總體基因體分析的主座標分析(縱軸參數:Axis 2,橫軸Axis 3)。
[圖2A]是顯示在在例1中,基於盲腸的黏膜附著細菌叢總體基因體分析進行了菌叢結構分析的結果之圖表。
[圖2B]是顯示比較結果之圖表:在例1中,關於盲腸的黏膜附著細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中未分類的毛螺旋菌科(Unclassified f_Lachnospiraceae)的占有率,與在對照組(C組)中該占有率的比較結果。
[圖2C]是顯示比較結果的圖表:在例1中,關於盲腸的黏膜附著細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中黏液螺旋菌屬細菌(Mucispirillum)的占有率,與在對照組(C組)中的該占有率的比較結果。
[圖3A]是顯示在例1中,基於結腸的黏膜附著細菌叢總體基因體分析進行了菌叢結構分析之結果的圖表。
[圖3B]是顯示比較結果的圖表:在例1中,關於結腸的黏膜附著細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中之不動桿菌屬細菌(Acinetobacter)的占有率,與在對照組(C組)中的該占有率的比較結果。
[圖4A]是顯示在例1中,基於盲腸內容物之細菌叢總體基因體分析進行了菌叢結構分析之結果的圖表。
[圖4B]是顯示比較結果之圖表:在例1中,關於盲腸內容物的細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中之擬桿菌屬細菌(Bacteroides)的占有率,與在對照組(C組)中該占有率的比較結果。
[圖4C]是顯示比較結果的圖表:在例1中,關於盲腸內容物的細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中[普雷沃氏菌屬]細菌([Prevotella])的占有率,與在對照組(C組)中之該占有率的比較結果。
[圖4D]是顯示比較結果的圖表:在例1中,關於盲腸內容物的細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中毛螺旋菌科細菌(未分類的毛螺旋菌科(Unclassified Lachnospiraceae))的占有率,與在對照組(C組)中之該占有率的比較結果。
[圖4E]是顯示在例1中,基於結腸內容物之細菌叢總體基因體分析進行了菌叢結構分析之結果的圖表。
[圖4F]是顯示比較結果的圖表:在例1中,關於結腸內容物的細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中擬桿菌屬細菌(Bacteroides)的占有率,與在對照組(C組)中之該占有率的比較結果。
[圖4G]是顯示比較結果的圖表:在例1中,關於結腸內容物的細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中普雷沃氏菌屬細菌(Prevotella)的占有率,與在對照組(C組)中之該占有率的比較結果。
[圖4H]是顯示比較結果的圖表:在例1中,關於結腸內容物的細菌叢,在捲曲乳桿菌 CFF No.2031株投予組(L組)中[普雷沃氏菌屬]細菌([Prevotella])的占有率,與在對照組(C組)中之該占有率的比較結果。
[圖5A]是顯示在例2中受試小鼠體重變遷的圖表。顯示了把起始投予DSS第1日(d1)的體重令為100.00%,至起始投予DSS第11日(d11)為止的體重變遷(%)。
[圖5B]是顯示在例2中之受試小鼠的疾病活動性指標(Disease Activity Index)(DAI)之變遷的圖表。顯示了自起始投予DSS第1日(d1)至第11日(d11)為止的DAI分數。
Claims (8)
- 一種捲曲乳桿菌菌株,係寄存在寄存編號NITE BP-02869下的捲曲乳桿菌CFF No.2031。
- 一種組成物,包含如請求項1之菌株而成。
- 一種如請求項1之捲曲乳桿菌菌株於製造醫藥、飲食品、化妝品或飼料之用途。
- 一種如請求項1之捲曲乳桿菌菌株於製造組成物之用途,該組成物係用以經口投予至嬰幼兒或未成年。
- 一種如請求項1之捲曲乳桿菌菌株於製造組成物之用途,該組成物係用以抑制炎症相關之疾病或症狀。
- 一種如請求項1之捲曲乳桿菌菌株於製造組成物之用途,該組成物係用以保護消化道。
- 一種如請求項1之捲曲乳桿菌菌株於製造組成物之用途,該組成物係用以抑制體重減少。
- 一種如請求項1之捲曲乳桿菌菌株之用途,該用途係製造常居細菌叢中所含細菌之構成均衡的調節用組成物,前述常居細菌叢係黏膜附著細菌叢或腸內細菌叢,前述細菌之構成均衡的調節包含使毛螺旋菌科細菌、不動桿菌屬細菌、普雷沃氏菌屬細菌或擬桿菌屬細菌減少,或者使黏液螺旋菌屬細菌增加。
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