JP2021046391A - Plant lactobacillus extracellular polysaccharide and action thereof in preparing ace inhibitor composition - Google Patents
Plant lactobacillus extracellular polysaccharide and action thereof in preparing ace inhibitor composition Download PDFInfo
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- JP2021046391A JP2021046391A JP2020137287A JP2020137287A JP2021046391A JP 2021046391 A JP2021046391 A JP 2021046391A JP 2020137287 A JP2020137287 A JP 2020137287A JP 2020137287 A JP2020137287 A JP 2020137287A JP 2021046391 A JP2021046391 A JP 2021046391A
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- lactic acid
- plant
- extracellular polysaccharide
- derived lactic
- polysaccharide
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Abstract
Description
本発明は、微生物技術分野に関し、具体的には、植物性乳酸菌細胞外多糖および植物性乳酸菌細胞外多糖がＡＣＥ阻害薬製造中に果たす作用に関する。 The present invention relates to the field of microbial technology, and specifically to the action of plant-derived lactic acid bacteria extracellular polysaccharides and plant-derived lactic acid bacteria extracellular polysaccharides during the production of ACE inhibitors.
多糖は、自然界においてありふれたバイオポリマーであり、すべての動物、植物および微生物に存在し、同じ単糖から構成されるホモ多糖と異なる単糖から構成されるヘテロ多糖とに分けられる。近年、多糖の活性に対する研究が大きく前進し、レンチナン、霊芝多糖など多くな多糖が抗腫瘍活性を有することが証明され、人参多糖が免疫力を強化する機能を有することが証明される。微生物多糖は、生産量が安定で、気候や地理に影響されにくいため、研究ブームを迎える。多糖の生物活性多糖の生物活性は、自身の水溶性と、分子量（Ｍｗ）と、置換度と置換基の四つの要素によって影響修飾される。化学修飾によって多糖の生物活性に明らかな強化又は新しい活性が生じ、新型薬に開発できることが、多くの研究によって発見される。多糖の分子修飾と構造改造は重要意味を有する。 Polysaccharides are common biopolymers in nature and are present in all animals, plants and microorganisms and are divided into homopolysaccharides composed of the same monosaccharides and heteropolysaccharides composed of different monosaccharides. In recent years, research on the activity of polysaccharides has made great progress, and it has been proved that many polysaccharides such as lentinan and Ganoderma lucidum have antitumor activity, and that ginseng polysaccharide has a function of strengthening immunity. Microbial polysaccharides enter a research boom because their production is stable and they are not easily affected by climate and geography. Biological activity of polysaccharides The biological activity of polysaccharides is influenced and modified by four factors: their water solubility, their molecular weight (Mw), their degree of substitution and their substituents. Many studies have found that chemical modifications result in obvious enhancements or new activities in the biological activity of polysaccharides that can be developed into new drugs. Molecular modification and structural modification of polysaccharides are important.
微生物多糖の免疫調節活性は主に、マクロファージとナチュラルキラー細胞を活性化させることに体現される。中国には、良質な乳酸菌資源を有し、多くの伝統発酵食品には乳酸菌が含まれ、これらの資源に対し、研究や開発を行うことで、国民生活を改善することに役立てる。菌体外多糖乳酸菌の有益機能は主に自身の菌体外多糖によって決められると思う学者もいる。乳酸菌の菌体外多糖（Ｅｘｏｐｏｌｙｓａｃｃｈａｒｉｄｅｓ，□ＥＰＳ）は一般、菌体の表面に緊密に結合する細胞結合性の菌体外多糖と、周囲の液体環境に放出される放出多糖（Ｒｅｌｅａｓｅｄ□ｅｘｏｐｏｌｙｓａｃｃｈａｒｉｄｅｓ，□ｒ−ＥＰＳ）に分けられる。報道される大部分の乳酸菌は、ｒ−ＥＰＳだけを生じるが、Ｃ−ＥＰＳとｒ−ＥＰＳを同時に生じる乳酸菌もある。現在、報道された乳酸菌細胞外多糖乳酸菌は主に３０種類くらいある。乳酸菌細胞外多糖が免疫調節作用を強化させる機能を有することに対し、乳酸菌細胞外多糖の抽出が簡単であり、遠心によって菌体と液体を分離させ、そして液体を濃縮してエタノールを添加すればｒ−ＥＰＳが沈殿する。一部のウェルシュ菌は、Ｃ−ＥＰＳの抽出に使用されることができ、また、他の微生物多糖に比べ、乳酸菌細胞外多糖は、安全性がよく、各種食品と薬に応用できる。したがって、乳酸菌細胞外多糖に対する研究は非常に必要である。 The immunomodulatory activity of microbial polysaccharides is primarily embodied in the activation of macrophages and natural killer cells. China has high-quality lactic acid bacteria resources, and many traditional fermented foods contain lactic acid bacteria, and research and development of these resources will help improve people's lives. Some scholars believe that the beneficial functions of exopolysaccharide lactic acid bacteria are mainly determined by their own exopolysaccharide. Lactic acid bacteria exopolysaccharides (Exopolysaccharides, □ EPS) are generally cell-binding exopolysaccharides that bind tightly to the surface of the bacterium and released polysaccharides (Released □ exopolysaccharides, □) that are released into the surrounding liquid environment. It is divided into r-EPS). Most of the reported lactic acid bacteria produce only r-EPS, but some lactic acid bacteria produce C-EPS and r-EPS at the same time. Currently, there are mainly about 30 types of lactic acid bacteria extracellular polysaccharide lactic acid bacteria reported. Lactobacillus extracellular polysaccharide has a function of enhancing immunomodulatory action, whereas extraction of lactic acid bacterium extracellular polysaccharide is easy, if the cells and liquid are separated by centrifugation, and the liquid is concentrated and ethanol is added. r-EPS precipitates. Some Clostridium perfringens can be used for extraction of C-EPS, and lactic acid bacteria extracellular polysaccharides are safer than other microbial polysaccharides and can be applied to various foods and medicines. Therefore, research on lactic acid bacteria extracellular polysaccharides is very necessary.
本発明は、上記の技術的問題を解決するために、顕著なＡＣＥ阻害活性を示す、細胞傷害性を有しない植物性乳酸菌細胞外多糖を提供することを目的とする。 An object of the present invention is to provide a non-cytotoxic plant-derived lactic acid bacterium extracellular polysaccharide that exhibits remarkable ACE inhibitory activity in order to solve the above technical problems.
上記の目的を実現するために、本発明の植物性乳酸菌細胞外多糖は、１）と２）の少なくともいずれか一つの特徴を有し、
前記１）は、重量平均分子量は、１．３５×１０６Ｄａであり、
前記２）は、モル比が５．６：７．３：１のマンノースと、ガラクトースとブドウ糖から構成される。
In order to achieve the above object, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention has at least one of the characteristics of 1) and 2).
In 1), the weight average molecular weight is 1.35 × 106 Da.
The above 2) is composed of mannose having a molar ratio of 5.6: 7.3: 1, galactose and glucose.
前記植物性乳酸菌細胞外多糖は、アンジオテンシン変換酵素（ＡＣＥ）を阻害できる。本発明の植物性乳酸菌細胞外多糖は、アンジオテンシン変換酵素（ＡＣＥ）阻害活性を示し、血圧調節および高血圧予防の機能の作用を果たし、したがって、本発明の植物性乳酸菌細胞外多糖を心臓血管疾患予防・治療のための組成物の活性成分に使用できる。かつ、本発明の植物性乳酸菌細胞外多糖は、細胞傷害性を有しない。 The plant-derived lactic acid bacterium extracellular polysaccharide can inhibit angiotensin converting enzyme (ACE). The plant-derived lactic acid bacterium extracellular polysaccharide of the present invention exhibits angiotensin converting enzyme (ACE) inhibitory activity and fulfills the functions of blood pressure regulation and hypertension prevention. Therefore, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention can be used for cardiovascular disease prevention. -Can be used as an active ingredient in therapeutic compositions. Moreover, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention does not have cytotoxicity.
本発明は、植物性乳酸菌細胞外多糖がＡＣＥ阻害薬組成物の調製時の作用を更に提供する。本発明の細胞外多糖は、顕著なＡＣＥ阻害活性を示し、かつ細胞傷害性がなく、ＡＣＥ活性阻害剤組成物として有効に使用することができる。 The present invention further provides the action of plant-derived lactic acid bacteria extracellular polysaccharides in the preparation of ACE inhibitor compositions. The extracellular polysaccharide of the present invention exhibits remarkable ACE inhibitory activity, is not cytotoxic, and can be effectively used as an ACE activity inhibitor composition.
本発明は、植物性乳酸菌細胞外多糖のリン酸化誘導体を更に提供する。本発明の植物性乳酸菌細胞外多糖のリン酸化誘導体は、ＡＣＥ阻害活性を示し、血圧調節および高血圧予防の作用を有し、且つかつ細胞傷害性を有しない。 The present invention further provides a phosphorylated derivative of a plant-derived lactic acid bacterium extracellular polysaccharide. The phosphorylated derivative of the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention exhibits ACE inhibitory activity, has an action of regulating blood pressure and preventing hypertension, and has no cytotoxicity.
前記植物性乳酸菌細胞外多糖の置換度は、０．２４である。 The degree of substitution of the plant-derived lactic acid bacterium extracellular polysaccharide is 0.24.
本発明は、心臓血管疾患予防・治療用の組成物を更に提供し、前記細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを含む。前記組成物は、食品組成物又は医薬組成物であり、組成物全重量に対して、細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを０．１〜８０ｗｔ％含む。 The present invention further provides a composition for the prevention and treatment of cardiovascular diseases, and includes at least one of the extracellular polysaccharide and a phosphorylated derivative thereof. The composition is a food composition or a pharmaceutical composition, and contains 0.1 to 80 wt% of at least one of an extracellular polysaccharide and a phosphorylated derivative thereof based on the total weight of the composition.
前記心血管疾患は、高血圧、心疾患、脳卒中、血栓、動脈硬化、狭心症、心不全および心筋梗塞からなる群から選択される一種または複数種の疾患である。 The cardiovascular disease is one or more diseases selected from the group consisting of hypertension, heart disease, stroke, thrombosis, atherosclerosis, angina, heart failure and myocardial infarction.
前記組成物は、経口組成物である。 The composition is an oral composition.
前記組成物は、成人が一日あたりに０．１−５００ｍｇの植物性乳酸菌細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを摂取する方式で使用される。好ましくは、毎日５０−１００ｍｇ／ｋｇであり、毎日１−３回である。 The composition is used in such a manner that an adult ingests 0.1-500 mg of at least one of a plant-derived lactic acid bacterium extracellular polysaccharide and a phosphorylated derivative thereof per day. Preferably, it is 50-100 mg / kg daily, 1-3 times daily.
前記組成物は、９−１２週間又はもっと長い時間で摂取する方式で使用される。 The composition is used in a manner of ingestion for 9-12 weeks or longer.
本発明は、従来技術と比べ、下記のメリットを有する。 The present invention has the following merits as compared with the prior art.
本発明の植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は、いずれもＡＣＥ阻害活性を示し、血圧調節および高血圧予防の機能を有し、これによって本発明の植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は、心臓血管疾患の予防と治療のための組成物の活性成分に使用できる。また、本発明の植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は、細胞傷害性を有しなく、幅広く応用されることができる。 The plant-derived lactic acid bacterium extracellular polysaccharide of the present invention and its phosphorylated derivative all exhibit ACE inhibitory activity and have functions of blood pressure regulation and hypertension prevention, whereby the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention and its phosphorylation are exhibited. Derivatives can be used as active ingredients in compositions for the prevention and treatment of cardiovascular disease. In addition, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention and its phosphorylated derivative have no cytotoxicity and can be widely applied.
本発明は、上記技術的解決手段を採用して、植物性乳酸菌細胞外多糖および植物性乳酸菌細胞外多糖がＡＣＥ阻害薬製造中に果たす作用を提供し、従来技術の不足を補い、デザインが合理的で、操作が便利である。 The present invention employs the above technical solutions to provide the effects of plant-derived lactic acid bacteria extracellular polysaccharides and plant-derived lactic acid bacteria extracellular polysaccharides during the production of ACE inhibitors, compensating for the lack of prior art, and rationalizing the design. Targeted and convenient to operate.
下記に、本発明について、詳しく説明します。 The present invention will be described in detail below.
本実施方法は、植物性乳酸菌細胞外多糖を提供し、具体的には１）と２）の少なくともいずれか一つの特徴を有する。
１）重量平均分子量は、１．３５×１０６Ｄａであり、
２）モル比が５．６：７．３：１のマンノースと、ガラクトースとブドウ糖から構成される。
The present method provides a plant-derived lactic acid bacterium extracellular polysaccharide, and specifically has at least one of the characteristics of 1) and 2).
1) The weight average molecular weight is 1.35 × 106 Da.
2) It is composed of mannose with a molar ratio of 5.6: 7.3: 1, galactose and glucose.
好ましい具体例として、前記植物性乳酸菌細胞外多糖は、アンジオテンシン変換酵素（ＡＣＥ）を阻害できる。本発明の植物性乳酸菌細胞外多糖は、アンジオテンシン変換酵素（ＡＣＥ）阻害活性を示し、血圧調節および高血圧予防の機能の作用を果たし、したがって、本発明の植物性乳酸菌細胞外多糖を心臓血管疾患予防・治療のための組成物の活性成分に使用できる。かつ、本発明の植物性乳酸菌細胞外多糖は、細胞傷害性を有しない。 As a preferred embodiment, the plant-derived lactic acid bacterium extracellular polysaccharide can inhibit angiotensin converting enzyme (ACE). The plant-derived lactic acid bacterium extracellular polysaccharide of the present invention exhibits angiotensin converting enzyme (ACE) inhibitory activity and fulfills the functions of blood pressure regulation and hypertension prevention. Therefore, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention can be used for cardiovascular disease prevention. -Can be used as an active ingredient in therapeutic compositions. Moreover, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention does not have cytotoxicity.
本実施方法の植物性乳酸菌細胞外多糖は、
植物性乳酸菌を発酵させ、発酵液を収集するステップ１と、
トリクロロ酢酸水溶液と発酵液とを撹拌混合し、反応させ、反応液に含まれる上澄み液を収集するステップ２と、
上澄み液と無水エタノールとを撹拌混合し静置し、沈殿物を収集するステップ３と、
沈殿物を水に溶解させ、透析純化し、植物性乳酸菌細胞外多糖を得るステップ４と、
の方法によって調製される。
The plant-derived lactic acid bacteria extracellular polysaccharide of this method is
Step 3 of mixing the supernatant and absolute ethanol with stirring and allowing them to stand to collect the precipitate, and
It is prepared by the method of.
好ましい具体例として、植物性乳酸菌細胞外多糖の調製方法は、
植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、濃度０．０２〜０．１μＭのテトラエチルチウラムジスルフィド及び０．１〜０．５ｍＭのラクトースを含有するＭＲＳ培地に移し、３０〜４０℃で１２〜２４ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７０〜８０％のトリクロロ酢酸水溶液を均一に混合し、常温で１〜２ｈ撹拌反応させ、反応液中の上澄み液を採取し、発酵液中の細胞及びタンパク質を除去するステップ２と、
上澄み液に２?３倍体積の無水エタノールを加え、均一に混合し、４℃で１０―１５ｈ静置し、さらに４℃、８０００?１５０００ｒ／ｍｉｎで２０―３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を５〜１０ｈ毎に交換し、１２―２４ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖をイオン交換カラムクロマトグラフィー及びゲルカラムにより精製し、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、
を含み、
前記ステップ１において、テトラエチルチウラムジスルフィド及びラクトースを含有するＭＲＳ培地を使用することで、細胞外多糖の化学構造を変えて本発明の植物性乳酸菌細胞外多糖を得ることができるだけでなく、植物性乳酸菌細胞外多糖の対数期及び安定期の延長に有利であり、細胞外多糖を大量に合成して、植物性乳酸菌細胞外の多糖産生力を向上させ、最終的に細胞外多糖の収量を高めることができる。
As a preferred specific example, a method for preparing a plant-derived lactic acid bacterium extracellular polysaccharide is described.
The plant-derived lactic acid bacterium ATCC8014 was continuously activated for two generations, and then transferred to an MRS medium containing tetraethylthiuram disulfide having a concentration of 0.02 to 0.1 μM and lactose of 0.1 to 0.5 mM at 30 to 40 °
The fermentation broth and a 70 to 80% trichloroacetic acid aqueous solution are uniformly mixed, stirred and reacted at room temperature for 1 to 2 hours, the supernatant liquor in the reaction broth is collected, and cells and proteins in the fermented broth are removed. ,
Add 2 to 3 times the volume of absolute ethanol to the supernatant, mix uniformly, allow to stand at 4 ° C for 10 to 15 hours, and further centrifuge at 4 ° C and 8000 to 15000 r / min for 20 to 30 minutes to collect the precipitate. Step 3 and
The step of dissolving the precipitate in water, transferring to a dialysis bag, exchanging deionized water every 5 to 10 hours, dialyzing for 12 to 24 hours, then collecting the product and lyophilizing to obtain crude extracellular polysaccharide. 4 and
Including
In
本発明は、植物性乳酸菌細胞外多糖がＡＣＥ阻害薬組成物の調製時の作用を更に提供する。多糖分子は、リン酸化修飾された後、分岐鎖上の水酸基がリン酸基で置換され、多糖水溶性を高め、鎖立体配座を改変することにより、本発明の細胞外多糖は、顕著なＡＣＥ阻害活性を示し、かつ細胞傷害性がなく、ＡＣＥ活性阻害剤組成物として有効に使用することができる。 The present invention further provides the action of plant-derived lactic acid bacteria extracellular polysaccharides in the preparation of ACE inhibitor compositions. After phosphorylation modification of the polysaccharide molecule, the hydroxyl group on the branched chain is replaced with a phosphate group to increase the water solubility of the polysaccharide and modify the chain configuration, whereby the extracellular polysaccharide of the present invention is remarkable. It exhibits ACE inhibitory activity and is not cytotoxic, and can be effectively used as an ACE activity inhibitor composition.
本発明は、植物性乳酸菌細胞外多糖のリン酸化誘導体を更に提供する。 The present invention further provides a phosphorylated derivative of a plant-derived lactic acid bacterium extracellular polysaccharide.
好ましい具体例として、植物性乳酸菌細胞外多糖のリン酸化誘導体は、植物性乳酸菌細胞外多糖を、固液比１：１０―３０ｇ／ｍＬでジメチルスルホキシドに溶解し、５０―７０℃で２０―４０ｍｉｎ加熱して十分に溶解させた後、尿素およびリン酸を加え、５０―７０℃で２―５ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを使用して反応液を中和し、透析し、凍結乾燥することにより、調製される。前記植物性乳酸菌細胞外多糖のリン酸化誘導体の調製方法において、植物性乳酸菌細胞外多糖と尿素の重量比は、１：１５−２０であり、植物性乳酸菌細胞外多糖とリン酸の固液比は、１：５−１０ｇ／ｍＬであり、植物性乳酸菌細胞外多糖と脱イオン水の固液比は、１：８−１２ｇ／ｍＬである。 As a preferred embodiment, the phosphorylated derivative of the plant-derived lactic acid bacterium extracellular polysaccharide dissolves the plant-derived lactic acid bacterium extracellular polysaccharide in dimethyl sulfoxide at a solid-liquid ratio of 1:10 to 30 g / mL and 20 to 40 min at 50 to 70 ° C. After heating to sufficiently dissolve, urea and phosphoric acid are added, and the reaction is carried out at 50-70 ° C. for 2-5 hours, then deionized water is added to terminate the reaction, and the reaction solution is completed using 1 M NaOH. Is prepared by neutralizing, dialyzing, and lyophilizing. In the method for preparing a phosphorylated derivative of a plant-derived lactic acid bacterium extracellular polysaccharide, the weight ratio of the plant-derived lactic acid bacterium extracellular polysaccharide to urea is 1: 15-20, and the solid-liquid ratio of the plant-derived lactic acid bacterium extracellular polysaccharide to phosphoric acid. Is 1: 5-10 g / mL, and the solid-liquid ratio of plant-derived lactic acid bacteria extracellular polysaccharide to deionized water is 1: 8-12 g / mL.
好ましい具体例として、植物性乳酸菌細胞外多糖の置換度は、０．２４である。 As a preferred specific example, the degree of substitution of the plant-derived lactic acid bacterium extracellular polysaccharide is 0.24.
本発明は、心臓血管疾患予防・治療用の組成物を更に提供し、前記細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを含む。該組成物は、食品組成物又は医薬組成物であり、組成物全重量に対して、細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを０．１〜８０ｗｔ％含む。本発明の食品組成物に関し、本発明の細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを飲料、肉、ソーセージ、パン、ビスケット、餅、チョコレート、キャンディ、菓子、クッキー、パンケーキ、ラーメン、チューインガム、乳製品（アイスクリームを含む）、特殊栄養食（例えば、配合粉乳や幼児食）、加工肉製品、魚製品、豆腐、でんぷんゲル製品、健康補助食品、調味食品（例えば醤油、豆弁ジャム、唐辛子、かき揚げジャム）、ソース、その他の加工食品、漬物（例えば、漬物やソース野菜）、スープ、飲料、アルコール飲料及びビタミン複合体に添加することができ、広い意味では、健康食品の生産に適した食品がほとんど含まれているといえる。本発明の医薬組成物はさらに様々な栄養素、ビタミン、ミネラル、調味剤、着色剤、ペクチン酸及びその塩、アルギン酸及びその塩、有機酸、保護コロイド増粘剤、ｐＨ調整剤、安定剤、防腐剤、グリセリン、アルコール、ソーダ水に添加するための炭酸化剤等を含むことができる。前記担体、賦形剤又は希釈剤は、ラクトース、グルコース、スクロース、ソルビトール、マンニトール、キシリトール、エリスリトール、マルチトール、デンプン、アラビアガム、アルギン酸塩／エステル、ゼラチン、リン酸カルシウム、ケイ酸カルシウム、セルロース、メチルセルロース、微結晶セルロース、ポリビニルピロリドン、水、ヒドロキシ安息香酸メチル、ヒドロキシ安息香酸プロピル、タルク、ステアリン酸マグネシウム、鉱物油、デキストリン、炭酸カルシウム、プロピレングリコール、流動パラフィン及び塩水からなる群から選択されるが、必ずしもこれに限定されるものではない。なお、全ての成分は、単独で添加してもよく、また、共に添加してもよい。 The present invention further provides a composition for the prevention and treatment of cardiovascular diseases, and includes at least one of the extracellular polysaccharide and a phosphorylated derivative thereof. The composition is a food composition or a pharmaceutical composition and contains 0.1 to 80 wt% of at least one of an extracellular polysaccharide and a phosphorylated derivative thereof based on the total weight of the composition. With respect to the food composition of the present invention, at least one of the extracellular polysaccharide of the present invention and a phosphorylated derivative thereof is used as a beverage, meat, sausage, bread, biscuits, rice cake, chocolate, candy, confectionery, cookie, pancake, ramen. , Chewing gum, dairy products (including ice cream), special nutrition foods (eg compound milk powder and infant food), processed meat products, fish products, tofu, starch gel products, health supplements, seasoning foods (eg soy sauce, bean biscuits) Can be added to jams, peppers, fried jams), sauces and other processed foods, pickles (eg pickles and sauce vegetables), soups, beverages, alcoholic beverages and vitamin complexes, in a broad sense the production of health foods It can be said that most of the foods suitable for are contained. The pharmaceutical compositions of the present invention further include various nutrients, vitamins, minerals, seasonings, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives. It can contain agents, glycerin, alcohol, carbonating agents for addition to soda water, and the like. The carriers, excipients or diluents include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, martitol, starch, arabic gum, alginate / ester, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, etc. It is selected from the group consisting of microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin and salt water, but not necessarily. It is not limited to this. All the components may be added alone or together.
好ましい具体例として、心血管疾患は、高血圧、心疾患、脳卒中、血栓、動脈硬化、狭心症、心不全および心筋梗塞からなる群から選択される一種または複数種の疾患である。 As a preferred embodiment, cardiovascular disease is one or more diseases selected from the group consisting of hypertension, heart disease, stroke, thrombosis, atherosclerosis, angina, heart failure and myocardial infarction.
好ましい具体例として、前記組成物は、経口組成物である。 As a preferred embodiment, the composition is an oral composition.
好ましい具体例として、前記組成物は、成人が一日あたりに０．１−５００ｍｇの植物性乳酸菌細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを摂取する方式で使用される。好ましくは、毎日５０−１００ｍｇ／ｋｇであり、毎日１−３回である。 As a preferred embodiment, the composition is used in such a manner that an adult ingests 0.1-500 mg of a plant-derived lactic acid bacterium extracellular polysaccharide and at least one of a phosphorylated derivative thereof per day. Preferably, it is 50-100 mg / kg daily, 1-3 times daily.
好ましい具体例として、前記組成物は、９−１２週間又はもっと長い時間で摂取する方式で使用される。 As a preferred embodiment, the composition is used in a manner of ingestion for 9-12 weeks or longer.
下記に、実施例によって本発明を更に説明する。理解すべきは、前記実施例は本発明を実施する一案であり、本発明の範囲を制限しない。 The present invention will be further described below by way of examples. It should be understood that the above embodiment is an embodiment of the present invention and does not limit the scope of the present invention.
実施例１： Example 1:
本発明の植物性乳酸菌細胞外多糖の調製方法は、
上海北諾生物科技有限公司から購入の植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、濃度０．０５μＭのテトラエチルチウラムジスルフィド及び０．２ｍＭのラクトースを含有するＭＲＳ培地に移し、３７℃で１５ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７５％のトリクロロ酢酸水溶液を均一に混合し、常温で２ｈ撹拌反応させ、反応液中の上澄み液を採取するステップ２と、
上澄み液に２倍体積の無水エタノールを加え、均一に混合し、４℃で１２ｈ静置し、さらに４℃、１２０００ｒ／ｍｉｎで３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を８ｈ毎に交換し、１５ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖を蒸留水で溶解（１０ｍｇ／ｍＬ）した後、ＤＥＡＥ?Ｃｅｌｌｕｌｏｓｅ５２イオン交換カラムに１００ｍｇローディングし、蒸留水と、０．１ｍｏｌ／ＬのＮａＣｌと、０．３ｍｏｌ／ＬのＮａＣｌおよび０．５ｍｏｌ／ＬのＮａＣｌで直線的に勾配溶出し、溶出速度が１ｍＬ／ｍｉｎであり、チューブ毎に５ｍＬを採取し、多糖含有量（多糖含有量は硫酸フェノール法で検出して溶出曲線を描く）をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分をそれぞれ合わせて収集し、脱イオン水で透析、凍結乾燥するステップ４と、
０．３ｍｏｌ／ＬのＮａＣｌで溶出した多糖成分を蒸留水で溶解（５ｍｇ／ｍＬ）した後、ＳｅｐｈａｄｅｘＧ?１００ゲルカラムにローディングし、脱イオン水を用いて溶出し、溶出レートが０．２ｍＬ／ｍｉｎであり、溶出液を自動部分収集装置で収集し、チューブ毎に体積２ｍＬを収集し、多糖含有量をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分を合わせて収集し、脱イオン水で透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、
を含む。
The method for preparing the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention is:
The plant-derived lactic acid bacterium ATCC8014 purchased from Shanghai Hokujo Biotechnology Co., Ltd. was continuously activated for two generations, and then transferred to an MRS medium containing 0.05 μM of tetraethylthiuram disulfide and 0.2 mM lactose at 37 °
In
The precipitate is dissolved in water, transferred to a dialysis bag, the deionized water is replaced every 8 hours, dialyzed for 15 hours, and then the product is collected and lyophilized to obtain crude extracellular polysaccharide.
After dissolving the crude extracellular polysaccharide in distilled water (10 mg / mL), 100 mg was loaded on a DEAE? Cellulose 52 ion exchange column, and distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0 were loaded. Linear gradient elution with .5 mol / L NaCl, elution rate is 1 mL / min, 5 mL is collected for each tube, and polysaccharide content (polysaccharide content is detected by the phenol sulfate method and an elution curve is drawn. ) Is detected for each tube, single peak components are collected together according to the polysaccharide content detection value, dialyzed with deionized water, and freeze-dried.
The polysaccharide component eluted with 0.3 mol / L NaCl is dissolved in distilled water (5 mg / mL), loaded onto a Sephadex G-100 gel column, eluted with deionized water, and the elution rate is 0.2 mL / min. The eluate is collected by an automatic partial collector, a volume of 2 mL is collected for each tube, the polysaccharide content is detected for each tube, and a single peak component is collected together according to the polysaccharide content detection value, and then removed.
including.
植物性乳酸菌細胞外多糖の収率は、収率（％）＝（純成分質量／粗細胞外多糖質量）×１００で計算される。
算出した植物性乳酸菌細胞外多糖の収率は、５３．７２％である。
The yield of plant-derived lactic acid bacteria extracellular polysaccharide is calculated by yield (%) = (pure component mass / crude extracellular polysaccharide mass) × 100.
The calculated yield of plant-derived lactic acid bacterium extracellular polysaccharide is 53.72%.
実施例２ Example 2
植物性乳酸菌細胞外多糖のリン酸化誘導体の調整方法は、下記のようにである。 The method for preparing a phosphorylated derivative of a plant-derived lactic acid bacterium extracellular polysaccharide is as follows.
実施例１で得られた植物性乳酸菌細胞外多糖２ｇを１００ｍＬのジメチルスルホキシドに溶解し、６０℃で３０ｍｉｎ加熱して十分に溶解させた後、３６ｇの尿素と１５ｍＬのリン酸を加え、６０℃で３ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを用いて反応液を中和し、透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖のリン酸化誘導体を得る。 2 g of the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Example 1 was dissolved in 100 mL of dimethyl sulfoxide, heated at 60 ° C. for 30 minutes to sufficiently dissolve it, and then 36 g of urea and 15 mL of phosphoric acid were added to add 60 ° C. After reacting for 3 hours with, deionized water was added to terminate the reaction, the reaction solution was neutralized with 1 M NaOH, dialyzed, and freeze-dried to obtain a phosphorylated derivative of the extracellular polysaccharide of plant-derived lactic acid bacteria. obtain.
比較例１ Comparative Example 1
植物性乳酸菌細胞外多糖を、
上海北諾生物科技有限公司から購入の植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、濃度０．０５μＭのテトラエチルチウラムジスルフィドを含有するＭＲＳ培地に移し、３７℃で１５ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７５％のトリクロロ酢酸水溶液を均一に混合し、常温で２ｈ撹拌反応させ、反応液中の上澄み液を採取するステップ２と、
上澄み液に２倍体積の無水エタノールを加え、均一に混合し、４℃で１２ｈ静置し、さらに４℃、１２０００ｒ／ｍｉｎで３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を８ｈ毎に交換し、１５ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖を蒸留水で溶解（１０ｍｇ／ｍＬ）した後、ＤＥＡＥ?Ｃｅｌｌｕｌｏｓｅ５２イオン交換カラムに１００ｍｇローディングし、蒸留水と、０．１ｍｏｌ／ＬのＮａＣｌと、０．３ｍｏｌ／ＬのＮａＣｌおよび０．５ｍｏｌ／ＬのＮａＣｌで直線的に勾配溶出し、溶出速度が１ｍＬ／ｍｉｎであり、チューブ毎に５ｍＬを採取し、多糖含有量（多糖含有量は硫酸フェノール法で検出して溶出曲線を描く）をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分をそれぞれ合わせて収集し、脱イオン水で透析、凍結乾燥し、
０．３ｍｏｌ／ＬのＮａＣｌで溶出した多糖成分を蒸留水で溶解（５ｍｇ／ｍＬ）した後、ＳｅｐｈａｄｅｘＧ?１００ゲルカラムにローディングし、脱イオン水を用いて溶出し、溶出レートが０．２ｍＬ／ｍｉｎであり、溶出液を自動部分収集装置で収集し、チューブ毎に体積２ｍＬを収集し、多糖含有量をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分を合わせて収集し、脱イオン水で透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、によって調製し、収率が３６．７４％である。
Plant-derived lactic acid bacteria extracellular polysaccharide,
The plant-derived lactic acid bacterium ATCC8014 purchased from Shanghai Hokujo Biotechnology Co., Ltd. was continuously activated for two generations, then transferred to an MRS medium containing a tetraethylthiuram disulfide having a concentration of 0.05 μM, and allowed to ferment at 37 ° C. for 15 hours.
In
The precipitate is dissolved in water, transferred to a dialysis bag, the deionized water is replaced every 8 hours, dialyzed for 15 hours, and then the product is collected and lyophilized to obtain crude extracellular polysaccharide.
After dissolving the crude extracellular polysaccharide in distilled water (10 mg / mL), 100 mg was loaded on a DEAE? Cellulose 52 ion exchange column, and distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0 were loaded. Linear gradient elution with .5 mol / L NaCl, elution rate is 1 mL / min, 5 mL is collected for each tube, and polysaccharide content (polysaccharide content is detected by the phenol sulfate method and an elution curve is drawn. ) Is detected for each tube, single peak components are collected together according to the polysaccharide content detection value, dialyzed with deionized water, freeze-dried, and then dried.
The polysaccharide component eluted with 0.3 mol / L NaCl is dissolved in distilled water (5 mg / mL), loaded onto a Sephadex G-100 gel column, eluted with deionized water, and the elution rate is 0.2 mL / min. The eluate is collected by an automatic partial collector, a volume of 2 mL is collected for each tube, the polysaccharide content is detected for each tube, and a single peak component is collected together according to the polysaccharide content detection value, and then removed. It was prepared in
比較例２ Comparative Example 2
植物性乳酸菌細胞外多糖を、
上海北諾生物科技有限公司から購入の植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、濃度０．２ｍＭのラクトースを含有するＭＲＳ培地に移し、３７℃で１５ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７５％のトリクロロ酢酸水溶液を均一に混合し、常温で２ｈ撹拌反応させ、反応液中の上澄み液を採取するステップ２と、
上澄み液に２倍体積の無水エタノールを加え、均一に混合し、４℃で１２ｈ静置し、さらに４℃、１２０００ｒ／ｍｉｎで３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を８ｈ毎に交換し、１５ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖を蒸留水で溶解（１０ｍｇ／ｍＬ）した後、ＤＥＡＥ?Ｃｅｌｌｕｌｏｓｅ５２イオン交換カラムに１００ｍｇローディングし、蒸留水と、０．１ｍｏｌ／ＬのＮａＣｌと、０．３ｍｏｌ／ＬのＮａＣｌおよび０．５ｍｏｌ／ＬのＮａＣｌで直線的に勾配溶出し、溶出速度が１ｍＬ／ｍｉｎであり、チューブ毎に５ｍＬを採取し、多糖含有量（多糖含有量は硫酸フェノール法で検出して溶出曲線を描く）をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分をそれぞれ合わせて収集し、脱イオン水で透析、凍結乾燥し、
０．３ｍｏｌ／ＬのＮａＣｌで溶出した多糖成分を蒸留水で溶解（５ｍｇ／ｍＬ）した後、ＳｅｐｈａｄｅｘＧ?１００ゲルカラムにローディングし、脱イオン水を用いて溶出し、溶出レートが０．２ｍＬ／ｍｉｎであり、溶出液を自動部分収集装置で収集し、チューブ毎に体積２ｍＬを収集し、多糖含有量をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分を合わせて収集し、脱イオン水で透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、によって調製し、収率が４３．６８％である。
Plant-derived lactic acid bacteria extracellular polysaccharide,
The plant-derived lactic acid bacterium ATCC8014 purchased from Shanghai Hokuyo Biotechnology Co., Ltd. was continuously activated for two generations, then transferred to an MRS medium containing lactose at a concentration of 0.2 mM, and allowed to ferment at 37 ° C. for 15 hours for fermentation.
In
The precipitate is dissolved in water, transferred to a dialysis bag, the deionized water is replaced every 8 hours, dialyzed for 15 hours, and then the product is collected and lyophilized to obtain crude extracellular polysaccharide.
After dissolving the crude extracellular polysaccharide in distilled water (10 mg / mL), 100 mg was loaded on a DEAE? Cellulose 52 ion exchange column, and distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0 were loaded. Linear gradient elution with .5 mol / L NaCl, elution rate is 1 mL / min, 5 mL is collected for each tube, and polysaccharide content (polysaccharide content is detected by the phenol sulfate method and an elution curve is drawn. ) Is detected for each tube, single peak components are collected together according to the polysaccharide content detection value, dialyzed with deionized water, freeze-dried, and then dried.
The polysaccharide component eluted with 0.3 mol / L NaCl is dissolved in distilled water (5 mg / mL), loaded onto a Sephadex G-100 gel column, eluted with deionized water, and the elution rate is 0.2 mL / min. The eluate is collected by an automatic partial collector, a volume of 2 mL is collected for each tube, the polysaccharide content is detected for each tube, and a single peak component is collected together according to the polysaccharide content detection value, and then removed. It was prepared in
比較例３ Comparative Example 3
植物性乳酸菌細胞外多糖を、
上海北諾生物科技有限公司から購入の植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、ＭＲＳ培地に移し、３７℃で１５ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７５％のトリクロロ酢酸水溶液を均一に混合し、常温で２ｈ撹拌反応させ、反応液中の上澄み液を採取するステップ２と、
上澄み液に２倍体積の無水エタノールを加え、均一に混合し、４℃で１２ｈ静置し、さらに４℃、１２０００ｒ／ｍｉｎで３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を８ｈ毎に交換し、１５ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖を蒸留水で溶解（１０ｍｇ／ｍＬ）した後、ＤＥＡＥ?Ｃｅｌｌｕｌｏｓｅ５２イオン交換カラムに１００ｍｇローディングし、蒸留水と、０．１ｍｏｌ／ＬのＮａＣｌと、０．３ｍｏｌ／ＬのＮａＣｌおよび０．５ｍｏｌ／ＬのＮａＣｌで直線的に勾配溶出し、溶出速度が１ｍＬ／ｍｉｎであり、チューブ毎に５ｍＬを採取し、多糖含有量（多糖含有量は硫酸フェノール法で検出して溶出曲線を描く）をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分をそれぞれ合わせて収集し、脱イオン水で透析、凍結乾燥し、
０．３ｍｏｌ／ＬのＮａＣｌで溶出した多糖成分を蒸留水で溶解（５ｍｇ／ｍＬ）した後、ＳｅｐｈａｄｅｘＧ?１００ゲルカラムにローディングし、脱イオン水を用いて溶出し、溶出レートが０．２ｍＬ／ｍｉｎであり、溶出液を自動部分収集装置で収集し、チューブ毎に体積２ｍＬを収集し、多糖含有量をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分を合わせて収集し、脱イオン水で透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、によって調製し、収率が３５．０１％である。
Plant-derived lactic acid bacteria extracellular polysaccharide,
In
The precipitate is dissolved in water, transferred to a dialysis bag, the deionized water is replaced every 8 hours, dialyzed for 15 hours, and then the product is collected and lyophilized to obtain crude extracellular polysaccharide.
After dissolving the crude extracellular polysaccharide in distilled water (10 mg / mL), 100 mg was loaded on a DEAE? Cellulose 52 ion exchange column, and distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0 were loaded. Linear gradient elution with .5 mol / L NaCl, elution rate is 1 mL / min, 5 mL is collected for each tube, and polysaccharide content (polysaccharide content is detected by the phenol sulfate method and an elution curve is drawn. ) Is detected for each tube, single peak components are collected together according to the polysaccharide content detection value, dialyzed with deionized water, freeze-dried, and then dried.
The polysaccharide component eluted with 0.3 mol / L NaCl is dissolved in distilled water (5 mg / mL), loaded onto a Sephadex G-100 gel column, eluted with deionized water, and the elution rate is 0.2 mL / min. The eluate is collected by an automatic partial collector, a volume of 2 mL is collected for each tube, the polysaccharide content is detected for each tube, and a single peak component is collected together according to the polysaccharide content detection value, and then removed. It was prepared in
比較例４ Comparative Example 4
比較例１で得た植物性乳酸菌細胞外多糖２ｇを１００ｍＬのジメチルスルホキシドに溶解し、６０℃で３０ｍｉｎ加熱して十分に溶解させた後、３６ｇの尿素と１５ｍＬのリン酸を加え、６０℃で３ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを用いて反応液を中和し、透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖のリン酸化誘導体を得る。 2 g of the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Comparative Example 1 was dissolved in 100 mL of dimethyl sulfoxide, heated at 60 ° C. for 30 minutes to sufficiently dissolve it, and then 36 g of urea and 15 mL of phosphoric acid were added, and the temperature was increased to 60 ° C. After the reaction for 3 hours, deionized water is added to terminate the reaction, the reaction solution is neutralized with 1 M NaOH, dialyzed and freeze-dried to obtain a phosphorylated derivative of the extracellular polysaccharide of plant-derived lactic acid bacteria. ..
比較例５ Comparative Example 5
比較例２で得た植物性乳酸菌細胞外多糖２ｇを１００ｍＬのジメチルスルホキシドに溶解し、６０℃で３０ｍｉｎ加熱して十分に溶解させた後、３６ｇの尿素と１５ｍＬのリン酸を加え、６０℃で３ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを用いて反応液を中和し、透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖のリン酸化誘導体を得る。 2 g of the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Comparative Example 2 was dissolved in 100 mL of dimethyl sulfoxide, heated at 60 ° C. for 30 minutes to sufficiently dissolve it, and then 36 g of urea and 15 mL of phosphoric acid were added, and the temperature was increased to 60 ° C. After the reaction for 3 hours, deionized water is added to terminate the reaction, the reaction solution is neutralized with 1 M NaOH, dialyzed and freeze-dried to obtain a phosphorylated derivative of the extracellular polysaccharide of plant-derived lactic acid bacteria. ..
比較例６ Comparative Example 6
比較例３で得た植物性乳酸菌細胞外多糖２ｇを１００ｍＬのジメチルスルホキシドに溶解し、６０℃で３０ｍｉｎ加熱して十分に溶解させた後、３６ｇの尿素と１５ｍＬのリン酸を加え、６０℃で３ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを用いて反応液を中和し、透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖のリン酸化誘導体を得る。 2 g of the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Comparative Example 3 was dissolved in 100 mL of dimethyl sulfoxide, heated at 60 ° C. for 30 minutes to sufficiently dissolve it, and then 36 g of urea and 15 mL of phosphoric acid were added, and the temperature was increased to 60 ° C. After the reaction for 3 hours, deionized water is added to terminate the reaction, the reaction solution is neutralized with 1 M NaOH, dialyzed and freeze-dried to obtain a phosphorylated derivative of the extracellular polysaccharide of plant-derived lactic acid bacteria. ..
試験例１ Test Example 1
植物性乳酸菌ＡＴＣＣ８０１４の成長曲線の測定に関する： Regarding the measurement of the growth curve of the plant-derived lactic acid bacterium ATCC8014:
菌株を、２％の接種量で、実施例１と比較例１?３に使用の培地に、それぞれ接種し、振とうして振り混ぜた後３７℃で静置発酵し、２ｈおきに取り出し、培地をブランク溶液としてゼロ点調製を行い、６００ｎｍの波長で吸光値を検出する。時間を横軸、吸光値を縦軸にして、図１に示すように、前記菌株の成長曲線を得る。ＡＴＣＣ８０１４の成長曲線グラフから明らかなように、実施例１の植物性乳酸菌ＡＴＣＣ８０１４は、概ね２ｈ以降から対数増殖期に入り始め、６ｈ以降は定常期に入り始め、安定期が長く、比較例１及び比較例３の植物性乳酸菌ＡＴＣＣ８０１４は概ね３ｈ以降から対数増殖期に入り始め、１４ｈ以降は安定期に入り始め、比較例２の植物性乳酸菌ＡＴＣＣ８０１４は概ね３ｈ以降から対数増殖期に入り始め、１４ｈ以降は安定期に入り始めることから、実施例１の培地で植物性乳酸菌ＡＴＣＣ８０１４を培養すると、植物性乳酸菌の対数期と安定期が比較例１?３よりも長く、かつ植物性乳酸菌の密度も比較例１?３よりも明らかに高かくなる。 The strain was inoculated into the mediums used in Example 1 and Comparative Examples 1 to 3 at an inoculation amount of 2%, shaken and shaken, and then allowed to ferment at 37 ° C. and taken out every 2 hours. Zero point preparation is performed using the medium as a blank solution, and the absorption value is detected at a wavelength of 600 nm. As shown in FIG. 1, the growth curve of the strain is obtained with time on the horizontal axis and absorption value on the vertical axis. As is clear from the growth curve graph of ATCC8014, the plant-derived lactic acid bacterium ATCC8014 of Example 1 begins to enter the logarithmic growth phase after about 2h, enters the stationary phase after 6h, has a long stable phase, and has a long stable phase, and Comparative Example 1 and The plant-derived lactic acid bacterium ATCC8014 of Comparative Example 3 began to enter the logarithmic growth phase after about 3h, and started to enter the stable period after 14h, and the plant-derived lactic acid bacterium ATCC8014 of Comparative Example 2 began to enter the logarithmic growth phase from about 3h or later, 14h After that, since the stable period begins, when the plant-derived lactic acid bacterium ATCC8014 is cultured in the medium of Example 1, the logarithmic period and the stable period of the plant-derived lactic acid bacteria are longer than those of Comparative Examples 1 to 3, and the density of the plant-derived lactic acid bacteria is also increased. It is clearly higher than Comparative Examples 1 to 3.
植物性乳酸菌ＡＴＣＣ８０１４の成長曲線と、実施例１、比較例１−３で得られた細胞外多糖の収率から、テトラエチルチウラムジスルフィドおよびラクトースを含有するＭＲＳ培地で植物性乳酸菌ＡＴＣＣ８０１４を培養することにより、植物性乳酸菌の対数期および安定期の延長に有利となり、細胞外多糖を多く合成し、植物性乳酸菌の多糖産生力を向上させ、最終的に細胞外多糖の収量を向上させることができることがわかる。 From the growth curve of plant-derived lactic acid bacterium ATCC8014 and the yield of extracellular polysaccharides obtained in Example 1 and Comparative Example 1-3, by culturing the plant-derived lactic acid bacterium ATCC8014 in an MRS medium containing tetraethylthiuram disulfide and lactose. , It is advantageous for prolonging the logarithmic phase and stable phase of plant-derived lactic acid bacteria, it is possible to synthesize a large amount of extracellular lactose, improve the polysaccharide-producing ability of plant-derived lactic acid bacteria, and finally improve the yield of extracellular lactose. Understand.
試験例２ Test Example 2
植物性乳酸菌細胞外多糖の性能測定に関する： Regarding performance measurement of plant-derived lactic acid bacteria extracellular polysaccharide:
１．植物性乳酸菌細胞外多糖の分子量の測定 1. 1. Measurement of molecular weight of plant-derived lactic acid bacteria extracellular polysaccharide
分子量の異なるＤｅｘｔｒａｎ標準品を順次に測定対象多糖サンプルに注入し、保留時間（ＴＲ）を記録し、各標準品の保留時間（ＴＲ）を横軸とし、各多糖標準品のピーク分子量の対数（ｌｏｇ Ｍｏｌ Ｗｔ）を縦軸にして検量線（図２）を作成し、分子量と保留時間（ＴＲ）との回帰式を求め、ｙ＝１１．９９７２?０．４０６６２ｘ、Ｒ２＝０．９９８２４とする。測定対象多糖サンプルに上記手順でＤｅｘｔｒａｎ標準品を注入し、得られた保留時間（ＴＲ）に基づいて、標準分子量曲線から測定対象多糖サンプルの平均相対分子量を自動的に算出するとともに、多糖サンプルのクロマトグラム上のピークの数および形状から多糖の純度を同定することができる。実施例１、比較例１−３に係る細胞外多糖の高性能液体クロマトグラフィーは、図３に示すように、図から明らかなように、実施例１、比較例１−３に係る細胞外多糖は、いずれも単一ピーク形状であり、４種の多糖がいずれも均一の多糖であることを説明した。実施例１、比較例１−３の細胞外多糖の保留時間は順に１４．４２８ｍｉｎ、１５．６６０ｍｉｎ、１５．６６０ｍｉｎ及び１５．６６０ｍｉｎであり，対応する保留時間を標準分子量と保留時間の回帰式に代入して、実施例１、比較例１−３で得られた細胞外多糖の分子量は順に１．３５×１０６Ｄａ、４．２６×１０５Ｄａ、４．２６×１０５Ｄａ及び４．２６×１０５Ｄａである。 Dextran standard products with different molecular weights are sequentially injected into the polysaccharide sample to be measured, the hold time (TR) is recorded, the hold time (TR) of each standard product is on the horizontal axis, and the logarithmic peak molecular weight of each polysaccharide standard product ( A calibration curve (FIG. 2) is created with log Mol Wt) as the vertical axis, and the regression equation between the molecular weight and the holding time (TR) is obtained, and y = 11.9972 to 0.40662x and R2 = 0.99824. .. The Dextran standard product is injected into the polysaccharide sample to be measured according to the above procedure, and the average relative molecular weight of the polysaccharide sample to be measured is automatically calculated from the standard molecular weight curve based on the obtained hold time (TR), and the polysaccharide sample is automatically calculated. The purity of the polysaccharide can be identified from the number and shape of peaks on the chromatogram. As shown in FIG. 3, high performance liquid chromatography of the extracellular polysaccharide according to Example 1 and Comparative Example 1-3 is performed on the extracellular polysaccharide according to Example 1 and Comparative Example 1-3, as is clear from the figure. Explained that all of them had a single peak shape, and that all four types of polysaccharides were uniform polysaccharides. The holding times of the extracellular polysaccharides of Example 1 and Comparative Example 1-3 are 14.428min, 15.660min, 15.660min and 15.660min, respectively, and the corresponding holding times are expressed in the regression equation of the standard molecular weight and the holding time. Substituting, the molecular weights of the extracellular polysaccharides obtained in Example 1 and Comparative Example 1-3 are 1.35 × 106 Da, 4.26 × 105 Da, 4.26 × 105 Da and 4.26 × 105 Da, respectively.
２．植物性乳酸菌細胞外多糖の単糖構成分析 2. Monosaccharide composition analysis of plant-derived lactic acid bacteria extracellular polysaccharide
２．１多糖の加水分解 2.1 Hydrolysis of polysaccharides
細胞外多糖サンプル５ｍｇをアンプルに入れ、さらに２ｍｏｌ／Ｌトリフルオロ酢酸２ｍＬを加えてアルコールランプで封口し、オーブン中１２０℃で２ｈ加水分解し、加水分解液を減圧留去した後、さらに少量のメタノールを加えて減圧留去し（５回繰り返し）、残ったトリフルオロ酢酸を除去した後、少量の水を加えて溶解し、凍結乾燥すれば、完全酸加水分解された単糖サンプルを得る。 Put 5 mg of the extracellular polysaccharide sample in an ampol, add 2 mL of 2 mol / L trifluoroacetic acid, seal with an alcohol lamp, hydrolyze in an oven at 120 ° C. for 2 hours, distill off the hydrolyzate under reduced pressure, and then distill off a smaller amount. Methanol is added and distilled off under reduced pressure (repeated 5 times) to remove the remaining trifluoroacetic acid, and then a small amount of water is added to dissolve the mixture, and the mixture is freeze-dried to obtain a completely acid-hydrolyzed monosaccharide sample.
２．２糖のニトリル酢酸メンチル誘導体の調製 2.2 Preparation of nitrile mentyl acetate derivative of sugar
各単糖標準品５ｍｇ、ＮａＢＨ４３０ｍｇ及び５ｍｇのイノシトールをアンプルに量り取り、徐々に２ｍＬの蒸留水を滴下しながら振盪し、室温で糖アルコールに還元反応させ（２ｈ反応し）、気泡が発生しなくなるまで氷酢酸を滴下して過剰量のＮａＢＨ４を中和する。ロータリーエバポレーターを６０℃に温調してサンプルが完全に乾燥するまで真空回転蒸発させ、その後、０．１％（ｖ／ｖ）塩酸メタノール溶液２ｍＬで再溶解した後、再度蒸発乾固し、ホウ酸塩を除去するように４〜５回繰り返す。処理済みの生成物を１０５℃オーブンで１５ｍｉｎ脱水し、ピリジンと無水酢酸をそれぞれ０．５ｍＬ出し、アルコールジェットで封口し、沸騰水浴で１ｈ反応させ、得られた生成物を微孔膜（０．２２μｍ）を経て不純物を除去し、ガスクロマトグラフィー分析を行う。完全酸加水分解後の細胞外多糖サンプル誘導体の調製は同様にこの方法に従って調製される。 Weigh 5 mg of each monosaccharide standard, 430 mg of NaBH and 5 mg of inositol into an ampol, shake while gradually dropping 2 mL of distilled water, and reduce reaction to sugar alcohol at room temperature (react for 2 hours), and no bubbles are generated. Glacial acetic acid is added dropwise to neutralize the excess amount of NaBH4. The rotary evaporator was heated to 60 ° C. and vacuum rotary evaporated until the sample was completely dried, then redissolved in 2 mL of 0.1% (v / v) hydrochloric acid-methanol solution, evaporated to dryness again, and borate. Repeat 4-5 times to remove the acid salt. The treated product was dehydrated in an oven at 105 ° C. for 15 minutes, 0.5 mL each of pyridine and acetic anhydride were taken out, sealed with an alcohol jet, and reacted in a boiling water bath for 1 h, and the obtained product was subjected to a microporous membrane (0. Impurities are removed through 22 μm), and gas chromatography analysis is performed. Preparation of the extracellular polysaccharide sample derivative after complete acid hydrolysis is similarly prepared according to this method.
２．３ガスクロマトグラフィー条件クロマトグラフ 2.3 Gas Chromatography Condition Chromatography
Ａｇｉｌｅｎｔ７８９０Ａガスクロマトグラフを採用し、カラムは、ＤＢ２２５キャピラリカラム（３０ｍ×０．２５ｍｍ）であり、検出器は、酸素炎イオン検出器であり、流速は、１ｍＬ／ｍｉｎであり、スプリット比は、１：５０であり、注入量は、１μＬである。昇温プログラム：８０℃で３ｍｉｎ保持し、５℃／ｍｉｎで１９５℃まで昇温して１ｍｉｎ保持し、５℃／ｍｉｎで２１５℃まで昇温して１ｍｉｎ保持し、１０℃／ｍｉｎで２３０℃まで昇温して３ｍｉｎ保持する。 Adopting the Agent7890A gas chromatograph, the column is a DB225 capillary column (30 m x 0.25 mm), the detector is an oxygen flame ion detector, the flow velocity is 1 mL / min, and the split ratio is 1: It is 50 and the injection volume is 1 μL. Temperature rise program: Hold at 80 ° C for 3 min, raise to 195 ° C at 5 ° C / min for 1 min, raise to 215 ° C at 5 ° C / min for 1 min, hold at 230 ° C at 10 ° C / min The temperature is raised to 3 min.
実施例１、比較例１−３で得られた細胞外多糖をトリフルオロ酢酸で加水分解した後のガスクロマトグラフィー分析及び単糖標準品との比較結果は図４に示すとおりである。単糖標準品の保留時間（図４標準品）は左から右へ順にラムノース（２６．２１５ｍｉｎ）、フコース（２６．４２７ｍｉｎ）、アラビノース（２６．５３１ｍｉｎ）、キシロース（２６．９５１ｍｉｎ）、マンノース（３０．５５３ｍｉｎ）、フルクトース（３１．５５３ｍｉｎ）、グルコース（３１．７１４ｍｉｎ）、ガラクトース（３１．９２１ｍｉｎ）である。実施例１、比較例１−３の細胞外多糖はいずれも保留時間が３０．５５３ｍｉｎ、３１．７１４ｍｉｎ及び３１．９２１ｍｉｎの時にピークを出し（図４の実施例１、比較例１−３）、実施例１、比較例１−３の細胞外多糖はいずれも主にマンノース、ガラクトース及びグルコースの三種類の単糖から構成されることを示す。面積正規化法による定量分析により、実施例１に係る細胞外多糖におけるマンノース、ガラクトース及びグルコースのモル比は、５．６：７．３：１であり、比較例１−３に係る細胞外多糖におけるマンノース、ガラクトース及びグルコースのモル比は、１：２．６：４．２である。 The results of gas chromatography analysis after hydrolysis of the extracellular polysaccharides obtained in Example 1 and Comparative Example 1-3 with trifluoroacetic acid and comparison with the monosaccharide standard are shown in FIG. The holding time of the monosaccharide standard (Fig. 4 standard) is from left to right: rhamnose (26.215 min), fucose (26.427 min), arabinose (26.531 min), xylose (26.951 min), mannose (30). .553 min), fructose (31.553 min), glucose (31.714 min), galactose (31.921 min). The extracellular polysaccharides of Example 1 and Comparative Example 1-3 all peaked at the retention times of 30.535 min, 31.714 min and 31.921 min (Example 1 and Comparative Example 1-3 in FIG. 4). It is shown that the extracellular polysaccharides of Example 1 and Comparative Example 1-3 are mainly composed of three types of monosaccharides, mannose, galactose and glucose. According to the quantitative analysis by the area normalization method, the molar ratio of mannose, galactose and glucose in the extracellular polysaccharide according to Example 1 was 5.6: 7.3: 1, and the extracellular polysaccharide according to Comparative Example 1-3. The molar ratio of mannose, galactose and glucose in is 1: 2.6: 4.2.
細胞外多糖の分子量と単糖構成から、テトラエチルチウラムジスルフィドとラクトースを含有するＭＲＳ培地で植物性乳酸菌ＡＴＣＣ８０１４を培養することで、細胞外多糖の化学構造を変更し、本発明の細胞外多糖を得ることができることがわかる。 From the molecular weight and monosaccharide composition of the extracellular polysaccharide, the chemical structure of the extracellular polysaccharide is changed by culturing the plant-derived lactic acid bacterium ATCC8014 in an MRS medium containing tetraethylthiuram disulfide and lactose, and the extracellular polysaccharide of the present invention is obtained. You can see that you can.
試験例２ Test Example 2
植物性乳酸菌細胞外多糖リン酸化誘導体の置換度測定 Measurement of degree of substitution of plant-derived lactic acid bacteria extracellular polysaccharide phosphorylated derivative
リン元素の含有量は誘導結合−原子発光分光計を用いて測定し、実施例１、比較例１―３で得られたリン酸化誘導体のリン元素の含有量（Ｐ％）はそれぞれ４．１１％、２．３４％、２．３４％、２．３４％であり、置換度（リン酸化の程度）は、置換度＝（５．２２×Ｐ％）／（１−２．６１×Ｐ％）により算出する。 The phosphorus element content was measured using an induction bond-atomic emission spectrometer, and the phosphorus element content (P%) of the phosphorylated derivatives obtained in Example 1 and Comparative Example 1-3 was 4.11, respectively. %, 2.34%, 2.34%, 2.34%, and the degree of substitution (degree of phosphorylation) is the degree of substitution = (5.22 × P%) / (1-2.61 × P%). ).
実施例１、比較例１―３で得られたリン酸化誘導体のリン元素の含有量（Ｐ％）の算出結果は、それぞれ０．２４、０．１３、０．１３及び０．１３である。 The calculation results of the phosphorus element content (P%) of the phosphorylated derivatives obtained in Example 1 and Comparative Example 1-3 are 0.24, 0.13, 0.13 and 0.13, respectively.
試験例３ Test Example 3
１．植物性乳酸菌細胞外多糖及びそのリン酸化誘導体のＡＣＥ阻害性能測定 1. 1. Measurement of ACE inhibition performance of plant-derived lactic acid bacteria extracellular polysaccharide and its phosphorylated derivative
ＡＣＥは、人の血管を収縮させ、血圧を上昇させることができる。活性化されたＡＣＥは、高血圧病を致す主要原因である。したがって、阻害剤でＡＣＥに活性を失わせる又は活性を阻害することは、血圧を下降させる潜在的な手段の一つである。植物性乳酸菌細胞外多糖及びそのリン酸化誘導体のＡＣＥ阻害性能は、下記の方法によって測定する。 ACE can constrict a person's blood vessels and raise blood pressure. Activated ACE is a major cause of hypertension. Therefore, causing ACE to lose or inhibit activity with an inhibitor is one of the potential means of lowering blood pressure. The ACE inhibitory performance of plant-derived lactic acid bacteria extracellular polysaccharides and their phosphorylated derivatives is measured by the following method.
質量濃度１００ｍｇ／ｍＬの植物性乳酸菌細胞外多糖及びそのリン酸化誘導体のサンプル溶液を採取し、ＡＣＥ阻害活性を測定し、試験管に基質として６．５ｍｍｏｌ／ＬＨＨＬ溶液１００ｍＬ、サンプル溶液２００ｍＬ、１００ｍｍｏｌ／Ｌリン酸塩緩衝液（ｐＨ＝８．３）２００ｍＬを順次加え、３７℃恒温水浴で３〜５ｍｉｎ予熱した後、ＡＣＥ酵素液５００ｍＬを加えて反応を開始し、３７℃で３０ｍｉｎ水浴した後、１ｍｏｌ／ＬＨＣｌを１００ｍＬ加えて反応を終了する。そして酢酸エチルを１．５ｍＬ加え、均一に混合した後遠心分離（５２００ｒ／ｍｉｎ，１０ｍｉｎ）を行った後、上層の酢酸エチルを１ｍＬ吸引して別の試験管に移し、１２０℃のオーブンに入れて３０ｍｉｎ揮発させ、冷却後に蒸留水６ｍＬを加え、均一に混合した後にその吸光度値を２２８ｎｍで測定し、算出式は、ＡＣＥ阻害率（％）＝（Ａｂ−Ａａ）／（Ａｂ−Ａｃ）×１００とする。
A sample solution of a plant-derived lactic acid bacterium extracellular polysaccharide having a mass concentration of 100 mg / mL and a phosphorylated derivative thereof was collected, the ACE inhibitory activity was measured, and a 6.5 mmol / LHHL solution 100 mL, a
Ａａは、ＡＣＥとＨＨＬに阻害剤を添加して反応させた後のサンプルの吸光度値である。 Aa is the absorbance value of the sample after the inhibitor was added to ACE and HHL and reacted.
Ａｂは、阻害剤を添加せず、ＡＣＥとＨＨＬが完全に反応する対照群の吸光度値である。 Ab is the absorbance value of the control group in which ACE and HHL completely react with each other without adding an inhibitor.
Ａｃは、阻害剤を添加し、反応前に予め不活性化されたＡＣＥをＨＨＬと反応させるブランク対照群の吸光度値である。 Ac is the absorbance value of the blank control group in which the inhibitor is added and the ACE previously inactivated before the reaction is reacted with HHL.
植物性乳酸菌細胞外多糖及びそのリン酸化誘導体のＡＣＥへの阻害作用は、図５に示すとおりであり、実施例１で得られた植物性乳酸菌細胞外多糖および実施例２で得られた植物性乳酸菌細胞外多糖のリン酸化誘導体は、ＡＣＥに対し、顕著な阻害作用を有し、比較例１−６で得られた植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は、ＡＣＥに対する阻害作用が明らかでない。 The inhibitory effect of the plant-derived lactic acid bacterium extracellular polysaccharide and its phosphorylated derivative on ACE is as shown in FIG. 5, and the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Example 1 and the plant-derived product obtained in Example 2 are shown. The phosphorylated derivative of the lactic acid bacterium extracellular polysaccharide has a remarkable inhibitory effect on ACE, and the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Comparative Example 1-6 and its phosphorylated derivative have a clear inhibitory effect on ACE. Not.
２．植物性乳酸菌細胞外多糖及びそのリン酸化誘導体の細胞傷害性測定
ＭＴＴ法により、培養終了前４ｈ、ウェル当たり２０μＬＭＴＴ（５ｇ／Ｌ）を加え、４ｈ培養を継続する。培養終了後にジメチルスルホキシド１５０μｌを添加する。酵素結合免疫検出器は５７０ｎｍでＡ５７０値を測定する。その結果、図６に示すように、異なる濃度の植物性乳酸菌細胞外多糖及びそのリン酸化誘導体を添加した時の細胞培養液は、対照群と比較してＯＤ値の間に有意差がなく、実施例１?２、比較例１?６で得られた植物性乳酸菌細胞外多糖及びそのリン酸化誘導はいずれも細胞毒性を示さなかったことが明らかとなった。
2. Measurement of cytotoxicity of plant-derived lactic acid bacteria extracellular polysaccharide and its phosphorylated derivative By the MTT method, 20 μLMTT (5 g / L) per well is added 4 hours before the end of the culture, and the culture is continued for 4 hours. After completion of the culture, 150 μl of dimethyl sulfoxide is added. The enzyme-bound immunodetector measures the A570 value at 570 nm. As a result, as shown in FIG. 6, there was no significant difference in OD value between the cell culture solutions when different concentrations of plant-derived lactic acid bacterium extracellular polysaccharide and its phosphorylated derivative were added, as compared with the control group. It was clarified that none of the plant-derived lactic acid bacteria extracellular polysaccharides obtained in Examples 1 and 2 and Comparative Examples 1 to 6 and their phosphorylation induction showed cytotoxicity.
３．植物性乳酸菌細胞外多糖及びそのリン酸化誘導体の体内抗高血圧活性測定 3. 3. Measurement of in-vivo antihypertensive activity of plant-derived lactic acid bacteria extracellular polysaccharide and its phosphorylated derivative
動物実験は倫理基準に合致する（承認番号：２０１４０４０５）。高血圧自然発症ラットは恒温条件下（２２±２℃）で飼育し、１２ｈの明暗サイクルを与え、摂食を自由にする。実施例１の細胞外多糖及び実施例２の細胞外多糖のリン酸化誘導体を生理食塩水で０．０５ｇ／ｋｇの用量に調製し、毎回胃内に１ｍＬ投与し、対照群に等量の生理食塩水を投与する。第０、２、４、６、８、１２、２４ｈにおいて、それぞれラットの収縮期血圧と拡張期血圧を測定し、収縮期血圧（ＳＢＰ）と拡張期血圧（ＤＢＰ）の変化を観察して、植物性乳酸菌細胞外多糖及びそのリン酸化誘導体の降圧効果を評価する（（図７及び図８）。陰性対照群（生理食塩水）のラットのＳＢＰとＤＢＰは２４ｈ内に顕著に変化しない。実施例１の細胞外多糖群のラット血圧変化は明らかであり、実施例１の細胞外多糖を胃内に投与した８ｈ後、収縮期血圧は５３．８ｍｍＨｇ下降し、拡張期血圧は６８．１ｍｍＨｇ下降し、投与した２４ｈ後は初期レベルに対する収縮期血圧の差圧は２．１ｍｍＨｇ、初期レベルに対する拡張期血圧の差圧は２．１ｍｍＨｇであり、実施例２の細胞外多糖のリン酸化誘導体群のラット血圧変化は明らかであり、実施例１の細胞外多糖を胃内に投与した６ｈ後、収縮期血圧は６１．６ｍｍＨｇ下降し、拡張期血圧は６８．７ｍｍＨｇ下降し、投与した２４ｈ後は収縮期血圧の初期レベルに対する差圧は６．４ｍｍＨｇ、拡張期血圧の初期水平に対する差圧は２．５ｍｍＨｇである。体内の降圧結果はインビトロでＡＣＥの酵素に対する阻害作用結果と一致する。体内、インビトロ実験結果により、植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は高い降圧作用を有することを示す。 Animal studies meet ethical standards (approval number: 2014405). Spontaneous hypertensive rats are bred under homeothermic conditions (22 ± 2 ° C.) and given a 12 h light-dark cycle to free feeding. The extracellular polysaccharide of Example 1 and the phosphorylated derivative of the extracellular polysaccharide of Example 2 were prepared in physiological saline at a dose of 0.05 g / kg, and 1 mL was administered intragastrically each time, and the same amount of physiology was given to the control group. Administer saline solution. At the 0th, 2nd, 4th, 6th, 8th, 12th, and 24th hours, the systolic blood pressure and the diastolic blood pressure of the rats were measured, and the changes in the systolic blood pressure (SBP) and the diastolic blood pressure (DBP) were observed. The antihypertensive effect of plant-derived lactic acid bacterium extracellular polysaccharide and its phosphorylated derivative is evaluated ((FIG. 7 and 8). SBP and DBP of rats in the negative control group (physiological saline) do not change significantly within 24 hours. Changes in rat blood pressure in the extracellular polysaccharide group of Example 1 are clear, and after 8 hours of intragastric administration of the extracellular polysaccharide of Example 1, systolic blood pressure decreased by 53.8 mmHg and diastolic blood pressure decreased by 68.1 mmHg. However, 24 hours after administration, the differential pressure of systolic blood pressure with respect to the initial level was 2.1 mmHg, and the differential pressure of diastolic blood pressure with respect to the initial level was 2.1 mmHg. Changes in rat blood pressure are clear: systolic blood pressure decreased by 61.6 mmHg, diastolic blood pressure decreased by 68.7 mmHg after 6 hours of intragastric administration of the extracellular polysaccharide of Example 1, and contraction occurred 24 hours after administration. The differential pressure of systolic blood pressure with respect to the initial level is 6.4 mmHg, and the differential pressure of diastolic blood pressure with respect to the initial horizontal is 2.5 mmHg. Experimental results show that plant-derived lactic acid bacteria extracellular polysaccharides and their phosphorylated derivatives have a high antihypertensive effect.
上記実施例における従来技術は当業者に知られている従来技術であるため、ここでは詳細な説明を省略する。 Since the prior art in the above embodiment is a prior art known to those skilled in the art, detailed description thereof will be omitted here.
以上の実施形態は、本発明を説明するためのものであり、本発明を限定するものではなく、本発明の精神及び範囲から逸脱することなく、様々な変形や変形が可能であることは当業者に明らかである。したがって、すべての同等の技術的解決手段も本発明の範疇に含まれるものであり、本発明の特許請求の範囲は、特許請求の範囲によって制限されるべきである。 The above embodiments are for explaining the present invention, do not limit the present invention, and can be variously modified and modified without departing from the spirit and scope of the present invention. It is obvious to the trader. Therefore, all equivalent technical solutions are also included in the scope of the present invention, and the scope of claims of the present invention should be limited by the scope of claims.
本発明は、微生物技術分野に関し、具体的には、植物性乳酸菌細胞外多糖および植物性乳酸菌細胞外多糖がＡＣＥ阻害薬製造中に果たす作用に関する。 The present invention relates to the field of microbial technology, and specifically to the action of plant-derived lactic acid bacteria extracellular polysaccharides and plant-derived lactic acid bacteria extracellular polysaccharides during the production of ACE inhibitors.
多糖は、自然界においてありふれたバイオポリマーであり、すべての動物、植物および微生物に存在し、同じ単糖から構成されるホモ多糖と異なる単糖から構成されるヘテロ多糖とに分けられる。近年、多糖の活性に対する研究が大きく前進し、レンチナン、霊芝多糖など多くな多糖が抗腫瘍活性を有することが証明され、人参多糖が免疫力を強化する機能を有することが証明される。微生物多糖は、生産量が安定で、気候や地理に影響されにくいため、研究ブームを迎える。多糖の生物活性多糖の生物活性は、自身の水溶性と、分子量（Ｍｗ）と、置換度と置換基の四つの要素によって影響修飾される。化学修飾によって多糖の生物活性に明らかな強化又は新しい活性が生じ、新型薬に開発できることが、多くの研究によって発見される。多糖の分子修飾と構造改造は重要意味を有する。 Polysaccharides are common biopolymers in nature and are present in all animals, plants and microorganisms and are divided into homopolysaccharides composed of the same monosaccharides and heteropolysaccharides composed of different monosaccharides. In recent years, research on the activity of polysaccharides has made great progress, and it has been proved that many polysaccharides such as lentinan and Ganoderma lucidum have antitumor activity, and that ginseng polysaccharide has a function of strengthening immunity. Microbial polysaccharides enter a research boom because their production is stable and they are not easily affected by climate and geography. Biological activity of polysaccharides The biological activity of polysaccharides is influenced and modified by four factors: their water solubility, their molecular weight (Mw), their degree of substitution and their substituents. Many studies have found that chemical modifications result in obvious enhancements or new activities in the biological activity of polysaccharides that can be developed into new drugs. Molecular modification and structural modification of polysaccharides are important.
微生物多糖の免疫調節活性は主に、マクロファージとナチュラルキラー細胞を活性化させることに体現される。中国には、良質な乳酸菌資源を有し、多くの伝統発酵食品には乳酸菌が含まれ、これらの資源に対し、研究や開発を行うことで、国民生活を改善することに役立てる。菌体外多糖乳酸菌の有益機能は主に自身の菌体外多糖によって決められると思う学者もいる。乳酸菌の菌体外多糖（Ｅｘｏｐｏｌｙｓａｃｃｈａｒｉｄｅｓ，□ＥＰＳ）は一般、菌体の表面に緊密に結合する細胞結合性の菌体外多糖と、周囲の液体環境に放出される放出多糖（Ｒｅｌｅａｓｅｄ□ｅｘｏｐｏｌｙｓａｃｃｈａｒｉｄｅｓ，□ｒ−ＥＰＳ）に分けられる。報道される大部分の乳酸菌は、ｒ−ＥＰＳだけを生じるが、Ｃ−ＥＰＳとｒ−ＥＰＳを同時に生じる乳酸菌もある。現在、報道された乳酸菌細胞外多糖乳酸菌は主に３０種類くらいある。乳酸菌細胞外多糖が免疫調節作用を強化させる機能を有することに対し、乳酸菌細胞外多糖の抽出が簡単であり、遠心によって菌体と液体を分離させ、そして液体を濃縮してエタノールを添加すればｒ−ＥＰＳが沈殿する。一部のウェルシュ菌は、Ｃ−ＥＰＳの抽出に使用されることができ、また、他の微生物多糖に比べ、乳酸菌細胞外多糖は、安全性がよく、各種食品と薬に応用できる。したがって、乳酸菌細胞外多糖に対する研究は非常に必要である。 The immunomodulatory activity of microbial polysaccharides is primarily embodied in the activation of macrophages and natural killer cells. China has high-quality lactic acid bacteria resources, and many traditional fermented foods contain lactic acid bacteria, and research and development of these resources will help improve people's lives. Some scholars believe that the beneficial functions of exopolysaccharide lactic acid bacteria are mainly determined by their own exopolysaccharide. Lactic acid bacteria exopolysaccharides (Exopolysaccharides, □ EPS) are generally cell-binding exopolysaccharides that bind tightly to the surface of the bacterium and released polysaccharides (Released □ exopolysaccharides, □) that are released into the surrounding liquid environment. It is divided into r-EPS). Most of the reported lactic acid bacteria produce only r-EPS, but some lactic acid bacteria produce C-EPS and r-EPS at the same time. Currently, there are mainly about 30 types of lactic acid bacteria extracellular polysaccharide lactic acid bacteria reported. Lactobacillus extracellular polysaccharide has a function of enhancing immunomodulatory action, whereas extraction of lactic acid bacterium extracellular polysaccharide is easy, if the cells and liquid are separated by centrifugation, and the liquid is concentrated and ethanol is added. r-EPS precipitates. Some Clostridium perfringens can be used for extraction of C-EPS, and lactic acid bacteria extracellular polysaccharides are safer than other microbial polysaccharides and can be applied to various foods and medicines. Therefore, research on lactic acid bacteria extracellular polysaccharides is very necessary.
本発明は、上記の技術的問題を解決するために、顕著なＡＣＥ阻害活性を示す、細胞傷害性を有しない植物性乳酸菌細胞外多糖を提供することを目的とする。 An object of the present invention is to provide a non-cytotoxic plant-derived lactic acid bacterium extracellular polysaccharide that exhibits remarkable ACE inhibitory activity in order to solve the above technical problems.
上記の目的を実現するために、本発明の植物性乳酸菌細胞外多糖は、１）と２）の少なくともいずれか一つの特徴を有し、
前記１）は、重量平均分子量は、１．３５×１０６Ｄａであり、
前記２）は、モル比が５．６：７．３：１のマンノースと、ガラクトースとブドウ糖から構成される。
In order to achieve the above object, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention has at least one of the characteristics of 1) and 2).
In 1), the weight average molecular weight is 1.35 × 106 Da.
The above 2) is composed of mannose having a molar ratio of 5.6: 7.3: 1, galactose and glucose.
前記植物性乳酸菌細胞外多糖は、アンジオテンシン変換酵素（ＡＣＥ）を阻害できる。本発明の植物性乳酸菌細胞外多糖は、アンジオテンシン変換酵素（ＡＣＥ）阻害活性を示し、血圧調節および高血圧予防の機能の作用を果たし、したがって、本発明の植物性乳酸菌細胞外多糖を心臓血管疾患予防・治療のための組成物の活性成分に使用できる。かつ、本発明の植物性乳酸菌細胞外多糖は、細胞傷害性を有しない。 The plant-derived lactic acid bacterium extracellular polysaccharide can inhibit angiotensin converting enzyme (ACE). The plant-derived lactic acid bacterium extracellular polysaccharide of the present invention exhibits angiotensin converting enzyme (ACE) inhibitory activity and fulfills the functions of blood pressure regulation and hypertension prevention. Therefore, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention can be used for cardiovascular disease prevention. -Can be used as an active ingredient in therapeutic compositions. Moreover, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention does not have cytotoxicity.
本発明は、植物性乳酸菌細胞外多糖がＡＣＥ阻害薬組成物の調製時の作用を更に提供する。本発明の細胞外多糖は、顕著なＡＣＥ阻害活性を示し、かつ細胞傷害性がなく、ＡＣＥ活性阻害剤組成物として有効に使用することができる。 The present invention further provides the action of plant-derived lactic acid bacteria extracellular polysaccharides in the preparation of ACE inhibitor compositions. The extracellular polysaccharide of the present invention exhibits remarkable ACE inhibitory activity, is not cytotoxic, and can be effectively used as an ACE activity inhibitor composition.
本発明は、植物性乳酸菌細胞外多糖のリン酸化誘導体を更に提供する。本発明の植物性乳酸菌細胞外多糖のリン酸化誘導体は、ＡＣＥ阻害活性を示し、血圧調節および高血圧予防の作用を有し、且つかつ細胞傷害性を有しない。 The present invention further provides a phosphorylated derivative of a plant-derived lactic acid bacterium extracellular polysaccharide. The phosphorylated derivative of the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention exhibits ACE inhibitory activity, has an action of regulating blood pressure and preventing hypertension, and has no cytotoxicity.
前記植物性乳酸菌細胞外多糖の置換度は、０．２４である。 The degree of substitution of the plant-derived lactic acid bacterium extracellular polysaccharide is 0.24.
本発明は、心臓血管疾患予防・治療用の組成物を更に提供し、前記細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを含む。前記組成物は、食品組成物又は医薬組成物であり、組成物全重量に対して、細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを０．１〜８０ｗｔ％含む。 The present invention further provides a composition for the prevention and treatment of cardiovascular diseases, and includes at least one of the extracellular polysaccharide and a phosphorylated derivative thereof. The composition is a food composition or a pharmaceutical composition, and contains 0.1 to 80 wt% of at least one of an extracellular polysaccharide and a phosphorylated derivative thereof based on the total weight of the composition.
前記心血管疾患は、高血圧、心疾患、脳卒中、血栓、動脈硬化、狭心症、心不全および心筋梗塞からなる群から選択される一種または複数種の疾患である。 The cardiovascular disease is one or more diseases selected from the group consisting of hypertension, heart disease, stroke, thrombosis, atherosclerosis, angina, heart failure and myocardial infarction.
前記組成物は、経口組成物である。 The composition is an oral composition.
前記組成物は、成人が一日あたりに０．１−５００ｍｇの植物性乳酸菌細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを摂取する方式で使用される。好ましくは、毎日５０−１００ｍｇ／ｋｇであり、毎日１−３回である。 The composition is used in such a manner that an adult ingests 0.1-500 mg of at least one of a plant-derived lactic acid bacterium extracellular polysaccharide and a phosphorylated derivative thereof per day. Preferably, it is 50-100 mg / kg daily, 1-3 times daily.
前記組成物は、９−１２週間又はもっと長い時間で摂取する方式で使用される。 The composition is used in a manner of ingestion for 9-12 weeks or longer.
本発明は、従来技術と比べ、下記のメリットを有する。 The present invention has the following merits as compared with the prior art.
本発明の植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は、いずれもＡＣＥ阻害活性を示し、血圧調節および高血圧予防の機能を有し、これによって本発明の植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は、心臓血管疾患の予防と治療のための組成物の活性成分に使用できる。また、本発明の植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は、細胞傷害性を有しなく、幅広く応用されることができる。 The plant-derived lactic acid bacterium extracellular polysaccharide of the present invention and its phosphorylated derivative all exhibit ACE inhibitory activity and have functions of blood pressure regulation and hypertension prevention, whereby the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention and its phosphorylation are exhibited. Derivatives can be used as active ingredients in compositions for the prevention and treatment of cardiovascular disease. In addition, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention and its phosphorylated derivative have no cytotoxicity and can be widely applied.
本発明は、上記技術的解決手段を採用して、植物性乳酸菌細胞外多糖および植物性乳酸菌細胞外多糖がＡＣＥ阻害薬製造中に果たす作用を提供し、従来技術の不足を補い、デザインが合理的で、操作が便利である。 The present invention employs the above technical solutions to provide the effects of plant-derived lactic acid bacteria extracellular polysaccharides and plant-derived lactic acid bacteria extracellular polysaccharides during the production of ACE inhibitors, compensating for the lack of prior art, and rationalizing the design. Targeted and convenient to operate.
下記に、本発明について、詳しく説明します。 The present invention will be described in detail below.
本実施方法は、植物性乳酸菌細胞外多糖を提供し、具体的には１）と２）の少なくともいずれか一つの特徴を有する。
１）重量平均分子量は、１．３５×１０６Ｄａであり、
２）モル比が５．６：７．３：１のマンノースと、ガラクトースとブドウ糖から構成される。
The present method provides a plant-derived lactic acid bacterium extracellular polysaccharide, and specifically has at least one of the characteristics of 1) and 2).
1) The weight average molecular weight is 1.35 × 106 Da.
2) It is composed of mannose with a molar ratio of 5.6: 7.3: 1, galactose and glucose.
好ましい具体例として、前記植物性乳酸菌細胞外多糖は、アンジオテンシン変換酵素（ＡＣＥ）を阻害できる。本発明の植物性乳酸菌細胞外多糖は、アンジオテンシン変換酵素（ＡＣＥ）阻害活性を示し、血圧調節および高血圧予防の機能の作用を果たし、したがって、本発明の植物性乳酸菌細胞外多糖を心臓血管疾患予防・治療のための組成物の活性成分に使用できる。かつ、本発明の植物性乳酸菌細胞外多糖は、細胞傷害性を有しない。 As a preferred embodiment, the plant-derived lactic acid bacterium extracellular polysaccharide can inhibit angiotensin converting enzyme (ACE). The plant-derived lactic acid bacterium extracellular polysaccharide of the present invention exhibits angiotensin converting enzyme (ACE) inhibitory activity and fulfills the functions of blood pressure regulation and hypertension prevention. Therefore, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention can be used for cardiovascular disease prevention. -Can be used as an active ingredient in therapeutic compositions. Moreover, the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention does not have cytotoxicity.
本実施方法の植物性乳酸菌細胞外多糖は、
植物性乳酸菌を発酵させ、発酵液を収集するステップ１と、
トリクロロ酢酸水溶液と発酵液とを撹拌混合し、反応させ、反応液に含まれる上澄み液を収集するステップ２と、
上澄み液と無水エタノールとを撹拌混合し静置し、沈殿物を収集するステップ３と、
沈殿物を水に溶解させ、透析純化し、植物性乳酸菌細胞外多糖を得るステップ４と、
の方法によって調製される。
The plant-derived lactic acid bacteria extracellular polysaccharide of this method is
Step 3 of mixing the supernatant and absolute ethanol with stirring and allowing them to stand to collect the precipitate, and
It is prepared by the method of.
好ましい具体例として、植物性乳酸菌細胞外多糖の調製方法は、
植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、濃度０．０２〜０．１μＭのテトラエチルチウラムジスルフィド及び０．１〜０．５ｍＭのラクトースを含有するＭＲＳ培地に移し、３０〜４０℃で１２〜２４ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７０〜８０％のトリクロロ酢酸水溶液を均一に混合し、常温で１〜２ｈ撹拌反応させ、反応液中の上澄み液を採取し、発酵液中の細胞及びタンパク質を除去するステップ２と、
上澄み液に２‐３倍体積の無水エタノールを加え、均一に混合し、４℃で１０―１５ｈ静置し、さらに４℃、８０００‐１５０００ｒ／ｍｉｎで２０―３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を５〜１０ｈ毎に交換し、１２―２４ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖をイオン交換カラムクロマトグラフィー及びゲルカラムにより精製し、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、
を含み、
前記ステップ１において、テトラエチルチウラムジスルフィド及びラクトースを含有するＭＲＳ培地を使用することで、細胞外多糖の化学構造を変えて本発明の植物性乳酸菌細胞外多糖を得ることができるだけでなく、植物性乳酸菌細胞外多糖の対数期及び安定期の延長に有利であり、細胞外多糖を大量に合成して、植物性乳酸菌細胞外の多糖産生力を向上させ、最終的に細胞外多糖の収量を高めることができる。
As a preferred specific example, a method for preparing a plant-derived lactic acid bacterium extracellular polysaccharide is described.
The plant-derived lactic acid bacterium ATCC8014 was continuously activated for two generations, and then transferred to an MRS medium containing tetraethylthiuram disulfide having a concentration of 0.02 to 0.1 μM and lactose of 0.1 to 0.5 mM at 30 to 40 °
The fermentation broth and a 70 to 80% trichloroacetic acid aqueous solution are uniformly mixed, stirred and reacted at room temperature for 1 to 2 hours, the supernatant liquor in the reaction broth is collected, and cells and proteins in the fermented broth are removed. ,
Supernatant 2 - 3 volumes of absolute ethanol added and uniformly mixed, 10-15H allowed to stand at 4 ° C., further 4 ℃, 8000 - 20-30min centrifuged at 15,000 r / min, collecting the precipitate Step 3 and
The step of dissolving the precipitate in water, transferring to a dialysis bag, exchanging deionized water every 5 to 10 hours, dialyzing for 12 to 24 hours, then collecting the product and lyophilizing to obtain crude extracellular polysaccharide. 4 and
Including
In
本発明は、植物性乳酸菌細胞外多糖がＡＣＥ阻害薬組成物の調製時の作用を更に提供する。多糖分子は、リン酸化修飾された後、分岐鎖上の水酸基がリン酸基で置換され、多糖水溶性を高め、鎖立体配座を改変することにより、本発明の細胞外多糖は、顕著なＡＣＥ阻害活性を示し、かつ細胞傷害性がなく、ＡＣＥ活性阻害剤組成物として有効に使用することができる。 The present invention further provides the action of plant-derived lactic acid bacteria extracellular polysaccharides in the preparation of ACE inhibitor compositions. After phosphorylation modification of the polysaccharide molecule, the hydroxyl group on the branched chain is replaced with a phosphate group to increase the water solubility of the polysaccharide and modify the chain configuration, whereby the extracellular polysaccharide of the present invention is remarkable. It exhibits ACE inhibitory activity and is not cytotoxic, and can be effectively used as an ACE activity inhibitor composition.
本発明は、植物性乳酸菌細胞外多糖のリン酸化誘導体を更に提供する。 The present invention further provides a phosphorylated derivative of a plant-derived lactic acid bacterium extracellular polysaccharide.
好ましい具体例として、植物性乳酸菌細胞外多糖のリン酸化誘導体は、植物性乳酸菌細胞外多糖を、固液比１：１０―３０ｇ／ｍＬでジメチルスルホキシドに溶解し、５０―７０℃で２０―４０ｍｉｎ加熱して十分に溶解させた後、尿素およびリン酸を加え、５０―７０℃で２―５ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを使用して反応液を中和し、透析し、凍結乾燥することにより、調製される。前記植物性乳酸菌細胞外多糖のリン酸化誘導体の調製方法において、植物性乳酸菌細胞外多糖と尿素の重量比は、１：１５−２０であり、植物性乳酸菌細胞外多糖とリン酸の固液比は、１：５−１０ｇ／ｍＬであり、植物性乳酸菌細胞外多糖と脱イオン水の固液比は、１：８−１２ｇ／ｍＬである。 As a preferred embodiment, the phosphorylated derivative of the plant-derived lactic acid bacterium extracellular polysaccharide dissolves the plant-derived lactic acid bacterium extracellular polysaccharide in dimethyl sulfoxide at a solid-liquid ratio of 1:10 to 30 g / mL and 20 to 40 min at 50 to 70 ° C. After heating to sufficiently dissolve, urea and phosphoric acid are added, and the reaction is carried out at 50-70 ° C. for 2-5 hours, then deionized water is added to terminate the reaction, and the reaction solution is completed using 1 M NaOH. Is prepared by neutralizing, dialyzing, and lyophilizing. In the method for preparing a phosphorylated derivative of a plant-derived lactic acid bacterium extracellular polysaccharide, the weight ratio of the plant-derived lactic acid bacterium extracellular polysaccharide to urea is 1: 15-20, and the solid-liquid ratio of the plant-derived lactic acid bacterium extracellular polysaccharide to phosphoric acid. Is 1: 5-10 g / mL, and the solid-liquid ratio of plant-derived lactic acid bacteria extracellular polysaccharide to deionized water is 1: 8-12 g / mL.
好ましい具体例として、植物性乳酸菌細胞外多糖の置換度は、０．２４である。 As a preferred specific example, the degree of substitution of the plant-derived lactic acid bacterium extracellular polysaccharide is 0.24.
本発明は、心臓血管疾患予防・治療用の組成物を更に提供し、前記細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを含む。該組成物は、食品組成物又は医薬組成物であり、組成物全重量に対して、細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを０．１〜８０ｗｔ％含む。本発明の食品組成物に関し、本発明の細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを飲料、肉、ソーセージ、パン、ビスケット、餅、チョコレート、キャンディ、菓子、クッキー、パンケーキ、ラーメン、チューインガム、乳製品（アイスクリームを含む）、特殊栄養食（例えば、配合粉乳や幼児食）、加工肉製品、魚製品、豆腐、でんぷんゲル製品、健康補助食品、調味食品（例えば醤油、豆弁ジャム、唐辛子、かき揚げジャム）、ソース、その他の加工食品、漬物（例えば、漬物やソース野菜）、スープ、飲料、アルコール飲料及びビタミン複合体に添加することができ、広い意味では、健康食品の生産に適した食品がほとんど含まれているといえる。本発明の医薬組成物はさらに様々な栄養素、ビタミン、ミネラル、調味剤、着色剤、ペクチン酸及びその塩、アルギン酸及びその塩、有機酸、保護コロイド増粘剤、ｐＨ調整剤、安定剤、防腐剤、グリセリン、アルコール、ソーダ水に添加するための炭酸化剤等を含むことができる。前記担体、賦形剤又は希釈剤は、ラクトース、グルコース、スクロース、ソルビトール、マンニトール、キシリトール、エリスリトール、マルチトール、デンプン、アラビアガム、アルギン酸塩／エステル、ゼラチン、リン酸カルシウム、ケイ酸カルシウム、セルロース、メチルセルロース、微結晶セルロース、ポリビニルピロリドン、水、ヒドロキシ安息香酸メチル、ヒドロキシ安息香酸プロピル、タルク、ステアリン酸マグネシウム、鉱物油、デキストリン、炭酸カルシウム、プロピレングリコール、流動パラフィン及び塩水からなる群から選択されるが、必ずしもこれに限定されるものではない。なお、全ての成分は、単独で添加してもよく、また、共に添加してもよい。 The present invention further provides a composition for the prevention and treatment of cardiovascular diseases, and includes at least one of the extracellular polysaccharide and a phosphorylated derivative thereof. The composition is a food composition or a pharmaceutical composition, and contains 0.1 to 80 wt% of at least one of an extracellular polysaccharide and a phosphorylated derivative thereof based on the total weight of the composition. With respect to the food composition of the present invention, at least one of the extracellular polysaccharide of the present invention and a phosphorylated derivative thereof is used as a beverage, meat, sausage, bread, biscuits, rice cake, chocolate, candy, confectionery, cookie, pancake, ramen. , Chewing gum, dairy products (including ice cream), special nutrition foods (eg compound milk powder and infant food), processed meat products, fish products, tofu, starch gel products, health supplements, seasoning foods (eg soy sauce, bean biscuits) Can be added to jams, peppers, fried jams), sauces and other processed foods, pickles (eg pickles and sauce vegetables), soups, beverages, alcoholic beverages and vitamin complexes, in a broad sense the production of health foods It can be said that most of the foods suitable for are contained. The pharmaceutical compositions of the present invention further include various nutrients, vitamins, minerals, seasonings, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives. Agents, glycerin, alcohol, carbonates for addition to soda water and the like can be included. The carriers, excipients or diluents include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, martitol, starch, arabic gum, alginate / ester, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, It is selected from the group consisting of microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin and salt water, but not necessarily. It is not limited to this. All the components may be added alone or together.
好ましい具体例として、心血管疾患は、高血圧、心疾患、脳卒中、血栓、動脈硬化、狭心症、心不全および心筋梗塞からなる群から選択される一種または複数種の疾患である。 As a preferred embodiment, cardiovascular disease is one or more diseases selected from the group consisting of hypertension, heart disease, stroke, thrombosis, atherosclerosis, angina, heart failure and myocardial infarction.
好ましい具体例として、前記組成物は、経口組成物である。 As a preferred embodiment, the composition is an oral composition.
好ましい具体例として、前記組成物は、成人が一日あたりに０．１−５００ｍｇの植物性乳酸菌細胞外多糖とそのリン酸化誘導体の少なくともいずれか一つを摂取する方式で使用される。好ましくは、毎日５０−１００ｍｇ／ｋｇであり、毎日１−３回である。 As a preferred embodiment, the composition is used in such a manner that an adult ingests 0.1-500 mg of a plant-derived lactic acid bacterium extracellular polysaccharide and at least one of a phosphorylated derivative thereof per day. Preferably, it is 50-100 mg / kg daily, 1-3 times daily.
好ましい具体例として、前記組成物は、９−１２週間又はもっと長い時間で摂取する方式で使用される。 As a preferred embodiment, the composition is used in a manner of ingestion for 9-12 weeks or longer.
下記に、実施例によって本発明を更に説明する。理解すべきは、前記実施例は本発明を実施する一案であり、本発明の範囲を制限しない。 The present invention will be further described below by way of examples. It should be understood that the above embodiment is an embodiment of the present invention and does not limit the scope of the present invention.
実施例１： Example 1:
本発明の植物性乳酸菌細胞外多糖の調製方法は、
上海北諾生物科技有限公司から購入の植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、濃度０．０５μＭのテトラエチルチウラムジスルフィド及び０．２ｍＭのラクトースを含有するＭＲＳ培地に移し、３７℃で１５ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７５％のトリクロロ酢酸水溶液を均一に混合し、常温で２ｈ撹拌反応させ、反応液中の上澄み液を採取するステップ２と、
上澄み液に２倍体積の無水エタノールを加え、均一に混合し、４℃で１２ｈ静置し、さらに４℃、１２０００ｒ／ｍｉｎで３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を８ｈ毎に交換し、１５ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖を蒸留水で溶解（１０ｍｇ／ｍＬ）した後、ＤＥＡＥ‐Ｃｅｌｌｕｌｏｓｅ５２イオン交換カラムに１００ｍｇローディングし、蒸留水と、０．１ｍｏｌ／ＬのＮａＣｌと、０．３ｍｏｌ／ＬのＮａＣｌおよび０．５ｍｏｌ／ＬのＮａＣｌで直線的に勾配溶出し、溶出速度が１ｍＬ／ｍｉｎであり、チューブ毎に５ｍＬを採取し、多糖含有量（多糖含有量は硫酸フェノール法で検出して溶出曲線を描く）をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分をそれぞれ合わせて収集し、脱イオン水で透析、凍結乾燥するステップ４と、
０．３ｍｏｌ／ＬのＮａＣｌで溶出した多糖成分を蒸留水で溶解（５ｍｇ／ｍＬ）した後、ＳｅｐｈａｄｅｘＧ‐１００ゲルカラムにローディングし、脱イオン水を用いて溶出し、溶出レートが０．２ｍＬ／ｍｉｎであり、溶出液を自動部分収集装置で収集し、チューブ毎に体積２ｍＬを収集し、多糖含有量をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分を合わせて収集し、脱イオン水で透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、
を含む。
The method for preparing the plant-derived lactic acid bacterium extracellular polysaccharide of the present invention is:
The plant-derived lactic acid bacterium ATCC8014 purchased from Shanghai Hokujo Biotechnology Co., Ltd. was continuously activated for two generations, and then transferred to an MRS medium containing 0.05 μM of tetraethylthiuram disulfide and 0.2 mM lactose at 37 °
In
The precipitate is dissolved in water, transferred to a dialysis bag, the deionized water is replaced every 8 hours, dialyzed for 15 hours, and then the product is collected and lyophilized to obtain crude extracellular polysaccharide.
After dissolving the crude extracellular polysaccharide in distilled water (10 mg / mL) , 100 mg was loaded on a DEAE- Cellulose 52 ion exchange column, and distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0 were loaded. Linear gradient elution with .5 mol / L NaCl, elution rate is 1 mL / min, 5 mL is collected for each tube, and polysaccharide content (polysaccharide content is detected by the phenol sulfate method and an elution curve is drawn. ) Is detected for each tube, and single peak components are collected together according to the detection value of the polysaccharide content, dialyzed with deionized water, and freeze-dried.
After the polysaccharide components eluted at NaCl of 0.3 mol / L was dissolved in distilled water (5mg / mL), SephadexG - 100 loaded onto gel column, eluted with deionized water, elution rate is 0.2 mL / min The eluate is collected by an automatic partial collector, a volume of 2 mL is collected for each tube, the polysaccharide content is detected for each tube, and a single peak component is collected together according to the polysaccharide content detection value, and then removed.
including.
植物性乳酸菌細胞外多糖の収率は、収率（％）＝（純成分質量／粗細胞外多糖質量）×１００で計算される。
算出した植物性乳酸菌細胞外多糖の収率は、５３．７２％である。
The yield of plant-derived lactic acid bacteria extracellular polysaccharide is calculated by yield (%) = (pure component mass / crude extracellular polysaccharide mass) × 100.
The calculated yield of plant-derived lactic acid bacterium extracellular polysaccharide is 53.72%.
実施例２ Example 2
植物性乳酸菌細胞外多糖のリン酸化誘導体の調整方法は、下記のようにである。 The method for preparing a phosphorylated derivative of a plant-derived lactic acid bacterium extracellular polysaccharide is as follows.
実施例１で得られた植物性乳酸菌細胞外多糖２ｇを１００ｍＬのジメチルスルホキシドに溶解し、６０℃で３０ｍｉｎ加熱して十分に溶解させた後、３６ｇの尿素と１５ｍＬのリン酸を加え、６０℃で３ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを用いて反応液を中和し、透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖のリン酸化誘導体を得る。 2 g of the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Example 1 was dissolved in 100 mL of dimethyl sulfoxide, heated at 60 ° C. for 30 minutes to sufficiently dissolve it, and then 36 g of urea and 15 mL of phosphoric acid were added to add 60 ° C. After reacting for 3 hours with, deionized water was added to terminate the reaction, the reaction solution was neutralized with 1 M NaOH, dialyzed, and freeze-dried to obtain a phosphorylated derivative of the extracellular polysaccharide of plant-derived lactic acid bacteria. obtain.
比較例１ Comparative Example 1
植物性乳酸菌細胞外多糖を、
上海北諾生物科技有限公司から購入の植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、濃度０．０５μＭのテトラエチルチウラムジスルフィドを含有するＭＲＳ培地に移し、３７℃で１５ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７５％のトリクロロ酢酸水溶液を均一に混合し、常温で２ｈ撹拌反応させ、反応液中の上澄み液を採取するステップ２と、
上澄み液に２倍体積の無水エタノールを加え、均一に混合し、４℃で１２ｈ静置し、さらに４℃、１２０００ｒ／ｍｉｎで３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を８ｈ毎に交換し、１５ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖を蒸留水で溶解（１０ｍｇ／ｍＬ）した後、ＤＥＡＥ‐Ｃｅｌｌｕｌｏｓｅ５２イオン交換カラムに１００ｍｇローディングし、蒸留水と、０．１ｍｏｌ／ＬのＮａＣｌと、０．３ｍｏｌ／ＬのＮａＣｌおよび０．５ｍｏｌ／ＬのＮａＣｌで直線的に勾配溶出し、溶出速度が１ｍＬ／ｍｉｎであり、チューブ毎に５ｍＬを採取し、多糖含有量（多糖含有量は硫酸フェノール法で検出して溶出曲線を描く）をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分をそれぞれ合わせて収集し、脱イオン水で透析、凍結乾燥し、
０．３ｍｏｌ／ＬのＮａＣｌで溶出した多糖成分を蒸留水で溶解（５ｍｇ／ｍＬ）した後、ＳｅｐｈａｄｅｘＧ‐１００ゲルカラムにローディングし、脱イオン水を用いて溶出し、溶出レートが０．２ｍＬ／ｍｉｎであり、溶出液を自動部分収集装置で収集し、チューブ毎に体積２ｍＬを収集し、多糖含有量をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分を合わせて収集し、脱イオン水で透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、によって調製し、収率が３６．７４％である。
Plant-derived lactic acid bacteria extracellular polysaccharide,
The plant-derived lactic acid bacterium ATCC8014 purchased from Shanghai Hokujo Biotechnology Co., Ltd. was continuously activated for two generations, then transferred to an MRS medium containing a tetraethylthiuram disulfide having a concentration of 0.05 μM, and allowed to ferment at 37 ° C. for 15 hours.
In
The precipitate is dissolved in water, transferred to a dialysis bag, the deionized water is replaced every 8 hours, dialyzed for 15 hours, and then the product is collected and lyophilized to obtain crude extracellular polysaccharide.
After dissolving the crude extracellular polysaccharide in distilled water (10 mg / mL) , 100 mg was loaded on a DEAE- Cellulose 52 ion exchange column, and distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0 were loaded. Linear gradient elution with .5 mol / L NaCl, elution rate is 1 mL / min, 5 mL is collected for each tube, and polysaccharide content (polysaccharide content is detected by the phenol sulfate method and an elution curve is drawn. ) Is detected for each tube, and single peak components are collected together according to the detected value of polysaccharide content, dialyzed with deionized water, freeze-dried, and then dried.
After the polysaccharide components eluted at NaCl of 0.3 mol / L was dissolved in distilled water (5mg / mL), SephadexG - 100 loaded onto gel column, eluted with deionized water, elution rate is 0.2 mL / min The eluate is collected by an automatic partial collector, a volume of 2 mL is collected for each tube, the polysaccharide content is detected for each tube, and a single peak component is collected together according to the polysaccharide content detection value, and then removed. It was prepared in
比較例２ Comparative Example 2
植物性乳酸菌細胞外多糖を、
上海北諾生物科技有限公司から購入の植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、濃度０．２ｍＭのラクトースを含有するＭＲＳ培地に移し、３７℃で１５ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７５％のトリクロロ酢酸水溶液を均一に混合し、常温で２ｈ撹拌反応させ、反応液中の上澄み液を採取するステップ２と、
上澄み液に２倍体積の無水エタノールを加え、均一に混合し、４℃で１２ｈ静置し、さらに４℃、１２０００ｒ／ｍｉｎで３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を８ｈ毎に交換し、１５ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖を蒸留水で溶解（１０ｍｇ／ｍＬ）した後、ＤＥＡＥ‐Ｃｅｌｌｕｌｏｓｅ５２イオン交換カラムに１００ｍｇローディングし、蒸留水と、０．１ｍｏｌ／ＬのＮａＣｌと、０．３ｍｏｌ／ＬのＮａＣｌおよび０．５ｍｏｌ／ＬのＮａＣｌで直線的に勾配溶出し、溶出速度が１ｍＬ／ｍｉｎであり、チューブ毎に５ｍＬを採取し、多糖含有量（多糖含有量は硫酸フェノール法で検出して溶出曲線を描く）をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分をそれぞれ合わせて収集し、脱イオン水で透析、凍結乾燥し、
０．３ｍｏｌ／ＬのＮａＣｌで溶出した多糖成分を蒸留水で溶解（５ｍｇ／ｍＬ）した後、ＳｅｐｈａｄｅｘＧ‐１００ゲルカラムにローディングし、脱イオン水を用いて溶出し、溶出レートが０．２ｍＬ／ｍｉｎであり、溶出液を自動部分収集装置で収集し、チューブ毎に体積２ｍＬを収集し、多糖含有量をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分を合わせて収集し、脱イオン水で透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、によって調製し、収率が４３．６８％である。
Plant-derived lactic acid bacteria extracellular polysaccharide,
The plant-derived lactic acid bacterium ATCC8014 purchased from Shanghai Hokuyo Biotechnology Co., Ltd. was continuously activated for two generations, then transferred to an MRS medium containing lactose at a concentration of 0.2 mM, and allowed to ferment at 37 ° C. for 15 hours for fermentation.
In
The precipitate is dissolved in water, transferred to a dialysis bag, the deionized water is replaced every 8 hours, dialyzed for 15 hours, and then the product is collected and lyophilized to obtain crude extracellular polysaccharide.
After dissolving the crude extracellular polysaccharide in distilled water (10 mg / mL) , 100 mg was loaded on a DEAE- Cellulose 52 ion exchange column, and distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0 were loaded. Linear gradient elution with .5 mol / L NaCl, elution rate is 1 mL / min, 5 mL is collected for each tube, and polysaccharide content (polysaccharide content is detected by the phenol sulfate method and an elution curve is drawn. ) Is detected for each tube, and single peak components are collected together according to the detected value of polysaccharide content, dialyzed with deionized water, freeze-dried, and then dried.
After the polysaccharide components eluted at NaCl of 0.3 mol / L was dissolved in distilled water (5mg / mL), SephadexG - 100 loaded onto gel column, eluted with deionized water, elution rate is 0.2 mL / min The eluate is collected by an automatic partial collector, a volume of 2 mL is collected for each tube, the polysaccharide content is detected for each tube, and a single peak component is collected together according to the polysaccharide content detection value, and then removed. It was prepared in
比較例３ Comparative Example 3
植物性乳酸菌細胞外多糖を、
上海北諾生物科技有限公司から購入の植物性乳酸菌ＡＴＣＣ８０１４を、連続的に二世代活性化した後、ＭＲＳ培地に移し、３７℃で１５ｈ静置発酵させて発酵液を採取するステップ１と、
前記発酵液と７５％のトリクロロ酢酸水溶液を均一に混合し、常温で２ｈ撹拌反応させ、反応液中の上澄み液を採取するステップ２と、
上澄み液に２倍体積の無水エタノールを加え、均一に混合し、４℃で１２ｈ静置し、さらに４℃、１２０００ｒ／ｍｉｎで３０ｍｉｎ遠心分離し、沈殿物を採取するステップ３と、
沈殿物を水に溶解し、透析袋に移し、脱イオン水を８ｈ毎に交換し、１５ｈ透析した後、生成物を採取し、凍結乾燥して、粗細胞外多糖を得るステップ４と、
粗細胞外多糖を蒸留水で溶解（１０ｍｇ／ｍＬ）した後、ＤＥＡＥ‐Ｃｅｌｌｕｌｏｓｅ５２イオン交換カラムに１００ｍｇローディングし、蒸留水と、０．１ｍｏｌ／ＬのＮａＣｌと、０．３ｍｏｌ／ＬのＮａＣｌおよび０．５ｍｏｌ／ＬのＮａＣｌで直線的に勾配溶出し、溶出速度が１ｍＬ／ｍｉｎであり、チューブ毎に５ｍＬを採取し、多糖含有量（多糖含有量は硫酸フェノール法で検出して溶出曲線を描く）をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分をそれぞれ合わせて収集し、脱イオン水で透析、凍結乾燥し、
０．３ｍｏｌ／ＬのＮａＣｌで溶出した多糖成分を蒸留水で溶解（５ｍｇ／ｍＬ）した後、ＳｅｐｈａｄｅｘＧ‐１００ゲルカラムにローディングし、脱イオン水を用いて溶出し、溶出レートが０．２ｍＬ／ｍｉｎであり、溶出液を自動部分収集装置で収集し、チューブ毎に体積２ｍＬを収集し、多糖含有量をチューブ毎に検出し、多糖含有量検出値により単一ピーク成分を合わせて収集し、脱イオン水で透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖を得るステップ５と、によって調製し、収率が３５．０１％である。
Plant-derived lactic acid bacteria extracellular polysaccharide,
In
The precipitate is dissolved in water, transferred to a dialysis bag, the deionized water is replaced every 8 hours, dialyzed for 15 hours, and then the product is collected and lyophilized to obtain crude extracellular polysaccharide.
After dissolving the crude extracellular polysaccharide in distilled water (10 mg / mL) , 100 mg was loaded on a DEAE- Cellulose 52 ion exchange column, and distilled water, 0.1 mol / L NaCl, 0.3 mol / L NaCl and 0 were loaded. Linear gradient elution with .5 mol / L NaCl, elution rate is 1 mL / min, 5 mL is collected for each tube, and polysaccharide content (polysaccharide content is detected by the phenol sulfate method and an elution curve is drawn. ) Is detected for each tube, and single peak components are collected together according to the detected value of polysaccharide content, dialyzed with deionized water, freeze-dried, and then dried.
After the polysaccharide components eluted at NaCl of 0.3 mol / L was dissolved in distilled water (5mg / mL), SephadexG - 100 loaded onto gel column, eluted with deionized water, elution rate is 0.2 mL / min The eluate is collected by an automatic partial collector, a volume of 2 mL is collected for each tube, the polysaccharide content is detected for each tube, and a single peak component is collected together according to the polysaccharide content detection value, and then removed. It was prepared in
比較例４ Comparative Example 4
比較例１で得た植物性乳酸菌細胞外多糖２ｇを１００ｍＬのジメチルスルホキシドに溶解し、６０℃で３０ｍｉｎ加熱して十分に溶解させた後、３６ｇの尿素と１５ｍＬのリン酸を加え、６０℃で３ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを用いて反応液を中和し、透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖のリン酸化誘導体を得る。 2 g of the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Comparative Example 1 was dissolved in 100 mL of dimethyl sulfoxide, heated at 60 ° C. for 30 minutes to sufficiently dissolve it, and then 36 g of urea and 15 mL of phosphoric acid were added, and the temperature was increased to 60 ° C. After the reaction for 3 hours, deionized water is added to terminate the reaction, the reaction solution is neutralized with 1 M NaOH, dialyzed and freeze-dried to obtain a phosphorylated derivative of the extracellular polysaccharide of plant-derived lactic acid bacteria. ..
比較例５ Comparative Example 5
比較例２で得た植物性乳酸菌細胞外多糖２ｇを１００ｍＬのジメチルスルホキシドに溶解し、６０℃で３０ｍｉｎ加熱して十分に溶解させた後、３６ｇの尿素と１５ｍＬのリン酸を加え、６０℃で３ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを用いて反応液を中和し、透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖のリン酸化誘導体を得る。 2 g of the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Comparative Example 2 was dissolved in 100 mL of dimethyl sulfoxide, heated at 60 ° C. for 30 minutes to sufficiently dissolve it, and then 36 g of urea and 15 mL of phosphoric acid were added, and the temperature was increased to 60 ° C. After the reaction for 3 hours, deionized water is added to terminate the reaction, the reaction solution is neutralized with 1 M NaOH, dialyzed and freeze-dried to obtain a phosphorylated derivative of the extracellular polysaccharide of plant-derived lactic acid bacteria. ..
比較例６ Comparative Example 6
比較例３で得た植物性乳酸菌細胞外多糖２ｇを１００ｍＬのジメチルスルホキシドに溶解し、６０℃で３０ｍｉｎ加熱して十分に溶解させた後、３６ｇの尿素と１５ｍＬのリン酸を加え、６０℃で３ｈ反応させた後、脱イオン水を加えて反応を終了し、１ＭのＮａＯＨを用いて反応液を中和し、透析、凍結乾燥することにより、植物性乳酸菌細胞外多糖のリン酸化誘導体を得る。 2 g of the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Comparative Example 3 was dissolved in 100 mL of dimethyl sulfoxide, heated at 60 ° C. for 30 minutes to sufficiently dissolve it, and then 36 g of urea and 15 mL of phosphoric acid were added, and the temperature was increased to 60 ° C. After the reaction for 3 hours, deionized water is added to terminate the reaction, the reaction solution is neutralized with 1 M NaOH, dialyzed and freeze-dried to obtain a phosphorylated derivative of the extracellular polysaccharide of plant-derived lactic acid bacteria. ..
試験例１ Test Example 1
植物性乳酸菌ＡＴＣＣ８０１４の成長曲線の測定に関する： Regarding the measurement of the growth curve of the plant-derived lactic acid bacterium ATCC8014:
菌株を、２％の接種量で、実施例１と比較例１‐３に使用の培地に、それぞれ接種し、振とうして振り混ぜた後３７℃で静置発酵し、２ｈおきに取り出し、培地をブランク溶液としてゼロ点調製を行い、６００ｎｍの波長で吸光値を検出する。時間を横軸、吸光値を縦軸にして、図１に示すように、前記菌株の成長曲線を得る。ＡＴＣＣ８０１４の成長曲線グラフから明らかなように、実施例１の植物性乳酸菌ＡＴＣＣ８０１４は、概ね２ｈ以降から対数増殖期に入り始め、６ｈ以降は定常期に入り始め、安定期が長く、比較例１及び比較例３の植物性乳酸菌ＡＴＣＣ８０１４は概ね３ｈ以降から対数増殖期に入り始め、１４ｈ以降は安定期に入り始め、比較例２の植物性乳酸菌ＡＴＣＣ８０１４は概ね３ｈ以降から対数増殖期に入り始め、１４ｈ以降は安定期に入り始めることから、実施例１の培地で植物性乳酸菌ＡＴＣＣ８０１４を培養すると、植物性乳酸菌の対数期と安定期が比較例１‐３よりも長く、かつ植物性乳酸菌の密度も比較例１‐３よりも明らかに高かくなる。 Strains with an inoculum of 2%, Example 1 and Comparative Example 1 - the medium of use 3, respectively inoculated, shaken and allowed to stand fermented at 37 ° C. After shaking, it is taken out to 2h intervals, Zero point preparation is performed using the medium as a blank solution, and the absorption value is detected at a wavelength of 600 nm. As shown in FIG. 1, the growth curve of the strain is obtained with time on the horizontal axis and absorption value on the vertical axis. As is clear from the growth curve graph of ATCC8014, the plant-derived lactic acid bacterium ATCC8014 of Example 1 begins to enter the logarithmic growth phase after about 2h, enters the stationary phase after 6h, has a long stable phase, and has a long stable phase, and Comparative Example 1 and The plant-derived lactic acid bacterium ATCC8014 of Comparative Example 3 began to enter the logarithmic growth phase after about 3h, and began to enter the stable phase after 14h, and the plant-derived lactic acid bacterium ATCC8014 of Comparative Example 2 began to enter the logarithmic growth phase from about 3h or later, and entered the logarithmic growth phase for 14h. since the later begins to enter the stable period, the cultured plant lactic acid bacteria ATCC8014 with medium of example 1, comparison exponential phase and plateau vegetable lactic acid bacteria example 1 - longer than 3, and also the density of the plant lactic acid bacteria Comparative example 1 - 3 obviously becomes higher nuclear than.
植物性乳酸菌ＡＴＣＣ８０１４の成長曲線と、実施例１、比較例１−３で得られた細胞外多糖の収率から、テトラエチルチウラムジスルフィドおよびラクトースを含有するＭＲＳ培地で植物性乳酸菌ＡＴＣＣ８０１４を培養することにより、植物性乳酸菌の対数期および安定期の延長に有利となり、細胞外多糖を多く合成し、植物性乳酸菌の多糖産生力を向上させ、最終的に細胞外多糖の収量を向上させることができることがわかる。 From the growth curve of plant-derived lactic acid bacterium ATCC8014 and the yield of extracellular polysaccharides obtained in Example 1 and Comparative Example 1-3, by culturing the plant-derived lactic acid bacterium ATCC8014 in an MRS medium containing tetraethylthiuram disulfide and lactose. , It is advantageous for prolonging the logarithmic phase and stable phase of plant-derived lactic acid bacteria, it is possible to synthesize a large amount of extracellular lactose, improve the polysaccharide-producing ability of plant-derived lactic acid bacteria, and finally improve the yield of extracellular lactose. Understand.
試験例２ Test Example 2
植物性乳酸菌細胞外多糖の性能測定に関する： Regarding performance measurement of plant-derived lactic acid bacteria extracellular polysaccharide:
１．植物性乳酸菌細胞外多糖の分子量の測定 1. 1. Measurement of molecular weight of plant-derived lactic acid bacteria extracellular polysaccharide
分子量の異なるＤｅｘｔｒａｎ標準品を順次に測定対象多糖サンプルに注入し、保留時間（ＴＲ）を記録し、各標準品の保留時間（ＴＲ）を横軸とし、各多糖標準品のピーク分子量の対数（ｌｏｇ Ｍｏｌ Ｗｔ）を縦軸にして検量線（図２）を作成し、分子量と保留時間（ＴＲ）との回帰式を求め、ｙ＝１１．９９７２‐０．４０６６２ｘ、Ｒ２＝０．９９８２４とする。測定対象多糖サンプルに上記手順でＤｅｘｔｒａｎ標準品を注入し、得られた保留時間（ＴＲ）に基づいて、標準分子量曲線から測定対象多糖サンプルの平均相対分子量を自動的に算出するとともに、多糖サンプルのクロマトグラム上のピークの数および形状から多糖の純度を同定することができる。実施例１、比較例１−３に係る細胞外多糖の高性能液体クロマトグラフィーは、図３に示すように、図から明らかなように、実施例１、比較例１−３に係る細胞外多糖は、いずれも単一ピーク形状であり、４種の多糖がいずれも均一の多糖であることを説明した。実施例１、比較例１−３の細胞外多糖の保留時間は順に１４．４２８ｍｉｎ、１５．６６０ｍｉｎ、１５．６６０ｍｉｎ及び１５．６６０ｍｉｎであり，対応する保留時間を標準分子量と保留時間の回帰式に代入して、実施例１、比較例１−３で得られた細胞外多糖の分子量は順に１．３５×１０６Ｄａ、４．２６×１０５Ｄａ、４．２６×１０５Ｄａ及び４．２６×１０５Ｄａである。 Dextran standard products with different molecular weights are sequentially injected into the polysaccharide sample to be measured, the hold time (TR) is recorded, the hold time (TR) of each standard product is on the horizontal axis, and the logarithmic peak molecular weight of each polysaccharide standard product ( and a log Mol Wt) on the vertical axis to create a calibration curve (Fig. 2), a regression equation between the molecular weight and the hold time (TR), y = 11.9972 - and 0.40662x, R2 = 0.99824 .. The Dextran standard product is injected into the polysaccharide sample to be measured according to the above procedure, and the average relative molecular weight of the polysaccharide sample to be measured is automatically calculated from the standard molecular weight curve based on the obtained hold time (TR), and the polysaccharide sample is automatically calculated. The purity of the polysaccharide can be identified from the number and shape of peaks on the chromatogram. As shown in FIG. 3, high performance liquid chromatography of the extracellular polysaccharide according to Example 1 and Comparative Example 1-3 is performed on the extracellular polysaccharide according to Example 1 and Comparative Example 1-3, as is clear from the figure. Explained that all of them had a single peak shape, and that all four types of polysaccharides were uniform polysaccharides. The holding times of the extracellular polysaccharides of Example 1 and Comparative Example 1-3 are 14.428min, 15.660min, 15.660min and 15.660min, respectively, and the corresponding holding times are expressed in the regression equation of the standard molecular weight and the holding time. Substituting, the molecular weights of the extracellular polysaccharides obtained in Example 1 and Comparative Example 1-3 are 1.35 × 106 Da, 4.26 × 105 Da, 4.26 × 105 Da and 4.26 × 105 Da, respectively.
２．植物性乳酸菌細胞外多糖の単糖構成分析 2. Monosaccharide composition analysis of plant-derived lactic acid bacteria extracellular polysaccharide
２．１多糖の加水分解 2.1 Hydrolysis of polysaccharides
細胞外多糖サンプル５ｍｇをアンプルに入れ、さらに２ｍｏｌ／Ｌトリフルオロ酢酸２ｍＬを加えてアルコールランプで封口し、オーブン中１２０℃で２ｈ加水分解し、加水分解液を減圧留去した後、さらに少量のメタノールを加えて減圧留去し（５回繰り返し）、残ったトリフルオロ酢酸を除去した後、少量の水を加えて溶解し、凍結乾燥すれば、完全酸加水分解された単糖サンプルを得る。 Put 5 mg of the extracellular polysaccharide sample in an ampol, add 2 mL of 2 mol / L trifluoroacetic acid, seal with an alcohol lamp, hydrolyze in an oven at 120 ° C. for 2 hours, distill off the hydrolyzate under reduced pressure, and then distill off a smaller amount. Methanol is added and distilled off under reduced pressure (repeated 5 times) to remove the remaining trifluoroacetic acid, and then a small amount of water is added to dissolve the mixture, and the mixture is freeze-dried to obtain a completely acid-hydrolyzed monosaccharide sample.
２．２糖のニトリル酢酸メンチル誘導体の調製 2.2 Preparation of nitrile mentyl acetate derivative of sugar
各単糖標準品５ｍｇ、ＮａＢＨ４３０ｍｇ及び５ｍｇのイノシトールをアンプルに量り取り、徐々に２ｍＬの蒸留水を滴下しながら振盪し、室温で糖アルコールに還元反応させ（２ｈ反応し）、気泡が発生しなくなるまで氷酢酸を滴下して過剰量のＮａＢＨ４を中和する。ロータリーエバポレーターを６０℃に温調してサンプルが完全に乾燥するまで真空回転蒸発させ、その後、０．１％（ｖ／ｖ）塩酸メタノール溶液２ｍＬで再溶解した後、再度蒸発乾固し、ホウ酸塩を除去するように４〜５回繰り返す。処理済みの生成物を１０５℃オーブンで１５ｍｉｎ脱水し、ピリジンと無水酢酸をそれぞれ０．５ｍＬ出し、アルコールジェットで封口し、沸騰水浴で１ｈ反応させ、得られた生成物を微孔膜（０．２２μｍ）を経て不純物を除去し、ガスクロマトグラフィー分析を行う。完全酸加水分解後の細胞外多糖サンプル誘導体の調製は同様にこの方法に従って調製される。 Weigh 5 mg of each monosaccharide standard, 430 mg of NaBH and 5 mg of inositol into an ampol, shake while gradually dropping 2 mL of distilled water, and reduce reaction to sugar alcohol at room temperature (react for 2 hours), and no bubbles are generated. Glacial acetic acid is added dropwise to neutralize the excess amount of NaBH4. The rotary evaporator was heated to 60 ° C. and vacuum rotary evaporated until the sample was completely dried, then redissolved in 2 mL of 0.1% (v / v) hydrochloric acid-methanol solution, evaporated to dryness again, and borate. Repeat 4-5 times to remove the acid salt. The treated product was dehydrated in an oven at 105 ° C. for 15 minutes, 0.5 mL each of pyridine and acetic anhydride were taken out, sealed with an alcohol jet, and reacted in a boiling water bath for 1 h, and the obtained product was subjected to a microporous membrane (0. Impurities are removed through 22 μm), and gas chromatography analysis is performed. Preparation of the extracellular polysaccharide sample derivative after complete acid hydrolysis is similarly prepared according to this method.
２．３ガスクロマトグラフィー条件クロマトグラフ 2.3 Gas Chromatography Condition Chromatography
Ａｇｉｌｅｎｔ７８９０Ａガスクロマトグラフを採用し、カラムは、ＤＢ２２５キャピラリカラム（３０ｍ×０．２５ｍｍ）であり、検出器は、酸素炎イオン検出器であり、流速は、１ｍＬ／ｍｉｎであり、スプリット比は、１：５０であり、注入量は、１μＬである。昇温プログラム：８０℃で３ｍｉｎ保持し、５℃／ｍｉｎで１９５℃まで昇温して１ｍｉｎ保持し、５℃／ｍｉｎで２１５℃まで昇温して１ｍｉｎ保持し、１０℃／ｍｉｎで２３０℃まで昇温して３ｍｉｎ保持する。 Adopting the Agent7890A gas chromatograph, the column is a DB225 capillary column (30 m x 0.25 mm), the detector is an oxygen flame ion detector, the flow velocity is 1 mL / min, and the split ratio is 1: It is 50 and the injection volume is 1 μL. Temperature rise program: Hold at 80 ° C for 3 min, raise to 195 ° C at 5 ° C / min for 1 min, raise to 215 ° C at 5 ° C / min for 1 min, hold at 230 ° C at 10 ° C / min The temperature is raised to 3 min.
実施例１、比較例１−３で得られた細胞外多糖をトリフルオロ酢酸で加水分解した後のガスクロマトグラフィー分析及び単糖標準品との比較結果は図４に示すとおりである。単糖標準品の保留時間（図４標準品）は左から右へ順にラムノース（２６．２１５ｍｉｎ）、フコース（２６．４２７ｍｉｎ）、アラビノース（２６．５３１ｍｉｎ）、キシロース（２６．９５１ｍｉｎ）、マンノース（３０．５５３ｍｉｎ）、フルクトース（３１．５５３ｍｉｎ）、グルコース（３１．７１４ｍｉｎ）、ガラクトース（３１．９２１ｍｉｎ）である。実施例１、比較例１−３の細胞外多糖はいずれも保留時間が３０．５５３ｍｉｎ、３１．７１４ｍｉｎ及び３１．９２１ｍｉｎの時にピークを出し（図４の実施例１、比較例１−３）、実施例１、比較例１−３の細胞外多糖はいずれも主にマンノース、ガラクトース及びグルコースの三種類の単糖から構成されることを示す。面積正規化法による定量分析により、実施例１に係る細胞外多糖におけるマンノース、ガラクトース及びグルコースのモル比は、５．６：７．３：１であり、比較例１−３に係る細胞外多糖におけるマンノース、ガラクトース及びグルコースのモル比は、１：２．６：４．２である。 The results of gas chromatography analysis after hydrolysis of the extracellular polysaccharides obtained in Example 1 and Comparative Example 1-3 with trifluoroacetic acid and comparison with the monosaccharide standard are shown in FIG. The holding time of the monosaccharide standard (Fig. 4 standard) is from left to right: rhamnose (26.215 min), fucose (26.427 min), arabinose (26.531 min), xylose (26.951 min), mannose (30). .553 min), fructose (31.553 min), glucose (31.714 min), galactose (31.921 min). The extracellular polysaccharides of Example 1 and Comparative Example 1-3 all peaked at the retention times of 30.535 min, 31.714 min and 31.921 min (Example 1 and Comparative Example 1-3 in FIG. 4). It is shown that the extracellular polysaccharides of Example 1 and Comparative Example 1-3 are mainly composed of three types of monosaccharides, mannose, galactose and glucose. According to the quantitative analysis by the area normalization method, the molar ratio of mannose, galactose and glucose in the extracellular polysaccharide according to Example 1 was 5.6: 7.3: 1, and the extracellular polysaccharide according to Comparative Example 1-3. The molar ratio of mannose, galactose and glucose in is 1: 2.6: 4.2.
細胞外多糖の分子量と単糖構成から、テトラエチルチウラムジスルフィドとラクトースを含有するＭＲＳ培地で植物性乳酸菌ＡＴＣＣ８０１４を培養することで、細胞外多糖の化学構造を変更し、本発明の細胞外多糖を得ることができることがわかる。 From the molecular weight and monosaccharide composition of the extracellular polysaccharide, the chemical structure of the extracellular polysaccharide is changed by culturing the plant-derived lactic acid bacterium ATCC8014 in an MRS medium containing tetraethylthiuram disulfide and lactose, and the extracellular polysaccharide of the present invention is obtained. You can see that you can.
試験例２ Test Example 2
植物性乳酸菌細胞外多糖リン酸化誘導体の置換度測定 Measurement of degree of substitution of plant-derived lactic acid bacteria extracellular polysaccharide phosphorylated derivative
リン元素の含有量は誘導結合−原子発光分光計を用いて測定し、実施例１、比較例１―３で得られたリン酸化誘導体のリン元素の含有量（Ｐ％）はそれぞれ４．１１％、２．３４％、２．３４％、２．３４％であり、置換度（リン酸化の程度）は、置換度＝（５．２２×Ｐ％）／（１−２．６１×Ｐ％）により算出する。 The phosphorus element content was measured using an induction bond-atomic emission spectrometer, and the phosphorus element content (P%) of the phosphorylated derivatives obtained in Example 1 and Comparative Example 1-3 was 4.11, respectively. %, 2.34%, 2.34%, 2.34%, and the degree of substitution (degree of phosphorylation) is the degree of substitution = (5.22 × P%) / (1-2.61 × P%). ).
実施例１、比較例１―３で得られたリン酸化誘導体のリン元素の含有量（Ｐ％）の算出結果は、それぞれ０．２４、０．１３、０．１３及び０．１３である。 The calculation results of the phosphorus element content (P%) of the phosphorylated derivatives obtained in Example 1 and Comparative Example 1-3 are 0.24, 0.13, 0.13 and 0.13, respectively.
試験例３ Test Example 3
１．植物性乳酸菌細胞外多糖及びそのリン酸化誘導体のＡＣＥ阻害性能測定 1. 1. Measurement of ACE inhibition performance of plant-derived lactic acid bacteria extracellular polysaccharide and its phosphorylated derivative
ＡＣＥは、人の血管を収縮させ、血圧を上昇させることができる。活性化されたＡＣＥは、高血圧病を致す主要原因である。したがって、阻害剤でＡＣＥに活性を失わせる又は活性を阻害することは、血圧を下降させる潜在的な手段の一つである。植物性乳酸菌細胞外多糖及びそのリン酸化誘導体のＡＣＥ阻害性能は、下記の方法によって測定する。 ACE can constrict a person's blood vessels and raise blood pressure. Activated ACE is a major cause of hypertension. Therefore, causing ACE to lose or inhibit activity with an inhibitor is one of the potential means of lowering blood pressure. The ACE inhibitory performance of plant-derived lactic acid bacteria extracellular polysaccharides and their phosphorylated derivatives is measured by the following method.
質量濃度１００ｍｇ／ｍＬの植物性乳酸菌細胞外多糖及びそのリン酸化誘導体のサンプル溶液を採取し、ＡＣＥ阻害活性を測定し、試験管に基質として６．５ｍｍｏｌ／ＬＨＨＬ溶液１００ｍＬ、サンプル溶液２００ｍＬ、１００ｍｍｏｌ／Ｌリン酸塩緩衝液（ｐＨ＝８．３）２００ｍＬを順次加え、３７℃恒温水浴で３〜５ｍｉｎ予熱した後、ＡＣＥ酵素液５００ｍＬを加えて反応を開始し、３７℃で３０ｍｉｎ水浴した後、１ｍｏｌ／ＬＨＣｌを１００ｍＬ加えて反応を終了する。そして酢酸エチルを１．５ｍＬ加え、均一に混合した後遠心分離（５２００ｒ／ｍｉｎ，１０ｍｉｎ）を行った後、上層の酢酸エチルを１ｍＬ吸引して別の試験管に移し、１２０℃のオーブンに入れて３０ｍｉｎ揮発させ、冷却後に蒸留水６ｍＬを加え、均一に混合した後にその吸光度値を２２８ｎｍで測定し、算出式は、ＡＣＥ阻害率（％）＝（Ａｂ−Ａａ）／（Ａｂ−Ａｃ）×１００とする。
A sample solution of a plant-derived lactic acid bacterium extracellular polysaccharide having a mass concentration of 100 mg / mL and a phosphorylated derivative thereof was collected, the ACE inhibitory activity was measured, and a 6.5 mmol / LHHL solution 100 mL, a
Ａａは、ＡＣＥとＨＨＬに阻害剤を添加して反応させた後のサンプルの吸光度値である。 Aa is the absorbance value of the sample after the inhibitor was added to ACE and HHL and reacted.
Ａｂは、阻害剤を添加せず、ＡＣＥとＨＨＬが完全に反応する対照群の吸光度値である。 Ab is the absorbance value of the control group in which ACE and HHL completely react with each other without adding an inhibitor.
Ａｃは、阻害剤を添加し、反応前に予め不活性化されたＡＣＥをＨＨＬと反応させるブランク対照群の吸光度値である。 Ac is the absorbance value of the blank control group in which the inhibitor is added and the ACE previously inactivated before the reaction is reacted with HHL.
植物性乳酸菌細胞外多糖及びそのリン酸化誘導体のＡＣＥへの阻害作用は、図５に示すとおりであり、実施例１で得られた植物性乳酸菌細胞外多糖および実施例２で得られた植物性乳酸菌細胞外多糖のリン酸化誘導体は、ＡＣＥに対し、顕著な阻害作用を有し、比較例１−６で得られた植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は、ＡＣＥに対する阻害作用が明らかでない。 The inhibitory effect of the plant-derived lactic acid bacterium extracellular polysaccharide and its phosphorylated derivative on ACE is as shown in FIG. 5, and the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Example 1 and the plant-derived product obtained in Example 2 are shown. The phosphorylated derivative of the lactic acid bacterium extracellular polysaccharide has a remarkable inhibitory effect on ACE, and the plant-derived lactic acid bacterium extracellular polysaccharide obtained in Comparative Example 1-6 and its phosphorylated derivative have a clear inhibitory effect on ACE. Not.
２．植物性乳酸菌細胞外多糖及びそのリン酸化誘導体の細胞傷害性測定
ＭＴＴ法により、培養終了前４ｈ、ウェル当たり２０μＬＭＴＴ（５ｇ／Ｌ）を加え、４ｈ培養を継続する。培養終了後にジメチルスルホキシド１５０μｌを添加する。酵素結合免疫検出器は５７０ｎｍでＡ５７０値を測定する。その結果、図６に示すように、異なる濃度の植物性乳酸菌細胞外多糖及びそのリン酸化誘導体を添加した時の細胞培養液は、対照群と比較してＯＤ値の間に有意差がなく、実施例１‐２、比較例１‐６で得られた植物性乳酸菌細胞外多糖及びそのリン酸化誘導はいずれも細胞毒性を示さなかったことが明らかとなった。
2. Measurement of cytotoxicity of plant-derived lactic acid bacteria extracellular polysaccharide and its phosphorylated derivative By the MTT method, 20 μLMTT (5 g / L) per well is added 4 hours before the end of the culture, and the culture is continued for 4 hours. After completion of the culture, 150 μl of dimethyl sulfoxide is added. The enzyme-bound immunodetector measures the A570 value at 570 nm. As a result, as shown in FIG. 6, there was no significant difference in OD value between the cell culture solutions when different concentrations of plant-derived lactic acid bacterium extracellular polysaccharide and its phosphorylated derivative were added, as compared with the control group. example 1 - 2, Comparative example 1 - obtained plant lactic acid bacteria exopolysaccharides and its phosphorylated induced 6 clear that none cytotoxicity.
３．植物性乳酸菌細胞外多糖及びそのリン酸化誘導体の体内抗高血圧活性測定 3. 3. Measurement of in-vivo antihypertensive activity of plant-derived lactic acid bacteria extracellular polysaccharide and its phosphorylated derivative
動物実験は倫理基準に合致する（承認番号：２０１４０４０５）。高血圧自然発症ラットは恒温条件下（２２±２℃）で飼育し、１２ｈの明暗サイクルを与え、摂食を自由にする。実施例１の細胞外多糖及び実施例２の細胞外多糖のリン酸化誘導体を生理食塩水で０．０５ｇ／ｋｇの用量に調製し、毎回胃内に１ｍＬ投与し、対照群に等量の生理食塩水を投与する。第０、２、４、６、８、１２、２４ｈにおいて、それぞれラットの収縮期血圧と拡張期血圧を測定し、収縮期血圧（ＳＢＰ）と拡張期血圧（ＤＢＰ）の変化を観察して、植物性乳酸菌細胞外多糖及びそのリン酸化誘導体の降圧効果を評価する（（図７及び図８）。陰性対照群（生理食塩水）のラットのＳＢＰとＤＢＰは２４ｈ内に顕著に変化しない。実施例１の細胞外多糖群のラット血圧変化は明らかであり、実施例１の細胞外多糖を胃内に投与した８ｈ後、収縮期血圧は５３．８ｍｍＨｇ下降し、拡張期血圧は６８．１ｍｍＨｇ下降し、投与した２４ｈ後は初期レベルに対する収縮期血圧の差圧は２．１ｍｍＨｇ、初期レベルに対する拡張期血圧の差圧は２．１ｍｍＨｇであり、実施例２の細胞外多糖のリン酸化誘導体群のラット血圧変化は明らかであり、実施例１の細胞外多糖を胃内に投与した６ｈ後、収縮期血圧は６１．６ｍｍＨｇ下降し、拡張期血圧は６８．７ｍｍＨｇ下降し、投与した２４ｈ後は収縮期血圧の初期レベルに対する差圧は６．４ｍｍＨｇ、拡張期血圧の初期水平に対する差圧は２．５ｍｍＨｇである。体内の降圧結果はインビトロでＡＣＥの酵素に対する阻害作用結果と一致する。体内、インビトロ実験結果により、植物性乳酸菌細胞外多糖及びそのリン酸化誘導体は高い降圧作用を有することを示す。 Animal studies meet ethical standards (approval number: 2014405). Spontaneous hypertensive rats are bred under homeothermic conditions (22 ± 2 ° C.) and given a 12 h light-dark cycle to free feeding. The extracellular polysaccharide of Example 1 and the phosphorylated derivative of the extracellular polysaccharide of Example 2 were prepared in physiological saline at a dose of 0.05 g / kg, and 1 mL was administered intragastrically each time, and the same amount of physiology was given to the control group. Administer saline solution. At the 0th, 2nd, 4th, 6th, 8th, 12th, and 24th hours, the systolic blood pressure and the diastolic blood pressure of the rats were measured, and the changes in the systolic blood pressure (SBP) and the diastolic blood pressure (DBP) were observed. The antihypertensive effect of plant-derived lactic acid bacterium extracellular polysaccharide and its phosphorylated derivative is evaluated ((FIG. 7 and 8). SBP and DBP of rats in the negative control group (physiological saline) do not change significantly within 24 hours. Changes in rat blood pressure in the extracellular polysaccharide group of Example 1 are clear, and after 8 hours of intragastric administration of the extracellular polysaccharide of Example 1, systolic blood pressure decreased by 53.8 mmHg and diastolic blood pressure decreased by 68.1 mmHg. However, 24 hours after administration, the differential pressure of systolic blood pressure with respect to the initial level was 2.1 mmHg, and the differential pressure of diastolic blood pressure with respect to the initial level was 2.1 mmHg. Changes in rat blood pressure are clear: systolic blood pressure decreased by 61.6 mmHg, diastolic blood pressure decreased by 68.7 mmHg after 6 hours of intragastric administration of the extracellular polysaccharide of Example 1, and contraction occurred 24 hours after administration. The differential pressure of systolic blood pressure with respect to the initial level is 6.4 mmHg, and the differential pressure of diastolic blood pressure with respect to the initial horizontal is 2.5 mmHg. Experimental results show that plant-derived lactic acid bacteria extracellular polysaccharides and their phosphorylated derivatives have a high antihypertensive effect.
上記実施例における従来技術は当業者に知られている従来技術であるため、ここでは詳細な説明を省略する。 Since the prior art in the above embodiment is a prior art known to those skilled in the art, detailed description thereof will be omitted here.
以上の実施形態は、本発明を説明するためのものであり、本発明を限定するものではなく、本発明の精神及び範囲から逸脱することなく、様々な変形や変形が可能であることは当業者に明らかである。したがって、すべての同等の技術的解決手段も本発明の範疇に含まれるものであり、本発明の特許請求の範囲は、特許請求の範囲によって制限されるべきである。 The above embodiments are for explaining the present invention, do not limit the present invention, and can be variously modified and modified without departing from the spirit and scope of the present invention. It is obvious to the trader. Therefore, all equivalent technical solutions are also included in the scope of the present invention, and the scope of claims of the present invention should be limited by the scope of claims.
Claims (10)
前記１）は、重量平均分子量は、１．３５×１０６Ｄａであり、
前記２）は、モル比が５．６：７．３：１のマンノースと、ガラクトースとブドウ糖から構成される、
ことを特徴とする植物性乳酸菌細胞外多糖。 It is a plant-derived lactic acid bacterium extracellular polysaccharide and has at least one of the characteristics of 1) and 2).
In 1), the weight average molecular weight is 1.35 × 106 Da.
The above 2) is composed of mannose having a molar ratio of 5.6: 7.3: 1, galactose and glucose.
It is characterized by the fact that it is a plant-derived lactic acid bacterium extracellular polysaccharide.
ことを特徴とする請求項１に記載の植物性乳酸菌細胞外多糖。 Can inhibit angiotensin converting enzyme (ACE),
The plant-derived lactic acid bacterium extracellular polysaccharide according to claim 1.
ことを特徴とする請求項４に記載の植物性乳酸菌細胞外多糖。 The degree of substitution is 0.24.
The plant-derived lactic acid bacterium extracellular polysaccharide according to claim 4, wherein the polysaccharide is characterized by the above.
ことを特徴とする心臓血管疾患予防・治療用の組成物。 It comprises at least one of the plant-derived lactic acid bacterium extracellular polysaccharide according to claim 1 or 2 and the phosphorylated derivative of the plant-derived lactic acid bacterium extracellular polysaccharide according to claim 4 or 5.
A composition for the prevention and treatment of cardiovascular diseases.
ことを特徴とする請求項６に記載の心臓血管疾患予防・治療用の組成物。 The cardiovascular disease is one or more diseases selected from the group consisting of hypertension, heart disease, stroke, thrombosis, atherosclerosis, angina, heart failure and myocardial infarction.
The composition for preventing / treating cardiovascular disease according to claim 6.
ことを特徴とする請求項６に記載の心臓血管疾患予防・治療用の組成物。 The composition is an oral composition.
The composition for preventing / treating cardiovascular disease according to claim 6.
ことを特徴とする請求項６又は請求項８に記載の心臓血管疾患予防・治療用の組成物。 The composition is used in such a manner that an adult ingests 0.1-500 mg of at least one of a plant-derived lactic acid bacterium extracellular polysaccharide and a phosphorylated derivative thereof per day.
The composition for the prevention / treatment of cardiovascular disease according to claim 6 or 8.
ことを特徴とする請求項６又は請求項８に記載の心臓血管疾患予防・治療用の組成物。 The composition is used in a manner of ingestion for 9-12 weeks or longer.
The composition for the prevention / treatment of cardiovascular disease according to claim 6 or 8.
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| JP2021090884A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| JP2021090883A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| JP2021090881A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| JP2021090882A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| JP2021090880A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| CN116656577A (en) * | 2023-07-25 | 2023-08-29 | 内蒙古大学 | Lactobacillus plantarum extracellular polysaccharide and application thereof in preparation of immunoadjuvant |
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| CN113698503B (en) * | 2021-08-16 | 2022-05-24 | 华南理工大学 | Lactobacillus plantarum neutral exopolysaccharide and application thereof |
| CN114058510A (en) * | 2021-11-10 | 2022-02-18 | 广东丸美生物技术股份有限公司 | Extraction method of lactobacillus lysis active substance and lactobacillus lysis active substance obtained by same |
| WO2023230515A1 (en) * | 2022-05-26 | 2023-11-30 | Pouda Biotech Llc | Lactobacillus plantarum pdg8 and use in treating cardiovascular diseases |
| CN118006485B (en) * | 2024-01-23 | 2024-07-12 | 朗恒科技集团有限公司 | Lactobacillus plantarum ST10-7 and application thereof |
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| KR20110052808A (en) * | 2009-11-13 | 2011-05-19 | 충북대학교 산학협력단 | A polysaccharide producing lactobacillus paracasei and a use thereof |
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| AU2005211224A1 (en) * | 2004-02-04 | 2005-08-18 | Danisco A/S | Cheese products |
| CN100484963C (en) * | 2005-08-19 | 2009-05-06 | 光明乳业股份有限公司 | Exo-polysaccharide of lactobacillus casei and crude product, prepartion method and application |
| JP6524468B2 (en) * | 2013-09-19 | 2019-06-05 | 日東薬品工業株式会社 | Exopolysaccharides produced by lactic acid bacteria |
| CN105441357A (en) * | 2015-12-16 | 2016-03-30 | 北京工商大学 | Lactobacillus plantarum for producing antitumor activity exopolysaccharides |
| CN107058422B (en) * | 2017-05-17 | 2020-03-17 | 华南农业大学 | Method for high yield of exopolysaccharide by lactobacillus plantarum |
| CN109295126B (en) * | 2018-08-31 | 2021-01-08 | 四川农业大学 | Lactobacillus plantarum exopolysaccharide with immunoregulatory activity and preparation method thereof |
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| KR20110052808A (en) * | 2009-11-13 | 2011-05-19 | 충북대학교 산학협력단 | A polysaccharide producing lactobacillus paracasei and a use thereof |
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| BIOL. PHARM. BULL, vol. 40, JPN6020041827, 2017, pages 621 - 629, ISSN: 0004377197 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021090884A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| JP2021090883A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| JP2021090881A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| JP2021090882A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| JP2021090880A (en) * | 2021-03-19 | 2021-06-17 | 株式会社三洋物産 | Game machine |
| CN116656577A (en) * | 2023-07-25 | 2023-08-29 | 内蒙古大学 | Lactobacillus plantarum extracellular polysaccharide and application thereof in preparation of immunoadjuvant |
| CN116656577B (en) * | 2023-07-25 | 2023-09-26 | 内蒙古大学 | Lactobacillus plantarum extracellular polysaccharide and application thereof in preparation of immunoadjuvant |
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| Publication number | Publication date |
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| CN110714038B (en) | 2021-03-23 |
| CN110714038A (en) | 2020-01-21 |
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