TWI405849B - Lactic acid bacteria having a function of lowering uric acid in blood - Google Patents
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本發明係有關於具有降低血液中尿酸值之作用的乳酸菌及其應用,此外,係有關於含有該等乳酸菌之預防及/或治療高尿酸血症的食品或醫藥品。The present invention relates to a lactic acid bacterium having an effect of lowering uric acid value in blood and an application thereof, and a food or a pharmaceutical product containing the lactic acid bacteria for preventing and/or treating hyperuricemia.
高尿酸血症是由於環境因素(生活習慣)及遺傳因素,使尿酸排泄降低或尿酸產生過剩,而形成血液中尿酸過高的狀態。高尿酸血症也可能無自覺症狀,而係由痛風、腎機能不全、尿路結石、動脈硬化症等嚴重合併症所引起。高尿酸血症之代表性合併症為痛風,其主症狀顯現為伴隨劇痛的急性關節炎。過去將痛風稱為「帝王病」,也因頻繁且多量攝取肉、魚、或酒精等而發病而稱之為「鋪張病」,近年來,由於飲食生活變化而有年年增加病例的傾向。現在的日本痛風患者數為30~40萬人,高尿酸血症患者數推測600萬人,因此大眾也高度關切高尿酸血症之預防或治療法。Hyperuricemia is caused by environmental factors (living habits) and genetic factors, which leads to a decrease in uric acid excretion or an excess of uric acid, and a state in which blood uric acid is too high. Hyperuricemia may also have no symptoms, but is caused by severe complications such as gout, renal insufficiency, urinary calculi, and arteriosclerosis. A representative complication of hyperuricemia is gout, and the main symptom appears as acute arthritis accompanied by severe pain. In the past, gout was called "imperial disease", and it was called "spreading disease" because of frequent and large intake of meat, fish, or alcohol. In recent years, there has been a tendency to increase cases every year due to changes in dietary life. The current number of gout patients in Japan is 300,000 to 400,000, and the number of patients with hyperuricemia is estimated to be 6 million. Therefore, the public is also highly concerned about the prevention or treatment of hyperuricemia.
高尿酸血症之預防或治療係以食療法、運動療法、醫藥品及上述之組合進行血液中尿酸值之控制。特別是,以限制卡路里攝取量為最常選擇的高尿酸血症預防或治療方法之一,然而要長期嚴格限制卡路里攝取量相當困難,曾發表過改善此狀況的方法有經口攝取能分解嘌呤(或俗稱普林,purine)的乳酸菌、酵母等微生物的醫藥品或飲 食品,於腸管內分解由飲食攝取的嘌呤,而使體內吸收減少,進而降低血清尿酸值(專利文獻1、非專利文獻1)。自古以來就常將乳酸菌應用於食品或醫藥品,其對人體的安全性也高,乳酸菌攝取也少有令人擔心的副作用,故其為治療或預防高尿酸血症的有效方法。此外,如上述,高尿酸血症之預防或治療法的第一選擇是食療法,經由食品攝取可控制尿酸值的乳酸菌,是極為實際且有用的新式高尿酸血症預防及/或治療法。然而,上述文獻所揭露能分解嘌呤的乳酸菌有發酵乳酸桿菌(Lactobacillus fermentum)、戊糖乳酸桿菌(Lactobacillus pentosus),這些乳酸菌會產氣,就飲食品或醫藥品的應用觀點上,並非適當的菌種。The prevention or treatment of hyperuricemia is the control of blood uric acid value by a combination of food therapy, exercise therapy, pharmaceuticals, and combinations thereof. In particular, it is one of the most frequently selected methods for preventing or treating hyperuricemia by limiting caloric intake. However, it is quite difficult to strictly limit calorie intake for a long time. It has been published that the method of improving this condition can be decomposed by oral intake. (or commonly known as purin), lactic acid bacteria, yeast, and other microbial medicines or drinks In the food, the sputum ingested by the diet is decomposed in the intestine, and the absorption in the body is reduced, and the serum uric acid value is lowered (Patent Document 1 and Non-Patent Document 1). Since ancient times, lactic acid bacteria have been frequently used in foods or pharmaceuticals, and their safety to humans is high. Lactic acid bacteria intake has few side effects, which is an effective method for treating or preventing hyperuricemia. Further, as described above, the first choice for the prevention or treatment of hyperuricemia is food therapy, and lactic acid bacteria which can control uric acid value through food intake are highly practical and useful novel hyperuricemia prevention and/or treatment methods. However, the lactic acid bacteria which can decompose sputum disclosed in the above documents include Lactobacillus fermentum and Lactobacillus pentosus. These lactic acid bacteria produce gas, and it is not an appropriate bacteria from the viewpoint of application of foods and drinks or pharmaceuticals. Kind.
專利文獻1:WO2004/112809Patent Document 1: WO2004/112809
非專利文獻1:日本農藝化學會首頁日本農藝化學會年度大會演講發表資料庫(http://isbba.bioweb.ne.jp/jsbba_db/index.html )「日本農藝化學會2004.03.30一般演講、池永武、久米村惠 他:乳酸菌對飲食性高尿酸血症模式大鼠之血液尿酸值所致之影響」Non-Patent Document 1: Japanese Agrochemical Society Homepage Agronomic Chemistry Annual Conference Speech Presentation Database ( http://isbba.bioweb.ne.jp/jsbba_db/index.html ) "National Agrochemical Society 2004.03.30 General Lecture, Chi Yongwu, Jiu Mi Cun Hui: The effect of lactic acid bacteria on blood uric acid value in rats with dietary hyperuricemia
有鑑於上述狀況,本發明所欲解決之課題即係提供適於飲食品或醫藥品用途、且可用於預防及/或治療高尿酸 血症之乳酸菌,同時亦提供使用上述乳酸菌之預防及/或治療高尿酸血症的組合物。In view of the above circumstances, the problem to be solved by the present invention is to provide a food or food product for use in prevention and/or treatment of high uric acid. The lactic acid bacteria of the bacterium also provides a composition for preventing and/or treating hyperuricemia using the above lactic acid bacteria.
為解決上述課題,本發明人等經過反覆努力研究。首先,於肌苷(inosine)及鳥苷(guanosine)存在下培養各種乳酸菌,測定上述核苷消費量及分解物(次黃嘌呤(hypoxanthine)、鳥嘌呤(guanine))生產量,並選擇具有顯著核苷分解能力之複數種乳酸菌。將基於上述選擇而判斷為核苷分解能力高之乳酸菌,經口投與於以含有嘌呤飼料飼育之大鼠,測定該等大鼠之一般狀況及血清尿酸值,並觀察乳酸菌投與對於血清尿酸值的影響。其結果發現,對於血清尿酸值上升有顯著抑制作用之乳酸菌為:口乳酸桿菌OLL2779株(Lactobacillus oris OLL2779)及加氏乳酸桿菌OLL2959株(Lactobacillus gasseri OLL2959)。並且本發明人等使用上述乳酸菌調製優格,以確認上述乳酸菌是否適於含優格之食品加工用途。本發明之乳酸菌具有血清尿酸值上升的抑制作用,可有效利用於對於高尿酸值血症與痛風有預防及/或治療用途之醫藥品。此外,本發明之乳酸菌所具有血清尿酸值上升之抑制作用也由經口投與之實驗確認,並確認其適用於實際食品加工之使用,作為食品之應用將有顯著高的利用性。因此,本發明是有關於可預防及/或治療高尿酸血症之乳酸菌及其應用,具體而言,本發明係提供以下發明。In order to solve the above problems, the inventors of the present invention have made repeated efforts to study. First, various lactic acid bacteria were cultured in the presence of inosine and guanosine, and the above-mentioned nucleoside consumption and decomposition products (hypoxanthine, guanine) production amount were measured, and the selection was remarkable. A plurality of lactic acid bacteria that have the ability to decompose nucleosides. A lactic acid bacterium having a high nucleoside-decomposing ability based on the above selection is orally administered to a rat bred with a sputum feed, and the general condition and serum uric acid value of the rats are measured, and the lactic acid bacteria are administered for serum uric acid. The impact of the value. As a result, it was found that the lactic acid bacteria having a significant inhibitory effect on the increase in serum uric acid value were Lactobacillus oryzae OLL2779 strain and Lactobacillus gasseri OLL2959 strain. Further, the inventors of the present invention used the above-described lactic acid bacteria to prepare a yogurt to confirm whether or not the above lactic acid bacteria are suitable for food processing applications containing yoghurt. The lactic acid bacterium of the present invention has an inhibitory action against an increase in serum uric acid value, and can be effectively utilized for a pharmaceutical having a preventive and/or therapeutic use for high uric acidosis and gout. Further, the inhibitory effect of the increase in serum uric acid value of the lactic acid bacteria of the present invention is also confirmed by an experiment for oral administration, and it is confirmed that it is suitable for use in actual food processing, and it has a remarkable high use as a food application. Accordingly, the present invention relates to a lactic acid bacterium capable of preventing and/or treating hyperuricemia and an application thereof, and in particular, the present invention provides the following invention.
(1)一種乳酸桿菌屬(Lactobacillus)乳酸菌,其具有嘌 呤分解能力,並無產氣能力,(2)如上述(1)所載之乳酸菌,其為加氏乳酸桿菌(Lactobacillus gasseri)。(1) A Lactobacillus lactic acid bacterium having a cockroach 呤 Decomposition ability, no gas production capacity, (2) Lactobacillus gasseri, as shown in (1) above, is Lactobacillus gasseri.
(3)一種口乳酸桿菌(Lactobacillus oris),其具有嘌呤分解能力,(4)如上述(2)所載之乳酸桿菌屬乳酸菌,其為加氏乳酸桿菌OLL2959株(Lactobacillus gasseri OLL2959)(寄存編號:NITE BP224),(5)如上述(3)所載之乳酸桿菌屬乳酸菌,其為口乳酸桿菌OLL2779株(Lactobacillus oris OLL2779)(寄存編號:NITE BP-223),(6)一種對於血液中尿酸值上升具有抑制作用之飲食品,其含有如上述(1)至(5)中任一項所載之乳酸菌,上述乳酸菌含有物及/或其處理物,(7)一種對於血液中尿酸值上升具有抑制作用之醫藥品,其含有如上述(1)至(5)中任一項所載之乳酸菌,上述乳酸菌含有物及/或其處理物,(8)一種用於預防及/或治療高尿酸血症之醫藥品,其含有如上述(1)至(5)中任一項所載之乳酸菌,上述乳酸菌含有物及/或其處理物,(9)一種用於預防及/或治療高尿酸血症之飲食品,其含有如上述(1)至(5)中任一項所載之乳酸菌,上述乳酸菌含有物及/或其處理物,(10)一種抑制由食品所攝取之嘌呤量的方法,其特徵為 投與如上述(1)至(5)中任一項所載之乳酸菌,上述乳酸菌含有物及/或其處理物,(11)一種血液中尿酸值上升之抑制方法,其特徵為投與如上述(1)至(5)中任一項所載之乳酸菌,上述乳酸菌含有物及/或其處理物,(12)一種降低嘌呤量之飲食品的製造方法,其包括將飲食品之原料或中間製品接觸如上述(1)至(5)中任一項所載之乳酸菌,上述乳酸菌含有物及/或其處理物的步驟,(13)一種用於預防及/或治療由高尿酸血症、痛風、腎機能障害、尿路結石、及動脈硬化症所組成之群中任一項疾患或症狀的方法,其特徵為投與如上述(1)至(5)中任一項所載之乳酸菌,上述乳酸菌含有物及/或其處理物,(14)一種含有如上述(1)至(5)中任一項所載之乳酸菌,上述乳酸菌含有物及/或其處理物之使用,係製造對於投與對象之血液中尿酸值上升有抑制作用之飲食品及/或醫藥品。(3) A Lactobacillus oris having a decomposing ability, and (4) a Lactobacillus lactic acid bacterium as described in the above (2), which is a Lactobacillus gastrin OLL2959 strain (Lactobacillus gasseri OLL2959) : NITE BP224), (5) Lactobacillus lactic acid bacteria as described in (3) above, which is Lactobacillus kawaii OLL2779 (Lactobacillus oris OLL2779) (Accession No.: NITE BP-223), (6) one for blood A food or drink having an inhibitory effect on an increase in uric acid value, comprising the lactic acid bacterium according to any one of the above (1) to (5), the lactic acid bacteria-containing substance and/or a processed product thereof, and (7) a uric acid value in blood A medicinal product having an inhibitory effect, comprising the lactic acid bacterium according to any one of the above (1) to (5), the lactic acid bacteria-containing substance and/or a processed product thereof, and (8) one for prevention and/or treatment A uric acid bacterium according to any one of the above (1) to (5), the lactic acid bacteria-containing substance and/or a processed product thereof, and (9) a drug for prevention and/or treatment A food or drink of hyperuricemia, which contains as set forth in any one of the above (1) to (5) Lactic acid bacteria, lactic acid bacteria-containing composition described above and / or a processed material (10) an amount of one purine by the method of inhibition of food intake, wherein The lactic acid bacteria according to any one of the above (1) to (5), the lactic acid bacteria-containing substance and/or the processed product thereof, and (11) a method for suppressing an increase in uric acid value in blood, which is characterized in that The lactic acid bacteria according to any one of the above (1) to (5), the lactic acid bacteria-containing material and/or a processed product thereof, and (12) a method for producing a food or drink having a reduced amount of sputum, which comprises a raw material of a food or drink or The intermediate product is in contact with the lactic acid bacteria according to any one of the above (1) to (5), the lactic acid bacteria-containing substance and/or the processed product thereof, and (13) one for prevention and/or treatment of hyperuricemia And a method of any of the diseases or symptoms of a group consisting of gout, renal dysfunction, urinary calculi, and arteriosclerosis, characterized by administering as set forth in any one of the above (1) to (5) And a lactic acid bacterium according to any one of the above (1) to (5), wherein the lactic acid bacteria-containing substance and/or a processed product thereof is used. A food or drink and/or a pharmaceutical product which suppresses an increase in uric acid value in blood of a subject to be administered.
本發明係有關於具有嘌呤分解能力,並無產氣能力之乳酸桿菌屬(Lactobacillus)乳酸菌。本發明係基於本發明人等首次發現之具有嘌呤分解能力,並無產氣能力之乳酸桿菌屬(Lactobacillus)乳酸菌(以下簡稱為「本發明之乳酸桿菌屬乳酸菌」)。The present invention relates to a lactic acid bacterium of the genus Lactobacillus which has the ability to decompose hydrazine and has no gas generating ability. The present invention is a Lactobacillus lactic acid bacterium (hereinafter referred to as "the Lactobacillus lactic acid bacterium of the present invention" having the cockroach decomposition ability and having no gas generating ability, which was first discovered by the present inventors.
乳酸桿菌屬(Lactobacillus)係以乳酸菌為代表之一的菌屬,含有80種以上的菌種。乳酸桿菌屬所含菌種的例子為如德氏乳酸桿菌保加利亞種(Lactobacillus delbrueckii subsp.burgalicus)、德氏乳酸桿菌乳酸亞種(Lactobacillus delbrueckii subsp.lactis)、副乾酪乳酸桿菌副乾酪亞種(Lactobacillus paracasei subsp.paracasei)、瑞士乳酸桿菌(Lactobacillus helveticus)、瑞士乳酸桿菌約古特亞種(Lactobacillus helveticus subsp.jugurti)、嗜酸乳酸桿菌(Lactobacillus acidophilus)、卷曲嗜酸乳酸桿菌(Lactobacillus acidophilus crispatus)、食澱粉乳酸桿菌(Lactobacillus amylovorus)、雞乳酸桿菌(Lactobacillus gallinarum)、加氏乳酸桿菌(Lactobacillus gasseri)、口乳酸桿菌(Lactobacillus oris)、乾酪乳酸桿菌李糖亞種(Lactobacillus casei subsp.rhamnosus)、約氏乳酸桿菌(Lactobacillus johnsonii)、發酵乳酸桿菌(Lactobacillus fermentum)、短乳酸桿菌(Lactobacillus brevis)。本發明之乳酸桿菌屬乳酸菌具有嘌呤分解能力,卻無產氣能力,以乳酸桿菌屬中之任一種為佳,較佳為加氏乳酸桿菌。Lactobacillus is a genus of bacteria represented by lactic acid bacteria and contains more than 80 species of bacteria. Examples of strains contained in the genus Lactobacillus are Lactobacillus delbrueckii subsp. burgalicus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus subfamily (Lactobacillus). Paracasei subsp.paracasei), Lactobacillus helveticus, Lactobacillus helveticus subsp. jugurti, Lactobacillus acidophilus, Lactobacillus acidophilus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus oris, Lactobacillus casei subsp. rhamnosus, about Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus brevis. The lactic acid bacterium of the genus Lactobacillus of the present invention has a cockroach-decomposing ability, but has no gas-producing ability, and is preferably any one of the genus Lactobacillus, and is preferably Lactobacillus gasseri.
嘌呤係核酸構成成分,經由嘌呤再生(de novo)合成、回收途徑(salvage pathways)、食餌中核蛋白質等供給給生物體,不需要的嘌呤可經肝臟代謝排出。尿酸是人類、高等靈長類、鳥類、爬蟲類等動物體的嘌呤最終代謝產物。The quinone-based nucleic acid constituents are supplied to the living body via de novo synthesis, salvage pathways, nuclear proteins in the prey, and the like, and unnecessary sputum can be excreted by the liver. Uric acid is the ultimate metabolite of cockroaches in humans, higher primates, birds, reptiles and other animals.
本說明書中之嘌呤係指具有嘌呤結構的化合物。嘌呤之代表例為,例如,嘌呤核苷酸(腺嘌呤核苷酸、去氧腺嘌呤核苷酸、鳥嘌呤核苷酸、去氧鳥嘌呤核苷酸)、核苷(腺苷、去氧腺苷、鳥苷、去氧鳥苷)、嘌呤鹼基(腺嘌呤、鳥嘌呤)、含嘌呤鹼基之寡核苷酸與多核苷酸。嘌呤鹼基除為核酸構成成分,也是ATP、GTP、cAMP、cGMP、輔酶A、FAD、NAD等多種活體中之成份。本說明書所指涉之嘌呤並不限於具有嘌呤骨幹者,上述活體中之成分也全部包括在內。By hydrazine in the present specification is meant a compound having a fluorene structure. Representative examples of sputum are, for example, purine nucleotides (adenine nucleotides, deoxyadenosine nucleotides, guanine nucleotides, deoxyguanine nucleotides), nucleosides (adenosine, deoxygenation) Adenosine, guanosine, deoxyguanosine), purine bases (adenine, guanine), purine-containing oligonucleotides and polynucleotides. In addition to the nucleic acid constituents, the purine base is also a component of various living organisms such as ATP, GTP, cAMP, cGMP, coenzyme A, FAD, and NAD. The reference in this specification is not limited to those having a skeleton, and all the components in the above-mentioned living body are also included.
活體內的嘌呤係以尿酸形式代謝。嘌呤之尿酸形式代謝路徑係眾所周知的。AMP係經5’-核苷酸酶處理而成為腺苷,腺苷係經肌苷而成為次黃嘌呤。GMP係經5’-核苷酸酶處理而成為鳥苷後,再形成鳥嘌呤。次黃嘌呤又經黃嘌呤氧化酶處理,而鳥嘌呤又經鳥嘌呤去胺酶處理,分別代謝為黃嘌呤,黃嘌呤再經黃嘌呤氧化酶處理而成為尿酸。The lanthanide in the body is metabolized in the form of uric acid. The metabolic pathway of the uric acid form of sputum is well known. The AMP is treated with 5'-nucleotidase to form adenosine, and the adenosine is inosine to become hypoxanthine. The GMP is treated with 5'-nucleotidase to form guanosine, and then guanine is formed. Hypoxanthine is treated with xanthine oxidase, and guanine is treated with guanine deaminase, which is metabolized to xanthine, which is then treated with xanthine oxidase to form uric acid.
本發明所謂的嘌呤分解能力為具有至少一種嘌呤分解能力,而不問分解產物是否具有嘌呤骨幹。也就是說,分解嘌呤形成不具有嘌呤骨幹之化合物的能力,或分解嘌呤形成其他嘌呤(具有嘌呤骨幹之化合物)的能力,皆包括在本發明所謂的嘌呤分解能力中。The so-called cockroach decomposition ability of the present invention has at least one hydrazine decomposition ability regardless of whether the decomposition product has a sacral backbone. That is, the ability to decompose sputum to form a compound having no sacral stem, or to decompose sputum to form other sputum (a compound having a sacral backbone) is included in the so-called sputum decomposition ability of the present invention.
本發明所謂的乳酸桿菌屬(Lactobacillus)乳酸菌可以習知方法分離出來。例如,培養人類等哺乳類動物糞便取得的細菌,由所培養細菌之形狀、生理學特徵等分離出 乳酸桿菌屬細菌,檢測其是否具有嘌呤分解能力及產氣能力,撿選具有嘌呤分解能力且不具產氣能力的乳酸桿菌屬並可單離之。嘌呤分解能力的檢出與產氣能力的檢出可以習知方法進行,舉例而言,可以本實施例之方法進行。The so-called Lactobacillus lactic acid bacteria of the present invention can be isolated by a known method. For example, bacteria obtained by cultivating feces from mammals such as humans are separated from the shape and physiological characteristics of the cultured bacteria. A bacterium belonging to the genus Lactobacillus is tested for its ability to decompose and produce gas. It is selected from the genus Lactobacillus which has the ability to decompose sputum and has no gas producing ability and can be isolated. The detection of the decomposing ability and the detection of the gas generating ability can be carried out by a conventional method, and can be carried out, for example, by the method of the present embodiment.
本發明之乳酸桿菌屬乳酸菌的培養係以一般乳酸桿菌培養所適合的培養基為佳,可使用含有葡萄糖、乳糖、半乳糖、果糖、海澡糖、蔗糖、甘露糖、纖維二糖等碳源,肉萃取物、蛋白胴(peptone)、酵母菌萃取物、酪蛋白、乳清蛋白等氮源、硫酸鎂、硫酸鐵、硫酸錳等無機營養素之培養基。較佳的例子可舉乳酸桿菌MRS肉湯(Lactobacilli MRS broth)(Difco公司出品)。培養條件為腸內乳酸菌可繁殖的條件,並無特別限制,較佳的條件為,例如,pH5.0-pH8.0、溫度20℃-45℃,更佳的條件為,厭氧性環境、pH5.0-pH7.0、溫度30℃-40℃。The culture system of the Lactobacillus lactic acid bacteria of the present invention is preferably a medium suitable for general Lactobacillus culture, and a carbon source containing glucose, lactose, galactose, fructose, sea bath sugar, sucrose, mannose, cellobiose or the like can be used. A medium for extracting inorganic substances such as meat extract, peptone, yeast extract, casein, whey protein, or the like, and inorganic nutrients such as magnesium sulfate, iron sulfate, and manganese sulfate. A preferred example is Lactobacilli MRS broth (produced by Difco Co., Ltd.). The culture condition is a condition in which the lactic acid bacteria in the intestine can be propagated, and is not particularly limited. Preferred conditions are, for example, pH 5.0 to pH 8.0, temperature 20 ° C to 45 ° C, and more preferable conditions, an anaerobic environment, pH 5.0 - pH 7.0, temperature 30 ° C - 40 ° C.
如以下所述,本發明人等將本發明之乳酸桿菌屬乳酸菌經口投與模式動物,確認該等乳酸菌對血液中尿酸值上升的抑制效果。因此,由於本發明之乳酸桿菌屬乳酸菌可抑制血液中尿酸值之上升,故可應用於預防及/或治療高尿酸血症。此外,利用本發明之乳酸桿菌屬乳酸菌的嘌呤分解能力,可考慮製造嘌呤量降低之食品。As described below, the inventors of the present invention orally administered the Lactobacillus lactic acid bacteria of the present invention to a model animal, and confirmed the inhibitory effect of the lactic acid bacteria on the increase in uric acid value in the blood. Therefore, since the Lactobacillus lactic acid bacterium of the present invention can suppress an increase in uric acid value in blood, it can be applied to prevent and/or treat hyperuricemia. Further, by using the cockroach decomposition ability of the lactic acid bacterium of the lactic acid bacterium of the present invention, it is conceivable to manufacture a food having a reduced amount of mash.
本發明之「具有嘌呤分解能力、無產氣能力之乳酸桿菌屬乳酸菌」的具體例子為,寄存編號:NITE BP-224之特定加氏乳酸桿菌OLL2959株(Lactobacillus gasseri OLL2959)。本發明人等檢測多種乳酸菌之嘌呤分解能力與 產氣能力的有無,發現名為加氏乳酸桿菌OLL2959株(Lactobacillus gasseri OLL2959)的乳酸桿菌屬乳酸菌具有嘌呤分解能力、無產氣能力。且經體內(in vivo)試驗,發現加氏乳酸桿菌OLL2959株(Lactobacillus gasseri OLL2959)顯然對血液中尿酸值上升有抑制作用。本發明人等將上述菌株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。以下所載即為特定寄存之內容:A specific example of the "Lactobacillus lactic acid bacterium having a sputum-decomposing ability and a gas-producing ability" of the present invention is a specific L. lactis OLL2959 strain (Lactobacillus gasseri OLL2959) having the accession number: NITE BP-224. The present inventors have examined the decomposition ability of various lactic acid bacteria and The presence or absence of gas production ability revealed that Lactobacillus lactic acid bacteria named Lactobacillus gasseri OLL2959 had the ability to decompose and produce gas. And in vivo test, it was found that Lactobacillus kawaii OLL2959 strain (Lactobacillus gasseri OLL2959) obviously inhibited the increase of uric acid value in blood. The present inventors deposited the above-mentioned strains in the patent microbial deposit center of the independent administrative legal person product evaluation technology base institution. The following is the content of the specific deposit:
(1)寄存機關: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(地址:日本國千葉縣木更津市一樹鎌足2-5-8郵遞區號292-0818)(1) Depository authority: Patent Microbiology Depository Center for Product Evaluation Technology Basement Organization of Independent Administrative Corporation (Address: 2-5-8, Post Code, 292-0818, Kishozu, Kisarazu, Chiba Prefecture, Japan)
(2)原寄存日:2006年3月31日基於布達佩斯條約,由國內寄存機構移交管理日:2007年11月21日(2) Original registration date: March 31, 2006 based on the Budapest Treaty, transferred by the domestic depository management day: November 21, 2007
(3)寄存編號:Lactobacillus gasseri OLL2959株(寄存編號NITE BP-224)(3) Deposit number: Lactobacillus gasseri OLL2959 strain (registration number NITE BP-224)
上述之菌株並另寄存於中華民國財團法人食品工業發展研究所,寄存編號為BCRC 910379,寄存日期為民國96年12月24日。The above-mentioned strains are additionally deposited in the Food Industry Development Research Institute of the Republic of China. The deposit number is BCRC 910379, and the deposit date is December 24, 1996.
此外,本發明係有關於具有嘌呤分解能力之口乳酸桿菌(Lactobacillus oris)。本發明係基於本發明人等首次發現具有嘌呤分解能力之口乳酸桿菌(Lactobacillus oris)(以下簡稱為「本發明之口乳酸桿菌」)。Further, the present invention relates to Lactobacillus oris having a decomposing ability. The present invention is based on the first discovery by the present inventors that Lactobacillus oris (hereinafter referred to as "Lactobacillus brevisii" of the present invention) having a decomposing ability.
本發明之口乳酸桿菌係經由與上述說明方法相同之方法分離‧培養本發明之乳酸桿菌屬乳酸菌。此外,本發明之口乳酸桿菌也因具有血液中尿酸值上升抑制作用,可應用於治療及/或預防高尿酸血症。The Lactobacillus brevis strain of the present invention is isolated by the same method as described above. ‧ The Lactobacillus lactic acid bacterium of the present invention is cultured. Further, the Lactobacillus brevis of the present invention is also useful for treating and/or preventing hyperuricemia because it has an inhibitory effect on the increase in uric acid value in blood.
本發明之「具有嘌呤分解能力之口乳酸桿菌(Lactobacillus oris)」的具體例子為,寄存編號:NITE BP-223之特定口乳酸桿菌OLL2779株(Lactobacillus oris OLL2779)。本發明人等由多種乳酸菌中發現具有嘌呤分解能力之乳酸菌,口乳酸桿菌OLL2779株(Lactobacillus oris OLL2779),此外,經由體內(in vivo)試驗,確認口乳酸桿菌OLL2779株(Lactobacillus oris OLL2779)顯然具有血液中尿酸值上升之抑制作用。本發明人等將上述菌株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。以下所載即為特定寄存之內容:A specific example of the "Lactobacillus oris having a decomposing ability" of the present invention is a specific Lactobacillus oris OLL2779 strain (Lactobacillus oris OLL2779) having the accession number: NITE BP-223. The inventors of the present invention have found a lactic acid bacterium having a sputum-decomposing ability among various lactic acid bacteria, Lactobacillus oryzae OLL2779 strain (Lactobacillus oris OLL2779), and confirmed that the Lactobacillus oryzae OLL2779 strain (Lactobacillus oris OLL2779) has apparently passed through an in vivo test. Inhibition of increased uric acid levels in the blood. The present inventors deposited the above-mentioned strains in the patent microbial deposit center of the independent administrative legal person product evaluation technology base institution. The following is the content of the specific deposit:
(1)寄存機關: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(地址:日本國千葉縣木更津市一樹鎌足2-5-8郵遞區號292-0818)(1) Depository authority: Patent Microbiology Depository Center for Product Evaluation Technology Basement Organization of Independent Administrative Corporation (Address: 2-5-8, Post Code, 292-0818, Kishozu, Kisarazu, Chiba Prefecture, Japan)
(2)原寄存日:2006年3月31日基於布達佩斯條約,由國內寄存機構移交管理日:2007年11月21日(2) Original registration date: March 31, 2006 based on the Budapest Treaty, transferred by the domestic depository management day: November 21, 2007
(3)寄存編號:Lactobacillus oris OLL2779株(寄存編號:NITE BP-223)(3) Deposit number: Lactobacillus oris OLL2779 strain (registration number: NITE BP-223)
上述之菌株並另寄存於中華民國財團法人食品工業發展研究所,寄存編號為BCRC 910378,寄存日期為民國96年12月24日。The above-mentioned strain was deposited in the Food Industry Development Research Institute of the Republic of China, and the deposit number is BCRC 910378. The deposit date is December 24, 1996.
本發明之乳酸桿菌屬乳酸菌及本發明之口乳酸桿菌可應用於抑制血液中尿酸值上升之飲食品或醫藥品,或製造能預防及/或治療高尿酸血症之醫藥品或飲食品。The Lactobacillus lactic acid bacterium of the present invention and the Lactobacillus oral bacterium of the present invention can be used for a food or drink or a pharmaceutical product which suppresses an increase in uric acid value in blood, or a pharmaceutical or food or drink which can prevent and/or treat hyperuricemia.
將本發明之乳酸桿菌屬乳酸菌及本發明之口乳酸桿菌使用作飲食品時,並無限制其屬性與種類,機能性食品、特定保健用食品、健康食品、照護用食品等皆佳,此外,菓子、乳酸菌飲料、起司及優格等乳製品、調味料等亦佳。飲食品的形狀也無限制,固形、液狀、流動食狀、果凍狀、錠劑狀、顆粒狀、膠囊狀等,通常市面上流通的飲食品形狀皆可。上述飲食品的製作係以從業者一般常法進行製作。上述飲食品之製造時,限於不會影響乳酸菌生長繁殖者,可添加糖類、蛋白質、脂質、食物纖維、維生素類、活體所需微量金屬(硫酸錳、硫酸鋅、氯化鎂、碳酸鉀等)、香料及其他配合添加物。When the Lactobacillus lactic acid bacterium of the present invention and the Lactobacillus acidophilus of the present invention are used as foods and drinks, the properties and types thereof are not limited, and functional foods, specific health foods, health foods, and foods for care are preferable. Fruits, lactic acid bacteria beverages, cheese and yogurt, and other seasonings are also good. The shape of the food and drink is also unlimited, and it is solid, liquid, flowing, jelly, lozenge, granule, capsule, etc., and the shape of the food and beverage that is generally on the market is acceptable. The production of the above-mentioned foods and drinks is carried out by the usual methods of the practitioner. When manufacturing the above-mentioned food and beverage products, it is limited to those who do not affect the growth and reproduction of lactic acid bacteria, and may add sugars, proteins, lipids, dietary fibers, vitamins, and trace metals (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate, etc.) and spices required for living organisms. And other matching additives.
本發明之乳酸桿菌屬乳酸菌及本發明之口乳酸桿菌、及該等乳酸菌之含有物及/或其處理物(例如,培養物、濃縮物、膏狀物、噴霧乾燥物、冷凍乾燥物、真空乾燥物、滾筒乾燥物、液狀物、稀釋物、破碎物)係,利用含有上述乳製品、發酵乳之一般飲食品之加工流程,優格或起司等乳製品、發酵乳之製造用起始菌群(starter)等而製造。起始菌群限於對本發明之乳酸桿菌屬乳酸菌及本 發明之口乳酸桿菌之生長、增殖無阻礙者,此外,限於對乳製品製造不阻礙者,亦可與其他微生物混合。例如,與優格用乳酸菌的主要菌種德氏乳酸桿菌保加利亞種(Lactobacillus delbruekii subsp.bulgaricus)、嗜熱鏈球菌(Streptococcus thermophilus)、嗜酸乳酸桿菌(Lactobacillus acidophilus)等混合亦佳。此外,也可與一般優格用或起司用的菌種混合作為起始菌群。以上述起始菌群進行乳製品、發酵乳的製造可依一般常法進行。例如,加溫、混合、均質化、殺菌處理後冷卻的乳品或乳製品,與上述起始菌群混合,經發酵、冷卻後,製造出原味優格。The lactic acid bacterium of the genus Lactobacillus of the present invention, the lactic acid bacterium of the present invention, and the contents of the lactic acid bacteria and/or the processed material thereof (for example, culture, concentrate, paste, spray dried product, lyophilized product, vacuum) The dried product, the dried product of the drum, the liquid, the diluted product, and the crushed product are used in the processing of the general foods and drinks containing the above-mentioned dairy products and fermented milk, and the manufacture of dairy products and fermented milk such as Yogurt or cheese. It is manufactured by a starter or the like. The initial flora is limited to the Lactobacillus lactic acid bacteria and the present invention of the present invention. The growth and proliferation of Lactobacillus brevis is not inhibited, and it is also possible to mix with other microorganisms if it is not restricted to the production of dairy products. For example, it is also preferable to mix with Lactobacillus delbruekii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, and the like, which are the main species of lactic acid bacteria. In addition, it can also be mixed with a strain for general use or cheese as a starting flora. The production of the dairy product and the fermented milk by the above-mentioned starting flora can be carried out according to a usual method. For example, a dairy product or a dairy product which is heated, mixed, homogenized, and sterilized and cooled is mixed with the above-mentioned starting flora, fermented and cooled to produce an original flavor.
本發明之乳酸桿菌屬乳酸菌及本發明之口乳酸桿菌可與生理學可容許之載體、賦形劑、或稀釋劑等混合,作為醫藥組合物經口或非經口投與皆可,較佳的投與方法為經口投與。經口投與製劑可為週知的各種劑型,例如,可為顆粒劑、散劑、錠劑、丸劑、膠囊劑、液劑、糖漿劑、乳劑、懸濁劑、喉片等劑型。此外,以從業者周知方法做成腸溶性製劑,不受胃酸作用,可使本發明之乳酸桿菌屬乳酸菌有效率地到腸道運作。The lactic acid bacterium of the genus Lactobacillus of the present invention and the lactic acid bacterium of the present invention may be mixed with a physiologically acceptable carrier, excipient, or diluent, etc., and may be administered as a pharmaceutical composition by oral or parenteral administration, preferably. The method of investment is administered orally. The orally administered preparation may be in various known dosage forms, and may be, for example, a granule, a powder, a lozenge, a pill, a capsule, a liquid, a syrup, an emulsion, a suspension, a throat, or the like. Further, the enteric preparation prepared by a known method by a practitioner is not affected by gastric acid, and the Lactobacillus lactic acid bacteria of the present invention can be efficiently operated into the intestine.
使用本發明之乳酸桿菌屬乳酸菌及本發明之口乳酸桿菌所製造的醫藥品及飲食品,可期待其以飲食品中同樣細菌之作用,發揮對血液中尿酸值上升之抑制效果以及對高尿酸血症之預防及/或治療效果。By using the lactic acid bacteria of the lactic acid bacterium of the present invention and the medicinal products and foods and beverages produced by the lactic acid bacillus of the present invention, it is expected that the effect of the same bacteria in the food and beverage can be exerted to suppress the increase in uric acid value in the blood and the high uric acid. Prevention and/or treatment of blood.
此外,可利用本發明之乳酸桿菌屬乳酸菌及本發明之 口乳酸桿菌之嘌呤分解能力,製造嘌呤量降低之飲食品。本發明嘌呤量降低之飲食品的製造方法係,包括將本發明之乳酸桿菌屬乳酸菌或本發明之口乳酸桿菌與飲食品之原料或中間製品接觸的步驟。經由此步驟,該原料或中間製品中所含有嘌呤量可有效率地降低。上述步驟於本發明之乳酸桿菌屬乳酸菌及本發明之口乳酸桿菌生存可能之條件下進行為佳。本發明之製造方法係,將上述步驟包括在粉粹步驟、混合步驟、乾燥步驟、殺菌及充填步驟等,目的飲食品之通常製程之中。經由本發明方法製造之飲食品並無限制其屬性與種類,例如機能性食品、特定保健用食品、健康食品、照護用食品等皆佳,此外一般食品,例如以嗜好品分類的食品亦佳,需限制嘌呤攝取量之疾病或症狀的病患,作為預防上述疾病或症狀之日常或補助飲食品特別有用。本發明之方法係,對於嘌呤攝取量限制有必要性,或希望限制嘌呤攝取量的人們,得以攝取本來嘌呤量多的食品,並享受該食品的營養素與風味。Further, the Lactobacillus lactic acid bacterium of the present invention and the present invention can be used. The decomposition ability of Lactobacillus brevis is used to produce foods and drinks with reduced sputum. The method for producing a food or drink having a reduced amount of the present invention includes the step of bringing the Lactobacillus lactic acid bacteria of the present invention or the Lactobacillus oral bacterium of the present invention into contact with a raw material or an intermediate product of a food or beverage. Through this step, the amount of rhenium contained in the raw material or intermediate product can be effectively reduced. The above steps are preferably carried out under the conditions in which the Lactobacillus lactic acid bacteria of the present invention and the Lactobacillus oral bacterium of the present invention survive. In the manufacturing method of the present invention, the above steps are included in the usual steps of the pulverizing step, the mixing step, the drying step, the sterilizing and filling steps, and the like. The foods and drinks produced by the method of the present invention are not limited in terms of their properties and types, such as functional foods, specific health foods, health foods, and foods for care, and general foods such as foods classified as hobby goods are also preferred. A patient who needs to limit the disease or symptoms of sputum intake is particularly useful as a daily or supplementary food or drink for preventing the above diseases or symptoms. In the method of the present invention, it is necessary to limit the amount of sputum intake, or it is desirable to limit the amount of sputum intake, and it is possible to ingest a food having a large amount of food and enjoy the nutrients and flavor of the food.
尚且,本說明書中引用的全部先前技術文獻皆可作為本說明書之參考而列入本說明書中。Moreover, all of the prior art documents cited in the specification can be included in the present specification as a reference to the present specification.
以下將基於本發明之實施例更具體說明本發明。然而本發明並不限於下述實施例。尚且,實施例中,菌株名以JCM記載之菌株係獨立行政法人理化學研究所生物資源中心的微生物材料開發室取得之標準株,菌株名以ATCC記 載之菌株係美國菌種中心(American Type Culture Collection)取得之標準株,菌株名以MEP記載之菌株係明治乳業股份有限公司保有之菌株。Hereinafter, the present invention will be more specifically described based on examples of the invention. However, the invention is not limited to the embodiments described below. Further, in the examples, the strain whose strain name is JCM is a standard strain obtained by the Microbial Materials Development Laboratory of the Biological Resource Center of the Institute of Physical and Chemical Research, Institute of Physical and Chemical Research, and the strain name is recorded by ATCC. The strain contained is a standard strain obtained by the American Type Culture Collection, and the strain whose strain name is MEP is a strain possessed by Meiji Dairy Co., Ltd.
將各種乳酸菌以下述方法研究其嘌呤分解能力之有無。各種乳酸菌(菌體)係採用Difco牌乳酸桿菌MRS肉湯(Difco Lactobacilli MRS Broth)(BD製)作為培養基,與氧氣吸著劑「厭氧培養包」(三菱氣體(股份有限公司)製造)一併放入密閉容器中,於溫度37℃厭氧培養隔夜。培養後之菌體懸濁液以3000rpm轉速、溫度4℃離心10分鐘,並經沈澱回收(集菌)分離的菌體。Various lactic acid bacteria were studied for the presence or absence of their decomposition ability by the following method. Various lactic acid bacteria (bacteria) are made of Difco Lactobacilli MRS Broth (made by BD) as a medium, and an oxygen sorbent "anaerobic culture package" (manufactured by Mitsubishi Gas Co., Ltd.). It was placed in a closed container and anaerobic cultured overnight at a temperature of 37 °C. The cultured suspension of the cells was centrifuged at 3000 rpm for 10 minutes at a temperature of 4 ° C, and the isolated cells were recovered by precipitation (serum collection).
由此菌體以0.1M林酸鈉緩衝液調製1×109 CFU/mL之菌體懸濁液。This cell was prepared with a suspension of 1×10 9 CFU/mL of the cell suspension in 0.1 M sodium citrate buffer.
調製出各種菌體懸濁液後,將肌苷與鳥苷分別加入各種菌體懸濁液使分別為1.25mM濃度。將這些菌體懸濁液置入37℃恆溫槽,以水平轉速140rpm進行30分鐘或2小時振動培養。After preparing various cell suspensions, inosine and guanosine were separately added to various cell suspensions to a concentration of 1.25 mM. These cell suspensions were placed in a 37 ° C thermostat and shake cultured at a horizontal speed of 140 rpm for 30 minutes or 2 hours.
對於振動培養後之菌體懸濁液(反應液),以5-溴尿嘧啶作為內部標準、採用HPLC測定核苷消耗量、及核苷分解物鹼基(次黃嘌呤及鳥嘌呤)生成量。移動相A的780μL中,加入反應液200μL、作為內部標準之5-溴尿嘧啶(1.6mg/mL)20μL並混合之。此混合液以濾膜(孔徑0.45μm)過濾後,將50μL濾過液注入HPLC。HPLC之具 體操作條件依照以下。For the cell suspension (reaction solution) after vibration culture, 5-bromouracil was used as an internal standard, and the amount of nucleoside consumption and the amount of nucleoside-degrading base (hypoxanthine and guanine) were determined by HPLC. . In 780 μL of the mobile phase A, 200 μL of the reaction solution and 20 μL of 5-bromouracil (1.6 mg/mL) as an internal standard were added and mixed. After the mixture was filtered through a filter (pore size: 0.45 μm), 50 μL of the filtrate was poured into HPLC. HPLC The body operating conditions are as follows.
HPLC:Waters alliance 2690型HPLC: Waters alliance 2690
管柱:CAPCELL PAK C18 SG120、粒徑5μm、管柱大小4.6×250mm(資生堂)Column: CAPCELL PAK C 18 SG120, particle size 5μm, column size 4.6×250mm (Shiseido)
移動相:A:25mM KH2 PO4 (0.1%甲醇)B:25mM KH2 PO4 (0.1%甲醇)/甲醇(75:25)Mobile phase: A: 25 mM KH 2 PO 4 (0.1% methanol) B: 25 mM KH 2 PO 4 (0.1% methanol) / methanol (75: 25)
梯度A/B(min):100/0(0)-100/0(10)-20/80(20)-20/80(25)-100/0(26)-100/0(40)Gradient A/B(min): 100/0(0)-100/0(10)-20/80(20)-20/80(25)-100/0(26)-100/0(40)
檢驗器:光電二極體陣列(Waters 996型)檢出波長254nmVerifier: Photodiode array (Waters 996) detected wavelength 254nm
流速:1mL/minFlow rate: 1mL/min
管柱溫度:常溫Column temperature: normal temperature
結果如第1~3圖所示。各化合物之定量係基於HPLC圖中高峰期面積值進行。並且,第1圖與第2圖之分解率係依照下式算出。The results are shown in Figures 1 to 3. The quantification of each compound was based on the peak area values in the HPLC plot. Further, the decomposition rates of the first and second figures are calculated according to the following equation.
分解率=100-(肌苷或鳥苷量/空白對照組肌苷或鳥苷量)×100Decomposition rate = 100 - (inosine or guanosine amount / blank control group inosine or guanosine amount) × 100
此外,第3圖結果之計算方法係依照下式。In addition, the calculation method of the result of Fig. 3 is in accordance with the following formula.
(次黃嘌呤量+鳥嘌呤量)/5-溴尿嘧啶量(second dose of jaundice + amount of guanine) / 5 - bromo uracil amount
由第1~3圖之結果,選出判斷為具有顯著核苷分解能力之乳酸菌。From the results of Figs. 1 to 3, lactic acid bacteria which were judged to have significant nucleoside decomposition ability were selected.
依照先前文獻(非專利文獻1)中記載之方法,產生食 因性高尿酸血症的模式動物,研究微生物(乳酸菌)對於該動物之血清尿酸值帶來的影響。上述方法係,具體而言,先調製含有2.5重量%之苯胺羰酸鉀與1.0重量%之RNA的混餌,讓大鼠攝取該等混餌,比較攝取後陰性組與對照組的血液中尿酸值。已知對本方法之模式動物以尿酸生成阻斷藥別嘌醇(allopurinol)經口投與時,會顯著抑制該模式動物之血液中尿酸值(食品機能研究新聞(第14號)、2005年3月9日發行、(股份有限公司)美麗香克林太克(Mercian cleantec)環境檢查中心、http://www.m-cleantec.com/gizyutu/news_0503.htm )。顯示對高尿酸血症食品之有效性評價系統之上述方法係有效的。According to the method described in the prior literature (Non-Patent Document 1), a model animal of causative hyperuricemia is produced, and the effect of the microorganism (lactic acid bacteria) on the serum uric acid value of the animal is examined. In the above method, specifically, a mixed bait containing 2.5% by weight of potassium phenylaminocarbonate and 1.0% by weight of RNA was prepared, and the rats were ingested with the mixed baits, and the uric acid value in the blood of the negative group and the control group after ingestion was compared. It is known that the oral administration of the uric acid-producing blocker allopurinol to the model animal of the present method significantly inhibits the uric acid value in the blood of the model animal (Food Function Research News (No. 14), 2005 3 Issued on the 9th of June, (Mechanian Cleantec Environmental Inspection Center, http://www.m-cleantec.com/gizyutu/news_0503.htm ). The above method showing the effectiveness evaluation system for hyperuricemia foods is effective.
[2-1材料及實驗步驟][2-1 Materials and Experimental Procedures]
[微生物][microorganism]
上述體外試驗所判斷具有高度核苷分解能力者,為發酵乳酸桿菌MEP181504株(Lactobacillus fermentum MEP181504)(以下將「Lactobacillus」簡稱「L.」)、短乳酸桿菌MEP181507株(L.brevis MEP181507)、加氏乳酸桿菌JCM8787株(L.gasseri JCM8787)、加氏乳酸桿菌OLL2959株(L.gasseri OLL2959)、口乳酸桿菌OLL2779株(L.oris OLL2779)共5株。將各種乳酸菌調製出與體外試驗同樣的菌體懸濁液。將菌體懸濁液以1×109 CFU/10mL/kg的量經口投與大鼠。Those who have high nucleoside-dissolving ability in the above-mentioned in vitro test are Lactobacillus fermentum MEP181504 (hereinafter referred to as "Lactobacillus" or "L.") and Lactobacillus sp. MEP181507 (L. brevis MEP181507). There were 5 strains of Lactobacillus sp. JCM8787 strain (L. gasseri JCM8787), Lactobacillus delbrueckol OLL2959 strain (L. gasseri OLL2959), and Lactobacillus acidophilus OLL2779 strain (L. oris OLL2779). Various lactic acid bacteria were prepared into the same cell suspension as in the in vitro test. The bacterial suspension was orally administered to the rats in an amount of 1 × 10 9 CFU/10 mL / kg.
[實驗動物][experimental animal]
採用大鼠(Wister SPF、雄性、7週齡)。飼育(馴化與試驗)過程採用大鼠用塑膠籠,每籠收容1隻大鼠。明暗週期為明期上午7時~下午7時(12小時)。Rats (Wister SPF, male, 7 weeks old) were used. The breeding (domestication and test) process uses a plastic cage for rats, and each cage contains one rat. The light and dark cycle is from 7 am to 7 pm (12 hours) in the Ming Dynasty.
[預備飼育(馴化)與分組][prepared breeding (domestication) and grouping]
實驗動物遷入後進行1週的預備飼育(馴化)。馴化過程可自由攝食,以AIN-93G(東方酵母工業(股份有限公司))為食餌(飼料),以自來水為飲水。預備飼育之大鼠(遷入7日後、8週齡、第0日)於上午、非絕食的情形下由尾靜脈採血。此血液於室溫放置30分鐘以上,以10000rpm轉速離心10分鐘分離取出血清,以磷鎢酸法測定血清中之尿酸值。After the experimental animals were moved in, they were pre-incubated for 1 week (domestication). The domestication process can be freely fed, with AIN-93G (Oriental Yeast Industry Co., Ltd.) as the feeding ground (feed) and tap water as the drinking water. The rats that were prepared for breeding (after 7 days, 8 weeks, and 0 days) were collected from the tail vein in the morning, not in the case of non-fasting. The blood was allowed to stand at room temperature for 30 minutes or more, and the serum was separated by centrifugation at 10,000 rpm for 10 minutes, and the uric acid value in the serum was measured by the phosphotungstic acid method.
分組係以各組具同等血清尿酸值的方式進行。試驗中,1組有5隻大鼠,設定為陰性組(第1、5組)、對照組(第2、6組)、菌體投與組(第3、4、7-9組),共9組。組名、食餌、投與物(投與用量)、隻數等如以下所示。The grouping was performed in such a manner that each group had the same serum uric acid value. In the experiment, 5 rats in group 1 were set as the negative group (groups 1 and 5), the control group (groups 2 and 6), and the bacterial administration group (groups 3, 4, 7-9). A total of 9 groups. The group name, the prey, the dosing (dosing amount), and the number are as follows.
‧陰性組(第1、5組):給予「AIN-93G」食餌、投與「生理食鹽水」(10mL/Kg)、5隻‧ Negative group (Groups 1 and 5): Give "AIN-93G" prey, cast "physiological saline" (10mL/Kg), 5
‧對照組(第2、6組):給予「與2.5重量%之苯胺羰酸鉀、1.0重量%之RNA混合之AIN-93G」食餌、投與「生理食鹽水」(10mL/kg)、5隻‧ Control group (Groups 2 and 6): The "AIN-93G mixed with 2.5% by weight of phenylaminocarbonate and 1.0% by weight of RNA" was administered, and "physiological saline" (10 mL/kg) was administered. only
‧菌體投與組(第3、4、7~9組):每組給予「與2.5重量%之苯胺羰酸鉀、1.0重量%之RNA混合之AIN-93G」食餌、一組5隻。各組投與菌體及投與量為:第3組投與「發酵乳酸桿菌MEP181504株(L.fermentum MEP181504)懸濁液(1×108 CFU/mL)」(10mL/kg)、第4組投與「短乳酸桿菌MEP181507株(L.brevis MEP181507)懸濁液(1×108 CFU/mL)」(10mL/kg)、第7組投與「口乳酸桿菌OLL2779株(L.oris OLL2779)懸濁液(1×108 CFU/mL)」(10mL/kg)、第8組投與「加氏乳酸桿菌JCM8787株(L.gasseri JCM8787)懸濁液(1×108 CFU/mL)」(10mL/kg)、第9組投與「加氏乳酸桿菌OLL2959株(L.gasseri OLL2959)懸濁液(1×108 CFU/mL)」(10mL/kg)。‧ Bacterial administration group (Groups 3, 4, 7 to 9): Each group was given a "bait of AIN-93G mixed with 2.5% by weight of potassium phenylaminocarbonate and 1.0% by weight of RNA", and a group of five. The amount of cells administered in each group and the amount administered were as follows: Group 3 was administered "Lactobacillus fermentum MEP181504 strain (L. fermentum MEP181504) suspension (1 × 10 8 CFU/mL)" (10 mL/kg), 4th The group was administered with "Lacillus brevis MEP181507 strain (L. brevis MEP181507) suspension (1 × 10 8 CFU/mL)" (10 mL / kg), and the seventh group was administered with "L. oris OLL2779 strain (L. oris OLL2779). Suspension (1×10 8 CFU/mL)” (10 mL/kg), Group 8 was administered with “L. gasseri JCM8787 suspension (1×10 8 CFU/mL) (10 mL/kg) and the ninth group were administered "L. gasseri OLL2959 strain (L.gasseri OLL2959) suspension (1 × 10 8 CFU/mL)" (10 mL/kg).
[正式飼育(試驗)][Formal breeding (test)]
分組後的次日起為試驗期間,「AIN-93G」飼料(陰性組)與「AIN-93G+苯胺羰酸鉀+RNA」飼料(對照組、菌體投與組)分別以給餌器讓大鼠於8日間自由攝食。上述飼料的給餌開始日作為第1日,以後按日序起算日數。「AIN-93G+苯胺羰酸鉀+RNA」飼料係含有2.5重量%之苯胺羰酸鉀(100g、ALDRICH產品)、1.0重量%之RNA(500g、MP Biomedicals.Inc.產品)。菌體投與組之實驗動物係以上述菌體懸濁液強制經口投與1×109 CFU/10mL/kg。陰性組與對照組不投與菌體懸濁液,但將生理食鹽水強制經口投與10mL/kg。From the next day after grouping, the "AIN-93G" feed (negative group) and the "AIN-93G + aniline potassium carbonate + RNA" feed (control group, bacterial administration group) were given to the bait feeder. Free to eat on the 8th. The start date of the feed of the above feed is taken as the first day, and the number of days is counted in the order of the day. The "AIN-93G + potassium aniline potassium carbonate + RNA" feed contained 2.5% by weight of potassium phenylaminocarbonate (100 g, ALDRICH product) and 1.0% by weight of RNA (500 g, product of MP Biomedicals. Inc.). The experimental animals of the bacterial administration group were orally administered with 1 x 10 9 CFU/10 mL/kg by the above-mentioned bacterial suspension. The negative suspension group and the control group were not administered with the suspension of the cells, but the physiological saline was forcibly administered orally with 10 mL/kg.
[測定與檢查等][Measurement and inspection, etc.]
‧一般狀態觀察與體重測定‧General status observation and weight measurement
全體(所有組)皆從第1日開始到第8日連續每次投與時觀察一般狀態,第0日、第1日、第5日、第8日於午 前9~10時定時測量體重。All (all groups) observe the general state from the 1st to the 8th consecutive day, and the 0th, 1st, 5th, and 8th are in the afternoon. The weight is measured regularly from the first 9 to 10 o'clock.
‧攝食量與攝水量之測定‧ Determination of food intake and water intake
全體(所有組)在第1日(設定值)、第5日(殘值、設定值)、第8日(殘值)於午前9~10時定時測量攝食量與攝水量。The whole (all groups) measured the food intake and the amount of water intake at the first day (set value), the fifth day (residual value, the set value), and the eighth day (residual value) at 9:10 pm.
‧採血與生化學檢查‧ Blood collection and biochemical examination
全體(所有組)在第0日(午前)、第2日(投與1小時後)、第5日(投與1小時後)、第8日(投與前)由尾靜脈採血。採取的血液以轉速10000rpm離心10分鐘分離取出血清,以磷鎢酸法測定血清中之尿酸值。如前述,第0日測定當日血清中尿酸值,分組後再分別測定。All (all groups) were collected from the tail vein on day 0 (noon), day 2 (after 1 hour of administration), day 5 (after 1 hour of administration), and day 8 (before administration). The blood taken was centrifuged at 10,000 rpm for 10 minutes to separate the serum, and the uric acid value in the serum was measured by the phosphotungstic acid method. As described above, the uric acid value in the serum on the day of the measurement was measured on the 0th day, and then separately measured.
‧剖檢與生化學檢查‧ necropsy and biochemical examination
全體(所有組)在第8日由尾靜脈採血後,經口投與菌體懸濁液。投與1小時後以戊巴比妥(商品名Nembutal)(pentobarbital 40mg/kg)麻醉後,於腹大動脈全採血致死。採取之血液以轉速3000rpm離心15分鐘分離取出血清,測定血清中之肌酸酐、尿酸、尿素氮。All (all groups) were subjected to blood collection from the tail vein on the 8th day, and the suspension of the cells was orally administered. One hour later, after anesthesia with pentobarbital (trade name: Nembutal) (pentobarbital 40 mg/kg), blood was collected from the abdominal aorta. The blood taken was centrifuged at 3000 rpm for 15 minutes to remove the serum, and creatinine, uric acid, and urea nitrogen in the serum were measured.
‧臟器重量之測定‧ Determination of organ weight
摘出大鼠之腎臟,測定濕重量。The kidneys of the rats were removed and the wet weight was measured.
[統計處理][Statistical processing]
結果以平均值±標準差顯示,比較對照組與各菌體投與組。經數值化的檢查值分散比係以F檢定進行,等分散時以史氏T試驗(Student’s T test)進行分析,不等分散時以阿氏-惠氏T試驗(Aspin-Welch T test)進行分析。 統計處理係使用Excel 2004進行統計分析,最低顯著水準為兩側之5%。The results were shown as mean ± standard deviation, and the control group and each bacterial cell administration group were compared. The numerically-defined test value dispersion ratio is performed by F-test, and the dispersion is measured by Student's T test, and the unequal dispersion is analyzed by Aspin-Welch T test. . Statistical processing was performed using Excel 2004 for statistical analysis with a minimum significant level of 5% on both sides.
[2-2結果][2-2 results]
一般狀態的結果如表1(L.fermentum與L.brevis)與表2(L.oris與L.gasseri),血清尿酸值的推移則顯示於第4圖與第5圖。The results of the general state are shown in Table 1 (L. fermentum and L. brevis) and Table 2 (L. oris and L. gasseri), and the changes in serum uric acid values are shown in Figs. 4 and 5.
投與各種乳酸菌可使血清中尿酸值下降顯示於第5圖,L.oris OLL 2779株投與組與L.gasseri OLL 2959株投與組被認為具有統計上顯著差異。一般狀態顯示,不論何組的腎機能(肌酸酐值、血清中尿素氮、腎重量)皆無問題,且體重、攝食量、攝水量也沒有問題。此外,由一般狀態所見,各種乳酸菌間並無觀察出顯著差異。L.oris OLL 2779株與L.gasseri OLL 2959株的科學特徵如表3所示
(發酵乳的製造例1)(Production Example 1 of fermented milk)
以加氏乳酸桿菌OLL2959株(L.gasseri OLL2959)(NITE BP-224)、保加利亞乳酸桿菌JCM1002株(L.burgalicus JCM1002)、嗜熱鏈球菌ATCC19258株(S.thermophilus ATCC19258)調製原味優格。首先,採用脫脂奶粉10%之培養基,調製加氏乳酸桿菌OLL2959株(NITE BP-224)、保加利亞乳酸桿菌JCM1002株、嗜熱鏈球菌ATCC19258株之生產發酵劑(bulk starter)。接著,將優格混合物(無脂乳固形分(SNF):9.5%、脂肪分(FAT):3.0%)於95℃加熱處理5分鐘。對此加熱處理後的優格混合物接種保加利亞乳酸桿菌JCM1002株與嗜熱鏈球菌ATCC19258株之起始菌種各1%、加氏乳酸桿菌OLL2959株(NITE BP-224)之起始菌種5%,於43℃發酵4小時,得到原味優格。將此原味優格置於冷藏庫(5℃)冷卻後,確認其風味與物性。此時,風味與物性皆良好。The original yoghurt was prepared by Lactobacillus kawaii OLL2959 strain (L. gasseri OLL2959) (NITE BP-224), Lactobacillus bulgaricus JCM1002 strain (L. burgalicus JCM1002), and Streptococcus thermophilus ATCC19258 strain (S. thermophilus ATCC19258). First, a 10% medium of skim milk powder was used to prepare a bulk starter of Lactobacillus kawaii OLL2959 strain (NITE BP-224), Lactobacillus bulgaricus JCM1002 strain, and Streptococcus thermophilus ATCC19258 strain. Next, the Yogurt mixture (SLF-free: 9.5%, fat fraction (FAT): 3.0%) was heat-treated at 95 ° C for 5 minutes. The heat-treated yoghurt mixture was inoculated with 1% of the starting strains of Lactobacillus bulgaricus JCM1002 strain and Streptococcus thermophilus ATCC19258 strain, and 5% of the starting strain of Lactobacillus gasseri OLL2959 strain (NITE BP-224). The fermentation was carried out at 43 ° C for 4 hours to obtain the original yogurt. After the original yogurt was cooled in a refrigerator (5 ° C), the flavor and physical properties were confirmed. At this time, both flavor and physical properties were good.
(發酵乳的製造例2)(Production Example 2 of fermented milk)
以口乳酸桿菌OLL2779株(Lactobacillus oris OLL2779)(NITE BP-223)、保加利亞乳酸桿菌JCM1002株(L.burgalicus JCM1002)、嗜熱鏈球菌ATCC19258株(S.thermophilus ATCC19258)調製原味優格。首先,採用脫脂奶粉10%之培養基,調製口乳酸桿菌OLL2779株(NITE BP-223)、保加利亞乳酸桿菌JCM1002株、嗜熱鏈球菌ATCC19258株之生產發酵劑(bulk starter)。接著,將優 格混合物(無脂乳固形分(SNF):9.5%、脂肪分(FAT):3.0%)於95℃加熱處理5分鐘。對此加熱處理後的優格混合物接種保加利亞乳酸桿菌JCM1002株與嗜熱鏈球菌ATCC19258株之起始菌種各1%、口乳酸桿菌OLL2779株(NITE BP-223)之起始菌種5%,於43℃發酵4小時,得到原味優格。將此原味優格置於冷藏庫(5℃)冷卻後,確認其風味與物性。此時,風味與物性皆良好,確認口乳酸桿菌OLL2779株(NITE BP-223)極適於應用於食品製造。The original yoghurt was prepared by Lactobacillus oryzae OLL2779 strain (Lactobacillus oris OLL2779) (NITE BP-223), Lactobacillus bulgaricus JCM1002 strain (L. burgalicus JCM1002), and Streptococcus thermophilus ATCC19258 strain (S. thermophilus ATCC19258). First, a 10% medium of skim milk powder was used to prepare a bulk starter of Lactobacillus acidophilus OLL2779 strain (NITE BP-223), Lactobacillus bulgaricus JCM1002 strain, and Streptococcus thermophilus ATCC19258 strain. Then, will be excellent The mixture (SLF: 9.5%, fat fraction (FAT): 3.0%) was heat treated at 95 ° C for 5 minutes. The heat-treated yoghurt mixture was inoculated with 1% of the starting strains of Lactobacillus bulgaricus JCM1002 strain and Streptococcus thermophilus ATCC19258 strain, and 5% of the starting strain of Lactobacillus acidophilus OLL2779 strain (NITE BP-223). Fermentation was carried out at 43 ° C for 4 hours to obtain the original yogurt. After the original yogurt was cooled in a refrigerator (5 ° C), the flavor and physical properties were confirmed. At this time, both flavor and physical properties were good, and it was confirmed that Lactobacillus sp. OLL2779 strain (NITE BP-223) was extremely suitable for food production.
本發明提供可降低血液中尿酸值的乳酸菌。由於本發明之乳酸菌經口攝取可降低血液中尿酸值,本發明之乳酸菌可應用於預防及/或治療痛風及高尿酸血症之食品或醫藥品。特別是,本發明之乳酸菌在食品製造上,確認並無產氣之問題,適於將之應用於食品。此外,利用本發明之乳酸菌,可考慮製造降低嘌呤量之加工食品。因此,本發明之乳酸菌在食品及醫藥品產業上利用性極高。The present invention provides a lactic acid bacterium which can lower the uric acid value in blood. Since the lactic acid bacteria of the present invention can reduce the uric acid value in the blood by oral ingestion, the lactic acid bacteria of the present invention can be applied to foods or medicines for preventing and/or treating gout and hyperuricemia. In particular, the lactic acid bacteria of the present invention have been confirmed to have no problem of gas production in food production, and are suitable for use in foods. Further, with the lactic acid bacteria of the present invention, it is conceivable to produce a processed food having a reduced amount of mash. Therefore, the lactic acid bacteria of the present invention are highly utilized in the food and pharmaceutical industries.
第1圖係於嘌呤(肌苷)存在下培養各種乳酸菌時,各乳酸菌之嘌呤分解能力示意圖。認定為嘌呤分解能力高的菌株(星號)作為動物模式的實驗對象。Fig. 1 is a schematic diagram showing the decomposition ability of each lactic acid bacterium when various lactic acid bacteria are cultured in the presence of sputum (inosine). A strain (asterisk) which is considered to have a high decomposition ability is used as an experimental object of the animal model.
第2圖係於嘌呤(肌苷)存在下培養各種乳酸菌時,各乳酸菌之嘌呤分解能力示意圖。認定為嘌呤分解能力高的 菌株(星號)作為動物模式的實驗對象。Fig. 2 is a schematic diagram showing the decomposition ability of each lactic acid bacterium when various lactic acid bacteria are cultured in the presence of sputum (inosine). Recognized as high decomposing ability The strain (asterisk) was used as an experimental object of the animal model.
第3圖係發酵乳酸桿菌(L.fermentum)及短乳酸桿菌(L.brevis)之嘌呤分解能力以(次黃嘌呤量+鳥嘌呤量)/5-溴尿嘧啶進行評量之圖示。Fig. 3 is a graphical representation of the decomposing ability of Lactobacillus fermentum (L. fermentum) and Lactobacillus brevis (L. brevis) by (hypoxanthine + guanine) / 5-bromouracil.
第4圖係將嘌呤分解能力高之乳酸菌(發酵乳酸桿菌、短乳酸桿菌)經口投與食因性高尿酸血症動物模式,測定血清尿酸值之結果的示意圖。Fig. 4 is a schematic diagram showing the results of measuring the serum uric acid value by orally administering a lactic acid bacteria (fermentative lactobacillus, short Lactobacillus) having a high decomposing ability to an animal model of esophageal hyperuricemia.
第5圖係將嘌呤分解能力高之乳酸菌(口乳酸桿菌、加氏乳酸桿菌)經口投與食因性高尿酸血症動物模式,測定血清尿酸值之結果的示意圖。在口乳酸桿菌OLL2779株投與組(第7組)於投與開始後第2天及第5天,及在加氏乳酸桿菌OLL2959株投與組(第9組)於投與開始後第5天,可觀察到血清尿酸值上升的顯著抑制作用(圖中之#)。Fig. 5 is a schematic diagram showing the results of measuring the serum uric acid value by oral administration of a lactic acid bacteria (Lactobacillus brevis and Lactobacillus kawaii) having a high sputum decomposition ability to an animal model of esophageal hyperuricemia. In the Lactobacillus brevis OLL2779 strain administration group (Group 7) on the 2nd and 5th days after the start of administration, and in the Lactobacillus kawaii OLL2959 strain administration group (Group 9) after the start of administration 5th On the day, a significant inhibition of the increase in serum uric acid value (# in the figure) was observed.
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