TW201313234A - 口腔內細菌之增殖抑制劑 - Google Patents

口腔內細菌之增殖抑制劑 Download PDF

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TW201313234A
TW201313234A TW101123499A TW101123499A TW201313234A TW 201313234 A TW201313234 A TW 201313234A TW 101123499 A TW101123499 A TW 101123499A TW 101123499 A TW101123499 A TW 101123499A TW 201313234 A TW201313234 A TW 201313234A
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bacteria
oral
galactose
sorbitol
proliferation
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Yushi Kodama
Atsushi Narise
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Lotte Co Ltd
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Abstract

本發明係關於提供抑制口腔內細菌增殖之即使攝取亦無害之物質。尤其，對於併用糖與糖醇時之有效性，至今仍未明確，提供抑制口腔內細菌增殖之糖與糖醇之組合。含半乳糖及山梨糖醇為特徵之口腔內細菌之增殖抑制劑。關於本發明之口腔內細菌之增殖抑制劑係適用於改善及預防牙周病。

Description

口腔內細菌之增殖抑制劑
本發明係關於口腔內細菌之增殖抑制劑。
一般認為人的口腔內棲息達數百種細菌。此等中依據引起牙齦或牙周發炎，或產生酸而溶解骨骼或牙齒，亦包含許多成為牙周病或齲齒原因的菌。
具核酸桿菌(Fusobacterium nucleatum)(以後為F.nucleatum)係從人之健康、疾病部位兩方的牙垢，所大量檢測出之細菌種(非專利文獻1、2)。因為此菌係隨著牙周病惡化，其比率逐漸增加，所以認為幫助牙周病的發病及惡化(非專利文獻3~5)。另外，已知具有與其他許多細菌附著的能力，於牙菌斑中，擔任連接早期菌落形成菌種與含牙周病原菌之後期菌落形成菌種之橋樑角色(非專利文獻6)。進而，亦為產生成為口臭原因物質之揮發性硫化物之菌種(非專利文獻7)。因此，認為對F.nucleatum顯示活性之抗菌劑係對預防牙周病、減少口腔內細菌、及預防、減低口臭有效。
對於將此等菌活性化或抑制該活性之物質，亦進行研究。例如，糖類係眾知之齲齒原因菌製造酸時之原因物質。另一方面，已知糖醇具有對齲齒原因菌抑制增殖的作用，近年來亦報告對牙周病原菌亦有抑制增殖的作用。
先前技術文獻
非專利文獻
[非專利文獻1]Papapanou, P.N., Sellen, A., Wennstroem, J.L. and Dahlen, G., An analysis of the subgingival microflora in randomly selected subjects, Oral microbiology and immunology, 8, 24-29(1993).
[非專利文獻2]Moore, W.E. and Moore, L.V., The bacteria of periodontal diseases, Periodontology 2000, 5, 66-77 (1994).
[非專利文獻3]Dzink,J.L., Socransky, S.S. and Haffajee, A.D. The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases, Journal of clinical periocontology, 15, 316-323(1988).
[非專利文獻4]Socransky, S.S. Haffajee, A.D. and Dzink, J.L., Relationship of subgingival microbial complexes to clinical features at the sampled sites, Journal of clinical peridontology, 15, 440-444(1988).
[非專利文獻5]Tanner, A. and Bouldin, H., The microbiota of early periodontites lesions in adults, Journal of clinical periodontology, 16, 467-471(1989).
[非專利文獻6]Kolenbrander,P.E. Andersen, R.N., Blehert, D.S., Egland, P.G., Foster, J.S. and Palmer, R.J. Jr., Communication among oral bacteria, Microbiology and molecular biology reviews, 66, 486-505(2002).
[非專利文獻7]Krespi, Y.P., Shrime, M.G and Kacker, A., The relationship between oral malodor and volatile sulfur compound-producing bacteria, Otolaryngology-Head and Neck Surgery, 135, 671-676(2006).
[非專利文獻8]Bolstad, A.I., Jensen, H.B. and Bakken, V., Taxonomy, Biology, and Periodontal Aspects of Fusobacterium nucleatum, Clinical Microbiology Reviews, 9, 55-71 (1996).
[非專利文獻9]Kapartral, V., Anderson, I., Ivanova, N., Reznik, G., Los, T., Lykidis, A. and Overbeek, R., et al., Genome Sequence and Analysis of the Oral Bacterium Fusobacterium nucleatum Strain ATCC 25586, Journal of Bacteriology, 184, 2005-2018(2002).
[非專利文獻10]Kapatral, V., Ivanova, N., Anderson, I., Reznik, G., Bhattacharyya A., Gardner, W.L. and Kyrpides, N., et al., Genome Analysis of F. nucleatum sub spp vincentii and Its Comparison With the Genome of F. nucleastum ATCC25586, Genome Research, 6A, 1180-1189(2003).
已知作為牙周病原菌或齲齒病原菌係複數的口腔內細菌，尋求提供抑制此等菌增殖之物質。另外，作為如此物質，因為於口腔內作用，所以要求即使攝取於體內亦無害之物質。
有鑑於前述課題，本發明係以提供抑制口腔內細菌增 殖之即使攝取亦無害之物質為其目的。尤其，對於併用糖與糖醇時之有效性，至今仍未明確，提供抑制口腔內細菌增殖之糖與糖醇之組合為其目的。
本發明者等努力研究的結果係發現含半乳糖及山梨糖醇之培養基，明顯抑制口腔內細菌F.nucleatum之增殖，因此可完成本發明。亦即本發明係以含半乳糖及山梨糖醇為特徵之口腔內細菌之增殖抑制劑。
關於本發明之口腔內細菌之增殖抑制劑係藉由使含於食品或口腔組成物，可安全且日常地利用或攝取，使有效率地減少口腔內細菌，並且可改善及預防牙周病，進而亦可預防、減低口臭。
用以實施發明之最佳形態
本發明之含半乳糖及山梨糖醇之口腔內細菌之增殖抑制劑係具有相乘地抑制F.nucleatum增殖之效果。因為F.nucleatum係與其他許多細菌附著而成為形成牙菌斑的原因，所以抑制此菌增殖係可抑制成為牙周病或齲齒原因之口腔內其他細菌之增殖。另外，因為F.nucleatum係牙周病的病原菌之一，所以本發明係適用於改善及預防牙周病。進而，因為此菌係產生成為口臭原因物之揮發性硫化物 ，所以認為抑制F.nucleatum增殖，亦可有效地預防、減低口臭。
半乳糖及山梨糖醇之合計莫耳濃度係以0.5M以上，0.9M以下為宜。另外，半乳糖及山梨糖醇之莫耳濃度比率係以半乳糖：山梨糖醇=2：1~1：3為宜。
作為本發明中之半乳糖係以使用β-D-半乳糖為宜。另外，作為本發明中之山梨糖醇係以使用D-山梨糖醇(D-Sorbit)為宜。
另外，本發明之增殖抑制劑係可摻混於漱口劑、牙膏、吸入劑及口含劑等之口腔組成物、口香糖、糖果、糖錠、軟糖、巧克力、軟式小西餅及零食等之點心、冰淇淋、冰沙及冰果等之冷點、飲料、麵包、熱餅、乳製品、火腿及香腸等之畜肉製品類、或魚板及竹輪等之魚肉製品、熟食類、布丁、湯或果醬等之飲食品利用。
實施例1 試料
使用D(+)-半乳糖(和光純藥：特級)、D(-)-山梨糖醇(和光純藥：一級)。
試驗菌株
使用F.nucleatum JCM12990株作為口腔內細菌。
培養基
使用添加Yeast extract(3.0g/l)、Hemin(5.0mg/l)、Menadione(0.5mg/l)之Tripticase soy broth(以後為TSB培養基)，於37℃之厭氧條件下(10%CO2、10%H2、80%N2)進行培養。關於含半乳糖或山梨糖醇、或半乳糖及山梨糖醇之培養基，調製使半乳糖、山梨糖醇溶解之TSB培養基，添加3.0ml於7.0ml之TSB培養基時之最終濃度成為設定濃度。對於全部的培養基，進行過濾器(ADVANTEC，孔形0.20μm)滅菌。
測定菌數
添加口腔內細菌F.nucleatum JCM12990之前培養液於7.0ml之TSB培養基，培養至對數增殖期。去除與前培養液添加時等量之培養液，液量暫時成7.0ml後，添加3.0ml之含有半乳糖、山梨糖醇、或半乳糖及山梨糖醇之TSB培養基以調製10.0ml中之濃度成為設定濃度。此時添加3.0ml之不含半乳糖及山梨糖醇中任一種之通常的培養基者作為對照。將此等於厭氧條件培養1小時後，將培養試管移至小型振盪培養裝置(ADVANTEC東洋股份有限公司，Bio-Photo Recorder TVS062CA)內，以37℃，培養24小時後，進行測定(HITACHI U-3900H Spectrophotometer)於波長660nm之光學濃度(optical density：以後為OD值)。
實施例2
準備實施例1中，含0.1M或0.6M之半乳糖之添加半乳糖之TSB培養基、含0.2M、0.3M或0.6M之山梨糖醇之添加山梨糖醇之TSB培養基、及前述之含0.4M之半乳糖及0.2M之山梨糖醇、0.3M之半乳糖及0.3M之山梨糖醇、或0.3M之半乳糖及0.6M之山梨糖醇之混合TSB培養基，以及對照，進行試驗。對於各TSB培養基所得之OD值係如圖1所示。以含0.1M或0.6M之半乳糖之TSB培養基，培養24小時之結果，OD值大幅地高於對照之OD值。報告揭示F.nucleatum代謝半乳糖(非專利文獻8~10)，推測因其影響而促進增殖。另外，以含0.2M、0.3M或0.6M之山梨糖醇之TSB培養基，分別培養24小時時，確認以0.2M係高於對照之OD值，以0.3M則與對照相同程度之OD值，0.6M則低於對照之OD值。
在此，以含預測促進增殖之0.4M之半乳糖及經確認促進增殖之0.2M之山梨糖醇之混合添加TSB培養基，培養24小時之結果，確認低於對照之OD值。同樣地，對於含0.3M之半乳糖及0.3M之山梨糖醇、以及0.3M之半乳糖及0.6M之山梨糖醇之培養條件，確認低於對照之OD值，此值亦比0.3M之半乳糖、0.6M之山梨糖醇單獨條件之值低。
由以上結果，確認使培養基含半乳糖及山梨糖醇，抑制F.nucleatum之增殖。
實施例3
因為依據實施例2，半乳糖及山梨糖醇一同添加於培養基時，經確認有抑制F.nucleatum增殖之效果，所以改變此效果經確認之半乳糖及山梨糖醇之比率，檢討確認是否有同樣的抑制增殖效果。另外，設定半乳糖及山梨糖醇之合計莫耳濃度為0.6M。對於各TSB培養基所得之OD值係如圖2所示。
該結果係於半乳糖：山梨糖醇=2：1、1：1、1：2、1：3之各比率條件，確認低於對照之OD值。抑制增殖的程度係不依賴半乳糖或山梨糖醇中任一種之濃度，以半乳糖：山梨糖醇=1：2(半乳糖：山梨糖醇=0.2M：0.4M)之條件，確認為最強的增殖抑制效果。
實施例4
對於實施例3中經確認為最強的增殖抑制效果之半乳糖：山梨糖醇=1：2之條件，維持比率，改變合計莫耳濃度，進行相同的試驗。對於各TSB培養基所得之OD值係如圖3所示。
該結果係對於合計莫耳濃度為0.3M、0.4M之條件，確認高於對照之OD值。另一方面，對於合計莫耳濃度為0.5M、0.6M、0.9M之條件，確認低於對照之OD值。抑制增殖的程度係不依賴合計莫耳濃度，以0.6M(半乳糖：山梨糖醇=0.2M：0.4M)之條件，確認為最強的增殖抑制效果。
實施例5
本實施例係表示依據本發明代表之摻混例如下，但本發明並不局限於此等。
依據後述配方，製造含漱劑。
實施例6
依據後述配方，製造牙膏。
實施例7
依據後述配方，製造口臭用噴劑。
實施例8
依據後述配方，製造口含劑。
實施例9
依據後述配方，製造口香糖。
實施例10
依據後述配方，製造糖果。
實施例11
依據後述配方，製造糖錠。
實施例12
依據後述配方，製造軟糖。
實施例13
依據後述配方，製造飲料。
[圖1]表示對於實施例2中各培養基所得之OD值圖。
[圖2]表示對於實施例3中各培養基所得之OD值圖。
[圖3]表示對於實施例4中各培養基所得之OD值圖。

Claims (11)

  1. 一種口腔內細菌之增殖抑制劑，其特徵係含有半乳糖及山梨糖醇。
  2. 如申請專利範圍第1項之口腔內細菌之增殖抑制劑，其含有合計濃度為0.5M~0.9M之半乳糖及山梨糖醇。
  3. 如申請專利範圍第1項或第2項之口腔內細菌之增殖抑制劑，其中半乳糖：山梨糖醇之比率範圍係2：1~1：3。
  4. 如申請專利範圍第1項或第2項之口腔內細菌之增殖抑制劑，其中口腔內細菌係牙周病原菌。
  5. 如申請專利範圍第3項之口腔內細菌之增殖抑制劑，其中口腔內細菌係牙周病原菌。
  6. 如申請專利範圍第1項或第2項之口腔內細菌之增殖抑制劑，其中口腔內細菌係具核酸桿菌(Fusobacterium nucleatum)。
  7. 如申請專利範圍第3項之口腔內細菌之增殖抑制劑，其中口腔內細菌係Fusobacterium nucleatum。
  8. 如申請專利範圍第4項之口腔內細菌之增殖抑制劑，其中口腔內細菌係Fusobacterium nucleatum。
  9. 如申請專利範圍第5項之口腔內細菌之增殖抑制劑，其中口腔內細菌係Fusobacterium nucleatum。
  10. 一種口腔組成物，其特徵係含有如申請專利範圍第1項至第9項中任一項之口腔內細菌之增殖抑制劑。
  11. 一種食品，其特徵係含有如申請專利範圍第1項至 第9項中任一項之口腔內細菌之增殖抑制劑。
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