TW201701891A - Colitis inhibitor - Google Patents
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Abstract
Description
本專利申請案係基於屬先申請之日本專利申請案的日本專利特願2015-038302號(申請日:2015年2月27日)而伴隨優先權的主張,該先前專利申請案中之所有揭示內容係藉由引用而視為本案發明之揭示之一部分。 The present patent application is based on Japanese Patent Application No. 2015-038302 (filed on Feb. 27, 2015), which is hereby incorporated by reference. The content is considered as part of the disclosure of the present invention by reference.
本發明係關於以乳酸桿菌屬乳酸菌作為有效成分之大腸炎抑制劑。 The present invention relates to a colitis inhibitor having Lactobacillus lactic acid bacteria as an active ingredient.
大腸炎係發生在大腸中之炎症性疾病,在大腸炎中,包含屬自體免疫性疾病的炎症性腸疾病、屬特發性疾病的貝賽特氏症(Behcet’s syndrome)、缺血性腸炎、及感染症性腸炎等。炎症性腸疾病(Inflammatory Bowel Disease)係大致區分成潰瘍性大腸炎及克隆氏病(Crohn’s disease)2種,此等係共同被指定為日本的特定疾病。此等之病因不明,腸內細菌的參與或免疫機能的 異常等被認為是病因。目前,潰瘍性大腸炎的發病在日本係在美國的一半以下,人口每10萬人為100人左右,克隆氏病的發病在日本係在美國的約10分之1,人口每10萬人為27人左右,但一般認為在將來,在日本亦會隨著生活的歐美化,增加炎症性腸疾病的罹患率。 Colitis is an inflammatory disease that occurs in the large intestine. In colitis, it contains inflammatory bowel disease of autoimmune disease, Behcet's syndrome, and ischemic enteritis. And infectious enteritis. Inflammatory Bowel Disease is roughly classified into two types, ulcerative colitis and Crohn's disease, which are collectively designated as specific diseases in Japan. The cause of these diseases is unknown, the involvement of intestinal bacteria or immune function An abnormality or the like is considered to be the cause. At present, the incidence of ulcerative colitis is less than half of that in Japan, the population is about 100 per 100,000 people, the incidence of Crohn's disease is about one-tenth of that in Japan, and the population is 27 per 100,000 people. In the future, it is generally believed that in Japan, the incidence of inflammatory bowel disease will increase with the Europeanization of life.
就潰瘍性大腸炎及克隆氏病而言,治療係屬困難,並未確立根本的治療方法。作為內科療法,在潰瘍性大腸炎中,主體上係使用藥物療法(5-胺基水楊酸藥(5-ASA)等磺胺劑、普賴蘇穠(prednisolone)等類固醇劑、免疫調節劑/抑制劑等),在克隆氏病中,亦併用營養療法/飲食療法(完全靜脈營養療法、經腸營養療法、食餌療法)。 In the case of ulcerative colitis and Crohn's disease, the treatment is difficult and no fundamental treatment has been established. As a medical therapy, in ulcerative colitis, the main body is drug therapy (sulfuramide such as 5-aminosalicylic acid (5-ASA), steroids such as prednisolone, and immunomodulator/ Inhibitors, etc., in Crohn's disease, also combined with nutritional therapy/dietary therapy (complete intravenous nutrition therapy, enteral nutrition therapy, prey therapy).
另一方面,就以炎症性腸疾病為首之大腸炎而言,一般認為係由於為了從外部刺激中保護腸黏膜而在哺乳動物的腸管中運作之各種防禦機構產生破綻而發病,遂正進行腸管保護劑的開發。舉例而言,在專利文獻1:日本專利特開2010-83881號公報中,已報告藉由短乳酸桿菌(Lactobacillus brevis)SBC8803菌株之加熱死菌體的經口投予,由DSS(葡聚糖硫酸鈉)所誘發之類似潰瘍性大腸炎的腸管炎症係受到抑制。 On the other hand, in the case of colitis, which is caused by inflammatory bowel disease, it is generally considered that the various defensive mechanisms operating in the intestines of mammals in order to protect the intestinal mucosa from external stimuli are afflicted. Development of protective agents. For example, in the patent document 1: Japanese Patent Laid-Open Publication No. 2010-83881, oral administration of a heated dead cell of Lactobacillus brevis SBC8803 strain has been reported, by DSS (dextran) Intestinal inflammation similar to ulcerative colitis induced by sodium sulfate is inhibited.
此外,在專利文獻2:日本專利第5116194號公報中,已報告以乳酸桿菌屬細菌之酪蛋白乳酸桿菌(Lactobacillus casei)YIT9029、加氏乳酸桿菌(Lactobacillus gasseri)YIT0168之菌體或源自菌體之多 醣部分作為有效成分之炎症性腸疾病預防治療劑。 Further, in Patent Document 2: Japanese Patent No. 5116194, a bacterial cell of Lactobacillus casei YIT9029, Lactobacillus gasseri YIT0168, or a bacterial cell has been reported. Many A saccharide moiety as an active ingredient for the prophylactic treatment of inflammatory bowel diseases.
另一方面,此等乳酸桿菌屬之大腸炎抑制效果並不充分,此外,僅使用特定的菌株。如此,並未報告有可在日常上安全簡單地攝取之以乳酸菌作為有效成分且效果高之大腸炎抑制劑。 On the other hand, the colitis suppression effect of these Lactobacillus species is not sufficient, and in addition, only a specific strain is used. In this way, there is no report of a colitis inhibitor which is effective as a daily active ingredient and which is effective as a lactic acid bacteria and has a high effect.
〔專利文獻1〕日本專利特開2010-83881號公報 [Patent Document 1] Japanese Patent Laid-Open Publication No. 2010-83881
〔專利文獻2〕日本專利第5116194號公報 [Patent Document 2] Japanese Patent No. 5116194
本發明之目的為提供安全且具有優異的大腸炎抑制效果之大腸炎抑制劑及其製造方法。 An object of the present invention is to provide a colitis inhibitor which is safe and has an excellent colitis suppressing effect and a method for producing the same.
本發明者等人此次發現在於活體外所共同培養之大腸細胞中,特定而言,顯示出熱休克蛋白70的表現增強活性之乳酸桿菌屬乳酸菌係安全且具有優異的大腸炎抑制作用。本發明係基於該種見解而得。 The inventors of the present invention found that the Lactobacillus lactic acid bacteria which exhibit enhanced activity of heat shock protein 70 are safe and have an excellent colitis-inhibiting action in the large intestine cells which are co-cultured in vitro. The present invention is based on this finding.
根據本發明,係提供以下發明。 According to the present invention, the following invention is provided.
(1)一種大腸炎抑制劑,其係以具有大腸細胞中之熱休克蛋白(HSP)70的表現增強活性之乳酸桿菌屬乳酸菌作為有效成分。 (1) A colitis inhibitor comprising Lactobacillus lactic acid bacteria having an activity-enhancing activity of heat shock protein (HSP) 70 in colorectal cells as an active ingredient.
(2)如(1)所記載之大腸炎抑制劑,其中,乳酸桿菌屬乳酸菌為約氏乳酸桿菌(Lactobacillus johnsonii)。 (2) The colitis inhibitor according to (1), wherein the Lactobacillus lactic acid bacterium is Lactobacillus johnsonii.
(3)如(1)或(2)所記載之大腸炎抑制劑,其中,乳酸桿菌屬乳酸菌為選自約氏乳酸桿菌OLL203565株、約氏乳酸桿菌OLL204255株、及約氏乳酸桿菌OLL2978株者。 (3) The colitis inhibitor according to (1) or (2), wherein the Lactobacillus lactic acid bacterium is selected from the group consisting of Lactobacillus johnsonii OLL203565 strain, Lactobacillus johnsonii OLL204255 strain, and Lactobacillus johnsonii OLL2978 strain. .
(4)如(1)~(3)中任一項所記載之大腸炎抑制劑,其中,乳酸桿菌屬乳酸菌可使於活體外所共同培養之大腸細胞中之HSP70的表現量相較於未添加前述乳酸桿菌屬乳酸菌所培養之大腸細胞中之HSP70的表現量而言增加至1.5倍以上。 (4) The colitis inhibitor according to any one of (1) to (3), wherein the Lactobacillus lactic acid bacteria can express the amount of HSP70 in the large intestine cells co-cultured in vitro compared to The amount of HSP70 in the large intestine cells cultured with the Lactobacillus lactic acid bacteria added thereto was increased by 1.5 times or more.
(5)如(1)~(4)中任一項所記載之大腸炎抑制劑,其中,大腸炎為炎症性腸疾病。 (5) The colitis inhibitor according to any one of (1) to (4) wherein the colitis is an inflammatory bowel disease.
(6)如(5)所記載之大腸炎抑制劑,其中,炎症性腸疾病為潰瘍性大腸炎。 (6) The colitis inhibitor according to (5), wherein the inflammatory bowel disease is ulcerative colitis.
(7)如(1)~(6)中任一項所記載之大腸炎抑制劑,其係用於改善便性。 (7) The colitis inhibitor according to any one of (1) to (6), which is for improving the stool.
(8)一種大腸炎抑制劑之製造方法,其特徵為使其含有具有大腸細胞中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌。 (8) A method for producing a colitis inhibitor, which comprises a Lactobacillus lactic acid bacterium having an expression-enhancing activity of HSP70 in a large intestinal cell.
(9)一種具有HSP70的表現增強活性之乳酸桿菌屬乳酸菌之用途,其係用在大腸炎抑制劑的製造中。 (9) Use of a Lactobacillus lactic acid bacterium having an expression-enhancing activity of HSP70, which is used in the manufacture of a colitis inhibitor.
(10)一種具有HSP70的表現增強活性之乳酸桿菌屬乳酸菌之用途,其係用於抑制大腸炎。 (10) Use of a Lactobacillus lactic acid bacterium having an expression-enhancing activity of HSP70 for inhibiting colitis.
(11)一種具有HSP70的表現增強活性之乳酸桿菌屬乳酸菌,其係用於抑制大腸炎。 (11) A Lactobacillus lactic acid bacterium having an expression-enhancing activity of HSP70 for inhibiting colitis.
(12)一種大腸炎之抑制方法,其係包含使對此有所需要的對象攝取具有大腸細胞中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌的有效量而成。 (12) A method for inhibiting colitis comprising the step of ingesting an effective amount of a Lactobacillus lactic acid bacterium having an activity-enhancing activity of HSP70 in a large intestinal cell to a subject in need thereof.
根據本發明之大腸炎抑制劑,可有效地抑制大腸炎,特別是炎症性腸疾病。由於本發明之大腸炎抑制劑係以乳酸桿菌屬乳酸菌作為有效成分,故而可安全地抑制大腸炎,且在改善便性上有利地加以利用。 According to the colitis inhibitor of the present invention, colitis, particularly inflammatory bowel disease, can be effectively inhibited. Since the colitis inhibitor of the present invention contains Lactobacillus lactic acid bacteria as an active ingredient, it is possible to safely suppress colitis, and it is advantageously utilized for improving convenience.
〔圖1〕圖1係示出Caco-2細胞中之數種約氏乳酸桿菌株的投予所引發之HSPA1A基因表現誘導中之HSPA1A基因的表現量/GAPDH基因的表現量之比率的圖表。N=3。 Fig. 1 is a graph showing the ratio of the amount of expression of the HSPA1A gene/the amount of expression of the GAPDH gene in the induction of HSPA1A gene expression by administration of several strains of Lactobacillus johnsonii in Caco-2 cells. N=3.
〔圖2〕圖2係示出動物實驗之時程概略的概念圖。第1組(10隻):蒸餾水+PBS(磷酸緩衝液)之投予組、第2組(10隻):蒸餾水+約氏乳酸桿菌OLL203565株之投予組、第3組(10隻):DSS水溶液(2.0 w/v%)+PBS之投予組、第4組(10隻):DSS水溶液(2.0 w/v%)+約氏乳酸桿菌OLL203565株之投予組。 Fig. 2 is a conceptual diagram showing the outline of the time course of an animal experiment. Group 1 (10): the administration group of distilled water + PBS (phosphate buffer), the second group (10): the distilled water + the administration group of Lactobacillus johnsonum OLL203565 strain, the third group (10): DSS aqueous solution (2.0 w/v%) + PBS administration group, Group 4 (10): DSS aqueous solution (2.0 w/v%) + Lactobacillus johnsonii OLL203565 strained administration group.
〔圖3〕圖3係示出DSS的投予時之各組之平均體重之變化率的圖表(*p<0.05,與G1組比較)。其係將day 1(第1日)視為100之情況之變化率。G1:蒸餾水 +PBS之投予組、G2:蒸餾水+約氏乳酸桿菌OLL203565株之投予組、G3:DSS+PBS之投予組、G4:DSS+約氏乳酸桿菌OLL203565株之投予組。 Fig. 3 is a graph showing the rate of change of the average body weight of each group at the time of administration of DSS (*p < 0.05, compared with the G1 group). It is the rate of change of the day 1 (day 1) as 100. G1: distilled water + PBS administration group, G2: distilled water + Lactobacillus johnsonii OLL203565 strain administration group, G3: DSS + PBS administration group, G4: DSS + Lactobacillus johnsonii OLL203565 strain administration group.
〔圖4〕圖4係示出解剖時之DSS之投予組(G3及G4)之炎症評分的圖表(*p<0.05)。G3:DSS+PBS之投予組、G4:DSS+約氏乳酸桿菌OLL203565株之投予組。 Fig. 4 is a graph showing the inflammation score of the DSS administration group (G3 and G4) at the time of dissection (*p<0.05). G3: DSS + PBS administration group, G4: DSS + Lactobacillus johnsonii OLL203565 strain administration group.
〔圖5〕圖5係示出解剖時之各組之大腸的長度及每單位長度之重量的圖表(**p<0.01,與G1組比較)。G1:蒸餾水+PBS之投予組、G2:蒸餾水+約氏乳酸桿菌OLL203565株之投予組、G3:DSS+PBS之投予組、G4:DSS+約氏乳酸桿菌OLL203565株之投予組。 Fig. 5 is a graph showing the length of the large intestine and the weight per unit length of each group at the time of dissection (**p<0.01, compared with the G1 group). G1: administration group of distilled water + PBS, G2: administration group of distilled water + Lactobacillus johnsonii OLL203565 strain, administration group of G3: DSS + PBS, and administration group of G4: DSS + Lactobacillus johnsonii OLL203565 strain.
(大腸炎抑制劑) (colitis inhibitor)
本發明之大腸炎抑制劑之特徵為以具有大腸細胞中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌作為有效成分。具有HSP70的表現增強活性之乳酸桿菌屬乳酸菌具有顯著的大腸炎抑制作用實屬意外的事實。 The colitis inhibitor of the present invention is characterized in that Lactobacillus lactic acid bacteria having an activity-enhancing activity of HSP70 in large intestinal cells are used as an active ingredient. It is an accidental fact that the Lactobacillus lactic acid bacteria having the performance-enhancing activity of HSP70 have significant colitis suppression.
HSP70係會因熱或紫外線等而誘導表現之蛋白質之1種,例如由序列編號1的胺基酸序列所組成之蛋白質。此外,HSP70係HSPA1A所編碼出之蛋白質,HSPA1A的基因序列可藉由參照例如序列編號2而予以特 定。因此,本發明之大腸炎抑制劑之特徵亦可為以具有大腸細胞中之HSPA1A的表現增強活性之乳酸桿菌屬乳酸菌作為有效成分。 HSP70 is a protein which is induced by heat or ultraviolet rays, for example, a protein consisting of the amino acid sequence of SEQ ID NO: 1. In addition, HSP70 is a protein encoded by HSPA1A, and the gene sequence of HSPA1A can be specifically referred to by, for example, SEQ ID NO: 2 set. Therefore, the colitis inhibitor of the present invention may be characterized by having a Lactobacillus lactic acid bacterium having an activity-enhancing activity of HSPA1A in large intestinal cells as an active ingredient.
乳酸桿菌屬乳酸菌中之HSP70的表現增強活性之有無,如本案說明書之實施例之例1(活體外試驗)所記載,可藉由以編碼出HSP70之HSPA1A的表現量作為指標之比較試驗進行判定。於該種比較試驗中,在與乳酸桿菌屬乳酸菌所共同培養之大腸細胞中之HSPA1A的表現量相較於未添加前述乳酸桿菌屬乳酸菌所培養之大腸細胞中之HSPA1A的表現量而言係更高之情況,便認定為乳酸桿菌屬乳酸菌具有HSP70的表現增強活性。 The presence or absence of the performance-enhancing activity of HSP70 in the Lactobacillus lactic acid bacterium, as described in Example 1 (in vitro test) of the Example of the present specification, can be judged by a comparison test using the amount of HSPA1A encoding HSP70 as an index. . In this comparative test, the amount of HSPA1A in the large intestine cells co-cultured with the Lactobacillus lactic acid bacteria is more than the amount of HSPA1A in the large intestine cells cultured without the addition of the Lactobacillus lactic acid bacteria. In the case of high, it was confirmed that the Lactobacillus lactic acid bacteria had the performance enhancing activity of HSP70.
HSPA1A的表現量,如本案說明書之實施例之例1所記載,可以大腸細胞中之管家基因(甘油醛-3-磷酸脫氫酵素(GAPDH))的表現量為基準而予以算出。 The amount of expression of HSPA1A can be calculated based on the expression amount of the housekeeping gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) in the large intestine cells as described in Example 1 of the Example of the present specification.
根據本發明之較佳態樣,乳酸桿菌屬乳酸菌可使於活體外所共同培養之大腸細胞中之HSP70的表現量相較於未添加前述乳酸桿菌屬乳酸菌所培養之大腸細胞中之HSP70的表現量而言增加至較佳為1.5倍以上,更佳為1.7倍以上,再佳為1.8倍以上,再佳為1.9倍以上,再佳為2.0倍以上,再佳為2.5倍以上,再佳為3.0倍以上,再佳為3.5倍以上,再更佳為4.0倍以上。 According to a preferred embodiment of the present invention, the Lactobacillus lactic acid bacteria can express the amount of HSP70 in the large intestine cells co-cultured in vitro compared to the HSP70 in the large intestine cells cultured without the addition of the Lactobacillus lactic acid bacteria. The amount is preferably 1.5 times or more, more preferably 1.7 times or more, more preferably 1.8 times or more, more preferably 1.9 times or more, more preferably 2.0 times or more, and even more preferably 2.5 times or more. 3.0 times or more, more preferably 3.5 times or more, and even more preferably 4.0 times or more.
根據本發明之較佳態樣,乳酸桿菌屬乳酸菌可使於活體外所共同培養之大腸細胞中之HSPA1A的表現量相較於未添加前述乳酸桿菌屬乳酸菌所培養之大腸細胞 中之HSPA1A的表現量而言增加至較佳為1.5倍以上,更佳為1.7倍以上,再佳為1.8倍以上,再佳為1.9倍以上,再佳為2.0倍以上,再佳為2.5倍以上,再佳為3.0倍以上,再佳為3.5倍以上,再更佳為4.0倍以上。 According to a preferred embodiment of the present invention, the Lactobacillus lactic acid bacteria can express the amount of HSPA1A in the large intestine cells co-cultured in vitro compared to the large intestine cells cultured without the addition of the Lactobacillus lactic acid bacteria. In the performance of HSPA1A, it is preferably 1.5 times or more, more preferably 1.7 times or more, more preferably 1.8 times or more, more preferably 1.9 times or more, more preferably 2.0 times or more, and then preferably 2.5 times. The above is preferably 3.0 times or more, more preferably 3.5 times or more, and even more preferably 4.0 times or more.
根據本發明之更佳態樣,乳酸桿菌屬乳酸菌可使於活體外所共同培養之大腸細胞中之HSPA1A的表現量(藉由除以管家基因(GAPDH)的表現量而修正實驗組間之偏差所得之值)相較於未添加前述乳酸桿菌屬乳酸菌所培養之大腸細胞中之HSPA1A的表現量(藉由除以管家基因(GAPDH)的表現量而修正實驗組間之偏差所得之值)而言增加至較佳為1.5倍以上,更佳為1.7倍以上,再佳為1.8倍以上,再佳為1.9倍以上,再佳為2.0倍以上,再佳為2.5倍以上,再佳為3.0倍以上,再佳為3.5倍以上,再更佳為4.0倍以上。 According to a more preferred aspect of the present invention, the Lactobacillus lactic acid bacteria can express the amount of HSPA1A in the large intestine cells co-cultured in vitro (by dividing the expression of the housekeeping gene (GAPDH) to correct the deviation between the experimental groups The value obtained is compared with the amount of HSPA1A in the large intestine cells cultured without the addition of the aforementioned Lactobacillus lactic acid bacteria (the value obtained by dividing the deviation between the experimental groups by the expression amount of the housekeeping gene (GAPDH)) It is preferably 1.5 times or more, more preferably 1.7 times or more, more preferably 1.8 times or more, more preferably 1.9 times or more, more preferably 2.0 times or more, more preferably 2.5 times or more, and then preferably 3.0 times. The above is preferably 3.5 times or more, and more preferably 4.0 times or more.
HSP70的表現增強活性之測定中所使用之大腸細胞,如本案說明書之實施例之例1所記載,較佳為Caco-2細胞。 The colorectal cells used for the measurement of the activity-enhancing activity of HSP70 are preferably Caco-2 cells as described in Example 1 of the examples of the present specification.
根據本發明之更佳態樣,乳酸桿菌屬乳酸菌係例如為約氏乳酸桿菌、酪蛋白乳酸桿菌、加氏乳酸桿菌、瑞士乳酸桿菌(Lactobacillus helveticus)等,較佳為約氏乳酸桿菌(L.johnsonii)。且,乳酸桿菌屬乳酸菌較佳為生菌體,藉由攝取此等,腸內環境係獲得改善,可改善排便的次數、便量、其形狀、顏色或臭味等便性。 According to a more preferred aspect of the present invention, the Lactobacillus lactic acid bacteria are, for example, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus kawaii, Lactobacillus helveticus, etc., preferably Lactobacillus johnsonii (L. Johnsonii). Further, the Lactobacillus lactic acid bacterium is preferably a bacterium, and by ingesting, the intestinal environment is improved, and the number of times of defecation, the amount of stool, the shape, color, or odor can be improved.
約氏乳酸桿菌為革蘭氏陽性的桿菌,以同型 乳酸發酵之形式,在好氧條件下具有發育性。且,約氏乳酸桿菌,若將其塗佈於MRS瓊脂培養基(BD)平板上,在使用AnaeroPack Kenki(三菱氣體化學)之厭氧的條件下於37℃以48小時進行培養,則會形成圓形、白色、扁平狀菌落。此外,約氏乳酸桿菌在有效地抑制大腸炎上可特別有利地加以使用。 Lactobacillus johnsonii is a Gram-positive bacillus The form of lactic acid fermentation is developmental under aerobic conditions. Further, Lactobacillus johnsonii was applied to an MRS agar medium (BD) plate and cultured at 37 ° C for 48 hours under anaerobic conditions using AnaeroPack Kenki (Mitsubishi Gas Chemical) to form a circle. Shaped, white, flat colonies. Further, Lactobacillus johnsonii can be used particularly advantageously in effectively inhibiting colitis.
約氏乳酸桿菌較佳為約氏乳酸桿菌OLL203565株、約氏乳酸桿菌OLL204255株、約氏乳酸桿菌OLL2978株,更佳為約氏乳酸桿菌OLL203565株、約氏乳酸桿菌OLL204255株,再佳為約氏乳酸桿菌OLL203565株。此等可使用單獨1種作為有效成分,亦可使用混合有2種以上者作為有效成分。 The Lactobacillus johnsonii is preferably Lactobacillus johnsonii OLL203565 strain, Lactobacillus johnsonii OLL204255 strain, Lactobacillus johnsonii OLL2978 strain, more preferably Lactobacillus johnsonii OLL203565 strain, Lactobacillus johnsonii OLL204255 strain, and more preferably Yokogawa Lactobacillus OLL203565 strain. These may be used alone as one active ingredient, or two or more types may be used as an active ingredient.
約氏乳酸桿菌OLL203565株係於2015年2月3日以受託編號為NITE BP-02003(識別的標示:Lactobacillus johnsonii OLL203565)之形式寄存於日本獨立行政法人製品評估技術基礎機構專利微生物寄存中心(〒292-0818千葉縣木更津市上總鎌足2-5-8 122號室)。 The Lactobacillus johnsonii OLL203565 strain was deposited on the 3rd of February 2015 with the entrusted number NITE BP-02003 (identified by the logo: Lactobacillus johnsonii OLL203565) in the Patent Microbiology Depository Center of the Japan Independent Administrative Corporation Product Evaluation Technology Foundation. 292-0818 Kisatsuzu City, Chiba Prefecture, 2-5-8 Room 122.
約氏乳酸桿菌OLL204255株係於2015年2月3日以受託編號為NITE BP-02004(識別的標示:Lactobacillus johnsonii OLL204255)之形式寄存於日本獨立行政法人製品評估技術基礎機構專利微生物寄存中心(〒292-0818千葉縣木更津市上總鎌足2-5-8 122號室)。 The Lactobacillus johnsonii OLL204255 strain was deposited on February 3, 2015 in the form of a license number NITE BP-02004 (identified by the logo: Lactobacillus johnsonii OLL204255) at the Patent Microbiology Depository Center of Japan's Independent Administrative Corporation's Product Evaluation Technology Foundation. 292-0818 Kisatsuzu City, Chiba Prefecture, 2-5-8 Room 122.
約氏乳酸桿菌OLL2978株係於2015年2月3日以受託編號為NITE BP-02002(識別的標示:Lactobacillus johnsonii OLL2978)之形式寄存於日本獨立行政法人製品評估技術基礎機構專利微生物寄存中心(〒292-0818千葉縣木更津市上總鎌足2-5-8 122號室)。 The Lactobacillus johnsonii OLL2978 strain was deposited on the 3rd of February, 2015 in the form of the trustee number NITE BP-02002 (identified by the logo: Lactobacillus johnsonii OLL2978) at the Patent Microbiology Depository Center of Japan's independent administrative agency product evaluation technology infrastructure. 292-0818 Kisatsuzu City, Chiba Prefecture, 2-5-8 Room 122.
本發明之大腸炎抑制劑可依原樣使用乳酸桿菌屬乳酸菌,亦可與乳酸桿菌屬乳酸菌共同地併用經口上可容許之其他成分。 The colitis inhibitor of the present invention may be a Lactobacillus lactic acid bacterium as it is, or may be used in combination with other lactic acid bacteria of Lactobacillus.
經口上可容許之其他成分,在不會妨礙本發明之效果之前提下,並無特別限定,例如為賦形劑、安定劑、防腐劑、濕潤劑、乳化劑、潤滑劑、甜味料、著色料、香料、緩衝劑、抗氧化劑、pH調整劑等添加劑、或內科療法用之藥劑等。 The other components which can be tolerated by the mouth are not particularly limited, and are not particularly limited, and are, for example, excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, An additive such as a coloring matter, a fragrance, a buffer, an antioxidant, a pH adjuster, or a medicine for medical therapy.
本發明之大腸炎抑制劑的形態並無特別限制,可為粉末狀、錠劑狀、膠囊狀或飲劑狀。 The form of the colitis inhibitor of the present invention is not particularly limited, and may be in the form of a powder, a tablet, a capsule or a drink.
本發明之大腸炎抑制劑的形態較佳係以乳酸桿菌屬乳酸菌成為用於抑制大腸炎之有效量之方式,由1次的攝取量之單位所構成。本發明之大腸炎抑制劑之1次的攝取量之單位較佳係以1×107~1×109CFU/ml,且以100ml以上,更佳係以200ml以上含有乳酸桿菌屬乳酸菌。 The form of the colitis inhibitor of the present invention is preferably one in which the amount of the lactic acid bacteria of the genus Lactobacillus is an effective amount for suppressing colitis. The unit of the primary inoculation amount of the colitis inhibitor of the present invention is preferably 1 × 10 7 to 1 × 10 9 CFU/ml, and more preferably 100 ml or more, more preferably 200 ml or more, of Lactobacillus lactic acid bacteria.
本發明之大腸炎抑制劑的形態較佳係以每1次的攝取量之單位進行包裝而提供。本發明之大腸炎抑制劑之每1次的攝取量之單位包裝的形態係可列舉以軟包 裝、塑膠容器等包材規定一定量之形態,在該等包材等之表面,亦可附有每1次的攝取量之成分標示、或抑制大腸炎等之用途標示。該種單位包裝的形態可適當地例示補充品、醫藥製劑等。 The form of the colitis inhibitor of the present invention is preferably provided by packaging in units of intake per one time. The form of the unit package for each intake of the colitis inhibitor of the present invention is exemplified by a soft pack. A packaging material such as a plastic container or the like is provided in a predetermined amount, and the surface of the packaging material or the like may be marked with a component indicating the amount of intake per one time or a use index for suppressing colitis. The form of the unit package can be appropriately exemplified as a supplement, a pharmaceutical preparation, or the like.
本發明之大腸炎抑制劑的形態亦可作成緩釋性製劑。將本發明之大腸炎抑制劑的形態作成緩釋性製劑,就在經口攝取乳酸桿菌屬乳酸菌之後,使其活著到達大腸細胞之方面而言係較佳。 The form of the colitis inhibitor of the present invention can also be used as a sustained release preparation. The form of the colitis inhibitor of the present invention is preferably a sustained-release preparation, and is preferably in the form of orally ingesting Lactobacillus lactic acid bacteria to reach the large intestinal cells.
本發明之大腸炎抑制劑可將乳酸桿菌屬乳酸菌依所期望與經口上容許之其他成分共同進行混合而簡易地進行製造。從而,根據本發明之另一態樣,係提供大腸炎抑制劑之製造方法,其特徵為使其含有具有大腸細胞中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌。 The colitis inhibitor of the present invention can be easily produced by mixing Lactobacillus lactic acid bacteria as desired with other components which are orally allowed. Thus, according to another aspect of the present invention, there is provided a method for producing a colitis inhibitor, which comprises a Lactobacillus lactic acid bacterium having an expression-enhancing activity of HSP70 in a large intestinal cell.
本發明之製造方法亦可包含將乳酸桿菌屬乳酸菌與大腸細胞共同於活體外進行培養,並選擇大腸細胞中之HSP70的表現量高於預先設定之閾值的乳酸桿菌屬乳酸菌之步驟。 The production method of the present invention may further comprise the step of culturing the Lactobacillus lactic acid bacterium together with the large intestinal cells in vitro, and selecting a Lactobacillus lactic acid bacterium having a higher expression level of HSP70 in the large intestinal cells than a predetermined threshold.
在前述進行選擇之步驟中,測定HSP70的表現量之方法並無特別限制,可使用直接或間接地測定HSP70的表現量之手法。作為該種測定方法,可列舉例如即時PCR、北方墨點法(Northern blot)、RNA定序等。 In the above-described step of selecting, the method of measuring the amount of expression of HSP70 is not particularly limited, and a method of directly or indirectly measuring the amount of expression of HSP70 can be used. Examples of such a measurement method include real-time PCR, Northern blot, and RNA sequencing.
此外,在前述進行選擇之步驟中,預先設定之閾值,在不會妨礙本發明之效果之前提下,並無特別限定,可設為例如未添加乳酸桿菌屬乳酸菌所培養之大腸細 胞中之HSP70的表現量。該種HSP70的表現量之閾值,如本案說明書之實施例之例1所記載,亦可以HSPA1A的表現量作為指標而予以測定。再者,HSPA1A的表現量亦可以大腸細胞中之管家基因的表現量作為基準而予以算出。作為該種管家基因,舉例而言,如本案說明書之實施例之例1所記載,較佳係使用GAPDH,但亦可使用次黃嘌呤磷酸核糖基轉移酶1(HPRT1)、β-肌動蛋白(ACTB)等。 Further, in the step of selecting the above, the threshold value set in advance is not particularly limited, and is not particularly limited, and may be, for example, a large intestine finely cultured without adding Lactobacillus lactic acid bacteria. The amount of HSP70 in the cell. The threshold value of the amount of expression of the HSP70 can be measured by using the amount of expression of HSPA1A as an index as described in Example 1 of the embodiment of the present specification. Furthermore, the amount of expression of HSPA1A can also be calculated based on the amount of expression of the housekeeping gene in the large intestine cells. As such a housekeeping gene, for example, as described in Example 1 of the present specification, GAPDH is preferably used, but hypoxanthine phosphoribosyltransferase 1 (HPRT1), β-actin may also be used. (ACTB) and so on.
再者,在前述進行選擇之步驟中,將乳酸桿菌屬乳酸菌與大腸細胞共同進行培養之方法,在不會妨礙本發明之效果之前提下,並無特別限定,只要是熟習該項技術者即可適宜設定。作為該種培養條件(溫度、培養基、培養時間),舉例而言,在大腸細胞為Caco-2細胞之情況,可列舉37℃、高葡萄糖Dulbecco修飾Eagle培養基、於CO2環境下、24小時。 Further, in the step of selecting the above, the method of culturing the Lactobacillus lactic acid bacterium together with the large intestine cells is not particularly limited as long as the effect of the present invention is not impaired, as long as it is familiar to the skilled person. Can be set as appropriate. As such a culture condition (temperature, medium, culture time), for example, when the large intestine cells are Caco-2 cells, a high glucose Dulbecco-modified Eagle medium at 37° C. and a CO 2 atmosphere are used for 24 hours.
根據本發明之另一態樣,可提供就本發明之大腸炎抑制劑而言,包含大腸炎抑制劑而成之組成物。即,就本發明之大腸炎抑制劑而言,雖然亦可依原樣單獨使用,但在可顯現出大腸炎抑制機能之前提下,對於食品或醫藥品等各種經口攝取用(經口投予用)之組成物而言,亦可使其作為原料(素材)或添加劑等包含在內,便可獲得具有大腸炎抑制效果之組成物。因此,根據本發明之一實施態樣,係提供包含本發明之大腸炎抑制劑而成之組成物。 According to another aspect of the present invention, a composition comprising a colitis inhibitor for the colitis inhibitor of the present invention can be provided. In other words, the colitis inhibitor of the present invention can be used alone as it is, but it can be used for various oral ingestions such as foods and pharmaceuticals before it can exhibit colitis suppression function (oral administration) The composition of the composition may be obtained as a raw material (material) or an additive, and a composition having a colitis-inhibiting effect can be obtained. Therefore, according to an embodiment of the present invention, a composition comprising the colitis inhibitor of the present invention is provided.
根據本發明之一態樣,可提供以具有大腸細胞中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌作為有效成分之組成物。本發明之組成物可為食品組成物或醫藥組成物。該種態樣可按照上述之大腸炎抑制劑之記載予以實施。 According to an aspect of the present invention, a composition of Lactobacillus lactic acid bacteria having an activity-enhancing activity of HSP70 in large intestinal cells as an active ingredient can be provided. The composition of the present invention may be a food composition or a pharmaceutical composition. This aspect can be carried out in accordance with the description of the above-mentioned colitis inhibitor.
食品組成物係指醫藥組成物以外者,只要是溶液、懸浮液、乳濁液、粉末、固體成形物等可經口攝取之形態即可,並無特別限定。具體而言,可列舉例如即食麵、殺菌袋裝食品、罐頭、微波爐食品、即食湯品/醬湯類、冷凍乾燥食品等即時食品類;清涼飲料、果汁飲料、蔬菜飲料、豆乳飲料、咖啡飲料、茶飲料、粉末飲料、濃縮飲料、酒精飲料等飲料類;麵包、義大利麵、麵、蛋糕混料粉、麵包粉等小麥粉製品;飴糖、焦糖、口香糖、巧克力、甜餅乾、軟餅、蛋糕、餡餅、零食、薄脆餅乾、日式點心、甜點心等點心類;醬汁、蕃茄加工調味料、風味調味料、調理混合料、佐料汁類、沙拉醬類、羹湯類、咖哩/燉物之原料類等調味料;加工油脂、奶油、人造奶油、美乃滋等油脂類;乳飲料、優格類、乳酸菌飲料、冰淇淋類、鮮奶油類等乳製品;農產罐頭、果醬/柑橘醬類、穀片等農產加工品;冷凍食品等。食品係較佳為乳製品,更佳為乳飲料、優格類、乳酸菌飲料或冰淇淋類。 The food composition is not particularly limited as long as it is a form which can be orally ingested, such as a solution, a suspension, an emulsion, a powder, or a solid molded product. Specific examples include instant noodles such as instant noodles, sterilized bagged foods, canned foods, microwave foods, ready-to-eat soups/sauces, and freeze-dried foods; refreshing drinks, juice drinks, vegetable drinks, soy milk drinks, and coffee drinks. , tea drinks, powder drinks, concentrated drinks, alcoholic beverages and other beverages; bread, pasta, noodles, cake mix powder, bread flour and other wheat flour products; sugar, caramel, chewing gum, chocolate, sweet biscuits, soft cakes , cakes, pies, snacks, crackers, Japanese snacks, desserts and other snacks; sauces, tomato processing seasonings, flavorings, conditioning mixes, sauces, salad dressings, soups, Seasonings such as raw materials such as curry and stew; processing oils and fats, butter, margarine, mayonnaise and other oils; dairy beverages, yogurt, lactic acid bacteria beverages, ice creams, fresh creams, etc.; canned food, jam /Agricultural processed products such as citrus sauces and cereals; frozen foods, etc. The food product is preferably a dairy product, more preferably a milk beverage, a yogurt, a lactic acid bacteria beverage or an ice cream.
此外,在食品中,亦包含諸如健康食品(包含補充品)、機能性食品、營養補助食品、機能性標示食品、特定保健用食品、病者用食品、乳幼兒用調整奶粉、 妊產婦或授乳婦用奶粉、或附有減低疾病風險之標示的食品之分類者。 In addition, foods such as health foods (including supplements), functional foods, nutritional supplements, functional foods, specific health foods, foods for patients, milk powder for infants, A classification of foods for pregnant or maternal milk, or foods labeled with a reduced risk of disease.
根據本發明之較佳態樣,係提供以具有大腸細胞中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌作為有效成分之大腸炎抑制用乳製品,較佳為大腸炎抑制用乳飲料、優格類、乳酸菌飲料或冰淇淋類。該種態樣可按照上述之大腸炎抑制劑之記載予以實施。 According to a preferred aspect of the present invention, there is provided a lactose-inhibiting dairy product comprising Lactobacillus lactic acid bacteria having an activity-enhancing activity of HSP70 in a large intestinal cell as an active ingredient, preferably a milk for colitis suppression, and a yogurt Class, lactic acid bacteria drink or ice cream. This aspect can be carried out in accordance with the description of the above-mentioned colitis inhibitor.
醫藥組成物係指併用為了製劑化可容許之添加劑,並依照常法調製為經口製劑或非經口製劑者。在經口製劑之情況,可採取錠劑、散劑、細粒劑、顆粒劑、膠囊劑、丸劑、緩釋劑等固形製劑、溶液、懸浮液、乳濁液等液狀製劑之形態。此外,在非經口製劑之情況,可採取注射劑或栓劑之形態。從簡易性之方面而言,較佳為經口製劑。作為為了製劑化可容許之添加劑,可列舉例如賦形劑、安定劑、防腐劑、濕潤劑、乳化劑、潤滑劑、甜味料、著色料、香料、緩衝劑、抗氧化劑、pH調整劑等。在本發明之醫藥組成物中,係包含用於需要抑制大腸炎之疾病(例如炎症性腸疾病、貝賽特氏症、缺血性腸炎及感染症性腸炎)之醫藥組成物,更具體而言,預防用組成物及治療用組成物。 The pharmaceutical composition refers to a combination of an oral preparation or a non-oral preparation prepared in accordance with a conventional method in order to prepare an acceptable additive. In the case of the oral preparation, a solid preparation such as a tablet, a powder, a fine granule, a granule, a capsule, a pill, or a sustained release agent, or a liquid preparation such as a solution, a suspension or an emulsion may be used. Further, in the case of a parenteral preparation, an injection or a suppository may be employed. From the standpoint of simplicity, an oral preparation is preferred. Examples of the additives which can be tolerated for formulation include excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, coloring materials, perfumes, buffers, antioxidants, pH adjusters, and the like. . The pharmaceutical composition of the present invention comprises a pharmaceutical composition for a disease in which colitis is required to be suppressed (for example, inflammatory bowel disease, Behst's disease, ischemic enteritis, and infectious enteritis), and more specifically The composition for prevention and the composition for treatment.
此外,根據本發明之另一態樣,係提供大腸炎之抑制方法,其係包含使對此有所需要的對象攝取具有大腸細胞中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌的有效量。此處,在「抑制」中,不僅包含治療已確立 之病態,亦包含預防在將來有確立之可能性的病態之概念。再者,在本發明之「抑制」中,亦包含非治療性概念,例如改善或緩和起因於大腸中之炎症或與其相關連之症狀或狀態。從而,根據另一態樣,抑制大腸炎係改善或緩和起因於大腸中之炎症或與其相關連之症狀或狀態,較佳為非治療性。 Further, according to another aspect of the present invention, there is provided a method for inhibiting colitis which comprises an effective amount of a Lactobacillus lactic acid bacterium having a performance enhancing activity of HSP70 in a large intestinal cell for a subject in need thereof. Here, in "suppression", not only contains treatment has been established The morbid state also includes the concept of morbidity that prevents the possibility of establishing it in the future. Furthermore, in the "inhibition" of the present invention, non-therapeutic concepts are also included, such as improving or alleviating the symptoms or conditions associated with or associated with inflammation in the large intestine. Thus, according to another aspect, it is preferred that the colitis is inhibited from ameliorating or alleviating inflammation or associated symptoms or conditions associated with the large intestine, preferably non-therapeutic.
大腸炎之抑制方法中,可將乳酸桿菌屬乳酸菌的有效量與上述之大腸炎抑制劑中之1次的攝取量之單位同樣地進行設定或調整。 In the method for inhibiting colitis, the effective amount of the lactic acid bacterium of the genus Lactobacillus can be set or adjusted in the same manner as the unit of the intake amount of the colitis inhibitor described above.
使對此有所需要的對象攝取乳酸桿菌屬乳酸菌的有效量之方法,在不會妨礙本發明之效果之前提下,並無特別限定,例如為經口攝取或經腸攝取等,較佳為經口攝取。 The method of ingesting an effective amount of the Lactobacillus lactic acid bacterium for a subject in need thereof is not particularly limited, and is not particularly limited, and is, for example, oral ingestion or enteral ingestion, etc., preferably Ingested by mouth.
本發明之乳酸桿菌屬乳酸菌之攝取計畫並無特別限定,配合對象的年齡、性別、症狀及狀態,熟習該項技術者可適宜設定。對於本發明之對象,可例如以每1日1~10次投予1次的經口攝取量之單位,較佳為以每1日1~3次進行投予,更佳為以每1日1次進行投予。此外,本發明之大腸炎抑制劑可在進食之同時進行攝取。 The ingestion scheme of the Lactobacillus lactic acid bacteria of the present invention is not particularly limited, and the age, sex, symptoms, and state of the subject may be appropriately set by those skilled in the art. The object of the present invention can be administered, for example, in units of oral intake of 1 to 10 times per day, preferably 1 to 3 times per day, more preferably every 1 day. The administration was performed once. Further, the colitis inhibitor of the present invention can be ingested while eating.
作為本發明之大腸炎,係例如為炎症性腸疾病、貝賽特氏症、缺血性腸炎及感染症性腸炎,較佳為炎症性腸疾病(潰瘍性大腸炎、克隆氏病),更佳為潰瘍性大腸炎。 The colitis of the present invention is, for example, inflammatory bowel disease, Behst's disease, ischemic enteritis, and infectious enteritis, preferably inflammatory bowel disease (ulcerative colitis, Crohn's disease), and more Good for ulcerative colitis.
作為本發明之對象,係為哺乳動物,例如囓 齒類、犬、貓、牛、靈長類、人類,較佳為人類,更佳為患有大腸炎之患者。此外,本發明之對象亦可為健康正常者。作為該種健康正常者之例,較佳為儘管並未被診斷為患有大腸炎之患者,但具有起因於大腸中之炎症或與其相關連之症狀或狀態、或者有產生該等症狀或狀態之疑懼者。另外,是否為患有大腸炎之患者,舉例而言,潰瘍性大腸炎係可基於Katsuyoshi Matsuoka and Tsung-Chun Lee,Guidelines for the Management of ulcerative colitis in Japan,IBP Research 4:189-239,2010(特定而言,參照(14)頁之2、(16)頁之圖1)等,克隆氏病係可基於Fumiaki Ueno et.al.Evidence-based clinical practice guidelines for Crohn’s disease,integrated with formal consensus of experts in Japan,J Gastroenterol(2013)48:31-72(特定而言,參照38頁之II、43頁之II-8、44頁之表5)等公知的診斷方法進行診斷。 As a subject of the present invention, it is a mammal, such as a bite Teeth, dogs, cats, cows, primates, humans, preferably humans, more preferably patients with colitis. Further, the object of the present invention may also be a healthy person. As an example of such a healthy person, it is preferred that although it is not diagnosed as a patient suffering from colitis, it has symptoms or conditions resulting from or associated with inflammation in the large intestine, or is caused by such symptoms or states. Suspected. In addition, whether it is a patient suffering from colitis, for example, the ulcerative colitis system can be based on Katsuyoshi Matsuoka and Tsung-Chun Lee, Guidelines for the Management of ulcerative colitis in Japan, IBP Research 4: 189-239, 2010 (specific For example, refer to page 2 of page (14), Figure 1) of page 16 (16), etc., and the Crohn's disease system can be based on Fumiaki Ueno et. al. Evidence-based clinical practice guidelines for Crohn's disease, integrated with formal consensus of experts in Japan, J Gastroenterol (2013) 48: 31-72 (specifically, refer to page 38 of II, page 43 of II-8, page 44 of Table 5) and other known diagnostic methods for diagnosis.
根據本發明之一態樣,係提供具有大腸細胞中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌之用途,其係用在大腸炎抑制劑的製造中。此外,根據本發明之一態樣,係提供具有大腸細胞中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌用於抑制大腸炎之用途。根據本發明之一較佳態樣,該用途係非治療性用途。該種用途可按照上述之大腸炎抑制劑及大腸炎之抑制方法之記載予以實施。 According to an aspect of the present invention, there is provided a use of a Lactobacillus lactic acid bacterium having an expression-enhancing activity of HSP70 in a large intestinal cell, which is used in the manufacture of a colitis inhibitor. Further, according to an aspect of the present invention, there is provided a use of a Lactobacillus lactic acid bacterium having an expression-enhancing activity of HSP70 in a large intestinal cell for inhibiting colitis. According to a preferred aspect of the invention, the use is for non-therapeutic use. Such use can be carried out in accordance with the above-described methods for inhibiting colitis inhibitors and colitis.
根據本發明之一態樣,係提供具有大腸細胞 中之HSP70的表現增強活性之乳酸桿菌屬乳酸菌,其係用於抑制大腸炎。該種態樣可按照上述之大腸炎抑制劑之記載予以實施。 According to one aspect of the invention, a large intestinal cell is provided The Lactobacillus lactic acid bacteria whose activity enhances the activity of HSP70 is used for inhibiting colitis. This aspect can be carried out in accordance with the description of the above-mentioned colitis inhibitor.
藉由以下之例詳細地說明本發明,但本發明並不限定於此。 The present invention will be described in detail by the following examples, but the invention is not limited thereto.
〈例1:HSPA1A基因之表現誘導試驗(活體外試驗)〉 <Example 1: Expression induction test of HSPA1A gene (in vitro test)>
(1)生菌懸浮液及Caco-2細胞之調製 (1) Modulation of bacterial suspension and Caco-2 cells
將約氏乳酸桿菌OLL2978株、約氏乳酸桿菌OLL203565株、約氏乳酸桿菌OLL204255株使用Difco(商標)Lactobacilli MRS broth培養基(BD),在37℃、厭氧條件下以1次繼代進行培養。接著,將各菌進行離心分離處理(8,000rpm,5分鐘)並使其沉澱,以磷酸緩衝液(PBS,Phosphate Buffered Saline)洗淨後,使用分光光度計(U-2810 Spectrophotometer,HITACHI),於波長650nm測定濁度(OD)。此處,PBS係將PBS(Phosphate Buffered Salts)錠片(TaKaRa公司)溶解於蒸餾水中而予以調製。然後,使用PBS,以各菌成為OD=5之方式進行調整,調製生菌懸浮液。 Lactobacillus johnsonii OLL2978 strain, Lactobacillus johnsonii OLL203565 strain, and Lactobacillus johnsonii OLL204255 strain were cultured in a subculture at 37 ° C under anaerobic conditions using Difco (trademark) Lactobacilli MRS broth medium (BD). Next, each of the bacteria was subjected to centrifugation (8,000 rpm, 5 minutes), precipitated, washed with a phosphate buffer (PBS, Phosphate Buffered Saline), and then subjected to a spectrophotometer (U-2810 Spectrophotometer, HITACHI). The turbidity (OD) was measured at a wavelength of 650 nm. Here, PBS was prepared by dissolving PBS (Phosphate Buffered Salts) tablets (TaKaRa Co., Ltd.) in distilled water. Then, using PBS, the bacteria were adjusted so that each of the bacteria became OD=5, and the suspension of the bacteria was prepared.
將屬源自人類結腸癌之細胞的Caco-2細胞(DS Pharma Biomedical公司)使用以成為10% FBS、2mM麩醯胺酸之方式摻合而得之高葡萄糖Dulbecco修飾 Eagle培養基(Dulbecco’s Modified Eagle’s Medium-high glucose,D6546,Sigma-Aldrich公司),在37℃、5% CO2之環境下(NAPCO Series 5400 CO2 Incubator,Thermo Scientific)進行培養。 Caco-2 cells (DS Pharma Biomedical), which are cells derived from human colon cancer, were mixed with high glucose Dulbecco's Modified Eagle's Medium using 10% FBS, 2 mM glutamic acid. -high glucose, D6546, Sigma-Aldrich), cultured at 37 ° C, 5% CO 2 (NAPCO Series 5400 CO 2 Incubator, Thermo Scientific).
在投予生菌懸浮液之前日,以成為1.0×105個細胞/孔之方式,將Caco-2細胞繼代於6孔盤(TPP公司)中。 Caco-2 cells were subcultured into 6-well plates (TPP) in a manner of 1.0 × 10 5 cells/well on the day before administration of the suspension of the bacteria.
(2)HSP基因之表現誘導試驗 (2) HSP gene expression induction test
將生菌懸浮液以20μl/孔添加至前述包含繼代後之Caco-2細胞之6孔盤中,在37℃、5% CO2之環境下以24小時進行共培養。在結束此共培養後,緊接著將此等培養液(培養基)吸引去除,於其中添加RNA later(Ambion公司),浸漬細胞。藉由NucleoSpin RNA II(MACHEREY-NAGEL)自此等細胞萃取出RNA之後,使用PrimeScript RT Master Mix(Perfect Real Time,TaKaRa公司)進行反轉錄反應。使用SYBR Premix Ex Taq II(Tli RNaseH Plus,TaKaRa公司),藉由Thermal Cycler Dice Real Time System TP-800(TaKaRa),進行即時PCR,並進行Caco-2細胞之HSPA1A基因的表現解析。 The bacterial suspension was added to the aforementioned 6-well plate containing subcultured Caco-2 cells at 20 μl/well, and co-culture was carried out at 37 ° C under a 5% CO 2 atmosphere for 24 hours. After the completion of the co-culture, the culture solution (medium) was suction-removed, and RNA later (Ambion) was added thereto to impregnate the cells. After extracting RNA from these cells by NucleoSpin RNA II (MACHEREY-NAGEL), reverse transcription reaction was carried out using PrimeScript RT Master Mix (Perfect Real Time, TaKaRa). Real-time PCR was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus, TaKaRa) by Thermal Cycler Dice Real Time System TP-800 (TaKaRa), and the expression analysis of the HSPA1A gene of Caco-2 cells was performed.
將即時PCR所使用之引子及反應液的組成分別示於表1及表2。 The primers used in the real-time PCR and the compositions of the reaction solutions are shown in Tables 1 and 2, respectively.
即時PCR之反應條件係設為(95℃,30秒)→(95℃,5秒→60℃,30秒)×40個週期→95℃,15秒→60℃,30秒→95℃,15秒→4℃。 The reaction conditions of the real-time PCR were set to (95 ° C, 30 seconds) → (95 ° C, 5 seconds → 60 ° C, 30 seconds) × 40 cycles → 95 ° C, 15 seconds → 60 ° C, 30 seconds → 95 ° C, 15 Seconds → 4 ° C.
作為對照組,係使用投予PBS來代替生菌懸浮液並進行共培養而得者。 As a control group, PBS was used instead of the suspension of the bacteria and co-culture was carried out.
將結果示於圖1。 The results are shown in Fig. 1.
如圖1所示,相較於對照組而言,在約氏乳酸桿菌之生菌之投予組中,可見到HSPA1A基因的表現上升。 As shown in Fig. 1, the performance of the HSPA1A gene was observed to be increased in the administration group of the bacterium of Lactobacillus johnsonii compared to the control group.
〈例2:活體內試驗〉 <Example 2: In vivo test>
將約氏乳酸桿菌OLL203565株投予至小鼠,藉由動物實驗檢討對葡聚糖硫酸鈉(DSS,分子量:36,000~ 50,000Da,MP Biomedicals公司)所誘發之大腸炎之抑制效果。 Lactobacillus johnsonii OLL203565 strain was administered to mice, and the dextran sulfate sodium (DSS, molecular weight: 36,000~) was reviewed by animal experiments. 50,000 Da, MP Biomedicals) induced colitis suppression.
就生菌懸浮液而言,係將約氏乳酸桿菌OLL203565株1×108cfu懸浮於PBS 200μl中而予以調製。接著,藉由餵食管每日強制經口投予此生菌懸浮液。另外,將DSS水設為自由飲水。 In the case of the bacterial suspension, 1 × 10 8 cfu of Lactobacillus johnsonii OLL203565 strain was suspended in 200 μl of PBS to prepare a suspension. Next, the suspension of the bacterium is orally administered by daily feeding through a feeding tube. In addition, the DSS water was set to free drinking water.
使用BALB/c小鼠(4週齡,雄性,Charles River公司),將各組設為各10隻。自DSS水(2.0 w/v%)的投予之1週前起,對小鼠每日投予生菌懸浮液。將DSS水的投予之首日設為第1日,在1週內(直至DSS水的投予後之第7日)每日投予DSS水及生菌懸浮液之混合物。 BALB/c mice (4 weeks old, male, Charles River) were used, and each group was set to 10 each. The mice were administered a suspension of the bacteria daily from 1 week before the administration of DSS water (2.0 w/v%). The first day of the administration of DSS water was set to the first day, and the mixture of DSS water and the suspension of the bacteria was administered daily for one week (until the seventh day after the administration of DSS water).
作為對照組,係使用投予蒸餾水來代替DSS水者、投予PBS來代替生菌懸浮液者、以及投予蒸餾水及PBS來代替DSS及生菌懸浮液者(參照圖2)。 As a control group, distilled water was used instead of DSS water, PBS was added instead of the bacterial suspension, and distilled water and PBS were added instead of DSS and the bacterial suspension (see Fig. 2).
在DSS之投予期間中,每日記錄小鼠的體重之測定值及炎症評分。 During the administration of DSS, the measured values of the body weight of the mice and the inflammation score were recorded daily.
在炎症評分中,係附有下痢及血便的評分,依照表3,以0至3對評分進行評估。 In the inflammatory score, the scores of the lower jaw and the blood stool were attached, and according to Table 3, the scores were evaluated from 0 to 3.
在DSS的投予期間結束後(第8日),將小鼠進行解剖,摘取大腸,測定其長度及重量。針對將下痢及血便的評分之合計加以平均作為解剖時之炎症評分者,在DSS之投予組間進行比較。 After the end of the DSS administration period (Day 8), the mice were dissected, the large intestine was removed, and the length and weight were measured. The average of the scores of the lower jaw and the bloody stool was averaged as the inflammation score at the time of dissection, and the comparison was made between the DSS administration groups.
就小鼠的體重、大腸的長度/大腸的每單位長度之重量而言,係以Tukey檢定在組間進行比較。就炎症評分而言,係以Mann-Whitney U檢定在組間進行比較。統計處理之結果,p<0.05時,係判斷為顯著。在圖表中,係以平均值±標準偏差(SD)表示。 The weight of the mouse, the length of the large intestine, and the weight per unit length of the large intestine were compared between groups by Tukey's assay. In terms of inflammation scores, comparisons were made between groups using the Mann-Whitney U assay. The results of the statistical treatment were judged to be significant at p < 0.05. In the graph, it is expressed as mean ± standard deviation (SD).
將各自之結果示於圖3~5。 The results are shown in Figures 3 to 5.
如圖3所示,可確認由於DSS所誘發之大腸炎,小鼠的體重係減少,而藉由投予約氏乳酸桿菌OLL203565株,小鼠的體重減少係受到抑制。另外,G1:蒸餾水+PBS之投予組、G2:蒸餾水+約氏乳酸桿菌OLL203565株之投予組、G3:DSS+PBS之投予組、G4:DSS+約氏乳酸桿菌OLL203565株之投予組。 As shown in Fig. 3, it was confirmed that the body weight of the mouse was reduced by DSS-induced colitis, and the body weight loss of the mice was inhibited by administration of the Lactobacillus johnsonii OLL203565 strain. In addition, G1: distilled water + PBS administration group, G2: distilled water + Lactobacillus johnsonum OLL203565 strain administration group, G3: DSS + PBS administration group, G4: DSS + Lactobacillus johnsonii OLL203565 strain administration group .
如圖4所示,相較於DSS+PBS之投予組而言,在DSS+約氏乳酸桿菌OLL203565株之投予組中,炎症評分係顯著地減少。 As shown in Fig. 4, the inflammation score was significantly reduced in the administration group of DSS + Lactobacillus bulgaricus OLL203565 strain compared to the DSS + PBS administration group.
如圖5所示,在DSS之投予組中,大腸的長度係縮短。此外,相較於蒸餾水+PBS之投予組而言,在DSS+PBS之投予組中,每單位長度之重量係顯著地增加,而在DSS+約氏乳酸桿菌OLL203565株之投予組中,則並未見到顯著差異。 As shown in Fig. 5, in the DSS administration group, the length of the large intestine was shortened. In addition, the weight per unit length was significantly increased in the DSS + PBS administration group compared to the distilled water + PBS administration group, and in the DSS + Lactobacillus johnllii OLL203565 strain administration group, No significant differences were seen.
就結論而言,可確認藉由投予約氏乳酸桿菌OLL203565株之生菌,DSS所誘發之大腸炎係受到抑制。 As a result, it was confirmed that DSS-induced colitis was inhibited by administration of the bacterium of Lactobacillus johnsonii OLL203565 strain.
雖然未將數據示於此,但已確認在使用約氏乳酸桿菌OLL204255株來代替約氏乳酸桿菌OLL203565株之情況,亦與約氏乳酸桿菌OLL203565株同樣地,有DSS所誘發之大腸炎係受到抑制之傾向。 Although the data is not shown here, it has been confirmed that the Lactobacillus johnsonii OLL204255 strain is used instead of the Lactobacillus johnsonii OLL203565 strain, and similarly to the Lactobacillus johnsonii OLL203565 strain, DSS-induced colitis is affected. The tendency to suppress.
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國外生物材料【格式請依:寄存國家、機構、日期、號碼順序 Foreign biological materials [format please follow: deposit country, organization, date, number order
1.日本;獨立行政法人製品評價技術基盤機構特許 1. Japan; independent administrative legal person product evaluation technology base institution charter
微生物寄;2015/03/06;NITE BP-02002 Microbial delivery; 2015/03/06; NITE BP-02002
2.日本;獨立行政法人製品評價技術基盤機構特許 2. Japan; independent administrative legal person product evaluation technology base institution charter
微生物寄;2015/03/06;NITE BP-02003 Microbial delivery; 2015/03/06; NITE BP-02003
3.日本;獨立行政法人製品評價技術基盤機構特許 3.Japan; independent administrative legal person product evaluation technology base institution charter
微生物寄;2015/03/06;NITE BP-02004 Microbial delivery; 2015/03/06; NITE BP-02004
<110> 明治股份有限公司 <110> Meiji Co., Ltd.
<120> 大腸炎抑制劑 <120> colitis inhibitor
<130> 208829 <130> 208829
<160> 4 <160> 4
<170> PatentIn version 3.5 <170> PatentIn version 3.5
<210> 1 <210> 1
<211> 641 <211> 641
<212> PRT <212> PRT
<213> 智人(Homo sapiens) <213> Homo sapiens
<400> 1 <400> 1
<210> 2 <210> 2
<211> 2429 <211> 2429
<212> DNA <212> DNA
<213> 智人(Homo sapiens) <213> Homo sapiens
<400> 2 <400> 2
<210> 3 <210> 3
<211> 21 <211> 21
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 正向引子 <223> Forward introduction
<400> 3 <400> 3
<210> 4 <210> 4
<211> 24 <211> 24
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 反向引子 <223> Reverse primer
<400> 4 <400> 4
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