JP2019118281A - Substance produced by bacteria composing kefir grains - Google Patents
Substance produced by bacteria composing kefir grains Download PDFInfo
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- JP2019118281A JP2019118281A JP2017254100A JP2017254100A JP2019118281A JP 2019118281 A JP2019118281 A JP 2019118281A JP 2017254100 A JP2017254100 A JP 2017254100A JP 2017254100 A JP2017254100 A JP 2017254100A JP 2019118281 A JP2019118281 A JP 2019118281A
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- Prior art keywords
- food composition
- lactobacillus
- group
- composition according
- kefir
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
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- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 208000015670 renal artery disease Diseases 0.000 description 1
- 230000008085 renal dysfunction Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
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- 229940074386 skatole Drugs 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Botany (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、乳酸菌群および酵母群を含む微生物群の大豆由来発酵産物を含む食品組成物に関する。 The present invention relates to a food composition comprising a soybean-derived fermentation product of a microorganism group including a lactic acid bacteria group and a yeast group.
ケフィアは有史前より東ヨーロッパ（黒海周辺）で飲用されており、現在ではロシアを中心に東ヨーロッパで日常的に飲用されている。古来よりケフィアは種々の薬理作用を有することが経験的に知られている。このような薬理作用を示す物質として、牛乳ケフィアにおいて、発酵スターター（種菌）であるケフィア粒が産生する粘性多糖類およびその中に含まれているペプチドが報告されている（非特許文献１）。 Kefir has been consumed in Eastern Europe (around the Black Sea) since history, and is now routinely consumed in Eastern Europe, mainly in Russia. Since ancient times, kefir has been empirically known to have various pharmacological actions. As a substance exhibiting such a pharmacological action, viscous polysaccharides produced by kefir grains which are fermentation starters (inoculum) and peptides contained therein have been reported in milk kefir (Non-patent Document 1).
牛乳ケフィアにおけるそのような薬理作用としては、本発明者により、（１）大腸菌、ウエルシュ菌、黄色ブドウ球菌に対する抗菌作用（非特許文献２）、（２）抗ストレス作用（非特許文献３）が報告されている。 As such pharmacological actions in milk kefir, the present inventor (1) has an antibacterial action against E. coli, H. perfringens, S. aureus (non-patent document 2), (2) an anti-stress action (non-patent document 3) It has been reported.
その他のケフィアに関する従来の研究は、ケフィア粒構成菌の同定や発酵産物の経口投与による癌、動脈硬化、高血圧、関節炎等の酸化障害系の病理学的役割に関するもの（非特許文献４および５）および整腸作用（非特許文献６）が主であった。 Previous studies on other kefirs have identified the pathological role of oxidative damage systems such as cancer, arteriosclerosis, hypertension and arthritis by identification of kefir grain forming bacteria and oral administration of fermentation products (Non-patent Documents 4 and 5) And intestinal regulation (non-patent document 6) was the main.
加齢や生活習慣病に伴う変化とされている、生体臓器の機能障害による疾患の予防または進行の遅延に有用である日常的に摂取可能な食品はほとんど例がなく、そのような効能を謳う食品が稀にあるとしてもその作用機序は明らかとされていない。ケフィアに関しても、発酵産物中に含有される有用成分の役割については従来あまり検討されて来なかった。本発明は、生体臓器の機能障害による疾患の予防または進行の遅延に有用である日常的に摂取可能な食品組成物であり、その作用機序の一部が明らかである食品組成物を提供することを目的とする。 There are very few examples of food that can be taken on a daily basis, which is considered to be associated with aging and lifestyle-related diseases, and that is useful for preventing or delaying the progression of disease due to dysfunction of living organs. Even if food is rare, its mechanism of action is not clear. Also with regard to Kefir, the role of useful ingredients contained in the fermentation product has not been studied so far. The present invention provides a food composition which can be taken daily, which is useful for preventing or delaying the progression of a disease caused by dysfunction of a living organ, and for which a part of its action mechanism is apparent. The purpose is
本発明者は、果汁や牛乳を含む種々の食品原料を発酵スターター（種菌）であるケフィア粒で発酵させ、有用成分について鋭意研究したところ、大豆由来発酵産物中にイソフラボンの代謝物であるエクオール、グルタミンの代謝物であるγ−アミノ酪酸（ＧＡＢＡ）、ならびに、いわゆる悪玉菌であるクロストリウム属の細菌、大腸菌群（ｃｏｌｉｆｏｒｍ ｂａｃｔｅｒｉａ）、およびスタフィロコッカス属の細菌に対する強い抗菌作用を示し、いわゆる善玉菌である乳酸菌（ｌａｃｔｉｃ ａｃｉｄ ｂａｃｔｅｒｉａ）、酪酸菌（ｂｕｔｙｌａｔｅ−ｐｒｏｄｕｃｉｎｇ ｂａｃｔｅｒｉａ）、およびビフィズス菌（ｂｉｆｉｄｏｂａｃｔｅｒｉａ）などに対しては抗菌作用を示さない抗菌物質が含有されることを見出し、本発明を完成させた。 The inventor of the present invention fermented various food materials including fruit juice and milk with kefir grains which are fermentation starters (seeds), and studied intensively on useful components. It shows strong antibacterial activity against γ-aminobutyric acid (GABA), which is a metabolite of glutamine, and bacteria of the so-called bad bacteria Clostridium, coliform bacteria, and Staphylococcus bacteria, It has been found that the present invention contains an antibacterial substance which does not exhibit an antibacterial action against lactic acid bacteria (lactic acid bacteria), butylate-producing bacteria, bifidobacteria etc. which are bacteria. I let it go.
すなわち、本明細書は以下の発明の開示を包含する。
［１］ラクトバチルス属の少なくとも１種の乳酸菌およびラクトコッカス属の少なくとも１種の乳酸菌を含む乳酸菌群；ならびにサッカロミセス属の少なくとも１種の酵母およびクルイベロミセス属の少なくとも１種の酵母を含む酵母群；を含む微生物群の大豆由来発酵産物を含み、前記大豆由来発酵産物がエクオール、γ−アミノ酪酸、および抗菌物質を含有する、食品組成物
［２］乳酸菌群が、偏性ホモ型（グループＩ）の少なくとも１種、通性ヘテロ型（グループＩＩ）の少なくとも１種、および偏性ヘテロ型（グループＩＩＩ）の少なくとも１種のラクトバチルス属の乳酸菌を含む、［１］に記載の食品組成物。
That is, the present specification includes the disclosure of the following invention.
[1] A lactic acid bacteria group comprising at least one lactic acid bacterium of the genus Lactobacillus and at least one lactic acid bacterium of the genus Lactococcus; and a yeast containing at least one yeast of the genus Saccharomyces and at least one yeast of the genus Kluyveromyces A food composition comprising a soybean-derived fermentation product of a microorganism group comprising a group; and the soybean-derived fermentation product containing equol, γ-aminobutyric acid, and an antibacterial substance; Food composition according to [1], comprising at least one Lactobacillus lactic acid bacterium of at least one type of I, at least one type of facultative heterotype (group II), and at least one type of obligate heterotype (group III) object.
［３］乳酸菌群が、ラクトバチルス・アシドフィルス、ラクトバチルス・カゼイ カゼイ亜種、ラクトバチルスケフィリ、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、ラクトバチルスファーメンタム、ラクトコッカス・ラクティス ラクティス亜種、ラクトコッカス・ラクティス クレモリス亜種、およびストレプトコッカス・サーモフィルスを含む、［１］または［２］に記載の食品組成物。 [3] The lactic acid bacteria group is Lactobacillus acidophilus, Lactobacillus casei casei subsp., Lactobacillus kefili, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactococcus lactis subsp. The food composition according to [1] or [2], comprising Lactis cremoris subsp. And Streptococcus thermophilus.
［４］酵母群が、サッカロミセス・セルビシエ、カザフスタニア・チュリセンシス、カザフスタニア・ユニスポラ、およびクルイベロミセス・マルキシアヌスを含む、［１］〜［３］のいずれかに記載の食品組成物。 [4] The food composition according to any one of [1] to [3], wherein the yeast group comprises Saccharomyces cervissae, Kazakhstania thulensis, Kazakhstania unispora, and Kluyveromyces marxianus.
［５］大豆由来発酵産物が豆乳発酵産物である、［１］〜［４］のいずれかに記載の食品組成物。
［６］抗菌物質がバクテリオシンではない、［１］〜［５］のいずれかに記載の食品組成物。
[5] The food composition according to any one of [1] to [4], wherein the soybean-derived fermented product is a soymilk fermented product.
[6] The food composition according to any one of [1] to [5], wherein the antibacterial substance is not bacteriocin.
［７］生体臓器の機能障害による疾患の予防または進行の遅延に用いるための、［１］〜［６］のいずれかに記載の食品組成物。
［８］生体臓器の機能障害による疾患が腎臓の機能障害による疾患、肝臓の機能障害による疾患、または脳の機能障害による疾患である、［１］〜［７］のいずれかに記載の食品組成物。
[7] The food composition according to any one of [1] to [6], for use in preventing or delaying the progression of a disease due to dysfunction of a living organ.
[8] The food composition according to any one of [1] to [7], wherein the disease caused by dysfunction of a living organ is a disease caused by dysfunction of the kidney, a disease caused by dysfunction of the liver, or a disease caused by dysfunction of the brain object.
［９］固形である、［１］〜［８］のいずれかに記載の食品組成物。
［１０］液体である、［１］〜［８］のいずれかに記載の食品組成物。
[9] The food composition according to any one of [1] to [8], which is solid.
[10] The food composition according to any one of [1] to [8], which is a liquid.
本発明の食品組成物は、腸内において大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）やウエルシュ菌（Ｃｌｏｓｔｒｉｄｉｕｍ ｐｅｒｆｒｉｎｇｅｎｓ）などの悪玉菌の占有率を低下させることができる。悪玉菌はインドールなどの代謝物を産生する。そして、宿主の腸管から吸収されたインドールが宿主の肝臓においてシトクロームＰ４５０による酸化および硫酸転移酵素による硫酸抱合を受けてインドキシル硫酸を生成し、宿主の血中インドキシル硫酸濃度を増加させる（Niwa T., et. al., Am. J. Nephrol., 14(3), 207-212, 1994）。したがって、本発明の食品組成物は、悪玉菌の産生するインドールなどの代謝物を減少させ、宿主の体内でインドールから生成するインドキシル硫酸を減少させ、宿主の血中インドキシル硫酸濃度を減少させることができる。インドキシル硫酸は尿毒素として知られていることから、本発明の食品組成物は、これらの悪玉菌が腸内で増殖することにより引き起こされる、生体臓器の機能障害による疾患の予防または進行の遅延に有用である。これらの疾患は、加齢や生活習慣病に伴う変化とされていたことから、本発明の食品組成物は、いわゆる高齢化社会において健康長寿を全うする上での一助となり得る。 The food composition of the present invention can reduce the occupancy rate of bad bacteria such as Escherichia coli and Clostridium perfringens in the intestine. Bad bacteria produce metabolites such as indole. Then, indole absorbed from the intestinal tract of the host is oxidized by cytochrome P450 in the host liver and sulfated by sulfate transferase to form indoxyl sulfate and increase the blood indoxyl sulfate concentration in the host (Niwa T , et. al., Am. J. Nephrol., 14 (3), 207-212, 1994). Therefore, the food composition of the present invention reduces metabolites such as indole produced by bad bacteria, reduces indoxyl sulfate generated from indole in the host body, and reduces blood indoxyl sulfate concentration in the host. be able to. Since indoxyl sulfate is known as an urinary toxin, the food composition of the present invention prevents or delays the progression of disease caused by dysfunction of living organs, which is caused by the growth of these bad bacteria in the intestine. Useful for Since these diseases are considered to be changes associated with aging and lifestyle-related diseases, the food composition of the present invention can help to fulfill healthy longevity in the so-called aging society.
以下、非限定的に本発明の態様を説明する。
本発明の食品組成物は、乳酸菌群および酵母群を含む微生物群の大豆由来発酵産物を含む食品組成物である。
Hereinafter, aspects of the present invention will be described without limitation.
The food composition of the present invention is a food composition comprising a soybean-derived fermented product of a microorganism group including a lactic acid bacteria group and a yeast group.
本明細書において、「乳酸菌群」とは、ラクトバチルス属の少なくとも１種の乳酸菌およびラクトコッカス属の少なくとも１種の乳酸菌を含む乳酸菌群であり、好ましくは偏性ホモ型（グループＩ）の少なくとも１種、通性ヘテロ型（グループＩＩ）の少なくとも１種、および偏性ヘテロ型（グループＩＩＩ）の少なくとも１種のラクトバチルス属の乳酸菌を含み、さらに好ましくはストレプトコッカス属の少なくとも１種の乳酸菌も含む。 In the present specification, the “lactic acid bacteria group” is a lactic acid bacteria group including at least one lactic acid bacteria of the genus Lactobacillus and at least one lactic acid bacterium of the genus Lactococci, preferably at least at least one obligate homozygote (group I). It also comprises at least one lactobacillus of the genus Lactobacillus of at least one of the facultative heterozygotes (group II) and at least one of the heterozygous heterozygotes (group III), more preferably also of at least one lactobacilli of Streptococcus. Including.
ラクトバチルス属の乳酸菌としては、例えば、ラクトバチルス・アシドフィルス、ラクトバチルス・カゼイ、ラクトバチルスケフィリ、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、ラクトバチルスファーメンタム、ラクトバチルス・ラムノサスなどが挙げられ、好ましくはラクトバチルス・アシドフィルス、ラクトバチルス・カゼイ、ラクトバチルスケフィリ、ラクトバチルス・ブレビス、ラクトバチルス・ブフネリ、ラクトバチルスファーメンタムが挙げられる。また、ラクトバチルス・カゼイとしては、例えば、ラクトバチルス・カゼイ カゼイ亜種、ラクトバチルス・カゼイ アラクトサス亜種などが挙げられ、好ましくはラクトバチルス・カゼイ カゼイ亜種が挙げられる。 Examples of Lactobacillus lactic acid bacteria include Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefili, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus rhamnosus, etc. And Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefili, Lactobacillus brevis, Lactobacillus buchneri, and Lactobacillus fermentum. Examples of Lactobacillus casei include Lactobacillus casei subspecies, Lactobacillus casei alactosus subspecies, and the like, with preference given to Lactobacillus casei subspecies.
ラクトコッカス属の乳酸菌としては、例えば、ラクトコッカス・ラクティス、ラクトコッカス・プランタームなどが挙げられ、好ましくはラクトコッカス・ラクティスが挙げられる。また、ラクトコッカス・ラクティスとしては、例えば、ラクトコッカス・ラクティス ラクティス亜種、ラクトコッカス・ラクティス クレモリス亜種、ラクトコッカス・ラクティス デアセチラクティス亜種などが挙げられ、好ましくはラクトコッカス・ラクティス ラクティス亜種、ラクトコッカス・ラクティス クレモリス亜種が挙げられる。 Examples of the lactic acid bacteria of Lactococcus genus include Lactococcus lactis, Lactococcus plantum and the like, preferably Lactococcus lactis. In addition, as Lactococcus lactis, for example, Lactococcus lactis subgenus, Lactococcus lactis cremoris subgen, Lactococcus lactis deacetylactis subspecies and the like can be mentioned, and preferably Lactococcus lactis subgenus And Lactococcus lactis cremoris subspecies.
ストレプトコッカス属の乳酸菌としては、例えば、ストレプトコッカス・サーモフィルス、ストレプトコッカス・クリセツウスなどが挙げられ、好ましくはストレプトコッカス・サーモフィルスが挙げられる。 Examples of the lactic acid bacteria of the genus Streptococcus include Streptococcus thermophilus, Streptococcus chryseus, and the like, with preference given to Streptococcus thermophilus.
本明細書において、「酵母菌群」とは、サッカロミセス属の少なくとも１種の酵母およびクルイベロミセス属の少なくとも１種の酵母を含む酵母群であり、好ましくはカザフスタニア属の少なくとも１種の酵母も含む。 As used herein, the term "yeast group" refers to a group of yeasts including at least one yeast of the genus Saccharomyces and at least one yeast of the genus Kluyveromyces, preferably also at least one yeast of the genus Kazafstannia. Including.
サッカロミセス属の酵母としては、例えば、サッカロミセス・セルビシエ、サッカロミセス・パストリアヌスなどが挙げられ、好ましくはサッカロミセス・セルビシエが挙げられる。 Examples of the yeast belonging to the genus Saccharomyces include, for example, Saccharomyces cerevisiae, Saccharomyces pastorianus, and the like, with preference given to Saccharomyces cerevisiae.
クルイベロミセス属の酵母としては、例えば、クルイベロミセス・マルキシアヌス、クルイベロミセス・ラクチスなどが挙げられ、好ましくはクルイベロミセス・マルキシアヌスが挙げられる。 Examples of the Kluyveromyces genus yeast include Kluyveromyces marxianus, Kluyveromyces lactis, and the like, with preference given to Kluyveromyces marxianus.
カザフスタニア属の酵母としては、例えば、カザフスタニア・チュリセンシス、カザフスタニア・ユニスポラ、カザフスタニア・テルリスなどが挙げられ、好ましくはカザフスタニア・チュリセンシス、カザフスタニア・ユニスポラが挙げられる。 Examples of yeasts of the genus Kazakhstania include, for example, Kazakhstania churicensis, Kazakhstania unispora, Kazakhstanian terris and the like, with preference given to Kazakhstania thulensis and Kazakhstanian unispora.
一態様において、上記微生物群は、ケフィア粒などの保存されている発酵スターター（種菌）を構成するものであってもよく、種菌による発酵産物または該発酵産物を１回以上継代した発酵産物に含まれるものであってもよい。 In one aspect, the above-mentioned microorganism group may constitute a preserved fermentation starter (inoculum) such as kefir grains, and a fermentation product by the inoculum or a fermentation product obtained by passaging the fermentation product one or more times. It may be included.
本明細書において、「大豆由来発酵産物」とは、発酵基材である大豆原料または大豆加工品の上記微生物群による発酵産物であり、大豆原料としては、丸大豆、脱脂加工大豆、大豆全粒粉、大豆胚芽などが挙げられ、好ましくは大豆全粒粉、大豆胚芽などが挙げられる。大豆加工品としては、豆乳（成分調整および成分無調整）、豆腐、ゆば、おから、枝豆、大豆もやし、きな粉などが挙げられ、好ましくは豆乳などが挙げられる。また、大豆の品種はいずれの品種でもよい。 In the present specification, “soybean-derived fermented product” refers to a fermented product of a fermented soybean raw material or fermented soybean product as the fermented substrate by the above-mentioned microorganism group, and as a soybean raw material, whole soybean, defatted soybean, whole soybean flour, Examples thereof include soybean germ and the like, preferably whole soybean flour and soybean germ and the like. Examples of processed soybean products include soymilk (component adjustment and no component adjustment), tofu, yuba, okara, green soybeans, soybean sprouts, ground flour and the like, with preference given to soymilk and the like. Also, the variety of soybean may be any variety.
発酵基材である大豆原料または大豆加工品は、加熱殺菌されているものが好ましい。加熱殺菌は、例えば、６０〜１２０℃で、好ましくは８０〜１００℃で、例えば、０．５〜２．０時間、好ましくは０．５〜１．０時間加熱することにより行う。加熱殺菌は、加圧して行ってもよい。 The soybean raw material or soybean processed product which is a fermentation base material is preferably heat-sterilized. The heat sterilization is performed, for example, by heating at 60 to 120 ° C., preferably at 80 to 100 ° C., for example, for 0.5 to 2.0 hours, preferably 0.5 to 1.0 hours. Heat sterilization may be performed under pressure.
本発明において、発酵は発酵基材と上記微生物群を混ぜ、例えば２２〜５５℃、好ましくは２５〜４５℃、より好ましくは３０〜４０℃、さらに好ましくは３０〜３７℃、最も好ましくは３５℃で、例えば２０〜１００時間、好ましくは２４〜７５時間、より好ましくは３０〜５０時間、さらに好ましくは３２〜４０時間、最も好ましくは３６時間、例えば静置して、通気しながら撹拌して、撹拌して、好ましくは静置して行う。 In the present invention, fermentation is carried out by mixing the fermentation substrate and the above-mentioned microorganism group, for example, 22 to 55 ° C., preferably 25 to 45 ° C., more preferably 30 to 40 ° C., still more preferably 30 to 37 ° C., most preferably 35 ° C. , For example, for 20 to 100 hours, preferably 24 to 75 hours, more preferably 30 to 50 hours, still more preferably 32 to 40 hours, most preferably 36 hours, for example, leaving still and stirring with aeration, Stir, preferably leave at rest.
本発明において、上記大豆由来発酵産物は、液体（例えば、発酵濾液）であってもよく、固体（例えば、発酵濾液の凍結乾燥物）であってもよい。
本発明において、上記大豆由来発酵産物は、エクオール、γ−アミノ酪酸、抗菌物質などの有用成分を含有する。エクオールは、上記大豆由来発酵産物、好ましくは固体の上記大豆由来発酵産物中に、例えば、０．５〜２．０％（ｗ／ｗ）、好ましくは１．５〜２．０％（ｗ／ｗ）含有される。γ−アミノ酪酸は、上記大豆由来発酵産物、好ましくは固体の上記大豆由来発酵産物中に、例えば、０．５〜２．０％（ｗ／ｗ）、好ましくは１．５〜２．０％（ｗ／ｗ）含有される。抗菌物質は、液体の上記大豆由来発酵産物の希釈液を、大腸菌を１０６個含む培養液に１／２０の体積で添加して培養した場合に、５０％の増殖抑制率を示す希釈倍数が、例えば５倍以下、１０倍以下、２０倍以下、４０倍以下、８０倍以下、１６０倍以下、好ましくは２０倍以下、４０倍以下、８０倍以下、１６０倍以下、より好ましくは８０倍以下、１６０倍以下、最も好ましくは１６０倍以下となる程度に、１ｍｇ／ｍｌの固体の上記大豆由来発酵産物の溶液の希釈液を、大腸菌を１０６個含む培養液に１／２０の体積で添加して培養した場合に、５０％の増殖抑制率を示す希釈倍数が、例えば５倍以上、１０倍以上、２０倍以上、４０倍以上、８０倍以上、１６０倍以上、好ましくは２０倍以上、４０倍以上、８０倍以上、１６０倍以上、より好ましくは８０倍以上、１６０倍以上、最も好ましくは１６０倍以上となる程度に含有される。
In the present invention, the soybean-derived fermentation product may be a liquid (eg, a fermentation filtrate) or a solid (eg, a lyophilizate of the fermentation filtrate).
In the present invention, the soybean-derived fermentation product contains useful ingredients such as equol, γ-aminobutyric acid, and an antibacterial substance. The equol is, for example, 0.5 to 2.0% (w / w), preferably 1.5 to 2.0% (w / w) in the soybean-derived fermented product, preferably the solid soybean-derived fermented product. w) contained. The γ-aminobutyric acid is, for example, 0.5 to 2.0% (w / w), preferably 1.5 to 2.0%, in the soybean-derived fermented product, preferably the solid soybean-derived fermented product (W / w) is contained. The antibacterial substance is obtained by adding a dilution of liquid soy fermentation product to a culture broth containing 10 6 E. coli at a volume of 1/20, and a dilution ratio showing 50% growth inhibition rate is obtained. For example, 5 times or less, 10 times or less, 20 times or less, 40 times or less, 80 times or less, 160 times or less, preferably 20 times or less, 40 times or less, 80 times or less, 160 times or less, more preferably 80 times or less , 160 times or less, added to the degree most preferably as a 160 times or less, the dilution of a solution of 1mg / ml of the solid of the soybean-derived fermentation products, in a volume of 1/20 the culture solution containing 10 6 E. coli When culture is carried out, the dilution ratio showing 50% growth inhibition rate is, for example, 5 times or more, 10 times or more, 20 times or more, 40 times or more, 80 times or more, 160 times or more, preferably 20 times or more, 40 times or more, 80 times or more, 160 Or more, more preferably 80 times or more, 160 times or more, and most preferably contained to the extent that a 160 times or more.
本発明の食品組成物は、上記大豆由来発酵産物のみからなるものであってもよいし、上記大豆由来発酵産物以外の食品成分などを含むものであってもよい。そのような食品成分としては、例えば、ビタミン類、ミネラル類が挙げられる。 The food composition of the present invention may consist only of the above-mentioned fermented product derived from soybean, or may contain a food component other than the above-mentioned fermented product derived from soybean. Such food ingredients include, for example, vitamins and minerals.
本発明の食品組成物は、１日あたり、液体の上記大豆由来発酵産物の量に換算して、例えば、１〜１０ｇ、好ましくは２〜８ｇ、より好ましくは３〜５ｇ、固体の上記大豆由来発酵産物の量に換算して、例えば、１０〜５００ｍｇ、好ましくは５０〜３００ｍｇ、より好ましくは１００〜２００ｍｇの量を、例えば、７日以上、好ましくは１５日以上、より好ましくは３０日以上連続して摂取することで、腸内の大腸菌およびウエルシュ菌などの悪玉菌の数を、摂取前の例えば、１０％程度、好ましくは２５％程度、より好ましくは５０％程度減少することができる。また、糞便中のインドール含量を、摂取前の例えば、２％程度、好ましくは５％程度、より好ましくは１０％程度減少することができる。さらには、血中のインドキシル硫酸含量を、摂取前の例えば、２％程度、好ましくは５％程度、より好ましくは１０％程度減少することができる。 The food composition of the present invention is, for example, 1 to 10 g, preferably 2 to 8 g, more preferably 3 to 5 g of solid soybean origin, in terms of the amount of the liquid soy bean-derived fermentation product per day. In terms of the amount of fermentation product, for example, an amount of 10 to 500 mg, preferably 50 to 300 mg, more preferably 100 to 200 mg, for example, 7 days or more, preferably 15 days or more, more preferably 30 days or more Thus, the number of bad bacteria such as E. coli and Welsh bacteria in the intestine can be reduced, for example, by about 10%, preferably by about 25%, more preferably by about 50%, before ingestion. In addition, the indole content in feces can be reduced, for example, by about 2%, preferably by about 5%, more preferably by about 10%, before ingestion. Furthermore, the indoxyl sulfate content in blood can be reduced, for example, by about 2%, preferably by about 5%, more preferably by about 10%, before ingestion.
本発明の食品組成物は、健康食品、サプリメントなどとして使用することができる。例えば、錠剤、カプセル剤（軟カプセル剤、硬カプセル剤）、顆粒剤（ドライシロップを含む）、散剤（粉末剤）、細粒剤、丸剤などの固形の製剤、水性または非水性の内服用液（液剤、懸濁剤、シロップ剤を含む）などの液体の製剤として使用することができる。 The food composition of the present invention can be used as a health food, a supplement and the like. For example, tablets, capsules (soft capsules, hard capsules), granules (including dry syrup), powders (powders), fine granules, solid preparations such as pills, aqueous or non-aqueous internal use liquids It can be used as a liquid formulation (including solutions, suspensions and syrups).
上記の各種製剤は、本発明の食品組成物と、健康食品、サプリメントなどで通常許容され得る添加剤を混和して、周知の方法で製造することができる。そのような添加剤としては、例えば、賦形剤、滑沢剤（コーティング剤）、結合剤、崩壊剤、安定剤、矯味矯臭剤、基剤、分散剤、希釈剤、界面活性剤、乳化剤、経皮吸収促進剤、ｐＨ調整剤、保存剤、着色剤、油分（油脂、鉱物油など）、保湿剤、アルコール、増粘剤、ポリマー、皮膜形成剤、紫外線吸収剤、細胞賦活剤、酸化防止剤、防腐剤、清涼剤、消臭剤、顔料、染料、香料、糖類、アミノ酸類、ビタミン類、有機酸、有機アミン、植物抽出物などがあげられるが、これらに限定されるものではない。 The various formulations described above can be manufactured by known methods by mixing the food composition of the present invention with additives generally accepted for health foods, supplements and the like. As such additives, for example, excipients, lubricants (coating agents), binders, disintegrants, stabilizers, flavoring agents, bases, dispersants, diluents, surfactants, emulsifiers, Transdermal absorption promoter, pH adjuster, preservative, coloring agent, oil (oil and fat, mineral oil etc.), moisturizer, alcohol, thickener, polymer, film forming agent, UV absorber, cell activator, antioxidant Agents, preservatives, refreshing agents, deodorants, pigments, dyes, perfumes, saccharides, amino acids, vitamins, organic acids, organic amines, plant extracts and the like, but are not limited thereto.
一態様として、本発明の食品組成物は、血中インドキル硫酸の蓄積によって引き起こされると考えられる、生体臓器の機能障害による疾患、例えば、腎臓、肝臓、脳、心臓の機能障害による疾患、好ましくは腎臓、肝臓の機能障害による疾患、より好ましくは腎臓の機能障害による疾患の予防または進行の遅延に用いることができる。 In one embodiment, the food composition of the present invention is a disease caused by dysfunction of a living organ, which is considered to be caused by accumulation of blood indoxyl sulfate, for example, a disease caused by dysfunction of kidney, liver, brain, heart, preferably It can be used to prevent or delay the progression of a disease due to kidney or liver dysfunction, more preferably a disease due to kidney dysfunction.
腎臓の機能障害による疾患としては、例えば、慢性腎不全（ＣＤＫ）、腎血管疾患が挙げられる。
肝臓の機能障害による疾患としては、例えば、肝硬変、肝性脳症が挙げられる。
Diseases caused by renal dysfunction include, for example, chronic renal failure (CDK) and renal vascular disease.
Diseases due to liver dysfunction include, for example, liver cirrhosis and hepatic encephalopathy.
脳の機能障害による疾患としては、例えば、脳血管障害、認知症が挙げられる。
心臓の機能障害による疾患としては、例えば、心血管疾患（ＣＶＤ）が挙げられる。
さらなる一態様として、本発明の食品組成物は、口腔内の環境の改善、虫歯の予防または進行の遅延に用いることができる。
Diseases due to brain dysfunction include, for example, cerebrovascular disease and dementia.
Diseases due to cardiac dysfunction include, for example, cardiovascular disease (CVD).
As a further aspect, the food composition of the present invention can be used to improve the environment in the oral cavity, to prevent or delay the progression of dental caries.
さらなる一態様として、本発明の食品組成物は、精神ストレスの低減に用いることができる。
さらなる一態様として、本発明の食品組成物は、発がんリスクの低減に用いることができる。
In a further aspect, the food composition of the present invention can be used to reduce mental stress.
As a further aspect, the food composition of the present invention can be used to reduce cancer risk.
以下、本発明の具体的態様を実施例として説明する。
実施例１ 豆乳ケフィアの製造
まず、発酵スターター（種菌）の調製のため、ケフィア粒（５０g）に１０倍量の市販成分無調整牛乳を加え、２５℃で７２時間静置培養して発酵させた。ケフィア粒に含まれる、同定されている微生物は以下の表１に示すとおりである。
Hereinafter, specific embodiments of the present invention will be described as examples.
Example 1 Production of Soymilk Kefir Firstly, for preparation of a fermentation starter (seed fungus), 10 times amount of commercial component unadjusted milk was added to kefir grains (50 g) and fermented by standing culture at 25 ° C. for 72 hours . The identified microorganisms contained in Kefir grains are as shown in Table 1 below.
発酵後、網やザル等を用いて発酵産物をケフィア粒から分離し、分離した発酵産物を種菌とした。なお、発酵産物を分離した後のケフィア粒に再度牛乳を加えて継代培養することにより、さらに発酵産物を得ることができた。 After fermentation, the fermented product was separated from the kefir grains using a net or a monkey, and the separated fermented product was used as a seed fungus. In addition, a fermented product was able to be obtained by adding milk again to kefir grains after separation of the fermented product and subculturing.
次に、豆乳ケフィアの製造のため、全粒大豆粉末（５００ｇ、株式会社ペリカンより入手）、大豆胚芽（１０ｇ、株式会社ペリカンより入手）、市販の砂糖（２５ｇ）を水道水に混釈し、１，０００ｍｌにした。そして、９５℃で１時間加熱殺菌した後、２５℃に冷却し、上記方法にて得られた種菌を５０ｇ添加し、２５℃で３６時間静置して発酵させ、豆乳ケフィアを得た。また、比較対照として、市販成分無調整牛乳５００ｍｌを同様に種菌で発酵させ、牛乳ケフィアを製造した。 Next, for the production of soymilk kefir, whole grain soybean powder (500 g, obtained from pelican), soybean germ (10 g, obtained from pelican), commercially available sugar (25 g) is mixed with tap water, It was made into 1,000 ml. After heat sterilization at 95 ° C. for 1 hour, the mixture was cooled to 25 ° C., 50 g of the seed fungus obtained by the above method was added, and allowed to stand at 25 ° C. for 36 hours for fermentation to obtain soymilk kefir. In addition, 500 ml of commercially available component unadjusted milk was similarly fermented with the inoculum, and a milk kefir was produced as a control for comparison.
実施例２ 豆乳ケフィア中の成分の分析
１．イソフラボン類の配糖体、エクオール、およびγ−アミノ酪酸の分析
実施例１の豆乳ケフィアおよび比較対照の牛乳ケフィアについて、発酵前および発酵後のものをそれぞれ１５ｇ用い、５０％（ｖ／ｖ）エチルアルコール５００ｍｌを加え、室温下で振盪抽出した。この抽出液を試料として、試料中のイソフラボン類の配糖体、エクオール、およびγ−アミノ酪酸について、高速液体クロマトグラフ（ＨＰＬＣ）により分析した。
Example 2 Analysis of Ingredients in Soymilk Kefir
1. Analysis of glycosides of isoflavones, equol, and γ-aminobutyric acid For the soymilk kefir of Example 1 and the comparative milk milk kefir, 15 g of each before fermentation and after fermentation, 50% (v / v) ethyl 500 ml of alcohol was added and extraction was performed shaking at room temperature. This extract was used as a sample for analysis of glycosides of isoflavones, equol, and γ-aminobutyric acid in the sample by high performance liquid chromatography (HPLC).
（１）イソフラボン類の配糖体の分析
イソフラボン類の配糖体の分析に用いた条件は、以下のとおりである。
ＨＰＬＣシステム：ＬＣ−８０２０（東ソー株式会社製）
カラム：Ｈｙｐｅｒｓｉｌ ＨｙＰｕｒｉｔｙ Ｃ１８（内径２．１ｍｍ×長さ１００ｍｍ）（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ社製）
カラム温度：４０℃
移動相：アセトニトリル：水（７５：２５）
流速：０．２ｍｌ／分
試料注入量：１０μｌ
検出器：紫外可視検出器（ＵＶ−８０２０、東ソー株式会社製）
検出波長：２５４ｎｍ
標準品のダイジン、ゲニステイン、およびグリシチン（いずれも和光純薬工業株式会社より入手、製品番号：３０９−０５１６１、０７９−０５５３３、およびＧ６３５４１０）の分析結果との比較により、クロマトグラム上で試料中のイソフラボン類の配糖体のピークを同定した。
(1) Analysis of glycosides of isoflavones The conditions used for analysis of glycosides of isoflavones are as follows.
HPLC system: LC-8020 (manufactured by Tosoh Corporation)
Column: Hypersil HyPurity C18 (inner diameter 2.1 mm × length 100 mm) (Thermo Fisher company)
Column temperature: 40 ° C
Mobile phase: acetonitrile: water (75: 25)
Flow rate: 0.2 ml / min Sample injection volume: 10 μl
Detector: UV-visible detector (UV-8020, manufactured by Tosoh Corporation)
Detection wavelength: 254 nm
Comparison with the analysis results of standard products daidzin, genistein, and glycitin (all obtained from Wako Pure Chemical Industries, Ltd., product numbers: 309-05161, 079-05533, and G635410), it is in the sample on the chromatogram. The glycoside peak of isoflavones was identified.
（２）エクオールの分析
エクオールの分析に用いた条件は、以下のとおりである。
ＨＰＬＣシステム：ＬＣ−８０２０（東ソー株式会社製）
カラム：Ｈｙｐｅｒｓｉｌ ＨｙＰｕｒｉｔｙ Ｃ１８（内径２．１ｍｍ×長さ１００ｍｍ）（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ株式会社製）
カラム温度：４０℃
移動相：アセトニトリル：水（７５：２５）
流速：０．２０ｍｌ／分
試料注入量：１０μｌ
検出器：紫外可視検出器（ＵＶ−８０２０、東ソー株式会社製）
検出波長：２５４ｎｍ
標準品のエクオール（和光純薬工業株式会社より入手、製品番号：３８０−０４９９１）の分析結果との比較により、クロマトグラム上で試料中のエクオールのピークを同定した。標準品および豆乳ケフィア由来の試料より得られたクロマトグラムを、図１−１および図１−２にそれぞれ示す。
(2) Analysis of equol The conditions used for analysis of equol are as follows.
HPLC system: LC-8020 (manufactured by Tosoh Corporation)
Column: Hypersil HyPurity C18 (inner diameter 2.1 mm × length 100 mm) (manufactured by Thermo Fisher Inc.)
Column temperature: 40 ° C
Mobile phase: acetonitrile: water (75: 25)
Flow rate: 0.20 ml / min Sample injection volume: 10 μl
Detector: UV-visible detector (UV-8020, manufactured by Tosoh Corporation)
Detection wavelength: 254 nm
The peak of equol in the sample was identified on the chromatogram by comparison with the analysis result of standard product equol (obtained from Wako Pure Chemical Industries, Ltd., product number: 380-04991). Chromatograms obtained from a standard product and a sample derived from soymilk kefir are shown in FIGS. 1-1 and 1-2, respectively.
（３）γ−アミノ酪酸の分析
ＧＡＢＡの分析に用いた条件は、以下のとおりである。
ＨＰＬＣシステム：ＬＣ−８０２０（東ソー株式会社製）
カラム：ＴＳＫｇｅｌ ＯＤＳ−８０Ｔｓ（内径４．６ｍｍ×長さ２５０ｍｍ）（東ソー株式会社製）
カラム温度：４０℃
移動相：アセトニトリル：１０ｍＭ酢酸アンモニウム水溶液（５０：５）
流量：１．００ｍｌ／分
試料注入量：１０μｌ
誘導体化試薬：ニンヒドリン（プレカラム法）
検出器：紫外可視検出器（ＵＶ−８０２０、東ソー株式会社製）
検出波長：５７０ｎｍ
標準品のγ−アミノ酪酸（和光純薬工業株式会社より入手、製品番号：０１０−０２４４１）の分析結果との比較により、クロマトグラム上で試料中のγ−アミノ酪酸のピークを同定した。標準品および豆乳ケフィアより由来の試料より得られたクロマトグラムを、図２−１および図２−２にそれぞれ示す。
(3) Analysis of γ-aminobutyric acid The conditions used for analysis of GABA are as follows.
HPLC system: LC-8020 (manufactured by Tosoh Corporation)
Column: TSKgel ODS-80 Ts (inner diameter 4.6 mm × length 250 mm) (made by Tosoh Corporation)
Column temperature: 40 ° C
Mobile phase: acetonitrile: 10 mM aqueous ammonium acetate (50: 5)
Flow rate: 1.00 ml / min Sample injection amount: 10 μl
Derivatization reagent: ninhydrin (pre-column method)
Detector: UV-visible detector (UV-8020, manufactured by Tosoh Corporation)
Detection wavelength: 570 nm
The peak of γ-aminobutyric acid in the sample was identified on the chromatogram by comparison with the analysis result of the standard product γ-aminobutyric acid (obtained from Wako Pure Chemical Industries, Ltd., product number: 010-02441). Chromatograms obtained from a standard product and a sample derived from soymilk kefir are shown in FIGS. 2-1 and 2-2, respectively.
２．抗菌活性の分析
実施例１の豆乳ケフィアおよび比較対照の牛乳ケフィアについて、発酵前および発酵後のものからそれぞれ遠心分離により固形分を除去した液体部分を抗菌活性測定用試料とし、ディスク法で抗菌活性を測定することにより分析した。具体的には、ヒト糞便より調製した微生物にプレートカウントアガー培地（Ｍｅｒｃｋ社製）を加えて作製したプレート中央に、上記抗菌活性測定用試料をペーパーディスク（直径８ｍｍ、ＡＤＶＡＮＴＥＣ社製）に５０μｌ吸収させたものを載せ、３５℃で２４時間培養を行い、プレート上でペーパーディスクの周囲に形成された微生物の生育が阻止されている範囲（阻止円）の直径を観察した。
2. Analysis of antibacterial activity With respect to the soymilk kefir of Example 1 and the milk kefir of the comparative control, the liquid portion from which solids were removed by centrifugation from those before fermentation and after fermentation, respectively, was used as a sample for antibacterial activity measurement, and the antibacterial activity was achieved by the disc method It analyzed by measuring. Specifically, 50 μl of the above-mentioned sample for antibacterial activity measurement was absorbed in a paper disc (diameter 8 mm, manufactured by ADVANTEC) in the center of a plate prepared by adding plate count agar medium (manufactured by Merck) to microorganisms prepared from human feces The loaded product was placed and cultured at 35 ° C. for 24 hours, and the diameter of the range (inhibited circle) in which the growth of the microorganisms formed around the paper disc was blocked on the plate was observed.
上記「１．イソフラボン類の配糖体、エクオール、およびγ−アミノ酪酸の分析」および「２．抗菌活性の分析」により、実施例１の豆乳ケフィアおよび比較対照の牛乳ケフィアについて、イソフラボン類の配糖体、エクオール、およびγ−アミノ酪酸の含量、ならびに抗菌活性を分析した結果を、表２にまとめて示す。 According to the above-mentioned “1. Analysis of glycosides of isoflavones, equol and γ-aminobutyric acid” and “2. Analysis of antibacterial activity”, distribution of isoflavones with respect to soymilk kefir of Example 1 and milk kefir as a comparison control. The results of analysis of the contents of glycoside, equol, and γ-aminobutyric acid and antibacterial activity are summarized in Table 2.
抗菌活性において、−は陰性、＋は偽陽性（輪郭が不明瞭な阻止円を形成）、＋＋は弱陽性（輪郭が明瞭な阻止円を形成し、阻止円の直径が１０ｍｍ以下）、＋＋＋は中陽性（輪郭が明瞭な阻止円を形成し、阻止円の直径が１０〜１５ｍｍ）、および＋＋＋＋は強陽性（輪郭が明瞭な阻止円を形成し、阻止円のφが１５ｍｍ以上）をそれぞれ表す。 In the antibacterial activity,-is negative, + is false positive (obstacles with unclear outline forming a inhibition circle), ++ is weakly positive (obtrusive outline forming inhibition circle, diameter of inhibition circle is 10 mm or less), +++ is Moderately positive (forms a clear blocking circle and the diameter of the blocking circle is 10 to 15 mm), and ++++ shows strong positive (forms a clear blocking circle, φ of the blocking circle is 15 mm or more) .
実施例１の豆乳ケフィアは、発酵後にエクオールを２．０％（ｗ／ｗ）、γ−アミノ酪酸を２．０％（ｗ／ｗ）含有しており、抗菌活性も極めて高かったのに対し、牛乳ケフィアでは、発酵後にエクオールは検出されず、γ−アミノ酪酸の含有量も０．４％（ｗ／ｗ）であり、豆乳ケフィアと比較して低かった。また、豆乳ケフィアと比較して抗菌活性も低かった。このことから、実施例１で製造した豆乳ケフィアでは、発酵によりエクオール、γ−アミノ酪酸、および抗菌物質が効率良く産生されていることが示唆された。なお、エクオールは発酵基材である豆乳に含まれるイソフラボン類から、γ−アミノ酪酸は発酵基材である豆乳に含まれるグルタミン酸から産生されたものと考えられた。エクオールは発がんリスクを低減させる作用が、γ−アミノ酪酸は精神ストレスを低減させる作用があるとされている（Beg A. A., Jorgensen E. M., Nat. Neurosci., 6(11), 1145-1152, 2003）。 The soymilk Kefir of Example 1 contained 2.0% (w / w) of equol and 2.0% (w / w) of γ-aminobutyric acid after fermentation, while the antibacterial activity was also extremely high. In milk kefir, equol was not detected after fermentation, and the content of γ-aminobutyric acid was 0.4% (w / w), which was lower than that of soymilk kefir. Also, the antibacterial activity was lower than that of soymilk kefir. From this, it was suggested that in the soymilk kefir produced in Example 1, equol, γ-aminobutyric acid and an antibacterial substance were efficiently produced by fermentation. In addition, equol was considered to be produced from isoflavones contained in soymilk which is a fermentation base, and γ-aminobutyric acid was produced from glutamic acid contained in soymilk which is a fermentation base. Equol is thought to reduce carcinogenic risk, and γ-aminobutyric acid is thought to reduce mental stress (Beg AA, Jorgensen EM, Nat. Neurosci., 6 (11), 1145-1152, 2003) .
また、牛乳ケフィアでは、１０８ＣＦＵ／ｇ程度で乳酸菌の増殖が停止したのに対し、豆乳ケフィアでは１０１１ＣＦＵ／ｇ程度まで乳酸菌が増殖したことから、実施例１で製造した豆乳ケフィアと牛乳ケフィアのγ−アミノ酪酸の含有量の差は、発酵基材に含まれているグルタミン酸の量の差に加えて、増殖した乳酸菌の数の差によるものと考えられた。 In addition, in the case of milk kefir, the growth of lactic acid bacteria was stopped at about 10 8 CFU / g, whereas in soya milk kefir, the lactic acid bacteria grew to about 10 11 CFU / g, so the soy milk kefir and milk manufactured in Example 1 The difference in the content of γ-aminobutyric acid of Kefir was considered to be due to the difference in the number of lactic acid bacteria grown in addition to the difference in the amount of glutamic acid contained in the fermentation substrate.
実施例３ 豆乳ケフィアの抗菌活性の測定
実施例２で観察された豆乳ケフィアの抗菌活性について、以下の方法で測定することにより、さらに分析した。
Example 3 Measurement of antibacterial activity of soymilk kefir The antibacterial activity of soymilk kefir observed in Example 2 was further analyzed by measuring according to the following method.
試験材料
実施例１で得られた豆乳ケフィアから遠心分離または珪藻土濾過により固形分を除去した発酵濾液を凍結乾燥させたものを、抗菌活性の測定用の試験材料とした。また、牛乳ケフィアの発酵濾液を凍結乾燥させたものを比較対照とした。
Test Material A freeze-dried fermentation filtrate from which solid content was removed from the soymilk kefir obtained in Example 1 by centrifugation or filtration through diatomaceous earth was used as a test material for measurement of antibacterial activity. In addition, those obtained by freeze-drying the fermented filtrate of milk kefir were used as a control.
豆乳ケフィアの抗菌活性の濃度スクリーニング
液体培地における大腸菌の増殖抑制率を指標に、豆乳ケフィア由来の試験材料が抗菌活性を示す濃度について、スクリーニングを行った。まず、ＣＡＳＯ培地（Ｍｅｒｃｋ社より入手、製品番号：１．００５２５．５０００）２ｍｌに、大腸菌を１ｍｌあたり１０６個になるように添加した。豆乳ケフィア由来の試験材料を滅菌した生理食塩水で１ｍｇ／ｍｌの濃度に調製したものを基準試料とし、さらに滅菌した生理食塩水で２０〜６４０倍に段階希釈したものを希釈試料とした。
Concentration Screening of Antibacterial Activity of Soymilk Kefir A screening was carried out using a growth inhibition rate of E. coli in a liquid medium as an index, at a concentration at which a test material derived from soymilk Kefir shows antibacterial activity. First, to 2 ml of CASO medium (obtained from Merck, product number: 1.00525.5000), 10 6 E. coli were added per 1 ml. What prepared the test material derived from soymilk kefir to a concentration of 1 mg / ml with sterilized physiological saline was used as a reference sample, and the serial dilution of 20 to 640 times with sterilized physiological saline was used as a diluted sample.
基準試料および希釈試料をそれぞれ、上記の大腸菌を含むＣＡＳＯ培地に１００μｌずつ添加した。３７℃で６時間培養したものを各測定試料とし、６００ｎｍの波長における濁度（ＯＤ６００）を測定した。ＯＤ６００の測定結果、ならびに算出された大腸菌の増殖率（％）および増殖抑制率（％）を表３に示す。基準試料または希釈試料を添加せずに滅菌した生理食塩水１００μｌを添加して培養したものをコントロールとし、ＣＡＳＯ培地のみのものをブランクとした。大腸菌の増殖率（％）は、それぞれのＯＤ６００の値を基に、以下の式１で算出した。また、増殖抑制率（％）は、コントロールの増殖率１００％との差で算出した。なお、基準試料を添加した測定試料のＯＤ６００はブランクよりも低くなったため、増殖率を０％とした。 100 μl of each of the reference sample and the diluted sample was added to the CASO medium containing E. coli described above. What was cultured at 37 ° C. for 6 hours was used as each measurement sample, and the turbidity (OD 600) at a wavelength of 600 nm was measured. The measurement results of OD600 and the calculated growth rate (%) and growth suppression rate (%) of E. coli are shown in Table 3. A culture obtained by adding 100 μl of sterile physiological saline without adding a reference sample or a diluted sample was used as a control, and a control using CASO medium alone was used as a blank. The growth rate (%) of E. coli was calculated by the following equation 1 based on the respective OD600 values. Also, the growth inhibition rate (%) was calculated as the difference between the control and the growth rate of 100%. In addition, since OD600 of the measurement sample which added the reference sample became lower than a blank, growth rate was made into 0%.
式１
大腸菌の増殖率（％）＝
｛（各測定試料−ブランク）／（コントロール−ブランク）｝×１００
Formula 1
Growth rate of E. coli (%) =
{(Each measurement sample-blank) / (control-blank)} x 100
表３より、豆乳ケフィアの大腸菌の増殖に対する５０％阻害濃度は、豆乳ケフィア由来の試験材料濃度で６．２５μｇ／ｍｌ（１６０倍希釈試料を添加した測定試料）程度であると考えられた。 From Table 3, it was considered that the 50% inhibitory concentration of the soymilk kefir against the growth of E. coli was about 6.25 μg / ml (measured sample to which a 160-fold diluted sample was added) at the test material concentration derived from soymilk kefir.
豆乳ケフィアの抗菌活性スペクトルの分析
大腸菌、クロストリジウム属菌のウエルシュ菌、リステリア属菌のリステリア菌（Ｌｉｓｔｅｒｉａ ｍｏｎｏｃｙｔｏｇｅｎｅｓ）、緑膿菌（Ｐｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ）、およびサルモネラ属菌の腸炎菌（Ｓａｌｍｏｎｅｌｌａ ｅｎｔｅｒｉｔｉｄｉｓ）に対する、実施例１で得られた豆乳ケフィアの抗菌活性を以下の方法で試験した。
Analysis of antibacterial activity spectrum of soymilk kefir Carried out against Escherichia coli, Clostridium sp. Perchella, Listeria sp. Listeria monocytogenes, Pseudomonas aeruginosa and Salmonella enteritidis (Salmonella enteritidis) The antibacterial activity of the soymilk kefir obtained in Example 1 was tested by the following method.
大腸菌の増殖に対する５０％阻害濃度を参考にして、１０μｇ／ｍｌの濃度の豆乳ケフィア由来の試験材料の脱イオン水溶液を調製し、その５０μｌを直径８ｍｍのペーパーディスク（ＡＤＶＡＮＴＥＣ社製、製品番号：４９００５０１０）に含浸させ、１０ｍｌのプレートカウントアガー培地（Ｍｅｒｃｋ社製）の上に置いて、試験する細菌とともに培養した。大腸菌、クロストリジウム属菌、およびサルモネラ属菌は３７℃で２４時間、リステリア属菌および緑膿菌は３７℃で４８時間培養した。なお、クロストリジウム属菌は嫌気条件下で培養した。ディスクの周囲に形成された阻止円の直径を測定し、抗菌活性の程度を調べた。阻止円の直径が大きいほど、抗菌活性の程度が高いと考えられる。また、比較対照として、牛乳ケフィア由来の試験材料についても同様に試験した。結果を表４に示す。 Prepare a deionized water solution of a test material derived from soymilk Kefir at a concentration of 10 μg / ml with reference to 50% inhibitory concentration against the growth of E. coli and prepare 50 μl of the aqueous solution with a paper disc of 8 mm diameter (ADVANTEC, product number: 49005010 ) And placed on 10 ml of plate count agar medium (Merck) and cultured with the bacteria to be tested. E. coli, Clostridia, and Salmonella were cultured at 37 ° C. for 24 hours, and Listeria and P. aeruginosa were cultured at 37 ° C. for 48 hours. Clostridium bacteria were cultured under anaerobic conditions. The diameter of the blocking circle formed around the disc was measured to determine the degree of antibacterial activity. The larger the diameter of the blocking circle, the higher the degree of antibacterial activity is considered. In addition, as a comparative control, a test material derived from milk kefir was similarly tested. The results are shown in Table 4.
その結果、試験した細菌の全てに対して明瞭な阻止円が形成され、実施例１で得られた豆乳ケフィアはこれらの細菌の全てに対して抗菌活性を示すことが明らかとなった。また、これらの細菌の全てで、実施例１で得られた豆乳ケフィアの抗菌活性は牛乳ケフィアの抗菌活性よりも高かった。阻止円の直径の値の比で計算すると、実施例１で得られた豆乳ケフィアは牛乳ケフィアに比して、大腸菌で約１．７倍、クロストリジウム属菌で約１．５倍、リステリア属菌で約１．７倍、緑膿菌で約１．５倍、サルモネラ属菌で約３．３倍の抗菌活性を有することが示唆された。 As a result, a clear inhibition circle was formed for all the tested bacteria, and it was revealed that the soymilk kefir obtained in Example 1 exhibits antibacterial activity against all of these bacteria. Also, in all of these bacteria, the antibacterial activity of the soymilk kefir obtained in Example 1 was higher than that of milk kefir. The soymilk kefir obtained in Example 1 is about 1.7 times greater for E. coli, about 1.5 times greater for Clostridia than for milk kefir when calculated as the ratio of the values of the diameter of the inhibition circle, and the Listeria genus bacteria. It has been suggested that it has an antibacterial activity of about 1.7 times, about 1.5 times with Pseudomonas aeruginosa, and about 3.3 times with Salmonella spp.
このように、実施例１で得られた豆乳ケフィアの抗菌活性スペクトルは非常に広いことが明らかとなったが、乳酸菌などの細菌が産生する類縁の細菌に対する抗菌物質であるバクテリオシンの抗菌活性スペクトルが狭いことを考えると、実施例１で得られた豆乳ケフィアは、バクテリオシンとは異なる抗菌物質を含有することが示唆された。また、実施例１で得られた豆乳ケフィアの抗菌活性により生じた阻止円を生理食塩水で洗浄してから再度培養しても、試験した細菌の生育が一切認められなかったことから、この抗菌物質による作用は静菌作用ではなく、殺菌作用であることが示唆された。さらに、実施例１で得られた豆乳ケフィアにおいてケフィア粒が増殖したことから、この抗菌物質は、ケフィア粒にも含まれている乳酸菌やビフィズス菌などの有用細菌（いわゆる善玉菌）に対する抗菌活性を示さず、いわゆる悪玉菌に選択的に作用し得ると推定された。このような悪玉菌に対する選択的な抗菌作用は、日常生活における健康増進に大いに寄与することが期待できる。 Thus, the antibacterial activity spectrum of the soymilk kefir obtained in Example 1 was found to be very broad, but the antibacterial activity spectrum of bacteriocin, which is an antibacterial substance against similar bacteria produced by bacteria such as lactic acid bacteria In view of the narrowness, it was suggested that the soymilk kefir obtained in Example 1 contains an antibacterial substance different from bacteriocin. Also, even if the inhibition circle generated by the antibacterial activity of the soymilk kefir obtained in Example 1 was washed with physiological saline and then cultured again, no growth of the tested bacteria was observed, so this antibacterial It was suggested that the action by the substance was not bacteriostatic action but bactericidal action. Furthermore, since the kefir grains grew in the soymilk kefir obtained in Example 1, this antibacterial substance has an antibacterial activity against useful bacteria (so-called good bacteria) such as lactic acid bacteria and bifidobacteria which are also contained in the kefir grains. Not shown, it was estimated that it could act selectively on so-called bad bacteria. Such selective antibacterial action against bad bacteria can be expected to greatly contribute to health promotion in daily life.
なお、実施例１で得られた豆乳ケフィアの抗菌活性は、分子量１ｋＤａのカットオフ透析チューブの透析外液において認められ、トヨパールＨＷ−４０Ｓ（東ソー株式会社製）を用いたゲルろ過クロマトグラフィー（溶出液：７０％エタノール溶液）により、分子量４５０Ｄａおよび５００Ｄａ近傍に２つのピークで溶出されたことから、抗菌活性を有する抗菌物質は、分子量約４５０Ｄａおよび約５００Ｄａのグリセロール誘導体であることが推定された。 The antibacterial activity of the soymilk kefir obtained in Example 1 was observed in the dialysis outer solution of a cutoff dialysis tube with a molecular weight of 1 kDa, and gel filtration chromatography (elution using TOYO PEARL HW-40S (manufactured by Tosoh Corporation)) It was estimated that the antibacterial substance having antibacterial activity was a glycerol derivative having a molecular weight of about 450 Da and about 500 Da, since it was eluted with a liquid: 70% ethanol solution) and had two peaks at molecular weights of 450 Da and 500 Da.
実施例４ 豆乳ケフィア摂取の糞便中悪玉菌数および総インドール含量、ならびに血中インドキシル硫酸濃度に対する影響の評価
いわゆる悪玉菌である大腸菌群やクロストリジウム属菌は、トリプトファンを代謝してインドールを産生する。インドールはこれらの悪玉菌の宿主の肝臓で代謝され、インドキシル硫酸へと変換される。インドキシル硫酸は代表的な尿毒素であり、活性酸素を経由して転写因子である核内因子κＢ（ＮＦ−κＢ）を活性化させ、細胞老化マーカー遺伝子であるｐ５３とｐ２１を発現させて細胞の老化や繊維化を引き起こし、その結果、腎臓、肝臓、脳、心臓などの生体臓器の機能障害を来すと考えられている（Niwa T., et. al., Am. J. Nephrol., 14(3), 207-212, 1994; Niwa T. and Ise M., J. Lab. Clin. Med., 124(1), 96-104, 1994）。インドキシル硫酸は、血中でアルブミンと結合して、尿中に排泄されずに血中に蓄積されることにより、腎機能を始め、生体内の全ての臓器に悪影響を及ぼすと考えられる。例えば、血中インドキシル硫酸濃度は健常者で１０μＭ以下であるが、末期の慢性腎不全患者では最大で５５０μＭ、平均で２５０μＭ前後にまで達する（Niwa T. and Ise M., J. Lab. Clin. Med., 124(1), 96-104, 1994）。
Example 4 Evaluation of the influence of soymilk Kefir intake on fecal number and total indole content in stool and indoxyl sulfate concentration in blood, and so-called bad bacteria E. coli group and Clostridial bacteria metabolize tryptophan to produce indole . Indole is metabolized in the liver of these bad bacteria hosts and converted to indoxyl sulfate. Indoxyl sulfate is a typical urinary toxin, and activates the transcription factor nuclear factor κB (NF-κB) via active oxygen to express the cellular senescence marker genes p53 and p21, thereby It is believed that it causes aging and fibrosis, resulting in dysfunction of living organs such as kidney, liver, brain and heart (Niwa T., et. Al., Am. J. Nephrol., 14 (3), 207-212, 1994; Niwa T. and Ise M., J. Lab. Clin. Med., 124 (1), 96-104, 1994). Indoxyl sulfate is thought to adversely affect all organs in the living body, including renal function, by binding to albumin in the blood and accumulating in the blood without being excreted in the urine. For example, the blood indoxyl sulfate concentration is less than 10 μM in healthy subjects, but reaches up to 550 μM in patients with chronic renal failure in the end stage, and averages around 250 μM (Niwa T. and Ise M., J. Lab. Clin Med., 124 (1), 96-104, 1994).
また、上記悪玉菌は、タンパク質を分解し、インドール、スカトール、フェノール、アンモニアなどの有害物質を産生して腸管内をアルカリ性にしている。そのため、腸内環境を乱し、腸管内抗菌物質であるディフェンシンを無効化させるなどの悪影響を及ぼす（廣川 勝いく、漢方と最新治療、21（3）、235-242、2012）。さらに、「超悪玉菌」とされているウエルシュ菌は分泌型免疫グロブリンＡ（ｓＩｇ−Ａ）や腸管のバリアーであるムチン層を分解する酵素を分泌し、種々の疾患の要因にもなっている。 Moreover, the above-mentioned bad bacteria decompose proteins and produce harmful substances such as indole, skatole, phenol, ammonia and the like to make the intestinal tract alkaline. Therefore, the intestinal environment is disturbed, and adverse effects such as invalidation of the intestinal antibacterial agent defensin (Kashikawa Masaru, Kampo and the latest treatment, 21 (3), 235-242, 2012). Furthermore, C. perfringens, which is considered "super bad bacteria", secretes secretory immunoglobulin A (sIg-A) and an enzyme that degrades mucin layer, which is a barrier of the intestinal tract, and is a factor of various diseases. .
そこで、実施例１で得られた豆乳ケフィアが、糞便中の悪玉菌数、悪玉菌が産生するインドールの量、および血中のインドキシル硫酸の量にどのような影響を与えるかということについて、以下の方法で評価した。 Therefore, how the soymilk kefir obtained in Example 1 affects the number of bacteria in the feces, the amount of indole produced by the bacteria, and the amount of indoxyl sulfate in the blood, It evaluated by the following methods.
糞便中悪玉菌数に対する影響の評価
Ｗｉｓｔａｒ／ＳＴラット（６週齢、オス）を用い、２０ｍｇ／ｍｌに調製した塩化マグネシウムを胃ゾンデで強制的に投与して下痢を発症させ、消化管細菌を減少させた。便秘傾向のヒト大便を生理的食塩水に懸濁し、上記ラットに移植したものをヒト糞便移植ラットとした。そして、１００ｇの粉末飼料（ＣＥ-２、日本クレア株式会社製）に、加熱殺菌して凍結乾燥した豆乳ケフィア６ｍｇを混合した飼料を、ヒト糞便移植ラット１匹あたり１０ｇ摂取させた（豆乳ケフィア乾燥物として３．３ｍｇ／ｋｇ体重を投与）。ヒト糞便移植ラットの体重は１８０ｇとして計算した。比較対照として市販のプレーンヨーグルト（明治ブルガリアヨーグルト）を滅菌して凍結乾燥した後、同様の方法でヨーグルト乾燥物として３．３ｍｇ／ｋｇ体重を、ヒト糞便移植ラットに投与した。試験開始時、１５日目、および３０日目に糞便を集め、大腸菌、およびウエルシュ菌の数を、選択培地を用いて測定した。具体的には以下のとおりである：１ｇの糞便に滅菌した生理食塩水を９ｍｌ加えて１０倍希釈の原液を作製し、これに滅菌した生理食塩水を用いて１０倍の段階希釈を１０４倍まで行った希釈液を作製した。大腸菌用選択培地としてデソオキシコーレイト寒天培地（Ｍｅｒｃｋ社製）を、ウエルシュ菌用選択培地としてカナマイシン含有ＣＷ寒天基礎培地（ニッスイ社製）に５０％卵黄液を１０％添加したものを用い、各段階希釈液１ｍｌに選択培地を１５ｍｌ加えて混釈し、３７℃で４８時間培養した（各段階希釈液につきｎ=５で培養）。そして、各選択培地において生じたコロニー数から大腸菌およびウエルシュ菌の糞便中の数を算出した。なお、ウエルシュ菌は嫌気条件下で培養した。結果を図３に示す。豆乳ケフィア摂取により、大腸菌およびウエルシュ菌の数は、１５日目には摂取前の５０％程度まで減少した。
Evaluation of the effect on the number of bad bacteria in feces Using Wistar / ST rats (6 weeks old, male), magnesium chloride prepared to 20 mg / ml is forcibly administered with a gastric tube to cause diarrhea to develop digestive tract bacteria Reduced. Human stools prone to constipation were suspended in physiological saline, and those transplanted into the above rats were used as human feces-transplanted rats. Then, 10 g of a feed prepared by mixing 6 mg of freeze-dried soymilk Kefir mixed with 100 g of powdered feed (CE-2, manufactured by CLEA Japan, Inc.) was consumed per human fecal transplant rat (Soy milk Kefir dried) Dose of 3.3 mg / kg body weight). The weight of human feces-transplanted rat was calculated as 180 g. After commercially available plain yoghurt (Meiji Bulgaria yoghurt) was sterilized and lyophilized as a control, 3.3 mg / kg body weight as a yoghurt dried substance was administered to a human feces transplant rat in the same manner. At the beginning of the test, feces were collected on the 15th and 30th days, and the numbers of E. coli and Welsch were determined using a selective medium. Specifically as follows: The feces sterile saline 1g to prepare a stock solution of 9ml addition 10-fold dilution, 10-fold serial dilutions 10 4 with physiological saline and sterilized to The dilutions made up to double were made. Desoxycolate agar medium (Merck) is used as a selective medium for E. coli, and 50% egg yolk fluid is added to 10% of kanamycin-containing CW agar base medium (Nissui) as a selective medium for Welsh bacteria. 15 ml of selective medium was added to 1 ml of serial dilutions and mixed, and incubated at 37 ° C. for 48 hours (incubation at n = 5 for each serial dilution). And the number in the feces of E. coli and Welsch was calculated from the number of colonies generated in each selective medium. In addition, Welsch was cultured under anaerobic conditions. The results are shown in FIG. The intake of soymilk kefir reduced the number of E. coli and H. perfringens to about 50% before intake on the 15th day.
糞便中総インドール含量に対する影響の評価
糞便中の総インドールは、ヒト糞便移植ラット５匹より集めた糞便１ｇを破砕し、Ｂａｃｔｏ（商標） Ｈｅａｒｔ Ｉｎｆｕｓｉｏｎ Ｂｒｏｔｈ（ベクトン・ディッキンソン社製）培地に入れ、３７℃で培養し、培地中のインドール量を保田らの方法（保田正人ら、ｐ−ヂメチルアミノベンズアルデヒドによるインドール定量法の変法−ＩＩ、インドールの簡易定量法、Bulletin of the Faculty of Fisheries, Nagasaki University, No.7, pp.101-107, 1958）に従って定量した。すなわち、培養後の上記培地５０ｍｌに硫酸０．５ｍｌを加え、２０ｍｌの石油エーテルを加え、ボルテックスで激しく振盪して抽出した。石油エーテル層に呈色試薬として４０ｍｇのｐ−ヂメチルアミノベンズアルデヒドを加え、室温下で振盪しながら発色させた。遠心分離（１，０００ｒｐｍ、１０分）により呈色層を得て、その５６５ｎｍの吸光度を測定した。そして、標準品のインドール（和光純薬工業社製、製品番号：０９４−００１６１）を１０ｍｇ／ｍｌの濃度になるように脱イオン水に溶解した標準溶液を上記培地に添加して希釈したものについて、同様の測定を行って、吸光度とインドール量の検量線を作成し、作成した検量線から、上記糞便を培養した後の培地中のインドール量を定量し、糞便中の総インドール含量を算出した。結果を表５に示す。
Evaluation of the effect on total indole content in feces Total indole in feces was broken into 1 g of feces collected from 5 human feces-transplanted rats and placed in BactoTM Heart Infusion Broth (Becton Dickinson) medium, 37 C., and the amount of indole in the medium was determined by the method of Yasuda et al. (H. Masada et al., A modified method of determination of indole with p-dimethylaminobenzaldehyde-II, simplified determination of indole, Bulletin of the Faculty of Fisheries, Nagasaki It quantified according to University, No. 7, pp. 101-107, 1958). That is, 0.5 ml of sulfuric acid was added to 50 ml of the above-mentioned culture medium, 20 ml of petroleum ether was added, and the mixture was vigorously shaken and extracted with a vortex. To the petroleum ether layer, 40 mg of p-dimethylaminobenzaldehyde was added as a color reagent, and color was developed while shaking at room temperature. The colored layer was obtained by centrifugation (1,000 rpm, 10 minutes), and the absorbance at 565 nm was measured. And about what added and diluted the standard solution which melt | dissolved in deionized water so that it might become a density | concentration of 10 mg / ml so that it might become a density | concentration of 10 mg / ml of standard indole (made by Wako Pure Chemical Industries, product number: 094-00161) The same measurement was performed to create a calibration curve of absorbance and indole content, and from the calibration curve created, the indole content in the culture medium after culturing the above feces was quantified, and the total indole content in feces was calculated. . The results are shown in Table 5.
ヒト糞便移植ラットの糞便中総インドール含量は８３．２μｇ／ｍｌで、比較対照の市販ヨーグルト摂取群は７６．２μｇ／ｍｌであり、１０％未満程度の減少であったが、豆乳ケフィア摂取群では８．８μｇ／ｍｌであり、９０％程度も減少していた。 The total fecal indole content of human feces-transplanted rats was 83.2 μg / ml, and that of the control group commercially available yogurt intake group was 76.2 μg / ml, which was less than 10%, but in the soymilk kefir intake group It was 8.8 μg / ml and decreased by as much as 90%.
血中総インドキシル硫酸濃度に対する影響の評価
血中総インドキシル硫酸は、ＨＰＬＣを用いた丹羽らの方法（丹羽俊充ら、透析患者におけるインドキシル硫酸の動態、透析学会誌21(10):951-956, 1988）に従って定量した。糞便についての上記試験終了時に採決を行い、定法で血清を分離し、ＨＰＬＣによりインドキシル硫酸を分析した。ＨＰＬＣの分析条件は以下のとおりである。
Evaluation of the effect on blood total indoxyl sulfate concentration Blood total indoxyl sulfate was determined by the method of Niwa et al. Using HPLC (Niwa Toshimitsu et al., Dynamics of indoxyl sulfate in dialysis patients, Journal of Japanese Society for Dialysis 21 (10): 951 It quantified according to -956, 1988). At the end of the above test for feces, it was scored, serum was separated by a standard method, and indoxyl sulfate was analyzed by HPLC. The analytical conditions of HPLC are as follows.
ＨＰＬＣシステム：ＬＣ−８０２０（東ソー株式会社製）
カラム：ＣＡＰＣＥＬＬＰＡＫ ＭＧＩＩ（内径２．０ｍｍ×長さ１００ｍｍ）（株式会社資生堂製）
カラム温度：４０℃
移動相：０．２Ｍ酢酸／トリエチルアミン（ｐＨ６．５）：イソプロパノール：テトラヒドロフラン（８４：１０：６，ｖ／ｖ）
流量：０．２０ｍｌ／分
試料注入量：１０μｌ
検出器：紫外可視検出器（ＵＶ−８０２０、東ソー株式会社製）
検出波長：２７０ｎｍ
標準品のインドキシル硫酸カリウム（ナカライテスク株式会社より入手、製品番号：１９２０８−０４）の分析結果との比較により、クロマトグラム上で血液試料中のピークを同定した。標準品および豆乳ケフィアより由来の試料より得られたクロマトグラムを、図２−１および図２−２にそれぞれ示す。
HPLC system: LC-8020 (manufactured by Tosoh Corporation)
Column: CAPCELLPAK MGII (internal diameter 2.0 mm × length 100 mm) (manufactured by Shiseido Co., Ltd.)
Column temperature: 40 ° C
Mobile phase: 0.2 M acetic acid / triethylamine (pH 6.5): isopropanol: tetrahydrofuran (84: 10: 6, v / v)
Flow rate: 0.20 ml / min Sample injection amount: 10 μl
Detector: UV-visible detector (UV-8020, manufactured by Tosoh Corporation)
Detection wavelength: 270 nm
The peak in the blood sample was identified on the chromatogram by comparison with the analysis result of standard product indoxyl sulfate potassium (obtained from Nacalai Tesque, Inc., product number: 19208-04). Chromatograms obtained from a standard product and a sample derived from soymilk kefir are shown in FIGS. 2-1 and 2-2, respectively.
得られたクロマトグラムを図４−１および図４−２に示す。また、血中の総インドキシル硫酸濃度（ｎ＝５）を表６に示す。 The obtained chromatograms are shown in FIGS. 4-1 and 4-2. Also, the total indoxyl sulfate concentration in blood (n = 5) is shown in Table 6.
インドキル硫酸は、図４−１および図４−３のクロマトグラム上において約８．３分に溶出しており、ヒト糞便移植ラットの血中総インドキシル硫酸濃度は３２．６μｇ／ｍｌ（約１５３．０μＭ）であった。比較対照の市販ヨーグルト摂取群では３５．７μｇ／ｍｌ（約１６７．６μＭ）であり、影響がみられなかったのに対し、豆乳ケフィア摂取群では４．２μｇ／ｍｌ（約１９．７μＭ）であり、約８７％も減少していた。 Indoxyl sulfate elutes at approximately 8.3 minutes on the chromatograms in Figures 4-1 and 4-3, and the total indoxyl sulfate concentration in blood of human feces-transplanted rats is 32.6 μg / ml (approximately .0 μM). The effect was seen at 35.7 μg / ml (about 167.6 μM) in the commercially available yogurt intake group of the comparison control, whereas it was 4.2 μg / ml (about 19.7 μM) in the soymilk kefir intake group. , About 87% decreased.
以上の結果から、実施例１で得られた豆乳ケフィアを摂取することにより、大腸菌やウエルシュ菌などの悪玉菌数が減少し、これらの菌が宿主の腸内において産生するインドールが減少することが確認された。そして、腎臓、肝臓、脳、心臓などの生体臓器の機能障害の原因となる宿主の体内でのインドールからのインドキシル硫酸の生成が、実施例１で得られた豆乳ケフィアの摂取により減少することが確認された。 From the above results, when the soymilk kefir obtained in Example 1 is consumed, the number of bad bacteria such as E. coli and Welsh bacteria decreases, and the indole produced by these bacteria in the intestine of the host decreases. confirmed. Then, the production of indoxyl sulfate from indole in the host's body causing dysfunction of living organs such as kidney, liver, brain and heart is reduced by the intake of the soymilk kefir obtained in Example 1. Was confirmed.
また、大腸菌やウエルシュ菌などの悪玉菌数が減少したことから、実施例１で得られた豆乳ケフィアの摂取により、これらの悪玉菌が引き起こすその他の種々の疾患の予防も可能であることが示唆された。 In addition, since the number of bad bacteria such as E. coli and Welsh bacteria decreased, it is suggested that the intake of the soymilk kefir obtained in Example 1 can also prevent other various diseases caused by these bad bacteria. It was done.
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| 大人キレイ, ケフィア豆乳ヨーグルト, 2017.03.29 [検索日 2021.08.16], インターネット：<URL: HTTPS://OT, JPN6021036095, ISSN: 0004719468 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021024811A (en) * | 2019-08-05 | 2021-02-22 | クラシエフーズ株式会社 | Urine toxin production inhibitory composition |
| JP7397597B2 (en) | 2019-08-05 | 2023-12-13 | クラシエ株式会社 | Composition for suppressing uremic toxin production |
| JP7479833B2 (en) | 2019-12-18 | 2024-05-09 | 小林製薬株式会社 | Estrogenic Active Agent Compositions |
| JP7479832B2 (en) | 2019-12-18 | 2024-05-09 | 小林製薬株式会社 | Oral Compositions |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109965269A (en) | 2019-07-05 |
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