JP2015189731A - Mannopolyocyl fructose and production method thereof, improving agent for intestinal bacterial flora, culture medium for microbial cultivation, and anticancer agent - Google Patents
Mannopolyocyl fructose and production method thereof, improving agent for intestinal bacterial flora, culture medium for microbial cultivation, and anticancer agent Download PDFInfo
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- JP2015189731A JP2015189731A JP2014069592A JP2014069592A JP2015189731A JP 2015189731 A JP2015189731 A JP 2015189731A JP 2014069592 A JP2014069592 A JP 2014069592A JP 2014069592 A JP2014069592 A JP 2014069592A JP 2015189731 A JP2015189731 A JP 2015189731A
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- Prior art keywords
- oligosaccharide
- fructose
- mannose
- improving agent
- intestinal
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
Description
本発明は、マンノースとフラクトースを構成糖とする新規オリゴ糖、当該オリゴ糖の製造方法、当該オリゴ糖を有効成分とする腸内細菌叢改善剤、当該オリゴ糖を含有する微生物培養用培地、及び当該オリゴ糖を有効成分とする抗癌剤に関する。 The present invention relates to a novel oligosaccharide comprising mannose and fructose as constituent sugars, a method for producing the oligosaccharide, an intestinal flora improving agent comprising the oligosaccharide as an active ingredient, a culture medium for microbial culture containing the oligosaccharide, and The present invention relates to an anticancer agent containing the oligosaccharide as an active ingredient.
近年、Ｄ−マンノースがβ−１，４結合した化合物であるβ−１，４マンノビオースなどのβ−１，４−マンノオリゴ糖の持つ生理機能が注目されている。また、人間の糖タンパク質の糖鎖の重要な部分構造にはＤ−マンノースがβ−１，４結合したマンノオリゴ糖が含まれており、飲食品原料としてのみならず、医薬品の原料としての応用も期待されている。例えば、マンノースを構成糖とするオリゴ糖類を経口摂取することにより、血清中の総コレステロールや中性脂肪の量が低下することが報告されている（例えば、特許文献１参照。）。 In recent years, physiological functions of β-1,4-mannooligosaccharides such as β-1,4 mannobiose, which is a compound in which D-mannose is bonded to β-1,4, have attracted attention. In addition, an important partial structure of a sugar chain of human glycoprotein includes mannooligosaccharide in which D-mannose is linked by β-1,4, and it can be applied not only as a raw material for foods but also as a raw material for pharmaceuticals. Expected. For example, it has been reported that the amount of total cholesterol and neutral fat in serum decreases by ingesting oligosaccharides containing mannose as a constituent sugar (see, for example, Patent Document 1).
本発明は、マンノースとフラクトースを構成糖とする新規オリゴ糖、当該オリゴ糖の製造方法、当該オリゴ糖を有効成分とする腸内細菌叢改善剤、当該オリゴ糖を含有する微生物培養用培地、及び当該オリゴ糖を有効成分とする抗癌剤を提供することを目的とする。 The present invention relates to a novel oligosaccharide comprising mannose and fructose as constituent sugars, a method for producing the oligosaccharide, an intestinal flora improving agent comprising the oligosaccharide as an active ingredient, a culture medium for microbial culture containing the oligosaccharide, and It aims at providing the anticancer agent which uses the said oligosaccharide as an active ingredient.
本発明者らは、マンノースを主体とした単糖類が１〜１０分子結合したオリゴ糖のうち、還元末端がフラクトースである新規オリゴ糖を見出した。さらに、ビフィズス菌及び乳酸菌は当該新規オリゴ糖を資化し得るが、大腸菌等の腐敗菌は資化できないこと、及び癌細胞に対する増殖抑制能を有することを見出し、本発明を完成させた。 The present inventors have found a novel oligosaccharide having a reducing end of fructose among oligosaccharides in which 1 to 10 molecules of monosaccharide mainly composed of mannose are bonded. Further, the present invention was completed by finding that bifidobacteria and lactic acid bacteria can assimilate the novel oligosaccharide, but that rot bacteria such as Escherichia coli cannot be assimilated and have the ability to suppress the growth of cancer cells.
［１］ 本発明の第一の態様に係るオリゴ糖は、下記式（１）（式中、ｎは０〜８の整数を表す。）で表されるマンノポリオーシルフラクトースである。 [1] The oligosaccharide according to the first embodiment of the present invention is mannopolyosyl fructose represented by the following formula (1) (wherein n represents an integer of 0 to 8).
［２］ 本発明の第二の態様に係る腸内細菌叢改善剤は、前記式（１）で表されるマンノポリオーシルフラクトースを有効成分とする。
［３］ 前記［２］の腸内細菌叢改善剤は、さらに、マンノースを主体とした単糖類が１〜１０分子結合したオリゴ糖類を含有することが好ましい。
［４］ 前記［３］の腸内細菌叢改善剤としては、前記オリゴ糖類が、マンノース、グルコース、ガラクトース及びフルクトースからなる群より選択される１種以上の単糖類が１〜１０分子結合したものであることが好ましい。
［５］ 前記［３］又は［４］の腸内細菌叢改善剤においては、前記オリゴ糖類中のマンノース残基の割合が、７０質量％以上であることが好ましい。
［６］ 前記［３］〜［５］のいずれかの腸内細菌叢改善剤においては、前記オリゴ糖類が、マンナンを加水分解処理することによって得られるものであることが好ましい。
［７］ 前記［６］の腸内細菌叢改善剤においては、前記マンナンが、コーヒー豆及び／又はコーヒー抽出残渣から得られるものであることが好ましい。
［８］ 前記［３］の腸内細菌叢改善剤においては、前記オリゴ糖類が、マンノースが１〜１０分子結合したオリゴ糖類であることが好ましい。
［９］ 前記［３］の腸内細菌叢改善剤においては、前記オリゴ糖類が、マンノースが１〜１０分子結合したβ−１，４−マンノオリゴ糖類であることが好ましい。
［１０］ 前記［３］〜［９］のいずれかの腸内細菌叢改善剤においては、前記オリゴ糖類の含量割合が、総固形分に対して６０質量％以上であることが好ましい。
［１１］ 本発明の第三の態様に係る腸内細菌叢改善剤は、前記式（１）で表されるマンノポリオーシルフラクトースを含有し、ビフィズス菌又は乳酸菌の培養用として用いられる、微生物培養用培地である。
［１２］ 前記［１１］の微生物培養用培地としては、さらに、マンノースを主体とした単糖類が１〜１０分子結合したオリゴ糖類を含有することが好ましい。
［１３］ 前記［１１］の微生物培養用培地としては、資化性糖として、前記マンノポリオーシルフラクトースのみを含有することも好ましい。
［１４］ 本発明の第四の態様に係るマンノポリオーシルフラクトースの製造方法は、コーヒー豆及び／又はコーヒー豆絞り滓を１８０〜２５０℃で加熱処理した後、得られたオリゴ糖組成物から、前記式（１）で表されるマンノポリオーシルフラクトースを分離精製する。
［１５］ 本発明の第五の態様に係る抗癌剤は、前記式（１）で表されるマンノポリオーシルフラクトースを有効成分とする。
[2] The intestinal flora improving agent according to the second aspect of the present invention contains mannopolyosyl fructose represented by the formula (1) as an active ingredient.
[3] The intestinal flora improving agent of [2] preferably further contains an oligosaccharide in which 1 to 10 molecules of monosaccharides mainly composed of mannose are bonded.
[4] The intestinal bacterial flora-improving agent according to [3], wherein the oligosaccharide is a combination of 1 to 10 molecules of one or more monosaccharides selected from the group consisting of mannose, glucose, galactose and fructose. It is preferable that
[5] In the intestinal bacterial flora improving agent of [3] or [4], the proportion of mannose residues in the oligosaccharide is preferably 70% by mass or more.
[6] In the intestinal flora improving agent according to any one of [3] to [5], the oligosaccharide is preferably obtained by hydrolyzing mannan.
[7] In the intestinal flora improving agent of [6], it is preferable that the mannan is obtained from coffee beans and / or coffee extraction residues.
[8] In the intestinal flora improving agent of [3], the oligosaccharide is preferably an oligosaccharide having 1 to 10 molecules of mannose bonded thereto.
[9] In the intestinal flora improving agent of [3], the oligosaccharide is preferably a β-1,4-mannooligosaccharide having 1 to 10 molecules of mannose bound thereto.
[10] In the intestinal flora improving agent of any one of [3] to [9], the content ratio of the oligosaccharide is preferably 60% by mass or more based on the total solid content.
[11] The intestinal flora improving agent according to the third aspect of the present invention contains mannopolyosyl fructose represented by the above formula (1), and is used for culturing bifidobacteria or lactic acid bacteria. It is a culture medium for microorganisms.
[12] The culture medium for microbial culture of [11] preferably further contains an oligosaccharide in which 1 to 10 molecules of monosaccharides mainly composed of mannose are bonded.
[13] The microorganism culture medium of [11] preferably contains only the mannopolyocylfructose as an assimilating sugar.
[14] The method for producing mannopolyausyl fructose according to the fourth aspect of the present invention comprises heat treating coffee beans and / or coffee bean squeezed at 180 to 250 ° C., and then obtaining the resulting oligosaccharide composition. The mannopolyausyl fructose represented by the formula (1) is separated and purified.
[15] The anticancer agent according to the fifth aspect of the present invention contains mannopolyosyl fructose represented by the formula (1) as an active ingredient.
本発明に係る新規オリゴ糖であるマンノポリオーシルフラクトースは、ビフィズス菌及び乳酸菌は当該新規オリゴ糖を資化し得るが、大腸菌等の腐敗菌は資化できない。このため、マンノポリオーシルフラクトースを摂取することにより、腸内において、ビフィズス菌や乳酸菌を大腸菌等よりも優先的に増殖させることができ、腸内細菌叢を改善し得る。また、マンノポリオーシルフラクトースを資化性糖として含有する微生物培養用培地は、ビフィズス菌又は乳酸菌の培養用として好適である。
さらに、本発明に係る新規オリゴ糖であるマンノポリオーシルフラクトースは、癌細胞に対する増殖抑制能を有しており、このために抗癌剤の有効成分としても好適である。
Mannopolyocyl fructose, which is a novel oligosaccharide according to the present invention, can assimilate the novel oligosaccharide by bifidobacteria and lactic acid bacteria but cannot assimilate spoilage bacteria such as Escherichia coli. For this reason, by ingesting mannopolyosyl fructose, bifidobacteria and lactic acid bacteria can be preferentially grown in the intestine over E. coli and the like, and the intestinal bacterial flora can be improved. Moreover, the culture medium for microorganisms containing mannopolyosyl fructose as an assimilating sugar is suitable for culture of bifidobacteria or lactic acid bacteria.
Furthermore, mannopolyosyl fructose, which is a novel oligosaccharide according to the present invention, has the ability to suppress the growth of cancer cells, and is therefore suitable as an active ingredient of anticancer agents.
本発明に係る新規オリゴ糖は、下記式（１）で表されるマンノポリオーシルフラクトースである。式（１）中、ｎは０〜８の整数を表す。 The novel oligosaccharide according to the present invention is mannopolyosyl fructose represented by the following formula (1). In formula (1), n represents an integer of 0 to 8.
前記式（１）で表されるマンノポリオーシルフラクトース（以下、単に「マンノポリオーシルフラクトース」ということがある。）は、マンノースとフラクトースから公知の化学合成によって合成してもよく、天然のマンナン等を原料として得てもよい。 The mannopolyosyl fructose represented by the formula (1) (hereinafter sometimes simply referred to as “mannopolyocylfructose”) may be synthesized from mannose and fructose by a known chemical synthesis, Natural mannan or the like may be obtained as a raw material.
マンノポリオーシルフラクトースの製造方法としては、コーヒー豆（焙煎したコーヒー豆を含む。）及び／又はコーヒー豆絞り滓を１８０〜２５０℃で加熱処理した後、得られたオリゴ糖組成物（構成単糖の種類や数が異なる複数のオリゴ糖の混合物）から、カラムクロマトグラフィー法等により分離精製する方法が好ましい。コーヒー豆等を加熱処理することにより、マンノースのみが重合してなるマンノオリゴ糖と、マンノポリオーシルフラクトースの含有量が極めて高いオリゴ糖組成物が得られるため、当該オリゴ糖組成物から分離精製することにより、マンノポリオーシルフラクトースを容易に製造し得る。 As a method for producing mannopolyosyl fructose, a coffee bean (including roasted coffee bean) and / or coffee bean squeezed heat-treated at 180 to 250 ° C., and then an oligosaccharide composition (configuration) A method of separating and purifying from a mixture of a plurality of oligosaccharides having different types and numbers of monosaccharides by a column chromatography method or the like is preferable. By heat-treating coffee beans, etc., a manno-oligosaccharide obtained by polymerizing only mannose and an oligosaccharide composition having an extremely high content of mannopolyocyl fructose can be obtained, and thus separated and purified from the oligosaccharide composition. By doing so, mannopolyausyl fructose can be easily produced.
ビフィズス菌や乳酸菌はマンノポリオーシルフラクトースを資化できるが、大腸菌やクロストリジウム菌等は資化できない。このため、マンノポリオーシルフラクトースは、腸内細菌叢改善剤として有用である。 Bifidobacteria and lactic acid bacteria can assimilate mannopolyosyl fructose, but not Escherichia coli and Clostridium bacteria. For this reason, mannopolyosyl fructose is useful as an intestinal flora improving agent.
さらに、マンノポリオーシルフラクトースを含有する培地中では、癌細胞は顕著に増殖が抑制される。このため、マンノポリオーシルフラクトースは、抗癌剤として有用である。 Furthermore, the growth of cancer cells is markedly suppressed in a medium containing mannopolyosyl fructose. For this reason, mannopolyosyl fructose is useful as an anticancer agent.
本発明に係る腸内細菌叢改善剤及び抗癌剤は、マンノポリオーシルフラクトースのみからなるものであってもよく、他の成分を含むものであってもよい。例えば、本発明に係る腸内細菌叢改善剤は、有効成分のマンノポリオーシルフラクトースとして、前記コーヒー豆（焙煎したコーヒー豆を含む。）及び／又はコーヒー豆絞り滓を１８０〜２５０℃で加熱処理した後に得られたオリゴ糖組成物を用いてもよい。本発明に係る腸内細菌叢改善剤におけるマンノポリオーシルフラクトースの含量は、総固形分に対して３０質量％以上であることが好ましく、５０質量％以上であることがより好ましく、６０質量％以上であることがさらに好ましく、８０質量％以上であることがよりさらに好ましい。 The intestinal microflora improving agent and anticancer agent according to the present invention may be composed solely of mannopolyosyl fructose or may contain other components. For example, the intestinal microflora improving agent according to the present invention contains the coffee beans (including roasted coffee beans) and / or coffee bean squeezed at 180 to 250 ° C. as the active ingredient mannopolyosyl fructose. You may use the oligosaccharide composition obtained after heat-processing. The content of mannopolyausyl fructose in the intestinal flora improving agent according to the present invention is preferably 30% by mass or more, more preferably 50% by mass or more, based on the total solid content, and 60% by mass. % Or more, more preferably 80% by mass or more.
本発明に係る腸内細菌叢改善剤及び抗癌剤は、さらに、マンノースを主体とした単糖類が１〜１０分子結合したオリゴ糖類、すなわち、「マンノースを主体としたオリゴ糖類」のうち、構成単糖の数が１〜１０分子である組成物を含有することも好ましい。 The intestinal microflora improving agent and anticancer agent according to the present invention further comprise an oligosaccharide in which 1 to 10 molecules of monosaccharide mainly composed of mannose are bonded, that is, a constituent monosaccharide among “oligosaccharides mainly composed of mannose”. It is also preferable to contain the composition whose number is 1-10 molecules.
本発明及び本願明細書において、「マンノースを主体としたオリゴ糖類」とは、単糖であるマンノースを主たる構成要素とするオリゴ糖類を意味する。ここで「オリゴ糖類」なる語は、一般に単糖類と多糖類との間に位し、一定の小数量の単糖類分子のグリコシル結合からなる物質を指す。すなわち、結合している単糖の数が比較的少ないポリマーのことである。「オリゴ糖『類』」という場合、構成単糖の種類や数が様々である複数のオリゴ糖が含まれる組成物であることを意味する。「マンノースを主体としたオリゴ糖『類』」という場合は、オリゴ糖類のうち、特に、組成物全体の構成単糖に占めるマンノースの割合が５０％以上である組成物を指す。本発明及び本願明細書において「マンノオリゴ糖類」の語は、「マンノースを主体としたオリゴ糖類」の語と同様の意味において用いられる。 In the present invention and the specification of the present application, “an oligosaccharide mainly composed of mannose” means an oligosaccharide mainly composed of mannose which is a monosaccharide. Here, the term “oligosaccharide” generally refers to a substance that is located between a monosaccharide and a polysaccharide and consists of a glycosyl bond of a certain small number of monosaccharide molecules. That is, it is a polymer with a relatively small number of monosaccharides bound thereto. The term “oligosaccharide“ class ”” means a composition containing a plurality of oligosaccharides having different types and numbers of constituent monosaccharides. The term “oligosaccharide“ s ”” mainly composed of mannose refers to a composition in which the ratio of mannose to the constituent monosaccharides of the entire composition is 50% or more among oligosaccharides. In the present invention and the present specification, the term “mannooligosaccharide” is used in the same meaning as the term “oligosaccharide mainly composed of mannose”.
本発明及び本願明細書において、オリゴ糖類の重合度を表すために「ＤＰ」と記載することがある。ＤＰとは、オリゴ糖類を構成している単糖の数を意味する。すなわち単糖であるマンノースは「ＤＰ１」と表され、４つのマンノースから構成されたマンノオリゴ糖は重合度４、すなわち「ＤＰ４」と表される。学術的観点からは、重合度１（ＤＰ１）の糖は単糖であって、オリゴ糖ではない。しかし、本発明に用いるオリゴ糖類（組成物）中には、単糖が含まれる場合があるので、本願明細書においてはこのような場合であっても総称して「オリゴ糖類」と呼ぶものとする。すなわち、「１以上１０分子以下の、マンノースを主体とした単糖類が結合した、マンノースを主体としたオリゴ糖類」という場合には、この糖組成物中に重合度１の単糖も含まれている場合があると理解されたい。 In the present invention and the present specification, “DP” may be used to indicate the degree of polymerization of the oligosaccharide. DP means the number of monosaccharides constituting the oligosaccharide. That is, mannose, which is a monosaccharide, is expressed as “DP1”, and a mannooligosaccharide composed of four mannoses is expressed as a degree of polymerization of 4, that is, “DP4”. From an academic point of view, sugars with a degree of polymerization of 1 (DP1) are monosaccharides, not oligosaccharides. However, since the oligosaccharide (composition) used in the present invention may contain a monosaccharide, in this specification, even in such a case, the term “oligosaccharide” is collectively referred to. To do. That is, in the case of “an oligosaccharide mainly composed of mannose having 1 to 10 molecules bonded with mannose-based monosaccharides”, this saccharide composition also includes monosaccharides having a degree of polymerization of 1. Please understand that there are cases.
本発明に係る腸内細菌叢改善剤及び抗癌剤に含まれる「マンノースを主体としたオリゴ糖類」としては、構成単糖がマンノース、グルコース、ガラクトース及びフルクトースからなる群より選択される１種以上の単糖が１〜１０分子結合したオリゴ糖が複数種類含まれており、かつ構成単糖全体に占めるマンノースの割合が５０％以上であるオリゴ糖類（組成物）が好ましく、構成単糖がマンノース及びフルクトースからなる群より選択される１種以上の単糖が１〜１０分子結合したオリゴ糖が複数種類含まれており、かつ構成単糖全体に占めるマンノースの割合が５０％以上であるオリゴ糖類（組成物）がより好ましい。 The “oligosaccharide mainly composed of mannose” contained in the intestinal microbiota-improving agent and anticancer agent according to the present invention includes at least one monosaccharide selected from the group consisting of mannose, glucose, galactose and fructose. Oligosaccharides (compositions) that contain a plurality of oligosaccharides having 1 to 10 sugar-bonded saccharides and that contain 50% or more of mannose in the total constituent monosaccharides are preferred, and the constituent monosaccharides are mannose and fructose. An oligosaccharide (composition comprising a plurality of types of oligosaccharides in which 1 to 10 molecules of one or more types of monosaccharides selected from the group consisting of saccharides are combined, and the proportion of mannose in the total constituent monosaccharides is 50% or more Are more preferred.
本発明に係る腸内細菌叢改善剤及び抗癌剤に含まれる「マンノースを主体としたオリゴ糖類」としては、オリゴ糖類中のマンノース残基の割合（構成単糖全体に占めるマンノースの割合）が７０質量％以上であるオリゴ糖類（組成物）が好ましく、オリゴ糖類中のマンノース残基の割合が８０質量％以上であるオリゴ糖類（組成物）がより好ましい。マンノース残基の割合が充分に高いことにより、マンノオリゴ糖による作用をより効果的に得ることができ、また、グルコース等の他の単糖類の含有割合が比較的低いことにより、甘味度等を低く抑えることができ、飲食品やサプリメント、医薬品等へ幅広く適用しやすくなる。 As the “oligosaccharide mainly composed of mannose” contained in the intestinal microflora improving agent and anticancer agent according to the present invention, the proportion of mannose residues in the oligosaccharide (the proportion of mannose in the total constituent monosaccharides) is 70 mass. % Or more oligosaccharide (composition) is preferable, and the ratio of mannose residues in the oligosaccharide is more preferably 80% by mass or more. When the ratio of mannose residues is sufficiently high, the action of mannooligosaccharide can be obtained more effectively, and the content of other monosaccharides such as glucose is relatively low, thereby reducing the sweetness level. It can be suppressed, and it becomes easy to apply widely to foods and drinks, supplements, pharmaceuticals and the like.
本発明に係る腸内細菌叢改善剤及び抗癌剤に含有させる「マンノースを主体としたオリゴ糖類」としては、単糖のマンノース由来の苦味を抑えることができるため、遊離のマンノース含量が５０質量％以下に抑えられたものが好ましい。また、当該「マンノースを主体としたオリゴ糖類」としては、２〜９分子の単糖類が結合したオリゴ糖の含量が多いものが好ましく、２〜６分子の単糖類が結合したオリゴ糖の含量が多いものがより好ましい。また、当該「マンノースを主体としたオリゴ糖類」としては、各構成単糖がβー１，４結合したオリゴ糖の含有割合が５０質量％以上であるものが好ましい。 As the “oligosaccharide mainly composed of mannose” to be contained in the intestinal flora improving agent and anticancer agent according to the present invention, the bitter taste derived from mannose of a monosaccharide can be suppressed, so that the free mannose content is 50% by mass or less. What was suppressed to is preferable. The “oligosaccharide mainly composed of mannose” is preferably one having a high content of oligosaccharides bound with 2 to 9 molecules of monosaccharides, and has a content of oligosaccharides bound with 2 to 6 molecules of monosaccharides. Many are more preferable. In addition, the “oligosaccharide mainly composed of mannose” is preferably such that the content ratio of oligosaccharides in which each constituent monosaccharide is β-1,4-bonded is 50% by mass or more.
本発明において用いられる「マンノースを主体としたオリゴ糖類」としては、構成単糖がマンノースのみからなる（マンノースのみを構成単位とする）マンノオリゴ糖類、すなわち、マンノースが１〜１０分子結合したオリゴ糖類であることも好ましい。この場合には、マンノースが１〜１０分子結合したβ−１，４−マンノオリゴ糖類であることがより好ましい。 The “oligosaccharide mainly composed of mannose” used in the present invention is a mannooligosaccharide composed of only mannose (only mannose is a structural unit), that is, an oligosaccharide in which 1 to 10 molecules of mannose are bonded. It is also preferable that there is. In this case, β-1,4-mannooligosaccharide having 1 to 10 molecules of mannose bonded thereto is more preferable.
本発明において用いられる「マンノースを主体としたオリゴ糖類」としては、マンナンを加水分解処理することによって得られるものが好ましい。なお、本発明及び本願明細書において、単に「マンナン」という場合は、Ｄ−マンノースのみを構成単位とする多糖であるマンナンの他、マンノースとガラクトース又はグルコースと構成単位とした多糖であるガラクトマンナン、グルコマンナンも広義に含めるものとする。Ｄ−マンノースはアルドヘキソースであり、Ｄ−グルコース中のカルボキシル基に隣接する炭素に結合している水酸基の立体配置が逆になっているものである。 As the “oligosaccharide mainly composed of mannose” used in the present invention, those obtained by hydrolyzing mannan are preferable. In addition, in this invention and this-application specification, when only calling it "mannan", in addition to mannan which is a polysaccharide which only has D-mannose as a structural unit, galactomannan which is a polysaccharide which has mannose and galactose or glucose and a structural unit, Glucomannan shall be included in a broad sense. D-mannose is an aldohexose in which the configuration of the hydroxyl group bonded to the carbon adjacent to the carboxyl group in D-glucose is reversed.
ここで、原料のマンナンは、例えばココナッツ椰子から得られるコプラミール、フーク、南アフリカ産椰子科植物ＨｕａｃｒａＰａｌｍ、ツクネイモマンナン、ヤマイモマンナンより抽出することにより得ることができる。このように得たマンナンを、酸加水分解、高温加熱加水分解、酵素加水分解、微生物発酵の中から選択される１種又は２種以上の方法で処理し、好ましくは活性炭処理、吸着樹脂処理、イオン交換樹脂処理、イオン交換膜処理等の方法で精製して、糖混合物を得ることができる。かかる当混合物中には、上述した「マンノースを主体としたオリゴ糖類」が含まれている。したがって、このようにして得た組成物は、本発明に係る腸内細菌叢改善剤及び抗癌剤の有効成分として用いられる。さらに、「マンノースを主体としたオリゴ糖類」は、コンニャクイモ、ユリ、スイセン、ヒガンバナ等に含まれるグルコマンナン、ローカストビーンガム、グアーガム等に含まれるガラクトマンナンを酸加水分解、高温加熱加水分解、酵素加水分解、微生物発酵の中から選択される１種又は２種以上の方法で処理し、活性炭処理、吸着樹脂処理、イオン交換樹脂処理、イオン交換膜処理等の方法で分離精製し構成糖としてマンノースの比率を高めることにより製造したものであってもよい。 Here, the raw material mannan can be obtained by extraction from, for example, copra meal obtained from coconut coconut, fuchs, South African coconut plants Huacra Palm, tsukuneimo mannan, and yam mannan. Mannan thus obtained is treated by one or more methods selected from acid hydrolysis, high-temperature heat hydrolysis, enzyme hydrolysis, and microbial fermentation, preferably activated carbon treatment, adsorption resin treatment, A sugar mixture can be obtained by purification by an ion exchange resin treatment, an ion exchange membrane treatment or the like. This mixture contains the above-described “oligosaccharides mainly composed of mannose”. Therefore, the composition thus obtained is used as an active ingredient of the intestinal flora improving agent and anticancer agent according to the present invention. Furthermore, “oligosaccharides mainly composed of mannose” include glucomannan, locust bean gum, guar gum, etc. contained in cornflower, lily, daffodil, ganbana, etc., acid hydrolysis, high temperature heating hydrolysis, enzyme Treated by one or more methods selected from hydrolysis and microbial fermentation, separated and purified by activated carbon treatment, adsorption resin treatment, ion exchange resin treatment, ion exchange membrane treatment, etc., and mannose as a constituent sugar It may be produced by increasing the ratio.
本発明において用いられる「マンノースを主体としたオリゴ糖類」としては、コーヒー生豆又は焙煎したコーヒー豆を酸加水分解、高温加熱加水分解、酵素加水分解、微生物発酵の中から選択される１種又は２種以上の方法で処理し、活性炭処理、吸着樹脂処理、イオン交換樹脂処理、イオン交換膜処理等の方法で精製することによって得ることができる。あるいは、使用済みコーヒー残渣を酸加水分解、高温加熱加水分解、酵素加水分解、微生物発酵の中から選択される１種又は２種以上の方法で処理し、活性炭処理、吸着樹脂処理、イオン交換樹脂処理、イオン交換膜処理等の方法で精製することによって得ることも可能である。一般に、焙煎粉砕コーヒーを商業用の抽出器にて抽出すると、その際に焙煎コーヒーに含まれるガラクトマンナンの側鎖であるガラクトースが可溶化したり、アラビノガラクタンが加水分解によって可溶化する。従って、コーヒー残渣中にはマンナンが豊富であり、しかも直鎖構造をとっているものと推定される。一方、セルロースは分解されにくく残渣として残っているが、セルロースを分解せずにマンナンを特異的に加水分解する条件を適宜選択することにより、マンノースを主体とするオリゴ糖を得ることができる。 The “oligosaccharide mainly composed of mannose” used in the present invention is one kind selected from raw coffee beans or roasted coffee beans from acid hydrolysis, high-temperature heat hydrolysis, enzyme hydrolysis, and microbial fermentation. Or it can obtain by processing by 2 or more types of methods, and refine | purifying by methods, such as activated carbon processing, adsorption resin processing, ion-exchange resin processing, and ion-exchange membrane processing. Alternatively, the used coffee residue is treated by one or more methods selected from acid hydrolysis, high-temperature heat hydrolysis, enzyme hydrolysis, and microbial fermentation, and activated carbon treatment, adsorption resin treatment, ion exchange resin It can also be obtained by purification by a method such as treatment or ion exchange membrane treatment. In general, when roasted and ground coffee is extracted with a commercial extractor, galactose, which is a side chain of galactomannan contained in roasted coffee, is solubilized or arabinogalactan is solubilized by hydrolysis. . Therefore, it is presumed that the coffee residue is rich in mannan and has a linear structure. On the other hand, although cellulose is hardly decomposed and remains as a residue, an oligosaccharide mainly composed of mannose can be obtained by appropriately selecting conditions for specifically hydrolyzing mannan without decomposing cellulose.
特にコーヒー抽出残渣を分解する方法としては、酸及び／又は高温により加水分解する方法、酵素により分解する方法、微生物発酵により分解する方法が挙げられるが、これらに限定されない。酸及び／又は高温により加水分解する方法としては特開昭６１−９６９４７号公報、特開平２−２００１４７号公報等に開示されている。商業用のコーヒー多段式抽出系において出てくる使用済みコーヒー残渣を反応容器中において酸触媒を添加して加水分解することもでき、酸触媒を添加せずに高温で短時間処理して加水分解することによっても得ることができる。管形栓流反応器を使用する方法が便利であるが、比較的高温で短時間の反応を行わせる方法に向いているものであれば、いかなる反応器を使用しても良好な結果が得られる。反応時間と反応温度を調節し、可溶化して加水分解させることによってＤＰ１０〜４０のマンナンをＤＰ１〜１０のマンノオリゴ糖に分解し、その後コーヒー残渣と分離してマンノオリゴ糖類を得ることができる。なお、ここでコーヒー抽出残査とは、大気中あるいは加圧条件下で焙煎粉砕コーヒーを水などの溶媒で抽出した後の、いわゆるコーヒー抽出粕を意味する。 Particularly, the method for decomposing the coffee extraction residue includes, but is not limited to, a method of hydrolyzing with an acid and / or high temperature, a method of decomposing with an enzyme, and a method of decomposing by microbial fermentation. Methods for hydrolyzing with an acid and / or high temperature are disclosed in JP-A-61-96947, JP-A-2-200147, and the like. It is possible to hydrolyze spent coffee residue that comes out in a commercial coffee multistage extraction system by adding an acid catalyst in a reaction vessel and treating it at a high temperature for a short time without adding an acid catalyst. Can also be obtained. The method using a tubular plug flow reactor is convenient, but good results can be obtained using any reactor that is suitable for a method in which a reaction is performed at a relatively high temperature for a short time. It is done. Mannanoligosaccharides of DP10-40 are decomposed into DP1-10 manno-oligosaccharides by adjusting the reaction time and reaction temperature, solubilizing and hydrolyzing, and then separating from the coffee residue to obtain manno-oligosaccharides. Here, the coffee extraction residue means a so-called coffee extraction cake after the roasted and ground coffee is extracted with a solvent such as water in the air or under pressure.
「マンノースを主体としたオリゴ糖類」として、コーヒー豆（焙煎コーヒー豆、及び焙煎粉砕コーヒー豆を含む。）及び／又はコーヒー抽出残渣の加水分解処理により得られたものを用いる場合、使用するコーヒー豆の種類や産地に特に制限はなく、アラビカ種、ロバスタ種、リベリカ種等いずれのコーヒー豆でもよく、さらにブラジル、コロンビア産等いずれの産地のコーヒー豆も使用することができ、１種類の豆のみを単独で使用してもよく、ブレンドした２種以上の豆を使用してもよい。通常、商品価値がないとして廃棄処分されるような品質の悪いコーヒー豆又は小粒のコーヒー豆であっても使用することができる。上記コーヒー豆を一般的に用いられている焙煎機（直火、熱風、遠赤、炭火式など）による極浅炒り、浅炒り、中炒り、深炒りに焙煎したコーヒー豆、及びこの焙煎コーヒー豆を、一般的な粉砕機、ロールミルなどを用いて粉砕することにより得た、焙煎粉砕コーヒー（粗挽き、中粗挽き、中挽き、中細挽き、細挽きなどの種々の形状のものを含む）を用いることもできる。 Used as “oligosaccharides mainly composed of mannose” when coffee beans (including roasted coffee beans and roasted and ground coffee beans) and / or those obtained by hydrolysis treatment of coffee extraction residue are used. There are no particular restrictions on the type and production area of the coffee beans, and any coffee bean such as Arabica, Robusta, and Riberica can be used, and coffee beans from any origin such as Brazil and Colombia can be used. Only beans may be used alone, or two or more kinds of blended beans may be used. Usually, even low-quality coffee beans or small-sized coffee beans that are discarded as having no commercial value can be used. Coffee beans roasted in a shallow roast, shallow roast, medium roast, deep roast by a roasting machine (direct fire, hot air, far red, charcoal fire type, etc.) generally used for the above coffee beans, and this roast Roasted and ground coffee (coarse ground, medium ground, medium ground, medium ground, fine ground, etc.) Can also be used.
また、コーヒー抽出残渣としては、通常の液体コーヒーあるいはインスタントコーヒー製造工程において、焙煎粉砕コーヒーを抽出処理した後のものであれば、常圧下、加圧下抽出であろうと、またいかなる起源、製法のコーヒー抽出残渣であっても使用することができる。 In addition, as the coffee extraction residue, after the roasted and ground coffee is extracted in a normal liquid coffee or instant coffee manufacturing process, it may be extracted under normal pressure or under pressure, and any origin and production method may be used. Even coffee extraction residues can be used.
ここで、上記加水分解処理について、いくつか詳細に説明する。酵素により分解する方法としては、例えばコーヒー抽出残渣を水性媒体に懸濁させ、ここへ、例えば市販のセルラーゼ及びヘミセルラーゼ等を加えて撹拌しながら懸濁させればよい。酵素の量、作用させる温度及びその他の条件としては、通常の酵素反応に用いられる量、温度、条件であれば特に問題はなく、使用する酵素の最適作用量、温度、条件及びその他の要因によって適宜選択すればよい。
微生物発酵により分解する方法としては、例えば水性媒体に懸濁させたコーヒー抽出残渣にセルラーゼ、ヘミセルラーゼなどを産出する微生物を植菌して培養させればよい。使用する微生物は、細菌類や担子菌類などコーヒー抽出残渣中のマンナンを分解する酵素を産出するものであれば良く、使用する微生物によって培養条件などは適宜選択すればよい。
Here, some of the hydrolysis treatments will be described in detail. As a method of decomposing with an enzyme, for example, a coffee extraction residue may be suspended in an aqueous medium, and commercially available cellulase, hemicellulase, etc. may be added thereto and suspended with stirring. There are no particular problems with the amount of enzyme, the temperature at which it acts, and other conditions as long as it is the amount, temperature, and conditions used in normal enzyme reactions, depending on the optimum amount of enzyme used, temperature, conditions and other factors. What is necessary is just to select suitably.
As a method for decomposing by microbial fermentation, for example, a microorganism that produces cellulase, hemicellulase, etc. may be inoculated and cultured in a coffee extraction residue suspended in an aqueous medium. The microorganism to be used may be any microorganism that produces an enzyme that decomposes mannan in the coffee extraction residue such as bacteria and basidiomycetes, and the culture conditions and the like may be appropriately selected depending on the microorganism to be used.
上記の方法によって得られた「マンノースを主体としたオリゴ糖類」を含む反応液は、必要に応じて精製することができる。精製法としては、骨炭、活性炭、炭酸飽充法、吸着樹脂、マグネシア法、溶剤抽出法等で脱色・脱臭を行い、イオン交換樹脂、イオン交換膜、電気透析等で脱塩、脱酸を行うことが挙げられる。精製法の組み合わせ及び精製条件としては、マンノースを主体とするオリゴ糖類を含む反応液中の色素、塩、及び酸等の量や、その他の要因に応じて適宜選択すればよい。 The reaction solution containing “oligosaccharides mainly composed of mannose” obtained by the above method can be purified as necessary. As purification methods, bone charcoal, activated carbon, carbonic acid saturation method, adsorption resin, magnesia method, solvent extraction method, etc. are used for decolorization and deodorization, and ion exchange resin, ion exchange membrane, electrodialysis, etc. are used for desalting and deoxidation. Can be mentioned. The combination of the purification methods and the purification conditions may be appropriately selected according to the amount of pigment, salt, acid, etc. in the reaction solution containing the oligosaccharide mainly composed of mannose and other factors.
本発明において用いられる「マンノースを主体としたオリゴ糖類」は、原料として使用される前に、予め必要に応じて活性炭、イオン交換樹脂、溶剤等で脱色、脱臭、脱酸等の精製処理をしておいてもよい。 The “oligosaccharide mainly composed of mannose” used in the present invention is subjected to purification treatment such as decolorization, deodorization, deoxidation, etc. with activated carbon, ion exchange resin, solvent, etc., if necessary before use as a raw material. You may keep it.
本発明に係る腸内細菌叢改善剤及び抗癌剤が前記「マンノースを主体としたオリゴ糖類」を含む場合、マンノポリオーシルフラクトースと前記「マンノースを主体としたオリゴ糖類」の含量の総和は、総固形分に対して６０質量％以上であることが好ましく、８０質量％以上であることがより好ましい。 When the intestinal microflora improving agent and anticancer agent according to the present invention include the above-mentioned `` oligosaccharide mainly composed of mannose '', the sum of the contents of mannopolyosyl fructose and the above-mentioned `` oligosaccharide mainly composed of mannose '' is: It is preferably 60% by mass or more, more preferably 80% by mass or more, based on the total solid content.
本発明に係る腸内細菌叢改善剤は、マンノポリオーシルフラクトースによる腸内細菌叢改善効果を損なわない限り、その他の成分を含有していてもよい。例えば、賦形剤、結合剤、流動性改良剤（固結防止剤）、安定剤、保存剤、ｐＨ調整剤、溶解補助剤、懸濁化剤、乳化剤、粘稠剤、矯味剤、甘味料、酸味料、香料、着色料等として用いられている各種物質を、所望の製品品質に応じて適宜含有させてもよい。 The intestinal flora improving agent according to the present invention may contain other components as long as the effect of improving the intestinal flora by mannopolyocylfructose is not impaired. For example, excipient, binder, fluidity improver (anti-caking agent), stabilizer, preservative, pH adjuster, solubilizer, suspending agent, emulsifier, thickener, corrigent, sweetener Various substances used as acidulants, fragrances, colorants and the like may be appropriately contained depending on the desired product quality.
本発明に係る抗癌剤は、マンノポリオーシルフラクトースによる癌細胞増殖抑制効果を損なわない限り、その他の成分を含有していてもよい。その他の成分としては、腸内細菌叢改善剤において列挙したものが挙げられる。 The anticancer agent according to the present invention may contain other components as long as it does not impair the cancer cell growth inhibitory effect of mannopolyosyl fructose. Examples of other components include those listed in the intestinal flora improving agent.
本発明に係る腸内細菌叢改善剤及び抗癌剤の剤型は、特に限定されるものではなく、各種の剤型を適用できる。本発明に係る腸内細菌叢改善剤は、経口摂取することによって腸内細菌叢改善効果を奏するため、経口投与に適したものが好ましい。当該剤型としては、例えば、錠剤、カプセル剤、顆粒剤、散剤、シロップ剤等が挙げられる。 The dosage forms of the intestinal flora improving agent and anticancer agent according to the present invention are not particularly limited, and various dosage forms can be applied. The intestinal bacterial flora-improving agent according to the present invention exhibits an intestinal bacterial flora-improving effect when taken orally, and therefore is preferably suitable for oral administration. Examples of the dosage form include tablets, capsules, granules, powders, syrups and the like.
本発明に係る腸内細菌叢改善剤及び抗癌剤は、オリゴ糖類を主成分とするものであり、非常に安全に経口接種可能である。そこで、本発明に係る腸内細菌叢改善剤は、医薬品として以外にも、飲食品中に原料として配合させてもよい。例えば、本発明に係る腸内細菌叢改善剤を配合させた飲食品を継続的に摂取することにより、腸内において、ビフィズス菌や乳酸菌等野有用菌を優先的に増殖され、腸内細菌嚢のバランスが改善される。本発明に係る腸内細菌叢改善剤は、その他、飼料や化粧品の原料とすることも好ましい。 The intestinal microflora improving agent and anticancer agent according to the present invention are mainly composed of oligosaccharides and can be orally inoculated very safely. Therefore, the intestinal flora improving agent according to the present invention may be blended as a raw material in foods and drinks in addition to pharmaceuticals. For example, by continuously ingesting a food or drink containing the intestinal microflora improving agent according to the present invention, useful bacteria such as bifidobacteria and lactic acid bacteria are preferentially grown in the intestine, and the intestinal bacterial sac The balance is improved. In addition, the intestinal microflora improving agent according to the present invention is also preferably used as a raw material for feed and cosmetics.
例えば、コーヒー抽出残渣を酸及び／又は熱により加水分解しオリゴ糖類を高純度に含むように調製した「マンノースを主体としたオリゴ糖類」をそのまま、又は必要に応じて活性炭、イオン交換樹脂、溶剤等で脱色、脱臭、脱酸等の精製処理をした後に、液体コーヒー、インスタントコーヒー等に添加して使用することもできる。ここで、液体コーヒーとしては、缶又はいわゆるペットボトル容器に入れられて市販されているコーヒー飲料（若しくはコーヒー入り飲料と呼ばれるもの）が挙げられる。また、インスタントコーヒーとしては、焙煎粉砕コーヒーを熱湯で抽出した抽出液を噴霧又は凍結乾燥方法により水分を除去した可溶性粉末コーヒーと呼ばれるものが挙げられる。コーヒーミックス飲料としては、可溶性粉末コーヒーに砂糖、クリーミングパウダーなどを添加して混合した飲料などが挙げられる。 For example, the “oligosaccharides mainly composed of mannose” prepared by hydrolyzing the coffee extraction residue with acid and / or heat to contain oligosaccharides in high purity as it is or as necessary, activated carbon, ion exchange resin, solvent It can also be used after being subjected to purification treatment such as decolorization, deodorization, deoxidation, etc. and then added to liquid coffee, instant coffee and the like. Here, as liquid coffee, the coffee drink (or what is called a drink containing coffee) put into a can or what is called a PET bottle container and marketed is mentioned. Moreover, as instant coffee, what is called soluble powder coffee which removed the water | moisture content by spraying or freeze-drying the extract which extracted the roasting ground coffee with hot water is mentioned. Examples of the coffee mix beverage include beverages obtained by adding and mixing soluble powdered coffee with sugar, creaming powder, and the like.
また、マンノポリオーシルフラクトースは、その微生物による資化性の差を利用して、ビフィズス菌や乳酸菌を培養するための微生物培養用培地を有効成分とすることもできる。マンノポリオーシルフラクトースを含む培地は、大腸菌やクロストリジウム属菌等の腐敗菌よりも、ビフィズス菌や乳酸菌を優先的に増殖させることができる。 In addition, mannopolyosyl fructose can contain a microorganism culture medium for culturing bifidobacteria and lactic acid bacteria as an active ingredient by utilizing the difference in assimilation by microorganisms. A medium containing mannopolyocylfructose can preferentially grow bifidobacteria and lactic acid bacteria over spoilage bacteria such as Escherichia coli and Clostridium.
前記腸内細菌叢改善剤と同様、本発明に係る微生物培養用培地は、有効成分のマンノポリオーシルフラクトースとして、前記コーヒー豆（焙煎したコーヒー豆を含む。）及び／又はコーヒー豆絞り滓を１８０〜２５０℃で加熱処理した後に得られたオリゴ糖組成物を用いてもよく、マンノポリオーシルフラクトースに加えて、さらに前記「マンノースを主体としたオリゴ糖類」を含有していてもよい。 Similar to the intestinal microflora improving agent, the culture medium for microbial culture according to the present invention contains the coffee beans (including roasted coffee beans) and / or coffee bean squeezed as the active ingredient mannopolyosyl fructose. An oligosaccharide composition obtained after heat treatment at 180 to 250 ° C. may be used, and in addition to mannopolyocylfructose, the oligosaccharide composition may further contain the “oligosaccharide mainly composed of mannose”. Good.
本発明に係る微生物培養用培地は、資化性糖として、前記マンノポリオーシルフラクトースのみを含有することが好ましい。これにより、大腸菌等の増殖を充分に抑制し、ビフィズス菌や乳酸菌を効率よく選択的に増殖させることができる。 The culture medium for microbial culture according to the present invention preferably contains only the mannopolyosyl fructose as an assimilating sugar. Thereby, the growth of Escherichia coli and the like can be sufficiently suppressed, and Bifidobacteria and lactic acid bacteria can be efficiently and selectively grown.
次に、実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例等に限定されるものではない。 EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example etc.
［実施例１］
＜マンノオリゴ糖の調製＞
粉砕して粒径を約１ｍｍにしたコーヒー抽出残渣を、総固形分濃度が約１４質量％の水と粉砕物からなるスラリーに調製した後、４ｍの熱栓流反応器内において熱処理した。当該熱処理においては、当該スラリーを、滞留時間８分間に対応する速度で高圧蒸気とともに栓流反応器にポンプ輸送し、６．３５ｍｍφオリフィスを用いて約２１０℃に維持した。その後、当該スラリーを大気圧下に噴出することによって、反応を急止した。得られたスラリーを濾過して、不溶性固形分から可溶性固形分を含む液を分離した。この可溶性固形分含有液を、活性炭、次いで吸着樹脂で脱色し、さらにイオン交換樹脂で脱塩した後、濃縮、乾燥することによって、マンノースを主体とする単糖類が１〜１０分子結合したオリゴ糖類の混合物（オリゴ糖組成物）を得た。
[Example 1]
<Preparation of mannooligosaccharide>
A coffee extraction residue having a particle size of about 1 mm after pulverization was prepared as a slurry comprising water and a pulverized product having a total solid concentration of about 14% by mass, and then heat-treated in a 4 m hot plug flow reactor. In the heat treatment, the slurry was pumped to the plug flow reactor with high pressure steam at a rate corresponding to a residence time of 8 minutes and maintained at about 210 ° C. using a 6.35 mmφ orifice. Then, the reaction was stopped rapidly by ejecting the slurry under atmospheric pressure. The resulting slurry was filtered to separate a liquid containing soluble solids from insoluble solids. This soluble solid content-containing liquid is decolorized with activated carbon and then with an adsorption resin, further desalted with an ion exchange resin, concentrated and dried, and thereby an oligosaccharide in which 1 to 10 molecules of monosaccharides mainly composed of mannose are bonded. A mixture (oligosaccharide composition) was obtained.
＜マンノポリオーシルフラクトースの分離精製＞
このようにして得られたオリゴ糖組成物２００ｇを１０質量％濃度に溶解させた溶液を、活性炭充填した内径２０ｃｍ、長さ２ｍのカラムに負荷し、毎分１Ｌの水で溶出した。重合度の小さいものから先に溶出し、重合度２〜５については２つのピークが検出された。重合度４、５のものについては、一部にピークの重なりが見られたため、完全に分離した部分についてのみ分画乾燥して以下の実験に用いた。
<Separation and purification of mannopolyosyl fructose>
A solution in which 200 g of the oligosaccharide composition thus obtained was dissolved to a concentration of 10% by mass was loaded onto a column with an inner diameter of 20 cm and a length of 2 m packed with activated carbon, and eluted with 1 L of water per minute. Elution was performed first from the one having a small degree of polymerization, and two peaks were detected for the degree of polymerization of 2 to 5. Since those having a polymerization degree of 4 and 5 had peak overlap partially, only the completely separated portion was fractionally dried and used in the following experiments.
まず、二糖をβ−マンノシダーゼで処理したところ、前記活性炭充填カラムから先に溶出した二糖からは等量のマンノースとフルクトースが生成され、後から溶出した二糖からはマンノースのみが生成された。この結果より、先に溶出したものはβ−マンノシルフラクトースであることが確認された。また、三糖を同様の処理したところ、先に溶出したものからはマンノース２当量に対し１当量のフラクトースが生成され、マンノビオーシルフラクトースであることが確認された。同様に、重合度４、５の糖を処理したところ、先に溶出したものは、それぞれマンノトリオーシルフラクトース、マンノテトラオーシルフラクトースであることが確認された。 First, when the disaccharide was treated with β-mannosidase, equal amounts of mannose and fructose were produced from the disaccharide previously eluted from the activated carbon packed column, and only mannose was produced from the later eluted disaccharide. . From this result, it was confirmed that what was eluted earlier was β-mannosyl fructose. Further, when the same treatment was applied to the trisaccharide, 1 eq. Fructose was produced from 2 eq. Of mannose, and it was confirmed that it was mannobiosyl fructose. Similarly, when sugars having a degree of polymerization of 4 and 5 were treated, it was confirmed that those eluted first were mannotriosyl fructose and mannotetraoucyl fructose, respectively.
［実施例２］
実施例１で調製した還元末端をフラクトースとするオリゴ糖マンノポリオーシルフラクトースについて、腸内菌叢を構成する菌類に対する資化性を調べた。マンノポリオーシルフラクトースとしては、ＤＰ２のマンノシルフラクトースを用いた。
[Example 2]
The oligosaccharide mannopolyocyl fructose prepared in Example 1 with fructose at the reducing end was examined for assimilability to fungi constituting the intestinal flora. As the mannopolyausyl fructose, DP2 mannosyl fructose was used.
＜培地の調製＞
まず、１０ｇのトリプティケース（日本ベクトン・ディッキンソン社製）、５ｇの酵母エキス（日本ベクトン・ディッキンソン社製）、４０ｍＬのフィルズ溶液、４０ｍＬの塩類溶液、０．５ｇのＬ−システイン・ＨＣｌ・Ｈ２Ｏ、及び９２０ｍＬの精製水（ｐＨ７．２）を混合し、Ｐｅｐｔｏｎ−Ｙｅａｓｔ−Ｆｉｌｄｅｓ ｓｏｌｕｔｉｏｎ（ＰＹＦ）培地を調製した。
なお、塩類溶液は、０．２ｇの塩化カルシウム（無水）、０．２ｇの硫酸マグネシウム、１ｇのリン酸水素二カリウム、１ｇのリン酸二水素カリウムと、１０ｇの炭酸水素ナトリウム、２ｇの塩化ナトリウムを、１０００ｍＬの精製水に溶解させた溶液である。
また、フィルズ溶液は、１５０ｍＬの生理食塩水（０．８５（質量／容量）％ 塩化ナトリウム）、６ｍＬの濃塩酸、５０ｍＬの馬血液、及び１ｇのペプシン（１：１０００）（日本ベクトン・ディッキンソン社製）を混合し、５５℃の温浴槽水中で一夜保持し、消化させた後に、１２ｍＬの２０（質量／容量）％ 水酸化ナトリウム溶液を加えた後、水酸化ナトリウムによりｐＨ７．６になるように調整して得た。
<Preparation of medium>
First, 10 g of tripty case (manufactured by Nippon Becton Dickinson), 5 g of yeast extract (manufactured by Nippon Becton Dickinson), 40 mL of Phils solution, 40 mL of salt solution, 0.5 g of L-cysteine / HCl / H 2 O, and 920mL of purified water and (pH 7.2) were mixed to prepare a Pepton-Yeast-Fildes solution (PYF ) medium.
The salt solution was 0.2 g calcium chloride (anhydrous), 0.2 g magnesium sulfate, 1 g dipotassium hydrogen phosphate, 1 g potassium dihydrogen phosphate, 10 g sodium bicarbonate, 2 g sodium chloride. Is a solution in which 1000 ml of purified water is dissolved.
Fils's solution was 150 mL of physiological saline (0.85 (mass / volume)% sodium chloride), 6 mL of concentrated hydrochloric acid, 50 mL of horse blood, and 1 g of pepsin (1: 1000) (Nippon Becton Dickinson) ), And kept overnight in 55 ° C. warm bath water and digested, then 12 mL of 20 (mass / volume)% sodium hydroxide solution was added, and then adjusted to pH 7.6 with sodium hydroxide. It was obtained by adjusting.
次いで、マンノビオーシルフラクトースを１０（質量／容量）％溶液として調製して濾過滅菌した後、最終濃度０．５（質量／容量）％となるように、滅菌ＰＹＦ半流動寒天培地に添加した。また、マンノビオーシルフラクトースを１０（質量／容量）％溶液に代えて、グルコース１０（質量／容量）％溶液を添加したものを、対照とした。 Subsequently, mannobiosil fructose was prepared as a 10% (mass / volume) solution and sterilized by filtration, and then added to a sterilized PYF semi-fluid agar medium to a final concentration of 0.5% (mass / volume). . In addition, mannobiosil fructose was replaced with a 10 (mass / volume)% solution, and a glucose 10 (mass / volume)% solution was added as a control.
＜菌類に対する資化性＞
平板寒天板培地で培養した新鮮な菌を、前記で調製したマンノビオーシルフラクトース含有ＰＹＦ培地（マンノビオーシルフラクトースを０．５（質量／容量）％添加したもの）又はグルコース含有ＰＹＦ培地（グルコースを０．５（質量／容量）％添加したもの）に、各菌株が各々１０８ＣＦＵ／チューブとなるように接種し、３７℃で９６時間嫌気培養した。菌数の増加をｐＨの低下を測定することにより求めた。判定基準は、下記のようにした。
<Assimilation against fungi>
A fresh bacterium cultured on a plate agar plate was prepared by using the mannobiosil fructose-containing PYF medium (0.5% (mass / volume) added mannobiosil fructose) or the glucose-containing PYF medium ( Glucose (0.5% by mass / volume)) was inoculated so that each strain would be 10 8 CFU / tube, and anaerobically cultured at 37 ° C. for 96 hours. The increase in the number of bacteria was determined by measuring the decrease in pH. The judgment criteria were as follows.
「−」：培養液のｐＨが６．０以上である、
「±」：培養液のｐＨが５．５以上６．０未満である、
「＋」：培養液のｐＨが５．０以上５．５未満である、
「＋＋」：培養液のｐＨが４．５以上５．０未満である、
「＋＋＋」：培養液のｐＨが４．５未満である。
“-”: The pH of the culture solution is 6.0 or more,
“±”: pH of the culture solution is 5.5 or more and less than 6.0,
“+”: PH of the culture solution is 5.0 or more and less than 5.5,
“++”: pH of the culture solution is 4.5 or more and less than 5.0,
“++++”: The pH of the culture solution is less than 4.5.
判定結果を表１に示す。この結果、マンノポリオーシルフラクトースを含有する培地は、ビフィズス菌・乳酸菌等の有用菌を資化し、腐敗菌は資化しないことを確認した。マンノポリオーシルフラクトースは、対照のグルコースに比し、明らかに選択資化性が高いことが判明した。 The determination results are shown in Table 1. As a result, it was confirmed that the medium containing mannopolyocylfructose assimilated useful bacteria such as bifidobacteria and lactic acid bacteria, and did not assimilate spoilage bacteria. Mannopolyausyl fructose was clearly found to be highly selective as compared to control glucose.
［実施例３］
＜腸内細菌叢改善剤含有レギュラーコーヒーの製造＞
８ｇのレギュラーコーヒーに熱湯を加えてペーパードリップにて抽出して、１４０ｍＬのコーヒー液を得た。これに、実施例１で調整したマンノポリオーシルフラクトースを主体とするオリゴ糖類を含有する組成物を５ｇ添加した。本来のコーヒー味を持ち、僅かに甘味を有しコクの有るコーヒーが調製できた。このコーヒー液は腸内有用菌増殖促進作用を持つことが期待できる。
[Example 3]
<Manufacture of regular coffee containing intestinal flora improving agent>
Hot water was added to 8 g of regular coffee and extracted with a paper drip to obtain 140 mL of coffee liquid. To this, 5 g of a composition containing an oligosaccharide mainly composed of mannopolyocylfructose prepared in Example 1 was added. A coffee having an original coffee taste, slightly sweetness and richness could be prepared. This coffee liquid can be expected to have an effect of promoting the growth of useful intestinal bacteria.
［実施例４］
＜マンノポリオーシルフラクトースの癌細胞増殖抑制試験＞
実施例１で調製した還元末端をフラクトースとするオリゴ糖マンノポリオーシルフラクトースについて、癌細胞増殖抑制能を調べた。比較対象として、マンノビオース、フラクトオリゴ糖、及びキシロオリゴ糖を用いた。また、癌細胞として、マウス白血病病細胞Ｐ３８８（マウスリンパ球様がん由来）を用いた。
[Example 4]
<Mannopolyosyl fructose cancer cell growth inhibition test>
The oligosaccharide mannopolyausyl fructose prepared in Example 1 with fructose at the reducing end was examined for its ability to suppress the growth of cancer cells. For comparison, mannobiose, fructooligosaccharide, and xylo-oligosaccharide were used. Moreover, mouse leukemia disease cell P388 (derived from mouse lymphoid cancer) was used as a cancer cell.
具体的には、まず、マンノポリオーシルフラクトース、マンノビオース、フラクトオリゴ糖、及びキシロオリゴ糖を蒸留水に溶解させ、孔径０．２μｍのメンブレンフィルターで濾過した。その後、各濾液をＲＰＭＩ１６４０培地で１．０ｍｇ／ｍＬになるように濃度調整をしたものを、試験液とした。また、陽性対照液として、カンプトテシンを５．０ｎｇ／ｍＬとなるように含有させたＲＰＭＩ１６４０培地を用いた。 Specifically, first, mannopolyausyl fructose, mannobiose, fructooligosaccharide and xylo-oligosaccharide were dissolved in distilled water and filtered through a membrane filter having a pore size of 0.2 μm. Then, what adjusted the density | concentration of each filtrate so that it might become 1.0 mg / mL with RPMI1640 culture medium was made into the test liquid. As a positive control solution, RPMI1640 medium containing camptothecin at 5.0 ng / mL was used.
９６ウェルプレートにＰ３８８細胞を播種し、各試験液を培地として３日間培養を行った。培養後、各ウェルにおけるＰ３８８細胞の数を、生細胞数の酸化還元酵素により生成するホルマザン色素量として測定した。具体的には、Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ−８（（株）同仁化学研究所製）で処理を行った後、マイクロプレートリーダーで吸光度（４５０ｎｍ）を測定し、検体を共存させたときの色素生成量から未処置対照の生成量に対する相対値を計算し、細胞増殖抑制能を評価した。 P388 cells were seeded in a 96-well plate and cultured for 3 days using each test solution as a medium. After culture, the number of P388 cells in each well was measured as the amount of formazan dye produced by the oxidoreductase of the number of living cells. Specifically, after processing with Cell Counting Kit-8 (manufactured by Dojindo Laboratories Co., Ltd.), the absorbance (450 nm) is measured with a microplate reader, and the amount of dye produced when the sample coexists. The relative value with respect to the production amount of the untreated control was calculated, and the cytostatic ability was evaluated.
測定結果を図１に示した。マンノポリオーシルフラクトース（ＭＦ）は、陽性対照であるカンプトテシンと同等の細胞増殖抑制能を示した。一方、試験に供した他の糖類は、ほとんど細胞増殖抑制能を示さなかった。この結果は、ＭＦが癌細胞に対して高い増殖抑制能を有していることを示すものである。 The measurement results are shown in FIG. Mannopolyosyl fructose (MF) showed a cell growth inhibitory ability equivalent to that of camptothecin as a positive control. On the other hand, other saccharides subjected to the test showed almost no cell growth inhibitory ability. This result indicates that MF has a high growth inhibitory ability against cancer cells.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019181826A1 (en) * | 2018-03-23 | 2019-09-26 | ティーエフケイ株式会社 | Compound, agent for regulating composition ratios of intestinal microbiota, pharmaceutical product, food/beverage product, food additive, method for regulating composition ratios of intestinal microbiota, and compound production method |
| JP2021014415A (en) * | 2019-07-10 | 2021-02-12 | 味の素株式会社 | Method for producing composition having uric acid level reducing effect |
| CN115336724A (en) * | 2022-08-31 | 2022-11-15 | 西安诺众康健生物科技有限责任公司 | Konjac fermentation powder and preparation method and application thereof |
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| JP2001149041A (en) * | 1999-09-14 | 2001-06-05 | Ajinomoto General Foods Inc | Composition based on mannooligosaccharides |
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| JP2006169135A (en) * | 2004-12-14 | 2006-06-29 | Ajinomoto General Foods Inc | Anti-allergen composition containing mannooligosaccharide |
| WO2007086100A1 (en) * | 2006-01-24 | 2007-08-02 | Ajinomoto General Foods, Inc. | Composition having effect of lowering blood pressure and/or inhibiting increase in blood pressure and food and drink containing the same |
| JP2009275047A (en) * | 2009-06-30 | 2009-11-26 | Ajinomoto General Foods Inc | Composition having body fat reducing action and food and drink containing the same |
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| JP2001149041A (en) * | 1999-09-14 | 2001-06-05 | Ajinomoto General Foods Inc | Composition based on mannooligosaccharides |
| JP2006083127A (en) * | 2004-09-17 | 2006-03-30 | Ajinomoto General Foods Inc | Composition having body fat reduction action and food and drink containing the same |
| JP2006169135A (en) * | 2004-12-14 | 2006-06-29 | Ajinomoto General Foods Inc | Anti-allergen composition containing mannooligosaccharide |
| WO2007086100A1 (en) * | 2006-01-24 | 2007-08-02 | Ajinomoto General Foods, Inc. | Composition having effect of lowering blood pressure and/or inhibiting increase in blood pressure and food and drink containing the same |
| JP2009275047A (en) * | 2009-06-30 | 2009-11-26 | Ajinomoto General Foods Inc | Composition having body fat reducing action and food and drink containing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019181826A1 (en) * | 2018-03-23 | 2019-09-26 | ティーエフケイ株式会社 | Compound, agent for regulating composition ratios of intestinal microbiota, pharmaceutical product, food/beverage product, food additive, method for regulating composition ratios of intestinal microbiota, and compound production method |
| JP2021014415A (en) * | 2019-07-10 | 2021-02-12 | 味の素株式会社 | Method for producing composition having uric acid level reducing effect |
| CN115336724A (en) * | 2022-08-31 | 2022-11-15 | 西安诺众康健生物科技有限责任公司 | Konjac fermentation powder and preparation method and application thereof |
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| JP6287479B2 (en) | 2018-03-07 |
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